CN108148787A - A kind of method of pilot scale culture nostoc - Google Patents

A kind of method of pilot scale culture nostoc Download PDF

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CN108148787A
CN108148787A CN201810124680.6A CN201810124680A CN108148787A CN 108148787 A CN108148787 A CN 108148787A CN 201810124680 A CN201810124680 A CN 201810124680A CN 108148787 A CN108148787 A CN 108148787A
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culture
nostoc
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algae
culture vessel
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辛茂盛
申燕芝
逄勇
史春潇
贾如辉
贾欣欣
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QINGDAO HAO RAN ECOLOGICAL MARINE FISHERIES SCIENCE AND TECHNOLOGY Ltd Co
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QINGDAO HAO RAN ECOLOGICAL MARINE FISHERIES SCIENCE AND TECHNOLOGY Ltd Co
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract

The invention discloses a kind of method of nostoc pilot scale culture, the nostoc cultural method being produced on a large scale by the way that nostoc sphaeroides are purified and passed through with third stage culture, the advantageous effect of the present invention：The method of the invention nostoc pilot scale culture; it is easy to operate, equipment is simple, it is at low cost, be not subject to seasonal restrictions; primary culture amount is big; it can be suitble to the needs propagated artificially on a large scale; and the amplification culture Jing Guo three-level makes nostoc sphaeroides adequately be grown, product build is big, and nutritional ingredient is high; micronutrient levels is high, safe and non-toxic.

Description

A kind of method of pilot scale culture nostoc
Technical field
The present invention relates to nostoc to cultivate field, specifically a kind of method of pilot scale culture nostoc.
Background technology
Nostoc is Cyanophyta, and nostocaceae is a kind of many cells group, and principal shape is spherical shape, and minority is spindle Shape, oval row, dumb-bell shape or flat.Ancient name day rosetangle, beauty's rice, are commonly called as water agaric.It is long that nostoc eats medicinal history, With abundant nutritive value.According to《Mingyi Bielu》、《Compendium of Materia Medica》、《Pharmacological property is examined》、《National Chinese herbal medicine compilation》Wait historical data note It carries, nostoc has improving eyesight QI invigorating, antipyretic clear diaphragm, sharp stomach, eats macrobiosis long, dispelling fatigue, restrain, control yctalopia and other effects. Present scientific research has been acknowledged that protein content is high, and amino acid great variety of goods therein in nostoc, rational in infrastructure, base Originally meet the FAO/WHO Committee of Experts and general essential amino acid pattern was pushed away in 1973.It is a kind of low fat again simultaneously Content food, crude fat content are only 8.11%, the synthesis of body's cholesterol can be reduced, with prevention and cure of cardiovascular disease.In addition, Pueraria lobota Celestial rice is also rich in polysaccharide, multivitamin, the nutritional ingredients such as carotenoid, phycobilin, trace element.《Hefeng was state originally》Note It carries：" nostoc, produce away from Zhoucheng (Hefeng was state originally) it is over one hundred in, big rock is all raw everywhere outside the Pass in paddy field." Hubei Province Hefeng Area is the main original producton location of China's nostoc, and in natural environment, nostoc is common on the small stream stone of river or in rice terrace, in blackish green Chromosphere shape, it is extremely tight to growing environment and condition requirement, it is worldwide distributed also extremely narrow.The peak annual output of nostoc was once Up to 25 tons, in recent years, change to fertilize since peasant does not apply farm manure, seriously polluted soil, change the acid-base property of soil, And largely change growing environment that nostoc is depended on for existence etc. using herbicide, substantially reduce the yield of nostoc, Annual output falls sharply to 0.5 ton or even has a possibility of disappearance.Fully to develop and utilize nostoc resource, change growth by season The limitation that section and wild yield reduce year by year, meets market needs, it is necessary to carry out the artificial pilot scale culture research of nostoc.
At present, the acquisition of nostoc mainly has field cultivation and artificial culture two ways.But the Ge Xian of field cultivation Rice low output, growth cycle be short, by seasonal effect is big, nutrient composition content is low；The nostoc culture apparatus manually cultivated is complicated, Construction cost is high, culture scale is limited by reactor is difficult to mass produce.Therefore, it finds a kind of easy to operate, honest and clean Valency, efficient nostoc training mode are particularly important.
Invention content
Nostoc field cultured output is low, growth is subject to seasonal restrictions and manually cultivates construction cost in order to solve by the present invention High, reactor volume limitation is difficult to the present situation of large-scale production, provides a kind of to meet the pilot scale culture Pueraria lobota of the market demand The method of celestial rice
The purpose of the present invention is be achieved through the following technical solutions：
A kind of method of nostoc pilot scale culture, includes the following steps：
Step 1：The processing and culture of algae
By the disinfection of collected nostoc sphaeroides, washing, it is ground after processing, centrifuges and obtain Filamentous frond and hormocystangium is molten Liquid, and examine whether there is miscellaneous algae, algae after purification is obtained, then algae after purification is inoculated into culture vessel and is cultivated；
Step 2：Culture vessel
Third stage culture is carried out to the algae that step 1 obtains, algae after purification is inoculated into level-one culture vessel by level-one culture Culture obtains first order seed, and first order seed is inoculated into culture in two level culture vessel and obtains secondary seed, three-level by two level culture Secondary seed is inoculated into culture in third stage culture container and obtains three-level seed by culture；
Step 3：Pilot scale culture
Algae after purification will be handled as inoculum, be placed in culture vessel with the ratio of every liter of culture solution 3-5g weight in wet base, and CO is passed through with air inflator pump2, CO2Flow to ensure entire reaction solution be in mitigate flow regime；In level-one culture vessel Middle culture 25-35 days, by filamentous growth into fine particle shape, the culture solution in culture vessel becomes black for algae after purification； Two level culture vessel continues culture 25-35 days, and first order seed grows into subtle cylindric, and culture solution becomes black again；Three-level Nostoc of the culture vessel secondary seed culture to particle diameter for 1-5cm；
Step 4：Condition of culture
Cultivation temperature maintains 22-25 DEG C, and between 80-100 μ E.m-2.s-1, culture solution was replaced once every 10 days for illumination, Whether daily regular check inflation is normal, to ensure that nostoc particle is not sunk to the bottom；
Step 5：It harvests drying
Nostoc after third stage culture is handled by the sieves sieving that sieve pore is 2cm, dehydration, natural air drying.
It advanced optimizes, the BG-11 fluid nutrient mediums of improvement is placed in culture vessel described in step 1, entire molten Using dissolved in purified water, constant volume in the process for preparation of liquid, culture vessel handles 12h, the PH of culture solution with disinfection by ultraviolet light It is worth for 7.8-8.0.
It advanced optimizes, the level-one culture vessel is the volumetric flask that volume is 5L, and two level culture vessel is that volume is 17L Pure water barrel, third stage culture container for volume be more than 100L Homemade transparent container.
Advantageous effect of the present invention：The method of the invention nostoc pilot scale culture, it is easy to operate, equipment is simple, it is at low cost, It is not subject to seasonal restrictions, primary culture amount is big, can be suitble to the needs propagated artificially on a large scale, and the amplification culture Jing Guo three-level makes Pueraria lobota Celestial rice algae is adequately grown, product build is big, nutritional ingredient is high.
Description of the drawings
Fig. 1 is the front view for making the big volume culture vessel of all-transparent by oneself；
Fig. 2 is the cellular morphology microphoto of sample 1；
1st, culture vessel；2nd, iron wire；3rd, iron chains.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.It is unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.The present invention is described in further detail below according to embodiment：
Embodiment 1：
Step 1：The processing and culture of algae
By the disinfection of collected nostoc sphaeroides, washing, it is ground after processing, centrifuges and obtain Filamentous frond and hormocystangium is molten Liquid, and examine whether there is miscellaneous algae, algae after purification is obtained, then algae after purification is inoculated into culture vessel and is cultivated；
Step 2：Culture vessel
Third stage culture is carried out to the algae that step 1 obtains, algae after purification is inoculated into level-one culture vessel by level-one culture Culture obtains first order seed, and first order seed is inoculated into culture in two level culture vessel and obtains secondary seed, three-level by two level culture Secondary seed is inoculated into culture in third stage culture container and obtains three-level seed by culture；
Step 3：Pilot scale culture
Using the algae handled after purification as inoculum, it is placed in culture vessel, is used in combination with the ratio of every liter of culture solution 4g weight in wet base Air inflator pump is passed through CO2, CO2Flow to ensure entire reaction solution be in mitigate flow regime；In level-one culture vessel Culture 30 days, by filamentous growth into fine particle shape, the culture solution in culture vessel becomes black for algae after purification；Two level is trained Foster container continues culture 30 days, and first order seed grows into subtle cylindric, and culture solution becomes black again；Third stage culture container two Grade seed culture to the largest particles diameter 5cm nostoc；
Step 4：Condition of culture
Cultivation temperature maintains 22 DEG C, and illumination is between 80 μ E.m-2.s-1, and the pH value of culture solution is 7.8, and culture solution is every 10 It is replaced once, and whether daily regular check inflation is normal, to ensure that nostoc particle is not sunk to the bottom；
Step 5：It harvests drying
Nostoc after third stage culture is handled by the sieves sieving that sieve pore is 2cm, particle diameter is less than the Ge Xian of 2cm Rice, which is put into culture vessel, to be continued to cultivate, and a nostoc part of the grain size more than 2cm is used to process the raw material of cuisines, another portion Divide and be dried.Since the polysaccharide that nostoc spherical mantle has has high viscosity, easily one is bonded in holding vessel It rises, it is necessary to which the sphere of harvest is dried in time.It is dehydration, natural air drying that method, which is led to, used by drying.
Embodiment 2：
Step 1：The processing and culture of algae
By the disinfection of collected nostoc sphaeroides, washing, it is ground after processing, centrifuges and obtain Filamentous frond and hormocystangium is molten Liquid, and examine whether there is miscellaneous algae, algae after purification is obtained, then algae after purification is inoculated into culture vessel and is cultivated；
Step 2：Culture vessel
Third stage culture is carried out to the algae that step 1 obtains, algae after purification is inoculated into level-one culture vessel by level-one culture Culture obtains first order seed, and first order seed is inoculated into culture in two level culture vessel and obtains secondary seed, three-level by two level culture Secondary seed is inoculated into culture in third stage culture container and obtains three-level seed by culture；
Step 3：Pilot scale culture
Using the algae handled after purification as inoculum, it is placed in culture vessel, is used in combination with the ratio of every liter of culture solution 3g weight in wet base Air inflator pump is passed through CO2, CO2Flow to ensure entire reaction solution be in mitigate flow regime；In level-one culture vessel Culture 30 days, by filamentous growth into fine particle shape, the culture solution in culture vessel becomes black for algae after purification；Two level is trained Foster container continues culture 30 days, and first order seed grows into subtle cylindric, and culture solution becomes black again；Third stage culture container two Grade seed culture to the largest particles diameter 5cm nostoc；
Step 4：Condition of culture
Cultivation temperature maintains 23 DEG C, and illumination is between 100 μ E.m-2.s-1, and the pH value of culture solution is 7.9, and culture solution is every 10 It is replaced once, and whether daily regular check inflation is normal, to ensure that nostoc particle is not sunk to the bottom；
Step 5：It harvests drying
Nostoc after third stage culture is handled by the sieves sieving that sieve pore is 2cm, particle diameter is less than the Ge Xian of 2cm Rice, which is put into culture vessel, to be continued to cultivate, and a nostoc part of the grain size more than 2cm is used to process the raw material of cuisines, another portion Divide and be dried.Since the polysaccharide that nostoc spherical mantle has has high viscosity, easily one is bonded in holding vessel It rises, it is necessary to which the sphere of harvest is dried in time.It is dehydration, natural air drying that method, which is led to, used by drying.
Embodiment 3：
Step 1：The processing and culture of algae
By the disinfection of collected nostoc sphaeroides, washing, it is ground after processing, centrifuges and obtain Filamentous frond and hormocystangium is molten Liquid, and examine whether there is miscellaneous algae, algae after purification is obtained, then algae after purification is inoculated into culture vessel and is cultivated；
Step 2：Culture vessel
Third stage culture is carried out to the algae that step 1 obtains, algae after purification is inoculated into level-one culture vessel by level-one culture Culture obtains first order seed, and first order seed is inoculated into culture in two level culture vessel and obtains secondary seed, three-level by two level culture Secondary seed is inoculated into culture in third stage culture container and obtains three-level seed by culture；
Step 3：Pilot scale culture
Using the algae handled after purification as inoculum, it is placed in culture vessel, is used in combination with the ratio of every liter of culture solution 5g weight in wet base Air inflator pump is passed through CO2, CO2Flow to ensure entire reaction solution be in mitigate flow regime；In level-one culture vessel Culture 30 days, by filamentous growth into fine particle shape, the culture solution in culture vessel becomes black for algae after purification；Two level is trained Foster container continues culture 30 days, and first order seed grows into subtle cylindric, and culture solution becomes black again；Third stage culture container two Grade seed culture to the largest particles diameter 5cm nostoc；
Step 4：Condition of culture
Cultivation temperature maintains 24 DEG C, and illumination is between 95 μ E.m-2.s-1, and the pH value of culture solution is 8.0, and culture solution is every 10 It is replaced once, and whether daily regular check inflation is normal, to ensure that nostoc particle is not sunk to the bottom；
Step 5：It harvests drying
Nostoc after third stage culture is handled by the sieves sieving that sieve pore is 2cm, particle diameter is less than the Ge Xian of 2cm Rice, which is put into culture vessel, to be continued to cultivate, and a nostoc part of the grain size more than 2cm is used to process the raw material of cuisines, another portion Divide and be dried.Since the polysaccharide that nostoc spherical mantle has has high viscosity, easily one is bonded in holding vessel It rises, it is necessary to which the sphere of harvest is dried in time.It is dehydration, natural air drying that method, which is led to, used by drying.
Wherein since the algae particle of level-one, two level culture is smaller, the requirement to condition of culture is again harsher, therefore selects It selects and bioreactor is installed in glasshouse.The algae grain size of third stage culture is bigger, and the volume, illumination to reactor will Ask comparison high, therefore the big volume culture vessel of customization is installed in selection in vinyl house.Level-one culture vessel is that volume is 5L's Volumetric flask, two level culture vessel are the pure water barrel that volume is 17L, and third stage culture container is the big volume dress of the all-transparent of customization It puts（More than 100L）, and place the air inflator pump of respective model in the bottom of culture vessel of different cultivation stages and carry out Ventilation, makes the algae particle in entire culture vessel be in flow regime, and photosynthesis is carried out equably to receive illumination.
On the one hand it is because relatively good to the permeability of light, convenient for algae as culture vessel that first order reaction, which selects volumetric flask, Kind carries out photosynthesis using light intensity, is on the other hand more accurate using the solution of volumetric flask preparation, can meet experiment condition It is required that and occupy little space, be conducive to control Photosynthetic Conditions.
Second order reaction is by the use of pure water barrel as culture vessel, can both meet food stage reactor and the photosynthetic work of algae Needs, and can be cost-effective.
Third-order reaction is by the use of the big device in volume of homemade all-transparent as culture vessel.
As shown in Figure 1, the culture vessel is cylindrical, 1.5 meters high, 0.6 meter of diameter, periphery is fixed by iron wire, in iron wire Face is the cylinder of transparent food-grade PE materials, in one air air charging system of the bottom of culture vessel placement, then by using The device voluntarily built（Such as iron chains）Culture vessel is suspended in vinyl house, ensures that culture vessel adequately receives light According to.When reaction vessel diameter is more than 0.6 meter, has partial reaction object and sink to reactor bottom, photosynthesis can not be carried out；When straight Diameter be less than 0.6 meter when, reagent flow is violent, can cause part solution it is low-priced go out.
The effect of in order to illustrate the present invention, the fresh nostoc after 1 third stage culture of Example is sample 1, and is carried out such as Lower test：
First, Morphological Identification
Cellular morphology of the sheet 1 in the micro- Microscopic observation samples 1 of Olympus BX51 is sampled, and is acquired and schemed with Olympus DP71 Picture.Fig. 1 is the cellular morphology microphoto of sample 1, spherical in shape as can be seen from Figure 1, diameter about 1-15mm, irregular after ripe Gauffer nodular, group's glue is firm by outer layer, filament bending, be closely wound.4-5 microns of algal filament, cell is subsphaeroidal.According to above-mentioned Morphological feature analyzes and identifies the sample and belongs to level-one nostoc.
2nd, biochemical indicator detects
Take appropriate sample 1 and according to SNT1113-2002, SNT4260-2015, GB26405-2011, GB5009.3-2016 standard Phycocyanin, polysaccharides, lutein ester, the dry matter content of sample 1 are detected respectively, and testing result is shown in Table 1
The biochemical indicator of 1 sample 1 of table
Detection project Content Examination criteria
Phycocyanin 5.1g/100g SNT1113-2002
Polysaccharides 45% SNT4260-2015
Lutein ester 7.18mg/g GB26405-2011
Dry matter content 3% GB5009.3-2016
3rd, spectrum Buddhist nun test
The sample 1 of 600g is taken, utilizes liquid chromatograph, Atomic Absorption Spectrometer, Atomic Fluorescence Spectrometer, gas chromatograph, ammonia Base acid automatic analyzer carries out dependence test.Table 2 is the experimental result for composing Buddhist nun's test.
Spectrum Buddhist nun's test experiments result of 2 sample 1 of table
Detection project Testing result Detect foundation
Protein, g/100g 1.20 GB 5009.5-2016
Total dietary fiber, g/100g 1.34 GB 5009.88-2014
Total fat, g/100g 0.1 GB 5009.6-2016
Total reducing sugar, g/100g It does not detect（<0.25） GB 5009.8-2016
Vitamin E, mg/100g 0.142 GB 5009.82-2016
Lead, mg/kg It does not detect（<0.02） GB 5009.12-2017
Arsenic, mg/kg It does not detect（<0.010） GB 5009.11-2014
Mercury, mg/kg It does not detect（<0.003） GB 5009.17-2014
Cadmium, mg/kg It does not detect（<0.001） GB 5009.15-2014
Calcium, mg/kg 513 GB 5009.92-2016
Potassium, mg/kg 27.9 GB 5009.91-2017
Iron, mg/kg 34.3 GB 5009.90-2016
Selenium, mg/kg It does not detect（<0.006） GB 5009.93-2017
Zinc, mg/kg 1.7 GB 5009.14-2017
Magnesium, mg/kg 160 GB 5009.241-2017
The nostoc cultivated as can be seen from the above experimental data has the characteristics that：（1）Nutriment is comprehensive, and structure is closed Reason；（2）Special index content is high.Such as：The content of polysaccharides is 45%；（3）Micronutrient levels is high, such as：Containing abundant Ga, K, Fe, Mg etc.；（4）Safety, does not contain or the heavy metal containing denier.

Claims (3)

  1. A kind of 1. method of pilot scale culture nostoc, it is characterised in that：Include the following steps：
    Step 1：The processing and culture of algae
    By the disinfection of collected nostoc sphaeroides, washing, it is ground after processing, centrifuges and obtain Filamentous frond and hormocystangium is molten Liquid, and examine whether there is miscellaneous algae, algae after purification is obtained, then algae after purification is inoculated into culture vessel and is cultivated；
    Step 2：Culture vessel
    Third stage culture is carried out to the algae that step 1 obtains, algae after purification is inoculated into level-one culture vessel by level-one culture Culture obtains first order seed, and first order seed is inoculated into culture in two level culture vessel and obtains secondary seed, three-level by two level culture Secondary seed is inoculated into culture in third stage culture container and obtains three-level seed by culture；
    Step 3：Pilot scale culture
    Algae after purification will be handled as inoculum, be placed in culture vessel with the ratio of every liter of culture solution 3-5g weight in wet base, and CO is passed through with air inflator pump2, CO2Flow to ensure entire reaction solution be in mitigate flow regime；In level-one culture vessel Middle culture 25-35 days, by filamentous growth into fine particle shape, the culture solution in culture vessel becomes black for algae after purification； Two level culture vessel continues culture 25-35 days, and first order seed grows into subtle cylindric, and culture solution becomes black again；Three-level Nostoc of the culture vessel secondary seed culture to particle diameter for 1-5cm；
    Step 4：Condition of culture
    Cultivation temperature maintains 22-25 DEG C, and between 80-100 μ E.m-2.s-1, culture solution was replaced once every 10 days for illumination, Whether daily regular check inflation is normal, to ensure that nostoc particle is not sunk to the bottom；
    Step 5：It harvests drying
    Nostoc after third stage culture is handled by the sieves sieving that sieve pore is 2cm, dehydration, natural air drying.
  2. 2. the method for pilot scale culture nostoc according to claim 1, it is characterised in that：Culture vessel described in step 1 In the BG-11 fluid nutrient mediums of improvement are placed, using dissolved in purified water, constant volume in the process for preparation of entire solution, Culture vessel handles 12h with disinfection by ultraviolet light, and the pH value of culture solution is 7.8-8.0.
  3. 3. the method for pilot scale culture nostoc according to claim 1, it is characterised in that：The level-one culture vessel is Volume is the volumetric flask of 5L, and two level culture vessel is the pure water barrel that volume is 17L, and third stage culture container is more than 100L for volume Homemade transparent container.
CN201810124680.6A 2018-02-07 2018-02-07 A kind of method of pilot scale culture nostoc Pending CN108148787A (en)

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CN111040963A (en) * 2019-12-11 2020-04-21 常德炎帝牧源农业发展有限公司 Nostoc sphaeroids kutz algae section induction method
CN114410474A (en) * 2021-12-09 2022-04-29 湖北师范大学 Simple and efficient method for preparing nostoc mycosporine amino acid crude product

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