CN109097415A - A method of saxitoxin is prepared using malicious ocean sulfurous acid bacillus fermentation is produced - Google Patents
A method of saxitoxin is prepared using malicious ocean sulfurous acid bacillus fermentation is produced Download PDFInfo
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Abstract
The invention belongs to marine microorganism fermentation arts, more particularly, to a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced.The method is the following steps are included: (1) bacterial strain activates；(2) preparation of seed liquor；(3) fermentation tank culture is fermented；(4) extraction of fermentation liquid；(5) purifying of crude extract is to get saxitoxin.Compared with prior art, the invention has the following advantages that (1) the method for the present invention is production strain using marine microorganism bacterial strain sulfurous acidfast bacilli Z1-D, the strain fermentation saxitoxin yield is higher, up to 61.5-72.8 μ g/L, its fermentation time is 40-48 hours, saves energy consumption；(2) the method for the present invention step is simple, workload is small, and the period is shorter, and agents useful for same and material are microbial fermentation test common agents, harmless, environmentally friendly, at low cost, market prospects and economic value with higher.
Description
Technical field
The invention belongs to marine microorganism fermentation arts, utilize the malicious ocean sulfurous acid bacillus fermentation of production more particularly, to a kind of
The method for preparing saxitoxin.
Background technique
Paralytic shellfish poisoning (PSP) (paralytic shellfish posoning, PSP) belongs to guanamines toxoid, known
Red-tide toxin Poisoning is most strong, distribution is most wide, menace is maximum, it is most frequent to cause murder by poisoning event.It mainly include saxitoxin
(Saxitoxin, STX) and its derivative are typical Na-ion channel blockers.STX is in harmful algal detection, nerve biology
There is important application in the research such as, medical diagnosis, drug development, biochemical war agent.In terms of drug development, STX uniqueization
Structure and toxicological effect mechanism are learned, the important tool medicine in the research channel cell Na+ is become.It is with antitumor, antiviral, town
Bitterly, the multiple efficacies such as anesthesia, spasmolysis, decompression, Zhichuan.Currently, the acquisition of saxitoxin mainly passes through the scale of dinoflagellate algae strain
Culture and product extraction are prepared.Not only cultivation cycle is long, at high cost for this method, and needs special algae culture device.With
Dinoflagellate is compared, and there are marine bacteria the characteristics such as small genome, easy culture, genetic manipulation be simple therefore to utilize and produce the malicious micro- life in ocean
Object strain fermentation and separating product are an effective alternative solutions for saxitoxin.
Summary of the invention
The present invention be in order to overcome in the preparation method of saxitoxin in the prior art cultivation cycle long, at high cost, and
The problem of needing special algae culture device provides a kind of with yield is high, the period is short, at low cost a kind of using producing poison sea
The method that foreign sulfurous acid bacillus fermentation prepares saxitoxin.
The invention is realized by the following technical scheme:
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, and culture is lived
Change bacterial strain；
(2) preparation of seed liquor: the single colonie after picking activation is inoculated in the liquid seed culture medium in seeding tank, and spread cultivation bacterial strain
For use；
(3) fermentation tank culture is fermented: fermentation medium being fitted into fermentor, by the seed liquor access fermentation training in step (2)
It supports in base, fermentation obtains fermentation liquid；
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, obtaining bacterial sediment, extracted with 80% ethyl alcohol, combined extract, rotation
Inspissation contracting, obtains crude extract；
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, and rotation is steamed
Solvent is sent out, obtains white powder to get saxitoxin.
Saxitoxin in the present invention is prepared using ocean sulfurous acid bacillus fermentation method, with prior art phase
Than step of the invention is relatively simple, and whole preparation process time greatly reduces, the effective preparation effect for improving saxitoxin
Rate and yield.Saxitoxin can be effectively thus synthesized, is provided effectively to study the function and effect of saxitoxin
Approach.
Preferably, plating medium formula is as follows in the step (1): 0.8 ~ 1.2g of ammonium sulfate, yeast extract 0.2 ~
0.5g, 0.6 ~ 1.0g of sodium alginate, 0.1 ~ 0.5g of ferric phosphate, agar 2g, add natural sea-water to 100ml, pH6.9 ~ 7.3.
In the present invention sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is marine bacteria, therefore in incubation
In a certain amount of natural sea-water need to be added, with promote its activity.
Preferably, cultivation temperature is 27 ~ 31 DEG C in the step (1), incubation time is 2 ~ 4 days.
Preferably, liquid seed culture medium formula is as follows in the step (2): 5 g of yeast extract, 4 g of peptone, Portugal
1 g of grape sugar, natural sea-water 1000 mL, pH 6.2 ~ 7.1.
Preferably, the ventilatory capacity in the step (2) in seeding tank is 40-50 L/min, speed of agitator 160-180
Turn/min, 28 DEG C are cultivated 16 hours, and the OD600 to seed liquor reaches 0.6 ~ 0.8, and om observation thalli growth is good, without miscellaneous
Bacterium is stand-by when polluting.
Preferably, the formula of fermentation medium is as follows in the step (3): 10 g of yeast extract, 6 g of peptone, Portugal
4 g of grape sugar, 0.015 g of arginine, 0.002 g of calcium chloride and natural sea-water 1000 mL, pH 6.3 ~ 7.0.
As preferred.The charge weight of fermentation medium is fermentation tank capacity percent by volume in the step (3)
35 ~ 50%, seed liquor is by the inoculum concentration access fermentation medium of percent by volume 2.5% ~ 3.5%.
Preferably, fermentor described in the step (3) is flat-blade turbine stirred type fermentor, in fermentation process
It keeps tank to press 0.12 ~ 0.18MPa, stirring rate 140-160r/min, controls ventilatory capacity 40-60 L/min, fermentation temperature is
27 ~ 30 DEG C, fermentation time is 40-48 hours.
Preferably, the pH of 80% ethyl alcohol is 2.0 ~ 4.0 in the step (4), extraction time is 3 times.
Preferably, the saxitoxin yield is 61.5-72.8 μ g/L.
Compared with prior art, the invention has the following advantages that
(1) the method for the present invention is production strain, the strain fermentation Saxidomus using marine microorganism bacterial strain sulfurous acidfast bacilli Z1-D
Toxin yield is higher, and up to 61.5-72.8 μ g/L, fermentation time is 40-48 hours, saves energy consumption；
(2) the method for the present invention step is simple, workload is small, and the period is shorter, and agents useful for same and material are microbial fermentation test
Common agents, harmless, environmentally friendly, at low cost, market prospects and economic value with higher.
Specific embodiment
Below with reference to embodiment, the present invention is further described, following embodiments be it is illustrative, be not restrictive,
It cannot be limited the scope of protection of the present invention with following embodiments.
Embodiment 1
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, at 27 DEG C
Culture activated strains 2 days, wherein it is described the step of (1) in plating medium formula it is as follows: ammonium sulfate 0.8g, yeast extract 0.2g,
Sodium alginate 0.6g, ferric phosphate 0.1g, agar 2g, add natural sea-water to 100ml, pH6.9.
(2) preparation of seed liquor: preparing seed culture medium 1L, is added in 5L volume seeding tank, and steam heating, 121 DEG C go out
Bacterium 30 minutes, 28 DEG C are cooled to, the single colonie after picking activation, flame method is inoculated in the liquid seed culture medium in seeding tank
In, the ventilatory capacity in seeding tank is 40 L/min, and 160 turns/min of speed of agitator, 28 DEG C are cultivated 16 hours, to seed liquor
OD600 reaches 0.6, and om observation thalli growth is good, whens no living contaminants stand-by, described liquid seed culture medium formula
It is as follows: 5 g of yeast extract, 4 g of peptone, 1 g of glucose, natural sea-water 1000 mL, pH 6.2.
(3) fermentation tank culture is fermented: being prepared fermentation medium, is packed into according to the 35% of fermentation tank capacity percent by volume
Flat-blade turbine stirred type fermentor leads to steam heating, and 121 DEG C sterilize 30 minutes, is cooled to 27 DEG C, seed liquor presses percent by volume
In 2.5% inoculum concentration access fermentation medium, tank is kept to press 0.12MPa, stirring rate 140r/min, control in fermentation process
40 L/min of ventilatory capacity processed, fermentation temperature are 27 DEG C, and fermentation time is 40 hours, and fermentation obtains fermentation liquid, the fermentation training
Support base formula it is as follows: 10 g of yeast extract, 6 g of peptone, 4 g of glucose, 0.015 g of arginine, 0.002 g of calcium chloride and
Natural sea-water 1000 mL, pH 6.3.
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, bacterial sediment is obtained, the 80%(v/v for being 2.0 with pH) second
Extracting solution is filtered by 0.45 micron membrane filter, carries out second extraction to precipitating, obtain secondary raffinate by alcohol extracting；It will be secondary
The 80%(v/v that pH is 2.0 is added after the filtering of 0.45 micron membrane filter in clarified solution) ethanol solution extracted three times, merging three
Secondary extracting solution, concentrated by rotary evaporation obtain coarse extraction.
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, rotation
Turn evaporation solvent, obtains white powder to get saxitoxin, 61.5 μ g/L of yield.
Embodiment 2
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, at 31 DEG C
Culture activated strains 4 days, wherein it is described the step of (1) in plating medium formula it is as follows: ammonium sulfate 1.2g, yeast extract 0.5g,
Sodium alginate 1.0g, ferric phosphate 0.5g, agar 2g, add natural sea-water to 100ml, pH7.3.
(2) preparation of seed liquor: preparing seed culture medium 1L, is added in 5L volume seeding tank, and steam heating, 121 DEG C go out
Bacterium 30 minutes, 28 DEG C are cooled to, the single colonie after picking activation, flame method is inoculated in the liquid seed culture medium in seeding tank
In, the ventilatory capacity in seeding tank is 50 L/min, and 180 turns/min of speed of agitator, 28 DEG C are cultivated 16 hours, to seed liquor
OD600 reaches 0.8, and om observation thalli growth is good, whens no living contaminants stand-by, described liquid seed culture medium formula
It is as follows: 5 g of yeast extract, 4 g of peptone, 1 g of glucose, natural sea-water 1000 mL, pH 7.1.
(3) fermentation tank culture is fermented: being prepared fermentation medium, is packed into according to the 50% of fermentation tank capacity percent by volume
Flat-blade turbine stirred type fermentor leads to steam heating, and 121 DEG C sterilize 30 minutes, is cooled to 30 DEG C, seed liquor presses percent by volume
In 3.5% inoculum concentration access fermentation medium, tank is kept to press 0.18MPa, stirring rate 160r/min, control in fermentation process
60 L/min of ventilatory capacity processed, fermentation temperature are 30 DEG C, and fermentation time is 48 hours, and fermentation obtains fermentation liquid, the fermentation training
Support base formula it is as follows: 10 g of yeast extract, 6 g of peptone, 4 g of glucose, 0.015 g of arginine, 0.002 g of calcium chloride and
Natural sea-water 1000 mL, pH7.0.
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, bacterial sediment is obtained, the 80%(v/v for being 4.0 with pH) second
Extracting solution is filtered by 0.45 micron membrane filter, carries out second extraction to precipitating, obtain secondary raffinate by alcohol extracting；It will be secondary
The 80%(v/v that pH is 4.0 is added after the filtering of 0.45 micron membrane filter in clarified solution) ethanol solution extracted three times, merging three
Secondary extracting solution, concentrated by rotary evaporation obtain coarse extraction.
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, rotation
Turn evaporation solvent, obtains white powder to get saxitoxin, 71.2 μ g/L of yield.
Embodiment 3
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, at 28 DEG C
Culture activated strains 3 days, wherein it is described the step of (1) in plating medium formula it is as follows: ammonium sulfate 1.0g, yeast extract 0.4g,
Sodium alginate 0.8g, ferric phosphate 0.3g, agar 2g, add natural sea-water to 100ml, pH7.0.
(2) preparation of seed liquor: preparing seed culture medium 1L, is added in 5L volume seeding tank, and steam heating, 121 DEG C go out
Bacterium 30 minutes, 28 DEG C are cooled to, the single colonie after picking activation, flame method is inoculated in the liquid seed culture medium in seeding tank
In, the ventilatory capacity in seeding tank is 45L/min, and 170 turns/min of speed of agitator, 28 DEG C are cultivated 16 hours, the OD600 to seed liquor
Reach 0.7, om observation thalli growth is good, and whens no living contaminants is stand-by, and the liquid seed culture medium formula is as follows: ferment
Female 5 g of cream, 4 g of peptone, 1 g of glucose, natural sea-water 1000 mL, pH 6.8.
(3) fermentation tank culture is fermented: being prepared fermentation medium, is packed into according to the 40% of fermentation tank capacity percent by volume
Flat-blade turbine stirred type fermentor leads to steam heating, and 121 DEG C sterilize 30 minutes, is cooled to 28 DEG C, seed liquor presses percent by volume
In 2% inoculum concentration access fermentation medium, tank is kept to press 0.15MPa, stirring rate 150/min, control in fermentation process
Ventilatory capacity 50L/min, fermentation temperature are 28 DEG C, and fermentation time is 45 hours, and fermentation obtains fermentation liquid, the fermentation medium
Formula it is as follows: 10 g of yeast extract, 6 g of peptone, 4 g of glucose, 0.015 g of arginine, 0.002 g of calcium chloride and natural
Seawater 1000 mL, pH 6.5.
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, bacterial sediment is obtained, the 80%(v/v for being 4 with pH) ethyl alcohol
It extracts, extracting solution is filtered by 0.45 micron membrane filter, second extraction is carried out to precipitating, obtains secondary raffinate；It will be secondary clear
The 80%(v/v that pH is 4 is added after the filtering of 0.45 micron membrane filter in clear liquid) ethanol solution extracted three times, and merge and mentions three times
Liquid is taken, concentrated by rotary evaporation obtains coarse extraction.
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, rotation
Turn evaporation solvent, obtains white powder to get saxitoxin, 72.8 μ g/L of yield.
Embodiment 4
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, at 30 DEG C
Culture activated strains 3 days, wherein it is described the step of (1) in plating medium formula it is as follows: ammonium sulfate 0.9g, yeast extract 0.4g,
Sodium alginate 0.7g, ferric phosphate 0.2g, agar 2g, add natural sea-water to 100ml, pH7.2.
(2) preparation of seed liquor: preparing seed culture medium 1L, is added in 5L volume seeding tank, and steam heating, 121 DEG C go out
Bacterium 30 minutes, 28 DEG C are cooled to, the single colonie after picking activation, flame method is inoculated in the liquid seed culture medium in seeding tank
In, the ventilatory capacity in seeding tank is 50 L/min, and 175 turns/min of speed of agitator, 28 DEG C are cultivated 16 hours, to seed liquor
OD600 reaches 0.8, and om observation thalli growth is good, whens no living contaminants stand-by, described liquid seed culture medium formula
It is as follows: 5 g of yeast extract, 4 g of peptone, 1 g of glucose, natural sea-water 1000 mL, pH 6.9.
(3) fermentation tank culture is fermented: fermentation medium is prepared, according to 35 ~ 50% dresses of fermentation tank capacity percent by volume
Enter flat-blade turbine stirred type fermentor, lead to steam heating, 121 DEG C sterilize 30 minutes, are cooled to 29 DEG C, seed liquor presses volume basis
In inoculum concentration access fermentation medium than 2.8%, tank is kept to press 0.17MPa, stirring rate 145r/min in fermentation process,
55 L/min of ventilatory capacity is controlled, fermentation temperature is 29 DEG C, and fermentation time is 48 hours, and fermentation obtains fermentation liquid, the fermentation
The formula of culture medium is as follows: 10 g of yeast extract, 6 g of peptone, 4 g of glucose, 0.015 g of arginine, 0.002 g of calcium chloride with
And natural sea-water 1000 mL, pH 6.8.
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, bacterial sediment is obtained, the 80%(v/v for being 3.0 with pH) second
Extracting solution is filtered by 0.45 micron membrane filter, carries out second extraction to precipitating, obtain secondary raffinate by alcohol extracting；It will be secondary
The 80%(v/v that pH is 3.0 is added after the filtering of 0.45 micron membrane filter in clarified solution) ethanol solution extracted three times, merging three
Secondary extracting solution, concentrated by rotary evaporation obtain coarse extraction.
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, rotation
Turn evaporation solvent, obtains white powder to get saxitoxin, 66.8 μ g/L of yield.
Embodiment 5
A method of saxitoxin being prepared using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli is sulfurous
Acidfast bacilli (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, are bought from Chinese Typical Representative
Culture collection, the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, and 27 ~ 31 DEG C
Lower culture activated strains 2 days, wherein it is described the step of (1) in plating medium formula it is as follows: ammonium sulfate 1.2g, yeast extract
0.2g, sodium alginate 0.6g, ferric phosphate 0.5g, agar 2g, add natural sea-water to 100ml, pH7.1.
(2) preparation of seed liquor: preparing seed culture medium 1L, is added in 5L volume seeding tank, and steam heating, 121 DEG C go out
Bacterium 30 minutes, 28 DEG C are cooled to, the single colonie after picking activation, flame method is inoculated in the liquid seed culture medium in seeding tank
In, the ventilatory capacity in seeding tank is 45 L/min, and 180 turns/min of speed of agitator, 28 DEG C are cultivated 16 hours, to seed liquor
OD600 reaches 0.6, and om observation thalli growth is good, whens no living contaminants stand-by, described liquid seed culture medium formula
It is as follows: 5 g of yeast extract, 4 g of peptone, 1 g of glucose, natural sea-water 1000 mL, pH 6.3.
(3) fermentation tank culture is fermented: fermentation medium is prepared, according to 35 ~ 50% dresses of fermentation tank capacity percent by volume
Enter flat-blade turbine stirred type fermentor, lead to steam heating, 121 DEG C sterilize 30 minutes, are cooled to 28 DEG C, seed liquor presses volume basis
In inoculum concentration access fermentation medium than 3.0%, tank is kept to press 0.12 ~ 0.18MPa in fermentation process, stirring rate is
145r/min controls 45 L/min of ventilatory capacity, and fermentation temperature is 28 DEG C, and fermentation time is 46 hours, and fermentation obtains fermentation liquid, institute
The formula for the fermentation medium stated is as follows: 10 g of yeast extract, 6 g of peptone, 4 g of glucose, 0.015 g of arginine, calcium chloride
0.002 g and natural sea-water 1000 mL, pH6.6.
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, bacterial sediment is obtained, the 80%(v/v for being 4.0 with pH) second
Extracting solution is filtered by 0.45 micron membrane filter, carries out second extraction to precipitating, obtain secondary raffinate by alcohol extracting；It will be secondary
The 80%(v/v that pH is 4.0 is added after the filtering of 0.45 micron membrane filter in clarified solution) ethanol solution extracted three times, merging three
Secondary extracting solution, concentrated by rotary evaporation obtain coarse extraction.
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, rotation
Turn evaporation solvent, obtains white powder to get saxitoxin, 70.6 μ g/L of yield.
Claims (10)
1. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced, the sulfurous acidfast bacilli are Asia
Thiobacillus (SulfitobacterSp.) Z1-D bacterial strain, deposit number are CCTCC AB 2016296, the certainly Chinese allusion quotation of purchase
Type culture collection, which is characterized in that the method the following steps are included:
(1) bacterial strain activate: by sulfurous acidfast bacilli (SulfitobacterSp.) Z1-D is inoculated in slant medium, and culture is lived
Change bacterial strain；
(2) preparation of seed liquor: the single colonie after picking activation is inoculated in the liquid seed culture medium in seeding tank, and spread cultivation bacterial strain
For use；
(3) fermentation tank culture is fermented: fermentation medium being fitted into fermentor, by the seed liquor access fermentation training in step (2)
It supports in base, fermentation obtains fermentation liquid；
(4) extraction of fermentation liquid: by fermentation liquid plate-frame filtering, obtaining bacterial sediment, extracted with 80% ethyl alcohol, combined extract, rotation
Inspissation contracting, obtains crude extract；
(5) purifying of crude extract: crude extract carries out gradient elution through half preparation chromatography, collects saxitoxin component, and rotation is steamed
Solvent is sent out, obtains white powder to get saxitoxin.
2. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, plating medium formula is as follows in the step (1): 0.8 ~ 1.2g of ammonium sulfate, 0.2 ~ 0.5g of yeast extract, sea
0.6 ~ 1.0g of mosanom, 0.1 ~ 0.5g of ferric phosphate, agar 2g, add natural sea-water to 100ml, pH6.9 ~ 7.3.
3. a kind of side for preparing saxitoxin using the malicious ocean sulfurous acid bacillus fermentation of production according to claim 1 or 2
Method, which is characterized in that cultivation temperature is 27 ~ 31 DEG C in the step (1), and incubation time is 2 ~ 4 days.
4. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, liquid seed culture medium formula is as follows in the step (2): 5 g of yeast extract, 4 g of peptone, glucose 1
G, 1000 mL of natural sea-water, pH 6.2 ~ 7.1.
5. a kind of side for preparing saxitoxin using the malicious ocean sulfurous acid bacillus fermentation of production according to claim 1 or 4
Method, which is characterized in that ventilatory capacity in the step (2) in seeding tank is 40-50 L/min, speed of agitator 160-180 turns/
Min, 28 DEG C are cultivated 16 hours, and the OD600 to seed liquor reaches 0.6 ~ 0.8, and om observation thalli growth is good, and no miscellaneous bacteria is dirty
It is stand-by when dye.
6. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, the formula of fermentation medium is as follows in the step (3): 10 g of yeast extract, 6 g of peptone, glucose 4
G, 0.015 g of arginine, 0.002 g of calcium chloride and natural sea-water 1000 mL, pH 6.3 ~ 7.0.
7. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, the charge weight of fermentation medium is the 35 ~ 50% of fermentation tank capacity percent by volume in the step (3),
Seed liquor is by the inoculum concentration access fermentation medium of percent by volume 2.5% ~ 3.5%.
8. a kind of side for preparing saxitoxin using the malicious ocean sulfurous acid bacillus fermentation of production according to claim 1 or claim 7
Method, which is characterized in that fermentor is flat-blade turbine stirred type fermentor in the step (3), keeps tank pressure in fermentation process
0.12 ~ 0.18MPa, stirring rate are 140 ~ 160r/min, control 40 ~ 60 L/min of ventilatory capacity, and fermentation temperature is 27 ~ 30 DEG C,
Fermentation time is 40 ~ 48 hours.
9. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, the pH of 80% ethyl alcohol is 2.0 ~ 4.0 in the step (4), extraction time is 3 times.
10. a kind of method for preparing saxitoxin using malicious ocean sulfurous acid bacillus fermentation is produced according to claim 1,
It is characterized in that, the saxitoxin yield is 61.5 ~ 72.8 μ g/L.
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| CN102206270A (en) * | 2011-01-27 | 2011-10-05 | 中国水产科学研究院黄海水产研究所 | Saxitoxin artificial antigen, anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application |
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