Summary of the invention
The objective of the invention is for the problems referred to above, provide a kind of fermentation unit high, can stably manufactured, output is high and by product is few knob Kangding B not
0Produce bacterium.
Another object of the present invention is to provide this bacterial strain and is preparing not Kangding B of knob
0In application, and concrete fermentation preparation.
The objective of the invention is to realize by following technical proposal:
A kind of fungi FIM2006071, Classification And Nomenclature are Glarea lozoyensis FIM2006071, and by Chinese Typical Representative culture collection center preservation (being called for short CCTCC), preserving number is: CCTCC NO:M2012475, preservation date is: on November 23rd, 2012.
Described preserving number is that the bacterial strain of CCTCC NO:M2012475 obtains from the screening of Foochow, Fujian Province.
Described preserving number is the colony characteristics of the bacterial strain of CCTCC NO:M2012475: the bacterium colony subcircular, and edge white, irregular fan-shaped, colony diameter 7.5-17.8mm, bacterium colony protuberance, in be formed centrally knot or subside into the hole, middlely produce without secretory product and soluble pigment.
Description according to microbial morphology and external related data, be the various cultural characteristics of the bacterial strain of CCTCC NO:M2012475 in conjunction with preserving number, this preserving number is that the bacterial strain of CCTCC NO:M2012475 belongs to fungi, names the FIM2006071 into Glarea lozoyensis.
Described preserving number is that the bacterial strain of CCTCC NO:M2012475 can be applicable to not Kangding B of fermentation preparation knob
0This preparation method comprises the following steps:
A. bacterial strain employing deposit number is the bacterial strain of CCTCC NO:M2012475;
The 30% glycerine mycelia freeze pipe that b. will prepare according to a conventional method is by the inoculum size access shake-flask seed substratum of the 3-5% of shake-flask seed culture volume, and 220-260rpm cultivated 2-3 days, got shake-flask seed liquid; Shake-flask seed liquid is inoculated in seeding tank by the inoculum size that the seed tank culture matrix amasss 0.1-0.2%, and 120-200 rpm cultivated 3-4 days, got the tank seed liquor; The tank seed liquor is inoculated in fermentation tank culture medium by the inoculum size of the 5-15% of fermentation volume, and 150-200 rpm fermentation culture 9-12 days, collects fermented liquid; Wherein culture temperature is 22-26 ℃.
The ratio of wherein said each component of shake-flask seed substratum in substratum is: in every 100mL substratum, and carbon source 1-8g, nitrogenous source 2-8g, inorganic salt 0-2.5g, all the other are water;
The ratio of each component of seed tank culture base in substratum is: in every 100mL substratum, and carbon source 2-8g, nitrogenous source 1-7g, inorganic salt 0-2.5g, all the other are water;
The ratio of each component of fermention medium in substratum is: in every 100mL substratum, and carbon source 8-16g, nitrogenous source 1-5g, inorganic salt 0-1g, amino acid 0-2g, all the other are water.
Each component of wherein said substratum ratio in substratum is preferably:
The ratio of each component of shake-flask seed substratum is: in every 100mL substratum, and carbon source 2-4g, nitrogenous source 4-6g, inorganic salt 1-2g, all the other are water;
The ratio of each component of seed tank culture base is: in every 100mL substratum, and carbon source 4-6g, nitrogenous source 3-5g, inorganic salt 0.5-1.5g, all the other are water;
The ratio of each component of fermention medium is: in every 100mL substratum, and carbon source 10-14g, nitrogenous source 2-4g, inorganic salt 0.3-0.5g, amino acid 0.5-1.5g, all the other are water.
Wherein said carbon source is selected from potato starch, W-Gum, Zulkovsky starch, maltodextrin, potato dextrin, and glycerine, glucose, maltose, murphy juice, potato leach one or more in powder, N.F,USP MANNITOL; Described nitrogenous source is selected from soybean cake powder, gives birth to one or more in bean powder, dregs of beans, corn steep liquor, corn starch, silkworm chrysalis hydrolyzed solution, urea, ammonium sulfate, yeast powder, yeast extract powder, cottonseed meal, dried silkworm chrysalis meal; Inorganic salt are K
2HPO
4, MnSO
44H
2O, NaCl, Fe (NH
4)
2(SO
4)
26H
2O, NaNO
3, KNO
3, MgSO
47H
2O, FeSO
47H
2O, KH
2PO
4In one or more; Amino acid is one or more in 1B, L-Leu, ILE, TYR, L-threonine, Serine, L-PROLINE, L-arginine.
Wherein said medium optimization is: carbon source is one or more in glucose, N.F,USP MANNITOL; Nitrogenous source is yeast extract powder, corn starch, yeast powder, cottonseed meal, gives birth to one or more in bean powder; Inorganic salt are KH
2PO
4, K
2HPO
4, MnSO
44H
2O, FeSO
47H
2One or more in O; Amino acid is one or more in L-PROLINE, L-threonine.
Preserving number of the present invention is that the bacterial strain of CCTCC NO:M2012475 is the knob Kangding B not of a kind of natural high unit of being separated at present
0The generation bacterium, leavening property is good, can be used for scale operation.Fermenting process is not Kangding B of knob
0The important step of producing, its fermentation level is directly related with the technique quality, tests its stable production capacity in zymotechnique process shaking flask provided by the invention and 10 tons of fermentor tanks.After testing, with the knob of bacterial classification provided by the invention and fermentation culture method preparation Kangding B not
0Fermentation unit can up to 3000 about μ g/mL or more than, thereby provide good basis for follow-up suitability for industrialized production.Fermentation byproduct of the present invention is relatively less simultaneously, and has reduced the difficulty of rear extraction, thereby is conducive to the acquisition of high-quality Caspofungin, is being applied to have advantage on suitability for industrialized production.
Bacterial strain preservation situation: be preserved in Chinese Typical Representative culture collection center, Wuhan (being called for short CCTCC), deposit number is CCTCC NO:M2012475, and Classification And Nomenclature is fungi FIM2006071(Glarea lozoyensis FIM2006071).Preservation date is on November 23rd, 2012.
Embodiment
Be that the present invention conducts further description below in conjunction with specific embodiment, but method related in scheme and technical parameter can not be interpreted as limitation of the present invention.
Embodiment 1: preserving number is cultivation and physiological and biochemical property and the utilization of carbon source situation of the bacterial strain of CCTCC NO:M2012475
1, preserving number is the morphological feature of the strain culturing of CCTCC NO:M2012475
The inoculation that with preserving number is CCTCC NO:M2012475 is used for observing the substratum of morphological specificity in yeast malt extract nutrient agar (YME), potato glucose agar medium (PDA), sabouraud's agar (SMA) etc., cultivated for 3 weeks, observe and be described for 23 ℃.
Bacterium colony is 23 ℃ of cultivations on the YME nutrient agar, growth limitation, 3 all diameter 11.7-15.4mm; The bacterium colony subcircular, the edge is irregular fan-shaped; Bacterium colony protuberance, in be formed centrally knot or subside into the hole; The velvet-like double rope form of quality; The bacterium colony surface forms radioactive wrinkle; Mycelia color bois de rose is extremely rose pink, the light brown of reverse side; Produce without secretory product and soluble pigment.
Bacterium colony is 23 ℃ of cultivations on the PDA substratum, and the bacterium colony circular edge is slightly irregular, and the thicker central elevation of bacterium colony has irregular radioactivity wrinkle; Colony growth is slower, 3 all diameter 7.5-17.8mm, the velvet-like double rope form of quality.Bacterium colony is yellow-white the most at the beginning, white powder, and along with cell age increase to form dark bacterium colony, it is filbert that the center is, the adularescent edge; The reverse side color is yellow-white at the beginning, extends Vandyke brown with incubation time.Produce without secretory product and soluble pigment.
Bacterium colony is similar on the PDA substratum at cultural characteristic on the SMA substratum.The growth limitation, 3 all diameter 11.2-15mm; The bacterium colony subcircular, the edge is irregular; Bacterium colony protuberance, in be formed centrally knot; The velvet-like double rope form of quality; The bacterium colony surface forms radioactive wrinkle; The light pink of mycelia is to white powder, reverse side color yellow-white; Produce without secretory product and soluble pigment.
2, preserving number is the bacterial strain Physiology and biochemistry cultural characteristic of CCTCC NO:M2012475
Bacterial strain FIM2006071 can not degraded cellulose.Six kinds of two pH cultural characteristics of substratum are listed in table 1.
Six kinds of medium pHs 4 of table 1 and pH7 cultural characteristic
Substratum |
Growing state |
The base silk |
The gas silk |
Soluble pigment |
Bell potato agar glucose pH4 |
+++ |
Lead |
Light grey |
Olive brown |
Bell potato agar glucose pH7 |
+++ |
Tawny |
Pale pinkish grey |
Olive brown |
Yeast malt extract nutrient agar pH4 |
+++ |
Light yellow |
White powder |
— |
Yeast malt extract nutrient agar pH7 |
+++ |
Light yellow |
White powder |
— |
Sabouraud's agar pH4 |
+++ |
Light yellow |
White powder |
— |
Sabouraud's agar pH7 |
+++ |
Tawny |
White powder |
— |
Czapek agar medium pH4 |
++ |
Light tan |
— |
— |
Czapek agar medium pH7 |
+ |
White |
— |
— |
Glucose czapek agar medium pH4 |
+ |
Light tan |
— |
— |
Glucose czapek agar medium pH7 |
+ |
White |
— |
— |
Glycerine czapek agar medium pH4 |
+ |
White |
— |
— |
Glycerine czapek agar medium pH7 |
+ |
White |
— |
— |
[0036]+: growth is general; ++: grow medium; +++: growth is abundant;-: lack.
Soak at soil that on the juice nutrient agar, (10% soil soaks juice, and V/V), 23 ℃ of cultivations begin to form conidium in 2-3 week.Mycelia has tabula, diameter 2.5-3.5 μ m.Conidium budding type directly breaks up generation by mycelia, without conidiophore; Conidium group's size, shape differ and form the structure of chain, bunch shape or bulk; Conidium is spherical, subsphaeroidal, diameter 5.0-14.4 μ m * 4.8-12 μ m.
3, utilization of carbon source
This time test is take the PDA substratum as basic medium, and 26 ℃ of cultivations were observed at the 3rd, 7,10 day respectively.Result is as shown in table 2 below, and test-results shows, this bacterial strain can assimilate D-semi-lactosi, sweet and pure, pectinose, N.F,USP MANNITOL, inositol, rhamnosyl, sweet dew alcohol and glucose.
The utilization of carbon source ability of table 2 fungi CCTCC NO:M2012475
Carbon source |
3 days |
7 days |
10 days |
Blank |
﹣ |
﹣ |
﹣ |
The D-semi-lactosi |
﹢﹣ |
﹢ |
﹢ |
Sweet and pure |
﹢ |
﹢ |
﹢ |
Pectinose |
﹢﹣ |
﹢ |
﹢ |
Sucrose |
﹣ |
﹣ |
﹣ |
Sorbyl alcohol |
﹣ |
﹣ |
﹣ |
N.F,USP MANNITOL |
﹢﹣ |
﹢ |
﹢﹢ |
Inositol |
﹢﹣ |
﹢ |
﹢ |
Rhamnosyl |
﹢ |
﹢ |
﹢ |
The D-wood sugar |
﹣ |
﹣ |
﹣ |
Raffinose |
﹣ |
﹢﹣ |
﹢﹣ |
Seminose |
﹣ |
﹢ |
﹢ |
Glucose |
﹢ |
﹢ |
﹢﹢ |
+: growth is general; ++: grow medium;-: not long
In conjunction with morphological feature and physiological and biochemical property and the utilization of carbon source situation analysis of bacterial strain, identify that isolated strains FIM2006071 is Glarea lozoyensis.
Embodiment 2: fermentation culture prepares not Kangding B of knob
0
Adopting preserving number is the bacterial strain of CCTCC NO:M2012475.
1, seed spawn culture and preservation
Solid medium: glucose 4g, yeast extract powder 0.5g, KH
2PO
41g, soy peptone 0.5g, corn starch 1.5g, agar 2g adds water to 100mL, and pH transfers 7.0.
Solid culture method: inoculation was cultivated 10-12 days for 26 ℃ on culture medium slant.
After solid culture finished, it is standby that 4-10 ℃ of refrigeration is placed on the inclined-plane.
2, shake-flask seed is cultivated
Substratum: glucose 4g, yeast extract powder 2.5g, corn steep liquor 1.5g gives birth to bean powder 2g, KH
2PO
41g adds water to 100mL, and pH transfers 7.0.
Liquid amount: dress 100mL substratum in 500 mL triangular flasks
Inoculum size: 30% glycerine mycelia freeze pipe 5%(V/V)
Culture temperature: 26 ℃
Incubation time: 3 days
Shaking speed: 260rpm
Shake-flask seed cultural method: 30% glycerine mycelia freeze pipe is thawed rear according to inoculum size 5%(V/V) connect in the shake-flask seed substratum, treat that mycelia grows, having obvious wall cling phenomenon and bacterium dense is more than 25%, with 1L shake-flask seed nutrient solution, in access 1000L seeding tank.
The making of 30% glycerine mycelia freeze pipe: under sterile state, with the glycerine after sterilization approximately 30mL add in the good kind bottle of growth, shake up and make 30% glycerine mycelia suspension, then divide to be filled to (5mL/ props up) in small test tube, place-20 ℃ of refrigerations standby.
3, seeding tank seed culture medium
Substratum: glucose 30kg, give birth to bean powder 5kg, yeast powder 10kg, cottonseed meal 5kg, corn steep liquor 5kg, KH
2PO
45kg, NaCl 2.5kg adds water to 500L, and pH transfers 7.0.
Loading amount: the in-built substratum 500L of 1000L seeding tank, 121 ℃ of sterilization 30min.
The seed tank culture method: in cultured 1L shake-flask seed liquid access seeding tank, 26 ℃, 4 days.Control tank pressure: 0.04MPa in culturing process, stirring velocity 120rpm, air flow 1:1(V/V).
After cultivation, the microscopy mycelia is sturdy, and dyeing is dark, and without microbiological contamination, bacterium is dense 〉=and 15%
4, fermentor cultivation
Substratum: N.F,USP MANNITOL 400kg, W-Gum 100kg, cottonseed meal 100kg, yeast powder 50kg gives birth to bean powder 50kg, KH
2PO
45kg, FeSO
47H
2O 10kg, L-PROLINE 50kg, L-threonine 25kg, bubble enemy 7.5kg adds water to 5000L, and pH 7.0.
Loading amount: 5 tons of 10 tons of in-built substratum of fermentor tank
The fermentor cultivation method: on cultured 500L tank, seed liquor accesses in fermentor tank, and 26 ℃, 12 days.Control tank pressure: 0.04MPa in culturing process, stirring velocity 160rpm, air flow 1:1.3(V/V).
The fermentation termination judgement: mycelia dyeing is dark, and cavity is more, and mycelia has fracture, autolysis.
According to above-mentioned fermentation condition and technique, carry out 3 batch fermentation tests on 10 tons of tanks, fermentation unit is respectively: 3080ug/mL, 3100ug/mL, 3200ug/mL.
Fermented liquid can adopt another invention technology of the applicant, and (application number 201110266790.4, " a kind of knob is Kangding B not for denomination of invention
0Extracting and purifying method ") process, obtain the macroporous adsorbent resin stripping liquid, carry out chromatography, must purer flow point, collect liquid through concentrating under reduced pressure, resin concentration, flow point is condensed into solid, through pulverizing and being dried to pulverulent solids, is not Kangding B of knob
0Bacterial strain of the present invention is because fermentation unit is high, and the technique of extraction and purifying is simpler, is having more advantage on large production.
Embodiment 3: fermentation culture prepares not Kangding B of knob
0
Adopting deposit number is the bacterial strain of CCTCC NO:M2012475
Shake-flask seed is cultivated: contain N.F,USP MANNITOL 2g in every 100mL substratum, yeast powder 0.5g, corn steep liquor 0.5g, soybean cake powder 1g, KH
2PO
41g, MgSO
47H
2O 1g, all the other are water, the pH nature.Access 5%(V/V) 30% glycerine mycelia freeze pipe, 220rpm cultivated 3 days to get seed liquor for 26 ℃.
Seed tank culture: contain N.F,USP MANNITOL 4g in every 100mL substratum, give birth to bean powder 1.5g, cottonseed meal 0.5g, corn starch 1g, KH
2PO
40.5g all the other are water, the pH nature.Seed inoculum size 0.1%(V/V), 150rpm cultivated 4 days for 26 ℃.
Fermentor cultivation: contain N.F,USP MANNITOL 8g in every 100mL substratum, glucose 6g, cottonseed meal 3g, yeast powder 2g, KH
2PO
40.3g, FeSO
47H
2O 0.2g, Serine 0.3g, L-arginine 0.2g, all the other are water, pH7.0.Seed inoculum size 5% (V/V), 180rpm cultivated 9 days for 26 ℃.
Putting tank gained fermented liquid tires and is 2900ug/mL.
Embodiment 4: fermentation culture prepares not Kangding B of knob
0
Adopting deposit number is the bacterial strain of CCTCC NO:M2012475
Shake-flask seed is cultivated: contain maltodextrin 1g in every 100mL substratum, and yeast powder 1g, cottonseed meal 1g, urea 2g, all the other are water, pH7.0.Access 3%(V/V) 30% glycerine mycelia freeze pipe, 250rpm cultivated 3 days to get seed liquor for 24 ℃.
Seed tank culture: contain maltose 1g in every 100mL substratum, potato dextrin 1g, silkworm chrysalis hydrolyzed solution 0.3g, cottonseed meal 0.5g, corn starch 0.2g, all the other are water, pH7.0.Seed inoculum size 0.2%(V/V), 180rpm cultivated 4 days for 22 ℃.
Fermentor cultivation: contain glycerine 4g in every 100mL substratum, glucose 4g, cottonseed meal 0.5g, yeast extract powder 0.3g, corn starch 0.2g, all the other are water, pH7.0.Seed inoculum size 8% (V/V), 150rpm cultivated 10 days for 22 ℃.
Putting tank gained fermented liquid tires and is 3132ug/mL.
Embodiment 5: fermentation culture prepares not Kangding B of knob
0
Adopting deposit number is the bacterial strain of CCTCC NO:M2012475
Shake-flask seed is cultivated: contain glucose 4g in every 100mL substratum, and potato starch 4g, dried silkworm chrysalis meal 2g gives birth to bean powder 2g, corn starch 4g, MnSO
44H
2O 0.5g, NaNO
31g, K
2HPO
41g, all the other are water, the pH nature.Access 5%(V/V) 30% glycerine mycelia freeze pipe, 240rpm cultivated 2 days to get seed liquor for 26 ℃.
Seed tank culture: contain sucrose 4g in every 100mL substratum, potato leaches powder 4g, yeast powder 3g, dregs of beans 3g, corn starch 1g, Fe (NH
4)
2(SO
4)
26H
2O 1.5g, KNO
31g, all the other are water, the pH nature.Seed inoculum size 0.1%(V/V), 200rpm cultivated 3 days for 24 ℃.
Fermentor cultivation: contain N.F,USP MANNITOL 8g in every 100mL substratum, glucose 8g gives birth to bean powder 0.5g, yeast powder 1g, corn starch 0.5g, KH
2PO
40.5g, NaNO
30.5g, TYR 0.5g, L-threonine 0.5g, 1B 0.5g, L-Leu 0.5g, all the other are water, pH7.2.Seed inoculum size 15% (V/V), 200rpm cultivated 9 days for 26 ℃.
Putting tank gained fermented liquid tires and is 2902ug/mL.