CN110079569A - The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell - Google Patents

The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell Download PDF

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CN110079569A
CN110079569A CN201910354160.9A CN201910354160A CN110079569A CN 110079569 A CN110079569 A CN 110079569A CN 201910354160 A CN201910354160 A CN 201910354160A CN 110079569 A CN110079569 A CN 110079569A
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glue
fermentation
acid
cell
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CN110079569B (en
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张禹
张国沛
张少华
贾敬敏
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Hebei Xinhe Biochemical Co Ltd
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    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

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Abstract

The present invention relates to microbial fermentation, spy is standby to refer to a kind of method for praising glue as strain production microbial polysaccharide-three using Sphingol single-cell.It includes being inoculated into Sphingol single-cell (Sphingomonas sp.) CGMCC No.1650 after sterilizing and in the culture solution containing carbon source, nitrogen source and nutriment；The pH of culture solution is adjusted, aerobic fementation is carried out, fermentation liquid after fermentation is subjected to sedimentation threadiness is obtained by extraction three to praise glue；Carbon source in culture solution selects one of blackstrap, waste glucose mother liquor or its combination；Nitrogen source selects the compound nitrogen source of inorganic nitrogen and organic nitrogen composition；It is controlled in fermentation process using ventilation quantity and temperature section, fills into carbon source in aerobic fementation.The advantages such as the present invention, which solves, three praises the technical problems such as glue yield, gel strength be low in the prior art, and there are the technical indicators such as yield and gel strength to be substantially better than existing product, and production cost is low.

Description

The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell
Technical field
It is special a kind of using Sphingol single-cell as strain production microbial polysaccharide-for referring to the present invention relates to microbial fermentation Three methods for praising glue.
Background technique
Three to praise glue be biological polyoses glue, belongs to biopolymer.Three to praise glue safe and non-toxic, and physicochemical property is unique, Aqueous solution has viscosity high, and thickening properties are good, the energy some slightly solubility substances of even suspension, as thickener, stabilizer, emulsifier It has broad application prospects with gelling agent in multiple fields such as food, petroleum, chemical industry.
Current three industrialized productions for praising glue are with glucose and inorganic nitrogen-sourced to be for production strain with Sphingol single-cell Primary raw material is made using biology and Chemical Engineering Technology through techniques such as everfermentation, extraction, dryings.
A kind of Sphingol single-cell is disclosed in the patent of invention of Patent No. 200610048338.X and uses the bacterium The method that kind produces microbial polysaccharide, wherein with Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 with Portugal The carbon source of grape sugar or sucrose culture medium, in ventilation quantity 0.2vvm-0.5vvm, 120-160 revs/min of speed of agitator, cultivation temperature 35-40 DEG C, fermentation period is obtained three to praise glue fermentation liquid under conditions of 60-70 hour, and fermentation liquid after fermentation is with solvable Property neutral salt adjust and praise glue up to microbial polysaccharide three after isoelectric point, sedimentation extraction, dehydration, the three of above-mentioned technique praise glue yield about For 18g/L or so, prepared three gel strengths for praising glue are about 18g/cm2。
The application field for praising glue with three is increasingly expanded and for the raising that its properties of product requires, is effectively reduced three and praises The production cost of glue improves three and praises glue fermentation yield, and improving it includes the product performance indexs such as gelation, is not only expansion three and praises The current demand in the industrial applications field of glue, and be also one of the research hotspot of this field.
Summary of the invention
The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell it is an object of the invention to provide a kind of, Using as industrial waste blackstrap and waste glucose mother liquor as carbon source, by using filling into carbon source during the fermentation, have The technological means such as effect control pH value, pressure, temperature and ventilation quantity, can effectively improve three yields for praising glue, reduction is produced into This, prepared three praise glue with characteristics such as preferable gel strengths.
Overall technology design of the invention is:
The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell, is by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 is inoculated into after sterilizing and the culture solution containing carbon source, nitrogen source and nutriment In；The pH of culture solution is adjusted, aerobic fementation is carried out, fermentation liquid after fermentation is subjected to sedimentation threadiness is obtained by extraction three to praise Glue；Further include following process conditions:
A, the carbon source in culture solution selects one of blackstrap, waste glucose mother liquor or its combination；Nitrogen source is selected inorganic The compound nitrogen source of nitrogen and organic nitrogen composition；
B, pressure is 0.03MPa-0.05MPa, pH value=5.0-7.0, using logical in fermentation process during aerobic fementation Air quantity and temperature section control start to 20 hours temperature of fermentation to be 32 DEG C -34 DEG C from fermentation, tie from fermentation 20 hours to fermenting Shu Wendu is 28 DEG C -31 DEG C, starts to 30 hours ventilation quantities of fermenting to gradually increase from fermentation, ferments 30 hours certainly to fermentation ends Ventilation quantity gradually decreases；
C, carbon source was filled into respectively at aerobic fementation 11-20 hours and 21-30 hours, the carbon source is selected from blackstrap, gives up One of glucose mother liquid or its combination.
Particular technique of the invention is conceived also:
Thalli growth is easy for using the main purpose of Discrete control temperature and ventilation quantity in fermentation process, and in fermentation The dissolved oxygen in ventilation quantity adjustment fermentation liquid, the synthesis for praising glue in favor of three are controlled, wherein preferred technical solution is, the hair Temperature and ventilation quantity control condition during ferment is as follows:
0-10 hours: 32 DEG C -34 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm；
11-20 hours: 32 DEG C -34 DEG C of temperature, ventilation quantity 0.4vvm-0.5vvm；
21-30 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.7vvm-0.9vvm；
31-40 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.6vvm-0.7vvm；
41-50 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm.
The pH value of control fermentation liquid is one of the important technical in industrial fermentation, and main purpose is to meet thallus Growth conditions, to be conducive to the synthesis of fermentation end products, wherein preferred technical solution is, during the aerobic fementation PH=5.0-7.0 in fermentation process is adjusted using sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate.
More preferably technical solution is that the sodium hydroxide, potassium hydroxide, phosphoric acid, sulfuric acid or phosphate use matter Measure the solution that percent concentration is 10%-15%.
The main function of feed supplement is that carbon source disappears caused by supplementing because of reasons such as thalli growth, Product formations in fermentation process Consumption, to meet the needs of thalli growth and Product formation, preferred technical solution is, in aerobic fementation 11 hours to 20 hours and The carbon source for accounting for fermentation liquid gross mass 1%-2% is filled into respectively within 21 hours to 30 hours.
Three gel strengths and effect of extracting for praising glue are preferably improved to obtain, preferred technical solution is fermentation ends Fermentation liquid afterwards is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
The sodium hydroxide or hydroxide that mass percent concentration is 10%-20% are added in fermentation liquid after fermentation Potassium solution, additional amount are that solid NuaO or 2-4 grams of potassium hydroxide is added in every liter of fermentation liquid.
The main purpose that pH value adjusts in extraction process is to make three to praise glue to form fiberous material, and preferred technology is realized Mode is, organic acid or inorganic acid adjust fermentation liquid pH when extraction, makes the pH=0.5- of the mixture of extract liquor and fermentation liquid It is warming up to 55 DEG C -90 DEG C after 4.0, threadiness three is obtained by extraction and praises glue.
Wherein more preferably technical implementation way is, organic acid or inorganic acid select citric acid, formic acid, propionic acid, butyric acid, One of octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
Preferably to improve three solubility properties for praising glue, convenient for its effectively quickly application in the industrial production, obtain uniformly Plaster material, preferred technology realization rate be the threadiness three obtained after the extraction praise in glue be added strong base-weak acid salt or Uniform plaster material is made after adjusting pH=6.5-7.5 in diatomic base.
Production cost is effectively reduced under the premise of guaranteeing strain normal growth and being effectively synthesized final product to realize, preferably Technology realization rate be that the organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, bean powder and nitrate.
For the needs for meeting industrialized production, preferred technology realization rate is, described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 is after expanding culture, according to seed liquor: culture solution is connecing for 5%-20% Kind amount is inoculated in culture solution.
More preferably technical implementation way is that the expansion incubation includes:
(1) first order seed is made after expanding culture in slant strains；
(2) secondary seed is made after expanding culture in first order seed；
(3) by the culture solution after the access sterilizing of manufactured secondary seed, aerobic fementation is carried out；
Wherein step (1), the condition of culture of first order seed and secondary seed in (2) are as follows: inoculum concentration 5%-20%, air quantity are 0.2vvm-0.8vvm, 32 DEG C -34 DEG C of temperature, kind age is 20-30 hours.
Further, the primary-seed medium of culture first order seed includes following component in the step (1):
Carbon source 1%-2%；Yeast extract 1%-2%；Sodium nitrate 0.1%-1%；Magnesium sulfate 0.01%-0.15%；Phosphoric acid hydrogen Dipotassium 0.1%-0.5%；Ferrous sulfate 1ppm-10ppm；
Carbon source selects blackstrap, waste glucose mother liquor.
Further, the secondary seed medium for the secondary seed cultivated in the step (2) includes following component:
Carbon source 1%-2%；Yeast extract 0.03%-0.1%；Sodium nitrate 0.1%-0.5%；Magnesium sulfate 0.01%-0.15%； Dipotassium hydrogen phosphate 0.1%-0.3%；Peptone 0.05%-0.3%；Ferrous sulfate 1ppm-10ppm；
Carbon source selects blackstrap, waste glucose mother liquor.
Further, the culture solution includes following component:
Carbon source 2%-4%；Bean powder 0.05%-0.3%；Nitrate 0.05%-0.3%；Magnesium sulfate 0.01%-0.15%； Dipotassium hydrogen phosphate 0.1%-0.3%；Calcium carbonate 0.05%-0.4%；Ferrous sulfate 1ppm-10ppm；Defoaming agent 0.05%- 0.2%.
Three gelations for praising glue of the applicant final production yield to method of the invention and acquisition with the following method It can be carried out dependence test, specific as follows:
One, yield detects
Three detections for praising glue yield there is no national standard at present, the measurement in industry generally with the following method:
1, instrument
Insulating box, assay balance (0.001g)；
2, detection method
Fermentation liquid is poured into the small beaker of certain volume, makes its bubble-free with glass bar stirring, with glass bar surface It smoothes out, the fermentation liquid outside cup is cleaned up.Fermentation liquid is extracted with the ethyl alcohol of 90%-95%, is settled, filter cloth is extracted, will It is all thoroughly moved into culture dish after tearing up, and is then placed in 105 DEG C of baking ovens dry to constant weight (about 4 hours), takes out weighing.
Solid content=title sample grams/volume × 100%
Two, the test of gel strength
The detection for praising glue gel intensity about three there is no national standard at present, and applicant is using for reference with reference to national standard (GB28304- 2012) in about curdlan gel strength measuring method on the basis of, the physicochemical property for praising glue in conjunction with three, with the following method The gel strength for praising glue to three is measured.
1, instrument and equipment
Assay balance: it is accurate to 0.1mg；
Insulating box (temperature range: 5 DEG C -50 DEG C)；
Gel strength instrument；
Water-bath (temperature control range: -100 DEG C of room temperature)；
2, test condition
Shape of popping one's head in and size: 1.0cm2Stainless steel piston type cylindrical body；
Probe movement speed: 10mm/s；
3, analytical procedure
(1) sample preparation
2.0g (being accurate to 0.001g) sample is weighed, is slowly added under stirring conditions equipped with 200ml distilled water In stirring cup, sample solution is placed in 250ml beaker in tall form by moderate-speed mixer 15min, is weighed and is recorded total amount.Beaker is set It is heated under the conditions of water-bath, 95 DEG C, during which with glass bar occasional agitation 3 times, stirs under 5-10, taken after heating 30min every time Beaker out quickly dries outer wall, weighs, and supplements evaporated water to former total amount, stirs evenly.Sol solution is poured into flat appearance while hot Device, liquid level 2cm are stood, to be measured after placing 20h in 20 DEG C of insulating boxs after naturally cooling to gel.
(2) it measures
Three Duplicate Samples are measured with gel strength instrument, are averaged.
Substantive distinguishing features obtained by the present invention and significant technological progress are:
1, since the blackstrap of the invention for being employed as industrial waste and waste glucose mother liquor are as carbon source, guaranteeing industry Under the premise of fermentation is normally carried out and is effectively synthesized fermentation end products, production cost is considerably reduced.
2, the compound nitrogen source formed in fermentation process using inorganic nitrogen and organic nitrogen, effectively meets the needs of thalli growth And the needs of final product synthesis.
3, by using carbon source is filled into fermentation process, the techniques items such as pressure, temperature, pH value, ventilation quantity are effectively controlled Part makes earlier fermentation be conducive to thalli growth, and the fermentation middle and later periods is conducive to the synthesis of final product, while effectively increasing product Energy.
4, by the improvement to rear extraction process, three yields for praising glue are further improved.
5, it is tested through applicant, the yield for praising glue using method three of the invention compares prior art up to 25-32g/L Yield (about 18g/L) increase substantially, three prepared using method of the invention praise the gel strength of glue up to 25-30g/ cm2, it is significantly better than existing product (18g/cm2)。
Specific embodiment
The present invention is described further with reference to embodiments, but not as a limitation of the invention, guarantor of the invention Shield range is subject to the content of claim record, the replacement of equivalent manners that any specification according to the present invention is made, Protection scope of the present invention is not departed from
Embodiment 1
The method that microbial polysaccharide-three praises glue is produced by strain of Sphingol single-cell, is by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 is inoculated into after sterilizing and the culture solution containing carbon source, nitrogen source and nutriment In；The pH of culture solution is adjusted, aerobic fementation is carried out, fermentation liquid after fermentation is subjected to sedimentation threadiness is obtained by extraction three to praise Glue；Further include following process conditions:
A, the carbon source in culture solution selects blackstrap；Nitrogen source selects the compound nitrogen source of inorganic nitrogen and organic nitrogen composition；
B, pressure is 0.03MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity and temperature in fermentation process Spend Discrete control, start to 20 hours temperature of fermentation to be 32 DEG C from fermentation, from ferment 20 hours to fermentation ends temperature be 28 DEG C, Start to 30 hours ventilation quantities of fermenting to gradually increase since fermentation, be gradually decreased from fermenting to fermentation ends ventilation quantity within 30 hours；
C, the carbon source for accounting for fermentation liquid gross mass 1%, the carbon source were filled into respectively at aerobic fementation 11 hours and 21 hours Selected from blackstrap.
The organic nitrogen and inorganic nitrogen is selected from yeast extract, peptone, bean powder and nitrate.
Temperature and ventilation quantity control condition in the fermentation process is as follows:
0-10 hours: 32 DEG C of temperature, ventilation quantity 0.2vvm；
11-20 hours: 32 DEG C of temperature, ventilation quantity 0.4vvm；
21-30 hours: 28 DEG C of temperature, ventilation quantity 0.7vvm；
31-40 hours: 28 DEG C of temperature, ventilation quantity 0.6vvm；
41-50 hours: 28 DEG C of temperature, ventilation quantity 0.2vvm.
Sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate is used to adjust during the aerobic fementation PH=5.0-7.0 in fermentation process.
Sodium hydroxide, potassium hydroxide, phosphoric acid, sulfuric acid or the phosphate uses mass percent concentration for 10%- 15% solution.
Fermentation liquid after fermentation is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
It is molten for 10% sodium hydroxide or potassium hydroxide that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 2 grams of potassium hydroxide are added in every liter of fermentation liquid.
Organic acid or inorganic acid adjust fermentation liquid pH when extraction, make the pH=0.5 of the mixture of extract liquor and fermentation liquid It is warming up to 55 DEG C afterwards, threadiness three is obtained by extraction and praises glue.Organic acid or inorganic acid select citric acid, formic acid, propionic acid, fourth One of acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
The threadiness three obtained after the extraction is praised addition strong base-weak acid salt or diatomic base in glue and is made after adjusting pH=6.5-7.5 At uniform plaster material.
It is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: the inoculum concentration that culture solution is 5% is inoculated in culture solution.
The expansion incubation includes:
(1) first order seed is made after expanding culture in slant strains；
(2) secondary seed is made after expanding culture in first order seed；
(3) by the culture solution after the access sterilizing of manufactured secondary seed, aerobic fementation is carried out；
Wherein step (1), the condition of culture of first order seed and secondary seed in (2) are as follows: inoculum concentration 5%, ventilation quantity are 0.2vvm, 32 DEG C of temperature, kind age is 20 hours.
The primary-seed medium of culture first order seed includes following component in the step (1):
Carbon source 1%；Yeast extract 1%；Sodium nitrate 0.1%；Magnesium sulfate 0.01%；Dipotassium hydrogen phosphate 0.1%；Ferrous sulfate 1ppm；
Carbon source selects blackstrap, waste glucose mother liquor.
The secondary seed medium for the secondary seed cultivated in the step (2) includes following component:
Carbon source 1%；Yeast extract 0.03%；Sodium nitrate 0.1%；Magnesium sulfate 0.01%；Dipotassium hydrogen phosphate 0.1%；Peptone 0.05%；Ferrous sulfate 1ppm；
Carbon source selects blackstrap, waste glucose mother liquor.
The culture solution includes following component:
Carbon source 2%；Bean powder 0.05%；Nitrate 0.05%；Magnesium sulfate 0.01%；Dipotassium hydrogen phosphate 0.1%；Calcium carbonate 0.05%；Ferrous sulfate 1ppmm；Defoaming agent 0.05%.
It is tested through applicant, the yield of glue is praised up to 25g/L, using the side of the present embodiment using the method three of the present embodiment The three of method preparation praise the gel strength of glue up to 26g/cm2。
Embodiment 2
The present embodiment the difference from embodiment 1 is that:
Three to praise in the production of glue further include following process conditions:
A, the carbon source in culture solution selects waste glucose mother liquor；Nitrogen source selects the composite nitrogen of inorganic nitrogen and organic nitrogen composition Source；
B, pressure is 0.05MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity and temperature in fermentation process Spend Discrete control, start to 20 hours temperature of fermentation to be 34 DEG C from fermentation, from ferment 20 hours to fermentation ends temperature be 31 DEG C, Start to 30 hours ventilation quantities of fermenting to gradually increase since fermentation, be gradually decreased from fermenting to fermentation ends ventilation quantity within 30 hours；
C, the carbon source for accounting for fermentation liquid gross mass 2%, the carbon source were filled into respectively at aerobic fementation 20 hours and 30 hours Selected from waste glucose mother liquor.
The organic nitrogen and inorganic nitrogen is selected from yeast extract, peptone, bean powder and nitrate.
Temperature and ventilation quantity control condition in the fermentation process is as follows:
0-10 hours: 34 DEG C of temperature, ventilation quantity 0.3vvm；
11-20 hours: 34 DEG C of temperature, ventilation quantity 0.5vvm；
21-30 hours: 31 DEG C of temperature, ventilation quantity 0.9vvm；
31-40 hours: 31 DEG C of temperature, ventilation quantity 0.7vvm；
41-50 hours: 31 DEG C of temperature, ventilation quantity 0.3vvm.
Fermentation liquid after fermentation is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
It is molten for 20% sodium hydroxide or potassium hydroxide that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 4 grams of potassium hydroxide are added in every liter of fermentation liquid.
Organic acid or inorganic acid adjust fermentation liquid pH when extraction, make the pH=4.0 of the mixture of extract liquor and fermentation liquid It is warming up to 90 DEG C afterwards, threadiness three is obtained by extraction and praises glue.Organic acid or inorganic acid select citric acid, formic acid, propionic acid, fourth One of acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
It is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: the inoculum concentration that culture solution is 20% is inoculated in culture solution.
Expand the condition of culture of first order seed and secondary seed in culture are as follows: inoculum concentration 20%, ventilation quantity 0.8vvm, temperature 34 DEG C of degree, kind age are 30 hours.
The primary-seed medium for cultivating first order seed includes following component:
Carbon source 2%；Yeast extract 2%；Sodium nitrate 1%；Magnesium sulfate 0.15%；Dipotassium hydrogen phosphate 0.5%；Ferrous sulfate 10ppm；
Carbon source selects one of blackstrap, waste glucose mother liquor.
The secondary seed medium of the secondary seed of culture includes following component:
Carbon source 2%；Yeast extract 0.1%；Sodium nitrate 0.5%；Magnesium sulfate 0.15%；Dipotassium hydrogen phosphate 0.3%；Peptone 0.3%；Ferrous sulfate 10ppm；
Carbon source selects one of blackstrap, waste glucose mother liquor.
The culture solution includes following component:
Carbon source 4%；Bean powder 0.3%；Nitrate 0.3%；Magnesium sulfate 0.15%；Dipotassium hydrogen phosphate 0.3%；Calcium carbonate 0.4%；Ferrous sulfate 10ppm；Defoaming agent 0.2%.
It is tested through applicant, the yield of glue is praised up to 26g/L, using the side of the present embodiment using the method three of the present embodiment The three of method preparation praise the gel strength of glue up to 27g/cm2。
Embodiment 3
The present embodiment the difference from embodiment 1 is that:
Three to praise in the production of glue further include following process conditions:
A, the mixture that the carbon source in culture solution selects blackstrap and waste glucose mother liquor to compound according to equivalent；Nitrogen source choosing The compound nitrogen source formed with inorganic nitrogen and organic nitrogen；
B, pressure is 0.04MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity and temperature in fermentation process Spend Discrete control, start to 20 hours temperature of fermentation to be 33 DEG C from fermentation, from ferment 20 hours to fermentation ends temperature be 30 DEG C, Start to 30 hours ventilation quantities of fermenting to gradually increase since fermentation, be gradually decreased from fermenting to fermentation ends ventilation quantity within 30 hours；
C, the carbon source for accounting for fermentation liquid gross mass 1%-2% was filled into respectively at aerobic fementation 15 hours and 25 hours, it is described Carbon source is selected from the mixture after blackstrap, waste glucose mother liquor equivalent compounding.
The organic nitrogen and inorganic nitrogen is selected from yeast extract, peptone, bean powder and nitrate.
Temperature and ventilation quantity control condition in the fermentation process is as follows:
0-10 hours: 33 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm；
11-20 hours: 33 DEG C of temperature, ventilation quantity 0.4vvm-0.5vvm；
21-30 hours: 30 DEG C of temperature, ventilation quantity 0.7vvm-0.9vvm；
31-40 hours: 30 DEG C of temperature, ventilation quantity 0.6vvm-0.7vvm；
41-50 hours: 30 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm.
Fermentation liquid after fermentation is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
It is molten for 15% sodium hydroxide or potassium hydroxide that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 3 grams of potassium hydroxide are added in every liter of fermentation liquid.
Organic acid or inorganic acid adjust fermentation liquid pH when extraction, make the pH=3.0 of the mixture of extract liquor and fermentation liquid It is warming up to 75 DEG C afterwards, threadiness three is obtained by extraction and praises glue.Organic acid or inorganic acid select citric acid, formic acid, propionic acid, fourth One of acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
It is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: the inoculum concentration that culture solution is 15% is inoculated in culture solution.
Expand the condition of culture of first order seed and secondary seed in culture are as follows: inoculum concentration 12%, ventilation quantity 0.5vvm, temperature 33 DEG C of degree, kind age are 25 hours.
The primary-seed medium for cultivating first order seed includes following component:
Carbon source 1.5%；Yeast extract 1.5%；Sodium nitrate 0.5%；Magnesium sulfate 0.08%；Dipotassium hydrogen phosphate 0.3%；Sulfuric acid is sub- Iron 5ppm；
Carbon source select blackstrap compounded with waste glucose mother liquor equivalent after mixture.
The secondary seed medium of the secondary seed of culture includes following component:
Carbon source 1.5%；Yeast extract 0.06%；Sodium nitrate 0.3%；Magnesium sulfate 0.08%；Dipotassium hydrogen phosphate 0.2%；Albumen Peptone 0.15%；Ferrous sulfate 5ppm；
Carbon source select blackstrap compounded with waste glucose mother liquor equivalent after mixture.
The culture solution includes following component:
Carbon source 3%；Bean powder 0.15%；Nitrate 0.15%；Magnesium sulfate 0.08%；Dipotassium hydrogen phosphate 0.2%；Calcium carbonate 0.2%；Ferrous sulfate 5ppm；Defoaming agent 0.1%.
It is tested through applicant, the yield of glue is praised up to 28g/L, using the side of the present embodiment using the method three of the present embodiment The three of method preparation praise the gel strength of glue up to 28g/cm2。
Embodiment 4
The present embodiment the difference from embodiment 1 is that:
Three to praise in the production of glue further include following process conditions:
A, the carbon source in culture solution selects blackstrap；Nitrogen source selects the compound nitrogen source of inorganic nitrogen and organic nitrogen composition；
B, pressure is 0.035MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity and temperature in fermentation process Spend Discrete control, start to 20 hours temperature of fermentation to be 32 DEG C from fermentation, from ferment 20 hours to fermentation ends temperature be 30 DEG C, Start to 30 hours ventilation quantities of fermenting to gradually increase since fermentation, be gradually decreased from fermenting to fermentation ends ventilation quantity within 30 hours；
C, the carbon source for accounting for fermentation liquid gross mass 2%, the carbon source were filled into respectively at aerobic fementation 13 hours and 23 hours Selected from blackstrap.
The organic nitrogen and inorganic nitrogen is selected from yeast extract, peptone, bean powder and nitrate.
Temperature and ventilation quantity control condition in the fermentation process is as follows:
0-10 hours: 32 DEG C of temperature, ventilation quantity 0.3vvm；
11-20 hours: 32 DEG C of temperature, ventilation quantity 0.5vvm；
21-30 hours: 30 DEG C of temperature, ventilation quantity 0.7vvm；
31-40 hours: 30 DEG C of temperature, ventilation quantity 0.6vvm；
41-50 hours: 30 DEG C of temperature, ventilation quantity 0.3vvm.
Fermentation liquid after fermentation is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
It is molten for 12% sodium hydroxide or potassium hydroxide that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 2.5 grams of potassium hydroxide are added in every liter of fermentation liquid.
Organic acid or inorganic acid adjust fermentation liquid pH when extraction, after the pH=1 for making the mixture of extract liquor and fermentation liquid 65 DEG C are warming up to, threadiness three is obtained by extraction and praises glue.Organic acid or inorganic acid select citric acid, formic acid, propionic acid, butyric acid, One of octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
It is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: culture solution is that the inoculum concentration of 5%-20% is inoculated in culture solution.
Expand the condition of culture of first order seed and secondary seed in culture are as follows: inoculum concentration 10%, ventilation quantity 0.3vvm, temperature 33 DEG C of degree, kind age are 22 hours.
The primary-seed medium for cultivating first order seed includes following component:
Carbon source 1.2%；Yeast extract 1.2%；Sodium nitrate 0.3%；Magnesium sulfate 0.05%；Dipotassium hydrogen phosphate 0.2%；Sulfuric acid is sub- Iron 3ppm；
Carbon source selects blackstrap.
The secondary seed medium of the secondary seed of culture includes following component:
Carbon source 1.2%；Yeast extract 0.04%；Sodium nitrate 0.2%；Magnesium sulfate 0.05%；Dipotassium hydrogen phosphate 0.15%；Albumen Peptone 0.1%；Ferrous sulfate 3ppm；
Carbon source selects blackstrap.
The culture solution includes following component:
Carbon source 2.5%；Bean powder 0.1%；Nitrate 0.1%；Magnesium sulfate 0.05%；Dipotassium hydrogen phosphate 0.15%；Calcium carbonate 0.1%；Ferrous sulfate 3ppm；Defoaming agent 0.08%.
It is tested through applicant, the yield of glue is praised up to 29g/L, using the side of the present embodiment using the method three of the present embodiment The three of method preparation praise the gel strength of glue up to 30g/cm2。
Embodiment 5
The present embodiment the difference from embodiment 1 is that:
Three to praise in the production of glue further include following process conditions:
A, the carbon source in culture solution selects waste glucose mother liquor；Nitrogen source selects the composite nitrogen of inorganic nitrogen and organic nitrogen composition Source；
B, pressure is 0.05MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity and temperature in fermentation process Spend Discrete control, start to 20 hours temperature of fermentation to be 34 DEG C from fermentation, from ferment 20 hours to fermentation ends temperature be 28 DEG C, Start to 30 hours ventilation quantities of fermenting to gradually increase since fermentation, be gradually decreased from fermenting to fermentation ends ventilation quantity within 30 hours；
C, the carbon source for accounting for fermentation liquid gross mass 2%, the carbon source were filled into respectively at aerobic fementation 18 hours and 27 hours Selected from waste glucose mother liquor.
The organic nitrogen and inorganic nitrogen is selected from yeast extract, peptone, bean powder and nitrate.
Temperature and ventilation quantity control condition in the fermentation process is as follows:
0-10 hours: 34 DEG C of temperature, ventilation quantity 0.3vvm；
11-20 hours: 34 DEG C of temperature, ventilation quantity 0.4vvm；
21-30 hours: 28 DEG C of temperature, ventilation quantity 0.9vvm；
31-40 hours: 28 DEG C of temperature, ventilation quantity 0.7vvm；
41-50 hours: 28 DEG C of temperature, ventilation quantity 0.2vvm.
Fermentation liquid after fermentation is pre-processed before carrying out sedimentation extraction, pretreated process conditions are as follows:
It is molten for 18% sodium hydroxide or potassium hydroxide that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 3.5 grams of potassium hydroxide are added in every liter of fermentation liquid.
Organic acid or inorganic acid adjust fermentation liquid pH when extraction, make the pH=3.0 of the mixture of extract liquor and fermentation liquid It is warming up to 80 DEG C afterwards, threadiness three is obtained by extraction and praises glue.Organic acid or inorganic acid select citric acid, formic acid, propionic acid, fourth One of acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, carbonic acid etc. or at least two or more mixture.
It is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: culture solution is that the inoculum concentration of 5%-20% is inoculated in culture solution.
Expand the condition of culture of first order seed and secondary seed in culture are as follows: inoculum concentration 5%-20%, air quantity 0.6vvm, 34 DEG C of temperature, kind age is 28 hours.
The primary-seed medium for cultivating first order seed includes following component:
Carbon source 1.8%；Yeast extract 1.8%；Sodium nitrate 0.8%；Magnesium sulfate 0.12%；Dipotassium hydrogen phosphate 0.4%；Sulfuric acid is sub- Iron 8ppm；
Carbon source selects waste glucose mother liquor.
The secondary seed medium of the secondary seed of culture includes following component:
Carbon source 1.8%；Yeast extract 0.08%；Sodium nitrate 0.4%；Magnesium sulfate 0.13%；Dipotassium hydrogen phosphate 0.25%；Albumen Peptone 0.25%；Ferrous sulfate 8ppm；
Carbon source selects waste glucose mother liquor.
The culture solution includes following component:
Carbon source 3.5%；Bean powder 0.25%；Nitrate 0.25%；Magnesium sulfate 0.12%；Dipotassium hydrogen phosphate 0.25%；Carbonic acid Calcium 0.3%；Ferrous sulfate 8ppm；Defoaming agent 0.15%.
It is tested through applicant, the yield of glue is praised up to 32g/L, using the side of the present embodiment using the method three of the present embodiment The three of method preparation praise the gel strength of glue up to 30g/cm2。

Claims (15)

  1. It is by Sphingol single-cell 1. producing the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 is inoculated into after sterilizing and the culture solution containing carbon source, nitrogen source and nutriment In；The pH of culture solution is adjusted, aerobic fementation is carried out, fermentation liquid after fermentation is subjected to sedimentation threadiness is obtained by extraction three to praise Glue；Characterized by further comprising following process conditions:
    A, the carbon source in culture solution selects one of blackstrap, waste glucose mother liquor or its combination；Nitrogen source select inorganic nitrogen and The compound nitrogen source of organic nitrogen composition；
    B, pressure is 0.03MPa-0.05MPa during aerobic fementation, and pH value=5.0-7.0 uses ventilation quantity in fermentation process And temperature section control, start to 20 hours temperature of fermentation to be 32 DEG C -34 DEG C from fermentation, ferments 20 hours certainly to fermentation ends temperature Degree be 28 DEG C -31 DEG C, from fermentation start to ferment 30 hours ventilation quantities gradually increase, from ferment 30 hours to fermentation ends ventilation Amount gradually decreases；
    C, carbon source was filled into respectively at aerobic fementation 11-20 hours and 21-30 hours, the carbon source is selected from blackstrap, useless grape One of sugared mother liquor or its combination.
  2. 2. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special It levies the temperature being in the fermentation process and ventilation quantity control condition is as follows:
    0-10 hours: 32 DEG C -34 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm；
    11-20 hours: 32 DEG C -34 DEG C of temperature, ventilation quantity 0.4vvm-0.5vvm；
    21-30 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.7vvm-0.9vvm；
    31-40 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.6vvm-0.7vvm；
    41-50 hours: 28 DEG C -31 DEG C of temperature, ventilation quantity 0.2vvm-0.3vvm.
  3. 3. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Sign is to use sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sulfuric acid or phosphate to adjust hair during the aerobic fementation PH=5.0-7.0 during ferment.
  4. 4. according to claim 3 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Sign is that sodium hydroxide, potassium hydroxide, phosphoric acid, sulfuric acid or the phosphate use mass percent concentration for 10%-15% Solution.
  5. 5. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Sign, which is to fill into respectively at aerobic fementation 11 hours to 20 hours and 21 hours to 30 hours, accounts for fermentation liquid gross mass 1%-2%'s Carbon source.
  6. 6. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Sign is to be pre-processed before fermentation liquid after fermentation carries out sedimentation extraction, pretreated process conditions are as follows:
    It is molten for the sodium hydroxide or potassium hydroxide of 10%-20% that mass percent concentration is added in fermentation liquid after fermentation Liquid, additional amount are that solid NuaO or 2-4 grams of potassium hydroxide is added in every liter of fermentation liquid.
  7. 7. according to claim 6 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Organic acid or inorganic acid adjust fermentation liquid pH when sign is to extract, and make the pH=0.5- of the mixture of extract liquor and fermentation liquid It is warming up to 55 DEG C -90 DEG C after 4.0, threadiness three is obtained by extraction and praises glue.
  8. 8. according to claim 7 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, special Sign be organic acid or inorganic acid select citric acid, formic acid, propionic acid, butyric acid, octanoic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, boric acid, One of carbonic acid etc. or at least two or more mixture.
  9. 9. according to claim 1, producing microbial polysaccharide-three by strain of Sphingol single-cell described in any one of 6,7 or 8 The method for praising glue, it is characterised in that the threadiness three obtained after the extraction, which is praised, is added strong base-weak acid salt or diatomic base adjusting pH in glue Uniform plaster material is made after=6.5-7.5.
  10. 10. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, It is characterized in that the organic nitrogen and inorganic nitrogen are selected from yeast extract, peptone, bean powder and nitrate.
  11. 11. according to claim 1 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, Be characterized in that it is described by Sphingol single-cell (Sphingomonas sp.) CGMCCNo.1650 after expanding culture, according to Seed liquor: culture solution is that the inoculum concentration of 5%-20% is inoculated in culture solution.
  12. 12. according to claim 11 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, It is characterized in that the expansion incubation includes:
    (1) first order seed is made after expanding culture in slant strains；
    (2) secondary seed is made after expanding culture in first order seed；
    (3) by the culture solution after the access sterilizing of manufactured secondary seed, aerobic fementation is carried out；
    Wherein step (1), the condition of culture of first order seed and secondary seed in (2) are as follows: inoculum concentration 5%-20%, air quantity are 0.2vvm-0.8vvm, 32 DEG C -34 DEG C of temperature, kind age is 20-30 hours.
  13. 13. according to claim 12 produce the method that microbial polysaccharide-three praises glue by strain of Sphingol single-cell, The primary-seed medium for being characterized in that the middle culture first order seed of the step (1) includes following component:
    Carbon source 1%-2%；Yeast extract 1%-2%；Sodium nitrate 0.1%-1%；Magnesium sulfate 0.01%-0.15%；Dipotassium hydrogen phosphate 0.1%-0.5%；Ferrous sulfate 1ppm-10ppm；
    Carbon source selects blackstrap, waste glucose mother liquor.
  14. 14. producing microbial polysaccharide-three by strain of Sphingol single-cell described in any one of 2 or 13 according to claim 1 The method for praising glue, it is characterised in that the secondary seed medium for the secondary seed cultivated in the step (2) includes such as the following group Point:
    Carbon source 1%-2%；Yeast extract 0.03%-0.1%；Sodium nitrate 0.1%-0.5%；Magnesium sulfate 0.01%-0.15%；Phosphoric acid Hydrogen dipotassium 0.1%-0.3%；Peptone 0.05%-0.3%；Ferrous sulfate 1ppm-10ppm；
    Carbon source selects blackstrap, waste glucose mother liquor.
  15. 15. according to claim 1, producing microbial polysaccharide-by strain of Sphingol single-cell described in any one of 10 or 12 Three methods for praising glue, it is characterised in that the culture solution includes following component:
    Carbon source 2%-4%；Bean powder 0.05%-0.3%；Nitrate 0.05%-0.3%；Magnesium sulfate 0.01%-0.15%；Phosphoric acid Hydrogen dipotassium 0.1%-0.3%；Calcium carbonate 0.05%-0.4%；Ferrous sulfate 1ppm-10ppm；Defoaming agent 0.05%-0.2%.
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CN110760015A (en) * 2019-11-19 2020-02-07 河北鑫合生物化工有限公司 Improved extraction method of sanzan gum
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
CN111057711A (en) * 2019-12-25 2020-04-24 廊坊梅花生物技术开发有限公司 Sphingomonas engineering bacteria and construction method and application thereof
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
CN111500497A (en) * 2020-04-27 2020-08-07 河北鑫合生物化工有限公司 Sphingomonas with molecular marker and application thereof in preparation of sanzan
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
CN113248629A (en) * 2021-05-14 2021-08-13 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
CN113583904A (en) * 2021-07-24 2021-11-02 河北沣川生物科技有限公司 Extracellular polymer sphingomonas and application thereof in preparation of sanzan glue with high gel strength
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
CN114010533A (en) * 2021-11-05 2022-02-08 南开大学 Natural sanzang gel with alcohol solubility and moisture retention, no-clean disinfection gel and application

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