CN106676150A - Method for producing natamycin by using streptomyces gilvosporeus through fermentation - Google Patents
Method for producing natamycin by using streptomyces gilvosporeus through fermentation Download PDFInfo
- Publication number
- CN106676150A CN106676150A CN201710205059.8A CN201710205059A CN106676150A CN 106676150 A CN106676150 A CN 106676150A CN 201710205059 A CN201710205059 A CN 201710205059A CN 106676150 A CN106676150 A CN 106676150A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- brown
- streptomycete
- imperial robe
- fermented
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/626—Natamycin; Pimaricin; Tennecetin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for producing natamycin by using streptomyces gilvosporeus through fermentation. The method comprises the following steps of: 1) activating a streptomyces gilvosporeus spore liquid; 2) inoculating the streptomyces gilvosporeus spore liquid activated in the step 1) into a seed culture medium, and performing seed culture so as to obtain streptomyces gilvosporeus; and 3) inoculating the streptomyces gilvosporeus obtained in the step 2) into a fermentation culture medium, and performing aerobic fermentation culture, thereby obtaining natamycin. According to the method, an appropriate culture medium ratio is adopted, a synergist is added into basic materials, the growth activity of mycelia inn environment with a balanced carbon-to-nitrogen ratio and inorganic salts can be greatly improved, power conditions do not need to be increased merely, the mycelia can be relatively rapidly adaptive to the environment of a culture liquid, the lag phase can be shortened, and thus the fermentation cycle can be shortened. The whole fermentation cycle is 79-82 hours, the final concentration of the mycelia of streptomyces gilvosporeus is 44-45%, and the content of natamycin is 6.9-7g/L.
Description
Technical field
The present invention relates to technical field of microbial fermentation, more particularly to a kind of fermentation life of brown the imperial robe streptomycete natamycin
Product method.
Background technology
Natamycin is a kind of polyene macrolide antibiotics, by the Streptomyces natalensis of streptomyces, brown the imperial robe chain
The secondary metabolite that the bacterium such as mould produce in aerobic condition bottom fermentation, can suppress saccharomycete and filamentous fungi growth and
The generation of mycotoxin, has been widely used in food antiseptic (food-grade) and fungus-caused disease treatment (medical grade).
The biological preservative for using is allowed to be only three kinds of nisin, natamycin and polylysine in the world at present, wherein receiving
His mycin includes individual more than 50 including the U.S. as a kind of efficient, safe and nontoxic, low dosage biological preservative, by the whole world
Country uses, and becomes widely used food additives in global scope, with huge commercial value.
In current natamycin production, fermentation period is longer and fermentation level is relatively low becomes be badly in need of solving two
Problem, fermentation period is long, causes production cost too high, and fermentation level is low to cause final products amount less.Ask for the two
Topic, the solution for generally adopting is to improve the dynamic condition of fermentation starting, the excessive addition nutriment in zymotic fluid, especially
It is carbon nitrogen source, so as to shorten fermentation period to a certain extent, improves Product Level.
In existing solution, although increasing the amount of nutriment in zymotic fluid fermentation water can be to a certain extent improved
It is flat, but the nutriment of excess can make carbon-nitrogen ratio unbalance, and mycelium can affect its growth activity in excessive nutriment,
So as to affect zymophyte dense and product content；And increase the initial dynamic condition of fermentation, such as throughput is improved, increase stirring
Deng greatly improving production cost.
The content of the invention
In view of this, it is an object of the invention to provide a kind of brown the imperial robe streptomycete fermentation method for producing natamycin,
Shorten fermentation period, and improve natamycin yield.
In order to realize foregoing invention purpose, the invention provides a kind of brown the imperial robe streptomycete fermentation produces the side of natamycin
Method, comprises the following steps：
1) brown the imperial robe streptomycete spore liquid is activated；
2) by the step 1) activation after brown the imperial robe streptomycete spore inoculating seed culture is carried out in seed culture medium,
Obtain brown the imperial robe streptomycete；
3) by the step 2) the brown the imperial robe streptomycete that obtains is inoculated in fermentation medium and carries out aerobic fermentation culture, obtain
To natamycin；
The step 3) in fermentation medium include：60.0 ± 1.0g/L of glucose, 2.0 ± 0.5g/ of middle temperature soybean cake powder
L, 2.0 ± 0.5g/L of cottonseed meal, 10.0 ± 1.0g/L of dusty yeast, 15.0 ± 1.0g/L of corn steep liquor, 0.5 ± 0.1g/ of sodium chloride
L, 0.2 ± 0.1g/L of magnesium sulfate, 10.0 ± 1.0g/L of soya-bean oil, SCH-1 type 5.0 ± 0.5g/L of synergist, calcium carbonate 5.0 ±
The water of 0.5g/L and surplus.
Preferably, the step 2) in seed culture medium include：20.0 ± 1.0g/L of glucose, dusty yeast 3.0 ±
0.5g/L, 5.0 ± 0.5g/L of peptone, 5.0 ± 0.5g/L of corn steep liquor, 10.0 ± 1.0g/L of sodium chloride, potassium dihydrogen phosphate 0.5 ±
The water of 0.1g/L, 5.0 ± 0.5g/L of calcium carbonate and surplus.
Preferably, the step 2) in the condition of seed culture be：Shaking speed：210-230rpm, cultivation temperature is
25~30 DEG C, incubation time is 20~26h, and initial pH value is 6.9~7.0.
Preferably, by volume percentage, the step 3) in the inoculum concentration of fermented and cultured be fermentation medium volume
7.5~8.5%.
Preferably, by volume percentage, the step 3) in the initial addition of fermentation medium be fermenter volume
65~75%.
Preferably, the step 3) in the condition of fermented and cultured be：Cultivation temperature is 25~30 DEG C, and initial throughput is
0.25~0.35VVM, initial mixing speed is 140~160rpm, and initial ph value is 6.4~6.6, and fermentation is whole to maintain fermentation tank
Pressure is 0.04~0.06MPa.
Preferably, the step 3) in during fermented and cultured zymotic fluid dissolved oxygen amount more than 30%.
Preferably, the step 3) in during fermented and cultured the pH value of zymotic fluid be 6.9~7.1.
Preferably, by weight percentage, the step 3) in fermented and cultured during sugar concentration be 2.5~
3.5%.
Preferably, SCH-1 type synergist is added when the brown the imperial robe streptomycete mycelia bulk concentration in zymotic fluid reaches 30%.
The invention provides a kind of method that brown the imperial robe streptomycete fermentation produces natamycin, using the fermentation of suitable proportion
Culture medium, and add SCH-1 type synergist in basestocks, mycelium is raw under carbon-nitrogen ratio in a balanced way and inorganic ion environment
Long acting to greatly improve, natamycin yield is accordingly improved, while without the need for increasing dynamic condition simply, mycelium is more quick
The environment of nutrient solution is adapted to, lag phase shortens, so as to fermentation period shortening.Whole fermentation period is 79~82h, final brown the imperial robe
Streptomycete mycelia bulk concentration is 44~45%, the mycelia bulk concentration compared with the 30% of control group improves 50%, and natamycin contains
Amount brings up to 6.9~7g/L by the 5g/L of control group.
Specific embodiment
The invention provides a kind of method that brown the imperial robe streptomycete fermentation produces natamycin, comprises the following steps：
1) brown the imperial robe streptomycete spore liquid is activated；
2) by the step 1) the brown the imperial robe streptomycete spore liquid after activation is inoculated in seed culture medium and carries out seed training
Support, obtain brown the imperial robe streptomycete；
3) by the step 2) the brown the imperial robe streptomycete that obtains is inoculated in fermentation medium and carries out aerobic fermentation culture, obtain
To natamycin；
The step 3) in fermentation medium include：60.0 ± 1.0g/L of glucose, 2.0 ± 0.5g/ of middle temperature soybean cake powder
L, 2.0 ± 0.5g/L of cottonseed meal, 10.0 ± 1.0g/L of dusty yeast, 15.0 ± 1.0g/L of corn steep liquor, 0.5 ± 0.1g/ of sodium chloride
L, 0.2 ± 0.1g/L of magnesium sulfate, 10.0 ± 1.0g/L of soya-bean oil, SCH-1 type 5.0 ± 0.5g/L of synergist, calcium carbonate 5.0 ±
The water of 0.5g/L and surplus.
In the present invention, brown the imperial robe streptomycete spore liquid is activated.Preferably, the brown the imperial robe streptomycete spore liquid
Using front frozen under the conditions of -82~-78 DEG C.Bacterial strain and source no spy of the present invention to the brown the imperial robe streptomycete spore liquid
It is different to limit, using commercial goods, it is preferred that using Chinese microorganism strain collection is preserved in, bacterium numbering is
The bacterial strain of CGMCC NO.4628.
Preferably, first spore liquid is diluted before activation, it is furthermore preferred that the concentration after dilution is 10-5；Preferably adopt
Use ddH2O is diluted.Preferably, the activation is to cultivate the spore liquid after dilution in slant medium.Preferably, institute
Stating slant medium includes：10.0 ± 1.0g/L of glucose, malt leaching 3.0 ± 0.5g/L of powder, yeast extract powder 3.0 ±
The water of 0.5g/L, 5.0 ± 0.5g/L of peptone, 15.0 ± 1.0g/L of agar and surplus, more preferably glucose 10.0g/L, wheat
Bud leaches the water of powder 3.0g/L, yeast extract powder 3.0g/L, peptone 5.0g/L, agar 15.0 and surplus.
In the present invention, the condition of the activation is preferably：Activation temperature：26~30 DEG C, soak time is 6~8 days, just
Beginning pH value is 6.9~7.1；More preferably：Activation temperature：28 DEG C, soak time is 7 days, and initial pH value is 7.0.Activation terminates
Afterwards, inclined-plane is covered completely by spore and spore color switchs to grey by white.
The present invention is not particularly limited to the source of the component of above-mentioned slant medium, using those skilled in the art's routine
Prepare the medicine of culture medium.
In the present invention, the slant medium is preferred uses Jing after 120~130 DEG C sterilize 25~35min.In the present invention
In, the sterilising temp is more preferably 121 DEG C；The sterilization time is more preferably 30min.The present invention sets to medium sterilization
For without special restriction, using sterilizing installation well known to those skilled in the art.
In the present invention, by activation after brown the imperial robe streptomycete spore inoculating seed culture is carried out in seed culture medium,
Obtain brown the imperial robe streptomycete.Preferably, the inoculum concentration of the seed culture is 1.9~2.1%, more preferably 2%.Preferably,
The seed culture medium includes：20.0 ± 1.0g/L of glucose, 3.0 ± 0.5g/L of dusty yeast, 5.0 ± 0.5g/L of peptone, jade
5.0 ± 0.5g/L of Rice ＆ peanut milk, 10.0 ± 1.0g/L of sodium chloride, 0.5 ± 0.1g/L of potassium dihydrogen phosphate, 5.0 ± 0.5g/L of calcium carbonate and
The water of surplus, more preferably：Glucose 20.0g/L, dusty yeast 3.0g/L, peptone 5.0g/L, corn steep liquor 5.0g/L, chlorination
The water of sodium 10.0g/L, potassium dihydrogen phosphate 0.5g/L, calcium carbonate 5.0g/L and surplus.
In the present invention, the seed culture preferably adopts Shaking culture；Preferably, the starting of the seed culture medium adds
Dosage is the 9~11% of culture vessel volume, more preferably 10%.Preferably, the condition of the seed culture is：Shaking table turns
Speed：210~230rpm, cultivation temperature：25~30 DEG C, incubation time：20~26h, initial pH value is 6.9~7.0, more preferably
For：Shaking speed：220rpm, cultivation temperature：28 DEG C, incubation time：24h.Cultivate to brown the imperial robe strepto- bacteria concentration and be more than 10%.
The present invention is not particularly limited to the source of the component of above-mentioned seed culture medium, using those skilled in the art's routine
Prepare the medicine of culture medium.
In the present invention, the seed culture medium is preferred uses Jing after 120~130 DEG C sterilize 25~35min.In the present invention
In, the sterilising temp is more preferably 121 DEG C；The sterilization time is more preferably 30min.The present invention sets to medium sterilization
For without special restriction, using sterilizing installation well known to those skilled in the art.
The seed culture is obtained after brown the imperial robe streptomycete, and the brown the imperial robe streptomycete for obtaining is inoculated in fermentation training by the present invention
Aerobic fermentation culture is carried out in foster base, natamycin is obtained.Preferably, by volume percentage, the inoculation of the fermented and cultured
Measure as the 7.5~8.5% of fermentation medium volume, more preferably 8.0%.
Preferably, the initial addition of the fermentation medium is the 65~75% of fermenter volume, more preferably 70%.
In the present invention, the fermentation medium is preferably included：60.0 ± 1.0g/L of glucose, 2.0 ± 0.5g/L of middle temperature soybean cake powder,
2.0 ± 0.5g/L of cottonseed meal, 10.0 ± 1.0g/L of dusty yeast, 15.0 ± 1.0g/L of corn steep liquor, 0.5 ± 0.1g/L of sodium chloride,
0.2 ± 0.1g/L of magnesium sulfate, 10.0 ± 1.0g/L of soya-bean oil, SCH-1 type 5.0 ± 0.5g/L of synergist, 5.0 ± 0.5g/L of calcium carbonate
With the water of surplus, more preferably glucose 60.0g/L, middle temperature soybean cake powder 2.0g/L, cottonseed meal 2.0g/L, dusty yeast
10.0g/L, corn steep liquor 15.0g/L, sodium chloride 0.5g/L, magnesium sulfate 0.2g/L, soya-bean oil 10.0g/L, SCH-1 type synergist
The water of 5.0g/L, calcium carbonate 5.0g/L and surplus.
Preferably, the condition of the fermented and cultured is：Cultivation temperature be 25~30 DEG C, initial throughput be 0.25~
0.35VVM, initial mixing speed is 140~160rpm, and initial ph value is 6.4~6.6, and the whole maintenance fermentation pressure tank of fermentation is
0.04~0.06MPa, more preferably, cultivation temperature is 28 DEG C, and initial throughput is 0.3VVM, and initial mixing speed is
150rpm, initial ph value is 6.5, and fermentation is whole to maintain fermentation pressure tank to be 0.05MPa.
In the present invention, after fermented and cultured starts, with thalline acclimatizing culture medium and yeasting condition, dissolved oxygen amount meeting
It is gradually reduced.Preferably, dissolved oxygen amount is controlled by stirring and/or throughput；Preferably, the zymotic fluid during fermented and cultured
Dissolved oxygen amount more than 30%；Preferably, the dissolved oxygen amount is determined using the dissolved oxygen electrode that fermentation tank is carried.In the present invention,
During fermented and cultured, pH value can first rise more than 7.0, then be gradually reduced, and pH value is adjusted when dropping to 7.0, make pH
It is worth for 6.9~7.1.The preferred regulation pH value is to be adjusted from ammoniacal liquor, it is furthermore preferred that adopt mass fraction for 30% ammonia
Water.Preferably, the pH determination of electrode that the pH is carried using fermentation tank.In the present invention, during fermented and cultured, with battalion
The consumption of foster material, sugared concentration can be gradually reduced, and glucose is supplemented when dropping to 2.5%.Preferably, the glucose is dense
Spend for 200 ± 2g/L, more preferably 200g/L.Preferably, by weight percentage, the concentration of sugar is during fermented and cultured
2.5~3.5%.Preferably, the sugared concentration is determined using Fehling Regent method.
In the present invention, it is preferred to, during fermented and cultured per 3~5h to zymotic fluid in brown the imperial robe streptomycete mycelia
Bulk concentration once checked, more preferably 4h.Preferably, mend when the brown the imperial robe streptomycete mycelia bulk concentration reaches 30%
Plus SCH-1 type synergist.In the present invention, the concentration of the SCH-1 types synergist is preferably 20.0 ± 1g/L, more preferably
20.0g/L；The preferred synergist adopts ddH2O dilutes.Preferably, the addition of the SCH-1 types synergist is 0.25
~0.35ml/ (h.L), i.e., fill into per hour 0.25~0.35ml, more preferably 0.3ml/ (h.L) per 1L zymotic fluids.
In the present invention, with the carrying out of fermentation, the incrementss of natamycin start to slow down, and are adding natamycin precursor just
Butanol, can promote natamycin to continue to produce.Preferably, when fermentation proceeds to 58~62h, add in the fermentation medium
Normal propyl alcohol, more preferably 60h.Preferably, the normal propyl alcohol carries out before addition degerming；Preferably, the degerming mode was
Filter method, it is preferred that the filtration method adopts aperture for the bacteria filter of 0.22um.Preferably, the addition of the normal propyl alcohol is 4.5
~5.5ml/L, more preferably 5ml/L.When bacteria concentration is not further added by, terminate fermentation, whole fermentation period is about 80h.
The present invention is not particularly limited to the source of the component of above-mentioned culture medium, is formulated conventionally using those skilled in the art
The medicine of culture medium, it is preferred that the SCH-1 types synergist leads to bridge bioid plant produced for city of Leshan sichuan five.
In the present invention, the fermentation medium, supplement glucose and supplement the preferred Jing 120 of SCH-1 types synergist~
Use after 130 DEG C of 25~35min of sterilizing.In the present invention, the sterilising temp is more preferably 121 DEG C；The sterilization time is more
Preferably 30min.The present invention does not have special restriction to the equipment of medium sterilization, is gone out using well known to those skilled in the art
Bacterium equipment.
In the present invention, each nutrient ratio is suitable in the fermentation medium, and adds synergist in basestocks, bacterium
Filament growth activity under carbon-nitrogen ratio in a balanced way and inorganic ion environment is greatly improved, without the need for increasing dynamic condition, bacterium simply
Filament more quickly adapts to the environment of nutrient solution, and lag phase shortens, so as to fermentation period shortening；With the nutrition in basestocks
The consumption of material especially nitrogen source, mycelial growth becomes slow, now fills into SCH-1 type fermentation synergists, and thalline enters one
Step obtains flourish；When fermentation is carried out to a certain extent, the normal propyl alcohol of filtration sterilization is added, so as in the fermentation middle and later periods
The yield of natamycin has obtained further raising.Whole fermentation period about 80h, final brown the imperial robe streptomycete mycelia bulk concentration
For 44~45%, the content of natamycin is 6.9~7g/L.
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made all belongs to
In the scope of protection of the invention.
Embodiment 1
Brown the imperial robe streptomycete spore liquid activation：By the brown the imperial robe chain that -78 DEG C of frozen deposit numbers are CGMCCNO.4628
Mycotic spore liquid adopts ddH2O is diluted, and extension rate is 10-5, coat fill 20ml in 120 DEG C sterilize 35min after
In the plate of slant medium, 26 DEG C of cultures carry out activation culture in 8 days, and slant medium includes：Glucose 11.0g/L, malt
The water of powder 2.5g/L, yeast extract powder 3.5g/L, peptone 5.0g/L, agar 16.0g/L and surplus is leached, initial pH value is
6.9。
Seed culture：Seed culture medium after adding 40ml to sterilize 35min at 130 DEG C in the shaking flask of 500ml, picking is lived
In culture medium, inoculum concentration is 1.9% to brown the imperial robe streptomycete spore inoculating after change, in 210rpm, at 25 DEG C 26h is cultivated, and is obtained
To the brown the imperial robe streptomycete that concentration is 10.5%, seed culture medium includes：Glucose 21.0g/L, dusty yeast 3.5g/L, peptone
The water of 5.5g/L, corn steep liquor 5.0g/L, sodium chloride 9.0g/L, potassium dihydrogen phosphate 0.4g/L, calcium carbonate 5.0g/L and surplus, initially
PH value is 6.9.
Fermented and cultured：Add the fermented and cultured after 121 DEG C of sterilizing 35min of the volume of fermentation tank 75% in fermentation tank
Base, by the brown the imperial robe streptomycete for obtaining by weight percentage, is added in fermentation medium with 8.5% inoculum concentration, in training
Foster temperature is 25 DEG C, and initial throughput is 0.35VVM, and initial mixing speed is to be fermented under the conditions of 140rpm, and fermentation is whole
Fermentation pressure tank is maintained to be 0.05MPa.Fermentation medium includes：Glucose 60.0g/L, middle temperature soybean cake powder 2.3g/L, cottonseed
Cake powder 1.8g/L, dusty yeast 11.0g/L, corn steep liquor 15.0g/L, sodium chloride 0.6g/L, magnesium sulfate 0.3g/L, soya-bean oil 9.0g/L,
The water of SCH-1 type synergist 5.0g/L, calcium carbonate 5.5g/L and surplus, initial pH value is 6.5.In sweat, when under pH value
Mass fraction is 30% ammoniacal liquor regulation pH value when dropping to 7.0, makes pH value be 6.9~7.1.During the fermentation by stirring
And/or throughput is controlling dissolved oxygen amount, the dissolved oxygen amount of zymotic fluid determines sugared concentration more than 30% using Fehlings reagent, when
Sugared concentration adds concentration when dropping to 2.5% be the glucose after 200g/L sterilizes 35min in 121 DEG C, by weight percentage,
The concentration for making sugar during fermented and cultured is 2.5~3.5%.Brown the imperial robe streptomycete mycelia bulk concentration during often 3h is to zymotic fluid enters
Row one-time detection, it is 21.0g/LSCH-1 type synergist that concentration is added when brown the imperial robe streptomycete mycelia bulk concentration reaches 30%,
0.25ml is filled into per hour per 1L zymotic fluids.When fermentation proceeds to 58h, add adopt aperture for 0.22 in the fermentation medium
μm bacteria filter filtration sterilization n-butanol, addition is 5.5ml/L.The dissolved oxygen carried using fermentation tank in whole sweat
Determination of electrode dissolved oxygen amount, the pH determination of electrode pH carried using fermentation tank.After fermentation 82h, brown the imperial robe strepto- bacteria concentration no longer increases
Plus, concentration is 44%, and natamycin content is 6.9g/L, terminates fermentation.
Embodiment 2
Brown the imperial robe streptomycete spore liquid activation：By the brown the imperial robe chain that -82 DEG C of frozen deposit numbers are CGMCCNO.4628
Mycotic spore liquid adopts ddH2O is diluted, and extension rate is 10-5, coat fill 30ml in 130 DEG C sterilize 25min after
In the plate of slant medium, 30 DEG C of cultures carry out activation culture in 6 days, and slant medium includes：Glucose 9.0g/L, malt leaching
Go out the water of powder 3.5g/L, yeast extract powder 3.0g/L, peptone 5.5g/L, agar 14.0g/L and surplus, initial pH value is 7.1.
Seed culture：Seed culture medium after adding 60ml to sterilize 25min at 130 DEG C in the shaking flask of 500ml, picking is lived
In culture medium, inoculum concentration is 2.1% to brown the imperial robe streptomycete spore inoculating after change, in 230rpm, at 28 DEG C 20h is cultivated, and is obtained
To the brown the imperial robe streptomycete that concentration is 10.3%, seed culture medium includes：Glucose 19.0g/L, dusty yeast 3.0g/L, peptone
The water of 4.5g/L, corn steep liquor 4.5g/L, sodium chloride 10.5g/L, potassium dihydrogen phosphate 0.45g/L, calcium carbonate 5.5g/L and surplus, just
Beginning pH value is 7.0.
Fermented and cultured：Add the fermented and cultured after 130 DEG C of sterilizing 30min of the volume of fermentation tank 65% in fermentation tank
Base, by the brown the imperial robe streptomycete for obtaining by weight percentage, is added in fermentation medium with 7.5% inoculum concentration, in training
Foster temperature is 29 DEG C, and initial throughput is 0.25VVM, and initial mixing speed is to be fermented under the conditions of 160rpm, and fermentation is whole
Fermentation pressure tank is maintained to be 0.06MPa.Fermentation medium includes：Glucose 61.0g/L, middle temperature soybean cake powder 2.5g/L, cottonseed
Cake powder 2.5g/L, dusty yeast 11.0g/L, corn steep liquor 16.0g/L, sodium chloride 0.4g/L, magnesium sulfate 0.3g/L, soya-bean oil 11g/L,
The water of SCH-1 type synergist 5.5g/L, calcium carbonate 4.5g/L and surplus, initial pH value is 6.6.In sweat, when pH declines
PH value is adjusted to the ammoniacal liquor that mass fraction when 7.0 is 30%, makes pH value be 6.9~7.1.During the fermentation by stirring
And/or throughput is controlling dissolved oxygen amount, the dissolved oxygen amount of zymotic fluid determines sugared concentration more than 30% using Fehlings reagent, when
Sugared concentration adds concentration when dropping to 2.5% be the glucose after 202g/L sterilizes 35min in 121 DEG C, by weight percentage,
The concentration for making sugar during fermented and cultured is 2.5~3.5%.Brown the imperial robe streptomycete mycelia bulk concentration during often 5h is to zymotic fluid enters
Row one-time detection, it is 19.0g/LSCH-1 type synergist that concentration is added when brown the imperial robe streptomycete mycelia bulk concentration reaches 30%,
0.35ml is filled into per hour per 1L zymotic fluids.When fermentation proceeds to 62h, add adopt aperture for 0.22 in the fermentation medium
μm bacteria filter filtration sterilization n-butanol, addition is 4.5ml/L.The dissolved oxygen carried using fermentation tank in whole sweat
Determination of electrode dissolved oxygen amount, the pH determination of electrode pH carried using fermentation tank.After fermentation 79h, brown the imperial robe strepto- bacteria concentration no longer increases
Plus, concentration is 44.5%, and natamycin content is 6.9g/L, terminates fermentation.
Embodiment 3
Brown the imperial robe streptomycete spore liquid activation：By the brown the imperial robe chain that -80 DEG C of frozen deposit numbers are CGMCCNO.4628
Mycotic spore liquid adopts ddH2O is diluted, and extension rate is 10-5, coat fill 25ml in 121 DEG C sterilize 30min after
In the plate of slant medium, 28 DEG C of cultures carry out activation culture in 6 days, and slant medium includes：Glucose 10.0g/L, malt
The water of powder 3.0g/L, yeast extract powder 3.0g/L, peptone 5.0g/L, agar 15.0g/L and surplus is leached, initial pH value is
7.0。
Seed culture：Seed culture medium after adding 50ml to sterilize 30min at 121 DEG C in the shaking flask of 500ml, picking is lived
In culture medium, inoculum concentration is 2.0% to brown the imperial robe streptomycete spore inoculating after change, in 220rpm, at 28 DEG C 24h is cultivated, and is obtained
To the brown the imperial robe streptomycete that concentration is 10.4%, seed culture medium includes：Glucose 20.0g/L, dusty yeast 3.0g/L, peptone
The water of 5.0g/L, corn steep liquor 5.0g/L, sodium chloride 10.0g/L, potassium dihydrogen phosphate 0.5g/L, calcium carbonate 5.0g/L and surplus.
Fermented and cultured：Add the fermented and cultured after 121 DEG C of sterilizing 30min of the volume of fermentation tank 70% in fermentation tank
Base, by the brown the imperial robe streptomycete for obtaining by weight percentage, is added in fermentation medium with 7.0% inoculum concentration, in training
Foster temperature is 28 DEG C, and initial throughput is 0.3VVM, and initial mixing speed is to be fermented under the conditions of 150rpm, the whole dimension of fermentation
It is 0.05MPa to hold fermentation pressure tank.Fermentation medium includes：Glucose 60.0g/L, middle temperature soybean cake powder 2.0g/L, cottonseed cake
Powder 2.0g/L, dusty yeast 10.0g/L, corn steep liquor 15.0g/L, sodium chloride 0.5g/L, magnesium sulfate 0.2g/L, soya-bean oil 10.0g/L,
The water of SCH-1 type synergist 5.0g/L, calcium carbonate 5.0g/L and surplus, initial pH value is 6.5.In sweat, when pH declines
PH value is adjusted to the ammoniacal liquor that mass fraction when 7.0 is 30%, makes pH value be 6.9~7.1.During the fermentation by stirring
And/or throughput is controlling dissolved oxygen amount, the dissolved oxygen amount of zymotic fluid determines sugared concentration more than 30% using Fehlings reagent, when
Sugared concentration adds concentration when dropping to 2.5% be the glucose after 200g/L sterilizes 35min in 121 DEG C, by weight percentage,
The concentration for making sugar during fermented and cultured is 2.5~3.5%.Brown the imperial robe streptomycete mycelia bulk concentration during often 4h is to zymotic fluid enters
Row one-time detection, it is 20.0g/LSCH-1 type synergist that concentration is added when brown the imperial robe streptomycete mycelia bulk concentration reaches 30%,
0.3ml is filled into per hour per 1L zymotic fluids.When fermentation proceeds to 60h, add adopt aperture for 0.22 μ in the fermentation medium
The n-butanol of the bacteria filter filtration sterilization of m, addition is 5.0ml/L.The dissolved oxygen carried using fermentation tank in whole sweat
Determination of electrode dissolved oxygen amount, the pH determination of electrode pH carried using fermentation tank.After fermentation 80h, brown the imperial robe strepto- bacteria concentration no longer increases
Plus, concentration is 45%, and natamycin content is 7.0g/L, terminates fermentation.
Embodiment 4
Brown the imperial robe streptomycete spore liquid activation：By the brown the imperial robe chain that -80 DEG C of frozen deposit numbers are CGMCCNO.4628
Mycotic spore liquid adopts ddH2O is diluted, and extension rate is 10-5, coat fill 25ml in 121 DEG C sterilize 30min after
In the plate of slant medium, 28 DEG C of cultures carry out activation culture in 6 days, and slant medium includes：Glucose 10.0g/L, malt
The water of powder 3.0g/L, yeast extract powder 3.0g/L, peptone 5.0g/L, agar 15.0g/L and surplus is leached, initial pH value is
7.0。
Seed culture：Seed culture medium after adding 50ml to sterilize 30min at 121 DEG C in the shaking flask of 500ml, picking is lived
In culture medium, inoculum concentration is 2.0% to brown the imperial robe streptomycete spore inoculating after change, in 220rpm, at 28 DEG C 24h is cultivated, and is obtained
To the brown the imperial robe streptomycete that concentration is 10.4%, seed culture medium includes：Glucose 20.0g/L, dusty yeast 3.0g/L, peptone
The water of 5.0g/L, corn steep liquor 5.0g/L, sodium chloride 10.0g/L, potassium dihydrogen phosphate 0.5g/L, calcium carbonate 5.0g/L and surplus.
Fermented and cultured：Add the fermented and cultured after 121 DEG C of sterilizing 30min of the volume of fermentation tank 70% in fermentation tank
Base, by the brown the imperial robe streptomycete for obtaining by weight percentage, is added in fermentation medium with 7.0% inoculum concentration, in training
Foster temperature is 28 DEG C, and initial throughput is 0.3VVM, and initial mixing speed is to be fermented under the conditions of 150rpm, the whole dimension of fermentation
It is 0.05MPa to hold fermentation pressure tank.Fermentation medium includes：Glucose 60.0g/L, middle temperature soybean cake powder 2.0g/L, cottonseed cake
Powder 2.0g/L, dusty yeast 10.0g/L, corn steep liquor 15.0g/L, sodium chloride 0.5g/L, magnesium sulfate 0.2g/L, soya-bean oil 10.0g/L, carbon
The water of sour calcium 5.0g/L and surplus, initial pH value is 6.5.When pH drops to 7.0, mass fraction is 30% in sweat
Ammoniacal liquor adjust pH value, make pH value be 6.9~7.1.During the fermentation dissolved oxygen amount is controlled by stirring and/or throughput,
The dissolved oxygen amount of zymotic fluid determines sugared concentration more than 30% using Fehlings reagent, adds dense when sugared concentration drops to 2.5%
The glucose spent after sterilizing 35min in 121 DEG C for 200g/L, by weight percentage, makes the concentration of sugar during fermented and cultured
For 2.5~3.5%.Brown the imperial robe streptomycete mycelia bulk concentration during often 4h is to zymotic fluid carries out one-time detection.Whole sweat
The dissolved oxygen electrode that middle employing fermentation tank is carried determines dissolved oxygen amount, the pH determination of electrode pH carried using fermentation tank.After fermentation 100h,
Brown the imperial robe strepto- bacteria concentration no longer increases, and concentration is 30%, and natamycin content is 5.0g/L, terminates fermentation.
The method that the brown the imperial robe streptomycete fermentation that the application is provided produces natamycin, fermentation period is short, compared to enforcement
The control group of example 4, whole fermentation period shorten to 79~82h from 100h, and final brown the imperial robe streptomycete mycelia bulk concentration is 44~
45%, the mycelia bulk concentration compared with the 30% of control group improves 50%, and the content of natamycin is brought up to by the 5g/L of control group
6.9~7g/L.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method that brown the imperial robe streptomycete fermentation produces natamycin, comprises the following steps：
1) brown the imperial robe streptomycete spore liquid is activated；
2) by the step 1) activation after brown the imperial robe streptomycete spore inoculating seed culture is carried out in seed culture medium, obtain
Brown the imperial robe streptomycete；
3) by the step 2) the brown the imperial robe streptomycete that obtains is inoculated in fermentation medium and carries out aerobic fermentation culture, received
His mycin；
The step 3) in fermentation medium include：60.0 ± 1.0g/L of glucose, 2.0 ± 0.5g/L of middle temperature soybean cake powder, cotton
2.0 ± 0.5g/L of seed cake powder, 10.0 ± 1.0g/L of dusty yeast, 15.0 ± 1.0g/L of corn steep liquor, 0.5 ± 0.1g/L of sodium chloride, sulphur
Sour 0.2 ± 0.1g/L of magnesium, 10.0 ± 1.0g/L of soya-bean oil, SCH-1 type 5.0 ± 0.5g/L of synergist, 5.0 ± 0.5g/L of calcium carbonate and
The water of surplus.
2. fermentation method for producing according to claim 1, it is characterised in that the step 2) in seed culture medium bag
Include：20.0 ± 1.0g/L of glucose, 3.0 ± 0.5g/L of dusty yeast, 5.0 ± 0.5g/L of peptone, 5.0 ± 0.5g/L of corn steep liquor,
The water of 10.0 ± 1.0g/L of sodium chloride, 0.5 ± 0.1g/L of potassium dihydrogen phosphate, 5.0 ± 0.5g/L of calcium carbonate and surplus.
3. fermentation method for producing according to claim 1 and 2, it is characterised in that the step 2) in seed culture
Condition is：Shaking speed：210-230rpm, cultivation temperature is 25~30 DEG C, and incubation time is 20~26h, and initial pH value is 6.9
~7.0.
4. fermentation method for producing according to claim 1, it is characterised in that by volume percentage, the step 3) in
The inoculum concentration of fermented and cultured is the 7.5~8.5% of fermentation medium volume.
5. fermentation method for producing according to claim 1, it is characterised in that by volume percentage, the step 3) in
The initial addition of fermentation medium is the 65~75% of fermenter volume.
6. fermentation method for producing according to claim 1, it is characterised in that the step 3) in fermented and cultured condition
For：Cultivation temperature is 25~30 DEG C, and initial throughput is 0.25~0.35VVM, and initial mixing speed is 140~160rpm, is risen
Beginning pH value is 6.4~6.6, and fermentation is whole to maintain fermentation pressure tank to be 0.04~0.06MPa.
7. fermentation method for producing according to claim 1, it is characterised in that the step 3) in send out during fermented and cultured
The dissolved oxygen amount of zymotic fluid is more than 30%.
8. fermentation method for producing according to claim 1, it is characterised in that the step 3) in send out during fermented and cultured
The pH value of zymotic fluid is 6.9~7.1.
9. fermentation method for producing according to claim 1, it is characterised in that by weight percentage, the step 3) in
Fermented and cultured during sugar concentration be 2.5~3.5%.
10. fermentation method for producing according to claim 1, it is characterised in that the step 3) in when brown in zymotic fluid
The imperial robe streptomycete mycelia bulk concentration adds SCH-1 type synergist when reaching 30%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710205059.8A CN106676150B (en) | 2017-03-31 | 2017-03-31 | Method for producing natamycin by fermenting streptomyces fuscoporia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710205059.8A CN106676150B (en) | 2017-03-31 | 2017-03-31 | Method for producing natamycin by fermenting streptomyces fuscoporia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106676150A true CN106676150A (en) | 2017-05-17 |
| CN106676150B CN106676150B (en) | 2021-04-16 |
Family
ID=58825996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710205059.8A Active CN106676150B (en) | 2017-03-31 | 2017-03-31 | Method for producing natamycin by fermenting streptomyces fuscoporia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106676150B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337552A (en) * | 2021-06-03 | 2021-09-03 | 淮北师范大学 | Epsilon-polylysine-natamycin co-production method based on fermentation waste mushroom residue interactive recycling |
| CN114875100A (en) * | 2022-06-27 | 2022-08-09 | 山东第一医科大学(山东省医学科学院) | Method for improving natamycin fermentation yield by activating natamycin synthesis in advance |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515678A (en) * | 2003-08-25 | 2004-07-28 | 天津科技大学 | Preparation method of natamycin |
| CN1891834A (en) * | 2005-07-05 | 2007-01-10 | 广州市微生物研究所 | Fermenting method for producing natamycin |
| CN101062934A (en) * | 2007-05-31 | 2007-10-31 | 山东鲁抗医药股份有限公司 | Method for extracting natamycin from fermentation technique culture |
| CN103789379A (en) * | 2014-01-21 | 2014-05-14 | 绿康生化股份有限公司 | Method for increasing yield of natamycin |
-
2017
- 2017-03-31 CN CN201710205059.8A patent/CN106676150B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515678A (en) * | 2003-08-25 | 2004-07-28 | 天津科技大学 | Preparation method of natamycin |
| CN1891834A (en) * | 2005-07-05 | 2007-01-10 | 广州市微生物研究所 | Fermenting method for producing natamycin |
| CN101062934A (en) * | 2007-05-31 | 2007-10-31 | 山东鲁抗医药股份有限公司 | Method for extracting natamycin from fermentation technique culture |
| CN103789379A (en) * | 2014-01-21 | 2014-05-14 | 绿康生化股份有限公司 | Method for increasing yield of natamycin |
Non-Patent Citations (2)
| Title |
|---|
| 杨洋: "《发酵增效剂对阿维菌素发酵的影响》", 《试验研究》 * |
| 胡佩: "《新型发酵增效剂》", 《发酵工业网》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337552A (en) * | 2021-06-03 | 2021-09-03 | 淮北师范大学 | Epsilon-polylysine-natamycin co-production method based on fermentation waste mushroom residue interactive recycling |
| CN113337552B (en) * | 2021-06-03 | 2023-03-24 | 淮北师范大学 | Epsilon-polylysine-natamycin co-production method based on fermentation waste mushroom residue interactive recycling |
| CN114875100A (en) * | 2022-06-27 | 2022-08-09 | 山东第一医科大学(山东省医学科学院) | Method for improving natamycin fermentation yield by activating natamycin synthesis in advance |
| CN114875100B (en) * | 2022-06-27 | 2023-06-16 | 山东第一医科大学(山东省医学科学院) | Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106676150B (en) | 2021-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102653724B (en) | Lactobacillus casei and application thereof to produce L-lactic acid by fermentation | |
| CN104561154A (en) | Coenzyme Q10 fermentation process and control strategy | |
| CN102864114B (en) | Strain for highly yielding enramycin, and preparation method and application thereof | |
| CN105062947B (en) | A kind of production method of high gemma rate bacillus licheniformis | |
| CN102391034A (en) | Formula and preparation method of Pleurotus eryngii liquid strain culture medium | |
| CN102845225A (en) | Hypsizygus marmoreus liquid strain fermenting technique | |
| CN107201315A (en) | A kind of Paecilomyces thermaphila mutagenic strain and its method of mutagenesis and application | |
| CN105838645A (en) | Actinoplanes utahensis and application thereof in preparing acarbose | |
| CN103060405A (en) | Fermentation technique of A40926 | |
| CN109234346A (en) | A kind of fermentation process producing vitamin B2 | |
| CN109593807A (en) | A kind of method of high level fermenting and producing apramycin | |
| CN102381896B (en) | Crab-flavor mushroom liquid strain medium formula and preparation method thereof | |
| CN106676150A (en) | Method for producing natamycin by using streptomyces gilvosporeus through fermentation | |
| CN104388501B (en) | A kind of erythromycin preparation method using biology enzyme | |
| CN102127515B (en) | Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) | |
| CN108018324A (en) | A kind of fermentation medium for producing doractin and preparation method and application | |
| CN102550294B (en) | Method for liquid fermentation cultivation of Pleurotus cornucopiae strain | |
| CN102634553B (en) | Hainanmycin fermentation method | |
| CN106754401A (en) | A kind of production method of hirsutella sinensis fungal | |
| CN102787153A (en) | Method for producing enramycin by microbial fermentation supplement feed | |
| CN102649941A (en) | Phosphorus-dissolving pseudomonas putida L13 and fermentation process thereof | |
| CN101463370B (en) | Method for preparing L-lactic acid by fermenting potato starch by Rhizopus oryzae | |
| CN105586374B (en) | A method of sugar production doractin is mended based on metabolizing parameters reduced sugar | |
| CN107011396A (en) | A kind of method for reducing jinggangmeisu zymotic fluid remaining sugar concentration | |
| CN109810925B (en) | Improved polyoxin fermentation medium and fermentation process |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |