CN103834594A - Epsilon-polylysine bacterium and preparation and fermentation method thereof - Google Patents
Epsilon-polylysine bacterium and preparation and fermentation method thereof Download PDFInfo
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Abstract
The invention provides an epsilon-polylysine bacterium and a preparation and fermentation method thereof. The epsilon-polylysine bacterium (Streptomyces griseolusk6) is preserved in Sichuan Industrial Institute of Antibiotics on October 10, 2013, the preservation number is CCSIIA 51309, the epsilon-polylysine bacterium is inoculated into an active culture medium, cultured on a table concentrator of 37 DEG C for 18-20 hours and is centrifuged for 5 minutes under a condition of 8000 rpm/min, a precipitate is washed by sterile water and is prepared to bacterial liquid with concentration of 107 CFU/ml; the bacteria liquid is inoculated into a fermentation medium according to an inoculum size of 6% of the volume of the fermentation medium and a liquid volume of 20 mL/mL, and is cultured for 10-16 hours at a fermentation temperature of 30 DEG C, the initial pH of 7.0 and a rotating speed of the table concentrator of 200 r/min to obtain epsilon-polylysine fermentation liquid. The epsilon-polylysine bacterium and the fermentation method thereof provided by the invention can be used for greatly improving the yield of epsilon-PL, providing favorable information for the long-term even food industrial mass production of the epsilon-PL and laying a solid foundation and has a very important significance.
Description
Technical field
The invention belongs to fermentation technical field, relate in particular to a kind of ε-poly-lysine bacterium and preparation and fermentation process.
Background technology
Food preservatives is one of main class in foodstuff additive field, the food preservatives output of China has reached more than 40,000 ton, but the sanitas using is at present taking chemosynthesis sanitas as main, scientist is in long-term research, and the sanitas of finding some chemosynthesis has and lures carcinous, teratogenecity and easily cause the phenomenon that things is poisoning.Along with the raising of people to standard of living and the understanding to food safety and requirement, foodstuff additive have also started strong back to nature, have enjoyed green and healthy tide, and chemosynthesis sanitas is also more and more subject to people's repulsion.And ε-poly-lysine (ε-Poly-L-lysine, ε-PL) is to cultivate mainly with the aerobic fermentation of streptomyces a kind of natural microbial meta-bolites obtaining, it is the leavened prod obtaining through separation and purification.Epsilon-polylysine (ε-PL) is the homopolymerization polypeptide by Methionin monomer composition, has the advantages such as Heat stability is good, water-soluble strong, safe, has a broad antifungal spectrum, is widely used in food fresh keeping.In addition, in the field such as preparation, macromolecular material of gene therapy, microcapsule medicament, polylysine also has purposes widely, superior antiseptic property and huge business potential.ε-PL has broad-spectrum antibacterial, in acid and slightly acidic environment, gram-positive microorganism, Gram-negative bacteria and yeast, mould is all had to certain fungistatic effect, and it also has restraining effect to thermotolerance genus bacillus and some viruses.Therefore, ε-PL becomes the focus and emphasis that relevant industries personage pays close attention to.
Summary of the invention
The object of the present invention is to provide the fermentation process of a kind of ε-poly-lysine bacterium, aim to provide the fermented bacterium of a kind of ε of having-poly-lysine high yield potentiality.
A further object of the present invention is to provide the preparation method of above-mentioned ε-poly-lysine bacterium.
Another object of the present invention is to provide the fermentation process of above-mentioned ε-poly-lysine bacterium, be intended to solve the problem that output is very low, separation and purification is loaded down with trivial details of existing fermentation process ε-poly-lysine.
The present invention is achieved in that a kind of ε-poly-lysine bacterium (Streptomycesgriseolusk6), is preserved in Sichuan Industrial Institute of Antibiotics on October 10th, 2013, and deposit number is CCSIIA-51309.
The preparation method who the present invention further provides above-mentioned ε-poly-lysine bacterium, comprises the following steps:
(1) by dull and stereotyped methylene blue and the K of adding of SG
2cr
7o
7preliminary screening obtains epsilon-polylysine producing strains TUST-2;
(2) mutagenic treatment of the nitrosoguanidine by various dose to epsilon-polylysine producing strains TUST-2, determines mutagenesis dosage according to lethality rate, adopts dilution for many times method to stop mutagenesis when mutagenesis finishes;
(3) and bacterium colony growing way good mutagenesis bacterium large with the dull and stereotyped primary dcreening operation transparent circle of SGB, determines the concentration of glycine and ε-PL in SGB flat board, sifts out again the mutagenesis bacterium that upgrowth situation is good by glycine and ε-PL resistant panel;
(4) the mutagenesis bacterium of sifting out is again fermented and detect the output of ε-PL, finally filter out the mutagenic fungi that ε-PL output is the highest.
The fermentation process that the present invention further provides a kind of ε-poly-lysine, comprises the following steps:
(1) above-mentioned ε-poly-lysine bacterium bacterial classification is inoculated in activation medium, 37 DEG C of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid;
(2) described bacterium liquid is seeded in fermention medium by liquid amount 20mL/mL by the inoculum size of fermention medium 6% volume, under 30 DEG C of leavening temperatures, initial pH7.0, shaking speed 200r/min condition, cultivate 10～16h, obtain ε-poly-lysine fermented liquid.
Preferably, in step (1), activation medium comprises following each component by mass described in 1L:
Extracted beef powder 2～3g,
Sodium-chlor 4～5g.
Preferably, in step (1), fermention medium comprises following each component by mass described in 1L:
The present invention overcomes the deficiencies in the prior art, a kind of ε-poly-lysine bacterium and preparation and fermentation process are provided, this ε-poly-lysine bacterium (Streptomycesgriseolusk6), be preserved in Sichuan Industrial Institute of Antibiotics on October 10th, 2013, deposit number is CCSIIA-51309, by dull and stereotyped methylene blue and the K of adding of SG
2cr
7o
7preliminary screening obtains epsilon-polylysine producing strains TUST-2; The mutagenic treatment of nitrosoguanidine by various dose to epsilon-polylysine producing strains TUST-2, determines mutagenesis dosage according to lethality rate, adopts dilution for many times method to stop mutagenesis when mutagenesis finishes; The large and good mutagenesis bacterium of bacterium colony growing way with the dull and stereotyped primary dcreening operation transparent circle of SGB, determines the concentration of glycine and ε-PL in SGB flat board, sifts out again the mutagenesis bacterium that upgrowth situation is good by glycine and ε-PL resistant panel; Finally the mutagenesis bacterium of sifting out is again fermented and detect the output of ε-PL, obtain after finally filtering out the mutagenic fungi that ε-PL output is the highest.
Obtained ε-poly-lysine bacterium is carried out to fermentation condition optimization, by this ε-poly-lysine bacterium bacterial classification is inoculated in activation medium, 37 DEG C of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid; Described bacterium liquid is seeded in fermention medium by liquid amount 20mL/mL by the inoculum size of fermention medium 6% volume, under 30 DEG C of leavening temperatures, initial pH7.0, shaking speed 200r/min condition, cultivates 10～16h, obtain ε-poly-lysine fermented liquid.
By optimizing fermented bacterium and carrying out according to this bacterial classification after comprehensive optimization of substratum, fermentation condition, ε-PL output bacterium epsilon-polylysine producing strains TUST-2 that sets out has improved 10.02%.The long-range even food industrialization scale operation that this is ε-PL provides favourable data, and pad has descended solid basis, has very important meaning.
Brief description of the drawings
Fig. 1 is ε-poly-lysine canonical plotting in the embodiment of the present invention 1;
Fig. 2 is that in the embodiment of the present invention 1, glue purification reclaims DNA fragmentation electrophorogram;
Fig. 3 is that in the embodiment of the present invention 1, EcoR I enzyme is cut recombinant plasmid dna electrophorogram;
Fig. 4 is the phylogeny tree graph based on 16SDNA sequence homology in the embodiment of the present invention 1;
Fig. 5 is thalline weight in wet base and ε-PL Relationship with Yield figure in the embodiment of the present invention 1;
Fig. 6 is thalline weight in wet base and pH graph of a relation in the embodiment of the present invention 1.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
In following examples, the reagent that adopts, originate as follows: 1-methyl-3-nitro-1-nitrosoguanidine (Tokyo chemical industry Co., Ltd.); Acetone (Chengdu Ke Long chemical reagent factory); Glycine; ε-PL standard substance (Lanzhou Weiri Bio-Engineering Co., Ltd.); Bismuth subnitrate; Glacial acetic acid; Potassiumiodide; Na
2hPO
412H
2o; NaH
2pO
42H
2o; NaCl etc.Note: do not mark the medicine of producer all purchased from Tianjin Kermel Chemical Reagent Co., Ltd..
The instrument adopting and source are: LDZX-75KB vertical pressure steam sterilization pan (Shenan Medical Appliances Factory, Shanghai); TGL-16G whizzer (Anting Scientific Instrument Factory, Shanghai); LRH biochemical cultivation case (Shanghai Yiheng Scientific Instruments Co., Ltd); HH-4 digital display thermostat water bath (Changzhou Ao Hua Instrument Ltd.); ZHJHC1209B Bechtop (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.); UV-2600 type ultraviolet-visible pectrophotometer (Shanghai You Nike Instrument Ltd.); YLE-25 gene-amplificative instrament (Hangzhou Lang Ji scientific instrument company limited); DYY-4 type voltage stabilization and current stabilization electrophoresis apparatus (Beijing Liuyi Instrument Factory).
1, the dull and stereotyped primary dcreening operation of SGB
Refer to reference (Tan Zhilei, etc. the isolation identification [J] of epsilon-polylysine producing strains TUST-2 for Jia Shiru, Xu Chunying. microorganism journal, 2010,50(2): 191 – 196).
2, qualitative and quantitative detection
(1) qualitative detection
Drip DR(dragendorff to fermented supernatant fluid addend) reagent, if there is brick-red precipitation, positive, show to have ε-PL to produce.In addition, the ε-PL reference liquid with 0.5% in contrast, carries out paper chromatography detection to fermented supernatant fluid, if occur, the phenomenon identical with ε-PL reference liquid shows there is ε-PL generation.
(2) detection by quantitative, refer to reference (Liao Lijuan, improves [J] etc. the screening method of. epsilon-polylysine producing strains for Japanese plum, Chen Xusheng. food and biotechnology journal, 2010,29(2): 282-287).
3, biocidal property detects
Refer to reference (Yang Yuhong. the research [D] of screening, qualification and the fermentation of epsilon-polylysine producing strains. Shenyang: Agricultural University Of Shenyang, 2007. biology circulars, 2011,38(6): 871-877), taking intestinal bacteria as test organisms, filter paper method detects fermented liquid biocidal property.
4, the preparation of ε-poly-lysine typical curve
Refer to reference (Yang Yuhong. the research [D] of screening, qualification and the fermentation of epsilon-polylysine producing strains. Shenyang: Agricultural University Of Shenyang, 2007. biology circulars, 2011,38(6): 871-877), with OD
465for X-coordinate, the mass concentration of ε-PL be ordinate zou draw ε-PL typical curve as shown in Figure 1.And know coefficient of determination R by Fig. 1
2=0.9973, the dependency of curve is good; Fitting formula y=-5.1064x+3.4425(y is ε-PL mass concentration, and x is OD
465).
5, the molecular biology identification based on 16SrDNA
(1) pcr amplification target DNA fragment
The present invention chooses actinomycetes 16SrDNA universal primer as pcr amplification primer, wherein,
Forward primer P1:5'-AGAGTTTGATCCTGGCTCAG-3 ',
Reverse primer P2:5'-AAGGAGGTGATCCAGCCGCA-3 ';
Cellular lysate supernatant liquor is as PCR reaction template; Amplification condition is: 95 DEG C of denaturation 5min, and 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C are extended 2min, 30 circulations, last 72 DEG C are extended 10min.
(2) preparation of the recovery of target DNA fragment purifying and competent cell DH5a
Purify and reclaim test kit the target DNA fragment of about 1.5kb is carried out to glue purification recovery with the universal DNA of TIANGEN.Reclaim result as shown in Figure 2, in Fig. 2, swimming lane 1:Y# glue reclaims DNA solution, 5 μ L/Lane; Swimming lane 2:8# glue reclaims DNA solution, 5 μ L/Lane; Swimming lane 3:4# glue reclaims DNA solution, 5 μ L/Lane; Swimming lane M: λ Hind III digestMarker, 5 μ L/Lane.Only there is as can see from Figure 2 the single bright band of about 1500bp, illustrate that purifying recovering effect is better.
Prepare DH5a competent cell according to the CompetentCellPreparationKit test kit of TAKARA company.
(3) target DNA fragment is connected with PMD19-TVector and transforms
Carry out the connection of T carrier and transform escherichia coli DH5a according to the specification sheets of the pMD19-TVector of TAKARA company.Random cultivation transforms the single bacterium colony in LB-Amp flat board and extracts plasmid, then uses EcoR I digested plasmid DNA, selects recombinant plasmid for sequencing analysis.
By each reaction system, more than 37 DEG C of water-bath endonuclease reaction 1h, the agarose gel electrophoresis with 0.7% detects the result that obtains Fig. 3, in Fig. 3, and swimming lane M: λ Hind III digestMarker, 5 μ L/Lane; Swimming lane 1:4-7 plasmid DNA, 5 μ L/Lane; Swimming lane 2:4-5 enzyme is cut product, 5 μ L/Lane; Swimming lane 3:4-6 enzyme is cut product, 5 μ L/Lane; Swimming lane 4:4-7 enzyme is cut product, 5 μ L/Lane; Swimming lane 5:8-1 plasmid DNA; 5 μ L/Lane; Swimming lane 6:8-1 enzyme is cut product, 5 μ L/Lane; Swimming lane 7:8-3 enzyme is cut product, 5 μ L/Lane; Swimming lane 8:8-6 enzyme is cut product, 5 μ L/Lane; Swimming lane 9:Y-5 plasmid DNA, 5 μ L/Lane; Swimming lane 10:Y-5 enzyme is cut product, 5 μ L/Lane; Swimming lane 11:Y-8 enzyme is cut product, 5 μ L/Lane; Swimming lane 12:Y-10 enzyme is cut product, 5 μ L/Lane.
Because pcr amplified fragment size is about 1500bp, and PMD19-Tvector size is 2692bp, and therefore the clip size of successful connection is about 4100bp left and right, can see that by Fig. 3 minority is two bright bands, most three.And two less stripe size and be about 4100bp, illustrate all successful connections and have two EcoR I restriction enzyme sites, and screen 4-7#, 8-1#, Y-8# is sent to Zi Xi bio tech ltd, Beijing and carries out 16SrDNA order-checking.
(4) data analysis and processing
Screening enzyme is cut to 3 strain bacterium of detection successful connection and be sent to the order-checking of Zi Xi bio tech ltd, Beijing.The sequence recording is submitted to and in GenBank database, carries out homologous sequence search (Blast), then carry out Multiple Sequence Alignment analysis with ClustalW, finally by Mega4.0 phylogenetic tree construction, and analyze to determine and sieve to obtain the kind of bacterial strain.
Convert the sequence recording to forward sequence, be submitted to GenBank database after removing carrier sequence, obtain sequence accession number and be: Y-8(KC292488), 4-7(KC292489), 8-1(KC292490).Then enter NCBI Blast and carry out homologous sequence search, then carry out Multiple Sequence Alignment and Mega4.0 phylogenetic tree construction with ClustalW.Its result is as Fig. 4 and as shown in table 1 below:
Table 1Blast similarity searching result
From table 1 and Fig. 4,4# ranges Delftia and belongs to, and all reaches 99% with the similarity of known bacterium Delftia lacustris and Delftia acidovorans, 8# ranges Stenotrophomonas and belongs to, and reaches 99% with the similarity of known bacterium Stenotrophomonas maltophilia, Y# ranges Strep-tomyces and belongs to, and all reaches 99% with the similarity of known bacterium Streptomyces griseolus and Streptomyces chattanoogensis, by reference (Zhong Weihong. genetic engineering technique experiment instruction [M]. Beijing: Chemical Industry Press, 2007 (1): 95-102. and STACKEBRANDTE, GOEBELBM.Taxonomic note:a plce for DNA-DNA reassociation and16S rRNA sequence analysis in the present species definition in bacteriology[J] .International Journal of Systematic Bacteriology, 1994, 44 (4): 846-849.) can think a kind when sequence homology >=97% of proper 16SrRNA.And according to the distance analysis of growing tree branch situation, result shows that 4# may be Delftia acidovorans, and 8# may be stenotrophomonas maltophilias, and Y# may be Streptomyces griseolus.
6, monospore suspension preparation
By the phosphoric acid buffer replacement stroke-physiological saline solution of 0.1mol/LpH6.0, refer to reference (Li Shuanshuan, Wu Zhenqiang, Wu Qing equality. produce epsilon-polylysine streptomyces albus complex mutation breeding research [J]. China brewages, and 2010, (12): 108-111.) and (Zhang Chao, Wang Zhenggang, Duan Zuoying etc. heavy dose of ultraviolet mutagenesis seed selection epsilon-polylysine Producing Strain [J]. biological processing, 2007,8(3): 64-67.).
7, NTG mutagenic treatment
The mutagenic treatment of nitrosoguanidine by various dose to starting strain, determines mutagenesis dosage according to lethality rate, adopts dilution for many times method to stop mutagenesis when mutagenesis finishes.
Determining of NTG mutagenic condition carried out mutagenic treatment by the NTG of various dose to starting strain, and result is as shown in table 2 below:
Table 2 mutagenic treatment rear plate colony number
As can be seen from Table 2, in the time that NTG mutagenesis final concentration is 1.5mg/mL processing 60min, lethality rate has reached 96.25%, in high mutation rate region.Therefore determine that mutagenic condition is: NTG mutagenesis final concentration 1.5mg/mL, mutagenic treatment 60min.
8, the screening of object mutagenic strain
(1) screen the larger transparent circle bacterium colony of 200 strain numbering according to the principle of ε-PL and methylene blue formation transparent circle.
(2) concentration of determining glycine and ε-PL in resistant panel (refers to reference: (Li Shuanshuan, Wu Zhenqiang, Wu Qing equality. produce epsilon-polylysine streptomyces albus complex mutation breeding research [J]. China brewages, 2010,: 108-111.) and (Zhang Chao (12), Wang Zhenggang, Duan Zuoying etc. heavy dose of ultraviolet mutagenesis seed selection epsilon-polylysine Producing Strain [J]. biological processing, 2007,8(3): 64-67.)), sift out by glycine and ε-PL resistant panel the mutagenesis bacterium that 50 strain upgrowth situations are good.
Can obtain glycine according to the resistant panel the selection result of different concns gradient has certain bacteriostatic action, but DeGrain, ε-PL has obvious bacteria resistance function.In the time that in flat board, ε-PL concentration reaches 0.30mg/mL, test organisms almost can not be grown, and considers that glycine also has certain bacteria resistance function, therefore determine that ε-PL concentration is 0.25mg/mL.On colony number, impact is not obvious especially to glycine, but comparatively remarkable on the impact of bacterium colony size,, in the time that glycine concentration reaches 6mg/mL, colony diameter is all larger.Glycine and the difference analysis of ε-PL to bacteria resistance function of comprehensive different concns, finally in definite resistant panel, the concentration of glycine and ε-PL is respectively 6mg/mL and 0.25mg/mL.
(3) antagonism flat screen obtains the output of strain fermentation detection ε-PL, finally filters out the mutagenic fungi that ε-PL output improves.
The resistant panel screening of determining concentration for the 200 strain mutagenic strains that primary dcreening operation is obtained, obtain being numbered the good mutagenic strain of 50 strain colony growth situation of K-1～K-50, and it is carried out to shake flask fermentation detection, and the ε-PL output that obtains K-6 is the highest, and its mass concentration has reached 0.854g/L.
9, object mutagenic fungi study on the stability
K-6 is carried out to the cultivation of going down to posterity of 4 inclined-planes, and to per generation strain fermentation detect ε-PL output to measure the stability of the bacterial strain that goes down to posterity, its result is as shown in table 3 below:
Table 3 mutant strain K-6 4 impacts on ε-PL output of going down to posterity
As can be seen from Table 3, the mean value of the ε-PL content cultivated of obtaining going down to posterity for 4 times is 0.852g/L, is more or less the same, therefore can think that time mutant strain inherited character is comparatively stable with former mutant strain output.
10, the growth curve of object mutagenic strain is measured
Reflect its growing state taking thalline weight in wet base as index, in the fermenting process of 72h, measure a thalline weight in wet base every 8h, and measure the pH of fermented liquid and ε-PL output in order to understand the pH of fermented liquid in fermenting process and change and the production of ε-PL simultaneously, its measurement result is shown in Table 4:
Table 4 fermenting process K-6 growing state
According to table 4 data, taking incubation time as X-coordinate, thalline weight in wet base, ε-PL output and pH value have been drawn respectively K-6 strain growth curved line relation for ordinate zou, as illustrated in Figures 5 and 6.Can find out from Fig. 5 and Fig. 6,0～24h in adjustment period strain growth slow, 24～32h is that logarithmic phase increases sharply, 32～56h be stationary phase thalline weight in wet base substantially keep constant, 56～72h is decline phase, and therefore the seed activation time generally need to reach 24h.And obtain the content of ε-PL and the changing conditions of pH value is: in 0～32h stage, the content of ε-PL starts to increase, and during 32～56h, the content of ε-PL almost reaches the steady stage, and 56h starts to reduce later; And pH maintains 6.8 left and right in 0～24h, drop between 3.0～4.0 in moment between 24～32h, be slowly reduced to 3.0 thereafter.Suitable to pH4.0 left and right due to ε-PL accumulation, can find out with it and be consistent according to two figure above, between 32～56h, is therefore the best period of ε-PL accumulation.
11, by obtained K-6 bacterial strain called after ε-poly-lysine bacterium (Stre
ptom
yces
griseolusk6), and be preserved in Sichuan Industrial Institute of Antibiotics on October 10th, 2013, deposit number is CCSIIA-51309.
A fermentation process for ε-poly-lysine, comprises the following steps:
(1) above-mentioned ε-poly-lysine bacterium bacterial classification is inoculated in activation medium, 37 DEG C of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid;
In step (1), activation medium comprises following each component by mass described in 1L:
Extracted beef powder 2～3g,
Sodium-chlor 4～5g.
(2) described bacterium liquid is seeded in fermention medium by liquid amount 20mL/mL by the inoculum size of fermention medium 6% volume, under 30 DEG C of leavening temperatures, initial pH7.0, shaking speed 200r/min condition, cultivate 10～16h, obtain ε-poly-lysine fermented liquid;
In step (2), fermention medium comprises following each component by mass described in 1L:
ε-PL output in ε-poly-lysine fermented liquid is determined as to 0.895g/L.Fermentation culture experiment in triplicate, fermentation results is got to its mean value, and to have obtained ε-PL output be 0.889g/L.
The epsilon-polylysine producing strains TUST-2 obtaining by the dull and stereotyped primary dcreening operation of SGB in embodiment 1 is replaced to the ε-poly-lysine bacterium bacterial classification in the present embodiment, revision test, in ε-poly-lysine fermented liquid of acquisition, ε-PL output is determined as 0.808g/L.
In the present invention, by optimizing fermented bacterium and carrying out according to this bacterial classification after comprehensive optimization of substratum, fermentation condition, ε-PL output bacterium epsilon-polylysine producing strains TUST-2 that sets out has improved 10.02%.The long-range even food industrialization scale operation that this is ε-PL provides favourable data, and pad has descended solid basis, has very important meaning.
Compare the shortcoming and defect with prior art, the present invention has following beneficial effect: ε-poly-lysine bacterium of the present invention can significantly be improved the fermentation yield of epsilon-polylysine, for the long-range even food industrialization scale operation of ε-PL provides favourable data, pad has descended solid basis, has very important meaning.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. ε-poly-lysine bacterium (Streptomycesgriseolusk6), is preserved in Sichuan Industrial Institute of Antibiotics on October 10th, 2013, and deposit number is CCSIIA-51309.
2. the preparation method of ε-poly-lysine bacterium claimed in claim 1, is characterized in that comprising the following steps:
(1) by dull and stereotyped methylene blue and the K of adding of SG
2cr
7o
7preliminary screening obtains epsilon-polylysine producing strains TUST-2;
(2) mutagenic treatment of the nitrosoguanidine by various dose to epsilon-polylysine producing strains TUST-2, determines mutagenesis dosage according to lethality rate, adopts dilution for many times method to stop mutagenesis when mutagenesis finishes;
(3) and bacterium colony growing way good mutagenesis bacterium large with the dull and stereotyped primary dcreening operation transparent circle of SGB, determines the concentration of glycine and ε-PL in SGB flat board, sifts out again the mutagenesis bacterium that upgrowth situation is good by glycine and ε-PL resistant panel;
(4) the mutagenesis bacterium of sifting out is again fermented and detect the output of ε-PL, finally filter out the mutagenic fungi that ε-PL output is the highest.
3. a fermentation process for ε-poly-lysine, is characterized in that comprising the following steps:
(1) ε-poly-lysine bacterium bacterial classification claimed in claim 1 is inoculated in activation medium, 37 DEG C of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid;
(2) described bacterium liquid is seeded in fermention medium by liquid amount 20mL/mL by the inoculum size of fermention medium 6% volume, under 30 DEG C of leavening temperatures, initial pH7.0, shaking speed 200r/min condition, cultivate 10～16h, obtain ε-poly-lysine fermented liquid.
4. the fermentation process of ε-poly-lysine as claimed in claim 3, is characterized in that, in step (1), activation medium comprises following each component by mass described in 1L:
Peptone 9～10g,
Extracted beef powder 2～3g,
Sodium-chlor 4～5g.
5. the fermentation process of ε-poly-lysine as claimed in claim 4, is characterized in that, in step (1), fermention medium comprises following each component by mass described in 1L:
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