CN103789241B - One strain ι-carrageenin degradation bacteria and fermentation process thereof and application - Google Patents
One strain ι-carrageenin degradation bacteria and fermentation process thereof and application Download PDFInfo
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- CN103789241B CN103789241B CN201410043525.3A CN201410043525A CN103789241B CN 103789241 B CN103789241 B CN 103789241B CN 201410043525 A CN201410043525 A CN 201410043525A CN 103789241 B CN103789241 B CN 103789241B
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Abstract
One strain ι-carrageenin degradation bacteria and fermentation process thereof and application, relate to a kind of bacterium addicted to Mierocrystalline cellulose Pseudomonas.ι-carrageenin degradation bacteria (Cellulophaga? does is sp.) deposit number of KL-A CGMCC? No:8694.The production method of ι-carrageenan hydrolase: by the ι-carrageenin degradation bacteria (Cellulophaga of activation? sp.) KL-A coats on ι-carrageenin solid medium, cultivates, obtain seed liquor in picking list bacterium colony access seed culture medium; Seed liquor is inoculated in fermented liquid by the inoculum size by 1% ~ 5%, and shaking table is cultivated, and obtains fermented supernatant fluid; Under 4 DEG C of conditions, by gained fermented supernatant fluid through grade ammonium sulfate salting-out and DEAE-Mierocrystalline cellulose chromatography, dialysis desalination, vacuum lyophilization, obtains ι-carrageenan hydrolase.ι-carrageenan oligosaccharide can in preparation antibacterials, application in antiviral, immunomodulator, antioxidant etc.
Description
Technical field
The present invention relates to a kind of bacterium addicted to Mierocrystalline cellulose Pseudomonas, particularly relate to a strain ι-carrageenin degradation bacteria and fermentation process thereof and application.
Background technology
Carrageenin (Carrageenan), have another name called Deerhorn Glue, carrageenin etc., it is a kind of high molecular weight hydrophilic polysaccharide extracted from red algae, with repeat α-(1 → 4)-D-gala pyranose-β-(1 → 3)-D-gala pyranose (or 3,6 inner ether-D-gala pyranoses) disaccharide unit be that basic framework is formed by connecting.Content according to whether containing 3,6 inner ether-D-gala pyranoses, sulfate and sulfate position is in the molecule different, carrageenin is mainly divided into κ-, λ-, ι-3 race.
Carrageenin is because having excellent heat reversible gel, anti-protein gelatin, the hydrophilic performance such as nontoxic and being widely used in foodstuffs industry.Current carrageenin has been widely used in the foodstuffs industry such as jelly, soft sweets, ice-creams, meat product, beer, and Main Function shows gel, thickening and albumen test 3 aspects.That is familiar with carrageenin structure and function along with people gos deep into, and its Application Areas is constantly widened, and especially becomes increasingly extensive in the application of field of medicaments.
Existing achievement in research shows, carrageenin has multiple biological activity, as anticoagulation, antiviral, immunomodulatory, anti-oxidant etc., but due to carrageenin relative molecular mass excessive, its solvability and absorptivity are affected, limit its application at field of medicaments.By comparison, relative molecular mass is less for carrageenan oligosaccharide, and solvability, stability and security increase all to some extent, and biological activity also tool be improved to some extent.
The method of current degraded carrageenan has Physical, chemical method, enzymolysis process.Wherein enzyme liberating method is an important channel of preparing oligosaccharides, and because it has specificity, optionally enzymolysis cuts off the specific site on sugar chain, thus obtained specific oligosaccharides; And reaction conditions is gentle, degradation process is easy to control, and the utilization in polysaccharide degraded is increasing.At present, the research that Chinese scholars is carried out carrageenan oligosaccharide, existing achievement shows that carrageenan oligosaccharide has certain using value and DEVELOPMENT PROSPECT in functional foodstuff, medicine and other fields.Therefore, utilize the carrageenase extracted from marine microorganism to prepare carrageenin lower molecular weight active fragments, become the important directions of the high-valued research of carrageenin industry.It is very necessary for finding the bacterial classification with energy degraded carrageenan.
Chinese patent CN1544623 discloses a kind of kappa-carrageenan degrading enzyme and its preparation method and application, a kind of enzyme can make kappa-carrageenan degrade, for the preparation of carraoligose, can be degraded the β-1 of kappa-carrageenan, 4-glycosidic link, therefore being also called kappa-carrageenan degrading enzyme, the molecular weight of this enzyme is 30,000Da.When preparing this enzyme, by ocean Cytophaga with after 2216E substratum 28-35 DEG C of shake-flask culture, medium centrifugal is obtained fermenting enzyme liquid, after enzyme liquid ultrafiltration and concentration, to saltout collecting precipitation protein with (NH4) 2SO4 of 40%-80%, lyophilize.
Chinese patent CN102994408A discloses a kind of carrageenin degradation bacteria and fermentation process thereof and application, relate to the bacterium of a kind of new soil Bacillaceae, its called after carrageenin degradation bacteria 13-Q, i.e. Pedobacterhainanensissp.13-Q, this bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and deposit number is CGMCCNO.5564.
Summary of the invention
The first object of the present invention is to provide a strain can secrete the bacterial strain of carrageenase.
The second object of the present invention is the production method providing a kind of ι-carrageenan hydrolase.
The third object of the present invention is to provide ι-carrageenan oligosaccharide in preparation antibacterials, application in antiviral, immunomodulator, antioxidant etc.
The described bacterial strain secreting carrageenase is ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A, be separated from Fujian Province's Dongshan Island, bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo:8694.Colony morphology characteristic is: good at ι-carrageenin cultured on solid medium, and bacterium colony is yellow, circular, and surface is projection slightly, smooth, reflective, opaque, rod-short, neat in edge, Gram-negative, not raw spore, movable.
The physiologic character of bacterial strain is: growth needs NaCl, almost can not grow without during NaCl, and the NaCl of high density suppresses it to grow, and the ι-carrageenin of 0.2% can promote the generation of carrageenase.Optimum growth temperature is 28 ~ 32 DEG C.
According to 16SrRNA sequential analysis comparison result, described identification of strains is Cellulophagasp.KL-A.
Genetics characteristics: the 16SrRNA base sequence of described ι-carrageenin degradation bacteria, sequence is 1075bp altogether.
Be 99% to the similarity of the most similar bacterial strain Cellulophagalytica of the international geneseq database record of Genebank.
The production method of described a kind of ι-carrageenan hydrolase, comprises the following steps:
1) ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A of activation is coated on ι-carrageenin solid medium, cultivate in picking list bacterium colony access seed culture medium, obtain seed liquor;
2) be inoculated in fermented liquid by the inoculum size of 1% ~ 5% by seed liquor, shaking table is cultivated, and obtains fermented supernatant fluid;
3) under 4 DEG C of conditions, by step 2) gained fermented supernatant fluid is through grade ammonium sulfate salting-out and DEAE-Mierocrystalline cellulose chromatography, and dialysis desalination, vacuum lyophilization, obtains ι-carrageenan hydrolase.
In step 1), consisting of of described ι-carrageenin solid medium: mass concentration is the carbon source of 0.5% ~ 1%, the nitrogenous source of 0.3% ~ 0.5%, the sodium-chlor of 2%, the magnesium sulfate of 0.5%, the calcium chloride of 0.1%, the ferrous sulfate of 0.03%, the dipotassium hydrogen phosphate of 0.1%, the aqueous solution of the ι-carrageenin of 0.2%; Described carbon source can be selected from glucose, glycerine, semi-lactosi, sucrose, sodium acetate, the one in starch etc., preferred glucose; Described nitrogenous source can be selected from peptone, yeast powder, extractum carnis, ammonium sulfate, SODIUMNITRATE, the one in urea etc., preferred yeast powder; The condition of described cultivation can be 30 DEG C, and 200rpm cultivates 30h.
In step 2) in, the condition that described shaking table is cultivated can be 30 DEG C, and 30h cultivated by 200rpm shaking table.
Described ι-carrageenan hydrolase degraded ι-carrageenin, degraded product is 2 ι-carrageenan oligosaccharides, and the polymerization degree is respectively 2 ~ 4 sugar and 6 ~ 10 sugar.
Described ι-carrageenan oligosaccharide can in preparation antibacterials, application in antiviral, immunomodulator, antioxidant etc.In sea farming, the survival rate of prawn can improve 20% ~ 30%.
The present invention has following technique effect:
1, ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A synthesizes ι-carrageenan hydrolase under the induction of ι-carrageenin, and it is glucose and yeast powder respectively that single-factor screening obtains best carbon nitrogen source.By response surface optimization substratum and culture condition, the ι-carrageenan hydrolase vigor of acquisition reaches 226.73U/mL.Compared with having reported both at home and abroad, described ι-carrageenin degradation bacteria lytic enzyme throughput has greatly improved, and has certain DEVELOPMENT PROSPECT.
The lytic enzyme of the generation high level that 2, ι provided by the invention-carrageenin degradation bacteria energy is stable, utilizes this lytic enzyme can prepare the carrageenan oligosaccharide with other bacterium different polymerization degrees.
3, the molecular weight of ι-carrageenan hydrolase provided by the invention is 70kD, specificity hydrolysis ι-carrageenin, kappa-carrageenan, lambda-carrageenan, agar-agar can not be hydrolyzed, Mierocrystalline cellulose, chitin and starch, the product of hydrolysis is finally 2 ~ 8 sugar, and the ι-carrageenan oligosaccharide obtained has very high commercial application value.
4, ι-carrageenan hydrolase provided by the invention has certain thermostability, and 42 DEG C of thermal treatment 1h can keep the enzymic activity of 90%.
Accompanying drawing explanation
Fig. 1 is the thin-layer chromatogram of ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A degraded ι-carrageenin product.
Fig. 2 is the ι-degradation effect of carrageenin degradation bacteria (Cellulophagasp.) KL-A on ι-carrageenin flat board (degraded circle).
Embodiment
Embodiment 1
The separation and purification of ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A.
After gathering the new living body marine alga blade of Dongshan Island surrounding waters, Fujian Province, add antiseptic sea water cleaning, put into primary dcreening operation substratum, shake-flask culture 2 ~ 3 days.Nutrient solution is rule on the agar plate containing primary dcreening operation substratum, cultivates 3 days.The colony lift that agar plate is formed transparent circle is cultivated to new agar plate, repeats line, until obtain pure growth.Primary dcreening operation medium component (mass volume ratio): ι-carrageenin 0.2%, NaCl1.5%, NaNO
30.2%, MgSO
47H
2o0.05%, CaCl20.01%, tertiary iron phosphate 0.005%, pH value 7.5.
The pure growth be separated to by primary dcreening operation is inoculated in multiple sieve substratum, and shaking table is cultivated.Then nutrient solution is rule on multiple sieve solid medium, the bacterium colony picking producing most degradation circle is out carried out next step experiment.All bacterial strains screened, preserve after separation and purification.
Sieve medium component (mass volume ratio) again: ι-carrageenin 0.2%, yeast powder 0.1%, NaCl1.5%, NaNO
30.2%, MgSO
47H
2o0.05%, CaCl
20.01%, tertiary iron phosphate 0.005%, pH value 7.5.
Embodiment 2
Single-factor screening carbon nitrogen source
1, several different carbon source is chosen respectively, concentration is 10g/L, using the peptone of 5g/L as fixing nitrogenous source, separately adds the ι-carrageenin of 2g/L and the NaCl of 15g/L, preparation substratum, inoculum size is 3%, and culture temperature is 30 DEG C, and shaking speed is 200rpm, cultivate 48h, DNS method is surveyed enzyme and is lived, and the impact of carbon source on ι-carrageenan hydrolase fermentation is as shown in table 1, and glucose is optimum carbon source.
Table 1
| Carbon source | Glucose | Glycerine | Semi-lactosi | Sucrose | Sodium acetate | Starch |
| Enzyme activity (U/mg) | 287.6 | 201.3 | 234.7 | 168.2 | 171.1 | 168.2 |
2, with 15g/L glucose for fixing carbon source, choose several different nitrogenous source respectively, concentration is 5g/L, separately adds the ι-carrageenin of 2g/L and the NaCl of 15g/L, preparation substratum, inoculum size is 3%, and culture temperature is 30 DEG C, and shaking speed is 200rpm, cultivate 48h, DNS method is surveyed enzyme and is lived, and the impact of nitrogenous source on ι-carrageenan hydrolase fermentation is as shown in table 2, and yeast powder is optimum nitrogen source.
Table 2
| Nitrogenous source | Peptone | Yeast powder | Extractum carnis | Ammonium sulfate | SODIUMNITRATE | Urea |
| Enzyme activity (U/mg) | 277.5 | 213.6 | 198.6 | 178.8 | 201.4 | 205.7 |
Embodiment 3
ι-carrageenin degradation bacteria is utilized to prepare ι-carrageenan oligosaccharide
1) by described inoculation on ι-carrageenin solid medium, 30 DEG C cultivate 3 days;
2) by colony inoculation single on solid medium in 250mL Shake flask medium, 30 DEG C, 200rpm cultivates 3 days.
3) 3% access in 1L fermention medium by seed culture medium by volume, 30 DEG C, 200rpm cultivates 3 days, obtains fermented product.
4) by fermented liquid under 4 DEG C of conditions, the centrifugal 15min of 12000g, to remove thalline, stays supernatant liquor for subsequent use.
5) in supernatant liquor, add ammonium sulfate to 40% saturation ratio (adding solid ammonium sulfate by ammonium sulfate 4 DEG C of solubility tables) gradually, slowly stir and avoid producing bubble and cause enzyme deactivation, 4 DEG C staticly settle and spend the night after, 10000rpm frozen centrifugation removes foreign protein.In supernatant liquor, add ammonium sulfate to 70% saturation ratio gradually again, after 4 DEG C of hold over night, 10000rpm frozen centrifugation 10min collecting precipitation, meets cold deionized water dissolving with a small amount of 4 DEG C.
6) solution after dissolving is placed in dialysis tubing (MWCO:12000), and dialysis tubing is placed in the 20mMTris-HCl of precooling, desalination in the damping fluid of pH7.5, changes damping fluid several times more, then 4 DEG C of dialysed overnight.By solution stowage on DEAE-Mierocrystalline cellulose chromatography column, be 0.1MKCl eluant solution by concentration, collect the cut with ι-carrageenase activity.Cut is dialysed in identical damping fluid, vacuum lyophilization, obtains ι-carrageenan hydrolase.
7) above-mentioned ι-carrageenan hydrolase dry powder is dissolved in deionized water, enzyme liquid adds ι-carrageenin substrate solution (ι-carrageenin 0.5-2g in 100mL system, enzyme liquid 0.5-2mL, its surplus is water, pH7.5), enzyme digestion reaction 24h under 30 DEG C of conditions, reaction end is placed on boiling water 10min termination reaction, obtains ι-carrageenan oligosaccharide solution crude product.
8) dehydrated alcohol of 2 times of volumes is added to above-mentioned solution, the component that centrifugal segregation molecular weight is large, all the other soluble components carry out being concentrated into 20mL in 40 DEG C of rotary evaporations, and then the dehydrated alcohol adding 3 times of volumes makes oligose precipitate, centrifugal collecting precipitate, through desalination, rotary evaporation is concentrated obtains degraded product crude product.
9) by chloroform, amylalcohol (or propyl carbinol) is 4: 1 ratio mixing by volume, obtain mix reagent.Mix reagent and carrageenan oligosaccharide solution are mixed at 1: 3 by volume, the centrifugal 1min of 100rpm jolting 30min, 10000g removes insolubles, through vacuum rotary evaporator evaporative removal moisture, dries, obtains carrageenan oligosaccharide product.Namely product can be used as the sample of thin-layer chromatography.
Claims (8)
1. a strain ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A, bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo:8694.
2. a production method for ι-carrageenan hydrolase, is characterized in that comprising the following steps:
1) ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A of activation is coated on ι-carrageenin solid medium, cultivate in picking list bacterium colony access seed culture medium, obtain seed liquor; Consisting of of described ι-carrageenin solid medium: mass concentration is the carbon source of 0.5% ~ 1%, the nitrogenous source of 0.3% ~ 0.5%, the sodium-chlor of 2%, the magnesium sulfate of 0.5%, the calcium chloride of 0.1%, the ferrous sulfate of 0.03%, the dipotassium hydrogen phosphate of 0.1%, the aqueous solution of the ι-carrageenin of 0.2%; Described ι-carrageenin degradation bacteria (Cellulophagasp.) KL-A, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo:8694;
2) be inoculated in fermented liquid by the inoculum size of 1% ~ 5% by seed liquor, shaking table is cultivated, and obtains fermented supernatant fluid;
3) under 4 DEG C of conditions, by step 2) gained fermented supernatant fluid is through grade ammonium sulfate salting-out and DEAE-Mierocrystalline cellulose chromatography, and dialysis desalination, vacuum lyophilization, obtains ι-carrageenan hydrolase.
3. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 2, is characterized in that described carbon source is selected from glucose, glycerine, semi-lactosi, sucrose, sodium acetate, the one in starch.
4. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 3, is characterized in that described carbon source is glucose.
5. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 2, is characterized in that described nitrogenous source is selected from peptone, yeast powder, extractum carnis, ammonium sulfate, SODIUMNITRATE, the one in urea.
6. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 5, is characterized in that described nitrogenous source is yeast powder.
7. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 2, is characterized in that in step 1) in, the condition of described cultivation is 30 DEG C, and 200rpm cultivates 30h.
8. the production method of a kind of ι-carrageenan hydrolase as claimed in claim 2, is characterized in that in step 2) in, the condition that described shaking table is cultivated is 30 DEG C, and 30h cultivated by 200rpm shaking table.
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| CN1544623A (en) * | 2003-11-25 | 2004-11-10 | 中国海洋大学 | kappa- carrageenin catabolic enzymes , its preparing process and application |
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| CN1544623A (en) * | 2003-11-25 | 2004-11-10 | 中国海洋大学 | kappa- carrageenin catabolic enzymes , its preparing process and application |
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