CN103898012B - One strain sea is revolved bacterium and is prepared the method for agarase - Google Patents
One strain sea is revolved bacterium and is prepared the method for agarase Download PDFInfo
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Abstract
The present invention relates to a strain sea revolve bacterium (<i>thalassospira</i>sp.) fjfst-2013007, is being deposited in China typical culture collection center registration preservation on December 25th, 2013, does is its deposit number? CCTCC? M? 2013708, and the method for agarase is prepared with this strain fermentation. The present invention, using this bacterial strain as bacterial classification, adopts the fermention medium of carbon source, nitrogenous source, inorganic salt composition, and under culture condition after optimization, the vigor of agarase reaches 11.2U/ml. Fermented liquid is saltoutd by ammonium sulfate, centrifugal collecting precipitation, after dialyse, get dialyzate and cross ion exchange column and gel column, lyophilize can obtain agarase dry powder.
Description
Technical field
The invention belongs to technical field of food biotechnology, it relates to screen a strain sea and revolve bacterium (Thalassospirasp.) and cultivation and fermentation prepares the method for agarase.
Background technology
Agar-agar (agar), having another name called agar, be a kind of Sargassum polysaccharides obtained through hot water extraction in marine red alga, its resource is enriched, it is widely used in food, medicine, biotechnology field, agar-agar forms primarily of the agarose (agarose) of neutrality and the agaropectin (agar) of ion, agarose is by 1, the straight-chain polymer that the 3 ��-D-semi-lactosis connected and the 1,4 3,6-inner ether-��-L-galactose residues connected alternately connect repeatedly.
Agarase wide material sources, are mainly present in marine bacteria: alternately zygosaccharomyces, and phagocyte bacterium belongs to, Rhodopseudomonas, and pseudoalteromonas belongs to, streptomyces, and micro-bacterium that quivers belongs to, Vibrio. All being found in the animal taking marine alga as food belongs to, as sea hare belongs to, shore spiral shell belongs to, hat sea Zhan's genus etc.
Agaropectin oligose just refers to the oligose that agar polysaccharide is 2-10 through hydrolysis post polymerization degree (DP), also known as agar oligosaccharide, repeating unit primarily of fine jade disaccharides is formed by connecting, good water solubility, be conducive to absorption of human body, substantially increase its using value, it it is a kind of novel oceanic functional oligose, agaropectin oligose has a wide range of applications at cosmetic field, mainly stems from its skin care moisturizing, delays senility, the function of whitening and the effect that can prevent skin infections and allergic dermatitis that has as cosmetics additive. Simultaneously owing to it is similar to biological endogenous property sugar structure, therefore to organism toxicological harmless, the research of agaropectin oligose function is growing. And in foodstuff production, because the not digested enzyme of agaropectin oligose decomposes, and empty calory produces, the production for beverage, bread and low calorific food of the weighting agent of high sweeting agent and dispersion agent can be done; In addition, agaropectin oligose has stronger bacteriostatic action, is a kind of well natural antiseptic agent and antioxidant.
Fujian Province has wide marine site, has deepened the exploitation in oceanic resources in recent years. Marine algae resource abundant in ocean is that Sargassum polysaccharides provides sufficient source. The production of agar simultaneously depends on the industrialized utilization to marine algas such as gelidium, Pterocladia tenuis Okam., fragrant plant mentioned in ancient texts, Eucheuma muricatum (Gmel.) Web�� Van Bos��, lavers, and the production for agar is the Preliminary Exploitation to these marine algae resources, the product that its follow-up deep processing obtains commercially has very wide application prospect.If being processed further by agar, the agaropectin oligose obtained, is applied in the industries such as medicine, protective foods and makeup, and it is worth considerably beyond agar. Therefore making full use of resource is not only in the further intensive processing of marine alga, and has deepened the abundant understanding to oceanic resources and research, is all very important and necessary for production application and scientific research. Although Fujian Province's marine algae resource is quite abundant, but the green production of agaropectin oligose depends on the application of agarase. Owing to the source of agarase is relatively less, active low, expensive, production application has certain restriction. Microbe-derived agarase can be produced in enormous quantities, but generally screens the wild strain obtained, and its inulinase-producing activity often can not be very high. Therefore, it is to increase the product enzyme activity of bacterial strain, obtain the agarase that purity is relatively high, employing enzymolysis process is produced agaropectin oligose there is important meaning. Using microbe mutation breeding technologies improves the product enzyme activity of bacterial strain, and the agarase that fermentation obtains is carried out separation and purification, and the vigor of agarase and purity are improved all greatly, is conducive to agarase and product thereof in the application of industry and field of scientific study.
Agarase is mainly derived from sea mollusk and marine microorganism. the bacterium decomposing agar-agar is mainly present in ocean environment. since Gran first time in 1902 is separated to agarolytic bacteria one agar-agar pseudomonas (Psudomonasgalatica) from seawater, people have been separated to multiple agar-agar decomposer from sea water system. such as (Cytophaga (phagocyte bacterium genus) (DuckworthMetal., 1968), Vibrio (Vibrio) (Aokietal., 1990), Streptomyces (streptomyces) (BibbMJetal., 1987), Pseudomonas-like (class Rhodopseudomonas) (VonHofstenBetal., 1975), Alterococcus (alternately Coccus) (ShiehWYetal., 1998) Pseudomonas (Rhodopseudomonas) (BelasRetal., 1989) Pseudomanaswellantypicum (Jing Shi pseudomonas) (Wang Xuanliang, 1985), AltermonasagarlyticsGJ1B(pseudoalteromonas) (Potinetal., 1993), Thalassomonassp.JAMB-A33(ocean is addicted to cold Zymomonas mobilis) (Ohtaetal., 2005). therefore the new task that new microbial method is prepared agarase and is called investigator is screened.
Summary of the invention
It is an object of the invention to provide a strain sea and revolve algae, and a kind of method utilizing this bacterial strain to prepare agarase.
The technical scheme of the present invention: the seawater of a strain screening from offshore sea waters, Xiamen takes the bacterium in sample liquid, may be used for preparing agarase (agarase), bacterium (Thalassospirasp.) fjfst-2013007 is revolved in its classification called after sea, it is being deposited in China typical culture collection center registration preservation on December 25th, 2013, its deposit number is CCTCCM2013708, and preservation address is Wuhan University.
The selection systems of microorganism strains:
The present invention takes sample liquid from the seawater of offshore sea waters, Xiamen, coat in agar screening culture medium after being inoculated in enrichment medium to carry out illumination cultivation, at 25 DEG C, cultivate the bacterium colony that 48h selects significant depressions to enter agar screening culture medium carries out line separation and purification, purebred receive fermention medium 25 DEG C, 120rpm, shake-flask culture 24-72h, agarase vigor in detection substratum, finishing screen selects aimed strain, it is carried out physio-biochemical characteristics qualification and molecular biology experiment guide carries out 16srDNA sequential analysis, determine that it belongs to sea and revolves bacterium (Thalassospirasp.), intend called after sea and revolve bacterium (Thalassospirasp.) fjfst-2013007.
The method revolving bacterium (Thalassospirasp.) fjfst-2013007 production agarase with described sea comprises the steps:
(1) sea is revolved bacterium (Thalassospirasp.) fjfst-2013007 in seed culture medium, with the bottled 20-60mL seed culture medium of 250mL taper, sterilizing according to a conventional method, cooling, and inoculate bacterium colony, after 12-24h cultivated by 20-30 DEG C of 160rpm shaking table, obtain seed fermentation liquid after inoculation;
(2) with the bottled 30mL fermention medium of 250mL taper, sterilizing according to a conventional method, cooling, and press the inoculum size of 6%-10%, seed fermentation liquid is accessed fermention medium, and in 25-30 DEG C after inoculation, 160rpm shaking table obtains the fermented liquid containing agarase after cultivating 48-72h;
(3) then by fermention medium through 4 DEG C, after 6000-8000g frozen centrifugation 10-20min, get supernatant liquor;
(4) supernatant liquor adding the saturated ammonium sulphate that massfraction is 60%-80% to saltout, place refrigerator overnight, 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
(5) balance DEAE-SepharoseFastFlow post (�� 2cm �� 10cm) by buffer A, cross 4-5 bed volume. Taking 0.3g agarase powder is dissolved in 8mL buffer A, gets ion exchange column on supernatant liquor after the centrifugal 20min of 6000g. Cross 2-3 bed volume by buffer A after loading and wash the foreign protein not adsorbed, then carry out gradient elution in order to the NaCl solution of the 0-0.5mol/L of buffer A preparation. Flow rate control is at 0.5mL/min, and determined wavelength is 280nm. Elutriant fraction collection, 4.5mL/ manages. Measure the enzyme activity of solution in the test tube of wash-out pipe peak part, active part will be had to merge, after lyophilize, obtain enzyme work in-process;
(6), after SephcrylS-100 gel permeation chromatography post (�� 1.6cm �� 80cm) being balanced by buffer A, take work in-process agarase 0.05g and it is dissolved in 4mL level pad, after 6000g low-temperature centrifugation 20min, get chromatography column on supernatant liquor. Carrying out wash-out by buffer A, coutroi velocity is 0.1mL/min. Elutriant through ultraviolet detection and fraction collection (4.0mL/ pipe). Determine the enzyme activity of peak part test tube respectively, by there being active part to merge, concentrate through ultra-filtration membrane after enough hemodialysis desalination, to concentrated solution lyophilize, obtain finished product agarase.
Described fermention medium is: sea salt 20-30g/L, peptone 3-7g/L, yeast powder 0.5-2.5g/L, agar 1.5-2.5g/L, and adjustment pH is 7.2-8.4, and deionized water is prepared; Described seed culture medium: agar 2g/L, sea salt 30g/L, peptone 5g, yeast powder 1g, PH are 8.0, and deionized water is prepared.
Described buffer A is the Tris-HCl damping fluid of 0.02mol/L, pH7.5.
The detection method of agarase produced by the method:
Get above-mentioned steps (3) and obtain the fermented liquid supernatant liquid 1mL containing agarase, add the PBS (wherein containing agar-agar 0.2%) of 1mL, be placed in 40 DEG C of water-bath enzymolysis 30min, after add 1.5mLDNS, boiling water bath 15min. After being cooled to room temperature, mend neat scale with distilled water, it may also be useful to spectrophotometer detects under 520nm. 1U is defined as the value that 1min produces 1ug agaropectin oligose.
The useful effect of the present invention: filter out a kind of bacterial strain producing agarase for microbial method, bacterium (Thalassospirasp.) fjfst-2013007 is revolved in classification called after sea. The present invention using this bacterium as bacterial classification, domestic yet there are no with this bacterial classification produce agarase report. This bacterium is with common carbon source, and nitrogenous source, the fermention medium of inorganic salt composition, with short production cycle, 10h can reach logarithmic phase, this bacterial classification energy abduction delivering agarase, it is possible to reach maximum enzyme output in 24h.Can be collected in crude enzyme liquid by simple separation purification method and obtain active high agarase. Agarase used in the present invention preparation and purifying process are simple, with low cost, and it is short to produce the enzyme time, is suitable for industrial production or field of medicaments, the fields such as food chemical industry and mariculture.
Embodiment
The following is explanation specific embodiments of the invention, but the present invention is not limited to this.
Embodiment 1
The seawater getting offshore sea waters, Xiamen takes sample liquid, draws 1ml and carries out diluting (10-1,10-2,10-3,10-4), draw 0.5ml and coat in agar screening culture medium, at 25 DEG C, cultivate the bacterium colony that 48h selects significant depressions and enter agar screening culture medium carries out line separation and purification, receive liquid nutrient medium 25 DEG C by purebred, 100rpm, shake-flask culture 48h, centrifuging and taking supernatant, adopts the agarase in spectrophotometry substratum active, through just sieve, it is divided into from the bacterium obtaining the 3 strains long agarases of energy, in conjunction with the throughput of agarase, final selected as starting strain.
Embodiment 2
Through morphology and physio-biochemical characteristics research, the feature producing agarase bacterial strain is as follows:
Colonial morphology: bacterium colony presents circle, smooth surface, oyster white, edge has neatly.
Cellular form: this is Gram-negative bacteria, form is like vibrios shape.
Physiological and biochemical property: for strictly aerobic, starch can be utilized, glucose, sucrose, sodium-acetate, the multiple compounds such as D-semi-lactosi are as carbon source, it is also possible to utilize peptone, yeast extract paste, ammonium nitrate, ammonium sulfate, wait multiple compound as nitrogenous source.
The 16SrRNA gene of bacterial strain is carried out pcr amplification and sequencing, the Gene Partial fragment length finding its 16SrRNA is 1487bp, compare through NCBI and rrna database, it is accredited as sea and revolves bacterium (Thalassospirasp.), intending called after sea and revolve bacterium (Thalassospirasp.) fjfst-2013007, the 16SrRNA sequence of bacterial strain is visible sequence table specifically.
Embodiment 3
(1) sea is revolved bacterium (Thalassospirasp.) fjfst-2013007 in seed culture medium, with the bottled 30mL seed culture medium of 250mL taper, sterilizing according to a conventional method, cooling, and inoculate bacterium colony, in 25 DEG C after inoculation, 140rpm shaking table obtains seed fermentation liquid after cultivating 24h, described seed culture medium is: sea salt 25g/L, peptone 5g/L, yeast powder 1g/L, agar 2g/L, regulating pH to be 7.4, deionized water is prepared.
(2) with the bottled 30mL fermention medium of 250mL taper, sterilizing according to a conventional method, cooling, and the inoculum size by 4%, seed fermentation liquid is accessed fermention medium, and in 25 DEG C after inoculation, 140rpm shaking table obtains the fermented liquid containing agarase after cultivating 48h, described seed culture medium is: sea salt 25g/L, peptone 5g/L, yeast powder 1g/L, agar 2g/L, regulating pH to be 7.4, deionized water is prepared.
The vigor of the agarase of this embodiment gained is 1.71U/mL
Embodiment 4
(1) sea is revolved bacterium (Thalassospirasp) fjfst-2013007 in seed culture medium, with the bottled 30mL seed culture medium of 250mL taper, sterilizing according to a conventional method, cooling, and inoculate bacterium colony, in 25 DEG C after inoculation, 160rpm shaking table obtains seed fermentation liquid after cultivating 24h, described seed culture medium: agar 2g/L, sea salt 30g/L, peptone 5g, yeast powder 1g, PH is 8.0, and deionized water is prepared.
(2) with the bottled 30mL fermention medium of 250mL taper, sterilizing according to a conventional method, cooling, and the inoculum size by 6%, seed fermentation liquid is accessed fermention medium, and in 25 DEG C after inoculation, 160rpm shaking table obtains the fermented liquid containing agarase after cultivating 72h, described fermention medium is: sea salt 25g/L, peptone 5g/L, yeast powder 1g/L, agar 2.5g/L, regulating pH to be 7.5, deionized water is prepared;.
The vigor of the agarase of this embodiment gained is 11.2U/mL
Embodiment 5
(1) by above-mentioned fermention medium through 4 DEG C, after 8000g frozen centrifugation 15min, supernatant liquor is got.
(2) supernatant liquor is added massfraction be 80% saturated ammonium sulphate saltout, place refrigerator overnight, 4 DEG C, 10000g is centrifugal, and 30min gets precipitation.
(3) balance DEAE-SepharoseFastFlow post (�� 2cm �� 10cm) by buffer A (the Tris-HCl damping fluid of 0.02mol/L, pH7.5), cross 4-5 bed volume. Taking 0.3g agarase powder is dissolved in 8mL buffer A, gets ion exchange column on supernatant liquor after the centrifugal 20min of 6000g. Cross 2-3 bed volume by buffer A after loading and wash the foreign protein not adsorbed, then carry out gradient elution in order to the NaCl solution of the 0-0.5mol/L of buffer A preparation. Flow rate control is at 0.5mL/min, and determined wavelength is 280nm, elutriant fraction collection, and 4.5mL/ manages. Measure the enzyme activity of solution in the test tube of wash-out pipe peak part, active part will be had to merge, after lyophilize, obtain enzyme work in-process.
(4), after SephcrylS-100 gel permeation chromatography post (�� 1.6cm �� 80cm) being balanced by buffer A, take work in-process agarase 0.05g and it is dissolved in 4mL level pad, after 6000g low-temperature centrifugation 20min, get chromatography column on supernatant liquor. Carrying out wash-out by buffer A, coutroi velocity is 0.1mL/min. Elutriant through ultraviolet detection and fraction collection (4.0mL/ pipe). Determine the enzyme activity of peak part test tube respectively, by there being active part to merge, concentrate through ultra-filtration membrane after enough hemodialysis desalination, to concentrated solution lyophilize, obtain finished product agarase.
SEQUENCELISTING
<110>University Of Agriculture and Forestry In Fujian
<120>one strain seas are revolved bacterium and are prepared the method for agarase
<130>1
<160>1
<170>PatentInversion3.3
<210>1
<211>1487
<212>DNA
<213>16SrRNA
<400>1
cagagtttgatcctggctcagaacgaacgctggcggcaggcctaacacatgcaagtcgga60
cgagaaggttccttcgggaactggagagtggcgcacgggtgagtaacgcgtggggaccta120
cctcttagtgggggataacggttggaaacgaccgctaataccgcatacgcccttcggggg180
aaagatttatcgctaagagatggacccgcgttggattagatagttggtgaggtaacggct240
caccaagtcagcgatccatagctggtttgagaggatgatcagccacactgggactgagac300
acggcccagactcctacgggaggcagcagtggggaatattggacaatgggggcaaccctg360
atccagccatgccgcgtgagtgaagaaggccttcgggttgtaaagctctttcagatgcga420
agatgatgacggtaacatcagaagaagccccggctaatttcgtgccagcagccgcggtaa480
tacgaaaggggctagcgttgttcggatttactgggcgtaaagggcacgcaggcggtcttg540
ccagtcaggggtgaaagcccggggctcaaccccggaactgcctctgatactgcaagacta600
gagactaggagagggtggtggaattcccagtgtagaggtgaaattcgtagatattgggag660
gaacaccagaggcgaaggcggccacctggactagatctgacgctcaggtgcgaaagcgtg720
gggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctagttgt780
cgggacttcggtttcggtgacgcagctaacgcattaagcactccgcctggggagtacggt840
cgcaagattaaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtt900
taattcgaagcaacgcgcagaaccttaccaacccttgacatccctatcgcgattaccaga960
gatggttttcatcagttcggctggataggtgacaggtgctgcatggctgtcgtcagctcg1020
tgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccctgttcccagttgccagc1080
atttagttgggcactctggggagactgccggtgacaagccggaggaaggcggggatgacg1140
tcaagtcctcatggcccttacgggttgggctacacacgtgctacaatggtaactacagag1200
ggcagcgacttagcgataaggagccaatcccaaaaagttatctcagttcggattgcactc1260
tgcaactcgagtgcatgaagttggaatcgctagtaatcgtggatcagcatgccacggtga1320
atacgttcccgggccttatacacaccgcccgtcacaccatgggagttggttttacccgaa1380
gacggtgggctaaccttttaggaggcagccggccacggtaaggtcagcgactggggtgaa1440
gtcgtaacaaggtagccgtaggggaacctgcggctggatcacctcct1487
Claims (6)
1. bacterium is revolved in a strain sea, it is characterized in that: it is that bacterium (Thalassospirasp.) fjfst-2013007 is revolved in sea that bacterium is revolved in described sea, being deposited in China typical culture collection center registration preservation on December 25th, 2013, its deposit number is CCTCCM2013708.
2. bacterium is revolved in a strain sea according to claim 1, it is characterised in that: the sequence of the 16SrRNA of bacterium (Thalassospirasp.) fjfst-2013007 is revolved as shown in SEQIDNO.1 in described sea.
3. the purposes of bacterium is revolved in a sea as described in claim 1, it is characterised in that: for the preparation of agarase.
4. the method for bacterium for the preparation of agarase is revolved in a sea as described in claim 1, it is characterised in that: described method comprises the steps:
(1) with the bottled 20-60mL seed culture medium of 250mL taper, sterilizing according to a conventional method, cooling, and inoculate bacterium colony, in 20-30 DEG C after inoculation, 160rpm shaking table obtains seed fermentation liquid after cultivating 12-24h;
(2) with the bottled 20-60mL fermention medium of 250mL taper, sterilizing according to a conventional method, cooling, and press the inoculum size of 2%-10%, seed fermentation liquid is accessed fermention medium, after 20-30 DEG C of cultivation 24-60h, obtains the fermented liquid containing agarase;
(3) then by containing the fermented liquid of agarase through 4 DEG C, after 8000-10000g frozen centrifugation 10-20min, get supernatant liquor;
(4) supernatant liquor adding the saturated ammonium sulphate that massfraction is 60%-80% to saltout, place refrigerator overnight, 4 DEG C, 8000-10000g is centrifugal, and 20-40min gets precipitation;
(5) DEAE-SepharoseFastFlow post is balanced by buffer A, cross 4-5 bed volume, taking 0.3g agarase powder is dissolved in 8mL buffer A, ion exchange column on supernatant liquor is got after the centrifugal 20min of 6000g, cross 2-3 bed volume by buffer A after loading and wash the foreign protein not adsorbed, then carry out gradient elution in order to the NaCl solution of the 0-0.5mol/L of buffer A preparation;Flow rate control is at 0.5mL/min, and determined wavelength is 280nm, elutriant fraction collection, and 4.5mL/ manages, and measures the enzyme activity of solution in the test tube of wash-out pipe peak part, and active part will be had to merge, and obtains enzyme work in-process after lyophilize;
(6) after SephcrylS-100 gel permeation chromatography post being balanced by buffer A, taking work in-process agarase 0.05g is dissolved in 4mL level pad, chromatography column on supernatant liquor is got after 6000g low-temperature centrifugation 20min, wash-out is carried out by buffer A, coutroi velocity is 0.1mL/min, elutriant through ultraviolet detection and fraction collection, 4.0mL/ pipe, determine the enzyme activity of peak part test tube respectively, merge there being active part, concentrate through ultra-filtration membrane after enough hemodialysis desalination, to concentrated solution lyophilize, obtain finished product agarase.
5. according to method described in claim 4, it is characterised in that: described fermention medium is: sea salt 20-30g/L, peptone 3-7g/L, yeast powder 0.5-2.5g/L, agar 1.5-2.5g/L, and adjustment pH is 7.2-8.4, and deionized water is prepared; Described seed culture medium: agar 2g/L, sea salt 30g/L, peptone 5g/L, yeast powder 1g/L, PH are 8.0, and deionized water is prepared.
6. method according to claim 4, it is characterised in that: described buffer A is the Tris-HCl damping fluid of 0.02mol/L, pH7.5.
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| CN105349461B (en) * | 2015-11-26 | 2019-03-08 | 福建农林大学 | One plant of production agarase vibrio alginolyticus and application |
| CN106754538B (en) * | 2017-01-03 | 2019-12-20 | 青岛双瑞海洋环境工程股份有限公司 | Petroleum degrading bacterium Oil2-3, and separation and purification method and application of petroleum degrading bacterium Oil2-3 |
| CN111826297A (en) * | 2019-04-18 | 2020-10-27 | 新乡医学院 | Breeding method of agarase-producing strain |
| CN113817710B (en) * | 2021-11-09 | 2023-11-24 | 蓝脑科技(厦门)有限公司 | Agarase freeze-drying protective agent and agarase preservation method |
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| CN101691556B (en) * | 2009-09-25 | 2012-06-27 | 中国科学院南海海洋研究所 | Deep sea thalassospira and application thereof |
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