KR20030097166A - A method for production of Chitosan from Fungus and a method for preventing of turbid of bamboo-shoot by using thereof - Google Patents
A method for production of Chitosan from Fungus and a method for preventing of turbid of bamboo-shoot by using thereof Download PDFInfo
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Abstract
Description
본 발명은 균류를 이용한 키토산 생산방법 및 이을 이용한 늦죽순 병조림의 백탁방지방법에 관한 것이다. 더욱 상세하게는, 본 발명은 균류를 최적 조건하에 대량 배양하여 분리추출함으로써 제조공정이 간단하고 이차적인 수질오염 및 폐기물 처리를 줄일 수 있는 키토산 생산방법 및 이를 이용하여 늦죽순의 백탁을 방지하는 방법에 관한 것이다.The present invention relates to a method for producing chitosan using fungi and a method for preventing clouding of late bamboo shoots using the same. More specifically, the present invention is a chitosan production method that can reduce the secondary water pollution and waste treatment by simplifying the manufacturing process by separating and extracting a large amount of fungi under optimum conditions, and a method for preventing white bamboo shoots using the same It is about.
키토산은 D-glucosamine이 β-1,4 결합한 직쇄의 다당으로 자연계에는Mucor속이나Phycomtes속 등 균의 세포벽에 함유되어 있다. 키토산은 폐수처리에서 응집제와 금속이온의 킬레이트제, 종이 증강제, 점도조절제, 점착성 물질, 생분해성필름과 섬유의 생산을 위해 사용되는 생체 고분자이다. 해산 생체 고분자 특히 키토산이 가장 널리 사용되고 연구되는 것은 식품가공 폐수에서 부유물질의 응집제이다. 키토산은 계란 파손, 채소, 새우, 치즈, 육류 그리고 사과쥬스 가공을 포함한 매우 넓은 식품가공운전의 폐수처리에 사용되고 있다.Chitosan is a straight chain polysaccharide in which D-glucosamine is bound to β-1,4 and is naturally contained in cell walls of bacteria such as Mucor and Phycomtes . Chitosan is a biopolymer used in the production of chelating agents, paper enhancers, viscosity modifiers, adhesives, biodegradable films and fibers in flocculants and metal ions in wastewater treatment. The most widely used and studied biodissolved polymers, particularly chitosan, are flocculants of suspended solids in food processing wastewater. Chitosan is used in wastewater treatment in very wide food processing operations, including egg breakage, vegetables, shrimp, cheese, meat and apple juice processing.
현재 상업적으로 사용하는 키토산은 보통 강알칼리를 사용하여 탈아세틸화에 의해 게나 새우 껍질 키틴으로부터 유도된다. 그러나 이 제조법은 원료와 게, 새우 껍질의 집하중의 부패, 키틴과 키토산 제조공정에 요구되는 많은 양의 산, 알칼리 비용 및 제조공정 중에 생산되는 폐액은 하천과 해양의 오염원으로써 2차적인 문제를 야기시키고 있는 실정이다. 그리고 갑각류 가공 공정으로부터 생산되는 껍질 폐기물의 양은 점차 증가하고 있는데, 이는 생분해가 매우 느리므로 수산가공공장의 심각한 문제로 대두되고 있으며, 이는 생분해가 매우 느리므로 수산가공공장의 심각한 문제로 대두되고 있으며, 이러한 갑각류로부터 제조에서의 문제점을 보완하기 위하여 미생물, 효소를 이용한 바이오 수법에 의한 키틴, 키토산의 생합성이 절실히 필요한 상황이다.Currently commercially available chitosan is usually derived from crab or shrimp shell chitin by deacetylation using strong alkalis. However, this process is a secondary source of pollution for streams and marine pollutants due to the decay of the load of raw materials, crabs and shrimp shells, the large amount of acid, alkali costs required for the production of chitin and chitosan, and the waste liquor produced during the manufacturing process. It is causing. In addition, the amount of shell waste produced from crustacean processing is gradually increasing, which is a serious problem for fish processing plants because of its very slow biodegradation, which is a serious problem for fish processing plants because of its very slow biodegradation. In order to supplement the problems in the production from such shellfish, biosynthesis of chitin and chitosan by the bio technique using microorganisms and enzymes is urgently needed.
특히 키토산은 갑각류 이외에도 사상균 등의 세포벽에 많이 존재한다고 보고되고 있으며, 이렇게 생산한 키토산은 대량 균배양으로 키토산을 추출하므로 갑각류 껍질로부터 제조하는 경우에 비하여 탈회 및 탈아세틸화 등의 공정을 생략할 수 있고 균질한 제품의 획득이 가능한 장점이 있으므로, 우선 키토산을 대량 함유하고 있는 균주 및 배양조건을 발견한다면 키토산의 제조법으로써 활용 가능성이 크다고 할 수 있다.In particular, chitosan is reported to exist in cell walls such as filamentous fungus in addition to crustaceans.The chitosan produced in this way extracts chitosan by cultivation of a large amount of fungi, and thus, processes such as deliming and deacetylation can be omitted as compared to the case of preparation from shellfish shells. Since there is an advantage of obtaining a homogeneous product, it can be said that if a strain and culture conditions containing a large amount of chitosan are found first, it can be utilized as a manufacturing method of chitosan.
죽순(bamboo shoot)은 영양적인 가치가 높을 뿐만 아니라 약용으로도 옛부터 사용되어 왔으며, 단백질 함량이 풍부하고 칼슘, 철분, 인과 같은 무기질, 비타민 A, B군도 많이 함유되어 있다. 특히, 늦죽순의 성분조성은 수분 93%, 조단백질 2.8%, 당질 2.4%, 섬유질 0.9%, 지방 0.2%, 회분 0.7%이며, 아미노산 함량은 약 1600㎎(/100g 죽순)으로 그 중 티로신, 아스파라긴, 발린, 글루타민산 등이 전체 함량의 약 70%를 차지한다. 또한 죽순은 제6영양소로 분리되는 섬유질이 비교적 많아 장의 운동기능 촉진, 변비해소, 숙변제거 등에 효과가 있으므로 이를 이용한 건강식품 또는 전통가공식품으로 개발한다면 부가가치를 높일 수 있을 것이다.Bamboo shoots have high nutritional value and have been used for a long time in medicinal use. They are rich in protein and contain minerals such as calcium, iron and phosphorus, and vitamins A and B. In particular, the composition of late bamboo shoots is 93% moisture, crude protein 2.8%, sugar 2.4%, fiber 0.9%, fat 0.2%, ash 0.7%, amino acid content of about 1600mg (/ 100g bamboo shoot), among which tyrosine and asparagine , Valine and glutamic acid make up about 70% of the total content. In addition, bamboo shoots are relatively high in fiber separated by the sixth nutrient, which is effective in promoting intestinal exercise function, relieving constipation, and eliminating stool, so that the value added if developed as a health food or traditional processed food using the same.
죽순은 상온에서 쉽게 부패가 일어나 보관상의 어려움을 지니고 있다. 그리고 저온저장을 한다고 해도 연약한 어린죽순의 조직이 딱딱하게 변하는 등의 경화현상이 발생하여 식용으로 가공하기에 부적절한 상태가 될 뿐만아니라 영양성분의 분해에 의한 손실과 고유의 감칠맛 등이 감소하게 된다. 그러므로 이를 이용한 가공식품의 개발이 절실히 요구된다.Bamboo shoots are easily decayed at room temperature and have difficulty in storage. In addition, even if stored at low temperatures, the hardening of the soft bamboo shoots, such as hard tissue changes occurs, not only become inadequate for processing, but also due to the decomposition of nutrients loss and intrinsic umami taste is reduced. Therefore, the development of processed foods using this is urgently required.
본 발명자들은 상기와 같은 점을 감안하여 키토산 생산 균주를 선정하여 배양 최적 조건을 조사하였으며 그 최적 조건하에서 배양한 균체를 수세후 에탄올에 하룻밤 정도 처리한 뒤 여러번 수세한 후 동결건조시켜 건조 균체를 생산하고, 상기 건조 균체를 알칼리 추출 처리하여 알칼리 불용성 물질(alkali-insoluble materials, AIM)을 얻고, 이를 산처리한 후 pH를 8.5로 조절하여 4℃에서 12시간 방치한 뒤 침전물을 여과 후 수세 및 중화시킨 뒤 동결건조시켜 균류 키토산(fungal chitosan, FCS)를 생산하고, 상기 균류 키토산을 늦죽순 병조림에첨가하여 백탁방지효과를 조사함으로써 본 발명을 완성하였다.In view of the above, the present inventors selected chitosan-producing strains to investigate the optimum culture conditions, and the cells cultured under the optimum conditions were washed with ethanol overnight after washing with water several times and then lyophilized to produce dry cells. Alkali-insoluble materials (AIM) were obtained by alkali extraction of the dried cells, and after acid treatment, the pH was adjusted to 8.5 and left at 4 ° C. for 12 hours, followed by filtration and washing with water. After lyophilization to produce fungal chitosan (FCS), the fungus chitosan was added to the late bamboo shoots to investigate the anti-clouding effect was completed.
따라서, 본 발명의 목적은 균류를 최적 조건하에 대량 배양하여 분리추출함으로써 제조공정이 간단하고 이차적인 수질오염 및 폐기물 처리를 줄일 수 있는 키토산 생산방법을 제공함에 있다.Accordingly, an object of the present invention is to provide a chitosan production method that can simplify the production process and reduce secondary water pollution and waste treatment by cultivating and extracting the fungus in large quantities under optimum conditions.
본 발명의 다른 목적은 균류를 이용하여 생산한 균류 키토산을 이용한 늦죽순 병조림의 백탁방지방법을 제공함에 있다.Another object of the present invention is to provide a method of preventing cloudy turbidity of late bamboo shoots using fungus chitosan produced using fungus.
본 발명의 상기 목적은 키토산 생산 균주를 선정하여 배양 최적 조건을 조사하였으며 그 최적 조건하에서 배양한 균체를 수세후 에탄올에 하룻밤 정도 처리한 뒤 여러번 수세한 후 동결건조시켜 건조 균체를 생산하고, 상기 건조 균체를 알칼리 추출 처리하여 알칼리 불용성 물질(alkali-insoluble materials, AIM)을 얻고, 이를 산처리한 후 pH를 8.5로 조절하여 4℃에서 12시간 방치한 뒤 침전물을 여과 후 수세 및 중화시킨 뒤 동결건조시켜 균류 키토산(fungal chitosan, FCS)를 생산하고, 상기 균류 키토산을 늦죽순 병조림에 첨가하여 백탁방지효과를 조사함으로써 달성하였다.The object of the present invention is to select the chitosan-producing strains to investigate the optimum culture conditions, and the cells cultured under the optimum conditions after washing with ethanol overnight after washing several times and then lyophilized to produce dried cells, the drying Alkali-insoluble materials (AIM) were obtained by alkali extraction of the cells, and after acid treatment, the pH was adjusted to 8.5 and left at 4 ° C. for 12 hours. The precipitate was filtered, washed with water, neutralized, and lyophilized. It was achieved by producing fungal chitosan (FCS) and adding the fungus chitosan to late bamboo shoots to investigate the anti-clouding effect.
이하 본 발명의 구성을 설명한다.Hereinafter, the configuration of the present invention.
도 1은 본 발명 균류를 이용한 키토산의 생산에 사용하는 회분식 생물반응기의 개략적 구성도이다.1 is a schematic configuration diagram of a batch bioreactor used in the production of chitosan using the fungus of the present invention.
도 2는Absidia coeruleaIFO 5301,Gongronella butleriIFO 8080,Mucor tuberculisporusIFO 9256 및Rhizopus delemerIFO 4775 균주의 초기 pH에 따른 건조 균체량을 나타낸 결과이다.Figure 2 shows the dry cell weight according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.
도 3은Absidia coeruleaIFO 5301,Gongronella butleriIFO 8080,Mucor tuberculisporusIFO 9256 및Rhizopus delemerIFO 4775 균주의 초기 pH에 따른 알칼리 불용물질량을 나타낸 결과이다.Figure 3 shows the results of alkali insolubles according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.
도 4는Absidia coeruleaIFO 5301,Gongronella butleriIFO 8080,Mucor tuberculisporusIFO 9256 및Rhizopus delemerIFO 4775 균주의 초기 pH에 따른 균류 키토산량을 나타낸 결과이다.Figure 4 shows the results of fungal chitosan according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.
도 5는Absidia coeruleaIFO 5301에 의한 건조 균체 중량 생산에 대한 온도의 영향을 나타낸 것이다.5 shows the effect of temperature on dry cell weight production by Absidia coerulea IFO 5301.
도 6은Absidia coeruleaIFO 5301에 의한 건조 균체 중량 생산에 대한 배양 시간의 영향을 나타낸 것이다.Figure 6 shows the effect of incubation time on dry cell weight production by Absidia coerulea IFO 5301.
도 7은 본 발명 균류 키토산을 분리추출하는 과정을 간략하게 나타낸 흐름도이다.7 is a flowchart briefly illustrating a process of separating and extracting the fungus chitosan of the present invention.
도 8은 본 발명의 늦죽순 병조림을 제조하는 과정을 간략하게 나타낸 흐름도이다.8 is a flow chart briefly illustrating a process of producing a late bamboo shoot bottle of the present invention.
도 9는 균류 키토산의 농도에 따른 늦죽순 병조림의 백탁방지효과를 나타낸 그래프이다.9 is a graph showing the cloudiness prevention effect of late bamboo shoots according to the concentration of the fungus chitosan.
본 발명은 키토산 생산 균주를 준비하는 단계; 키토산 생산균주를 최적 조건하에서 대량 배양하는 단계; 상기 단계에서 얻은 균체에서 균류 키토산을 분리추출한 후 동결건조시키는 단계; 늦죽순 병조림을 제조하는 단계; 상기 단계에서 얻은 균류 키토산을 늦죽순 병조림에 첨가한 후 백탁방지 효과를 조사하는 단계로 구성된다.The present invention comprises the steps of preparing a chitosan producing strain; Culturing the chitosan producing strain under optimal conditions; Separating and extracting the fungus chitosan from the cells obtained in the step and lyophilizing; Preparing a late bamboo shoot bottle; The fungus chitosan obtained in the above step is added to the late bamboo shoot blight consisting of the step of investigating the anti-clouding effect.
본 발명에서 사용된 표준용 키토산은 미국 SIGMA사로부터 제공받은 C-3646을 필요시에 미세분말(200mesh)로 분쇄하여 사용하였다.The standard chitosan used in the present invention was used by grinding C-3646 provided from the US SIGMA company into fine powder (200mesh) as needed.
이하 본 발명의 구체적인 방법을 실시예를 들어 단계별로 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.
실시예 1 : 키토산 생산균주의 준비Example 1 Preparation of Chitosan Producing Strains
본 실험에서 사용한 4균주는 키토산 생산 균주인Gongronella butleriIFO 8080,Absidia coeruleaIFO 5301,Rhizopus delemerIFO 4775,Mucor tuberculisporusIFO 9256으로써 일본의 발효학 연구소에서 분양 받았다.The four strains used in this experiment were distributed as chitosan producing strains such as Gongronella butleri IFO 8080, Absidia coerulea IFO 5301, Rhizopus delemer IFO 4775, and Mucor tuberculisporus IFO 9256.
균주의 보존을 위하여 PDA배지에서 30일마다 계대배양하여 4℃에서 보존하였다(표 1).For preservation of the strains were subcultured every 30 days in PDA medium and stored at 4 ℃ (Table 1).
실시예 2: 키토산 생산균주 배양Example 2: Chitosan Production Strain Culture
제 1단계 : 전배양Stage 1: Preculture
전배양은 PDA 39g과 효모 추출물 3g을 탈이온수 1,000㎖에 가열용해하여 각 시험관에 10㎖씩 분주한 후, 121℃에서 15분간 고압증기 멸균하여 만든 PDA 사면배지에 각각 접종하여 27℃의 항온기에서 7∼9일간 배양시킨 후, 다시 4℃의 냉장고에 보존하거나 본배양에 사용하였다.In pre-culture, 39 g of PDA and 3 g of yeast extract were dissolved in 1,000 ml of deionized water, and each 10 ml was dispensed into each test tube, and then inoculated in PDA slope medium prepared by autoclaving at 121 ° C. for 15 minutes in an incubator at 27 ° C. After 7 to 9 days of incubation, it was again stored in a refrigerator at 4 ℃ or used in the main culture.
제 2단계 : 본배양Step 2: Main Culture
키토산 생산균주의 배지조성은 Rane (Rane, K. D., and D. G. Hoover. 1993.) 등의 방법에 준하여 행하였으며, 글루코스(glucose) 20g, 펩톤(peptone) 10g, 효모 추출물(yeast extract) 1.0g, (NH4)2SO45.0g, K2HPO41.0g, NaCl 1.0g, MgSO4·7H2O 0.5g 및 CaCl2·2H2O 0.1g을 증류수 1,000㎖에 용해하여 1N-HCl로 초기 pH 5.0으로 조절하였고 500㎖-평저 플라스크에 배지 200㎖를 분주하였으며, 생물반응기에서의 배양은 5ℓ의 반응조내에 3ℓ의 액체배지를 가한 다음, 121℃에서 15분간 고압증기 멸균하였다. 실온에 방냉한 다음, 1N HCl로 초기 pH를 조절하였으며, 여기에 균현탁액을 2㎖ 접종하여 본배양하였다.Medium composition of chitosan producing strains was performed according to the method of Rane (Rane, KD, and DG Hoover. 1993.), glucose 20g, peptone 10g, yeast extract 1.0g, ( 5.0 g of NH 4 ) 2 SO 4 , 1.0 g of K 2 HPO 4, 1.0 g of NaCl, 0.5 g of MgSO 4 · 7H 2 O and 0.1 g of CaCl 2 · 2H 2 O were dissolved in 1,000 ml of distilled water and the initial pH was adjusted to 1N-HCl. It was adjusted to 5.0 and 200 ml of medium was dispensed into a 500 ml flat bottom flask, and the culture in the bioreactor was added with 3 liter of liquid medium in a 5 liter reaction tank, followed by autoclaving at 121 ° C. for 15 minutes. After cooling to room temperature, the initial pH was adjusted with 1N HCl, and the main culture was inoculated with 2 ml of the suspension.
균류 키토산 생산균주의 플라스크 배양에서는 회전형 진탕배양기(C-SKI-2, 한영기계)를 사용하였으며, 진폭 7㎝이고 회전수는 130rpm으로 운전하였다.In the flask culture of the fungus chitosan producing strain, a rotary shaker (C-SKI-2, Hanyoung Machinery) was used, and the amplitude was 7 cm and the rotation speed was 130 rpm.
생물반응기는 회분식 생물반응기(KF-25L; 한국발효기(주))를 사용하였다(도 1).The bioreactor used a batch bioreactor (KF-25L; Korea fermenter Co., Ltd.) (Fig. 1).
실험예 1 : 배지성분의 영향 조사Experimental Example 1: Investigation of the influence of the media components
1. 탄소원의 영향1. Influence of carbon source
초기 pH 6.5, 130 rpm, 27℃, 6일 배양에서A. coeruleaIFO 5301의 탄소원인 글루코스의 농도를 0.5%, 1%, 2.0%로 하여 실험한 결과 2.0%에서 가장 높은 수율을 나타내었다.In the initial pH 6.5, 130 rpm, 27 ℃, 6 days incubation, the concentration of glucose, the carbon source of A. coerulea IFO 5301 at 0.5%, 1%, 2.0% experiments showed the highest yield at 2.0%.
2. 유기질소원의 영향2. Influence of Organic Nitrogen Sources
초기 pH 6.5, 130 rpm, 27℃, 6일 배양에서A. coeruleaIFO 5301의 질소원인 펩톤(peptone)의 농도를 0.3%, 0.7%, 1.0%로 하여 실험한 결과 1.0%에서 가장 높은 수율을 나타내었다.In the initial pH 6.5, 130 rpm, 27 ℃, 6 days cultivation of the concentration of peptone (0.3%, 0.7%, 1.0%) of the nitrogen source of A. coerulea IFO 5301 showed the highest yield at 1.0% It was.
3. 무기 질소원의 영향3. Influence of inorganic nitrogen sources
A. coeruleaIFO 5301의 배지조성 중 무기 질소원 (NH4)2HPO4, NaNO3, NH4Cl, NH4H2PO4, NH4NO3을 각각 배지의 0.5% 농도씩 첨가하여 초기 pH 6.5으로 조절한 다음, 130 rpm, 27℃에서 6일간 배양하여 균체량을 조사한 결과, 황산암모늄((NH4)2SO4)이 가장 높은 수율을 나타내었다.Inorganic Nitrogen Source (NH 4 ) 2 HPO 4 , NaNO 3 , NH 4 Cl, NH 4 H 2 PO 4 , NH 4 NO 3 was added at 0.5% concentration in the medium for A. coerulea IFO 5301. After adjusting to, and incubated at 130 rpm, 27 ℃ for 6 days to check the cell mass, ammonium sulfate ((NH 4 ) 2 SO 4 ) showed the highest yield.
4. 황산암모늄 농도의 영향4. Effect of Ammonium Sulfate Concentration
키토산 생산배지에서 질소원으로써 가장 균체생산이 많았던 황산암모늄을 배지의 0.1%∼0.5%의 농도별로 첨가하여 27℃에서 6일간 배양하여 얻은 균체량을 조사한 결과, 키토산 생산배지의 약 0.5% 농도로 황산암모늄을 첨가하였을 때 균체 생산량이 가장 많았다.The amount of ammonium sulfate, which was the most cell production as a nitrogen source in chitosan producing medium, was added to each concentration of 0.1% to 0.5% of the medium, and the cell mass obtained by incubating at 27 ° C for 6 days was examined. The highest yield was obtained when added.
5. 인산 및 칼륨원 농도의 영향5. Effect of Phosphoric Acid and Potassium Source Concentration
키토산 생산배지의 조성에서 인산 및 칼륨원인 K2HPO4와 KH2PO4를 각각 배지의 0.1∼0.3% 농도로 첨가하여 27℃에서 6일간A. coeruleaIFO 5301을 배양시켜 얻은 균체생산량을 조산 결과 인산 및 칼륨원은 K2HPO4가 KH2PO4보다 대체적으로 균체생산량이 많았으며 그 중 배지의 농도를 0.1%로 하였을 때 가장 높은 수율을 나타냈다.Cause chitosan phosphate and potassium in the composition of the production medium K 2 HPO 4 and KH 2 PO 4 was added to 0.1~0.3% in concentration of each culture medium 6 days at 27 ℃ A. coerulea IFO result the cell yield obtained by culturing the immature birth 5301 Phosphoric acid and potassium sources showed higher yields of K 2 HPO 4 than KH 2 PO 4 , and showed the highest yield when the concentration of medium was 0.1%.
6. 염화나트륨 농도의 영향6. Effect of sodium chloride concentration
A. coeruleaIFO 5301의 배지에 0.1∼0.3% 농도의 염화나트륨을 첨가하여 27℃에서 6일간 배양하여 얻은 균체량을 조사한 결과, 염화나트륨을 첨가하지 않은 대조군에 비하여 배지의 0.1%를 첨가하였을 때 균체생산량이 가장 높게 나타났다. 그러나 그 이상 첨가하였을 때에는 균체생산량이 감소하였다. The cell mass obtained by adding 0.1-0.3% sodium chloride to the medium of A. coerulea IFO 5301 was incubated for 6 days at 27 ° C. As a result, when the 0.1% of the medium was added compared to the control group without adding sodium chloride, The highest. However, when added more, the cell yield decreased.
7. 금속염 농도의 영향7. Effect of metal salt concentration
금속염류 MgSO4·7H2O, FeCl3·6H2O, MnCl2·4H2O, CaCl2·2H2O를 키토산 생산배지에 각각 0.01∼0.05%의 농도로 첨가하여A. coeruleaIFO 5301 균주를 접종한 다음, 27℃, 130 rpm에서 6일간 배양시켜 얻은 균체량을 조사한 결과, CaCl2·2H2O와 MgSO4·7H2O의 경우에 각각 배지의 0.01%와 0.05% 농도에서 균체생산량이 높게 나타났다.Metal salts MgSO 4 · 7H 2 O, FeCl 3 · 6H 2 O, MnCl 2 · 4H 2 O, CaCl 2 · 2H 2 O were added to chitosan production medium at a concentration of 0.01 to 0.05%, respectively, to A. coerulea IFO 5301 strain. and then, 27 ℃, at 130 rpm 6 ilgan research gyuncheryang obtained by culturing a result, CaCl 2 · 2H 2 O and MgSO 4 · cell yield from 0.01% and 0.05% concentration of each medium in case of 7H 2 O inoculated with the High.
CaCl2·2H2O와 MgSO4·7H2O를 각각 단독으로 첨가하였을 때보다 두 금속염을 같이 첨가하여 배양하였을 때 건초균체량의 수율이 더 높았다.The yield of haye cell mass was higher when two metal salts were incubated together than when CaCl 2 · 2H 2 O and MgSO 4 · 7H 2 O were added alone.
실험예 2 : 최적 초기 pH 조사Experimental Example 2 Optimal Initial pH Irradiation
키토산 생산배지의 초기 pH에 따른 4균주Absidia coeruleaIFO 5301,Gongronella butleriIFO 8080,Rhizopus delemarIFO 4775,Mucor tuberculisporusIFO 9256의 DCW, AIM 및 FCs의 결과를 표 2와 도 2, 3 및 4에 나타내었다.The results of DCW, AIM and FCs of four strains Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Rhizopus delemar IFO 4775, Mucor tuberculisporus IFO 9256 according to the initial pH of chitosan production medium are shown in Table 2 and FIGS. 2, 3 and 4. .
[주] 1) 건조 균체 중량(Dry cell weight);NOTE 1) Dry cell weight;
130rpm으로 교반하면서 27.0℃에서 5일간 발효Fermentation at 27.0 ° C. for 5 days with stirring at 130 rpm
2) 알칼리 불용물질(Alkali insoluble materials);2) Alkali insoluble materials;
121℃에서 15분동안 1N NaOH로 처리Treated with 1N NaOH at 121 ° C. for 15 minutes
3) 균류 키토산(Fungal chitosan);3) fungal chitosan;
95℃에서 12시간동안 2% 아세트산으로 처리Treated with 2% acetic acid for 12 hours at 95
키토산 생산균주들 중에서Absidia coeruleaIFO 5301과Gongronella butleriIFO 8080이 초기 pH에 따른 건초균체 생산량이 가장 높았으며,Absidia coeruleaIFO 5301은 초기 pH 6.5에서 건조균체량이 1,514㎎,Gongronella butleriIFO 8080은 초기 pH 5.5에서 건조균체량이 200㎖ 키토산 생산 배지 중 2,044㎎으로써 가장 수율이 높았다(도2).Among chitosan producing strains, Absidia coerulea IFO 5301 and Gongronella butleri IFO 8080 showed the highest yield of hay cell according to initial pH. Absidia coerulea IFO 5301 had a dry cell mass of 1,514 mg at initial pH 6.5 and Gongronella butleri IFO 8080 at an initial pH of 5.5. The dry cell mass at 2,044 mg in 200 ml chitosan production medium was the highest (Fig. 2).
그러나 초기 pH에 따른 균류키토산의 중간물질인 알칼리 불용물질의 수율은Absidia coeruleaIFO 5301이 초기 pH 6.5에서 06㎎/200㎖로 4균주 중 가장 높은 수율을 나타냈으며 그 다음으로는Gongronella butleriIFO 8080이 초기 pH 5.5에서 547㎎/200㎖이었다.However, the yield of alkali-insoluble substance, which is an intermediate of fungal chitosan according to the initial pH, showed the highest yield among four strains with Absidia coerulea IFO 5301 at the initial pH of 6.5 mg / 200ml, followed by Gongronella butleri IFO 8080. 547 mg / 200 ml at an initial pH of 5.5.
균류키토산의 생산은Absidia coeruleaIFO 5301의 경우 205∼258㎎/200㎖로써 초기 pH 6.5에서 가장 수율이 좋았다.The production of the fungus chitosan was 205-258 mg / 200 ml for Absidia coerulea IFO 5301, which was the highest yield at initial pH 6.5.
초기 pH 조건 실험에서Absidia coeruleaIFO 5301이 타균주에 비하여 키토산 생산이 우수하였으므로 이하 배양온도와 배양시간에 따른 균체생산 실험에서는 이 균주를 사용하였다. Absidia coerulea IFO 5301 produced chitosan better than other strains in the initial pH condition experiments.
실험예 3 : 최적 배양온도 조사Experimental Example 3 investigation of the optimum culture temperature
초기 pH 실험에서 키토산 수율이 가장 높은Absidia coeruleaIFO 5301을 1N HCl로 초기 pH 6.5으로 조절한 다음, 배양온도에 따른 균체생산의 효과를 알아보기 위하여 24℃, 27℃ 및 30℃로 배양온도를 달리하여 5일동안 130rpm에서 진탕배양하였다.In the initial pH experiment, Absidia coerulea IFO 5301, which has the highest yield of chitosan, was adjusted to initial pH 6.5 with 1N HCl, and then the culture temperature was changed to 24 ° C, 27 ° C and 30 ° C in order to examine the effect of cell production according to the culture temperature. Was shaken at 130 rpm for 5 days.
Absidia coeruleaIFO 5301은 27℃에서 가장 많은 균체를 생산하였다(도 5). Absidia coerulea IFO 5301 produced the most cells at 27 ° C. (FIG. 5).
실험예 4 : 최적 배양시간 조사Experimental Example 4 investigation of the optimum incubation time
Absidia coeruleaIFO 5301의 균현탁액을 2㎖을 키토산 생산배지 200㎖가 들어있는 플라스크에 1N HCl로 초기 pH를 6.5로 조절하여 접종한 다음 최적 배양온도 27℃, 130rpm의 진탕배양기에서 배양시간을 각각 4일, 5일, 6일, 7일 동안 배양시켰다. 실험 결과, 6일에서 가장 높은 수율을 얻었다(도6). 2ml of Absidia coerulea IFO 5301 was inoculated into a flask containing 200ml of chitosan production medium with 1N HCl, adjusted to an initial pH of 6.5, and then incubated at an optimum incubation temperature of 27 ℃ and 130rpm in a shaker. Incubated for days, 5 days, 6 days, and 7 days. As a result, the highest yield was obtained at 6 days (FIG. 6).
실험예 5 : 회분식 생물반응기의 운전조건 조사Experimental Example 5: Investigation of the operating conditions of the batch bioreactor
1. 균농도의 영향1. Effect of Bacterial Concentration
플라스크 배양에서 찾은Absidia coeruleaIFO 5301의 배양 최적조건을 회분식 생물반응기에 적용시켜 균체를 생산하였다. 회분식 생물반응기에서 운전은 공기유입량 1.0ℓ/min, 교반속도 600rpm, 배양시간 24시간, 배지량 3ℓ로 하였으며, 균현탁액 농도에 따른 균체수율을 조사한 결과, 70㎖까지는 균체생산량이 뚜렷이 증가하였으며 그 이상에서는 거의 변화가 없었다.Optimal culture conditions of Absidia coerulea IFO 5301 found in the flask culture were applied to a batch bioreactor to produce cells. Operation of the batch bioreactor was performed with air inflow rate of 1.0ℓ / min, agitation speed 600rpm, incubation time 24 hours, medium volume 3ℓ. As a result of checking the cell yield according to the concentration of the suspension, the cell yield was markedly increased up to 70ml. There was little change.
2. 교반속도의 영향2. Effect of Stirring Speed
회분식 생물반응기에서 교반속도에 따른Absidia coeruleaIFO 5301균주로부터 균체수율을 400rpm, 600rpm, 800rpm, 1,000rpm으로 달리하여 조사한 결과, 800rpm에서 균체수율이 가장 높았다.In the batch bioreactor, the cell yields of Absidia coerulea IFO 5301 strains were varied at 400 rpm, 600 rpm, 800 rpm, and 1,000 rpm in the batch bioreactor. The yield was highest at 800 rpm.
3. 공기유입량의 영향3. Effect of air inflow
회분식 생물반응기내로 유입되는 공기의 양을 0.5∼2.0ℓ/min로 달리하여 균체량을 조사한 결과, 0.5ℓ/min에서 가장 수율이 높았다.The amount of air introduced into the batch bioreactor was varied from 0.5 to 2.0 l / min, and the yield was the highest at 0.5 l / min.
4. 배양시간의 영향4. Effect of incubation time
Absidia coeruleaIFO 5301의 회분식 생물반응기에서 생육최적 조건으로 선정된 배양온도 27℃, 초기 pH 6.5에서 균현탁액 70㎖를 3ℓ의 키토산 생산배지가 들어있는 반응조에 접종하였으며 공기유입량 0.5ℓ/min, 교반속도 800rpm으로 조절하여 배양시간을 24시간, 36시간, 48시간으로 하여 균체를 생산하였다. 실험 결과, 36시간 동안 배양시 가장 수율이 높았다. In a batch bioreactor of Absidia coerulea IFO 5301, 70 ml of the suspension was inoculated into a reactor containing 3 liters of chitosan production medium at an incubation temperature of 27 ° C and an initial pH of 6.5. Cells were produced by adjusting the incubation time to 24 hours, 36 hours, and 48 hours at 800 rpm. As a result, the highest yield was obtained when incubated for 36 hours.
상기와 같은 실험 결과, 회분식 생물반응기에서의 균체생산시 최적 운전조건이 하기 표 3과 같음을 확인할 수 있었다.As a result of the experiment, it was confirmed that the optimum operating conditions in the production of the cells in the batch bioreactor as shown in Table 3.
실시예 3 : 알칼리불용물질 및 균류 키토산의 분리Example 3 Separation of Alkali Insolubles and Fungus Chitosan
Kobayashi(Kobayashi, T., Takkiguchi, Y., Shimahara, K. and Sannan, T. 1988.)와 Rane(Rane, K.D. and Hoover, D. G. 1993) 등의 방법에 따라 균류의 세포벽으로부터 키토산을 추출하였다. 플라스크와 생물반응기에서 생산된 균체 덩어리를 수세한 다음, G3 글라스 필터(glass filter)로 여과하고 에탄올에 하룻밤 정도 처리하였으며, 여러번 수세한 후 동결건조하여 DCW를 얻었다. 균체의 세포벽의 성분인 단백질 등의 골격적 성분을 분리해 내기 위하여 알칼리 처리하였는데 이는 1N aOH를 균체량에 대해 1:40(w/v)의 비로 가한 다음, 121℃에서 15분간 반응시켰다. 이렇게 알칼리 처리된 것을 중화하기 위하여 반복하여 수세한 후, 탈수 및 동결건조하여 AIM을 얻었다.Chitosan was extracted from the cell walls of fungi according to the methods of Kobayashi (Kobayashi, T., Takkiguchi, Y., Shimahara, K. and Sannan, T. 1988.) and Rane (Rane, K.D. and Hoover, D. G. 1993). The cell mass produced in the flask and the bioreactor was washed with water, filtered through a G3 glass filter, treated with ethanol overnight, washed several times, and lyophilized to obtain DCW. Alkali treatment was performed to separate skeletal components such as proteins, which are components of cell walls of cells, and 1N aOH was added at a ratio of 1:40 (w / v) to the cell weight, followed by reaction at 121 ° C. for 15 minutes. After repeated washing with water to neutralize the alkali treatment, it was dehydrated and lyophilized to obtain AIM.
알칼리 불용물질에는 키토산외에 다른 물질들도 함께 존재하고 있으므로 키토산을 분리해 내기 위하여 키토산이 묽은 초산에 녹는 성질을 이용하였다. 알칼리 불용물질을 플라스크에 적당량 가한 다음, 여기에 2%의 초산을 1:50(w/v)의 비로 가하였다. 이 플라스크에 냉각관과 온도계 그리고 교반기(stirrer)를 장착하여 기름욕조상에서 95℃와 40℃에서 12시간 반응시켰다. 이렇게 산처리한 후, 불용물질과 용해물질을 분리하기 위하여 10,000 rpm에서 20분간 원심분리하여 상징액을 회수하였고 불용물질은 다시 2, 3차 산처리하여 상징액을 회수하였다. 회수된 상징액을 G3 글라스 필터로 여과하였고 여과된 액을 진한 NaOH 용액을 사용하여 pH 8.5로 조절하여 4℃에서 12시간 방치하여 키토산을 침전시켰다. 이 침전물을 원심분리시켜 증류수로 수세 및 중화를 반복하여 동결건조시켜 FCS를 얻었다(도 7).In addition to chitosan, alkali insolubles were also present, so that chitosan was dissolved in dilute acetic acid to separate chitosan. An alkali insoluble substance was added to the flask in an appropriate amount, and then 2% acetic acid was added in a ratio of 1:50 (w / v). The flask was equipped with a cooling tube, a thermometer, and a stirrer to react at 95 ° C. and 40 ° C. for 12 hours in an oil bath. After acid treatment, the supernatant was recovered by centrifugation at 10,000 rpm for 20 minutes to separate the insoluble and dissolved substances, and the supernatant was recovered by the second and third acid treatment again. The collected supernatant was filtered through a G3 glass filter, and the filtered solution was adjusted to pH 8.5 using a concentrated NaOH solution and left at 4 ° C. for 12 hours to precipitate chitosan. The precipitate was centrifuged and washed with distilled water and neutralized repeatedly to freeze-dry to obtain an FCS (FIG. 7).
실험예 6 : 균류 키토산의 일반성분 및 아미노산 분석Experimental Example 6: Analysis of General Components and Amino Acids of the Fungus Chitosan
에이. 코에룰레아 IFO5301(A. coeruleaIFO5301)균주로부터 건조균체를 회수하여 알칼리 처리에 의하여 알칼리 불용물질을 얻었으며, 이로부터 산처리하여 균류키토산을 생산하였다. 산처리의 온도에 따른 균류 키토산의 수율은 저온에서보다 고온에서 다소 높게 나타났지만, 색깔이 저온처리에 비하여 다소 갈색을 나타내어 성분 및 구조상의 차이가 없는지를 알아보고자 하였다. 알칼리 불용물질로부터 2% 초산을 40℃에서 12시간 처리하여 제조한 균류키토산을 FCs-40으로, 95℃에서 12시간 처리한 것을 FCs-95로 하여 각각의 성분을 분석한 것은 표 4와 같다.a. The dry cells were recovered from the A. coerulea IFO5301 strain to obtain an alkali insoluble substance by alkali treatment, and acid-treated therefrom to produce fungal chitosan. The yield of fungus chitosan was slightly higher at high temperature than at low temperature, but the color was slightly brown compared to the low temperature treatment. The fungus chitosan prepared by treating 2% acetic acid at 40 ° C. for 12 hours with an alkali insoluble substance was treated with FCs-40 and 12 hours at 95 ° C. as an FCs-95.
실험예 7 : 균류 키토산의 물리화학적 특성 조사Experimental Example 7 Investigation of Physicochemical Properties of Fungus Chitosan
두 균류 키토산의 용해도, 점도, 아세틸화도 및 분자량 등의 물리화학적 특성을 분석한 결과를 표 5에 나타내었다.Table 5 shows the results of analyzing physicochemical properties such as solubility, viscosity, acetylation and molecular weight of the two fungus chitosan.
[주][week]
1) 25℃에서 1% 용액(2% 아세트산을 용매로 사용)으로 측정됨1) Measured as 1% solution (using 2% acetic acid as solvent) at 25 ° C
2) 적외선 흡착 스펙트럼(infrared adsorption spectrum)으로 측정됨2) measured by the infrared adsorption spectrum
3) 인트린식 점도법(intrinsic viscosity method)3) intrinsic viscosity method
실험예 8 : 균류 키토산의 I.R. 및 NMR 스펙트럼 분석Experimental Example 8: I.R. of the fungus chitosan. And NMR spectral analysis
본 발명의 균류 키토산의 화학 구조식을 IR과 NMR 스펙트럼을 이용하여 표준키토산의 것과 비교분석한 결과, IR 스펙트럼상의 흡수 밴드가 거의 일치하고 NMR 스펙트럼도 거의 일치하는 것으로 나타났다.The chemical structural formula of the fungus chitosan of the present invention was compared with that of standard chitosan using IR and NMR spectra, and the absorption bands on the IR spectrum were almost identical and the NMR spectrum was also almost identical.
실시예 4 : 늦죽순 병조림의 제조Example 4 Preparation of Late Bamboo Shoot Bottles
늦죽순은 경남 하동지방에서 5월 중순에서 6월 중순에 생산된 것을 구입하였으며 정제염은 순도 99% 이상의 한주소금, 키토산은 1%초산에 용해시켜 용액상태로 사용하였다.Late bamboo shoots were produced in mid-May to mid-June in Hadong, Gyeongnam. Purified salt was used as a solution by dissolving Hanjugeum with purity over 99% and chitosan in 1% acetic acid.
늦죽순을 수세, 다듬기 및 속껍질 벗기기를 한 다음, 적절한 농도의 소금 용액에서 블렌칭을 하였다. 이를 흐르는 물에서 급냉하여 재다듬기하였으며, 탁하게 하는 원인물질인 티로신을 제거하기 위하여 30분동안 물에 담구어 두었다. 블렌칭한 늦죽순 100g을 선별하여 225㎖의 유리병에 담고 98℃의 뜨거운 물을 부어 10분간 탈기 시킨다음, 105℃의 건조기에서 60분간 살균하고 실온에서 냉각하였다. 이를 실링 필름(sealing film)으로 뚜껑을 밀봉하여 늦죽순 병조림을 제조하였다(도 8).The late bamboo shoots were washed, trimmed and peeled, and then blotted in an appropriate concentration of salt solution. The mixture was quenched in running water and refined, and soaked in water for 30 minutes to remove tyrosine, the causative agent of turbidity. 100 g of the blanched late bamboo shoots were screened, placed in a 225 ml glass bottle, poured with 98 ° C. hot water, degassed for 10 minutes, sterilized for 60 minutes in a 105 ° C. dryer, and cooled at room temperature. The lid was sealed with a sealing film (sealing film) to prepare a late bamboo shoot bottle (Fig. 8).
실험예 9 : 키토산 첨가농도에 따른 백탁영향 조사Experimental Example 9: Investigation of turbidity effect according to chitosan concentration
늦죽순 병조림의 장기저장에 따른 성분의 용출로 인한 백탁현상으로 인하여 부패한 것으로 오인할 수 있기 때문에 이를 방지하기 위하여 늦죽순 병조림 제조공정중에 사용하는 소금의 농도를 1%, 2%, 3%로 달리하였으며, 이들을 60분간 블렌칭한 후, 저장에 따른 백탁현상방지 효과를 관찰하였다. 관찰 결과, 소금 농도를 1%로 하였을 경우에 가장 백탁현상이 낮게 나타났다. 따라서 이후 실험에서는 소금 농도를 1%로 하여 수행하였다. 그리고 블렌칭에 따른 백탁방지효과가 가장 우수한 조건을 선정하여 균류키토산을 10㎎/225㎖, 20㎎/225㎖, 40㎎/225㎖ 농도별로 각각 첨가하여 실온저장에 따른 백탁방지효과를 알아보기 위하여 일정시간마다 시료액을 채취하여 탁도를 측정하였다. 대조군은 1%의 소금물 농도에서 늦죽순을 60분간 블렌칭한 것으로 하였다.In order to prevent this, the salt concentrations used during the manufacturing process of late bamboo shoots were changed to 1%, 2%, and 3% in order to prevent this. After 60 minutes of blending, the effect of the whitening on storage was observed. As a result, when the salt concentration was 1%, the haze was the lowest. Therefore, the experiment was performed with a salt concentration of 1%. In addition, by selecting the conditions with the best anti-clouding effect according to the blending and adding fungal chitosan by concentrations of 10mg / 225ml, 20mg / 225ml and 40mg / 225ml, respectively, to find out the anti-clouding effect according to room temperature storage. In order to measure the turbidity by taking a sample of the liquid at regular intervals. The control group was made by blending late bamboo shoots for 60 minutes at 1% brine concentration.
실험 결과, 균류 키토산의 농도에 따른 백탁현상의 영향은 40㎎/225㎖, 20㎎/225㎖, 10㎎/225㎖의 순서로 나타났으며, 저장기간이 길수록 백탁현상은 큰 변화를 보이지 않았으며 오히려 감소하는 경향이 있었는데 이는 균류 키토산의 응집효과에 기인한다(도 9). 따라서, 상기 결과에서 확인할 수 있듯이 균류 키토산을 식품제조공정중에 첨가함으로써 저장성을 향상시킬 수 있음을 알 수 있었다.As a result of the experiment, the effect of turbidity according to the concentration of fungus chitosan was shown in the order of 40mg / 225ml, 20mg / 225ml, 10mg / 225ml. Rather, it tended to decrease due to the aggregation effect of the fungus chitosan (FIG. 9). Therefore, as can be seen from the above results, it was found that the shelf life can be improved by adding the fungus chitosan to the food manufacturing process.
이상, 상기 실시예 및 실험예를 통하여 설명한 바와 같이 본 발명의 균류를 이용한 키토산 생산방법은 제조공정이 간단하며 기존의 갑각류로부터의 키토산 생산방법에 비하여 제조공정 중 요구되는 산 및 알칼리의 양을 줄일 수 있어 제조공정 중 발생하는 폐액 및 폐기물을 줄임으로써 이차적인 환경오염을 줄일 수 있을 뿐만 아니라 균류 키토산을 대량으로 생산할 수 있으며 생산된 키토산을 늦죽순 병조림의 백탁방지에 사용함으로써 식품산업상 매우 유용한 발명인 것이다.As described above, the chitosan production method using the fungus of the present invention, as described in the above Examples and Experimental Examples, has a simple manufacturing process and reduces the amount of acid and alkali required during the manufacturing process as compared to the conventional chitosan production method from shellfish. It is possible to reduce secondary environmental pollution by reducing waste and waste generated during the manufacturing process, as well as to produce a large amount of fungus chitosan, and to use the produced chitosan to prevent clouding of late bamboo shoots. will be.
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