CN104726445A - Culture method for improving yield of botryococcus polysaccharides - Google Patents
Culture method for improving yield of botryococcus polysaccharides Download PDFInfo
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Abstract
The invention discloses a culture method for improving the yield of botryococcus polysaccharides, comprising the following steps: culturing purified botryococcus to a logarithmic growth period, selecting botryococcus liquid in logarithmic growth, performing induced mutation under the irradiation of argon ion laser, then inoculating to a BG11 liquid medium, and culturing under the conditions that the temperature is 28-33 DEG C, the illumination intensity is 43-86mumol.m<-2>.s<-1> and the illumination period is 14 hours every day; performing ultrasonic radiation treatment after culturing 2-3 days, continuously culturing the botryococcus subjected to the ultrasonic radiation treatment for 12-13 days under the conditions that the culture temperature is 28-33 DEG C, the illumination intensity is 43-86mumol.m<-2>.s<-1> and the illumination period is 14 hours every day, and performing centrifugal separation and sterile water washing on the obtained culture solution to obtain botryococcus rich in polysaccharides. By combining the argon ion laser induced mutation with the ultrasonic treatment, the yield of the cultured botryococcus is high, many polysaccharides are accumulated, and the polysaccharide content of the botryococcus can reach 35.99% of the dry weight.
Description
(1) technical field
The present invention relates to a kind of cultural method with the Wild Vitis species of the high polysaccharide of anti-microbial effect.
(2) background technology
The environmental safety that the excessive exploitation of fishery and the fast development of aquaculture bring, aquaculture security problems are more and more outstanding, and the drawback such as bacterial drug resistance increase, drug residue, environmental pollution that long-term Drug abuse produces becomes the outstanding problem in food safety day by day.Meanwhile, along with the development of batch production high-density breeding, throw in artificial diet in a large number, make in water body, to dissolve a large amount of bait remnants and the movement of aquatic animal, cause serious aquaculture water to pollute, result in growing of cultivated animals and be subject to serious harm.Use feeding antibiotic substitute mainly probiotics or traditional plant medicament extract on the market at present, wherein plant amedica extract is by resource limit, and probiotics is worldwide popularized from the eighties in 20th century.
The research of China's feeding micro-ecological preparation starts from the eighties, in succession has the nineties product to occur, 2010 registered has feeding micro-ecological preparation hundreds of.Current China market supplies feeding micro-ecological preparation various in style, comprise single microbial inoculum and composite fungus agent, registered external imported product has tens kinds, and constantly has new product introduction.Current domestic microbiological fodder additives industry is still in the starting stage, be engaged in the enterprise of living microorganism fodder additives Application and Development at about 400, obtain about 140 that the Ministry of Agriculture produces certification, domestic microbiological fodder additives annual sales amount is greatly about 2,000,000,000 Renminbi, but sales volume more than 100,000,000 yuan less than 5, the annual sales amount of most enterprises is below 1,000 ten thousand yuan.According to current internal feed year production and pig, fowl, ruminate and calculate with the cultivation total amount of aquatic animal, the market capacity of domestic microbiological fodder additives is between 18,000,000,000 yuan to 20,000,000,000 yuan, existing market popularizes rate and is about 10%, the visible probiotics market space is very big, and prospect of industrial development is very wide.
On the other hand, the various microbiotic major parts of widespread use come from terrestrial actinomycetes and fungi, and long-term exploitation has made terrestrial Microbial resources increasingly exhausted.Take up an area the ocean of ball surface-area 71%, there is with natural condition such as the high pressure of its uniqueness, high salt, low nutrition, low light photographs the ability of producing special construction and functionally active material, more and more receive the concern of investigator.Except containing natural bacteriostatic material in marine microalgae, also containing large number of biological active substance, as active polysaccharide, unsaturated fatty acids, astaxanthin, phycobiliprotein, hidden algae element, ciguatoxin etc., be used as fodder additives, have stomach invigorating invigorating the spleen concurrently, increase appetite, extra-nutrition, growth promoting effects and the effect such as to improve food conversion ratio, some is not second to microbiotic.And there is the advantages such as noresidue, not easily generation resistance, be a large focus of international animal Nutritional studies in recent years.
Due in recent years to the attention of healthy aquaculture, micro-algae is used more and more to be familiar with by people the advantage that healthy aquaculture brings.There is the material of strengthening immunity and anti-microbial activity in micro-algae, fish intestinal environment can be improved, strengthen the resistance against diseases of fish body.Especially microalgae cell wall contain acidic polysaccharose body can cause produce a large amount of Interferon, rabbit (Interferon), Interferon, rabbit can increase phagocytic cell in body, gulps down external bacterium, mould or other virus etc., has again anticancer effect.Anti-microbial effect about microalgal polysaccharide has more bibliographical information, as leaf brocade woodss in 2004 etc. have studied Porphyridium cruentum extracting solution and polysaccharide soln pathogenic bacteria, fungi performance, shows both comparatively obvious to the bacteriostatic action of gram positive bacterium.Yin Hong duckweeds in 2006 etc. are to mouse peritoneal infectable infection streptococcus aureus, again to its administration of salt polysaccharides, found that the polysaccharide from Dunaliella salina of 100mg/kg and 200mg/kg dosage group significantly can increase survival number in infecting mouse 24h, show certain antibacterial ability.The broken alkaline extraction of the using ultrasounds such as Xu Tao an ancient unit of weight in 2010 prepares autotrophy chlorella polysaccharide, culture condition in vitro, with the chlorella polysaccharide process liver cancer cell of different concns, and detected by methods such as MTT, bisbenzimidazole (Hoechst33258) dyeing, immunocytochemistries.Result shows, chlorella polysaccharide effect Hepatocellular carcinoma cell line 1 2h of 1g/L, has remarkable restraining effect to its propagation, and induces it that apoptosis occurs by the expression of lowering inhibitor of apoptosis protein Bcl-2 and upregulation of apoptosis execution PROTEIN C apase-3.It is research object that Liu Si light in 2012 etc. choose autotrophy chlorella (Chlorella autotrophica), have chosen 4 kinds of bacterial strains common in ocean environment or medical science for as strain subject, carry out the experiment of chlorella polysaccharide PCA2-1 and PCA2-2 bacteriostatic activity respectively, wherein alginic acid vibrios (Vibrio alginolyticus) conciliate by intestinal bacteria microbiotic calibrating strain (Escherichia coli) is Gram-negative bacteria, staphylococcus epidermidis (Staphytococcus epidermidis) and micrococcus lysodeikticus (Micrococcus lysodeikticus) are gram-positive microorganism.Result shows, these 2 kinds of polysaccharide all go out certain restraining effect to certain specific strains expressed.Wherein, PCA2-2 conciliates alginic acid vibrios to micrococcus lysodeikticus stronger bacteriostatic action, and KI value can reach more than 70%.
Therefore, the outstanding problems such as bacterial drug resistance increase, drug residue are produced for fishing feeding antibiotic, the chlorella short period of time is utilized to accumulate the feature of biomass in a large number, optimize the high polysaccharide chlorella culture technique with anti-microbial activity, the biological action of its immunity moderation can be played, improve cultivated animals resistance against diseases, reduce the generation of disease.
(3) summary of the invention
The object of the invention is to provide one with Wild Grape algae for raw material, after removal living contaminants obtains pure strain, after comparing determine optimum medium and antibiotic concentration by screening, with total candy output and the speed of growth for index, in conjunction with argon laser and ultrasonic technique, the photosynthetic carbon fixation approach of this algae kind is optimized, and adopt Caenorhabditis elegans to verify produce the anti-microbial activity of polysaccharide, obtain the cultural method of the obvious high polysaccharide Wild Vitis species of antibacterial effect.
The technical solution used in the present invention is:
Improve a cultural method for Wild Vitis species polysaccharide yield, described method is:
The Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, to take the logarithm the algae liquid of growing up, adopt argon laser to irradiate after mutagenesis, inoculate in BG11 liquid nutrient medium, culture temperature 28 ~ 33 DEG C (preferably 28 DEG C), intensity of illumination 43 ~ 86 μm of olm
-2s
-1(preferably 65 μm of olm
-2s
-1), 14 hours every days of periodicity of illumination; Cultivate the Wild Vitis species after 2 ~ 3 days and carry out Ultrasonic Radiation process, the Wild Vitis species after Ultrasonic Radiation process continues at culture temperature 28 ~ 33 DEG C (preferably 28 DEG C), intensity of illumination 43 ~ 86 μm of olm
-2s
-1(preferably 65 μm of olm
-2s
-1), cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal is separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
The Wild Vitis species BG11 liquid nutrient medium of described purifying is cultured to the logarithm growth stage, and culture condition is culture temperature 28 ~ 33 DEG C (preferably 28 DEG C), intensity of illumination 43 ~ 86 μm of olm
-2s
-1(preferably 65 μm of olm
-2s
-1), 14 hours every days of periodicity of illumination.)
Described algae liquid irradiates after mutagenesis through argon laser, and be seeded in BG11 liquid nutrient medium, inoculum size is generally the 1/3-1/2 of nutrient solution volume.
The Wild Vitis species of described purifying is the Wild Vitis species list bacterium colony algae kind obtained by line partition method purification process by Wild Grape algae, generally can operate by the following method: abandon supernatant liquor by after Wild Grape algae algae liquid centrifugal concentrating, the sterilized water added containing 0.5wt% mycillin solution is resuspended, supernatant liquor is abandoned after recentrifuge, the algae mud obtained after washing 3 times as stated above carries out line and is separated: in Bechtop, algae mud is lined on BG11 solid medium, be inverted in illumination box and cultivate, culture temperature 28 DEG C, illumination 3000lx, 14 hours every days of periodicity of illumination, every 10 days is one-period, after cultivating one-period, picking list bacterium colony carries out second time line, the third generation is obtained after experiencing 2 line, the Wild Vitis species algae kind of purifying can be obtained.This well known to a person skilled in the art separation purification method.
Described mycillin solution is the mixing solutions of 100X, namely containing penicillin 10000U/ml, Streptomycin sulphate 10000 μ g/ml.
The formula of described BG11 solid medium is: in every 1000mL distilled water, containing 1.5gNaNO
3, 0.04g K
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036g CaCl
2.7H
2o, 0.02g Na
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution (comprises 2.86g H
3bO
4, 1.81g MnCl
2.4H
2o, 0.222g ZnSO
4, 0.39gNa
2moO
4, 0.079g CuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o), 20.0g agar, pH value 7.0, for subsequent use after pouring 6cm culture dish into.
The formula of BG11 liquid nutrient medium is: in every 1000mL distilled water, containing 1.5g NaNO
3, 0.04g K
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036g CaCl
2.7H
2o, 0.02gNa
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution (comprises 2.86g H
3bO
4, 1.81g MnCl
2.4H
2o, 0.222g ZnSO
4, 0.39g Na
2moO
4, 0.079g CuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o), pH value 7.0.
This is all the substratum that this area is commonly used.
When described argon laser irradiates mutagenesis, the preferred 90mw of power of argon laser, irradiation time 10 ~ 40 seconds, preferably 30 seconds.
When described argon laser irradiates mutagenesis, the distance of Wild Vitis species and light source is preferably 80cm.
During described Ultrasonic Radiation process, the preferred 20kHz of hyperacoustic frequency, power 4 ~ 8W, preferred 8W, 30 ~ 90 seconds treatment times, preferably 60 seconds.
Further, the method for the invention is preferably undertaken by following operation: the Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; The algae liquid of growth stage of taking the logarithm carries out argon laser and irradiates mutagenesis, the distance of Wild Vitis species and light source is made to be 80cm, by the argon laser radiation treatment 10 ~ 40 seconds of 90mw, then the Wild Vitis species after mutation induced by laser is inoculated and cultivate into BG11 liquid nutrient medium, culture temperature 28 ~ 33 DEG C (preferably 28 DEG C), intensity of illumination 43 ~ 86 μm of olm
-2s
-1(preferably 65 μm of olm
-2s
-1), 14 hours every days of periodicity of illumination; Cultivate the Ultrasonic Radiation process 30 ~ 90 seconds of the Wild Vitis species frequency 20kHz after 2 ~ 3 days, power 4 ~ 8W, the Wild Vitis species after Ultrasonic Radiation process continues at culture temperature 28 ~ 33 DEG C (preferably 28 DEG C), intensity of illumination 43 ~ 86 μm of olm
-2s
-1(preferably 65 μm of olm
-2s
-1), cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal is separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
Further, the method for the invention is most preferably undertaken by following operation: the Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; The algae liquid of growth stage of taking the logarithm carries out argon laser and irradiates mutagenesis, the distance of Wild Vitis species and light source is made to be 80cm, by the argon laser radiation treatment 30 seconds of 90mw, then the Wild Vitis species after mutation induced by laser is inoculated and cultivate into BG11 liquid nutrient medium, culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; Cultivate the Ultrasonic Radiation process 60 seconds of the Wild Vitis species frequency 20kHz after 2 ~ 3 days, power 8W, the Wild Vitis species after Ultrasonic Radiation process continues culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal be separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
Described argon laser irradiation mutagenesis more specifically operation steps is: on the label paper consistent with slide glass size dimension, get with punch tool multiple circular holes that diameter is 0.6cm, then be attached on clean slide, circular hole is numbered, and irradiate 20 ~ 30 minutes under slide glass being placed in ultraviolet lamp, obtain sterilizing slide glass, the Wild Vitis species algae liquid of logarithm growth stage, be applied in the circular hole on sterilizing slide glass with the dose uniformity in 0.05ml/ hole, then sterilizing slide glass is fixed on the 80cm place of the light source of the argon laser of distance 90mw, make slide glass vertical with light source direction, the circular hole front of coating algae liquid is just to hot spot, argon laser is made to irradiate circular hole on slide glass successively each 30 seconds, obtain the Wild Vitis species after mutation induced by laser.
The present invention take Wild Vitis species as raw material, adopts argon laser and supersonic treatment successively, is cultivating in optimum medium, illumination temperature and intensity of illumination to Wild Vitis species, obtains the Wild Vitis species cultural method of the obvious high polysaccharide of antibacterial effect.Wild Vitis species itself has certain polysaccharide content (about 17%), technical essential of the present invention is that argon laser is in conjunction with supersonic treatment, and under the suitableeest illumination temperature and intensity of illumination, Wild Vitis species is processed, to accelerate the metabolic processes of Wild Vitis species cell, improve the Enzyme activity of Wild Vitis species glucose phosphate isomerase and hexokinase, thus the accumulation of Promote cell's growth and raising polysaccharide.
The present invention adopts BG11 substratum, and the basic medium used with SE substratum and factory large scale culturing is compared, and its Wild Vitis species dry weight reached the highest 15 days time.Use BG11 substratum, without any under microbiotic existent condition, the biomass of Wild Vitis species reaches the highest.
The growth conditions of Wild Vitis species the best is temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, the biomass of Wild Vitis species is the highest; The product sugar condition of Wild Vitis species the best is temperature 33 DEG C, intensity of illumination 65 μm of olm
-2s
-1.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
(1) for the feature of Wild Vitis species nutritive ingredient, first by a kind of bio-sensing light source--the method for argon laser mutagenesis, accelerate the metabolic processes of Wild Vitis species cell, the cavatition that recycling ultrasonic wave produces, mechanical effect improve the activity of synthesizing relevant enzyme in grape gonidium with polysaccharide, and under the culture condition of effective photosynthetic carbon fixation, the accumulation of Promote cell's growth and raising polysaccharide.The present invention adopts argon laser, belongs to Ultra-Violet Laser, and wavelength can at below 360nm, and power can reach more than 90mw, easily makes cell produce mutagenesis.Compared with He-Ne laser, He-Ne laser belongs to visible laser, and wavelength is at about 630nm, and power is generally no more than 1mw, and time of its process material require is longer, short then tens minutes, long then several hours.And argon laser mutation time is very short, just can complete in the several seconds, efficiency of inducing mutation and effectiveness comparison high.Wild Vitis species is after above-mentioned pre-treatment, and polysaccharide yield increases, and Caenorhabditis elegans streptococcus aureus and colibacillary fungistatic effect is all better than to the microbiotic of 0.5%.And whole mutagenesis culture process process is simple physical method, has the advantages such as energy consumption is low, efficiency is high, effective constituent polysaccharide accumulation is many, the Wild Vitis species good anti-bacterial effect of acquisition.
(2) it is high that the present invention obtains Wild Vitis species output, polysaccharide accumulation is many, its polysaccharide content can up to 35.99% of dry weight, improve 2.14 times than polysaccharide before mutagenesis, improve 1.39 times than the polysaccharide only adopting biochemical culture condition optimizing to obtain, the polysaccharide obtained than argon laser irradiation biochemical culture condition optimizing improves 1.32 times, improves 1.23 times than supersound process in conjunction with the polysaccharide that biochemical culture condition optimizing obtains; Processing condition are gentle, and environmental protection, meets the production requirement of heath food.
(3) the high yield Wild Vitis species that the present invention obtains extracts the polysaccharide obtained and has good antimicrobial property, the In vivo antibacterial experiment of Caenorhabditis elegans is adopted in embodiment, the fungistatic effect of Wild Vitis species polysaccharide to beautiful hidden bar line Salmonellas of 5mg/ml is better than the microbiotic of same concentrations (5mg/ml), and the fungistatic effect of the Wild Vitis species polysaccharide of 2.5mg/ml to beautiful hidden bar line streptococcus aureus is better than the microbiotic that concentration is 5mg/ml.
(4) accompanying drawing explanation
Fig. 1 Wild Vitis species purifying cultivates the s-generation, the third generation and unpurified Wild Vitis species photo; In figure, 1 is the Wild Vitis species be contaminated by bacterial before separation and purification, and 2 is by the Wild Vitis species of fungal contamination before separation and purification; 3 is s-generation Wild Vitis species after once line is separated, and 4 to rule the Wild Vitis species of the separation and purification third generation for secondary.
Fig. 2 Wild Vitis species polysaccharide is to the fungistatic effect graphic representation of Caenorhabditis elegans Salmonellas, and in Fig. 2, K represents negative control group, 100% represents concentration 5mg/ml group, 50% represents 2.5mgml group, and 10% represents 0.5mg/ml group, and 0.5% anti-ly represents 0.5% mycillin group.。
Fig. 3 Wild Vitis species polysaccharide is to the fungistatic effect graphic representation of Caenorhabditis elegans streptococcus aureus, and in Fig. 3, K represents negative control group, 100% represents concentration 5mg/ml group, 50% represents 2.5mgml group, and 10% represents 0.5mg/ml group, and 0.5% anti-ly represents 0.5% mycillin group.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) the Wild Vitis species culture process research of high polysaccharide
1 materials and methods
1.1 experiment material
Wild Vitis species is provided by Hangzhou Hua Dan agricultural-food company limited;
Mycillin solution: containing penicillin 10000U/ml, Streptomycin sulphate 10000U/ml;
VS-840K-U super clean bench, SuZhou Antai Air Tech Co., Ltd.;
722S visible spectrophotometer, Prism Optical Technology Co;
TGL-16G table model high speed centrifuge, Hunan Xingke Scientific Instrument Co., Ltd.;
ZHWY-2102C constant-temperature table incubator, Shanghai Zhi Cheng analytical instrument Manufacturing Co., Ltd;
HH-2 digital display thermostat water bath, Shanghai Chang Si Trade Co., Ltd.;
PGX multistage illumination box, Ningbo Lai Fu Science and Technology Ltd.;
Argon ion laser, U.S. Coherent Innova 308C;
CY-5D type ultrasound biological growth promoting effects instrument, Ningbo Xin Zhike device institute;
MINI-PAM chlorophyll fluorescence instrument, German WALZ company;
All chemical reagent are analytical pure.
1.2 experimental technique
1.2.1 the separation and purification of Wild Vitis species: it is BG11 solid medium [1.5g NaNO that Wild Grape algae is separated used medium
3, 0.04g K
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036gCaCl
2.7H
2o, 0.02g Na
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution (comprises 2.86g H
3bO
4, 1.81g MnCl
2.4H
2o, 0.222g ZnSO
4, 0.39g Na
2moO
4, 0.079g CuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o), 1000ml distilled water, 20.0g agar], pH value 7.0, for subsequent use after being poured into 6cm culture dish.
The formula of BG11 liquid nutrient medium is: in every 1000mL distilled water, containing 1.5g NaNO
3, 0.04g K
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036g CaCl
2.7H
2o, 0.02gNa
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution (comprises 2.86g H
3bO
4, 1.81g MnCl
2.4H
2o, 0.222g ZnSO
4, 0.39g Na
2moO
4, 0.079g CuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o), pH value 7.0.
The purifying of algae kind adopts partition method of repeatedly ruling: by 200ml Wild Grape algae algae liquid centrifugal concentrating (4000r/min, 10min) abandon supernatant liquor, add 20ml resuspended containing the sterilized water of 0.5% mycillin solution, abandon supernatant liquor after recentrifuge, the algae mud obtained after the same washing 3 times is in order to line.In Bechtop, lined on BG11 solid medium by algae mud, be inverted in and cultivate in illumination box (28 DEG C, 3000lx, periodicity of illumination 14h/d), 10d is one-period.After one-period, picking list bacterium colony carries out second time line, can obtain the Wild Vitis species list bacterium colony algae kind of purifying after experiencing for 3 generations.
1.2.2 Wild Vitis species Screening of Media: the three kinds of liquid nutrient mediums adopted in experiment are respectively the basic medium (NaNO that BG11 liquid nutrient medium, SE substratum and factory's large scale culturing use
337.5mg/L, K
2hPO
41mg/L, Citric acid 0.16mg/L, Na
2cO
30.5mg/L, Na
2eDTA0.026mg/L, MgSO
47H
2o 1.875mg/L, CaCl
22H2O 0.68mg/L, FeCl
30.14mg/L).Adjust three kinds of medium pHs 7.0 ± 0.5, be placed in aseptic working platform for subsequent use through high pressure steam sterilization.Get the algae kind of purifying in 1.2.1, in three kinds of liquid nutrient mediums, being cultured to logarithmic growth after date respectively, (culture temperature 28 DEG C, light intensity is 65 μm of olm
-2s
-1, periodicity of illumination is 14h/d, and rotating speed is 150r/min shaking table), be inoculated in three kinds of substratum more respectively, Wild Vitis species is inoculated in 500ml Erlenmeyer flask, adds three kinds of substratum respectively, obtains 200ml nutrient solution, inoculum size is the 1/3-1/2 of nutrient solution volume, in an experiment, because the dependency of absorbancy and biomass is fine, generally represent by absorbancy in experiment, adjustment inoculum size, makes the absorbance A under nutrient solution 667nm wavelength
667be 0.3, now inoculum size is probably 1/3 of nutrient solution volume, is placed in 28 DEG C, and light intensity is 65 μm of olm
-2s
-1, periodicity of illumination is 14h/d, and rotating speed is cultivate in 150r/min shaking table, and often kind of substratum experimental group all arranges 3 parallel group.Sampling and measuring algae liquid A every three days
667, utilize Wild Vitis species biomass typical curve to calculate algae liquid biomass (the algae liquid dry weight contained in often liter of nutrient solution, unit g/L).
SE substratum: NaNO
30.25g/L, K
2hPO
43H
2o 0.075g/L, MgSO
47H
2o0.075g/L, CaCl
22H
2o 0.025g/L, KH
2pO
40.175g/L, NaCl 0.025g/L, Soilextract 40mL, FeCl
36H
2o 0.005g/L, Fe-EDTA 1mL, A
5solution 1mL.[wherein, Soil Extract (soil extract) collocation method: get garden soil and do not execute overfertilization 0.5kg and be placed in beaker triangular flask, add distilled water 1000ml, bottleneck porous plug seals, in a water bath boiling water heating 2h, cooling number hour, aseptically filter, get supernatant liquor, sterile purified water added supernatant to cumulative volume 1000ml, soil extract be kept at 4 DEG C for subsequent use.A5 solution composition: containing 286mg H in 100ml distilled water
3bO
3, 181mg MnCl
24H
2o, 22mgZnSO
47H
2o, 7.9mg CuSO
45H
2o, 3.9mg (NH
4)
6mo
7o
244H
2o.The collocation method of Fe-EDTA: by Na-EDTA and FeCl
36H
2o is water-soluble and HCl (0.1mol/L) respectively, mixes.Composition is: 1g Na-EDTA, 50ml distilled water, 81mg FeCl
36H
2o, 50ml HCl (0.1mol/L).]
Wild Vitis species biomass typical curve obtains according to the following steps: the algae kind of getting purifying in 1.2.1 is inoculated into BG11 liquid nutrient medium, is placed in 28 DEG C, and light intensity is 65 μm of olm
-2s
-1periodicity of illumination is 14h/d, rotating speed is cultivate in 150r/min shaking table, timing sampling measures cell quantity, ordinate zou is done with the logarithm of cell number, X-coordinate is done with incubation time, draw Wild Vitis species growth curve, get adaptive phase, logarithmic phase, stationary phase about 333mL mixing separately (after mixing cumulative volume 1L) respectively, after mixing, algae liquid absorbancy is about 0.9, mixed algae liquid is concentrated or obtain five concentration gradient algae liquid (namely 2 times altogether with substratum dilution, 1 times, 1/2,1/4,1/8) A of different concns gradient algae liquid, is measured
667then by 1L algae liquid 8000r/min, 10min is centrifugal, collect algae mud after in 80 DEG C of baking ovens, be dried to constant weight after weigh, obtain algae liquid dry weight, draw the typical curve between dry weight/volume (unit g/L) (the algae liquid dry weight in 1L nutrient solution) and absorbancy, calculate regression equation, obtain Wild Vitis species biomass typical curve.The dependency of absorbancy and biomass is very good.
1.2.3 the screening of antibiotic concentration in Wild Vitis species substratum: use BG11 substratum in experiment, the volumetric concentration adjusting mycillin solution in substratum is respectively 0%, 0.1% and 0.3%, carry out inoculating, cultivating according to step 1.2.2, and tracking monitor Wild Vitis species biomass.(mycillin solution is the mixing solutions of 100X, namely containing penicillin 10000U/ml, Streptomycin sulphate 10000 μ g/ml.In substratum, the volumetric concentration of mycillin solution refers to mycillin solution concentration in the medium)
The screening that temperature and light when 1.2.4 cultivating is strong: select BG11 substratum, inoculate according to step 1.2.2, cultivates, and changes illumination level and culture temperature: arrange three kinds of illumination levels in experiment: low light intensity 43 μm of olm
-2s
-1, medium light intensity 65 μm of olm
-2s
-1with high light intensity 86 μm of olm
-2s
-1.Be placed in 28 DEG C and 33 DEG C of illumination constant-temperature tables are cultivated, periodicity of illumination is 14h/d, and rotating speed is that all the other conditions of 150r/min remain unchanged in batches.Every 2d sampling, survey its OD value under 667nm and total sugar content respectively, MINI-PAM chlorophyll fluorescence instrument is used when logarithmic phase, the maximum Photochemical quantum yield of its PS II (Fv/Fm) is surveyed after 15min dark adatpation, photochemistry fluorescent quenching coefficient (qP) and non-photochemistry fluorescent quenching coefficient (qN), calculate the possibility (1-qP/qN) of Wild Vitis species generation Xanthophyll cycle.
Wild Vitis species total sugar content measuring method adopts Phenol sulfuric acid procedure.
The dry weight of the total reducing sugar quality/Wild Vitis species of total sugar content=record
Data analysis adopts SPSS 19.0 software, and use one-way ANOVA, DUNCAN LSD carries out significant difference analysis.
1.2.5 argon laser mutagenesis: with punch tool with slide glass size dimension close to consistent label paper being got multiple circular holes that diameter is 0.6cm, then be attached on clean slide, after circular hole numbering, irradiate 20 minutes under slide glass being placed in ultraviolet lamp, obtain sterilizing slide glass.Get the Wild Vitis species BG11 culture medium culturing of step 1.2.1 purifying to the logarithm growth stage, culture temperature is 28 DEG C, and illumination is 65 μm of olm
-2s
-1, periodicity of illumination is 14h/d, and the algae liquid of growth stage of taking the logarithm, is applied to the dose uniformity in 0.05ml/ hole in the circular hole of sterilizing slide glass.Under the argon laser of 90mw, apart from light source 80cm place, fixing slide glass, makes it vertical with light source direction, and the circular hole front being coated with algae liquid, just to hot spot, makes argon laser irradiate circular hole on slide glass successively each 30 seconds, obtains the Wild Vitis species after mutation induced by laser.Then be seeded in BG11 liquid nutrient medium by the algae liquid in each aperture after mutation induced by laser in super clean bench, inoculum size makes the absorbance A of gained nutrient solution under 667nm wavelength
667be 0.3, culture temperature is 28 DEG C, and illumination is 65 μm of olm
-2s
-1, periodicity of illumination is cultivate after 3 days under 14h/d condition, adopts the ultrasonic wave of frequency 20kHz, power 8W to Wild Vitis species liquid radiation 60s, then continues to cultivate 12 days under these conditions, survey Wild Vitis species biomass and polysaccharide content.
2 experimental results
The separation and purification result of 2.1 Wild Vitis species
Through 2 times line purifying, single Wild Vitis species bacterium colony can be obtained, see accompanying drawing 1, by with non-purifying before Wild Vitis species contrast, separation and purification successful.In figure, 1 is the Wild Vitis species be contaminated by bacterial before separation and purification, and 2 is by the Wild Vitis species of fungal contamination before separation and purification; 3 is s-generation Wild Vitis species after once line is separated, and 4 to rule the Wild Vitis species of the separation and purification third generation for secondary.Fig. 1 can find out that the Wild Vitis species color after separation and purification is dark green, surface wettability, and algae falls single, can think the object having reached Wild Vitis species separation and purification.
2.2 Wild Vitis species Screening of Media results
1.2.2 experimental result show, Wild Vitis species dry weight in BG11 liquid nutrient medium reached the highest 15 days time, for 0.81g/L, and SE and the highest 0.70g/L and 0.48g/L that only can reach of basic medium, there is significant difference (P<0.01).Therefore the appropriateness that three kinds of substratum grow for Wild Vitis species is: BG11>SE> basic medium.Subsequent experimental all adopts BG11 liquid nutrient medium.
In 2.3 substratum, antibiotic concentration is on the impact of Wild Vitis species biomass
1.2.3 experimental result show, without any under microbiotic existent condition, the biomass of Wild Vitis species reaches the highest, for 0.81g/L, and the biomass that with the addition of 0.1% and 0.3% mycillin is only 0.44g/L and 0.37g/L, there is significant difference (P<0.01).Therefore in after this experiment, each group all not added with antibiotic.
2.4 culture temperature and light intensity are on the impact of Wild Vitis species biomass and total sugar content
The maximum Photochemical quantum yield of PS II (Fv/Fm), reflects light energy use efficiency in PS II reactive center, because it declines obviously under stress conditions, and can as reflection Wild Vitis species well-grown whether important indicator.1.2.4 experimental result as shown in table 1, result shows: the Wild Vitis species Fv/Fm under 28 DEG C of medium light intensity is 0.177, be greater than 0.169 under 0.104 and low light intensity under high light intensity, and the Wild Vitis species Fv/Fm under 33 DEG C of medium light intensity is 0.232, be greater than 0.215 under 0.172 and low light intensity under high light intensity equally, therefore under medium light intensity, Wild Vitis species has best growth conditions.Can find equally, when 28 DEG C, the possibility (1-qP/qN) that Wild Vitis species issues third contact of a total solar or lunar eclipse suppression in medium light intensity be 17.8%, be less than 58.4% under high light intensity, and when 33 DEG C, what Wild Vitis species issued that the third contact of a total solar or lunar eclipse suppresses in medium light intensity may (1-qP/qN) be 41.9%, is less than 71.2% under high light intensity.Therefore, Wild Vitis species has less Xanthophyll cycle possibility under medium light intensity.Can also find, with under isocandela, the possibility of 33 DEG C of group Wild Vitis species generation Xanthophyll cycle is all greater than 28 DEG C of group Wild Vitis species simultaneously.Therefore, the illumination condition of this Wild Vitis species the best should be medium light intensity, i.e. 65 μm of olm
-2s
-1, 28 DEG C.Experimental result shows, and when temperature is 28 DEG C, light intensity is medium (65 μm of olm
-2s
-1) under the biomass the highest (P<0.01) of Wild Vitis species, be 0.86g/L; Be greater than the 0.82g/L under low light intensity and the 0.70g/L under high isocandela.When temperature is 33 DEG C, light intensity is medium (65 μm of olm
-2s
-1) under the biomass of Wild Vitis species the highest, be 0.81g/L; Be greater than the 0.70g/L under low light intensity and the 0.63g/L under high isocandela.Under equal illumination condition, the Wild Vitis species biomass of 28 DEG C of groups is all higher than 33 DEG C of groups.Therefore, can obtain the growth conditions of Wild Vitis species the best: temperature is 28 DEG C, illumination is 65 μm of olm
-2s
-1.
When temperature is 28 DEG C, medium light intensity (65 μm of olm
-2s
-1) under the sugar degree the highest (P<0.01) of Wild Vitis species, be 24.7%; Be greater than 18.1% under 19.7% and high isocandela under low light intensity.When temperature is 33 DEG C, light intensity is medium (65 μm of olm
-2s
-1) under the sugar degree of Wild Vitis species the highest, be 26.0%; Be greater than 18.9% under 20.2% and high isocandela under low light intensity.And under equal illumination condition, the Wild Vitis species sugar degree of 33 DEG C of groups is all higher than 28 DEG C of groups.Therefore, the product sugar condition of Wild Vitis species the best can be obtained: temperature is 33 DEG C, and illumination is 65 μm of olm
-2s
-1.
The photosynthesis of Wild Vitis species at table 1 different light intensity and temperature
The best biochemical culture condition of 2.5 argon lasers, combination of ultrasound is on the impact of Wild Vitis species biomass and total sugar content
1.2.5 experimental result: select BG11 substratum, not added with antibiotic, the argon laser mutagenesis of employing 90mw 30 seconds, be 33 DEG C in culture temperature, illumination is 65 μm of olm
-2s
-1cultivate under condition after 3 days, adopt the ultrasonic wave of frequency 20kHz, power 8W to Wild Vitis species liquid radiation 60s, continue cultivation 12 days, Wild Vitis species biomass can reach 0.97g/L, and now polysaccharide content is 35.99%.
(2) bacteriostatic activity of Wild Vitis species polysaccharide
This experiment is with Caenorhabditis elegans (wild-type N2) for model animal (each experimental group 50), and research Wild Vitis species polysaccharide is to the effect of Caenorhabditis elegans anti-salmonella and streptococcus aureus.
1 experiment material and method
1.1 experiment material
Caenorhabditis elegans (wild-type N2) is provided by animal science institute of Zhejiang University;
Salmonellas and streptococcus aureus: Microbiological Lab of this institute provides;
VS-840K-U super clean bench, SuZhou Antai Air Tech Co., Ltd.;
The intelligent biochemical cultivation case of PHX, Ningbo Lai Fu Science and Technology Ltd.;
All chemical reagent are analytical pure.
1.2 experimental technique
1.2.1 Caenorhabditis elegans is cultivated
Take a morsel intestinal bacteria OP50 bacterium liquid, with its OD of normal saline dilution
600for 1.0-1.2, draw 100 μ L bacterium drops and to be added on NGM flat board and to smear evenly (application area accounts for the 50%-60% of total surface area, dull and stereotyped wall about the 20mm of coating bacterium Edge Distance), 37 DEG C cultivate 12h after, 4 DEG C of storages are for subsequent use.The cultivation of nematode wild-type-N2: adopt Brenne method succeeding transfer culture on NGM substratum, select nematode more and the normal flat board of growth, cut with one block of more agar of nematode with scalper, by its back-off on the NGM substratum scribbling intestinal bacteria OP50, being placed in 16 DEG C of biochemical cultivation cases cultivates after 3 days, namely visible substratum has a large amount of adult and larva.
1.2.2 the antibacterial model of nematode is set up
Synchronization: collect the flat board that nematode growth is good, with M9 damping fluid, nematode whole in flat board is washed in EP pipe; After centrifugal (3000rpm, 1min), with the resuspended polypide of 0.25ml ddH2O, add 0.15mL nematode lysate (3.4mL distilled water, 0.6mL 5%NaClO, 1mL 8mol/LNaOH, matching while using), after vortex 5min cracking, obtain worm's ovum; After M9 buffer solution for cleaning worm's ovum several, worm's ovum is placed in blank NGM flat board and can obtains L1 nematode in period after 16 DEG C of incubator incubated overnight; Washed in the NGM flat board being coated with OP50 and can be obtained synchronized L4 nematode in period after 16 DEG C of incubator growth 48h.
Contamination: picking nematode, in EP pipe, with M9 buffer solution pipe center line borer population time rear transfer nematode to being coated with on the NGM substratum of streptococcus aureus and Salmonellas in advance, cultivates 12h.
Treatment: picking has contaminated nematode in EP pipe, washes nematode in blank NGM flat board after washing for several times, lets alone the 2-3h that creeps; Nematode is chosen be coated with sample to be evaluated in advance be coated with in the NGM flat board of OP50, every 12h records living nematodes number.
1.2.3 Wild Vitis species polysaccharide fungistatic effect
Experimental group is set for being coated with 5mg/ml, 2.5mg/ml, 0.5mg/ml Wild Vitis species polysaccharide 50 μ L, positive controls is coated with 50 μ L 0.5% mycillins, and negative control group is coated with normal saline, arranges parallel group 5 groups, Continuous Observation 144h, result adopts SPSS19.0 to analyze.
Wild Vitis species polysaccharide extracts by the following method and obtains:
1) alkaline process broken wall: measure the Wild Vitis species algae liquid that 100ml cultivates by 1.2.5 step, centrifugal (4000rpm, 10min) obtains algae mud.After pure water algae mud several, resuspended according to solid-liquid ratio 1:20 (w/v).In stirring, at the uniform velocity add NaOH solid to its concentration is 4%, after NaOH dissolves completely, and ultrasonic 60min;
2) polysaccharide water extraction: regulate algae liquid PH to 7,80 DEG C of water extraction 1h, centrifugal (2000rpm, 15min) gets supernatant.On Rotary Evaporators, concentrated supernatant is to original 1/3;
3) polysaccharide alcohol precipitation: concentrated solution is pressed solid-liquid ratio 1:4 and mix with 95v% alcohol, glass stick stirs 1min, and after natural sedimentation, centrifugal (2000rpm, 15min) removes supernatant, obtains polysaccharide and slightly puies forward precipitation.
4) Sevage deproteinated: precipitation is used a small amount of water dissolution, adds the sevage reagent of 1/4 volume, thermal agitation 20min in sample, 3500rpm, 10min centrifuging and taking supernatant, and repeat about 5 times, frozen drying obtains Wild Vitis species Crude polysaccharides.For in above-mentioned bacteriostatic experiment.
2 experimental results
2.1 Wild Vitis species polysaccharide are to the fungistatic effect of Caenorhabditis elegans Salmonellas
The fungistatic effect of Wild Vitis species polysaccharide to Caenorhabditis elegans Salmonellas is shown in accompanying drawing 2.Result shows, the fungistatic effect of Wild Vitis species polysaccharide to Caenorhabditis elegans Salmonellas has dose-dependently, and Wild Vitis species polysaccharide concentration is higher, more obvious to the fungistatic effect of Salmonellas, and the survival rate of Caenorhabditis elegans is higher.After Caenorhabditis elegans cultivates 84h, different concns polysaccharide group more all has significant difference (P<0.05) with positive controls and negative control group, when continuing to be cultured to 144h, different concns polysaccharide group more still has significant difference (P<0.05) with positive controls and negative control group, and the fungistatic effect of Wild Vitis species polysaccharide to Caenorhabditis elegans Salmonellas of 5mg/ml is better than the microbiotic of same concentrations (5mg/ml).Illustrate that Wild Vitis species polysaccharide has significant protective effect to the nematode infected after Salmonellas, inhibit the growth of Salmonellas in its body.
2.2 Wild Vitis species polysaccharide are to the fungistatic effect of Caenorhabditis elegans streptococcus aureus
The fungistatic effect of Wild Vitis species polysaccharide to Caenorhabditis elegans streptococcus aureus is shown in accompanying drawing 3.Result shows, there is not dose-dependently in the fungistatic effect of Wild Vitis species polysaccharide to Caenorhabditis elegans streptococcus aureus, but there is optimal dose, namely when Wild Vitis species polysaccharide concentration is 2.5mg/ml, the most obvious to the fungistatic effect of streptococcus aureus, the survival rate of Caenorhabditis elegans is the highest.After Caenorhabditis elegans cultivates 72h, different concns polysaccharide group more all has significant difference (P<0.05) with positive controls and negative control group, when continuing to be cultured to 144h, different concns polysaccharide group more still has significant difference (P<0.05) with positive controls and negative control group, and the fungistatic effect of the Wild Vitis species polysaccharide of 2.5mg/ml to beautiful hidden bar worm line streptococcus aureus is better than the microbiotic that concentration is 5mg/ml.Illustrate that Wild Vitis species polysaccharide has significant protective effect equally to the nematode infected after streptococcus aureus, the growth of streptococcus aureus in its body can be suppressed.
The above results shows, at the Gram of Wild Vitis species polysaccharide to beautiful hidden bar line contamination
+with Gram
-all there is significant fungistatic effect, and when the working concentration of Wild Vitis species polysaccharide is equal to or less than antibiotic concentration, its effect is better than microbiotic, there is the effect significantly improving beautiful hidden bar line survival rate, illustrating that Wild Vitis species polysaccharide is at substitute antibiotics, for strengthening cultivated animals disease resistance aspect, there is great potentiality and value.
Comparative example 1:
Get Wild Vitis species algae kind after purifying, after being cultured to the logarithm growth stage with BG11 liquid nutrient medium, (culture temperature 28 DEG C, light intensity is 65 μm of olm
-2s
-1, periodicity of illumination is 14h/d, and rotating speed is 150r/min shaking table), do not use argon laser mutagenesis and supersound process, be directly seeded to the BG11 substratum (not adding microbiotic) filtered out in embodiment 1, nutrient solution A after inoculation
667be 0.3, the optimum culturing temperature (28 DEG C) filtered out in embodiment 1 and intensity of illumination (65 μm of olm
-2s
-1), periodicity of illumination is 14h/d, and rotating speed is cultivate 15 days under 150r/min shaking table condition, calculates Wild Vitis species biomass and polysaccharide content, and contrasts with the Wild Grape algae not carrying out any optimization, the results are shown in shown in following table 2 by embodiment 1:
Table 2: the Wild Vitis species biomass that different culture process obtains and polysaccharide yield
Wild Grape algae gets Wild Vitis species algae kind after purifying, and with after the base culture base in embodiment 1 to logarithm growth stage, (culture temperature 28 DEG C, light intensity is 65 μm of olm
-2s
-1, periodicity of illumination is 14h/d, and rotating speed is 150r/min shaking table), be seeded to basic medium, nutrient solution A after inoculation
667be 0.3, at 28 DEG C and intensity of illumination (65 μm of olm
-2s
-1), periodicity of illumination is 14h/d, and rotating speed is cultivate under 150r/min shaking table condition to cultivate for 15 days to obtain.
As shown in Table 2, different culture process affects very large on the biomass of Wild Vitis species and polysaccharide yield, compared with Wild Grape algae, argon laser mutagenesis, supersound process can make the biomass of Wild Vitis species and polysaccharide yield greatly increase in conjunction with training systern, the biomass of Wild Vitis species and polysaccharide yield increase respectively 2.36 times with 2.14 times; Compared with single optimization culture condition, the biomass of Wild Vitis species and polysaccharide yield increase respectively 1.13 times with 1.39 times.
Comparative example 2:
Get Wild Vitis species algae kind after purifying, operate according to the 1.2.5 step of embodiment 1 (one) part, difference is, change the argon laser mutagenic treatment time be respectively 10,20,30,40,50, after 60s, and follow-uply do not carry out Ultrasonic Radiation process, adopt the BG11 substratum (not adding microbiotic) filtered out in embodiment 1, the optimum culturing temperature filtered out in embodiment 1 (28 DEG C) and intensity of illumination (65 μm of olm
-2s
-1) under cultivate 15 days, calculate Wild Vitis species biomass and polysaccharide content by embodiment 1, and contrast in conjunction with supersound process Wild Vitis species with argon laser mutagenesis, the results are shown in shown in following table 3:
Table 3: the Wild Vitis species biomass that different culture process obtains and polysaccharide yield
As shown in Table 3, different treatment technique affects comparatively large on the biomass of Wild Vitis species and polysaccharide yield, and argon laser mutation time also produces considerable influence to the biomass of Wild Vitis species and polysaccharide yield.Argon laser mutagenesis Best Times is 30s, and the time is too short all unfavorable with the accumulation of overlong time to Wild Vitis species biomass and polysaccharide.Argon laser mutagenesis and supersound process combine and are used alone compared with argon laser mutagenesis, the biomass of Wild Vitis species and polysaccharide yield increase respectively 1.10 times with 1.32 times.
Comparative example 3:
Get Wild Vitis species algae kind after purifying, operate according to the 1.2.5 step of embodiment 1 (one) part, difference is, do not carry out argon laser mutagenic treatment, and change supersound process condition, under frequency is 20kHz condition, set 1 group (4W, 60s), 2 groups (4W, 90s), 3 groups of (8W respectively, 30s), 4 groups of (8W, 60s), 5 groups (16W, 30s), 6 groups (16W, 60s), other conditions are constant, the optimum culturing temperature (28 DEG C) filtered out in embodiment 1 and intensity of illumination (65 μm of olm
-2s
-1) under cultivate 15 days, calculate Wild Vitis species biomass and polysaccharide content by embodiment 1, and contrast in conjunction with supersound process Wild Vitis species with argon laser mutagenesis, the results are shown in shown in following table 4:
Table 4: the Wild Vitis species biomass that different culture process obtains and polysaccharide yield
As shown in Table 4, different treatment technique affects comparatively large on the biomass of Wild Vitis species and polysaccharide yield, and supersound process condition also produces considerable influence to the biomass of Wild Vitis species and polysaccharide yield.Supersound process best power and time are 8W and 60s, and power is excessive extremely unfavorable with the accumulation of overlong time to Wild Vitis species biomass and polysaccharide.Argon laser mutagenesis and supersound process combine and are used alone compared with supersound process, the biomass of Wild Vitis species and polysaccharide yield increase respectively 1.07 times with 1.23 times.
Claims (9)
1. improve a cultural method for Wild Vitis species polysaccharide yield, it is characterized in that described method is:
The Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, the algae liquid of growth stage of taking the logarithm, and after adopting argon laser to irradiate mutagenesis, inoculates in BG11 liquid nutrient medium, culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; Cultivate the Wild Vitis species after 2 ~ 3 days and carry out Ultrasonic Radiation process, the Wild Vitis species after Ultrasonic Radiation process continues culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal be separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
2. the method for claim 1, it is characterized in that the Wild Vitis species BG11 liquid nutrient medium of described purifying is cultured to the logarithm growth stage, culture condition is culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination.
3. the method for claim 1, it is characterized in that the Wild Vitis species of described purifying is the Wild Vitis species list bacterium colony algae kind obtained by line partition method purification process by Wild Grape algae, purifying obtains by the following method: abandon supernatant liquor by after Wild Grape algae algae liquid centrifugal concentrating, the sterilized water added containing 0.5wt% mycillin solution is resuspended, supernatant liquor is abandoned after recentrifuge, the algae mud obtained after washing 3 times as stated above carries out line and is separated: in Bechtop, algae mud is lined on BG11 solid medium, be inverted in illumination box and cultivate, culture temperature 28 DEG C, illumination 3000lx, 14 hours every days of periodicity of illumination, every 10 days is one-period, after cultivating one-period, picking list bacterium colony carries out second time line, the third generation is obtained after experiencing 2 line, namely the Wild Vitis species algae kind of purifying is obtained.
4. method as claimed in claim 1 or 2, is characterized in that the formula of described BG11 liquid nutrient medium is: in every 1000mL distilled water, containing 1.5g NaNO
3, 0.04gK
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036g CaCl
2.7H
2o, 0.02gNa
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution, the composition of pH value 7.0,1ml trace element solution comprises 2.86g H
3bO
4, 1.81gMnCl
2.4H
2o, 0.222g ZnSO
4, 0.39g Na
2moO
4, 0.079gCuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o.
5. method as claimed in claim 3, is characterized in that the formula of described BG11 solid medium is: in every 1000mL distilled water, containing 1.5g NaNO
3, 0.04g K
2hPO
4, 0.075g MgSO
4.7H
2o, 0.036g CaCl
2.7H
2o, 0.02g Na
2cO
3, 0.006g citric acid, 0.006g ironic citrate, 1ml trace element solution, 20.0g agar, the composition of pH value 7.0,1ml trace element solution comprises 2.86g H
3bO
4, 1.81gMnCl
2.4H
2o, 0.222g ZnSO
4, 0.39g Na
2moO
4, 0.079gCuSO
4.5H
2o, 49.4g Co (NO
3)
2.6H
2o.
6. the method for claim 1, when it is characterized in that described argon laser irradiates mutagenesis, the power 90mw of argon laser, irradiation time 10 ~ 40 seconds, the distance of Wild Vitis species and light source is 80cm.
7. the method for claim 1, when it is characterized in that described Ultrasonic Radiation process, hyperacoustic frequency is 20kHz, power 4 ~ 8W, 30 ~ 90 seconds treatment times.
8. the method for claim 1, is characterized in that described method is undertaken by following operation: the Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; The algae liquid of growth stage of taking the logarithm carries out argon laser and irradiates mutagenesis, the distance of Wild Vitis species and light source is made to be 80cm, by the argon laser radiation treatment 10 ~ 40 seconds of 90mw, then the Wild Vitis species after mutation induced by laser is inoculated and cultivate into BG11 liquid nutrient medium, culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; Cultivate the Ultrasonic Radiation process 30 ~ 90 seconds of the Wild Vitis species frequency 20kHz after 2 ~ 3 days, power 4 ~ 8W, the Wild Vitis species after Ultrasonic Radiation process continues culture temperature 28 ~ 33 DEG C, intensity of illumination 43 ~ 86 μm of olm
-2s
-1, cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal be separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
9. the method for claim 1, is characterized in that described method is undertaken by following operation: the Wild Vitis species BG11 liquid nutrient medium of purifying is cultured to the logarithm growth stage, culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; The algae liquid of growth stage of taking the logarithm carries out argon laser and irradiates mutagenesis, the distance of Wild Vitis species and light source is made to be 80cm, by the argon laser radiation treatment 30 seconds of 90mw, then the Wild Vitis species after mutation induced by laser is inoculated and cultivate into BG11 liquid nutrient medium, culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, 14 hours every days of periodicity of illumination; Cultivate the Ultrasonic Radiation process 60 seconds of the Wild Vitis species frequency 20kHz after 2 ~ 3 days, power 8W, the Wild Vitis species after Ultrasonic Radiation process continues culture temperature 28 DEG C, intensity of illumination 65 μm of olm
-2s
-1, cultivate 12-13 days under the condition of 14 hours every days of periodicity of illumination, gained medium centrifugal be separated, sterilized water washing obtains the Wild Vitis species being rich in polysaccharide.
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| CN112391293A (en) * | 2020-11-25 | 2021-02-23 | 宁波大学 | Method for preparing squalene by autotrophic culture of botryococcus through heterotrophic biomembrane adherence |
| CN112608956A (en) * | 2020-12-10 | 2021-04-06 | 浙江大学 | Method for improving yield of extracellular polysaccharide of bacteria by using ultrasonic waves |
| CN112608956B (en) * | 2020-12-10 | 2024-03-15 | 浙江大学 | Method for improving bacterial extracellular polysaccharide yield by utilizing ultrasonic waves |
| GB2607838A (en) * | 2021-08-09 | 2022-12-14 | Univ Jiangsu | Bacillus Velezensis strain bred through ultrasound-assisted adaptive evolution, and use thereof |
| GB2607838B (en) * | 2021-08-09 | 2024-03-20 | Univ Jiangsu | Mutated Bacillus velezensis strain bred through ultrasound and cold-shock assisted adaptive evolution, and use thereof |
| CN114634877A (en) * | 2022-04-14 | 2022-06-17 | 浙江大学 | Synthetic method of biological photosynthetic organelle, product and application thereof |
| CN114634877B (en) * | 2022-04-14 | 2023-11-24 | 浙江大学 | Synthesis method of biological photosynthetic organelle, product and application thereof |
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