CN101481718A - Preparation of chitosan in fungal cell wall - Google Patents
Preparation of chitosan in fungal cell wall Download PDFInfo
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- CN101481718A CN101481718A CNA2009100459238A CN200910045923A CN101481718A CN 101481718 A CN101481718 A CN 101481718A CN A2009100459238 A CNA2009100459238 A CN A2009100459238A CN 200910045923 A CN200910045923 A CN 200910045923A CN 101481718 A CN101481718 A CN 101481718A
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Abstract
The invention relates to the culture of fungi and a preparing method of separating and extracting chitosan from mycelium. Actinomucor taiwanensis fungi are used as raw materials. After plate inscription, shaking culture and culture in fermentor, bacterial liquid is obtained. Then, the centrifugation is carried out on the obtained bacterial liquid to obtain wet cells. After separating, drying, crushing, alkali treatment, acid treatment and drying, the chitosan is obtained. The cell culturing method is simple; the process for preparing the chitosan is easy, the alkali treatment with low concentration during the deacetylation process greatly reduces pollutions on the environment, the production cost is low, and the yield is stable.
Description
Technical field
The invention belongs to biological chemical field, relate to the method for from the fungal cultures of Mucoraceae, extracting chitosan.
Background technology
Chitosan (chitosan) has another name called chitosan, chitosan, poly-glucosamine; it is the deacetylated product that chitin has another name called chitin (chitin); for D-glucosamine β-1～4 bonded straight-chain polysaccharide, be alkaline polysaccharide unique in the natural polysaccharide.The distribution of occurring in nature chitosan than chitin lack many, only deposit on a small quantity at cell walls of zygomycota etc., be white or canescence pearliness slightly, the shape chip solid is translucent, good, nontoxic, the water insoluble and alkaline solution of its chemical stability dissolves in most of diluted acid example hydrochloric acids, acetic acid, citric acid etc.
It is the starting material that glucosamine and chitosan are produced in conduct that the primary commercial of chitosan is worth.Chitosan has physics, chemical property and the biological function of many uniquenesses, uses very extensive in a plurality of fields such as textile printing and dyeing, sewage disposal, papermaking, food, makeup, medical treatment, biotechnology and pharmacy.
Chitosan is to be only second to cellulosic second largest natural high moleculer eompound in the world, and the traditional mode of production chitin is that starting material extract chitin with shrimp and crab shell etc. mainly, then drying, pulverizing, again through machinery and chemical treatment gained.Its commercial production process is limited by raw-material supply.In addition, its production peak of extraction chitosan from the Mycophyta cell walls of reporting for work both at home and abroad is 1.1g/L, and productivity ratio is lower.Simultaneously because the difference of crustaceans raw material; be difficult to obtain unified high-quality chitosan; the degree of deacetylation of its product is different and different with raw-material source and treating processes, for example the size of Crustacean raw material, age, kind, growing environment, collecting location or the like.In addition, need to use a large amount of strong acid and strong bases and pyroprocessing in the traditional technology preparation process, the treatment time is long, big energy-consuming, and environment had bigger pollution.
Summary of the invention
Technical problem to be solved by this invention provides a kind of low power consuming, low pollute, be easy to the chitosan biological preparation method cultivating, extract.The present invention adopts suitable medium to cultivate Actinomucor taiwanensis class fungi, gather in the crops mycelium in suitable strain growth time, from thalline, isolate chitosan, a kind of method that such fungi makes its high yield chitosan of cultivating is provided.
Actinomucor taiwanensis class fungi (ATCC deposit number 52360) is cultivated the characteristic with unusual high yield chitosan in the E2 substratum.The substratum useful to the present invention can comprise the PDA substratum, E2 substratum, the Adlerika of about 0.01～30g/L and other proper composition.Can be used for concrete Pseudomonas of the present invention also comprises: Mucor rouxii, Aspergillus niger, Aspergillus terreus, Aspergillusoryzae, Lactarius vellereus, Penicillium chrysogenum, Penicilliumnotatum, Saccharomyces cerevisiae and portion C andidaguillermondi, Aspergillus niger, Aspergillus terreus.
It is as follows with the technical scheme of separating chitosan that the present invention cultivates fungi:
The method that the present invention prepares chitosan may further comprise the steps:
(1): Actinomucor taiwanensis class fungal bacterial strain is drawn plate, shake-flask culture, fermentor cultivation, and the bacterium liquid that obtains is through centrifugal acquisition wet thallus;
(2): from mycelium, separate and extract chitosan.
It is the PDA substratum that stroke plate in the step (1) is cultivated used substratum, and it consists of: potato 300g/L, glucose 20g/L, agar 15g/L.
Described in the step (1) the used basic medium of shake-flask culture be the E2 substratum, it consists of NaNH
4HPO
44H
2O:3.5g/L; K
2HPO
43H
2O:7.5g/L; KH
2PO
4: 3.7g/L; MgSO
4: 0.12g/L; Carbon source: carbon content 20g/L; Described carbon source comprises glucose, beef extract, yeast extract; Shake-flask culture adds the CaCl of 0.56g/L on the basis of E2
2, the peptone of 0～5g/L.
The initial pH value of the fermention medium that fermentor cultivation adopted described in the step (1) is 3～8; Its composition comprises the MgSO of 20～200g/L carbon source, 10～80g/L nitrogenous source, 1.0～20g/L yeast extract, 0.01～40g/L
4, 0～5.6g/L CaCl
2, described carbon source comprises glucose, beef extract; Nitrogenous source comprises peptone, NaNH
4HPO
44H
2O, nitrogen water.
Cultivate fungi and adopted specific substratum, fungi is inoculated in the PDA substratum, will take over the agar plate of planting or shake the biochemical cultivation of bottle 4～10 days in 25 ℃ to 37 ℃.The fungal spore that obtains is suspended in the E2 liquid nutrient medium, and shake-flask culture obtains original seed.Be seeded in the fermentor tank with this original seed, cultivate to obtain a large amount of mycelium.
Use standard method that fungi is heavily separated and purifying, be generally and reclaim cell lump from fermented liquid, filtered through gauze or centrifugal was handled cell 20 minutes in 121 ℃ of 2N NaOH, and the centrifugation solids is also used distilled water and washing with alcohol.Handle washed throw out with 2% hydrochloric acid soln, and in 95 ℃ of insulations 12～18 hours, centrifugation gained suspension produced acid supernatant liquor and sour insoluble precipitate.
With 2N NaOH supernatant liquor is adjusted to the pH value more than 10, thereby is settled out cotton-shaped chitosan.The centrifugal solid matter that gets of 12000rpm, lyophilize gets product.
Beneficial effect of the present invention:
Adopt a kind of less energy-consumption provided by the invention, low pollute, be easy to the chitosan biological preparation method cultivating and extract, can cultivate such fungi and make its high yield chitosan.Technology is simple, cost is low, stable yield and be easy to industrialization, greatly reduces production cost.
Embodiment
Following example will further specify the present invention, but they are not limitation of the invention, and the result of embodiment sketches in table 1.
Embodiment 1
Slant culture (PDA substratum) direct inoculation of the Actinomucor taiwanensis of 4 ℃ of preservations is shaken in the bottle in the 500ml that contains 100ml fresh seeds nutrient solution, in 30 ℃ and cultivate 48h with the speed oscillation of 280rpm.Seed culture medium adopts the E2 substratum, contains 40g glucose (being the 20g carbon source), 3.5gNaNH in every liter of seed culture medium
4HPO
44H
2O (being the 0.23g nitrogenous source), 7.5g K
2HPO
43H
2O, 3.7gKH
2PO
4, 0.12g MgSO
4, 0.56g CaCl
2, shake-flask culture obtained original seed after 2 days, was inoculated in the 5L fermentor tank with this original seed.
Adorn liquid by 40% liquid amount in the fermentor tank, every liter of central seed culture medium content that uses 10 times of amounts of fermented liquid, 5% inoculum size inoculation, temperature is controlled at 25～30 ℃, and rotating speed is 100～400rpm, fermentation culture 6d.
Reclaim cell lump from fermented liquid, filtered through gauze or centrifugal was handled cell 20～30 minutes in 121 ℃ of 1NNaOH, and the centrifugation solids is also used distilled water and washing with alcohol.Oven dry is also pulverized solids, and 95 ℃ of insulations of the hydrochloric acid soln with 2% were handled 12 hours, and centrifugation gained suspension produces acid supernatant liquor and sour insoluble precipitate.
With 2N NaOH supernatant liquor is adjusted to the pH value more than 10, thereby is settled out cotton-shaped chitosan.12000rpm centrifugal solids material, lyophilize gets product.Use this seed culture medium combined ferment substratum, the highest dry cell weight 14.6g/L that gets gets chitosan 0.85g/L.Productive rate is 5.8% (with respect to dry cell weight, the results are shown in table 1, down together).
Embodiment 2
The slant culture direct inoculation of the Actinomucor taiwanensis of 4 ℃ of preservations is shaken in the bottle in the 500ml that contains 100ml fresh seeds nutrient solution.By the embodiment 1 described shake-flask culture that carries out, different is to contain 20g carbon source (beef extract), 0.23g nitrogenous source (3.5g NaNH in every liter of seed culture medium
4HPO
44H
2O), 7.5g K
2HPO
43H
2O, 3.7g KH
2PO
4, 0.12g MgSO
4, 0.56g CaCl
2Behind the shake-flask culture 2 days, shake and obtain original seed in the bottle, be inoculated in the fermentor cultivation liquid with this original seed.
The fermentor tank liquid amount is operated by embodiment 1 liquid amount, adorns liquid by 40% liquid amount in the fermentor tank, every liter of central seed culture medium that uses 5 times of amounts of fermented liquid.5% inoculum size inoculation, temperature is controlled at 25～30 ℃, and rotating speed is 100～400rpm, fermentation culture 6d.
Extracting method is extracted from the separation chitosan according to the method described in the embodiment 1, and different is to adopt the NaOH of 2N to carry out the alkali lye processing.Use this seed culture medium combined ferment substratum, the highest dry cell weight 8.5g/L that gets gets chitosan 0.4g/L.Productive rate is 4.7%.
Embodiment 3
By the embodiment 1 described shake-flask culture that carries out, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is operated by embodiment 1 liquid amount, and different is in the fermented liquid with yeast extract as carbon source, and the amount that yeast extract adds is 25.0g/L.Press the method separation and Extraction product of embodiment 2, dry cell weight be 7.1g/L, chitosan 0.32g/L.Productive rate is 4.5%.
Embodiment 4
By the embodiment 3 described shake-flask culture that carry out, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is by the embodiment 1 liquid amount line operate of going forward side by side, and different is not have inorganic salt CaCl in the fermented liquid
2, press the method separation and Extraction product of embodiment 2, dry cell weight be 8.6g/L, chitosan 0.48g/L.Productive rate is 5.6%.
Embodiment 5
By the embodiment 1 described shake-flask culture that carries out, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is by the embodiment 4 liquid amounts line operate of going forward side by side, different is to regulate the pH value that sets with ammoniacal liquor in the ferment tank culturing process, press the method separation and Extraction product of embodiment 1, the adjusting of ammoniacal liquor makes dry cell weight reach 13.4g/L, obtain chitosan 0.86g/L, productive rate has reached 6.4%.
Embodiment 6
By the embodiment 1 described shake-flask culture that carries out, different is adds the peptone of 5g/L in shaking bottle, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is operated by implementing 5 liquid amounts, and different is in fermention medium, has increased Mg
2+Consumption and increased peptone.MgSO
4Consumption be 0.60g/L, the add-on of peptone is 10g/L.Press the method separation and Extraction product of embodiment 2, peptone, Mg
2+Adding make dry cell weight reach 16.8g/L, obtain chitosan 1.27g/L, productive rate has reached 6.6%.
Embodiment 7
By the embodiment 1 described shake-flask culture that carries out, different is adds the peptone of 5g/L in shaking bottle, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is operated by implementing 6 liquid amounts, and different is in fermention medium, has added CaCl
2, CaCl
2Consumption be the method separation and Extraction product of 0.56g/L by embodiment 2, peptone, Mg
2+, Ca
2+Adding make dry cell weight reach 19.2g/L, obtain chitosan 1.21g/L, productive rate has reached 6.3%.
Embodiment 8
By the embodiment 1 described shake-flask culture that carries out, different is adds the peptone of 5g/L in shaking bottle, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is by implementing the 6 liquid amounts line operate of going forward side by side, and different is in fermention medium, removes CaCl
2Adding but increased the adding of carbon source beef extract, its add-on is 20g/L.Press the method separation and Extraction product of embodiment 2, peptone, extractum carnis, Mg
2+Adding make dry cell weight reach 27.1g/L, obtain chitosan 1.84g/L, productive rate has reached 6.8%.
Embodiment 9
By the embodiment 1 described shake-flask culture that carries out, different is adds the peptone of 5g/L in shaking bottle, cultivate suitable concentration, be generally the gained mycelium dry weight when reaching between 10.5mg/ml～15.0mg/ml as the fermentor tank seed.
The fermentor tank liquid amount is by implementing the 6 liquid amounts line operate of going forward side by side, and different is in fermention medium, removes CaCl
2Adding but increased the adding of carbon source beef extract, yeast extract, its add-on is beef extract 10g/L, yeast extract is 25.0g/L.Press the method separation and Extraction product of embodiment 2, peptone, extractum carnis, yeast extract, Mg
2+Adding make dry cell weight reach 26.9g/L, obtain chitosan 1.8g/L, productive rate has reached 6.7%.
The result of above-mentioned example 1 to 9 shows: (1) PDA cultivate and through E2 liquid culture shaking table cultivate Actinomucor taiwanensis can unusual high yield chitosan.
(2) add MgSO among the E2
4, peptone, beef extract half complex medium can increase the biomass of hypha fermentation, also increase the productive rate of chitosan simultaneously.
(3) in the deacetylation process, adopt lower concentration NaOH (2N) to handle mycelium, can increase the productive rate that from thalline, extracts chitosan.
Claims (5)
1. the preparation method of chitosan in the fungal cell wall may further comprise the steps:
(1): Actinomucor taiwanensis class fungal bacterial strain is drawn plate, shake-flask culture, fermentor cultivation, and the bacterium liquid that obtains is through centrifugal acquisition wet thallus;
(2): from mycelium, separate and extract chitosan.
2. the preparation method of chitosan according to claim 1 is characterized in that, it is the PDA substratum that stroke plate in the step (1) is cultivated used substratum, and it consists of: potato 300g/L, glucose 20g/L, agar 15g/L.
3. the preparation method of chitosan according to claim 1 is characterized in that, described in the step (1) the used basic medium of shake-flask culture be the E2 substratum, it consists of NaNH
4HPO
44H
2O:3.5g/L; K
2HPO
43H
2O:7.5g/L; KH
2PO
4: 3.7g/L; MgSO
4: 0.12g/L; Carbon source: carbon content 20g/L; Described carbon source comprises glucose, beef extract, yeast extract; Shake-flask culture adds the CaCl of 0.56g/L on the basis of E2
2, the peptone of 0～5g/L.
4. the preparation method of chitosan according to claim 1 is characterized in that, the initial pH value of the fermention medium that fermentor cultivation adopted described in the step (1) is 3～8; Its composition comprises the MgSO of 20～200g/L carbon source, 10～80g/L nitrogenous source, 1.0～20g/L yeast extract, 0.01～40g/L
4, 0～5.6g/L CaCl
2, described carbon source comprises glucose, beef extract; Nitrogenous source comprises peptone, NaNH
4HPO
44H
2O, nitrogen water.
5. the preparation method of chitosan according to claim 1, it is characterized in that step (2) is isolated behind the mycelium filter and done, handled cell 15～30 minutes in 110～130 ℃, 1～2N NaOH, centrifugation solids and with distilled water and washing with alcohol is dried and is pulverized solid; Hydrochloric acid soln with 1～4% is handled the throw out of washing, makes its pH value between 5.0 to 6.5, and in 90～100 ℃ of insulations 10～14 hours, centrifugation produced acid supernatant liquor and sour insoluble precipitate; With 2N NaOH supernatant liquor is adjusted to the pH value more than 10, thereby is settled out cotton-shaped chitosan, centrifugal, lyophilize gets product.
Priority Applications (1)
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|---|---|---|---|
| CNA2009100459238A CN101481718A (en) | 2009-01-22 | 2009-01-22 | Preparation of chitosan in fungal cell wall |
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|---|---|---|---|
| CNA2009100459238A CN101481718A (en) | 2009-01-22 | 2009-01-22 | Preparation of chitosan in fungal cell wall |
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| Publication Number | Publication Date |
|---|---|
| CN101481718A true CN101481718A (en) | 2009-07-15 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104379001A (en) * | 2012-06-25 | 2015-02-25 | 戴姆有限公司 | Fat binder obtained from biomass resulting from beer production |
-
2009
- 2009-01-22 CN CNA2009100459238A patent/CN101481718A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104379001A (en) * | 2012-06-25 | 2015-02-25 | 戴姆有限公司 | Fat binder obtained from biomass resulting from beer production |
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Application publication date: 20090715 |