CN110079569B - Method for producing microbial polysaccharide-sanzan gum by taking sphingosine monad as strain - Google Patents
Method for producing microbial polysaccharide-sanzan gum by taking sphingosine monad as strain Download PDFInfo
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Abstract
The invention relates to microbial fermentation, in particular to a method for producing microbial polysaccharide sanzan gum by using sphingomonas as a strain. Inoculating Sphingomonas (Sphingomonas sp.) CGMCC No.1650 into a sterilized culture solution containing a carbon source, a nitrogen source and nutrient substances; adjusting the pH value of the culture solution, performing aeration fermentation, and performing sedimentation extraction on fermentation liquor after fermentation is finished to obtain fibrous sanzan gum; the carbon source in the culture solution is one or a combination of waste molasses and waste glucose mother liquor; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen; the ventilation and temperature are controlled in a segmented manner in the fermentation process, and a carbon source is supplemented during aeration fermentation. The invention solves the technical problems of low yield, low gel strength and the like of sanzan glue in the prior art, and has the advantages of obviously superior technical indexes such as yield, gel strength and the like to the prior product, low production cost and the like.
Description
Technical Field
The invention relates to microbial fermentation, in particular to a method for producing microbial polysaccharide sanzan gum by using sphingomonas as a strain.
Background
Sanzan gum is a biological polysaccharide gum, belonging to a biological high molecular polymer. The sanzan gum is safe and non-toxic, has unique physical and chemical properties, has high viscosity and good thickening performance in an aqueous solution, can uniformly suspend some insoluble substances, and has wide application prospects in various fields of food, petroleum, chemical industry and the like as a thickening agent, a stabilizing agent, an emulsifying agent and a gelling agent.
Currently, the industrial production of sanzan gum is prepared by using sphingomonas as a production strain, using glucose and an inorganic nitrogen source as main raw materials, and using biological and chemical technologies through processes of fermentation, extraction, drying and the like.
The invention patent with the patent number of 200610048338.X discloses a Sphingomonas and a method for producing microbial polysaccharide by adopting the same, wherein, sphingomonas (Sphingomonas sp.) CGMCC No.1650 is used as a carbon source in a glucose or sucrose culture medium, the ventilation quantity is 0.2vvm-0.5vvm, the stirring speed is 120-160 r/min, the culture temperature is 35-40 ℃, and the fermentation period is 60-70 hoursPreparing Trizanol fermentation liquor, adjusting isoelectric point of the fermentation liquor with soluble neutral salt, settling, extracting, and dehydrating to obtain microbial polysaccharide Trizanol, wherein the yield of Trizanol obtained by the above process is about 18g/L, and the gel strength of the obtained Trizanol is about 18g/cm 2 。
With the increasing expansion of the application field of sanzan glue and the improvement of the product performance requirement of the sanzan glue, the production cost of the sanzan glue is effectively reduced, the fermentation yield of the sanzan glue is improved, the product performance indexes including gel property and the like are improved, the practical requirement of the industrialized application field of the sanzan glue is expanded, and the sanzan glue is one of the research hotspots in the field.
Disclosure of Invention
The invention aims to provide a method for producing microbial polysaccharide-sanzhan gum by taking sphingosine monad as a strain, waste molasses and waste glucose mother liquor which are industrial waste materials are used as carbon sources, and the technical means of supplementing the carbon sources, effectively controlling the pH value, the pressure, the temperature, the ventilation quantity and the like in the fermentation process are adopted, so that the yield of the sanzhan gum can be effectively improved, the production cost is reduced, and the prepared sanzhan gum has the characteristics of better gel strength and the like.
The overall technical concept of the invention is as follows:
a method for producing microbial polysaccharide-sanzan gum with Sphingomonas sp as strain comprises inoculating Sphingomonas sp (CGMCC No. 1650) into sterilized culture solution containing carbon source, nitrogen source and nutrient substances; adjusting the pH value of the culture solution, performing aeration fermentation, and performing sedimentation extraction on fermentation liquor after fermentation is finished to obtain fibrous sanzan gum; the method also comprises the following process conditions:
A. the carbon source in the culture solution is selected from one or a combination of waste molasses and waste glucose mother liquor; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.03MPa-0.05MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 32-34 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 28-31 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. respectively supplementing carbon source selected from one or combination of waste molasses and waste glucose mother liquor in 11-20 hours and 21-30 hours of aeration fermentation.
The specific technical concept of the invention is as follows:
the method mainly aims to facilitate the growth of thalli by controlling the temperature and the ventilation quantity in a segmented manner in the fermentation process, and controls the ventilation quantity to adjust the dissolved oxygen in fermentation liquor in the fermentation process so as to be beneficial to the synthesis of sanzajiao, wherein the preferable technical scheme is that the temperature and ventilation quantity control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 32-34 ℃, and the ventilation rate is 0.2vvm-0.3vvm;
11-20 hours: the temperature is 32-34 ℃, and the ventilation volume is 0.4vvm-0.5vvm;
21-30 hours: the temperature is 28-31 ℃, and the ventilation rate is 0.7vvm-0.9vvm;
31-40 hours: the temperature is 28-31 ℃, and the ventilation rate is 0.6vvm-0.7vvm;
41-50 hours: the temperature is 28-31 ℃, and the ventilation rate is 0.2vvm-0.3vvm.
The pH value of the fermentation liquor is controlled to be one of important technical means in industrial fermentation, the main purpose of the pH value control method is to meet the growth conditions of thalli to be beneficial to the synthesis of a fermentation end product, and the preferable technical scheme is that sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate is adopted to adjust the pH value =5.0-7.0 in the fermentation process.
More preferably, the sodium hydroxide, the potassium hydroxide, the phosphoric acid, the sulfuric acid or the phosphate adopts a solution with the mass percentage concentration of 10-15%.
The main effect of the feeding in the fermentation process is to supplement the consumption of carbon source caused by the growth of thalli, product synthesis and other reasons so as to meet the requirements of the growth of thalli and the product synthesis, and the preferable technical proposal is that the carbon source accounting for 1-2 percent of the total mass of the fermentation liquid is respectively fed in 11-20 hours and 21-30 hours of aerated fermentation.
In order to better improve the gel strength and the extraction effect of sanzan gum, the preferable technical scheme is that the fermentation liquor after fermentation is subjected to sedimentation extraction is pretreated, and the pretreatment process conditions are as follows:
adding 10-20% sodium hydroxide or potassium hydroxide solution into fermentation liquor after fermentation, wherein the addition amount is 2-4 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
The main purpose of adjusting the pH value in the extraction process is to enable the sanzan gum to form fibrous materials, and the preferable technical realization mode is that organic acid or inorganic acid is used for adjusting the pH value of fermentation liquor during extraction, the pH value of a mixture of extraction liquid and the fermentation liquor is increased to 55-90 ℃ after the pH value of the mixture is =0.5-4.0, and the fibrous sanzan gum is obtained through extraction.
The more preferable technical implementation mode is that the organic acid or the inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, caprylic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid and the like.
In order to better improve the solubility of sanzan gum and facilitate the effective and rapid application of sanzan gum in industrial production to obtain a uniform paste material, the preferable technical implementation means is that strong base, weak acid salt or divalent base is added into the fibrous sanzan gum obtained after extraction to adjust the pH to be between 6.5 and 7.5, and then the fibrous sanzan gum is prepared into the uniform paste material.
In order to effectively reduce the production cost on the premise of ensuring the normal growth of the strains and effectively synthesizing the final product, the preferable technical implementation means is that the organic nitrogen and the inorganic nitrogen are selected from yeast extract, peptone, bean flour and nitrate.
In order to meet the requirement of industrial production, the preferable technical implementation means is that after the Sphingomonas (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, the culture medium is prepared by the following steps: the inoculum size of the culture solution is 5-20% and is inoculated in the culture solution.
More preferably, the expanded culture process comprises:
(1) Performing amplification culture on the slant strains to prepare first-stage seeds;
(2) Carrying out amplification culture on the first-stage seeds to prepare second-stage seeds;
(3) Inoculating the prepared second-stage seeds into the sterilized culture solution, and performing ventilation fermentation;
wherein the culture conditions of the first-stage seeds and the second-stage seeds in the steps (1) and (2) are as follows: the inoculation amount is 5-20%, the air volume is 0.2-0.8 vvm, the temperature is 32-34 ℃, and the seed age is 20-30 hours.
Furthermore, the primary seed culture medium for culturing the primary seeds in the step (1) comprises the following components:
1% -2% of a carbon source; 1% -2% of yeast extract; 0.1 to 1 percent of sodium nitrate; 0.01 to 0.15 percent of magnesium sulfate; dipotassium hydrogen phosphate 0.1-0.5%; 1ppm to 10ppm of ferrous sulfate;
the carbon source is selected from waste molasses and waste glucose mother liquor.
Further, the secondary seed culture medium of the secondary seeds cultured in step (2) comprises the following components:
1% -2% of a carbon source; 0.03 to 0.1 percent of yeast extract; 0.1 to 0.5 percent of sodium nitrate; 0.01 to 0.15 percent of magnesium sulfate; 0.1 to 0.3 percent of dipotassium hydrogen phosphate; 0.05 to 0.3 percent of peptone; 1ppm to 10ppm of ferrous sulfate;
the carbon source is waste molasses and waste glucose mother liquor.
Furthermore, the culture solution comprises the following components:
2% -4% of a carbon source; 0.05 to 0.3 percent of bean flour; 0.05 to 0.3 percent of nitrate; 0.01 to 0.15 percent of magnesium sulfate; dipotassium hydrogen phosphate 0.1-0.3%; 0.05 to 0.4 percent of calcium carbonate; 1ppm to 10ppm of ferrous sulfate; 0.05 to 0.2 percent of defoaming agent.
The applicant carried out relevant tests on the yield of the final product of the method of the invention and the gel properties of the obtained sanzan gum by the following methods:
1. detection of yield
The detection of the yield of sanzan glue has no national standard at present, and the following method is generally adopted for the determination in the industry:
1. the instruments used
Incubator, analytical balance (0.001 g);
2. detection method
Pouring the fermentation liquor into a small beaker with a certain volume, stirring with a glass rod to make the fermentation liquor bubble-free, troweling the surface with the glass rod, and cleaning the fermentation liquor outside the cup. Extracting the fermentation liquor with 90-95% ethanol, settling, squeezing with filter cloth, shredding, transferring into culture dish, oven drying at 105 deg.C to constant weight (about 4 hr), taking out, and weighing.
Solids content = weight grams/volume x 100%
2. Measurement of gel Strength
The detection of the gel strength of sanzan glue has no national standard at present, and the applicant determines the gel strength of the sanzan glue by combining the physicochemical characteristics of the sanzan glue on the basis of the gel strength determination method of the curdlan in reference to the national standard (GB 28304-2012).
1. Instrument and apparatus
Analytical balance: accurate to 0.1mg;
a constant temperature box (temperature range: 5 ℃ -50 ℃);
a gel strength meter;
a water bath (temperature control range: room temperature-100 ℃);
2. test conditions
Probe shape and size: 1.0cm 2 A stainless steel piston cylinder;
probe moving speed: 10mm/s;
3. analytical procedure
(1) Sample preparation
2.0g (exactly to 0.001 g) of the sample is weighed, slowly added into a stirring cup filled with 200ml of distilled water under stirring, stirred at medium speed for 15min, the sample solution is placed into a 250ml beaker, and the total amount is weighed and recorded. Placing the beaker in a water bath kettle, heating at 95 deg.C while stirring with a glass rod intermittently for 3 times, each time stirring for 5-10 times, heating for 30min, taking out the beaker, quickly wiping off the outer wall, weighing, supplementing the evaporated water to the original total amount, and stirring well. Pouring the glue solution into a flat-bottom container with the liquid level height of 2cm while the glue solution is hot, standing, naturally cooling to gel, and placing in a thermostat at 20 ℃ for 20 hours for testing.
(2) Measurement of
Three replicates were measured using a gel strength instrument and averaged.
The invention achieves the substantive characteristics and obvious technical progress that:
1. because the invention adopts the waste molasses and the waste glucose mother liquor as the industrial waste as the carbon source, the production cost is greatly reduced on the premise of ensuring the normal operation of industrial fermentation and effectively synthesizing the final fermentation product.
2. In the fermentation process, a composite nitrogen source consisting of inorganic nitrogen and organic nitrogen is adopted, so that the requirements of thallus growth and the requirements of final product synthesis are effectively met.
3. In the fermentation process, the carbon source is supplemented, and the process conditions such as pressure, temperature, pH value, ventilation quantity and the like are effectively controlled, so that the growth of thalli in the early stage of fermentation is facilitated, the synthesis of final products is facilitated in the middle and later stages of fermentation, and the product performance is effectively improved.
4. The yield of sanzan gum is further improved by improving the post extraction process.
5. The yield of the sanzan gum prepared by the method can reach 25-32g/L, is greatly improved compared with the yield (about 18 g/L) of the prior art, and the gel strength of the sanzan gum prepared by the method can reach 25-30g/cm 2 Is obviously superior to the prior product (18 g/cm) 2 )。
Detailed Description
The present invention is further described with reference to the following examples, which are not intended to limit the scope of the invention, the claims are only to be interpreted as referring to the content of the claims, and any equivalent means can be substituted without departing from the scope of the present invention
Example 1
A method for producing microbial polysaccharide-sanzan gum with Sphingomonas sp as strain comprises inoculating Sphingomonas sp (CGMCC No. 1650) into sterilized culture solution containing carbon source, nitrogen source and nutrient substances; adjusting the pH of the culture solution, performing aerated fermentation, and performing sedimentation extraction on fermentation liquor after the fermentation is finished to obtain fibrous sanzan gum; the method also comprises the following process conditions:
A. the carbon source in the culture solution is waste molasses; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.03MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 32 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 28 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. and respectively supplementing a carbon source accounting for 1 percent of the total mass of the fermentation liquor into the fermentation liquor after 11 hours and 21 hours of aeration fermentation, wherein the carbon source is selected from waste molasses.
The organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, soybean powder and nitrate.
The temperature and ventilation control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 32 ℃, and the ventilation rate is 0.2vvm;
11-20 hours: the temperature is 32 ℃, and the ventilation rate is 0.4vvm;
21-30 hours: the temperature is 28 ℃, and the ventilation rate is 0.7vvm;
31-40 hours: the temperature is 28 ℃, and the ventilation rate is 0.6vvm;
41-50 hours: the temperature was 28 ℃ and the ventilation was 0.2vvm.
And in the aeration fermentation process, sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate is adopted to adjust the pH =5.0-7.0 in the fermentation process.
The sodium hydroxide, the potassium hydroxide, the phosphoric acid, the sulfuric acid or the phosphate adopts a solution with the mass percentage concentration of 10-15%.
And (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows:
and adding 10% by mass of sodium hydroxide or potassium hydroxide solution into the fermentation liquor after the fermentation is finished, wherein the addition amount is 2g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
And (3) adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 55 ℃ after the pH of the mixture is =0.5, and extracting to obtain the fibrous sanzan gum. The organic acid or inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid, etc.
Adding strong base weak acid salt or divalent base into the fibrous sanzang gum obtained after extraction to adjust the pH =6.5-7.5, and then preparing into uniform pasty material.
After Sphingomonas sp (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, according to the following seed liquid: the culture solution was inoculated in an inoculum size of 5%.
The process of the expanded culture comprises the following steps:
(1) Performing amplification culture on the slant strains to prepare first-grade seeds;
(2) Carrying out amplification culture on the first-stage seeds to prepare second-stage seeds;
(3) Inoculating the prepared second-level seeds into the sterilized culture solution, and performing ventilation fermentation;
wherein the culture conditions of the first-stage seeds and the second-stage seeds in the steps (1) and (2) are as follows: the inoculation amount is 5 percent, the ventilation amount is 0.2vvm, the temperature is 32 ℃, and the seed age is 20 hours.
The primary seed culture medium for culturing the primary seeds in the step (1) comprises the following components:
1% of carbon source; 1% of yeast extract; 0.1 percent of sodium nitrate; magnesium sulfate 0.01%; dipotassium phosphate 0.1%; 1ppm of ferrous sulfate;
the carbon source is waste molasses and waste glucose mother liquor.
The secondary seed culture medium of the secondary seeds cultured in the step (2) comprises the following components:
1% of carbon source; 0.03% of yeast extract; 0.1 percent of sodium nitrate; magnesium sulfate 0.01%; dipotassium phosphate 0.1%; 0.05% of peptone; 1ppm of ferrous sulfate;
the carbon source is selected from waste molasses and waste glucose mother liquor.
The culture solution comprises the following components:
2% of a carbon source; 0.05% of bean flour; 0.05 percent of nitrate; 0.01 percent of magnesium sulfate; dipotassium phosphate 0.1%; 0.05 percent of calcium carbonate; ferrous sulfate 1ppmm; 0.05 percent of defoaming agent.
Through the test of the applicant, the yield of the sanzang glue prepared by the method in the embodiment can reach 25g/L, and the gel strength of the sanzang glue prepared by the method in the embodiment can reach 26g/cm 2 。
Example 2
This example differs from example 1 in that:
the production of sanzang glue also comprises the following process conditions:
A. selecting waste glucose mother liquor as a carbon source in the culture solution; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.05MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 34 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 31 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. respectively supplementing carbon sources accounting for 2 percent of the total mass of the fermentation liquor into the fermentation liquor after 20 hours and 30 hours of aeration fermentation, wherein the carbon sources are selected from waste glucose mother liquor.
The organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, soybean powder and nitrate.
The temperature and ventilation control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 34 ℃, and the ventilation rate is 0.3vvm;
11-20 hours: the temperature is 34 ℃, and the ventilation rate is 0.5vvm;
21-30 hours: the temperature is 31 ℃, and the ventilation rate is 0.9vvm;
31-40 hours: the temperature is 31 ℃, and the ventilation rate is 0.7vvm;
41-50 hours: the temperature was 31 ℃ and the ventilation was 0.3vvm.
And (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows:
and adding 20 percent by mass of sodium hydroxide or potassium hydroxide solution into the fermentation liquor after the fermentation is finished, wherein the addition amount is 4 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
And (3) adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 90 ℃ after the pH of the mixture is =4.0, and extracting to obtain the fibrous sanzan gum. The organic acid or inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid, etc.
After the Sphingomonas (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, according to the seed liquid: the culture solution is inoculated in the culture solution with the inoculum size of 20 percent.
The culture conditions of the first-level seeds and the second-level seeds in the amplification culture are as follows: the inoculation amount is 20 percent, the ventilation amount is 0.8vvm, the temperature is 34 ℃, and the seed age is 30 hours.
The primary seed culture medium for culturing the primary seeds comprises the following components:
2% of a carbon source; 2% of yeast extract; 1% of sodium nitrate; 0.15 percent of magnesium sulfate; dipotassium phosphate 0.5%; 10ppm of ferrous sulfate;
the carbon source is selected from one of waste molasses and waste glucose mother liquor.
The secondary seed culture medium of the cultured secondary seeds comprises the following components:
2% of a carbon source; 0.1% of yeast extract; 0.5 percent of sodium nitrate; 0.15 percent of magnesium sulfate; dipotassium phosphate 0.3%; 0.3% of peptone; 10ppm of ferrous sulfate;
the carbon source is selected from one of waste molasses and waste glucose mother liquor.
The culture solution comprises the following components:
4% of a carbon source; 0.3 percent of bean flour; 0.3 percent of nitrate; 0.15 percent of magnesium sulfate; dipotassium phosphate 0.3%; 0.4 percent of calcium carbonate; 10ppm of ferrous sulfate; 0.2 percent of defoaming agent.
Through the test of the applicant, the yield of the sanzan gum adopting the method of the embodiment can reach 26g/L, and the sanzan gum adopting the method can reach 26g/LThe gel strength of the sanzan glue prepared by the method of the embodiment can reach 27g/cm 2 。
Example 3
The present example differs from example 1 in that:
the production of sanzang glue also comprises the following process conditions:
A. the carbon source in the culture solution is a mixture of waste molasses and waste glucose mother liquor which are compounded according to the equal amount; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.04MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 33 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 30 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. respectively supplementing carbon sources accounting for 1-2% of the total mass of the fermentation liquor in 15 hours and 25 hours of aerated fermentation, wherein the carbon sources are selected from a mixture of equivalently compounded waste molasses and waste glucose mother liquor.
The organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, soybean powder and nitrate.
The temperature and ventilation control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 33 ℃, and the ventilation volume is 0.2vvm-0.3vvm;
11-20 hours: the temperature is 33 ℃, and the ventilation volume is 0.4vvm-0.5vvm;
21-30 hours: the temperature is 30 ℃, and the ventilation rate is 0.7vvm-0.9vvm;
31-40 hours: the temperature is 30 ℃, and the ventilation volume is 0.6vvm-0.7vvm;
41-50 hours: the temperature is 30 ℃, and the ventilation rate is 0.2vvm-0.3vvm.
And (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows:
and adding 15% by mass of sodium hydroxide or potassium hydroxide solution into the fermentation liquor after the fermentation is finished, wherein the addition amount is 3 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
And (3) adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 75 ℃ after the pH of the mixture is =3.0, and extracting to obtain the fibrous sanzan gum. The organic acid or inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid, etc.
After Sphingomonas sp (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, according to the following seed liquid: the culture solution is inoculated in an inoculum size of 15 percent.
The culture conditions of the first-stage seeds and the second-stage seeds in the amplification culture are as follows: the inoculation amount is 12 percent, the ventilation amount is 0.5vvm, the temperature is 33 ℃, and the seed age is 25 hours.
The primary seed culture medium for culturing the primary seeds comprises the following components:
1.5% of a carbon source; 1.5% of yeast extract; 0.5 percent of sodium nitrate; magnesium sulfate 0.08%; dipotassium phosphate 0.3%; 5ppm of ferrous sulfate;
the carbon source is a mixture of waste molasses and waste glucose mother liquor which are compounded in equal amount.
The secondary seed culture medium of the cultured secondary seeds comprises the following components:
carbon source 1.5 percent; 0.06% of yeast extract; 0.3 percent of sodium nitrate; magnesium sulfate 0.08%; dipotassium phosphate 0.2%; 0.15 percent of peptone; 5ppm of ferrous sulfate;
the carbon source is a mixture of waste molasses and waste glucose mother liquor which are compounded in equal amount.
The culture solution comprises the following components:
3% of a carbon source; 0.15 percent of bean flour; 0.15 percent of nitrate; magnesium sulfate 0.08%; dipotassium phosphate 0.2%; 0.2 percent of calcium carbonate; 5ppm of ferrous sulfate; 0.1 percent of defoaming agent.
Through the test of the applicant, the yield of the sanzang glue prepared by the method in the embodiment can reach 28g/L, and the gel strength of the sanzang glue prepared by the method in the embodiment can reach 28g/cm 2 。
Example 4
This example differs from example 1 in that:
the production of sanzan glue also comprises the following process conditions:
A. the carbon source in the culture solution is waste molasses; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.035MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 32 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 30 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. and respectively supplementing a carbon source accounting for 2 percent of the total mass of the fermentation liquor into the fermentation liquor after 13 hours and 23 hours of aeration fermentation, wherein the carbon source is selected from waste molasses.
The organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, soybean powder and nitrate.
The temperature and ventilation control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 32 ℃, and the ventilation rate is 0.3vvm;
11-20 hours: the temperature is 32 ℃, and the ventilation rate is 0.5vvm;
21-30 hours: the temperature is 30 ℃, and the ventilation rate is 0.7vvm;
31-40 hours: the temperature is 30 ℃, and the ventilation rate is 0.6vvm;
41-50 hours: the temperature was 30 ℃ and the ventilation was 0.3vvm.
And (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows:
and adding a 12 mass percent sodium hydroxide or potassium hydroxide solution into the fermentation liquor after the fermentation is finished, wherein the addition amount is 2.5 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
And (3) adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 65 ℃ after the pH of the mixture of the extraction liquid and the fermentation liquor is =1, and extracting to obtain the fibrous sanzan gum. The organic acid or inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid, etc.
After the Sphingomonas (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, according to the seed liquid: the inoculum size of the culture solution is 5-20% and is inoculated in the culture solution.
The culture conditions of the first-level seeds and the second-level seeds in the amplification culture are as follows: the inoculation amount is 10 percent, the ventilation amount is 0.3vvm, the temperature is 33 ℃, and the seed age is 22 hours.
The primary seed culture medium for culturing the primary seeds comprises the following components:
1.2% of a carbon source; 1.2% of yeast extract; 0.3 percent of sodium nitrate; magnesium sulfate 0.05%; dipotassium phosphate 0.2%; 3ppm of ferrous sulfate;
the carbon source is waste molasses.
The secondary seed culture medium of the cultured secondary seeds comprises the following components:
1.2% of a carbon source; 0.04% of yeast extract; 0.2 percent of sodium nitrate; 0.05 percent of magnesium sulfate; dipotassium phosphate 0.15%; 0.1% of peptone; 3ppm of ferrous sulfate;
the carbon source is waste molasses.
The culture solution comprises the following components:
2.5 percent of carbon source; 0.1 percent of bean flour; 0.1 percent of nitrate; 0.05 percent of magnesium sulfate; dipotassium phosphate 0.15%; 0.1 percent of calcium carbonate; 3ppm of ferrous sulfate; 0.08 percent of defoaming agent.
Through the test of the applicant, the yield of the sanzan gum prepared by adopting the method of the embodiment can reach 29g/L, and the gel strength of the sanzan gum prepared by adopting the method of the embodiment can reach 30g/cm 2 。
Example 5
This example differs from example 1 in that:
the production of sanzang glue also comprises the following process conditions:
A. selecting waste glucose mother liquor as a carbon source in the culture solution; the nitrogen source is a compound nitrogen source consisting of inorganic nitrogen and organic nitrogen;
B. the pressure is 0.05MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 34 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 28 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, and the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation;
C. respectively supplementing a carbon source which accounts for 2 percent of the total mass of the fermentation liquor in 18 hours and 27 hours of aeration fermentation, wherein the carbon source is selected from waste glucose mother liquor.
The organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, soybean powder and nitrate.
The temperature and ventilation control conditions in the fermentation process are as follows:
0-10 hours: the temperature is 34 ℃, and the ventilation rate is 0.3vvm;
11-20 hours: the temperature is 34 ℃, and the ventilation rate is 0.4vvm;
21-30 hours: the temperature is 28 ℃, and the ventilation rate is 0.9vvm;
31-40 hours: the temperature is 28 ℃, and the ventilation rate is 0.7vvm;
41-50 hours: the temperature was 28 ℃ and the ventilation was 0.2vvm.
And (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows:
adding 18 percent by mass of sodium hydroxide or potassium hydroxide solution into the fermentation liquor after the fermentation is finished, wherein the adding amount is 3.5 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor.
And (3) adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 80 ℃ after the pH of the mixture is =3.0, and extracting to obtain the fibrous sanzan gum. The organic acid or inorganic acid is one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, caprylic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid, etc.
After the Sphingomonas (Sphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, according to the seed liquid: the inoculum size of the culture solution is 5-20 percent and is inoculated in the culture solution.
The culture conditions of the first-level seeds and the second-level seeds in the amplification culture are as follows: the inoculation amount is 5-20%, the air volume is 0.6vvm, the temperature is 34 ℃, and the seed age is 28 hours.
The primary seed culture medium for culturing the primary seeds comprises the following components:
1.8% of a carbon source; 1.8 percent of yeast extract; 0.8 percent of sodium nitrate; 0.12 percent of magnesium sulfate; dipotassium phosphate 0.4%; 8ppm of ferrous sulfate;
the carbon source is waste glucose mother liquor.
The secondary seed culture medium of the cultured secondary seeds comprises the following components:
1.8% of a carbon source; 0.08 percent of yeast extract; 0.4 percent of sodium nitrate; 0.13 percent of magnesium sulfate; dipotassium phosphate 0.25%; 0.25% of peptone; 8ppm of ferrous sulfate;
the carbon source is waste glucose mother liquor.
The culture solution comprises the following components:
3.5% of a carbon source; 0.25 percent of bean flour; 0.25 percent of nitrate; 0.12 percent of magnesium sulfate; dipotassium phosphate 0.25%; 0.3 percent of calcium carbonate; 8ppm of ferrous sulfate; 0.15 percent of defoaming agent.
Through the test of the applicant, the yield of the sanzang glue prepared by the method in the embodiment can reach 32g/L, and the gel strength of the sanzang glue prepared by the method in the embodiment can reach 30g/cm 2 。
Claims (10)
1. A process for preparing microbial polysaccharide-sanzan gum from sphingomonas includes such steps as preparing the bacterial strain of sphingomonas (Sphingomonas)Sphingomonas spNo.1650 of CGMCC is inoculated into the sterilized culture solution containing carbon source, nitrogen source and nutrient substance; adjusting the pH value of the culture solution, performing aeration fermentation, and performing sedimentation extraction on fermentation liquor after fermentation is finished to obtain fibrous sanzan gum; the method is characterized by also comprising the following process conditions:
A. the carbon source in the culture solution is selected from one or a combination of waste molasses and waste glucose mother liquor; the nitrogen source is a compound nitrogen source consisting of organic nitrogen and inorganic nitrogen;
B. the pressure is 0.03MPa-0.05MPa, the pH value is =5.0-7.0 in the aeration fermentation process, the ventilation quantity and the temperature are controlled in a segmented mode in the fermentation process, the temperature is 32-34 ℃ from the beginning of fermentation to 20 hours of fermentation, the temperature is 28-31 ℃ from 20 hours of fermentation to the end of fermentation, the ventilation quantity is gradually increased from the beginning of fermentation to 30 hours of fermentation, the ventilation quantity is gradually reduced from 30 hours of fermentation to the end of fermentation, and the control conditions of the temperature and the ventilation quantity in the fermentation process are as follows:
0-10 hours: the temperature is 32-34 ℃, and the ventilation rate is 0.2vvm-0.3vvm;
11-20 hours: the temperature is 32-34 ℃, and the ventilation rate is 0.4vvm-0.5vvm;
21-30 hours: the temperature is 28-31 ℃, and the ventilation volume is 0.7vvm-0.9vvm;
31-40 hours: the temperature is 28-31 ℃, and the ventilation volume is 0.6-0.7 vvm;
41-50 hours: the temperature is 28-31 ℃, and the ventilation rate is 0.2vvm-0.3vvm;
C. respectively supplementing carbon sources accounting for 1-2% of the total mass of the fermentation liquor into the fermentation liquor after 11-20 hours and 21-30 hours of aeration fermentation, wherein the carbon sources are selected from one or combination of waste molasses and waste glucose mother liquor;
and (3) pretreating the fermentation liquor after fermentation is finished before sedimentation extraction, wherein the pretreatment process conditions are as follows: adding 10-20% sodium hydroxide or potassium hydroxide solution by mass percent into fermentation liquor after fermentation is finished, wherein the addition amount is 2-4 g of solid sodium hydroxide or potassium hydroxide added into each liter of fermentation liquor;
adjusting the pH of the fermentation liquor by using organic acid or inorganic acid during extraction, heating the mixture of the extraction liquid and the fermentation liquor to 55-90 ℃ after the pH of the mixture is =0.5-4.0, and extracting to obtain fibrous sanzan gum;
adding strong base and weak acid salt or divalent base into the fibrous sanzang gum obtained after extraction to adjust the pH =6.5-7.5, and preparing into uniform pasty material.
2. The method of claim 1, wherein the pH =5.0-7.0 is adjusted by sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate during the aeration fermentation process.
3. The method for producing microbial polysaccharide-sanzan gum using sphingomonas as a strain according to claim 2, wherein the sodium hydroxide, potassium hydroxide, phosphoric acid, sulfuric acid or phosphate is a solution with a concentration of 10-15% by mass.
4. The method of claim 1, wherein the organic acid or inorganic acid is selected from one or a mixture of at least two of citric acid, formic acid, propionic acid, butyric acid, caprylic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, and carbonic acid.
5. The method of claim 1, wherein the organic nitrogen and the inorganic nitrogen are selected from yeast extract, peptone, soybean meal and nitrate.
6. The method of claim 1 for producing microbial polysaccharide Trizanol using Sphingomonas spSphingomonas sp.) CGMCC No.1650 is subjected to amplification culture, and then the culture medium is prepared according to the following steps: the inoculum size of the culture solution is 5-20 percent and is inoculated in the culture solution.
7. The method of claim 6, wherein the expanded culture process comprises:
(1) Performing amplification culture on the slant strains to prepare first-grade seeds;
(2) Carrying out amplification culture on the first-stage seeds to prepare second-stage seeds;
(3) Inoculating the prepared second-level seeds into the sterilized culture solution, and performing ventilation fermentation;
wherein the culture conditions of the first-stage seeds and the second-stage seeds in the steps (1) and (2) are as follows: the inoculation amount is 5-20%, the air volume is 0.2-0.8 vvm, the temperature is 32-34 ℃, and the seed age is 20-30 hours.
8. The method for producing microbial polysaccharide Trizanol as claimed in claim 7, wherein the primary seed culture medium for culturing the primary seed in step (1) comprises the following components:
1% -2% of a carbon source; 1% -2% of yeast extract; 0.1 to 1 percent of sodium nitrate; 0.01 to 0.15 percent of magnesium sulfate; 0.1 to 0.5 percent of dipotassium hydrogen phosphate; 1ppm to 10ppm of ferrous sulfate;
the carbon source is waste molasses and waste glucose mother liquor.
9. The method for producing microbial polysaccharide Trizanol as claimed in any one of claims 7 or 8, wherein the secondary seed culture medium for culturing the secondary seed in step (2) comprises the following components:
1% -2% of a carbon source; 0.03% -0.1% of yeast extract; 0.1 to 0.5 percent of sodium nitrate; 0.01 to 0.15 percent of magnesium sulfate; 0.1 to 0.3 percent of dipotassium hydrogen phosphate; 0.05 to 0.3 percent of peptone; 1ppm to 10ppm of ferrous sulfate;
the carbon source is selected from waste molasses and waste glucose mother liquor.
10. The method for producing microbial polysaccharide Trizanol as claimed in any one of claims 1, 6 or 7, wherein the culture medium comprises the following components:
2% -4% of a carbon source; 0.05 to 0.3 percent of bean flour; 0.05 to 0.3 percent of nitrate; 0.01 to 0.15 percent of magnesium sulfate; 0.1 to 0.3 percent of dipotassium hydrogen phosphate; 0.05 to 0.4 percent of calcium carbonate; 1ppm to 10ppm of ferrous sulfate; 0.05 to 0.2 percent of defoaming agent.
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| CN110760015B (en) * | 2019-11-19 | 2022-07-22 | 河北鑫合生物化工有限公司 | Improved extraction method of sanzan gum |
| CN111057711B (en) * | 2019-12-25 | 2022-01-18 | 廊坊梅花生物技术开发有限公司 | Sphingomonas engineering bacteria and construction method and application thereof |
| CN111500497B (en) * | 2020-04-27 | 2022-09-06 | 河北鑫合生物化工有限公司 | Sphingomonas, a synthetic bacterium of sanzan glue with molecular marker, and application thereof in preparation of sanzan glue |
| CN113248629B (en) * | 2021-05-14 | 2023-04-25 | 廊坊梅花生物技术开发有限公司 | Method for extracting sanzan gum from fermentation liquor and product thereof |
| CN113583904B (en) * | 2021-07-24 | 2023-09-12 | 河北沣川生物科技有限公司 | Extracellular multimeric Sphingomonas and application thereof in preparation of high gel strength sanzan gum |
| CN114010533B (en) * | 2021-11-05 | 2022-08-05 | 南开大学 | Natural sanzang gel with alcohol solubility and moisture retention, no-clean disinfection gel and application |
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