CN104498565A - Method for co-production of alpha-arbutin and xanthan gum by xanthomonas - Google Patents
Method for co-production of alpha-arbutin and xanthan gum by xanthomonas Download PDFInfo
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- CN104498565A CN104498565A CN201410458385.6A CN201410458385A CN104498565A CN 104498565 A CN104498565 A CN 104498565A CN 201410458385 A CN201410458385 A CN 201410458385A CN 104498565 A CN104498565 A CN 104498565A
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- arbutin
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Abstract
Belonging to the field of biotechnologies, the invention in particular relates to a method of utilizing the thalli and supernatant of xanthomonas campestris fermentation liquor centrifugation or microfiltration membrane filtration to conduct catalytic synthesis of alpha-arbutin and cell-free fermentation xanthan gum by a free cell technique respectively. The method includes: preparation of xanthomonas campestris oblique plane seeds and first level liquid fermentation, catalytic synthesis of alpha-arbutin with sucrose and hydroquinone as the substrates by the free cell technique, and cell-free fermentation production of xanthan gum. According to the method, the transformation rate of alpha-arbutin synthesis reaches 75-90%, the tolerance of hydroquinone is increased to 50-67mmol/L. The cell-free process xanthan gum yield reaches 10.0-20.0g/L, and the viscosity is 4500-5380mPa.S. The method has the advantages of high enzyme activity, easily controllable conditions, and easy product separation, etc.
Description
Technical field
The invention belongs to biological technical field, be specifically related to utilize xanthomonas campestris (
xanthomonas campestris) thalline after the centrifugal or micro-filtrate membrane filtration of CGMCC NO.1243 fermented liquid and upper clear enzyme solution, take free cell method to catalyze and synthesize the method for alpha-arbutin and cell free fermentation xanthan gum respectively.
Background technology
Arbutin (Arbutin) is a kind of composition in the uva ursi daughter cell of Ericaceae black bearberry genus, has good skin whitening effects, has many uses in cosmetic industry.Research shows that the chemical property of alpha-arbutin is more stable than β type, and whitening effect is better, and less to the side effect of human body cell.The synthetic method of present alpha-arbutin is also is mostly add substrate by fermentable to carry out.
Xanthan gum (Xanthan gum) is a kind of polymer exocellular polysaccharide, be mostly by xanthomonas campestris (
xanthomnas campestris) fermentation generation.Xanthan gum has three grades of special helical molecular structures, make it have good water-soluble, pseudo-plasticity, often be used to do emulsifying agent, thickening material etc., be widely used in the industrial circles such as food, oil, geology, medicine, agricultural chemicals, light industry, printing and dyeing, pottery, had very large market potential.
Forefathers have certain research respectively for Xanthomonas campestris catalytic production alpha-arbutin and production xanthan gum both direction.Xu Xilin etc. utilize centrifugal method to obtain except cell fermentation liquid, and the yield of xanthan gum obtained after adding substratum comparatively general method is slightly high, proves the feasibility of this method.The condition that free cell method produces alpha-arbutin has been probed in Wang Xiu handful etc., and its transformation efficiency is about 80%, but Resorcinol tolerance level is lower, only has 30mmol/L.Free cell method and cell free fermentation have respective advantage: the feature that free cell has enzymic activity high, and easy control of reaction conditions is also beneficial to arbutin separation and purification, but owing to lacking the protection of xanthan gum, on the low side to the tolerance level of Resorcinol.The benefit of cell free fermentation is less for the demand of oxygen, in separation and purification, also more easily obtain product, reduces supplies consumption.But because the enzyme amount of discharging outside born of the same parents is little, enzymatic reaction requires high, make the xanthan gum that obtains in output and these two important indicators of viscosity all lower than the level of cell fermentation.But utilize the research of same bacterial classification coproduction two kinds of products to be not reported so far.
The present invention adopts same bacterial classification, utilizing the coproduction mode of innovation, adopting the method for free cell and cell free fermentation respectively, to reaching maximum output ratio and minimum energy consumption in the production of two kinds of products.
Summary of the invention
The object of this invention is to provide a kind of method utilizing Xanthomonas campestris coproduction alpha-arbutin and these two kinds of products of xanthan gum, adopt free cell to catalyze and synthesize alpha-arbutin, cell free fermentation xanthan gum respectively, there is the advantage that enzymic activity is high, easy control of reaction conditions, product are easy to separation and purification.The present invention utilizes same bacterial classification, after high speed centrifugation or micro-filtrate membrane filtration process, be respectively used to coproduction two kinds of products, the object of saving material, reducing costs can be reached, bring good economic benefit and social benefit, there is novelty, for industrial production is laid a good foundation.
Overall technology design of the present invention is:
Utilize xanthomonas campestris (
xanthomonas campestris) method of CGMCC NO.1243 coproduction alpha-arbutin and xanthan gum, the method is made up of following process steps:
(1) xanthomonas campestris (
xanthomonas campestris) preparation of CGMCC NO.1243 inclined-plane seed and level liquid fermentation;
(2) step (1) fermented liquid high speed centrifugation or micro-filtrate membrane filtration are obtained thalline and upper clear enzyme solution;
(3) free cell method synthesis alpha-arbutin;
(4) cell free fermentation production xanthan gum.
Concrete technology step of the present invention and processing condition are:
In step (1), medium component is:
Sucrose 15 ~ 30g/L, beef peptone 1 ~ 10g/L, MgS0
47H
201 ~ 10g/L, yeast powder 1 ~ 10g/L, K
2hP0
43H
201 ~ 10g/L, NaCl 1 ~ 10g/L, agar 5 ~ 20g/L.(slant medium adds, and liquid nutrient medium does not add)
The preparation technology of slant strains is: be equipped with in the test tube of solid medium by Xanthomonas campestris access, bevel bacterial classification, cultivate 12 ~ 48 hours under 20 ~ 35 DEG C of conditions.
The preparation technology of level liquid fermentation is: after the physiological saline solution after the Xanthomonas campestris sterilizing grow inclined-plane, be inoculated in 50ml liquid nutrient medium, inoculum size is 1% ~ 10%, 20 ~ 35 DEG C, cultivate in the shaking table of 100 ~ 200r/min, adopt the mode of aerobic cultivation to cultivate 12 ~ 48 hours.
Step (2) high speed is centrifugal:
By step (1) fermented liquid high speed centrifugation 10 ~ 30min under 5000 ~ 20000r/min condition, or 0.20 μm ~ 0.45 μm micro-filtrate membrane filtration is adopted to collect thalline and upper clear enzyme solution.
In step (3), free cell method synthesis alpha-arbutin concrete technology is:
Collect centrifugal after thalline, in phosphate buffer solution, add gelatin and xitix, addition is respectively 1 ~ 10% and 0.1 ~ 2.0g/L, and washing thalline forms uniform bacterium liquid, add sucrose and Resorcinol as reaction substrate, react 12 ~ 48 hours under temperature 20 ~ 35 DEG C of conditions.
In step (4), cell free fermentation production xanthan gum concrete technology is:
Centrifugal upper clear enzyme solution joins equal-volume and produces glue substratum, and temperature is 15-35 DEG C, cultivates 15-45 hour.
Producing glue medium component is: sucrose 10 ~ 30g/L, yeast extract paste 0.08g/L, CaCO
31 ~ 5g/L, MgS0
41.2g/L, KH
2p0
40.5g/L, KCl 5 ~ 30g/L, citric acid 0.01g/L, pH6.0 ~ 8.0.
The present invention's xanthomonas campestris used (
xanthomonas campestris) CGMCC NO.1243 is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation address is No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica (100080), preserving number is xanthomonas campestris CGMCC NO.1243, and the preservation time is on November 8th, 2004.
Technical progress acquired by the present invention is:
The method of coproduction alpha-arbutin of the present invention and xanthan gum is utilized to have the following advantages:
(1) method of free cell catalytic production alpha-arbutin that adopts of the present invention, has the advantages that enzymic activity is high, easy control of reaction conditions, product purity is high, arbutin is easy to separation and purification.
(2) the present invention effectively reduces in free cell damping fluid that Resorcinol is to the toxic action of thalline, and the water-soluble polymer of interpolation and antioxidant significantly improve building-up reactions transformation efficiency.
(3) method of cell free fermentation xanthan gum that adopts of the present invention, the demand for oxygen is less, solves the problem that product that the high viscosity due to xanthan gum causes is difficult to be separated, reduces supplies consumption.
(4) the present invention utilizes the coproduction mode of innovation, uses same bacterial classification, adopts the method for free cell and cell free fermentation in the production of two kinds of products respectively, to reaching maximum output ratio and minimum energy consumption.
Embodiment: the invention will be further described below in conjunction with embodiment.
Embodiment 1
(1) preparation of xanthomonas campestris inclined-plane seed and level liquid fermentation:
Sucrose 25g/L, beef peptone 6.0g/L, MgS0
47H
20 1.0g/L, yeast powder 2.5g/L, K
2hP0
43H
20 2.0g/L, NaCl 2.0g/L, agar 15g/L.(slant medium adds, and liquid nutrient medium does not add)
The preparation technology of slant strains is: be equipped with in the test tube of solid medium by Xanthomonas campestris access, bevel bacterial classification, cultivate 24 hours under 25 DEG C of conditions.
The preparation technology of level liquid fermentation is: after the physiological saline solution after the Xanthomonas campestris sterilizing grow inclined-plane, be inoculated in 50ml liquid nutrient medium, inoculum size is 2%, 28 DEG C, cultivate in the shaking table of 150r/min, adopt the mode of aerobic cultivation to cultivate 12 hours.
(2) step (1) fermented liquid high speed centrifugation is obtained thalline and upper clear enzyme solution:
By step (1) fermented liquid high speed centrifugation 30min under 15000r/min condition, collect thalline and upper clear enzyme solution.
(3) free cell method synthesis alpha-arbutin:
Collect centrifugal after thalline, in phosphate buffer solution, add gelatin and xitix, addition is respectively 2% and 0.1g/L, and washing thalline forms uniform bacterium liquid, adds sucrose and Resorcinol as reaction substrate, reacts 48 hours under temperature 22 DEG C of conditions.At this point in the reaction, alpha-arbutin transformation efficiency is 75.43%, and Resorcinol tolerance level is 37mmol/L.
(4) cell free fermentation production xanthan gum:
Centrifugal upper clear enzyme solution joins equal-volume and produces glue substratum, and temperature is 15-35 DEG C, cultivates 15-45 hour.Yield of xanthan gum reaches 12.4g/L, viscosity 4520 mPas.
Producing glue medium component is: sucrose 10g/L, yeast extract paste 0.08g/L, CaCO
31.0g/L, MgS0
41.2g/L, KH
2p0
40.5g/L, KCl 10g/L, citric acid 0.01g/L, pH7.5.
Embodiment 2
(1) preparation of xanthomonas campestris inclined-plane seed and level liquid fermentation:
Sucrose 25g/L, beef peptone 6.0g/L, MgS0
47H
20 1.0g/L, yeast powder 2.5g/L, K
2hP0
43H
20 2.0g/L, NaCl 2.0g/L, agar 15g/L.(slant medium adds, and liquid nutrient medium does not add)
The preparation technology of slant strains is: be equipped with in the test tube of solid medium by Xanthomonas campestris access, bevel bacterial classification, cultivate 24 hours under 25 DEG C of conditions.
The preparation technology of level liquid fermentation is: after the physiological saline solution after the Xanthomonas campestris sterilizing grow inclined-plane, be inoculated in 50ml liquid nutrient medium, inoculum size is 2%, 28 DEG C, cultivate in the shaking table of 150r/min, adopt the mode of aerobic cultivation to cultivate 10 hours.
(2) step (1) fermented liquid micro-filtrate membrane filtration is obtained thalline and upper clear enzyme solution:
Step (1) fermented liquid is used 0.20 μm of micro-filtrate membrane filtration, collects thalline and upper clear enzyme solution.
(3) free cell method synthesis alpha-arbutin:
Collect the thalline after filtering, in phosphate buffer solution, add gelatin and xitix, addition is respectively 6% and 0.4g/L, and washing thalline forms uniform bacterium liquid, adds sucrose and Resorcinol as reaction substrate, reacts 48 hours under temperature 22 DEG C of conditions.At this point in the reaction, alpha-arbutin transformation efficiency is 90%, and Resorcinol tolerance level is 40mmol/L.
(4) cell free fermentation production xanthan gum:
Centrifugal upper clear enzyme solution joins equal-volume and produces glue substratum, and temperature is 32 DEG C, cultivates 36 hours.Yield of xanthan gum reaches 13.6g/L, viscosity 5120 mPas.
Producing glue medium component is: sucrose 10g/L, yeast extract paste 0.08g/L, CaCO
33.0g/L, MgS0
41.2g/L, KH
2p0
40.5g/L, KCl 5g/L, citric acid 0.01g/L, pH7.5.
Embodiment 3
(1) preparation of xanthomonas campestris inclined-plane seed and level liquid fermentation:
Sucrose 25g/L, beef peptone 6.0g/L, MgS0
47H
20 1.0g/L, yeast powder 2.5g/L, K
2hP0
43H
20 2.0g/L, NaCl 2.0g/L, agar 15g/L.(slant medium adds, and liquid nutrient medium does not add)
The preparation technology of slant strains is: be equipped with in the test tube of solid medium by Xanthomonas campestris access, bevel bacterial classification, cultivate 24 hours under 25 DEG C of conditions.
The preparation technology of level liquid fermentation is: after the physiological saline solution after the Xanthomonas campestris sterilizing grow inclined-plane, be inoculated in 50ml liquid nutrient medium, inoculum size is 2%, 28 DEG C, cultivate in the shaking table of 150r/min, adopt the mode of aerobic cultivation to cultivate 14 hours.
(2) step (1) fermented liquid micro-filtrate membrane filtration is obtained thalline and upper clear enzyme solution:
Step (1) fermented liquid is used 0.45 μm of micro-filtrate membrane filtration, collects thalline and upper clear enzyme solution.
(3) free cell method synthesis alpha-arbutin:
Collect the thalline after filtering, in phosphate buffer solution, add gelatin and xitix, addition is respectively 8% and 1.0g/L, and washing thalline forms uniform bacterium liquid, adds sucrose and Resorcinol as reaction substrate, reacts 48 hours under temperature 24 DEG C of conditions.At this point in the reaction, alpha-arbutin transformation efficiency is 80%, and Resorcinol tolerance level is 67mmol/L.
(4) cell free fermentation production xanthan gum:
Centrifugal upper clear enzyme solution joins equal-volume and produces glue substratum, and temperature is 32 DEG C, cultivates 36 hours.Yield of xanthan gum reaches 20g/L, viscosity 5380 mPas.
Producing glue medium component is: sucrose 10g/L, yeast extract paste 0.08g/L, CaCO
35.0g/L, MgS0
41.2g/L, KH
2p0
40.5g/L, KCl 15g/L, citric acid 0.01g/L, pH7.5.
Claims (8)
1. utilize a method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum, it is characterized by: utilize xanthomonas campestris (
xanthomonas campestris) thalline after CGMCC NO.1243 liquid fermentation liquid high speed centrifugation or micro-filtrate membrane filtration and upper clear enzyme solution, use free cell method synthesis alpha-arbutin and cell free fermentation production xanthan gum respectively, reach the object using same bacterial classification coproduction two kinds of products; The transformation efficiency of free cell method synthesis alpha-arbutin reaches 75 ~ 90%, and Resorcinol tolerance level brings up to 50 ~ 67mmol/L; Cell-free method produces yield of xanthan gum and reaches 10.0 ~ 20.0g/L, viscosity 4500 ~ 5380mPas.
2. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum as claimed in claim 1, it is characterized in that: its preparation flow is: xanthomonas campestris (
xanthomonas campestris) preparation of CGMCC NO.1243 inclined-plane seed and level liquid fermentation; Step (1) fermented liquid high speed centrifugation or micro-filtrate membrane filtration are obtained thalline and upper clear enzyme solution;
Free cell method synthesis alpha-arbutin; Cell free fermentation production xanthan gum.
3. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum as claimed in claim 2, it is characterized in that: the medium component in described (1) step is sucrose 15 ~ 30g/L, beef peptone 1 ~ 10g/L, MgS0
47H
201 ~ 10g/L, yeast powder 1 ~ 10g/L, K
2hP0
43H
201 ~ 10g/L, NaCl 1 ~ 10g/L, agar 5 ~ 20g/L.
4. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum according to claim 2, it is characterized in that: the free cell method synthesis alpha-arbutin in described (3) step is specially: form uniform bacterium liquid with the thalline that phosphate buffer solution washing is collected, add sucrose and Resorcinol as reaction substrate, catalyze and synthesize alpha-arbutin; Reaction conditions is 15 ~ 30 DEG C, shaking speed 100 ~ 200r/min, 12 ~ 60 hours reaction times.
5. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum according to claim 2, it is characterized in that: the phosphoric acid buffer in described (3) step needs to add gelatin, polyvinyl alcohol, sodium alginate, polyacrylamide and xitix, vitamin-E, and addition is respectively 1 ~ 10% and 0.1 ~ 2.0g/L.
6. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum according to claim 2, it is characterized in that: the cell free fermentation production xanthan gum in described (4) step is specially: centrifugal upper clear enzyme solution joins equal-volume and produces glue substratum, temperature is 15-35 DEG C, cultivates 15-45 hour.
7. utilize the method for Xanthomonas campestris coproduction alpha-arbutin and xanthan gum according to claim 2, it is characterized in that: the product glue medium component in described (4) step is: sucrose 10 ~ 30g/L, yeast extract paste 0.08g/L, CaCO
31 ~ 5g/L, MgS0
41.2g/L, KH
2p0
40.5g/L, KCl 5 ~ 30g/L, citric acid 0.01g/L, pH6.0 ~ 8.0.
8. xanthomonas campestris (
xanthomonas campestris) CGMCC NO.1243.
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Cited By (1)
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| CN117286082A (en) * | 2023-11-24 | 2023-12-26 | 内蒙古工业大学 | Xanthomonas campestris and method for producing low-viscosity xanthan gum by fermentation |
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