KR20000001915A - Method of manufacturing oligo chitosan - Google Patents
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- KR20000001915A KR20000001915A KR1019980022394A KR19980022394A KR20000001915A KR 20000001915 A KR20000001915 A KR 20000001915A KR 1019980022394 A KR1019980022394 A KR 1019980022394A KR 19980022394 A KR19980022394 A KR 19980022394A KR 20000001915 A KR20000001915 A KR 20000001915A
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- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
Description
본 발명은 키토산을 이용한 올리고 키토산의 제조방법에 관한 것이다. 보다 상세하게는 주요 중합도가 5-10인 올리고키토산의 제조방법에 관한 것이다.The present invention relates to a method for producing oligo chitosan using chitosan. More specifically, the present invention relates to a method for preparing oligochitosan having a main polymerization degree of 5-10.
키토산의 전구체인 키틴은 아미노당으로 구성되어 있는 다당류의 일종으로서, 산업적으로 이용되는 원료는 게나 새우이다. 예전에는 이들의 껍질에 붙어 있는 단백질이 부패하여 악취 뿐만 아니라 그들의 강한 결합으로 인하여 용해성이 좋지 않기 때문에 산업 폐기물로 취급되어 왔으나 최근에는 여러 분야에서의 소재로 주목을 받고 있는 관계로 폐기물 처리 문제와 천연자원의 이용 면에서 키틴에 대한 많은 연구가 진행 중에 있다.Chitin, a precursor of chitosan, is a kind of polysaccharide composed of amino sugars. The raw material used industrially is crab or shrimp. In the past, the proteins attached to their shells were decayed, and they were treated as industrial wastes because of their poor solubility due to their strong binding as well as odors, but recently they have been attracting attention as materials in various fields. Much research is underway on chitin in terms of resource use.
키토산은 키틴을 탈아세틸화시킨 물질로서 분자량에 따라 동맥경화와 심장병을 예방하는 탈콜레스테롤 작용(Lipids, 1988, 23, 187-91), 간장기능의 강화작용, 상처치유 및 세포재생 효과와 세균 억제효과 등의 효능·효과(Advances in chitin and chitosan, 5th international conference on chitin and chitosan held in princeton)가 있는 것으로 알려져 있다. 그러나 키토산은 이러한 여러 생리활성 및 물리화학적 특성을 지니고 있지만 키토산을 녹일 수 있는 용매는 몇 가지 용매를 제외하고는 극히 제한되어 있다.Chitosan is a deacetylation of chitin, a decholesterol action (Lipids, 1988, 23, 187-91) that prevents arteriosclerosis and heart disease according to its molecular weight, hepatic function enhancement, wound healing and cell regeneration and bacterial inhibition It is known that there are advances in chitin and chitosan, 5th international conference on chitin and chitosan held in princeton. However, although chitosan has various physiological activities and physicochemical properties, solvents capable of dissolving chitosan are extremely limited except for a few solvents.
최근에는 키토산을 화학적 방법 및 효소적 방법에 의하여 가수분해시켜 저분자량의 올리고키토산을 제조하는 방법이 개발됨에 따라서 이의 효능·효과가 알려지고 있으며, 용도개발에 대한 연구가 활발히 진행되고 있다. 특히 현재까지 밝혀진 연구 결과에 의하면 올리고키토산중 효능을 나타낼 수 있는 당은 중합도가 5이상이 되야 한다. 그러나 현재 화학적 방법 및 효소적 방법에 의하여 제조되는 올리고키토산은 중합도가 2-4인 것이 대부분이거나 또는 중합도가 30이상이다.Recently, as a method of producing low molecular weight oligochitosan by hydrolyzing chitosan by chemical and enzymatic methods has been developed, its efficacy and effects are known, and research on the use development is being actively conducted. In particular, according to the research results so far, the oligochitosan can be effective in the degree of polymerization of 5 or more. However, currently, oligochitosan prepared by chemical and enzymatic methods mostly have a degree of polymerization of 2-4 or a degree of polymerization of 30 or more.
기존의 올리고 키토산을 제조하는 방법으로서는 농염산을 이용한 가수분해 방법, HF를 이용한 방법 및 NaNO2를 이용한 방법이 있다. 그러나 이러한 화학적 방법들은 반응조건이 까다로울 뿐만 아니라 불순물의 제거가 어렵기 때문에 대량생산이 어렵고, 분리에 따른 비용이 많이 소요된다. 뿐만 아니라 화장품 및 식품에의 응용에는 어려움이 있다.Existing methods for producing oligo chitosan include hydrolysis using concentrated hydrochloric acid, HF, and NaNO 2 . However, these chemical methods are difficult to mass-produce because of the difficult reaction conditions and the difficulty of removing impurities. In addition, there are difficulties in application to cosmetics and food.
최근에는 키토산나아제를 이용한 효소적 가수분해 방법에 대한 많은 연구가 이루어지고 있으나 이 방법에 의한 대부분의 올리고키토산은 중합도가 2-4당이 주를 이루며, 아직은 키토산나아제의 활성 및 기질에 대한 특이성에 대한 것은 연구 단계에 있다. 또한 이러한 키토산나아제의 가격이 상당히 비싸기 때문에 대량생산에의 적용에 많은 어려움이 수반된다.Recently, many studies on enzymatic hydrolysis using chitosanase have been conducted, but most of the oligochitosans by this method have a degree of polymerization of 2-4 sugars. Specificity is in the research stage. In addition, since the price of such chitosanase is quite expensive, it is accompanied by many difficulties in the application to mass production.
본 발명자는 상기와 같은 문제점을 해결하기 위하여 예의 연구 검토한 결과, 구조적으로 유사한 셀룰로오즈(cellulose)의 가수분해 효소인 셀룰라제(cellulase)를 이용하여 2당에서 10당의 올리고 키토산을 분리 및 제조하는 방법을 개발하였으며 이를 대량생산에 적용하여 순도 높은 올리고 키토산을 생산하고 또한 공정을 최적화함으로써 수율을 극대화시키는데 성공함으로써 본 발명을 완성하였다.The present inventors have diligently studied to solve the above problems, and as a result, a method for separating and preparing oligo chitosan of 10 to 10 sugars using cellulase, a structurally similar cellulose hydrolase The present invention was completed by applying this product to mass production to produce high purity oligo chitosan and optimizing the process to maximize the yield.
본 발명은 주요 중합도가 5-10인 올리고키토산의 제조 및 분리하는 방법과 이의 대량생산방법에 기초를 두고 있다.The present invention is based on the method for preparing and separating oligochitosan having a major degree of polymerization of 5-10 and its mass production method.
본 발명을 보다 상세히 설명하면 키틴을 40-50%(w/v) 수산화나트륨수용액에 침적시킨후 90-120℃에서 탈아세틸화시켜 키토산을 얻고, 이후 충분히 수세된 저분자량의 키토산을 이용하여 묽은 염산용액과 혼합하여 키토산 용액을 제조한다(고분자량의 키토산은 용매에 대한 용해도가 저분자량의 키토산과 비교하여 현저히 낮으며 용액의 점도가 높기 때문에 교반의 어려움이 있으며 이에 따른 수율의 저하를 가져오므로 올리고 키토산을 제조하기에는 저분자량의 키토산을 이용함이 적당하다). 키토산이 완전히 용해된후 용액의 온도를 45-50℃부근으로 조절하고 이후 알칼리수용액(소듐 바이카보네이트 수용액)을 서서히 첨가하여 pH를 4.5-5.0로 조절한다. 이는 셀루라제의 최적 활성을 띄는 조건이다. 본 발명에서 사용한 셀룰라제는 트리코더마 비리드(Trichoderma viride)에서 기원된 것으로써 엔도(endo)형이며, 가수분해반응에 의한 올리고 키토산의 분자량은 셀룰라제의 기질 즉, 키토산에 대한 농도와 반응시간, 용액의 pH에 따라 좌우된다.In more detail, the chitin was deposited in 40-50% (w / v) sodium hydroxide solution and deacetylated at 90-120 ° C. to obtain chitosan, which was then diluted using sufficiently washed low molecular weight chitosan. A chitosan solution is prepared by mixing with a hydrochloric acid solution. (The high molecular weight chitosan has a low solubility in solvents compared to a low molecular weight chitosan and a high viscosity of the solution, making it difficult to stir, resulting in a decrease in yield. It is suitable to use low molecular weight chitosan to prepare oligochitosan. After the chitosan is completely dissolved, the temperature of the solution is adjusted to around 45-50 ° C., and then the pH is adjusted to 4.5-5.0 by slowly adding an alkaline aqueous solution (aqueous sodium bicarbonate solution). This is the condition that shows the optimal activity of cellulase. Cellulase used in the present invention originated from Trichoderma viride (endorich) is an endo (endo) type, the molecular weight of the oligo chitosan by the hydrolysis reaction is the substrate of the cellulase, that is, the concentration and reaction time for chitosan, It depends on the pH of the solution.
효소의 농도는 기질 대비 0.1-3%로 사용되며 시간은 6-48시간으로 조절하는 것이 적절하다. 효소실활을 위하여 80-100℃로 가열하여 미세여과를 통하여 효소를 제거하고, 이후 결정화 공정을 통하여 물을 제거한후 고순도의 미황색 올리고 키토산을 얻는다. 반응시간이 6시간 이하일 경우 최종제품에서 단백질의 함량이 높아져 적합하지 않고 48시간이 넘을 경우 너무 심한 분해로 인한 이당류, 삼당류의 분포가 많아지며 제품의 성상에도 불리하다.Enzyme concentration is used as 0.1-3% of the substrate and the time is appropriately adjusted to 6-48 hours. For enzymatic deactivation, the enzyme was removed by microfiltration by heating to 80-100 ° C., and then water was removed through crystallization to obtain a pale yellow oligo chitosan of high purity. If the reaction time is less than 6 hours, the content of protein in the final product is not suitable, and if it is more than 48 hours, the distribution of disaccharides and trisaccharides due to too much decomposition increases, and is also disadvantageous in the properties of the product.
이하 실시예를 통해 본 발명을 보다 상세히 설명한다.The present invention will be described in more detail with reference to the following examples.
(비교예 1)(Comparative Example 1)
본 실험은 화학적 가수분해방법에 의한 올리고키토산의 제조방법이다.This experiment is a method for producing oligochitosan by chemical hydrolysis.
저분자량 키토산을 묽은 염산용매에 용해시켜 NaNO2수용액(10% w/v)을 서서히 첨가시켜 반응을 진행시켰다. 이때 NaNO2의 양은 키토산과의 몰비를 계산하여 첨가한다. 본 비교예에서는 올리고 키토산의 DP(degree of polymerization)2와 DP6을 기준으로 제조하였다. 반응 온도는 6-10℃를 유지시켰으며 반응시간은 6시간으로 정하였다. 반응 종료시 최종 pH는 4-5이며 이후 중화반응을 거쳐 여과후 NaBH4를 서서히 첨가시켰다. 이것은 용액중 올리고 키토산의 말단기에 존재하는 알데히드기를 환원시키기 위함이다. 이후 2-3시간 동안 충분히 교반후, 분리·정제를 거쳐 올리고 키토산을 회수하였다.The low molecular weight chitosan was dissolved in dilute hydrochloric acid solvent, and NaNO 2 aqueous solution (10% w / v) was added slowly to proceed with the reaction. At this time, the amount of NaNO 2 is added by calculating the molar ratio with chitosan. In this comparative example was prepared based on DP (degree of polymerization) 2 and DP6 of oligo chitosan. The reaction temperature was maintained at 6-10 ℃ and the reaction time was set to 6 hours. At the end of the reaction, the final pH was 4-5, followed by neutralization, followed by gradual addition of NaBH 4 . This is to reduce the aldehyde group present in the terminal group of the oligo chitosan in the solution. After sufficiently stirring for 2-3 hours, the oligo chitosan was recovered through separation and purification.
회수된 올리고키토산을 완전히 건조시킨 후 올리고키토산 수용액을 제조하여 박층 크로마토그래피로 분자량 분포를 확인하였다. 박층 크로마토그래피의 플레이트는 실리카겔이 도포되어 있는 것을 사용하였으며 전개는 이소프로판올, 피리딘, 아세트산, H2O를 10 : 4 : 6 : 10의 중량비로 혼합한 용매를 사용하였으며 발색시약은 닌히드린 시액을 사용하였다.After the dried oligochitosan was completely dried, an aqueous solution of oligochitosan was prepared, and molecular weight distribution was confirmed by thin layer chromatography. The thin layer chromatography plate was coated with silica gel, and the development was carried out using a solvent in which isopropanol, pyridine, acetic acid and H 2 O were mixed at a weight ratio of 10: 4: 6: 10. It was.
전개결과 DP2와 DP6 모두 전개가 안되었다. 따라서 DP2, DP6의 분자중합도는 실제로는 중합도 6을 훨씬 넘는 것으로 보인다.As a result of development, neither DP2 nor DP6 was deployed. Therefore, the degree of molecular polymerization of DP2 and DP6 actually seems to be much higher than degree of polymerization 6.
(실시예 1)(Example 1)
저분자량 키토산, 농염산, 이온수의 질량비를 3:2:95의 비율로 혼합하여 키토산 3%용액으로 제조하였다. 키토산을 완전히 용해시킨후 온도를 50℃로 고정시키고, 이 상태에서 소듐 바이카보네이트 수용액(10w/v%)을 서서히 첨가하여 pH를 4.5-5.0로 맞추었다. 이 조건은 셀룰라제의 최적 활성 조건이다. 셀룰라제를 5-10w/v%수용액으로 제조한 후 키토산에 대하여 0.1%의 양을 키토산용액에 가한후 서서히 교반하여 6시간 반응시켰다. 이후 반응액을 90-100℃에서 20-30분 동안 가열하여 효소를 실활시켜 반응을 종료시킨후 규조토여과를 하여 불순물을 제거하였다. 여액을 1/5이상 농축한후 95% 에탄올을 농축액의 4-5배부피 가하여 올리고 키토산을 회수하였다.The mass ratio of low molecular weight chitosan, concentrated hydrochloric acid and ionized water was mixed in a ratio of 3: 2: 95 to prepare a chitosan 3% solution. After completely dissolving the chitosan, the temperature was fixed at 50 ° C, and in this state, an aqueous sodium bicarbonate solution (10w / v%) was slowly added to adjust the pH to 4.5-5.0. This condition is the optimal activity condition of the cellulase. Cellulase was prepared in 5-10 w / v% aqueous solution, and 0.1% of chitosan was added to the chitosan solution, followed by agitation for 6 hours. Thereafter, the reaction solution was heated at 90-100 ° C. for 20-30 minutes to inactivate the enzyme to terminate the reaction, and then, diatomaceous earth filtration was performed to remove impurities. After concentrating the filtrate 1/5 or more, 95% ethanol was added 4-5 times the volume of the concentrate to recover the oligo chitosan.
이후 완전히 건조된 올리고 키토산을 수용액으로 제조한후 박층크로마토그래피로 전개시킨 결과, 전개가 안되었다(전개조건은 비교예 1과 동일하다). 따라서 이 조건에서 제조된 올리고 키토산의 중합도는 DP10이상인 것으로 예상된다.After fully dried oligo chitosan was prepared as an aqueous solution and then developed by thin layer chromatography, the development was not possible (development conditions are the same as in Comparative Example 1). Therefore, the degree of polymerization of oligo chitosan prepared under these conditions is expected to be at least DP10.
(실시예 2)(Example 2)
키토산에 대한 셀룰라제의 농도를 1%로 한 것을 제외한 나머지 반응조건은 실시예 1과 동일하게 하였다.The reaction conditions were the same as in Example 1 except that the concentration of cellulase to chitosan was 1%.
회수된 올리고 키토산을 박층 크로마토그래피로 전개시킨 결과 전개가 안되었다. 실시예 1과 마찬가지로 중합도가 DP10이상인 것으로 예상된다.The recovered oligo chitosan was developed by thin layer chromatography, resulting in no development. As in Example 1, the degree of polymerization is expected to be at least DP10.
(실시예 3)(Example 3)
효소를 키토산에 대하여 3% 첨가하고, 나머지 반응조건은 실시예1과 동일하게 하였다.The enzyme was added 3% to chitosan, and the remaining reaction conditions were the same as in Example 1.
박층 크로마토그래피로 전개시킨 결과 DP2-10까지 고른 분포를 나타내었다. 박층 크로마토그래피 전개 결과는 아래와 같다.Thin layer chromatography showed even distribution up to DP2-10. The thin layer chromatography development results are as follows.
(실시예 4)(Example 4)
효소를 키토산에 대하여 3% 첨가하고 반응시간을 15, 24, 48시간으로 변화시켜 가며 올리고 키토산을 제조하였다.The enzyme was added to the chitosan 3% and the reaction time was changed to 15, 24, 48 hours to prepare an oligo chitosan.
제조된 각각의 올리고키토산에 대하여 박층 크로마토그래피를 이용하여 분자량 분포를 조사하였다. 전개 결과 15, 24, 48시간의 분자량은 6시간동안 반응시킨 올리고 키토산에 비하여 DP10 부근이 희미한 것으로 보아 DP10이상의 분자가 상대적으로 적은 것으로 예상된다.For each oligochitosan prepared, molecular weight distribution was investigated using thin layer chromatography. As a result of the development, the molecular weight of 15, 24, 48 hours is expected to be relatively near DP10 compared to oligo chitosan reacted for 6 hours.
박층 크로마토그래피 전개 결과는 아래와 같다.The thin layer chromatography development results are as follows.
이상의 결과를 바탕으로 공장생산에 적용할 경우 다음의 3가지 문제가 발생할 수 있다.Based on the above results, the following three problems can occur when applied to factory production.
첫째, 키토산의 농도가 3%이기 때문에 배치(batch)당 생산수율이 3%를 넘을수 없다. 따라서 생산수율이 현저히 낮기 때문에 제조원가면에서 다른 제품에 비하여 경쟁력이 없다.First, because the concentration of chitosan is 3%, the yield per batch cannot exceed 3%. Therefore, the production yield is significantly lower, so it is not competitive in comparison with other products in terms of manufacturing cost.
둘째, 제조된 올리고키토산의 분리,정제공정중 효소실활공정후 여과공정을 거친 여액에서 올리고키토산을 분리하기 위하여 에탄올을 첨가하여 올리고키토산의 용해도를 저하시켜 침전을 형성시킨다. 그러나 올리고키토산은 분자량이 작기 때문에 침전에 사용되는 에탄올의 양은 상당한 양을 필요로 한다. 따라서 이에 따른 원료의 비용이 상승하여 제조원가에 상당한 영향을 미치게 된다.Second, ethanol is added to separate oligochitosan from the filtrate after enzymatic inactivation during the separation and purification of the prepared oligochitosan, thereby lowering the solubility of the oligochitosan to form a precipitate. However, since oligochitosan has a low molecular weight, the amount of ethanol used for precipitation requires a considerable amount. As a result, the cost of raw materials rises, which significantly affects manufacturing costs.
셋째, 키토산의 용해액에 완충액(buffer)인 탄산수소나트륨수용액을 첨가하여 pH를 조절하는데 이때 해리된 나트륨이온과 염소이온이 결합하여 올리고키토산의 생성시 염을 형성하여 같이 침전되게 된다. 따라서 생성된 올리고키토산의 맛을 보면 짠맛을 강하게 느낄 수 있다. 이는 올리고키토산의 순도를 떨어뜨릴 수 있다.Third, the pH is adjusted by adding a buffered sodium hydrogen carbonate solution to the solution of chitosan. At this time, the dissociated sodium ions and chlorine ions are combined to form salts in the formation of oligochitosan to precipitate. Therefore, if you look at the taste of the oligochitosan produced, you can feel the saltiness strongly. This can reduce the purity of oligochitosan.
위의 문제점은 다음과 같은 키토산의 특성을 이용함으로써 해결할 수 있다.The above problem can be solved by using the following chitosan properties.
키토산의 말단기에 있는 아민기는 키토산이 염산수용액에 용해될 때 -NH3 +의 형태로 용해된다. 따라서 키토산이 용해됨에 따라 자연히 pH는 상승하게 된다. 이런 원리를 이용하여 키토산의 용해도한계범위에서 함량을 높이면 완충액(buffer)를 사용하지 않고 pH를 조절할 수 있다. 이에 따라서 생산수율을 높일 수 있으며, 완충액(buffer)를 사용하지 않음으로써 염의 생성을 방지하여 제품의 순도를 향상시킬 수 있다.Amine group in the terminal groups of the chitosan as chitosan is soluble in the hydrochloric acid solution is dissolved in the form of -NH 3 +. Therefore, the pH naturally rises as chitosan is dissolved. Using this principle, it is possible to adjust the pH without using a buffer by increasing the content within the solubility limit of chitosan. As a result, the production yield can be increased and the purity of the product can be improved by preventing the formation of salt by using no buffer.
다음의 실시예를 통하여 본 발명을 보다 상세히 설명한다.The present invention will be described in more detail with reference to the following examples.
(실시예 5)(Example 5)
염산 2.05%수용액을 제조한후 키토산을 서서히 첨가하여 용액의 pH를 50℃에서 4.7-4.8을 유지시켰다. 이때 키토산의 농도는 4.67%이다. 이후 셀룰라제를 기질에 대하여 3%첨가한 후 15시간동안 가수분해를 시켰다. 이 조건에서 셀룰라제는 최적의 활성을 띄어 DP 6-10의 올리고키토산을 생성할 수 있게 된다. 가수분해후 효소를 실활시킨후 제거하고 농축을 하여 부피를 반으로 줄인후에 분무건조를 하여 미세한 분말의 올리고키토산을 얻었다. 이때 분무건조 입구의 온도는 120℃로 조절하였는데 입구온도가 너무 높게되면 제품이 갈변화되기 때문에 분무건조기입구의 온도를 적절하게 조절함은 제품의 질을 결정함에 중요한 요인이 된다. 이와같이 수득된 올리고키토산에서는 짠맛을 전혀 느낄 수 없었다.After preparing 2.05% hydrochloric acid solution, chitosan was slowly added to maintain the pH of the solution at 50 ° C. at 4.7-4.8. At this time, the concentration of chitosan is 4.67%. Cellulase was then added to the substrate 3% and hydrolyzed for 15 hours. Under these conditions, cellulase has the optimal activity to produce oligochitosan of DP 6-10. After hydrolysis, the enzyme was inactivated, then removed, concentrated to reduce the volume in half, and then spray dried to obtain a fine powder of oligochitosan. At this time, the temperature of the spray drying inlet was adjusted to 120 ° C. However, if the inlet temperature is too high, the product changes brown, so that the proper control of the spray dryer inlet temperature is an important factor in determining the product quality. In the oligochitosan thus obtained, no salty taste was felt.
위의 실시예 1-4에서는 올리고키토산을 회수하기위해서 1/5농축을 한후 4-5배의 에탄올을 가하여 침전시키며, 미립자의 분말을 얻기위해 다시 분쇄공정을 거친후 수분을 줄이기 위해 건조공정을 해야하는 번거로움과 동시에, 공정의 복잡화로 인한 제조경비가 상승하게 된다. 그러나 실시예 5에서와 같이 분무건조공정을 통하여 이러한 여러 공정을 한 공정으로 단순화시켜 제조경비의 상승을 줄일 수 있을 뿐만 아니라 최종제품의 수율도 에탄올을 이용한 공정보다 높은 80-90%로 높일 수 있다.In Example 1-4, to recover the oligochitosan, concentrate 1/5 to recover 4-5 times of ethanol, and then settle, and after drying again to obtain fine powder, dry process is performed to reduce moisture. At the same time, the manufacturing cost increases due to the complexity of the process. However, as in Example 5, the spray drying process simplifies these processes to a single process to reduce the manufacturing cost and increases the yield of the final product to 80-90% higher than the process using ethanol. .
(실시예6)Example 6
실시예 5에서와 같은 비율로 키토산을 용해시켜 50℃에서 pH를 4.7-4.9로 조절한후 셀룰라제를 2%첨가하여 4시간동안 부분가수분해시킨 다음 염산 2%수용액을 서서히 가하여 용액의 pH를 1-2정도로 맞춘후 키토산을 가하여 pH를 4.7-4.9로 상승시켰다. 이때 pH 4.7-4.9까지 올리는데 소요된 키토산의 양은 초기 투여된 키토산의 약 60-70%정도이다. 이후 셀루라제를 3%첨가하여 15시간동안 가수분해를 진행시킨 다음 효소를 실활시켜 반응을 끝낸후 효소를 제거하고 농축공정을 거치지 않고 바로 분무건조를 하였다. 고형분의 함량이 8%이상이면 분무건조를 위한 조건이 되므로 농축을 할 필요가 없는 것이다. 이 방법에 의하여 실시예 5에서보다 약 1.7배 높은 수율을 얻을 수 있었다. 박층크로마토그래피 전개결과 실시예 4의 15시간 결과와 동일하였다.The chitosan was dissolved in the same ratio as in Example 5, and the pH was adjusted to 4.7-4.9 at 50 ° C., followed by partial hydrolysis of cellulase for 2 hours by adding 2% of cellulase, and then slowly adding 2% aqueous hydrochloric acid solution to adjust the pH of the solution. After adjusting to 1-2, chitosan was added to increase the pH to 4.7-4.9. At this time, the amount of chitosan required to raise the pH to 4.7-4.9 is about 60-70% of the initially administered chitosan. After hydrolysis for 15 hours by adding 3% of cellulase, the enzyme was inactivated to complete the reaction, and then the enzyme was removed and spray-dried immediately without going through the concentration process. If the content of solids is more than 8% it is not necessary to concentrate because it is a condition for spray drying. By this method, a yield about 1.7 times higher than in Example 5 was obtained. The thin layer chromatography development result was the same as the 15 hour result of Example 4.
이상에서 설명한 바와 같이, 본 발명에 따른 올리고 키토산의 제조방법은 대량생산에 적용하여 순도 높은 올리고 키토산을 생산하고 또한 공정을 최적화함으로써 수율을 극대화시킬 수 있다.As described above, the method for preparing oligo chitosan according to the present invention may be applied to mass production to produce high purity oligo chitosan and further optimize the yield.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20010058591A (en) * | 1999-12-30 | 2001-07-06 | 김동준 | Scorched Laver Added Chitosan Oligosaccharide and the Preparing Method Thereof |
| KR100370929B1 (en) * | 2000-03-28 | 2003-02-05 | 조훈형 | Preparing Methode for Aqueous Chitosan |
| KR100402889B1 (en) * | 2000-12-04 | 2003-10-22 | 주식회사 한국메디 | Method for Preparing Concentrated Solutuion Including Chitosan with Higher Antimicrobial Activity |
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| JPS6363388A (en) * | 1986-09-04 | 1988-03-19 | Nitta Zerachin Kk | Production of low molecular weight chitosan |
| JPS6398395A (en) * | 1986-10-16 | 1988-04-28 | Katakura Chitsukarin Kk | Production of chitosan oligosaccharide |
| JPH0220292A (en) * | 1988-07-06 | 1990-01-23 | Agency Of Ind Science & Technol | Production of depolymerized chitosan |
| JPH07155193A (en) * | 1993-12-07 | 1995-06-20 | Toyobo Co Ltd | Production of chitosan oligosaccharide derived from microorganism |
| KR0145132B1 (en) * | 1995-01-28 | 1998-07-01 | 이용현 | Enzymatic Preparation of Chitooligosaccharides |
| KR100200547B1 (en) * | 1997-05-27 | 1999-06-15 | 유향자 | Method of separation and purification for low molecular weight chitosan using multi-step membrane process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20010058591A (en) * | 1999-12-30 | 2001-07-06 | 김동준 | Scorched Laver Added Chitosan Oligosaccharide and the Preparing Method Thereof |
| KR100370929B1 (en) * | 2000-03-28 | 2003-02-05 | 조훈형 | Preparing Methode for Aqueous Chitosan |
| KR100402889B1 (en) * | 2000-12-04 | 2003-10-22 | 주식회사 한국메디 | Method for Preparing Concentrated Solutuion Including Chitosan with Higher Antimicrobial Activity |
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