CN1260253C

CN1260253C - Method for extracting and separating chitosan from waste citric acid mycelium

Info

Publication number: CN1260253C
Application number: CN 200410060983
Authority: CN
Inventors: 杜予民; 蔡俊; 杨建红; 邱雁临
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2004-10-19
Filing date: 2004-10-19
Publication date: 2006-06-21
Anticipated expiration: 2024-10-19
Also published as: CN1629197A

Abstract

The present invention relates to a method for extracting and separating chitosan from a citric acid waste mycelium. A waste mycelium as a byproduct in the process of the production of citric acid is treated in a colloidal mill for 1 to 1.5 hours; the mycelium treated by the colloidal mill is put into 0.2 M of Na2 HPO4-NaH2 PO4 buffer solution, and the pH value of the Na2 HPO4-NaH2 PO4 buffer solution is from 6.0 to 8.0; neutral proteinase generated by bacillus subtilis is added and continuously stirred at the temperature of 50 to 55DEG C for 2 to 3 hours; solid matters are collected and put into 50 m of Tris-HCl buffer solution, and the pH value of the Tris-HCl buffer solution is from 6.0 to 8.5; chitin is added to remove aceto enzyme and continuously stirred at the temperature of 50 to 55DEG C for 6 to 8 hours, and solid matters are collected; the chitosan is extracted with 10 to 30 g/liter of acetic acid solution, the pH value of the chitosan is adjusted between 7.5 and 8.0 by NaOH, and the chitosan is precipitated and obtained through centrifugal separation; precipitates are washed with deionized water and 75 to 95% of ethanol to obtain the chitosan after the precipitates are centrifugally separated and dried. Compared with the traditional acid and base hydrolyzing extraction method, the product obtained by the present invention has the advantages of higher molecular weight, higher extraction yield and wider product application range; in addition, compared with the traditional acid and base hydrolyzing extraction method, the technology has the advantages of less wastewater generation in the extraction process, and environmental pollution reduction.

Description

The method of extraction separation chitosan from the lemon acid waste mycelium

Technical field

The present invention relates to the method for extraction separation chitosan from the lemon acid waste mycelium.

Background technology

Chitosan and derivative thereof can be widely used in many fields such as medicine, makeup, food, chemical industry, agricultural, biotechnology, macromolecular material.The chitosan of selling on the current market is mainly from the shells of shrimp, crab etc., mature preparation process, suitability for industrialized production, but this technology exists many inherent shortcomings such as otherness of the places of origin of raw materials and dispersiveness, seasonality, quality, and these have brought difficulty for extractions of chitin.On the raw material sources of chitin and chitosan production, people have ignored another important channel, be chitin and the chitosan in the microorganism, the content in the fungal cell walls such as its tangible Ascomycetes, Basidiomycetes, algae guiding principle and deuteromycetes is very considerable (chitin content be dry cell weight 20%～22%).The chitosan that from mycelium, extracts with compare very approachingly with the chitosan of shrimp shell production, but it is specially adapted to contain the more waste water of heavy metal ion to the adsorptive power of metal ion (as Cu, the Hg) chitosan much larger than shrimp shell source; And antibiotic (to milk-acid bacteria, the withered grass bacterium etc.) of the food preservative made from the mycelia chitosan can force rate shrimp shell source chitosan high 1～2 times.

Citric acid factory is the specialized factory that produces citric acid, in the citric acid production process, has a large amount of by product waste thallus to produce, the chitin that contains larger proportion in these waste thallus, if these waste thallus are discarded, not only can cause the waste of resource, and can bring serious environmental to pollute.Chitin and chitosan content from various microorganisms, the chitin content of aspergillus niger is higher, the used bacterial strain of citric acid fermentation is an aspergillus niger, dried with potato be in the fermenting process of raw material, 1 ton of citric acid of every production can produce the dried filter residue about 500kg, wherein contains the dry mycelium about 160kg, and promptly mycelial content is about 20g/L in fermented liquid, chitosan is by 20% in the black-koji mould filament, and its chitosan productive rate can reach 4.0g/L.China is a large-scale citric acid production state, and manufacturer is also concentrated relatively, and can produce nearly 1,000,000 tons waste thallus every year, and the discharging of these thalline not only can bring serious environmental to pollute, and can cause the huge waste of chitosan raw materials for production.

Abroad, more and more about the report of chitin extraction from fungi and chitosan, but do not see the bibliographical information that utilizes the fermentation plant waste thallus to prepare chitin and chitosan.At home, Lou Yongjiang adopts milling treatment of colloid citric acid factory waste residue, and hyphostoma particle is attenuated, and water-soluble substances is stripping to greatest extent, adopts concentrated base staging treating chitin extraction and chitosan simultaneously, and the molecular weight of products obtained therefrom is 7.89 * 10 ⁴, deacetylation is 96.82%, the yield of the relative dry mycelium of chitosan is 3.35%; Employing soda acid alternative methods such as Zhao Jilun take off the albumen of citric acid fermentation factory waste thallus, utilize the high alkali liquid deacetylation to prepare chitosan then; People such as Cao Jian study chitin and the chitosan that adopts in electrolytic process and extraction method extraction at the intermittence citric acid fermentation factory waste thallus.But the molecular weight of these research products obtained therefroms is low, and extract yield is low, and leaching process can bring more environmental pollutant.

Summary of the invention

The invention provides a kind of from the lemon acid waste mycelium method of extraction separation chitosan, chitosan molecule amount, extract yield that this method is extracted are higher, and be low to the pollution that environment brings.

The applied range of product, and this technology is compared the waste water that the leaching process generation is less with traditional acid and alkali hydrolysis extraction method.

Technical scheme provided by the invention is: the method for extraction separation chitosan from the lemon acid waste mycelium, by product waste mycelia in the citric acid production process was handled in colloidal mill 1～1.5 hour, the mycelium that to cross with milling treatment of colloid is by 1 gram mycelium: 2-5mL Na ₂HPO ₄-NaH ₂PO ₄The ratio of buffered soln, the Na that inserts 0.2M pH6.0～8.0 ₂HPO ₄-NaH ₂PO ₄In the buffered soln, add the neutral protease that producing bacillus subtilis is given birth to, in 50～55 ℃ of continuously stirring 2～3 hours, collect solid substance, restrain solid substance in 1: the ratio of 2-5mLTris-HCl buffered soln, insert in the Tris-HCl buffered soln of 50mM pH6.0～8.5, add chitin deacetylase, in 50～55 ℃ of continuously stirring 6～8 hours, collect solid substance, rise the acetic acid extraction chitosan with 10～30g/, transfer pH7.5～8.0 precipitations with NaOH, centrifugation gets chitosan, and throw out promptly gets chitosan with deionized water and 75～95% washing with alcohol after the centrifugation drying.

The consumption of the neutral protease that above-mentioned producing bacillus subtilis is given birth to is 0.05～0.30wt% of waste mycelia.

Above-mentioned chitin deacetylase is the chitin deacetylase that blue colter mould (Absidia coerulae) produces, and the consumption of chitin deacetylase is that every hectogram waste mycelia adds 800～2000units.

The present invention adopts colloidal mill smudge cells and plurality of enzymes continuous action to combine and handles citric acid fermentation factory waste thallus; make that material in the cytolemma discharges, the protein in the cell walls is degraded removal, slough the N-ethanoyl of chitin in the cell walls etc., extracts chitosan at last.The molecular weight of this technology products obtained therefrom, extract yield be than traditional acid and alkali hydrolysis extraction method height, and this technology compares with traditional acid and alkali hydrolysis extraction method, and leaching process produces less waste water, and environmental pollution will significantly reduce.

Embodiment

(raw material is from the Hunan extra large petrochemical complex of silver company limited with fresh citric acid factory waste thallus.Because it is bacterial classification that the producer of present domestic production citric acid all adopts aspergillus niger, and technology is similar substantially, so the waste thallus of different manufacturers composition is similar, and the result is as broad as long substantially.) repeated treatments 1～1.5 hour in colloidal mill, then, join the Na of 0.2MpH6.0～8.0 in the ratio of 1: 3 (g/ml) ₂HPO ₄-NaH ₂PO ₄Buffered soln in; add the neutral protease deproteinated that 0.17% (g/g waste mycelia) producing bacillus subtilis is given birth to; change 20 minutes centrifugal collection solid substances in 12000; wash three times; ratio in 1: 3 (g/ml) joins in the buffered soln of 50mM pH6.0～8.5Tris-HCl; add 1200units% (1unit/g waste mycelia; 1unit is meant that the poly-N-acetylglucosamines of every milligram of enzyme per minute catalysis six produce 1 μ mol ethanoyl) the chitin deacetylase that produces of Absidia coerulae (blue colter is mould) carry out deacetylation; change 20 minutes centrifugal collection solid substances in 12000; water washing three times; with 1～3% (g/ml) acetic acid extraction chitosan; transfer pH7.5～8.0 precipitations with NaOH; centrifugation gets chitosan; throw out respectively washs three times with deionized water and 75～95% (ml/ml) ethanol, promptly gets chitosan after the centrifugation drying.

According to the said extracted process, proteinic decreasing ratio is 58.9% in the raw material, the extraction yield of chitosan is 50.0%, molecular-weight average is 267.97kDa, the content of chitosan is 84.4% in the product, deacetylation is 73.6%, (proteinic decreasing ratio is 62.3% with the chemical extraction method, the extraction yield of chitosan is 41.7%, molecular-weight average is 84.04kDa, the content of chitosan is 82.7% in the product, and deacetylation is 76.8%) to compare, the extraction yield of Enzymatic Extraction products obtained therefrom, molecular weight, chitosan content all extract high than chemical method.

Claims

1. the method for extraction separation chitosan from the lemon acid waste mycelium is characterized in that: the by product waste mycelia in the citric acid production process was handled in colloidal mill 1～1.5 hour, and the mycelium that will cross with milling treatment of colloid is by 1 gram mycelium: 2-5mL Na ₂HPO ₄-NaH ₂PO ₄The ratio of buffered soln, the Na that inserts 0.2M pH6.0～8.0 ₂HPO ₄-NaH ₂PO ₄In the buffered soln, add the neutral protease that producing bacillus subtilis is given birth to, in 50～55 ℃ of continuously stirring 2～3 hours, collect solid substance, restrain solid substance in 1: the ratio of 2-5mLTris-HCl buffered soln, insert in the Tris-HCl buffered soln of 50mM pH6.0～8.5, add chitin deacetylase, in 50～55 ℃ of continuously stirring 6～8 hours, collect solid substance, rise the acetic acid extraction chitosan with 10～30g/, transfer pH7.5～8.0 precipitations with NaOH, centrifugation gets chitosan, and throw out promptly gets chitosan with deionized water and 75～95% washing with alcohol after the centrifugation drying; The consumption of the neutral protease that producing bacillus subtilis is given birth to is the 0.17wt% of waste mycelia; The consumption of chitin deacetylase is that every hectogram waste mycelia adds 1200units.