KR20180057235A - Method of biosurfactant using candida bombicola strain - Google Patents
Method of biosurfactant using candida bombicola strain Download PDFInfo
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- 239000003876 biosurfactant Substances 0.000 title claims abstract description 22
- 241001278026 Starmerella bombicola Species 0.000 title claims abstract description 9
- 238000000034 method Methods 0.000 title claims description 17
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical compound OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 claims description 8
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Abstract
Description
본 발명은 캔디다 봄비콜라 균주(Candida bombicola strain) ATCC 22214를 이용한 바이오 계면활성제를 제공하는 것으로, 더욱 자세하게는, 디에틸렌 글리콜에 프로필렌 옥사이드를 첨가하여 만든 합성오일인 DEG(PO)4를 포함하는 배지에서 캔디다 봄비콜라 균주를 배양하여, 바이오 계면활성제를 제공하는 것이다.The present invention relates to Candida sp. The present invention relates to a biosurfactant using bombicola strain ATCC 22214, and more particularly to a biosurfactant using a bacterium Bacillus coli strain in a medium containing DEG (PO) 4, a synthetic oil made by adding propylene oxide to diethylene glycol, And to provide a biosurfactant.
바이오 계면활성제란, 효모, 곰팡이, 박테리아 등 미생물에 의해 생산되는 생분해성 계면활성제를 의미하며 균주에 따라 세포외 또는 세포 내에 생성이 된다.A biosurfactant is a biodegradable surfactant produced by microorganisms such as yeast, fungi, and bacteria, and is produced extracellularly or intracellularly depending on the strain.
다양한 미생물이 세포 외 또는 세포 내에 생산하는 바이오 계면활성제는 화학합성 계면활성제에 비해 무독성으로 생분해가 용이한 친환경적인 물질일 뿐만 아니라, 다양한 온도와 pH에 의해 계면활성제의 물리 화학적 성상을 안정하게 유지하므로 그 사용가치가 매우 높아 최근에 관심을 갖는 물질이다.Biosurfactants produced by various microorganisms extracellularly or intracellularly are not only environmentally friendly substances which are non-toxic and biodegradable as compared with chemically synthesized surfactants, but also stably maintain the physicochemical properties of the surfactant by various temperatures and pH It is a substance that has recently attracted attention because of its high use value.
실험에 사용한 효모는 소포로리피드(sophorolipid)를 생산하는 캔디다 봄비콜라 균주로 캔디다에 속하는 효모이다(Cavalero 2002). 소포로리피디는 캔디다 봄비콜라, 캔디다 아피콜라, 캔디다 바티스태, 위커라미넬라 도메리키에 및 로도토룰라 보고리엔시스와 같은 일부 효모 종에 의해 합성되는 글리코리피드(glycolipid) 형태의 계면활성제 물질이다.The yeast used in the experiment was candida bovis cola producing sophorolipid, which belongs to Candida (Cavalero 2002). Sophorolipid is a glycolipid-type surfactant material synthesized by some yeast species, such as Candida bombycola, Candida Apicola, Candida battista, Wicker laminella dermatique, and Rhodotorula borealisis.
이들이 생산한 소포로리피드는 친수성과 소수성을 모두 가지고 있다.The soporolipids produced by them have both hydrophilicity and hydrophobicity.
바이오 계면활성제의 소수성 부분은 대개 선형 포화 탄화수소로 이루어지며 친수성 부분은 당 또는 아미노산으로 구성된다.The hydrophobic part of the biosurfactant is usually composed of linear saturated hydrocarbons and the hydrophilic part is composed of sugars or amino acids.
바이오 계면활성제의 구분은 생산 균주에 의해 구분되며, 계면활성제 형태에 따라 나뉠 수 있다.The classification of biosurfactants is divided by production strains and can be divided according to the type of surfactant.
바이오 계면활성제는 표면장력이 25-35dyne/cm 범위에 있고, 마이셀 형성농도(CMC; Criticla Micelle Concentration)는 0.1-150mg/L의 광범위한 특성을 보이고 있다.The surface tension of the biosurfactant is in the range of 25-35 dyne / cm and the CMC (Criticla Micelle Concentration) shows a wide range of 0.1-150 mg / L.
따라서, 계면활성제가 갖는 이런 독특한 성질 때문에 생물 계면활성제는 식품, 제약, 화장품, 농산물 가공업, 생물정화 등에 다양하게 활용되고 있고, 최근에는 일부 생물 계면활성제가 병을 치료하는 효과를 갖고 있어, 의약품으로도 활용할 수 있고, 또 병원성 미생물이 인체에 부착되지 못하게 하는 효과도 있는 것으로 확인되는 등 많은 연구가 진행되고 있다.Because of this unique property of surfactants, biosurfactants are widely used in foods, pharmaceuticals, cosmetics, agricultural products processing, biological purification, etc. Recently, some biosurfactants have the effect of treating diseases, And it is confirmed that there is also an effect of preventing the pathogenic microorganisms from adhering to the human body.
본 발명은 친환경 농약용 계면활성제를 개발하기 위해 캔디다 봄비콜라(Candida bombicola)를 이용하여 바이오 계면활성제를 생산하고 이를 상업적으로 활용하고자 하는 것이다.The present invention is to produce a biosurfactant using Candida bombicola to develop a surfactant for environmentally friendly agrochemicals and to commercialize it.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자는 디에틸렌 글리콜에 프로필렌 옥사이드를 첨가하여 만든 합성오일인 DEG(PO)4를 포함하는 배지에서 캔디다 봄비콜라 균주를 배양하여, 배양액으로부터 소포로리피드를 추출하는 단계를 포함하는 바이오 계면활성제를 제공하는 것이다.DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-described needs, and the present inventors have found that a Candida albicans coli strain is cultured in a medium containing DEG (PO) 4, a synthetic oil prepared by adding propylene oxide to diethylene glycol, And a step of extracting the lorifide.
본 발명의 일 실시예에 따르면, 디에틸렌 글리콜에 프로필렌 옥사이드를 첨가하여 만든 합성오일인 DEG(PO)4를 포함하는 배지에서 효모를 배양하여, 제조된 배양액으로부터 소포로리피드를 추출하는 단계를 포함하여, 하기 화학식 1로 표시되는 바이오 계면활성제인 것을 특징으로 하는 바이오 계면활성제의 제조방법을 제공한다.According to one embodiment of the present invention, yeast is cultured in a medium containing DEG (PO) 4, which is a synthetic oil made by adding propylene oxide to diethylene glycol, and the step of extracting the soporolipid from the prepared culture Wherein the biosurfactant is a biosurfactant represented by the following formula (1).
[화학식 1] [Chemical Formula 1]
본 발명에서 이용할 수 있는 효모는 당업계에 알려진 다양한 균주를 이용할 수 있으며, 바람직하게는 캔디다 봄비콜라 균주(Candida bombicola strain), 보다 바람직하게는 수탁번호 ATCC22214의 캔디다 봄비콜라 균주이다.Yeast that can be used in the present invention may use a variety of strains known in the art, preferably a spring rain Candida coke strain (Candida bombicola strain, more preferably Candida albicans strains of accession number ATCC22214.
상기 캔디다 봄비콜라(Candida bombicola) 균주 배양은 일반 배지 및 생산 배지의 배양인 것을 특징으로 한다. 보다 구체적으로, 상기 일반 배지는 D-글루코즈(D-glucose) 10 g/l, 효모 추출액(yeast extract) 3 g/l, 맥아 추출액(Malt extrct) 3 g/l, 펩톤(Peptone) 5g /l 이고, 상기 생산 배지는 D-글루코즈(D-glucose) 100 g/l, 효모 추출액(yeast extract) 5 g/l. 펩톤(Peptone 0.7 g/l), 인산이수소칼륨(KH2PO4) 1 g/l, 황산마그네숨 7수화물(MgSO47H2O) 0.5 /l, 염화칼슘 2수화물(CaCl22H2O) 0.1 g/l, 염화나트륨(NaCl) 0.1 g/l, 펩톤(Peptone) 0.7 g/l, 오일(Oil) 100 g/l 인 것을 특징으로 한다.The Candida spring rain coke (Candida bombicola ) is characterized in that it is a culture of normal medium and production medium. More specifically, the general medium was prepared by adding 10 g / l of D-glucose, 3 g / l of yeast extract, 3 g / l of malt extract, 5 g / l of peptone , And the production medium was 100 g / l of D-glucose and 5 g / l of yeast extract. 1 g / l of peptone (0.7 g / l Peptone), potassium dihydrogenphosphate (KH 2 PO 4 ), 0.5 l of magnesium sulfate heptahydrate (MgSO 4 7H 2 O), calcium chloride dihydrate (CaCl 2 2H 2 O) 0.1 g / l of sodium chloride, 0.1 g / l of sodium chloride, 0.7 g / l of peptone and 100 g / l of oil.
상기 소포로리피드는 52~55%의 C12 지방산 및 20~23%의 C14 지방산을 포함하는 소포도리피드일 수 있으며, 바람직하게는 52~55%의 C12 지방산 및 20~23%의 C14 지방산을 포함하는 소포로리피드 일 수 있으며, 더욱 바람직하게는 약 53%의 C12 지방산 및 약 21%의 C14 지방산을 포함하는 소포로리피드일 수 있으나, 이에 제한되지 않는다.The soporolipid may be a soporidipid comprising 52 to 55% C12 fatty acid and 20 to 23% C14 fatty acid, preferably containing 52 to 55% C12 fatty acid and 20 to 23% C14 fatty acid , More preferably about 53% C12 fatty acid and about 21% C14 fatty acid, but is not limited thereto.
또한, 본 발명은 소포로리피드를 유효성분으로 함유하는 항균용 조성물을 제공하는 것을 특징으로 한다.Further, the present invention is characterized by providing an antimicrobial composition containing a sophorolipid as an active ingredient.
또한, 본 발명에서 상기 소포로리피드는 황색포도상구균(Staphylococcus aureus) 및 대장균(Escherichia coli)에 대한 항균 활성을 갖는것을 특징으로 한다.Further, in the present invention, the sophorolipid is characterized by having antimicrobial activity against Staphylococcus aureus and Escherichia coli .
본 발명의 바이오 계면활성제는 다양한 용도로 이용이 가능하며, 예컨대 세정 및 정화용 조성물 등으로 이용할 수 있으며, 그 외에도 의약품, 식품, 화장품 등 화학 합성 계면활성제가 사용되는 대부분의 다양한 산업분야에서 사용할 수 있다.The biosurfactant of the present invention can be used for various purposes, for example, as a composition for cleaning and purifying, and in addition, it can be used in most various industrial fields where a chemical synthetic surfactant such as medicines, foods, and cosmetics is used .
도 1은 디에틸렌 글리콜과 프로필렌 옥사이드가 반응하여 바이오 계면활성제가 제조되는 화학반응을 나타낸 도면이다.
도 2는 배지 만드는 순서를 나타낸 도면이다.
도 3은 고압멸균기 작동방법 순서를 나타낸 도면이다.
도 4는 발효기 설치 순서 및 균 접종을 나타낸 도면이다.
도 5는 발효기 세척 및 배양액 보관 방법을 나타낸 도면이다.
도 6은 균 배양 순서를 나타낸 도면이다.
도 7은 발효조 접종을 나타낸 도면이다.
도 8은 소포리피드 후 처리공정을 나타낸 도면이다.
도 9는 SUNPOL-BSDE1_IR을 나타낸 도면이다.
도 10은 DEG(PO)4_IR을 나타낸 도면이다.1 is a view showing a chemical reaction in which a diethylene glycol and propylene oxide are reacted to produce a biosurfactant.
FIG. 2 is a view showing a procedure for producing a medium. FIG.
3 is a view showing the procedure of the operation method of the high-pressure sterilizer.
Fig. 4 is a diagram showing a fermenter installation sequence and a homogenization method.
5 is a view showing a method of washing a fermenter and storing a culture medium.
Fig. 6 is a view showing the sequence of bacterial culturing.
7 is a view showing the fermentation tank inoculation.
Fig. 8 is a view showing a process for post-treatment of a siporite.
9 is a diagram showing SUNPOL-BSDE1_IR.
10 is a diagram showing DEG (PO) 4_IR.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
실시예Example 1: 균주, 배지 및 배양 조건 1: strain, medium and culture conditions
스포로리피드를 생산하기 위해 캔디다 봄비콜라(Candida bombicola , ATCC 22214)를 이용하였다. 캔디다 봄비콜라 균주를 YM 브로스(broth) 배지에서 일반 배지인 D-글루코즈(D-glucose) 10 g/l, 효모 추출액(yeast extract) 3 g/l, 맥아 추출액(Malt extrct) 3 g/l, 펩톤(Peptone) 5g /l 와 생산 배지인 D-글루코즈(D-glucose) 100 g/l, 효모 추출액(yeast extract) 5 g/l. 펩톤(Peptone 0.7 g/l), 인산이수소칼륨(KH2PO4) 1 g/l, 황산마그네슘 7수화물(MgSO47H2O) 0.5 /l, 염화칼슘 2수화물(CaCl22H2O) 0.1 g/l, 염화나트륨(NaCl) 0.1 g/l, 펩톤(Peptone) 0.7 g/l, 오일(Oil) 100 g/l 에 DEG(PO)4에서 배양하였다. Spokane Candida spring rain coke for the production of feed Lori (Candida bombicola , ATCC 22214) was used. Candida bovis cola was cultured in YM broth medium with 10 g / l of D-glucose, 3 g / l of yeast extract, 3 g / l of malt extract (Malt extrct) 5 g / l of peptone, 100 g / l of D-glucose as a production medium, and 5 g / l of yeast extract. 1 g / l of peptone (Peptone 0.7 g / l), potassium dihydrogenphosphate (KH 2 PO 4 ), 0.5 l of magnesium sulfate 7 hydrate (MgSO 4 7H 2 O), calcium chloride dihydrate (CaCl 2 2H 2 O) 0.1 (PO) 4 to 0.1 g / l of sodium chloride (NaCl), 0.7 g / l of peptone and 100 g / l of oil.
(1) 균주관리(1) Strain management
테스트 튜브(Test tube)에 YM 배지 5ml을 제조 후, 121 15분간 오토 클레이브(auto clave)로 멸균 후 무균실험대(clean-bench)에 배지를 식히면서 보관한 후, 균주 stock(-80)을 YM 배지에 2%(v/v) 접종 시킨 후, 30, 200rpm, 48 시간 동안 흔들면서 인큐베이터에에서 배양한다.5 ml of YM medium was prepared in a test tube, sterilized by an auto clave for 121-15 minutes, and stored in a clean-bench while cooling the medium. The strain stock (-80) was transferred to YM medium (V / v), and incubated in an incubator with shaking at 30, 200 rpm for 48 hours.
(2) 전배양(1차 종균배양)(2) Pre-culture (primary seed culture)
250ml 플라스크(Flask)에 YM배지 50ml을 제조 후, 121 15분간 오토 클레이브(auto clave)로 멸균 후 무균실험대(clean-bench)에 배지를 식히면서 보관한다, 이후에 테스트 튜브(Test tub)e에서 배양한 균주를 플라스크(flask) YM 배지로 2%(v/v) 접종 후 30, 200rpm, 48 시간 동안 흔들어 인큐베이터에 배양 후, 플라스크(Flask) YM 배지에 2%(v/v) 균주 접종함으로 계대 배양 후, 30, 200rpm, 48시간 동안 흔들어 인큐베이터에서 배양 후 플라스크(Flask) YM 배지에 2%(v/v) 접종함으로 균주 계대 배양 후 30, 200rpm, 48시간동안 흔들어 인큐베이터에서 배양한다.50 ml of YM medium is prepared in a 250 ml flask, sterilized by an auto clave for 121-15 minutes, and stored in a clean-bench while cooling the medium. Then, the culture is incubated in a test tube (e) One strain was inoculated with 2% (v / v) in a flask YM medium, shaken at 30, 200 rpm for 48 hours, incubated in an incubator, and inoculated with 2% (v / v) After incubation, shake at 30, 200 rpm for 48 hours, incubate in an incubator, and subculture by 2% (v / v) inoculation in Flask YM medium and incubate at 30, 200 rpm for 48 hours in an incubator.
(3) 본배양 (Main culture)(3) Main culture
5L jar 에 3L의 생산배지를 제 후, jar를 조립 후 각각의 연결 부 및 노출부분을 호일로 감싸고 121 15분간 접종구는 열어놓고 auto clave로 멸균시킨다. Auto clave 멸균 후 꺼내기 전에 접종구 잠그고 꺼낸다. 컨덴서(Condenser)와 증류수 자켓(water jacket)의 주입구(inlet)와 배출구(outlet)를 연결한다. 공기 주입구(Air inlet)을 air line과 연결 후 공기(air)를 넣어주고 air가 충분히 들어가면 air outlet의 밸브(valve)를 열어주고 식힌다. 온도가 적당히 내려가면 DO 프로브(probe), pH 프로브(probe)를 본체와 연결 하고, 30, pH=3.5, 500rpm, 1vvm 조건 설정 후 작동시킨다. 접종구를 통해 5%(V/V) 균주 접종 후 8일간 배양한다.After 3L production medium is prepared in 5L jar, the jar is assembled, and each connection part and the exposed part are wrapped with foil, and the inoculation area for 121-15 minutes is opened and sterilized by auto clave. Auto sterilize the clave and lock the inoculum before removing it. Connect the inlet and outlet of the condenser and distilled water jacket. Connect the air inlet to the air line, and then add air. If the air is enough, open the valve of the air outlet and cool. When the temperature falls moderately, connect the DO probe and pH probe to the instrument and set it to 30, pH = 3.5, 500 rpm, 1 vvm. After inoculation with 5% (V / V) strain, cultivate for 8 days.
실시예Example 2: 2: 후 공정Post-process
원심분리기(Centrifuge) 8000rpm, 25, 20min을 통한 배양액 수거하는데, 가라앉은 세포(cell)를 제거한 상등액을 수거한다.Centrifuge Collect the supernatant from which the cells have been removed by collecting the culture solution at 8000 rpm, 25, 20 min.
배양액을 에틸 아세테으트로 3회 추출하고 증발 농축을 통해 소포로리피드를 수득한다.The culture was extracted three times with ethyl acetate and concentrated by evaporation to obtain the sophorolipid.
실시예Example 3: 수분측정 3: Moisture measurement
Karlfischer method (KEM,ELECTRONICSMFG.CO.,LTD)을 사용해 수분측정을 했다.The moisture was measured using the Karlfischer method (KEM, ELECTRONICSMFG.CO., LTD).
실시예Example 4: 표면장력 측정 4: Surface tension measurement
Wilhemy Plate Method (Rhesca)을 사용해 표면장력을 측정했다.The surface tension was measured using the Wilhemy Plate Method (Rhesca).
실시예Example 5: 5: 적외선분광분석기Infrared spectrometer (FT-IR) 측정 (FT-IR) measurement
전반사 측정법/Attenuated Total Reflection (Varian)으로 측정했다.And measured with an Attenuated Total Reflection (Varian).
실시예Example 6: 독성 시험 6: Toxicity test
경구독성/2000mg/kg (안전성평가연구소), 경피독성/2000mg/kg (한국화학융합시험연구원) 의 독성시험 결과를 나타낸다.Oral toxicity / 2000mg / kg (Safety Evaluation Research Institute), dermal toxicity / 2000mg / kg (Korea Chemical Fusion Research Institute).
실시예Example 7: 생분해성 시험 7: Biodegradability test
OECD 301/310 70% 이상이면 통과이다.OECD 301/310 70% or more is a pass.
실시예Example 8: 8: 향균Antibiotic 활성 시험 Activity test
황색포도상구균(Staphylococcus aureus) 및 대장균(Escherichia coli)에 대한 소포로리피드의 항균 능력은 Shin 등에 의해 기술된 방법에 따라 측정되었다(Shin et al., Bioresour . Technol. 101(9), 3170-3174, 2010), DF-100(포도씨 추출물)이 표준물질로 사용되었다.(Defer et al., Aquaculture, 293(1-2), 1-7, 2009). Staphylococcus aureus and Escherichia coli antibacterial ability of the vesicles to the Lowry feed coli) was measured according to the method described by Shin (Shin et al., Bioresour . Technol. 101 (9), 3170-3174, 2010), DF-100 ( grape seed extract) Were used as reference materials (Defer et al ., Aquaculture , 293 (1-2), 1-7, 2009).
Claims (7)
[화학식 1]
[Chemical Formula 1]
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102053280B1 (en) * | 2018-06-05 | 2019-12-06 | 주식회사 유나이티드엑티브 | Fermented emulsifier forming reverse micell and composition comprising thereof and preparing method for fermented emulsifier |
| WO2020085857A1 (en) * | 2018-10-26 | 2020-04-30 | 주식회사 유나이티드엑티브 | Transdermal delivery system comprising reverse micelle |
| WO2023250308A1 (en) * | 2022-06-22 | 2023-12-28 | Locus Solutions Ipco, Llc | Compositions and methods for controlling foam |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102053280B1 (en) * | 2018-06-05 | 2019-12-06 | 주식회사 유나이티드엑티브 | Fermented emulsifier forming reverse micell and composition comprising thereof and preparing method for fermented emulsifier |
| WO2019235761A1 (en) * | 2018-06-05 | 2019-12-12 | 주식회사 유나이티드엑티브 | Fermented emulsifier forming reverse micelles, composition containing same and method for preparing fermented emulsifier |
| WO2019235840A3 (en) * | 2018-06-05 | 2020-02-06 | 주식회사 유나이티드엑티브 | Fermented emulsifier for forming reverse micelle, composition comprising same and method for producing fermented emulsifier |
| WO2020085857A1 (en) * | 2018-10-26 | 2020-04-30 | 주식회사 유나이티드엑티브 | Transdermal delivery system comprising reverse micelle |
| KR20200047430A (en) * | 2018-10-26 | 2020-05-07 | 주식회사 유나이티드엑티브 | Transepidermal delivery system comprising reverse micell |
| WO2023250308A1 (en) * | 2022-06-22 | 2023-12-28 | Locus Solutions Ipco, Llc | Compositions and methods for controlling foam |
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