CN1539987A - Fermentation technique for producing xanthan gum - Google Patents

Abstract

A process for preparing xanthan gum by fermentation features that the beet molasses culture medium whose primary component is beet molasses is used, and the Xant homonas campestris of wold rape is used for fermentation. Its advantages are high output, and high quality of product.

Description

The fermentation manufacturing technique of xanthan gum

Technical field

The present invention relates to a kind of fermentation process of Microbial exopolysaccharides, the present invention relates to a kind of fermentation manufacturing technique of xanthan gum specifically.

Background technology

Xanthan gum (Xanthan gum), claim yellow glue, xanthan gum, yellow unicellular polysaccharide again, be that bird rape Xanthomonas campestris (Xanthomonas campestris) is main raw material with the carbohydrate, a kind of broad-spectrum Microbial exopolysaccharides through fermentation engineering is produced is called for short XC.Because xanthan gum has good solubility, tackifying, stability, and can acid and alkali-resistance, hypersaline environment, high temperature resistance, cryogenic freezing, readily biodegradable, contamination resistance is strong, have favorable compatibility and dissemination simultaneously, be a kind of in petroleum industry wide seam fracturing technology oil recovery ideal " rheology control liquid ".Xanthan gum can be widely used in more than 20 industries such as food, oil, pottery, weaving, printing and dyeing, medicine, papermaking, coating, makeup, is the maximum and broad-spectrum microbial polysaccharide of industrial scale in the world at present.

Xanthan gum is that the USDA northern territory institute fifties (NRRL) at first finds.Kelco companies in 1969 obtain the approval of food and drug administration (FDA), begin to sell the food grade xanthan gum.Afterwards, states such as Japan, Denmark, Britain, Holland, Spain, Canada, France, Sweden, Belgium ratify to use in succession.Nineteen eighty-three the World Health Organization and the World Food Programme (FAO/WHO) approval xanthan gum as foodstuff additive, and announced the quality standard of xanthan gum in 1986 as foodstuff additive.From then on, xanthan gum begins worldwide to be developed widely and use, and the output of world's xanthan gum in 2000 reaches 1,000,000 tons, and now the whole world is to the speed increment with 5-7% of the demand of xanthan gum.In China, in August, 1988, the Ministry of Health ratified the hygienic standard of food grade xanthan gum, and was put into foodstuff additive list (GB 2760-86 foodstuff additive use hygienic standard (amendments in 1988)).China is only in foodstuffs industry year 4000 tons of demands, and with annual 8% speed increase.Current, new and high technology is advanced by leaps and bounds, especially bionic great development, development to xanthan gum has played great pushing effect, many new and high technologies in the biotechnology are swarmed into the development and the production field, particularly genetic engineering technique, membrane separation technique etc. of xanthan gum, make the xanthan gum productive rate improve, quality-improving, cost reduces.Along with the increase of external xanthan gum manufacturer, competition is growing more intense, and general scale is more than 2000t/a, and what have reaches ten thousand tons.

At present, the production technique of xanthan gum mainly is to utilize the mode of microbial fermentation, comprise that mainly a series of processes such as seed culture, strain fermentation and fermented liquid aftertreatment obtain xanthan gum, wherein mainly be carbon source with the W-Gum in the Guan Jian fermentation step, and it is carbon source that employing sucrose is also arranged.In the vast zone of northern China, mainly based on the W-Gum fermentation, suitability for industrialized production aspect cost is higher.Though known xanthan gum has the good characteristics that a lot of other polysaccharide are not had, but at present, because be subjected to the restriction of starting material and technology, the xanthan gum production cost can be in any more, product always can not long run supply, limited the widespread use of xanthan gum, therefore, how further to reduce production costs, carry out scale operation, become xanthan gum production, the gordian technique of especially extensively promoting the use of.

Beet is a kind of 3 years living cross pollinated plants, and beet sirup is the liquid glucose molasses in the sugar industry,, low price abundant in China source, its main component is to contain sucrose 52-62%, colloid 8.5-10%, carbonic acid ash content 7-8%, total nitrogen 1.9-2.1% is a kind of nutritious microbial fermentation raw material, at present, 8,500,000 tons of whole nation sugar total amounts, sugaring sugar futures amount is 8,700,000 tons.The production of beet in China based on northeast, northwest, North China, cultivated area is wide, output is abundant, with Xinjiang is example, beet is one of Xinjiang three high industries, cultivated area increases to 1,270,000 mu, per mu yield is up to 2 tons, more than 300 ten thousand tons of beet output produce about 180,000 tons of waste molasses, are used to produce alcohol, the L-glutamic acid except that about 140,000 tons, also have an appointment 40,000 tons and pour off in vain, not only wasted resource but also polluted environment, and its main products alcohol, L-glutamic acid also hastens towards saturation, so the utilization again of molasses also is instant problem.

Summary of the invention

At the problems referred to above, one of main purpose of the present invention is to provide a kind of technology of utilizing beet sirup substratum fermenting and producing xanthan gum, and the comprehensive utilization by raw material reaches the purpose that reduces cost, economizes on resources, reduces environmental pollution.

Xanthan gum production technique provided by the invention, it comprises:

A:1.1781 bird rape Xanthomonas campestris (Xanthomonas campestris) seed culture step;

B: utilize the beet sirup substratum of beet sirup, the step of fermenting with 1.1781 bird rape Xanthomonas campestris seeds for main raw material;

C: utilize the fermented liquid aftertreatment technology to obtain xanthan gum;

Wherein leavening temperature is 20 ℃-35 ℃ in the described seed culture step of steps A, and stir speed (S.S.) is 200-300rpm, air flow 0.8-0.9v/v/m, and the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, the sturdy quantity of bacterial classification is many;

Inoculum size is 1%-15% among the step B, and temperature is 20 ℃-35 ℃, and air flow is 0.6-1.0v/v/m, and when the microscopy thalline wears out, divides, diminishes, liquid thickness during fermentation, reducing sugar be below 1.0, fermentation ends when viscosity no longer increases.

The present invention utilizes beet sirup to prepare fermention medium for main raw material, be applicable to 1.1781 bird rape Xanthomonas campestris fermenting and producing xanthan gums, used 1.1781 bird rape Xanthomonas campestris (Xanthomonas campestris) belong to xanthomonas, can be available from common micro-organisms DSMZ of Chinese microorganism strain management committee.The xanthan gum of utilizing zymotechnique of the present invention to obtain not only has the characteristics and the advantage of existing xanthan gum, and quality reaches national gold standard (GB13886-92 xanthan gum product quality standard and GB2760-1996 foodstuff additive use hygienic standard), can be widely used in each field of national economy.Thereby can be with a kind of substitute of beet sirup as W-Gum, sucrose, this not only can be avoided because of being the raw materials consumption that carbon source through fermentation production xanthan gum causes with W-Gum, sucrose, problems such as production cost height, can also turn waste into wealth, save a large amount of fund and grain, thereby reach the purpose that reduces cost, economizes on resources, reduces environmental pollution, for the perfect development of China's xanthan gum production technology provides a new approach.

In the fermentation manufacturing technique of above-mentioned xanthan gum, the technology controlling and process of fermenting with 1.1781 bird rape Xanthomonas campestris seeds is committed step of the present invention.The present invention is through prolonged and repeated research and experiment, determine that preferred inoculum size is about 1%-15% in the fermentation step, temperature is about 25 ℃-35 ℃, air flow is about 0.6-1.0v/v/m, when the microscopy thalline wears out, divides, diminishes, liquid thickness during fermentation, reducing sugar be below 1.0, fermentation ends when viscosity no longer increases.More preferably leavening temperature is about 25-35 ℃, and air flow is about 0.8-0.9v/v/m, and mixing speed is about 100-400rpm, and fermentation time is about 50-80 hour usually.

The present invention carries out small-scale test on the shake flask fermentation experimental basis, and the processing condition of 5 liters of stirring-type aerobic fermentation cylinders have been studied, determine that its optimum condition is as follows: leavening temperature is about 30 ℃, stir speed (S.S.) is about 300rpm, the about 0.9v/v/m of air flow, fermented about 54-56 hour, xanthan gum output is about 26-28g/L, and the sucrose inversion rate is about 69-73%.It will be further appreciated that, the present invention is on the basis of seeding tank (100 liters) fermentation test, 2300 liters of stirring-type aerobic fermentation pilot plant tests have been carried out, final determine that its optimum condition is as follows: the long-pending capacity of dress is about 60%, and leavening temperature is about 30 ℃, and stir speed (S.S.) is about 300rpm, the about 0.9v/v/m of air flow, fermented about 52-58 hour, more than the xanthan gum output 23g/L, to the sucrose inversion rate more than 65%.

The composition of the beet sirup substratum in the fermentation step of step B of the present invention and content also are important parameters of fermenting process, the contriver is by a large amount of experiments, studied of the influence of factors such as molasses concentration, pH to xanthan gum output, thereby the preferred composition and the content of beet sirup substratum have been established, wherein the molasses that contain in the beet sirup substratum amount to sucrose about 25-60g/L, medium pH is about 6-8, more preferably the molasses that contain in the beet substratum amount to sucrose about 38-53g/L, and pH is about 7.In addition, the contriver has also studied the influence of different nitrogen sources to xanthan gum output, and on this basis, according to feed stock conversion and xanthan gum output, the mode of conversion xanthan gum raw materials cost, propose two kinds of beet sirup culture medium prescriptions, use for actual production, output is directly calculated after can adopting fermented liquid thin film concentration (about 5-12%).Wherein contain the about 60-80g/L of molasses (sugar 60%), the about 0.5-1.5g/L of urea, K for a kind of molasses culture medium (prescription one) ₂HPO ₄About 0.5-1.5g/L, the more preferably about 80g/L of molasses, the about 1g/L of urea, K ₂HPO ₄About 1g/L, the pilot scale average result is the about 22g/L of xanthan gum output, and transformation efficiency is about 62.8%, and conversion xanthan gum raw materials cost is about 2160 yuan/ton, and though this prescription feed stock conversion, xanthan gum output are lower slightly, the raw materials for production cost obviously reduces; Another kind of molasses culture medium (prescription two) contains molasses amounts to sucrose about 60-80g/L, the about 2-4g/L of yeast extract paste, K ₂HPO ₄About 0.5-1.5g/L, MgSO ₄7H ₂The about 0.1-1g/L of O, the about 80g/L of molasses more preferably, the about 3g/L of yeast extract paste, K ₂HPO ₄About 1g/L, MgSO ₄7H ₂The about 0.2g/L of O, xanthan gum output is about 25.6g/L, transformation efficiency about 68.3%, conversion xanthan gum raw materials cost is about 5160 yuan/ton, and feed stock conversion in this prescription, xanthan gum output are all higher, but the raw materials for production cost obviously raises, therefore, in actual production process, should determine different producing and manufacturing technique, in the hope of maximum economic benefits according to different xanthan gum uses.The present invention further on the suitableeest nitrogen, inorganic salt and concentration range basis that one-factor experiment is determined, selects the optimal medium prescription by orthogonal experiment method, and optimum formula is as follows: contain molasses: about 70g/L; Yeast extract paste is about: 3g/L; K ₂HPO ₄Approximately: 1g/L; PH is about: 7.0; And ferment in about 30 ℃ temperature.

In xanthan gum production technique of the present invention, the spawn culture of steps A is another committed step, can utilize the female a series of cascade amplification process such as cultivation, shake-flask seed cultivation, seed tank culture of planting in inclined-plane known in the art, obtains required seed liquor.The contriver is by repetition test, and leavening temperature is 20 ℃-35 ℃ in the described seed culture step of determining step A, and stir speed (S.S.) is 200-300rpm, air flow 0.8-0.9v/v/m, and the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, the sturdy quantity of bacterial classification is many.In the seed culture process, can be according to fermentation scale difference, adopt different cascade amplification process, for example carry out large scale fermentation, can be on the basis of inclined-plane seed culture, shake-flask culture, first order seed fermentation, increase the secondary seed fermentation, its substratum can use the first order seed fermention medium, in order to save the raw materials cost of substratum, also can adopt beet sirup is the beet sirup substratum of main raw material, for example: the about 50-80g/L of molasses, the about 5-15g/L of sucrose, the about 2-4g/L of yeast extract paste, K ₂HPO ₄About 0.5-2.5g/L, MgSO ₄7H ₂The about 0.1-1g/L of O, the about 6-8 of pH; For the secondary seed fermentation, the seed inspection standard is similarly microscopy does not have assorted bacterium, no pod membrane, and the sturdy quantity of bacterial classification is many, generally ferments 16-18 hour, gets final product.

In xanthan gum production technique of the present invention, the beet sirup in the beet sirup substratum can directly use without any pre-treatment; Preferably pass through following method pre-treatment,, handle back xanthan gum rate ratio and handle preceding high about 0.5-1.8g/L after treatment for identical substratum and fermentation condition.Concrete grammar is as follows: transferred about 4.0 heated and boiled of pH about 10 minutes, temperature is neutralized to pH about 7.0 and filters when reducing to about 70-80 ℃.

Can utilize production technique of the present invention, just through comprising abovementioned steps A: the seed culture step; With step B: the fermented liquid that obtains after the seed fermentation step; With step C: the fermented liquid aftertreatment technology obtains xanthan gum; Wherein extraction process can use those skilled in the art to know technology after the related fermented liquid of step C, serial of methods such as the KCl dissolving that for example quaternary amine-methyl alcohol method, or embodiment of the present invention provides, alcohol precipitation, alcohol wash-out, oven dry obtain high-quality xanthan gum of the present invention.Also can will directly use behind the xanthan gum fermentation broth thin film concentration that obtain according to production technique of the present invention.Especially in petroleum industry,, directly use behind the employing fermented liquid thin film concentration, satisfy stifled well in the oil recovery, well workover, pressure break use, reduce the xanthan gum production cost greatly according to the handling characteristics of petroleum industry.Directly use behind the preferred employing fermented liquid thin film concentration 5-12%.

In a specific embodiment of the present invention, the xanthan gum production technique comprises:

A:1.1781 bird rape Xanthomonas campestris seed culture step;

B: utilize the beet sirup substratum of beet sirup, the step of fermenting with 1.1781 bird rape Xanthomonas campestris seeds for main raw material;

C: utilize fermented liquid thin film concentration technology to obtain xanthan gum;

Leavening temperature in the seed culture step described in the steps A is 20 ℃-35 ℃, and stir speed (S.S.) is 200-300rpm, air flow 0.8-0.9v/v/m, and the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, the sturdy quantity of bacterial classification is many; And seed culture is cultivated through secondary seed, and its substratum is molasses 50-80g/L, sucrose 5-15g/L, yeast extract paste 2-4g/L, K ₂HPO ₄0.5-2.5g/L, MgSO ₄7H ₂O 0.1-1g/L, pH6-8; The secondary seed fermentation time is 16-18 hour;

Inoculum size is 1%-15% among the step B, and temperature is 20 ℃-35 ℃, and air flow is 0.8-0.9v/v/m, and mixing speed is 100-400rpm, and fermentation time is 50-60 hour, and the molasses that substratum contains are amounted to sucrose 25-60g/L, pH=7.

Beneficial effect:

The present invention utilizes the beet sirup substratum of beet sirup for main raw material, ferment with 1.1781 bird rape Xanthomonas campestris, and utilize the fermented liquid aftertreatment technology to obtain xanthan gum, thereby can be with beet sirup as W-Gum, a kind of substitute of sucrose, this not only can be avoided because of using W-Gum, sucrose is the raw materials consumption that carbon source through fermentation production xanthan gum causes, problems such as production cost height, can also turn waste into wealth, save a large amount of fund and grain, reach the purpose that reduces cost, what solved waste molasses simultaneously utilizes problem again, reaches the purpose that reduces environmental pollution, for the perfect development of China's xanthan gum production technology provides a new approach.The xanthan gum of utilizing zymotechnique of the present invention to obtain not only has the characteristics and the advantage of existing xanthan gum, and quality reaches national gold standard.

The present invention to xanthan gum fermentation strain fermentation process condition optimizing and control, compares with the technology that with starch is the raw material production xanthan gum by further according to the screening and the spawn culture of raw material, has the fermentation time weak point, transformation efficiency height, the advantage that cost obviously reduces.The present invention is raw material beet sirup substratum with the beet sirup, utilizes the fermentation of 1.1781 bird rape Xanthomonas campestris to carry out xanthan gum production, and its technical indicator is as shown in table 1:

Classification	With starch is raw material	With the beet sirup is raw material
			Output	25-27g/L	22g/L
Transformation efficiency	65-70％	68.3％
			Fermentation time	72-80 hour	54 hours

Produce one ton of xanthan gum supplies consumption and raw materials cost: prescription one

Title	Specification	Consumption (ton)	Unit price (unit/ton)	Price (unit)
					Beet sirup (60 grams per liter)		????4.5	???350	????1575
Dipotassium hydrogen phosphate	Food grade	????0.046	???7000	????322
					Urea		????0.046	???1300	????59.8
Water		????200	???1	????200

Add up to: 2156.8 yuan

Prescription two:

Title	Specification	Consumption (ton)	Unit price (unit/ton)	Price (unit)
					Beet sirup (60 grams per liter)		????3.9	???350	??1365
Dipotassium hydrogen phosphate	Food grade	????0.046	???7000	??322
					Sal epsom	Food grade	????0.01	???10000	??100
Yeast extract paste		????0.138	???23000	??3174
					Water		????200	???1	??200

Add up to: 5161 yuan

As mentioned above, produce xanthan gum per ton and need about 4.5 tons of beet sirup approximately, by about 350 yuan of calculating per ton, be roughly equal to about 1365 yuan of Renminbi, the cost market value is lower; And from fermentation time, owing to need pass through hydrolysis for the raw material production xanthan gum with starch, it is polysaccharide, and beet sirup is a disaccharide, does not need hydrolysis time, and with the beet sirup being like this raw material production xanthan gum fermentation time needs 54 hours, in order to starch is the raw material production xanthan gum fermentation time to need nearly 80 hours, it is nearly 1/3rd to shorten fermentation time, thereby reduces production costs, and visible garden beet waste molasses production xanthan gum production technique has good market outlook and advantage.

In order more to be expressly understood essence of the present invention, it is made an explanation referring now to following drawings and Examples.Drawings and Examples are for the present invention is described, and do not limit the present invention in any way.

Description of drawings

Fig. 1 shows the concrete technological process of production figure of xanthan gum.

Fig. 2 is the sticking dense relation curve of polymkeric substance.

Fig. 3 is that polymkeric substance is at NaCl solution medium viscosity change curve.

Fig. 4 is that polymkeric substance is at CaCl ₂Solution medium viscosity change curve.

Fig. 5 is that polymkeric substance is at salts solution medium viscosity change curve.

Fig. 6 is the susceptibility of polymkeric substance to temperature.

Fig. 7 is polymer solution shear speed and viscosity change curve.

Embodiment

See also Fig. 1, Fig. 1 is the concrete production technique of a kind of xanthan gum of the present invention, mainly comprises: strain expanded culture, extraction step after seed liquor fermentation culture and the fermented liquid.Wherein the spawn culture step is committed step of the present invention, can utilize a series of cascade amplification process such as the female kind in inclined-plane known in the art, shake-flask seed, seeding tank, obtain required seed liquor, the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, and the sturdy quantity of bacterial classification is many; Fermentation culture stage, it is another committed step of the present invention, the present invention is the beet substratum of main raw material by selecting beet, utilize 1.1781 bird rape Xanthomonas campestris seeds to ferment, liquid aftertreatment technology by fermentation, obtain xanthan gum cheaply, reached and reduce cost, reduced the purpose of environmental pollution.The present invention again further to fermentation strain optimization of fermentation condition and control, reaches the purpose that fermentation time is short, transformation efficiency is high, cost obviously reduces on the basis of culture medium raw material screening; Extraction step can adopt method well known in the art after the fermented liquid, through ethanol or quaternary salt deposit, washing, drying and crushing or a series of well known technology such as concentrate, obtains finished industrial product.Those skilled in the art are known, and Fig. 1 is the concrete example to overall plan of the present invention, and limits the present invention never in any form.

Need to prove: the consumption of molasses can be converted and be sucrose content in the culture medium prescription of the present invention, also can directly adopt molasses to put down in writing, the content of sucrose is incomplete same in the beet sirup in the different places of production, but can measure, when adopting the molasses consumption to represent, the sucrose amount of bringing in the substratum can be converted by the sucrose content in these molasses.Otherwise also be the same.No matter adopt which kind of mode, all should be considered as clearly.

Embodiment 1 utilizes beet sirup production xanthan gum production craft step

1. bacterial classification: 1.1781 bird rape Xanthomonas campestris (Xanthomonas campestris), trade(brand)name: 1.1781 bird rape Xanthomonas campestris, available from common micro-organisms DSMZ of Chinese microorganism strain management committee, Beijing, China.

2. substratum:

Beet sirup: from sugar refinery, Shihezi, Xinjiang, major ingredient sucrose: 54-55%, total hydrogen: 1.9-2.1%, colloid 9.5-11%, carbonic acid ash content: 7.8-8.1%, pH:7.3-7.4.

Beet sirup pre-treatment: transfer pH about 4.0 with yellow soda ash, add yellow soda ash when heated and boiled 10 minutes, temperature are reduced to about 70-80 ℃ and be neutralized to pH7.0, filtration.

(1) slant medium (100ml):

Sucrose: 1g; Peptone: 0.2g; Extractum carnis: 0.1g; Yeast extract paste: 0.3g; Agar: 2.0g; PH:7.0; 1.0kg/cm ²Sterilized 30 minutes.

(2) first order seed, use Qie Shi flask culture (100ml):

Sucrose: 2.0g; Peptone: 0.2g; Yeast extract paste: 0.3g; K ₂HPO ₄: 0.1g; Agar: 2.0g; PH:7.0.

(3) secondary seed (100 liters of jars):

Beet sirup (being untreated): 60g/L; Sucrose: 10g/L; Yeast extract paste: 3g/L; K ₂HPO ₄: 1g/L; MgSO ₄7H ₂O:0.2g/L; PH:7.0.

(4) pilot scale fermentation substratum:

Beet sirup: 70g/L; Yeast powder: 3g/L; K ₂HPO ₄: 1g/L; MgSO ₄7H ₂O:0.2g/L; Defoamer: 1g/L; PH:7.0.

3. culture condition:

(1) inclined-plane seed: Qie Shi bottle seed, cultivated 48 hours for 30 ℃.

(2) secondary seed: 100 liters of fermentor tanks, loading amount 60%, energy air quantity 0.8-0.9 (v/v/m), tank pressure 1kg/cm ², stirring velocity 200rpm cultivated 16-18 hour for 30 ℃.

(3) pilot scale fermentation: 2300 liters of ventilation stirred fermentors, height: diameter=2: 1, loading amount 60%, air flow 0.8 (v/v/m), tank pressure 1kg/cm ², stirring velocity 200rpm, 30 ℃ fermented 56-80 hour.

4. fermenting process inspection:

(1) seed inspection: microscopy does not have assorted bacterium, no pod membrane, and the sturdy quantity of bacterial classification is many.

(2) fermented liquid: the microscopy thalline wears out, divides, diminishes, and liquid thickness during fermentation is surveyed reducing sugar below 1.0, and viscosity no longer increases.

5. plant and instrument:

Shaking table: the desk-top constant temperature oscillator of THZ-03 type

5 liters of jars: Switzerland Bei Lang-05 type automatic glass fermentor tank

Seeding tank, fermentor tank: for stainless steel stirs the ventilated type fermentor tank

6. measuring method:

(1) viscosimetric analysis: NDT-79 type viscosity juice, with glycerine check and correction, 25 ℃.

(2) xanthan gum assay: the liquid dilution is 5 times during fermentation, adds 1%KCl, and the dissolving back adds the alcohol of 2 times of volumes 95%, makes the xanthan gum precipitation, removes supernatant liquor, and precipitation is used 95% alcohol wash-out again, and 60 ℃ of oven dry claim dry weight.

(3) residual sugar is measured: film reagent method

(4) pH value: METTLER TOLEDO pH2100 instrumentation is fixed

Product meets GB13886-92 xanthan gum product quality standard and the GB2760-1996 foodstuff additive use hygienic standard.

Embodiment 2:5 rises the test of automatic glass fermentor tank

On the shake flask fermentation experimental basis, carried out the air flow test, rotating speed 300rpm with Switzerland Bei Lang-05 type automatic glass fermentor tank.(5 batches of mean value) is referring to table 2:

By in the table as can be seen, the suitableeest air flow of xanthan gum fermentation is about: 0.8-1.0v/v/m, further test can be determined at 0.8-0.9v/v/m.

Embodiment 3: middle fermenting experiment

On the basis of seeding tank (100 liters) fermentation test, carry out: 2300 liters of fermentor tank expanding tests, carry out altogether: 6 batches of tests such as tables 3:

Batch	Fermentation time (hour)	Fermentation broth viscosity	Output (g/L)	Transformation efficiency (%)
					??1	??56	??7180	???25.6	??68.3
??2	??58	??8900	???26.6	??71.2
					??3	??54	??6500	???24.1	??64.6
??4	??55	??7200	???25.8	??68.5
					??5	??52	??8100	???26.1	??69.6
On average	??54	??7920	???25.6	??68.3

The result shows: under the suitable culture condition, 1.1781 bird rape Xanthomonas campestris utilize the not treated direct fermentation of beet sirup 54 hours, and xanthan gum output reaches 25.6g, transformation efficiency 68.3%.

Embodiment 4: different nitrogen sources is to the influence of xanthan gum fermentation:

Molasses with 6% are carbon source, add the influence of different nitrogen sources to xanthan gum fermentation, result such as table 4:

Nitrogenous source	Content (g/L)	Xanthan gum output (g/L) (on average)	Transformation efficiency (%)
				Yeast extract paste	????3	????11.8	??40.3
Peptone	????3	????8.8	??31.2
				Fish meal	????3	????2.5	??8.6
Soya-bean cake	????3	????1.2	??3.98
				Urea	????1	????6.1	??20.6
Cotton rouge and powder	????3	????1.1	??3.7
				??(NH 4) 2SO 4	????1	????1.6	??5.4
Contrast (not adding nitrogen)	????-	????0.9	??2.7

As can be seen from the table, yeast extract paste has clearly effect to xanthan gum output, concentration can 2-4g/L for well, the nitrogen that molasses itself contain also can be utilized a part.

Embodiment 5: inorganic salt are to the influence of xanthan gum fermentation:

Molasses with 70g/L are carbon source, are nitrogenous source with the yeast extract paste of 3g/L, add various inorganic salt and see its influence to xanthan gum fermentation, result such as table 5:

Inorganic salt	Xanthan gum output (g/L)	Transformation efficiency (%)
			????K 2HPO 4	???23.8	????66.1
????KH 2PO 4	???15.2	????39.2
			????MgSO 4·7H 2O	???20.2	????53.9
????CaCO 3	???19.1	????50.1
			Contrast (not with salt)	???17.9	????46.2

As can be seen from the table, K2HPO4, MgSO47H2O have more significantly effect to xanthan gum output, and KH2PO4 has certain restraining effect.

Embodiment 6: fermented liquid pH is to the influence of xanthan gum output:

Molasses: 70g/L; Yeast powder: 3g/L; K2HPO4:1g/L; MgSO47H2O:0.2g/L; Transfer different pH values to yield effect such as following table 6:

L27 (3) 13 orthogonal table

Gauge outfit design A B A*B C D E
	Row numbers 123456789 10 11 12 13
A. molasses: A1 4 A2 6 A3 8 B. yeast extract paste: B1 0.1 B2 0.3 B3 0.6 C.K 2HPO 4: C1 0.05 C2 0.1 C3 0.2 D.pH:D1 6 D2 7 D3 8 E. temperature: 35 ℃ of 30 ℃ of E3 of E1 Z5 ℃ E2

Test-results, referring to table 7:

Test number result experiment result experiment result

??1?????0.88?????10?????1.60??????18??????1.76 ??2?????1.36?????11?????1.71??????20??????2.15 ??3?????0.98?????12?????1.29??????21??????1.52 ??4?????1.17?????13?????1.42??????22??????1.72 ??5?????0.80?????14?????1.98??????23??????2.78 ??6?????1.06?????15?????2.71??????24??????2.23 ??7?????0.77?????16?????2.41??????25??????2.31 ??8?????0.70?????17?????1.61??????26??????1.64 ??9?????0.751????18?????1.67??????27??????1.87

Test-results is: xanthan gum output in the 100g fermented liquid, referring to table 8:

????pH	Xanthan gum output (g/L)	Transformation efficiency (%)
			????5	???4.2	????12.3
????6	???19.4	????60.6
			????7	???20.0	????74.1
????8	???19.7	????61.1
			????9	???6.9	????19.2

As can be seen from the table, when pH＜6 or pH＞8, xanthan gum output sharply reduces, and is the best with pH about 7.0.

Embodiment 7: the selection of Optimal compositions of fermentation medium

One-factor experiment has been determined the suitableeest nitrogen, inorganic salt and concentration range, on this basis, selects optimum carbon nitrogen ratio by orthogonal experiment method, pH and inorganic salt, and amount ratio is examined or check 5 factors, molasses concentration A, yeast extract paste concentration B, K ₂HpO ₄Concentration C, pH value D, temperature E, each factor is got 3 levels, and therefore A * the B that interacts between the Consideration selects orthogonal table (orthogonal experiment, Peking University's mathematics group, 1975) L for use ₂₇(3) ¹³Experiment arrangement.

Data sum and the average of xanthan gum under each level of factor, referring to table 9:

The row level	A???????B??????A*B???????????????????D????????????????????E 1??????2?????3?????4?????6?????7??????8?????9?????10????11????12????13
		I II III I?????9 II????9 III???9	8.57???11.2??12.8??12.7??7.6???6.8????12.8??8.8???10.3??11.2??14.1??13.2 17.2???15.4??14.1??13.9??22.1??21.2???19.3??16.2??14.3??20.3??14.1??16.4 16.21??14.3??13.6??13.4??12.2??15.31??11.1??16.7??18.1??9.2???16.9??12.9 0.84???1.23??1.42??1.41??0.83??0.75???1.37??0.9???1.1???1.22??1.23??1.47 1.9????1.6???1.58??1.56??2.41??2.4????2.18??1.8???1.5???2.3???1.53??1.5 1.7????1.51??1.51??1.5???1.36??1.7????1.31??1.82??2.01??1.31??1.7???1.36

Analysis of variance table, referring to table 10:

Find out by table 7 test-results, the best A3B2C2D2E1 that is combined as, A2B2C3D4E2, xanthan gum output is respectively 27.8g/L and 27.1g/L.Best of breed is that this combination of A2B2D2E2 does not occur in 27 groups of experiments of table 7 as can be seen from Table 9, but by the variance analysis gained, therefore with 15 groups preferably of this combinations, 23 groups of comparative experiments results.

Repeat the comparison test result, referring to table 11:

Result combinations	Xanthan gum output (g/L)	On average	Transformation efficiency
				?A2B2C2D2E1	????28.6????27.9????28.1	????28.3	????73.2
?A2B2C3D1E2	????27.2????28.8????27.4	????27.8	????72.6
				?A2B2C2D2E2	????28.4????28.1????29.9	????27.8	????74.8

By last table molasses are described: about 70g/L; Yeast powder: about 3g/L; K ₂HPO ₄: about 1g/L; PH: about 7.0; About 30 ℃ of temperature is best substratum.

Embodiment 8: the research of production formula:

According to the petroleum industry handling characteristics,, reduce the xanthan gum production cost greatly for satisfying stifled well in the oil recovery, well workover, pressure break use.The report that fermented liquid thin film concentration (5-12%) back is directly used is all arranged at present both at home and abroad.The present invention has carried out the research selection of production formula in view of the above, and one of them fill a prescription preferably (prescription one) is: the about 80g/L of molasses, the about 1g/L of urea, K ₂HpO ₄About 1g/L; The pilot scale average result is the about 22g/L of xanthan gum output, and transformation efficiency is about 62.8%, and conversion xanthan gum raw materials cost is about 2160 yuan/ton.

Pilot scale xanthan gum prescription (prescription two): the about 80g/L of molasses, the about 3g/L of yeast extract paste, K ₂HPO ₄About 1g/L, MgSO ₄7H ₂The about 0.2g/L of O; Xanthan gum output is about 25.6g/L, transformation efficiency 68.3%, and conversion xanthan gum raw materials cost is 5130 yuan of/ton xanthan gum.

Above-mentioned two prescriptions as can be seen, though prescription one feed stock conversion and xanthan gum output is lower slightly, the raw materials for production cost obviously reduces; And fill a prescription in two feed stock conversion and xanthan gum output are all higher, but the raw materials for production cost obviously raises, and should determine different culture medium prescriptions according to different xanthan gum uses in actual production process, in the hope of maximum economic benefits.

Embodiment 9: the xanthan gum evaluation test

1. sample title and material:

XC1: adopt production technique of the present invention, the xanthan gum of acquisition, fermented liquid about 2.2%

XC2: adopt production technique of the present invention, the xanthan gum of acquisition, fermented liquid about 2.1%

XC3:Flocon4800, xanthan gum concentrated broth about 12.3%

XC4: about 1.709%, the 1500PPM64.4C43CP of Kodak, xanthan gum fermentation broth

XC5: about 1.700%, the 1500PPM64.12C of Kodak, xanthan gum fermentation broth

XC6: the Xinhe River, xanthan gum fermentation broth about 2.2%

HPAM1: Dalian 1,020 ten thousand polyacrylamides

HPAM2: Fushun 500-600 ten thousand polyacrylamides

HPAM3: U.S. FLOAAM2330S 1,000 ten thousand polyacrylamides

Sodium-chlor: analytical pure

Calcium chloride: analytical pure

Water: first water

2. instrument:

The RV-100 viscometer

Brookfieid viscometer UL rotor

Filterable agent determinator (filter membrane aperture: 1.2 μ m)

The screen factor viscometer

3. test-results

3.1 the tackifying of polymkeric substance

Polyacrylamide and xanthan gum all have good tackifying in the aqueous solution, this from test-results Fig. 2 as can be seen, Fig. 2 is the tackify effect comparison diagrams of 8 kinds of polymkeric substance in the aqueous solution, wherein, HPAM1, HPAM2 and HPAM3 are Industrial products (Powdered); XC1, XC4, XC5 are xanthan gum thickness fermented liquid; XC3 and XC6 are the xanthan gum liquid that contracts deeply, find out from test: the tackifying order of 8 kinds of polymkeric substance in the aqueous solution is as follows:

HPAM1＞XC1＞HPAM3＞XC3＞HPAM2＞XC6＞XC5＞XC4

That the tackify effect of 6 kinds of xanthan gum in the aqueous solution is best is product XC1 of the present invention, and its tackifying and high-molecular weight polypropylene amine in the aqueous solution is very nearly the same, and than other products remarkable advantages is arranged.

3.2 the salt tolerance of polymkeric substance

Xanthan gum has good salt tolerance, and this is the most outstanding advantage with respect to partially hydrolyzed polyacrylamide, below we will discuss respectively in NaCl solution, CaCl ₂The tackifying of polymkeric substance in the solution, in the salts solution.

3.2.1 the tackify effect of polymkeric substance in the NaCl aqueous solution as shown in Figure 3, wherein, polymers soln is 2000mg/L.As seen from Figure 3,3 kinds of polyacrylamide solutions are with the increase of NaCl concentration in the system, and basic viscosity number descends gradually; And 6 kinds of xanthan gum are highly stable in NaCl solution medium viscosity value, and their viscosity number and the NaCl concentration in the system are irrelevant, and their tackifying orders in the NaCl aqueous solution are as follows: XC1＞XC2＞XC3＞XC5＞XC4＞XC6.That 6 kinds of xanthan gum tackify effect in the NaCl aqueous solution is best is product XC1 of the present invention, XC2.

3.2.2 polymkeric substance CaCl ₂Tackify effect in the solution as shown in Figure 4.Wherein, polymer concentration is 2000mg/L, and as seen from the figure, 3 kinds of polyacrylamide solutions are with CaCl in the system ₂The increase of concentration, its viscosity number sharply descends; And plant xanthan gum at CaCl ₂Solution medium viscosity value is more stable, their viscosity number and the CaCl in the system ₂Concentration is irrelevant substantially, and that more special is product XC1 of the present invention, XC2, and they are at CaCl ₂When concentration ratio is low (≤100mg/L), its viscosity number is subjected to CaCl ₂The influence of concentration is bigger, works as CaCl ₂Concentration is during greater than 100mg/L, their viscosity number and the CaCl in the system ₂Concentration is irrelevant, and they are at CaCl ₂Tackifying order in the aqueous solution is as follows: XC1＞XC2＞XC3＞XC5＞XC4＞XC6.6 kinds of xanthan gum are at CaCl ₂That tackifying tackify effect is best in the aqueous solution is product XC1 of the present invention, XC2.

3.2.3 the tackifying of polymkeric substance in salts solution

The tackify effect of 5 kinds of xanthan gum in salts solution as shown in Figure 5, NaCl concentration 1000mg/L wherein, CaCl ₂Concentration is 150mg/L.Compare with the result of Library, tackifying and their the tackifying in the aqueous solution of 5 kinds of xanthan gum in salt is molten, and viscosity variation tendency basically identical, test explanation thus, xanthan gum has extraordinary anti-salt property (comprising monovalent ion and divalent ion).

3.3 biological polymer is to the susceptibility of temperature

Fig. 6 is that the sticking of 3 kinds of xanthan gum---kelvin relation curve, XC11, XC21, XC31 are that (NaCl concentration is 1000mg/L to xanthan gum, CaCl at salts solution ₂Concentration is 150mg/L) in sticking---the kelvin relation curve, wherein, xanthan gum concentration is 2000mg/L, as seen from Figure 6 along with the rising of temperature, XC1 of the present invention, XC2 are not too responsive to variation of temperature, and the salinity in the late into the night does not have tangible influence to them therein yet; Flocon4800 is relatively more responsive to variation of temperature, and along with the rising of temperature, its viscosity number is linear and descends, and illustrates that it is comparatively responsive to temperature.

3.4 the rheological of biological polymer

Xanthan gum has bigger shear shinning performance, but this and polyacrylamide shear shinning are inconsistent, polyacrylamide is that the xanthan gum molecule has rigidity because molecule is subjected to the effect of shear-stress to produce degraded, and the reduction of viscosity and rise are irreversible, under shear stress, produce the release of spirane structure, promptly from order to unordered transformation, when shear-stress failed, it was again from the transformation of disorder to order, be a kind of reversible phenomenon, can not cause the degraded of molecule.Fig. 7 is the flow curve figure of 3 kinds of xanthan gum, and wherein XC1, XC2, XC3 are the flow curve of xanthan gum in the aqueous solution, and XC11, XC21 are that (NaCl concentration is 1000mg/L to xanthan gum, CaCl at salts solution ₂Concentration is 150mg/L) in flow curve, wherein, xanthan gum concentration is 2000mg/L, we are as seen from Figure 7 along with the raising of shearing rate, the viscosity of its solution reduces gradually, XC1 of the present invention, XC2 viscosity degradation amplitude are greater than Flocon4800 viscosity degradation amplitude, and this anti-mechano-degradation and shear shinning have crucial meaning in actual applications.

3.5 the mensuration of biological polymer filterable agent and screen factor

Measure the filterable agent of biological polymer by the filterable agent determinator.

Xanthan gum filterable agent measurement result

The biological polymer title	????XC5	????XC4	????XC3	????XC2	????XC1
						????T1(MIN)	????14.75	????28.38	????6.01(SEC)	????143.0	????130.55
????T2(MIN)	????254.19	????121.19	????6.51(SEC)	????346.9	????237.37
						Filterable agent (FF)	????1.72	????4.27	????1.08	????2.43	????1.66

From test, find out, XC2, XC4 filterable agent value are bigger, their relatively wider or their intermolecular easy bondings of molecular weight distribution are described, also have, except that XC3, other biological polymer filterable agent minute is oversize, and polyacrylamide is according to the molecular weight difference, minute was generally 0.5-1 hour, and no longer than 2 hours, filterable agent value maximum also was no more than 2.Explanation thus, xanthan gum molecule distribute and relatively extensively or between them bond easily most.

By the screen factor of screen factor viscometer determining biological polymer, the result is as showing:

Xanthan gum screen factor measurement result

The biological polymer title	????XC1	????XC2	????XC3	????XC4	????XC5
						????T1(SEC)	????10.59	????10.59	????10.59	????10.59	????10.59
????T2(SEC)	????138.16	????123.63	????155.74	????103.63	????112.16
						Screen factor (SF)	????13.05	????11.69	????14.71	????9.79	????10.59

Find out that from test XC1 of the present invention, XC2 screen factor value are less than Flocon4800, greater than the XC4 of Kodak company, XC5.XC1, XC2 tackifying are better than XC4, XC5.

4. conclusion

Draw by performance test and tackify effect assessment to xanthan gum, XC1 xanthan gum product tackifying of the present invention is strong, and molecular weight distribution is smaller, and over-all properties can be used for the oil field well workover apparently higher than other xanthan gum product and polyacrylamide, water blockoff and fracturing liquid.

Claims (10)

1, the production technique of xanthan gum is characterized in that comprising:

Steps A: the cultivation of 1.1781 bird rape Xanthomonas campestris (Xanthomonas campestris) seed;

Step B: utilize the beet sirup substratum of beet sirup, ferment with 1.1781 bird rape Xanthomonas campestris seeds for main raw material;

Step C: fermented liquid is carried out aftertreatment.

2, xanthan gum production technique as claimed in claim 1 is characterized in that:

The leavening temperature of steps A is 20 ℃-35 ℃, and stir speed (S.S.) is 200-300rpm, air flow 0.8-0.9v/v/m, and the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, the sturdy quantity of bacterial classification is many;

Inoculum size is 1%-15% among the step B, and leavening temperature is 20 ℃-35 ℃, and air flow is 0.6-1.0v/v/m, and when the microscopy thalline wears out, divides, diminishes, liquid thickness during fermentation, reducing sugar be below 1.0, fermentation ends when viscosity no longer increases.

3, xanthan gum production technique as claimed in claim 2 is characterized in that the leavening temperature among the described step B is 25 ℃-35 ℃, and air flow is 0.8-0.9v/v/m, and mixing speed is 100-400rpm, and fermentation time is 50-80 hour.

4, as each described xanthan gum production technique of claim 1-3, it is characterized in that it is 25-60g/L that the molasses that contain in the described beet sirup substratum of step B are converted into sucrose, the pH of substratum is 6-8.

5, xanthan gum production technique as claimed in claim 4 is characterized in that described molasses culture medium is a liquid nutrient medium, and the molasses that contain are amounted to sucrose 38-53g/L, urea 1.5-2.5g/L, K ₂HPO ₄0.5-2.5g/L; Or the molasses that contain are amounted to sucrose 38-53g/L, yeast extract paste 2-4g/L, K ₂HPO ₄0.5-2.5g/L, MgSO ₄7H ₂O 0.1-1g/L.

6, xanthan gum production technique as claimed in claim 5 is characterized in that described molasses culture medium contains molasses: 70g/L; Yeast powder: 3g/L; K ₂HPO ₄: 1g/L; PH:7.0; And ferment 30 ℃ temperature.

7, xanthan gum production technique as claimed in claim 1 or 2 is characterized in that seed culture comprises the secondary seed cultivation in the described steps A, and its substratum is molasses 50-80g/L, sucrose 5-15g/L, yeast extract paste 2-4g/L, K ₂HPO ₄0.5-2.5g/L, MgSO ₄7H ₂O 0.1-1g/L, pH6-8; The secondary seed fermentation time is 16-18 hour.

8, xanthan gum production technique as claimed in claim 1 or 2, it is characterized in that the molasses process pre-treatment of the molasses culture medium of described step B, pretreatment process is: transfer pH3.5-4.5, heated and boiled 5-15 minute, be neutralized to pH6-8 when temperature is reduced to 70-80 ℃ again and filter.

9, xanthan gum production technique as claimed in claim 1 obtains xanthan gum after the aftertreatment technology that it is characterized in that described step C adopts fermented liquid thin film concentration 5-12%.

10, xanthan gum production technique as claimed in claim 1 is characterized in that:

Leavening temperature in the seed culture step described in the steps A is 20 ℃-35 ℃, and stir speed (S.S.) is 200-300rpm, air flow 0.8-0.9v/v/m, and the seed inspection standard is that microscopy does not have assorted bacterium, no pod membrane, the sturdy quantity of bacterial classification is many; And seed culture is cultivated through secondary seed, and its substratum is molasses 50-80g/L, sucrose 5-15g/L, yeast extract paste 2-4g/L, K ₂HPO ₄0.5-2.5g/L, MgSO ₄7H ₂O 0.1-1g/L, pH6-8; The secondary seed fermentation time is 16-18 hour;

Inoculum size is 1%-15% among the step B, and temperature is 20 ℃-35 ℃, and air flow is 0.8-0.9v/v/m, and mixing speed is 100-400rpm, and fermentation time is 50-60 hour, and the molasses that substratum contains are amounted to sucrose 25-60g/L, pH=7.

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