KR102682973B1 - Lactic acid bacteria-Chitosan Composite for the Removal of Phthalate Endocrine Hormone and method of preparing the same - Google Patents
Lactic acid bacteria-Chitosan Composite for the Removal of Phthalate Endocrine Hormone and method of preparing the same Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 156
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 100
- 239000004310 lactic acid Substances 0.000 title claims abstract description 78
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 78
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 title claims description 10
- 238000000034 method Methods 0.000 title description 8
- 239000005556 hormone Substances 0.000 title description 2
- 229940088597 hormone Drugs 0.000 title description 2
- 239000002131 composite material Substances 0.000 title 1
- 230000002124 endocrine Effects 0.000 title 1
- 241000894006 Bacteria Species 0.000 claims abstract description 56
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims description 28
- FLKPEMZONWLCSK-UHFFFAOYSA-N diethyl phthalate Chemical compound CCOC(=O)C1=CC=CC=C1C(=O)OCC FLKPEMZONWLCSK-UHFFFAOYSA-N 0.000 claims description 26
- 239000002245 particle Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000003431 cross linking reagent Substances 0.000 claims description 10
- IRIAEXORFWYRCZ-UHFFFAOYSA-N Butylbenzyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCC1=CC=CC=C1 IRIAEXORFWYRCZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- HBGGXOJOCNVPFY-UHFFFAOYSA-N diisononyl phthalate Chemical compound CC(C)CCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCC(C)C HBGGXOJOCNVPFY-UHFFFAOYSA-N 0.000 claims description 4
- 241000194035 Lactococcus lactis Species 0.000 claims description 3
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 3
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 claims description 2
- 239000004971 Cross linker Substances 0.000 claims 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical class OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 abstract description 5
- 238000001179 sorption measurement Methods 0.000 description 34
- 239000003463 adsorbent Substances 0.000 description 28
- 230000000052 comparative effect Effects 0.000 description 20
- 239000000243 solution Substances 0.000 description 19
- 239000002105 nanoparticle Substances 0.000 description 17
- 239000000126 substance Substances 0.000 description 9
- 125000000524 functional group Chemical group 0.000 description 8
- 125000005498 phthalate group Chemical class 0.000 description 8
- 235000019832 sodium triphosphate Nutrition 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000004014 plasticizer Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000001878 scanning electron micrograph Methods 0.000 description 3
- 239000010409 thin film Substances 0.000 description 3
- 229920002101 Chitin Polymers 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 229920000388 Polyphosphate Chemical class 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- KKEYFWRCBNTPAC-UHFFFAOYSA-N benzene-dicarboxylic acid Natural products OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 231100000507 endocrine disrupting Toxicity 0.000 description 1
- 231100000049 endocrine disruptor Toxicity 0.000 description 1
- 239000000598 endocrine disruptor Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000001205 polyphosphate Chemical class 0.000 description 1
- 235000011176 polyphosphates Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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Abstract
본 발명은 키토산이 유산균 표면에 섬(island) 형태로 결합되어 프탈레이트 화합물을 흡착 제거할 수 있는 유산균-키토산 복합체 및 이의 제조방법에 관한 것이다. The present invention relates to a lactic acid bacteria-chitosan complex in which chitosan is bonded to the surface of lactic acid bacteria in the form of islands and can adsorb and remove phthalate compounds, and a method for producing the same.
Description
본 발명은 프탈레이트 환경 호르몬 제거를 위한 유산균-키토산 복합체 및 이의 제조방법에 관한 것으로서, 보다 상세하게는 키토산이 유산균 표면에 섬(island) 형태로 결합되어 프탈레이트 화합물을 흡착 제거할 수 있는 유산균-키토산 복합체 및 이의 제조방법에 관한 것이다. The present invention relates to a lactic acid bacteria-chitosan complex for removing phthalate environmental hormones and a method for producing the same. More specifically, the lactic acid bacteria-chitosan complex is capable of adsorbing and removing phthalate compounds by binding chitosan in the form of an island to the surface of lactic acid bacteria. and its manufacturing method.
플라스틱의 제조 공정에서 반드시 함유되는 물질인 가소제는 유연성과 내구성 등 플라스틱의 가소성을 높이기 위하여 오랫동안 사용되어 왔다. 프탈레이트(Phthalates)는 가장 일반적으로 사용되는 가소제로서 Dibutyl phthalate(DBP), Diethyl phthalate(DEP) 등이 있다. 매년 전 세계적으로 약 810만 톤의 가소제가 생산되고 있다. 이러한 프탈레이트는 플라스틱 matrix에 공유결합되지 않기 때문에 결합력이 약하여 생산 과정이나 실제 사용하는 플라스틱 등에서 환경 또는 사람에게 쉽게 노출될 수 있다. Plasticizers, which are essential substances in the plastic manufacturing process, have been used for a long time to increase the plasticity of plastics, such as flexibility and durability. Phthalates are the most commonly used plasticizers and include dibutyl phthalate (DBP) and diethyl phthalate (DEP). About 8.1 million tons of plasticizers are produced worldwide every year. Since these phthalates are not covalently bonded to the plastic matrix, their bonding strength is weak and they can be easily exposed to the environment or people during the production process or in actual plastics used.
프탈레이트의 기본 구조는 두 개의 곁사슬을 가지는 benzene dicarboxylic acid 로, 곁 사슬은 alkyl, benzyl, cycloalkyl, phenyl 또는 alkoxy 등의 작용기로 이루어져 있다. 저 분자량의 프탈레이트는 생식 건강에 해를 끼치는 등 내분비 교란 물질로서 European Union’s REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) 에서 위험 물질로 분류하고 있다. The basic structure of phthalate is benzene dicarboxylic acid with two side chains, and the side chains consist of functional groups such as alkyl, benzyl, cycloalkyl, phenyl, or alkoxy. Low molecular weight phthalates are endocrine disruptors that cause harm to reproductive health and are classified as hazardous substances by the European Union’s REACH (Registration, Evaluation, Authorization and Restriction of Chemicals).
현재 프탈레이트 제거를 위한 많은 연구가 진행되고 있으며, 특히 흡착을 이용한 제거가 많이 이용되고 있는데, 흡착제로는 활성탄, zeolite, 실리카겔 등이 있다. 그 중 활성탄은 유기 물질과 비극성 흡착물의 흡착에 주로 사용되며, 표면 작용기 및 기공 크기 등의 물리적 특성을 조절할 수 있기 때문에 가장 널리 사용되고 있는 흡착제이다. 활성탄의 흡착 능력이 뛰어나지만, 비용이 비싸서 광범위한 사용에는 제한적이다. Currently, much research is being conducted to remove phthalates, and in particular, removal using adsorption is widely used. Adsorbents include activated carbon, zeolite, and silica gel. Among them, activated carbon is mainly used for adsorption of organic substances and non-polar adsorbents, and is the most widely used adsorbent because it can control physical properties such as surface functional groups and pore size. Although activated carbon has excellent adsorption capacity, its widespread use is limited due to its high cost.
경제적인 흡착제에 대한 필요성이 증가함에 따라 biomass를 사용한 흡착제 연구가 활발히 진행되고 있다. Biomass로써 사용 가능한 물질로는 chitin, chitosan, cellulose 등이 있으며, 풍부한 생물 자원으로서 쉽게 저렴하게 구할 수 있다. 또한 다양한 표면 작용기를 가지고 있어 흡착제로서 사용 가능성이 크다. As the need for economical adsorbents increases, research on adsorbents using biomass is actively underway. Substances that can be used as biomass include chitin, chitosan, and cellulose, which are abundant biological resources and can be obtained easily and inexpensively. In addition, it has a variety of surface functional groups, so it has great potential for use as an adsorbent.
경제적인 흡착제 기술에 대한 필요성이 증가함에 따라 바이오매스의 흡착 능력을 이용한 오염물질 제거에 대한 연구가 진행되고 있다. 셀룰로오스 다음으로 가장 풍부한 생물 자원인 키토산은 갑각류의 껍질에서 얻을 수 있는 키틴에서 유래된 바이오 폴리머이다. 키토산은 말단기에 존재하는 아민기(-NH2)와 히드록시기(-OH) 로 인하여 변형이 용이하고, 높은 흡착 능력을 지니고 있어 금속, 양이온성 및 음이온성 염료 분자를 흡착하여 제거하는 다양한 연구가 진행되고 있다. As the need for economical adsorbent technology increases, research on pollutant removal using the adsorption capacity of biomass is underway. Chitosan, the most abundant biological resource after cellulose, is a biopolymer derived from chitin, which can be obtained from the shell of crustaceans. Chitosan is easy to modify due to the amine group (-NH2) and hydroxy group (-OH) present at the terminal group, and has a high adsorption capacity, so various studies are being conducted to adsorb and remove metal, cationic, and anionic dye molecules. It is becoming.
생물학적 처리에도 사용되고 있는 미생물은 세포벽에 존재하는 카르복실기, 인산기, 아민 및 히드록시기 등의 여러 작용기의 존재로 생흡착이 가능한 물질이다. 이때의 생흡착 메커니즘은 흡착, 침착, 이온 교환과 같은 물리적 및 화학적 상호 작용을 포함한다. 미생물 생흡착은 높은 흡착 능력, 빠른 정상상태 도달 및 낮은 공정비용 등의 장점이 있다. 하지만 기본적으로 밀도가 낮고 강도가 약한 입자로 공정 과정에서의 발생할 수 있는 입자의 팽윤, 재생 및 재사용이 불가능하다는 문제점이 있다. Microorganisms that are also used in biological treatment are substances capable of biosorption due to the presence of various functional groups such as carboxyl groups, phosphate groups, amine groups, and hydroxy groups present in the cell walls. The biosorption mechanism at this time includes physical and chemical interactions such as adsorption, deposition, and ion exchange. Microbial biosorption has advantages such as high adsorption capacity, rapid reaching steady state, and low process cost. However, it is basically a low-density and weak-strength particle, and there is a problem in that it is impossible to swell, regenerate, or reuse the particles that may occur during the process.
한국등록특허 10-2210922호(본 발명자의 출원 특허임)는 구형의 키토산 나노입자 내부에 복수개의 유산균이 담지된 키토산 나노입자를 개시하고 있다. 상기 등록특허는 염료나 금속 이온이 키토산 나노입자 내부로 이동하여 내부의 유산균에 흡착되거나 키토산 표면 기능기에 흡착되는 염료나 금속이온 흡착제를 언급하고 있다. 하지만, 상기 등록특허는 유산균을 키토산 나노입자 내부에 담지하고 있어 입자 크기가 물질이 키토산 내부로 이동되기가 쉽지 않을 뿐만 아니라 흡착대상 물질이 정전기적 인력으로 표면의 키토산에 상당수가 흡착되므로 키토산 내부에 위치하는 유산균의 활용도가 높지 않다는 문제점이 제기되었다. Korean Patent No. 10-2210922 (a patent filed by the present inventor) discloses chitosan nanoparticles containing a plurality of lactic acid bacteria inside spherical chitosan nanoparticles. The registered patent refers to a dye or metal ion adsorbent in which dye or metal ions move inside chitosan nanoparticles and are adsorbed on internal lactic acid bacteria or adsorbed on chitosan surface functional groups. However, in the above registered patent, lactic acid bacteria are supported inside chitosan nanoparticles, so the particle size makes it difficult for the material to move into the chitosan, and a significant portion of the adsorption target material is adsorbed to the chitosan on the surface due to electrostatic attraction, so it is not easy to move the material into the chitosan. A problem was raised that the utilization of the lactic acid bacteria located there is not high.
본 발명은 유산균의 흡착 효율을 높인 복합체를 제공하는 것이다. The present invention provides a complex with increased adsorption efficiency of lactic acid bacteria.
본 발명은 내분비 교란 물질인 프탈레이트를 제거할 수 있는 흡착제를 제공하는 것이다. The present invention provides an adsorbent capable of removing phthalate, an endocrine disrupting substance.
하나의 양상에서 본 발명은 In one aspect, the present invention
키토산 분말을 물에 용해시키는 단계 ; Dissolving chitosan powder in water;
유산균 용액을 상기 키토산 용액에 넣어 혼합하는 단계 ; Adding and mixing the lactic acid bacteria solution into the chitosan solution;
가교제를 상기 키토산 용액에 적하하는 단계 ; Adding a cross-linking agent dropwise to the chitosan solution;
생성된 입자를 상기 용액으로부터 분리하는 단계를 포함하는 유산균-키토산 복합체의 제조방법에 관련된다. It relates to a method for producing a lactic acid bacteria-chitosan complex including the step of separating the generated particles from the solution.
다른 양상에서, 본 발명은 In another aspect, the present invention
응집된 유산균 : Agglomerated lactic acid bacteria:
상기 유산균 주변을 섬 구조로 둘러싸 위치된 키토산 나노입자를 포함하고, Contains chitosan nanoparticles surrounded by an island structure around the lactic acid bacteria,
상기 생균과 키토산 나노입자는 아미드 결합, 수소결합 또는 정전기적 결합을 통해 프탈레이트계 화합물을 흡착하는 유산균-키토산 복합체에 관련된다. The live bacteria and chitosan nanoparticles are related to a lactic acid bacteria-chitosan complex that adsorbs phthalate compounds through amide bonds, hydrogen bonds, or electrostatic bonds.
본 발명의 유산균-키토산 복합체는 유산균과 그 주변을 섬 구조로 둘러싼 키토산 나노입자로 이루어진 흡착제로서, 키토산뿐만 아니라 유산균 표면에도 다량의 프탈레이트 화합물이 흡착될 수 있다. The lactic acid bacteria-chitosan complex of the present invention is an adsorbent composed of lactic acid bacteria and chitosan nanoparticles surrounding them in an island structure, and a large amount of phthalate compounds can be adsorbed not only on chitosan but also on the surface of the lactic acid bacteria.
본 발명의 유산균-키토산 복합체는 쉽고 빠른 방법으로 제조 가능하며, 프탈레이트를 효과적으로 제거할 수 있는 저비용의 흡착제로서 사용할 수 있다. The lactic acid bacteria-chitosan complex of the present invention can be prepared in an easy and fast manner and can be used as a low-cost adsorbent that can effectively remove phthalates.
도 1은 본 발명에서 사용되는 유산균을 준비하는 과정을 도시한 것이다.
도 2는 본 발명의 유산균-키토산 복합체를 제조하는 단계를 도시한 것이다.
도 3은 본 발명의 유산균-키토산 복합체의 개념도이다.
도 4는 키토산 농도에 따른 비교예와 실시예의 입자 직경을 나타낸 그래프이다.
도 5는 제조된 유산균, 비교예 1(키토산 나노입자)와 실시예 1(유산균-키토산 복합체)의 SEM 이미지이다.
도 6은 유산균(L.ac), 비교예 1, 실시예 1의 작용기를 IR spectroscope 를 통하여 측정한 결과이다.
도 7은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 농도(즉, 흡착제의 농도)에 따른 DBP에 대한 흡착능(A)과 제거효율(B)을 나타낸다.
도 8은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 농도(흡착제의 농도)에 따른 DEP에 대한 흡착능(A)과 제거효율(B)을 나타낸다.
도 9는 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 흡착 시간에 따른 DBP에 대한 흡착능(A)과 제거효율(B)을 나타낸다.
도 10은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 흡착 시간에 따른 DEP에 대한 흡착능(A)과 제거효율(B)을 나타낸다. Figure 1 shows the process of preparing lactic acid bacteria used in the present invention.
Figure 2 shows the steps for preparing the lactic acid bacteria-chitosan complex of the present invention.
Figure 3 is a conceptual diagram of the lactic acid bacteria-chitosan complex of the present invention.
Figure 4 is a graph showing particle diameters of comparative examples and examples according to chitosan concentration.
Figure 5 is an SEM image of the prepared lactic acid bacteria, Comparative Example 1 (chitosan nanoparticles) and Example 1 (lactic acid bacteria-chitosan complex).
Figure 6 shows the results of measuring the functional groups of lactic acid bacteria ( L.ac) , Comparative Example 1, and Example 1 through an IR spectroscope.
Figure 7 shows the adsorption capacity (A) and removal efficiency (B) for DBP according to the concentration (i.e., concentration of adsorbent) of lactic acid bacteria ( L.ac) , Comparative Example 1 (CNPs), and Example 1 ( L.ac -CNPs). ).
Figure 8 shows the adsorption capacity (A) and removal efficiency (B) for DEP according to the concentration (adsorbent concentration) of lactic acid bacteria ( L.ac) , Comparative Example 1 (CNPs), and Example 1 ( L.ac -CNPs). indicates.
Figure 9 shows the adsorption capacity (A) and removal efficiency (B) for DBP according to the adsorption time of lactic acid bacteria (L.ac), Comparative Example 1 (CNPs), and Example 1 (L.ac-CNPs).
Figure 10 shows the adsorption capacity (A) and removal efficiency (B) for DEP according to the adsorption time of lactic acid bacteria (L.ac), Comparative Example 1 (CNPs), and Example 1 (L.ac-CNPs).
이하에서는 첨부한 도면을 참조하여 본 발명의 실시 예를 상세히 설명한다. 본 발명의 실시 예를 설명함에 있어 관련된 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 것이다. 또한, 후술 되는 용어들은 본 발명에서의 기능을 고려하여 정의된 용어들로서 이는 사용자, 운용자의 의도 또는 관례 등에 따라 달라질 수 있다. 그러므로 그 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In describing embodiments of the present invention, if it is determined that a detailed description of a related known function or configuration may obscure the gist of the present invention, the detailed description will be omitted. In addition, the terms described below are terms defined in consideration of functions in the present invention, and may vary depending on the intention or custom of the user or operator. Therefore, the definition should be made based on the contents throughout this specification.
본 발명은 유산균-키토산 복합체 및 이의 제조방법을 제공한다. 본 발명의 일 구현예를 상세히 설명한다. The present invention provides a lactic acid bacteria-chitosan complex and a method for producing the same. An embodiment of the present invention will be described in detail.
도 1은 본 발명에서 사용되는 유산균을 준비하는 과정을 도시한 것이고, 도 2는 본 발명의 유산균-키토산 복합체를 제조하는 단계를 도시한 것이고, 도 3은 본 발명의 유산균-키토산 복합체의 개념도이고, 도 4는 키토산 농도에 따른 비교예와 실시예의 입자 직경을 나타낸 그래프이고, 도 5는 제조된 유산균, 비교예 1(키토산 나노입자)와 실시예 1(유산균-키토산 복합체)의 SEM 이미지이다. Figure 1 shows the process of preparing lactic acid bacteria used in the present invention, Figure 2 shows the steps for preparing the lactic acid bacteria-chitosan complex of the present invention, and Figure 3 is a conceptual diagram of the lactic acid bacteria-chitosan complex of the present invention. , Figure 4 is a graph showing the particle diameter of comparative examples and examples according to chitosan concentration, and Figure 5 is an SEM image of the prepared lactic acid bacteria, Comparative Example 1 (chitosan nanoparticles) and Example 1 (lactic acid bacteria-chitosan complex).
도 1 내지 도 2를 참고하면, 본 발명의 유산균-키토산 복합체 제조 방법은 키토산 수용액 제조, 혼합 단계, 적하 단계, 분리단계를 포함한다. Referring to Figures 1 and 2, the method for producing a lactic acid bacteria-chitosan complex of the present invention includes preparing an aqueous chitosan solution, a mixing step, a dropping step, and a separation step.
키토산 수용액 제조는 키토산을 물에 용해시키는 단계이다. 상기 키토산은 공지된 키토산이 제한없이 사용될 수 있다. 또한, 상기 키토산으로 변형된 키토산 유도체도 포함될 수 있다. Preparation of chitosan aqueous solution involves dissolving chitosan in water. The chitosan may be any known chitosan without limitation. Additionally, chitosan derivatives modified with the above chitosan may also be included.
키토산 수용액은 키토산 분말을 물에 용해시켜 수득할 수 있다. Chitosan aqueous solution can be obtained by dissolving chitosan powder in water.
상기 키토산은 1,000 내지 2,000,000, 바람직하게는 5,000 내지 500,000의 중량 평균 분자량을 가질 수 있다. The chitosan may have a weight average molecular weight of 1,000 to 2,000,000, preferably 5,000 to 500,000.
상기 키토산 분말은 증류수에 0.1~5g/L로 용해될 수 있다. 키토산 분말이 5g/L 보다 높은 경우 키토산이 완전히 증류수에 녹지 않을 수 있다. The chitosan powder can be dissolved in distilled water at 0.1 to 5 g/L. If the chitosan powder is higher than 5g/L, the chitosan may not completely dissolve in distilled water.
상기 키토산 수용액의 pH가 3~6, 바람직하게는 5가 되도록 조절할 수 있다. The pH of the chitosan aqueous solution can be adjusted to 3 to 6, preferably 5.
상기 혼합 단계는 유산균 용액을 상기 키토산 용액에 넣어 혼합하는 단계이다. The mixing step is a step of mixing the lactic acid bacteria solution into the chitosan solution.
상기 유산균 용액은 유산균이 식염수나 물에 분산된 용액이다. The lactic acid bacteria solution is a solution in which lactic acid bacteria are dispersed in saline solution or water.
상기 유산균은 락토바실러스속(Lactobacillus), 락토코커스속(Lactococcus), 스트렙토코커스속(Streptococcus), 쌍구균인 류코노스록(Leuconostoc) 또는 비피더스속(Bifidobacterium) 등일 수 있고, 바람직하게는, 상기 유산균은 락토코쿠스 락티스(Lactococcus lactis)일 수 있다. The lactic acid bacteria may be Lactobacillus, Lactococcus, Streptococcus, Leuconostoc or Bifidobacterium, etc. Preferably, the lactic acid bacteria are lactobacilli. It may be Lactococcus lactis.
상기 유산균 용액은 유산균이 식염수에 상기 유산균은 식염수 용액에 50~200g/L, 바람직하게는 70~200g/L, 더욱 바람직하게는 100~200g/L로 희석될 수 있다. The lactic acid bacteria solution may be diluted in saline solution to 50 to 200 g/L, preferably 70 to 200 g/L, and more preferably 100 to 200 g/L.
상기 유산균의 함량이 50g/L 미만, 또는 10g/L 이하인 경우, 혼합용액 중에 소량의 유산균이 분산된 상태에서 키토산이 가교되므로 키토산 나노입자 내부에 하나 이상(복수개)의 유산균이 담지된 구형의 키토산 나노입자가 제조될 수 있다. When the content of lactic acid bacteria is less than 50 g/L or less than 10 g/L, chitosan is crosslinked with a small amount of lactic acid bacteria dispersed in the mixed solution, so that spherical chitosan is formed with one or more (multiple) lactic acid bacteria supported inside the chitosan nanoparticles. Nanoparticles can be manufactured.
이와 달리, 본 발명은 종래 기술(한국등록특허 10-2210922호)에 비해 유산균의 함량을 10 배 정도 많이 첨가함에 따라 복수 개의 유산균이 응집되어 존재하고, 따라서 키토산이 가교되더라도 응집된 유산균을 선행 1과 같이 둘러싸기 어렵고, 섬 구조(듬성 듬성)로 유산균 표면에 부착되는 구조를 나타낼 수 있다. On the other hand, in the present invention, as the content of lactic acid bacteria is added about 10 times higher than in the prior art (Korean Patent No. 10-2210922), a plurality of lactic acid bacteria exist in aggregate, and therefore, even if chitosan is cross-linked, the aggregated lactic acid bacteria are formed first. It is difficult to surround, and can show a structure attached to the surface of lactic acid bacteria in an island structure (sparse).
상기 키토산 용액과 상기 유산균 용액의 부피비는 20ml : 0.1 ~ 5ml 범위일 수 있다. The volume ratio of the chitosan solution and the lactic acid bacteria solution may be in the range of 20ml: 0.1 to 5ml.
상기 적하(drop) 단계는 가교제를 상기 키토산 용액에 떨어뜨리는 단계이다. 상기 적하단계는 0.5~5 ml 의 0.05~1(w/v)% 가교제, 예를 들면, 1ml의 0.1(w/v)% 가교제를 상기 키토산 용액에 첨가할 수 있다. The dropping step is a step of dropping the cross-linking agent into the chitosan solution. In the dropping step, 0.5 to 5 ml of 0.05 to 1 (w/v)% cross-linking agent, for example, 1 ml of 0.1 (w/v)% cross-linking agent, may be added to the chitosan solution.
상기 가교제는 키토산을 가교시킬 수 있는 공지된 가교제를 제한없이 사용할 수 있다. 예를 들면, 상기 가교제로 알데히드, 폴리포스페이트 염, 바람직하게는 나트륨 트리폴리포스페이트(TPP)를 사용할 수 있다. The crosslinking agent may be any known crosslinking agent capable of crosslinking chitosan without limitation. For example, aldehydes and polyphosphate salts, preferably sodium tripolyphosphate (TPP), can be used as the crosslinking agent.
도 3과 도 5를 참고하면. 키토산 용액에 가교제를 적하하면, 고분자 키토산이 가교제에 의해 상호 가교되어 나노 입자화되고, 입자화 과정 중에 유산균 표면에 섬 구조로 부착될 수 있으며, 또한, 유산균 표면에 박막층이 형성될 수 있다. Referring to Figures 3 and 5. When a cross-linking agent is added dropwise to the chitosan solution, the polymeric chitosan is cross-linked with each other by the cross-linking agent to form nanoparticles, and may be attached to the surface of the lactic acid bacteria in an island structure during the particle formation process. Additionally, a thin film layer may be formed on the surface of the lactic acid bacteria.
상기 유산균-키토산 복합체의 크기는 키토산과 유산균의 함량 제어를 통해 조절할 수 있다. 예를 들면, 상기 유산균-키토산 나노입자 크기는 500~2000nm 일 수 있다. The size of the lactic acid bacteria-chitosan complex can be adjusted by controlling the contents of chitosan and lactic acid bacteria. For example, the size of the lactic acid bacteria-chitosan nanoparticles may be 500 to 2000 nm.
상기 유산균-키토산 복합체는 키토산 입자가 유산균 표면의 일부에 응집되어 위치하거나 박막을 형성하므로, 키토산 나노입자나 키토산 박막층뿐만 아니라 유산균도 외부에 노출될 수 있다. In the lactic acid bacteria-chitosan complex, chitosan particles are aggregated on a portion of the surface of the lactic acid bacteria or form a thin film, so not only the chitosan nanoparticles or chitosan thin film layer but also the lactic acid bacteria can be exposed to the outside.
유체 내에 존재하는 프탈레이트 등 화합물이 키토산과 유산균에 존재하는 기능기에 흡착될 수 있다. Compounds such as phthalate present in the fluid may be adsorbed to functional groups present in chitosan and lactic acid bacteria.
즉, 상기 유산균-키토산 복합체는 수소결합, 아미드 결합 또는 정전기적 인력으로 프탈레이트계 화합물을 흡착하는 흡착제로 사용될 수 있다. 상기 프틀레이트계 화합물은 디부틸프탈레이트(DBP), 디에틸프탈레이트(DEP), 디(2-에틸헥실)프탈레이트(DEHP), 디이소노닐프탈레이트(DINP) 또는 벤질부틸프탈레이트(BBP)일 수 있으며, 바람직하게는 디부틸프탈레이트(DBP)일 수 있다. That is, the lactic acid bacteria-chitosan complex can be used as an adsorbent that adsorbs phthalate-based compounds through hydrogen bonding, amide bonding, or electrostatic attraction. The phthalate-based compound may be dibutyl phthalate (DBP), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), or benzylbutyl phthalate (BBP), Preferably, it may be dibutyl phthalate (DBP).
이하 본 발명을 다음의 실시 예에 의해 좀더 상세하게 설명하겠으나, 하기 실시 예는 본 발명을 예시하기 위한 것이며 본 발명이 범위를 한정하는 것은 아니다. Hereinafter, the present invention will be described in more detail through the following examples. However, the following examples are intended to illustrate the present invention and do not limit the scope of the present invention.
유산균 준비Probiotic preparation
200 ml MRS 배지에 L.lac를 접종하여 호기성 조건에서 37℃, 12시간 동안 배양하였다. 배양된 균은 8000 rpm, 4℃ 에서 10분 동안 원심 분리하여 0.9 % 의 멸균된 saline solution 으로 2회 세척하고, 캡슐화 공정을 위하여 멸균된 saline solution 에 분산 (0.1 g/ml) 시켜 보관하였다. L.lac was inoculated into 200 ml MRS medium and cultured at 37°C for 12 hours under aerobic conditions. The cultured bacteria were centrifuged at 8000 rpm at 4°C for 10 minutes, washed twice with 0.9% sterilized saline solution, and dispersed (0.1 g/ml) in sterilized saline solution for the encapsulation process and stored.
실시예 1Example 1
양이 다른 키토산 분말(1, 2, 3, 4, 5 g/L )을 50 ul의 아세트산, HPLC water 19 ml에 넣고 완전히 녹여주었다. Chitosan solution의 온도를 상온으로 낮춰 준 후, 1 ml 의 L. lac.-saline solution (0.1 g/ml) 을 일정하게 교반하며 섞어주었다. 1 시간 동안 교반 후, 일정하게 교반을 해주며 10ml 의 0.1 (w/v)% TPP 용액을 뷰렛을 사용하여 천천히 한방울씩 첨가하였다. 1 시간동안 반응을 시켜준 후, 5500 rpm, 20℃에서 20 분 동안 원심 분리를 한 뒤, HPLC water 20 ml 에 고르게 재분산하였다. 그 결과, CNPs 보다 약간 더 흰색의 Lactococcus lactis-chitosan nanoaprticles (L. lac-CNPs) 현탁액이 생성되는 것을 확인할 수 있었다. Different amounts of chitosan powder (1, 2, 3, 4, 5 g/L) were added to 50 ul of acetic acid and 19 ml of HPLC water and completely dissolved. After lowering the temperature of the chitosan solution to room temperature, 1 ml of L. lac. -saline solution (0.1 g/ml) was mixed with constant stirring. After stirring for 1 hour, 10 ml of 0.1 (w/v)% TPP solution was slowly added drop by drop using a burette while stirring constantly. After reacting for 1 hour, centrifugation was performed at 5500 rpm and 20°C for 20 minutes, and then evenly redispersed in 20 ml of HPLC water. As a result, it was confirmed that a slightly whiter Lactococcus lactis -chitosan nanoaprticles ( L. lac -CNPs) suspension was produced than CNPs.
비교예 1 Comparative Example 1
양이 다른 Chitosan powder(1, 2, 3, 4, 5 g/L )를 60 ℃ 에서 1 시간 동안 Acetic acid 50 ㎕, HPLC water 20 ml에 넣고 완전히 녹여주었다. Chitosan solution의 온도를 상온으로 낮춰 준 후, 일정하게 교반을 해주며 10 ml 의 0.1 (w/v)% TPP solution 을 뷰렛을 사용하여 천천히 한 방울씩 첨가하였다. 1 시간 동안 반응을 시켜준 후, 5500 rpm, 20 ℃에서 20 분 동안 원심 분리를 한 뒤, HPLC water 20 ml 에 고르게 재분산하였다. 이와 같은 조건에서 유백색의 현탁액이 생성되며 이는 Chitosan nanoparticles (CNPs)이 제조되었음을 나타낸다. Different amounts of Chitosan powder (1, 2, 3, 4, 5 g/L) were added to 50 ㎕ of Acetic acid and 20 ml of HPLC water at 60°C for 1 hour and completely dissolved. After lowering the temperature of the chitosan solution to room temperature, 10 ml of 0.1 (w/v)% TPP solution was slowly added drop by drop using a burette while stirring constantly. After reacting for 1 hour, centrifugation was performed at 5500 rpm and 20°C for 20 minutes, and then evenly redispersed in 20 ml of HPLC water. Under these conditions, a milky white suspension is produced, indicating that Chitosan nanoparticles (CNPs) have been prepared.
프탈레이트 제거 실험Phthalate removal experiment
CNPs(비교예 1) L.lac(유산균), L. lac.-CNPs(실시예 1)의 phthalates 의 제거 효율을 확인하기 위하여 DBP, DEP를 사용하였다. 제거 실험은 batch mode 에서 반응 속도 (450rpm), 반응 온도 (room temperature), 반응 부피 (10ml), 흡착제의 농도 (2 ~ 50 g/L), 오염 물질의 농도 (5 ~ 5000 ppm), 반응 시간 (0 ~ 48 hr) 의 조건으로 진행하였다. CNPs (Comparative Example 1) L. lac (lactic acid bacteria) , L. lac. -DBP and DEP were used to confirm the removal efficiency of phthalates of CNPs (Example 1). Removal experiments were performed in batch mode based on reaction speed (450rpm), reaction temperature (room temperature), reaction volume (10ml), adsorbent concentration (2 ~ 50 g/L), contaminant concentration (5 ~ 5000 ppm), and reaction time. It was conducted under the conditions of (0 ~ 48 hr).
이전 과정에서 준비한 CNPs, L. lac., L. lac.-CNPs 와 phthalates 를 반응한 뒤, 450 nm syringe filter 를 사용하여 오염 물질의 흡착이 완료된 입자를 걸러주어 GC-FID 를 사용하여 흡착 전후의 차이를 측정하였다. After reacting the CNPs, L. lac., L. lac.-CNPs prepared in the previous process with phthalates, filter out particles for which contaminant adsorption has been completed using a 450 nm syringe filter, and use GC-FID to analyze before and after adsorption. The difference was measured.
흡착제의 흡착능(adsorption capacity)과 제거효율(Removal efficiency) 은 흡착제의 능력을 판단하는 중요한 척도이다. 다음은 흡착능과 제거효율을 구하는 식을 나타낸 것이다: The adsorption capacity and removal efficiency of an adsorbent are important measures to determine the ability of an adsorbent. The following shows the equations for calculating adsorption capacity and removal efficiency:
여기서, Q (mg/L) 는 흡착능으로 흡착제의 단위 중량 당 흡착된 흡착물질의 양, q (%) 는 제거 효율로 흡착제에 흡착된 흡착물질의 농도와 흡착전 흡착물질의 초기 농도의 비를 백분율로 나타낸 것이다. Ci (mg/L) 는 흡착 전 흡착물질의 초기 농도, Cf (mg/L) 는 흡착제에 흡착된 흡착물질의 농도, V (L)는 흡착제와 흡착물질의 반응 부피, m (g) 은 흡착제의 양을 의미한다Here, Q (mg/L) is the adsorption capacity, which is the amount of adsorbed material per unit weight of the adsorbent, and q (%) is the removal efficiency, which is the ratio of the concentration of the adsorbed material adsorbed on the adsorbent and the initial concentration of the adsorbed material before adsorption. It is expressed as a percentage. Ci (mg/L) is the initial concentration of the adsorbent before adsorption, Cf (mg/L) is the concentration of the adsorbent adsorbed on the adsorbent, V (L) is the reaction volume of the adsorbent and the adsorbent, and m (g) is the adsorbent. means the amount of
도 4는 키토산 농도에 따른 비교예와 실시예의 입자 직경을 나타낸 그래프이다. 1, 2, 3, 4, 5 g/L Chitosan 과 0.1 wt% TPP가 사용되었다. 일정한 부피의 TPP 를 사용하였을 때, Chitosan 의 농도에 따라 CNPs 는 365.3 ± 3.254 nm 부터 914.2 ± 2.987 nm, L. lac.-CNPs 는 544.6 ± 5.576 nm 부터 1617.1 ± 7.570 nm 의 입자가 형성된 것을 확인하였다. 따라서 Chitosan 의 농도가 입자의 크기에 영향을 미친다는 것을 알 수 있다. 또한 같은 농도의 Chitosan 을 사용하여 나노 입자를 제조하였을 때, CNPs 에 비하여 L. lac.-CNPs 의 입자가 더욱 크게 형성된 것으로 보아 로딩된 L. lac. 에 의한 것으로 생각된다. Figure 4 is a graph showing particle diameters of comparative examples and examples according to chitosan concentration. 1, 2, 3, 4, and 5 g/L Chitosan and 0.1 wt% TPP were used. When a constant volume of TPP was used, it was confirmed that particles were formed from 365.3 ± 3.254 nm to 914.2 ± 2.987 nm for CNPs and from 544.6 ± 5.576 nm to 1617.1 ± 7.570 nm for L. lac.-CNPs, depending on the concentration of chitosan. Therefore, it can be seen that the concentration of chitosan affects the size of particles. In addition, when nanoparticles were manufactured using the same concentration of Chitosan, the particles of L. lac.-CNPs were formed larger than those of CNPs, indicating that the loaded L. lac. It is thought to be caused by
도 5는 제조된 유산균, 비교예 1(키토산 나노입자)와 실시예 1(유산균-키토산 복합체)의 SEM 이미지이다. 도 5를 참고하면, 5 g/L 의 chitosan 을 사용하여 제조한 CNPs(비교예 1) 는 약 400 nm 의 찌그러진 구형으로 형성된 것을 알 수 있다. L. lac. 는 약 500 nm 의 구형의 쌍, 짧은 단편의 사슬이 모여있는 상태로 존재하며, 세포는 매끈한 표면으로 이루어진 것을 확인하였다. L. lac.-CNPs(실시예 1)는 약 1000 nm 의 대부분의 입자가 L. lac. 와 동일하게 구형의 쌍 혹은 짧은 단편의 사슬이 모인 무리 형태로 존재하였다. L. lac.-CNPs 표면은 L. lac. 에 chitosan 층이 형성되었고, 일부 표면에는 CNPs 가 부분적으로 뭉쳐진 형태로 존재하는 것을 확인하였다. Figure 5 is an SEM image of the prepared lactic acid bacteria, Comparative Example 1 (chitosan nanoparticles) and Example 1 (lactic acid bacteria-chitosan complex). Referring to Figure 5, it can be seen that CNPs (Comparative Example 1) prepared using 5 g/L of chitosan were formed into distorted spheres of about 400 nm. L. lac. It was confirmed that a pair of spherical shapes of about 500 nm, a chain of short fragments, existed in a state gathered together, and that the cells had a smooth surface. In L. lac.-CNPs (Example 1), most particles of about 1000 nm are L. lac. Likewise, it existed in the form of a spherical pair or a group of short chains. The surface of L. lac.-CNPs is L. lac. A chitosan layer was formed, and CNPs were confirmed to exist in a partially aggregated form on some surfaces.
도 6은 유산균(L.ac), 비교예 1, 실시예 1의 작용기를 IR spectroscope 를 통하여 측정한 결과이다. 도 6을 참고하면, CNPs 그래프에서는 chitosan 의 특성 피크인 3600~3200 cm-1 (-OH, NH2 stretch (overlap)), 1537 cm-1 와 1376 cm-1 (N-H stretch), 2890 cm-1 (C-H stretch), 1058 cm-1 (C-O stretch) 가 나타났다. 그리고 TPP 의 특성 피크인 887 cm-1 (P-O stretch) 이 나타난 것으로 보아 chitosan 과 TPP 의 ionic cross-lining gelation 에 의한 L. lac.-CNPs가 잘 합성된 것을 알 수 있다. Figure 6 shows the results of measuring the functional groups of lactic acid bacteria (L.ac), Comparative Example 1, and Example 1 through an IR spectroscope. Referring to Figure 6, in the CNPs graph, the characteristic peaks of chitosan are 3600~3200 cm-1 (-OH, NH2 stretch (overlap)), 1537 cm-1 and 1376 cm-1 (N-H stretch), and 2890 cm-1 ( C-H stretch), 1058 cm-1 (C-O stretch) appeared. In addition, the characteristic peak of TPP, 887 cm-1 (P-O stretch), appeared, indicating that L. lac.-CNPs were well synthesized by ionic cross-lining gelation of chitosan and TPP.
도 7은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 농도(즉, 흡착제의 농도)에 따른 DBP에 대한 흡착능(A)과 제거효율(B)을 나타내고, 도 8은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 농도(흡착제의 농도)에 따른 DEP에 대한 흡착능(A)과 제거효율(B)을 나타낸다.. 도 7과 도 8을 참고하면, CNPs, L. lac., L. lac.-CNPs 의 농도가 2, 4, 6, 8, 10, 20, 30, 40, 50 g/L 로 증가함에 따라, 흡착능은 점점 감소하였으며 그에 따른 제거 효율은 점점 증가하였다. Figure 7 shows the adsorption capacity (A) and removal efficiency (B) for DBP according to the concentration (i.e., concentration of adsorbent) of lactic acid bacteria ( L.ac) , Comparative Example 1 (CNPs), and Example 1 ( L.ac -CNPs). ), and Figure 8 shows the adsorption capacity (A) and removal efficiency for DEP according to the concentration (adsorbent concentration) of lactic acid bacteria ( L.ac) , Comparative Example 1 (CNPs), and Example 1 ( L.ac -CNPs). (B). Referring to Figures 7 and 8, the concentrations of CNPs, L. lac., and L. lac.-CNPs are 2, 4, 6, 8, 10, 20, 30, 40, and 50 g. As /L increased, the adsorption capacity gradually decreased and the removal efficiency gradually increased.
특히, 유산균(L. lac.)이나 CNPs 단독으로 사용한 경우보다, 실시예 1의 흡착능과 제거 효율이 증가하였음을 확인할 수 있다. In particular, it can be seen that the adsorption capacity and removal efficiency of Example 1 increased compared to the case where lactic acid bacteria (L. lac.) or CNPs were used alone.
도 9는 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 흡착 시간에 따른 DBP에 대한 흡착능(A)과 제거효율(B)을 나타내고, 도 10은 유산균(L. ac), 비교예 1(CNPs), 실시예 1(L.ac-CNPs)의 흡착 시간에 따른 DEP에 대한 흡착능(A)과 제거효율(B)을 나타낸다. Figure 9 shows the adsorption capacity (A) and removal efficiency (B) for DBP according to the adsorption time of lactic acid bacteria (L.ac), Comparative Example 1 (CNPs), and Example 1 (L.ac-CNPs), and Figure 10 shows the adsorption capacity (A) and removal efficiency (B) for DEP according to the adsorption time of lactic acid bacteria (L.ac), Comparative Example 1 (CNPs), and Example 1 (L.ac-CNPs).
도 9와 도 10을 참고하면, 실시예 1의 흡착제가 비교예 1이나 유산균에 비해 높은 흡착능과 제거효율을 보여준다. 정전기적 인력과 수소 결합에 의한 화학적 흡착만 발생했을 경우, phthalates 의 제거 실험 결과는 비교적 분자량이 적은 DEP의 제거가 DBP의 제거보다 효율이 높았을 것으로 생각하였다. 그러나 모든 실험 조건에서 DBP의 제거가 DEP의 제거보다 효율이 높은 것으로 확인되었으며(도 10 참고), 이것은 또 다른 화학적 흡착인 아미드 결합(amide bonding)을 통하여 추가로 흡착이 발생했을 것으로 판단된다. 아미드 결합은 프탈레이트의 ester(R-COO-R’)와 L.ac-CNPs의 amine (R”-NH2)이 amide bond(R-CONH-R”)로 결합하고 부산물로 alcohol (R’-OH)을 생성하는 결합이다. Referring to Figures 9 and 10, the adsorbent of Example 1 shows higher adsorption capacity and removal efficiency than Comparative Example 1 or lactic acid bacteria. If only chemical adsorption occurred due to electrostatic attraction and hydrogen bonding, the phthalates removal experiment results showed that the removal of DEP, which has a relatively low molecular weight, would have been more efficient than the removal of DBP. However, it was confirmed that the removal of DBP was more efficient than the removal of DEP in all experimental conditions (see Figure 10), and it is believed that additional adsorption occurred through amide bonding, another chemical adsorption. The amide bond combines the ester (R-COO-R') of phthalate and the amine (R"-NH2) of L.ac-CNPs with an amide bond (R-CONH-R"), and alcohol (R'-OH) is produced as a by-product. ) is a combination that produces.
이상에서, 본 발명의 바람직한 구현 예에 대하여 상세하게 설명하였으나, 이들은 단지 설명의 목적을 위한 것으로 본 발명의 보호 범위가 이들로 제한되는 것은 아니다. In the above, preferred embodiments of the present invention have been described in detail, but these are for illustrative purposes only and the scope of protection of the present invention is not limited thereto.
Claims (9)
유산균 용액을 상기 키토산 용액에 넣어 혼합하는 단계 ;
가교제를 상기 키토산 용액에 적하하는 단계 ;
생성된 입자를 상기 용액으로부터 분리하는 단계를 포함하는 유산균-키토산 복합체의 제조방법으로서,
상기 키토산 분말은 0.1~5g/L이고, 상기 유산균은 식염수 용액에 50~200g/L로 희석되고, 상기 키토산 용액과 상기 유산균 용액의 부피비는 20ml : 0.1 ~ 5ml 범위이고,
상기 적하단계는 5~20 ml 의 0.05~0.5(w/v)% 가교제 용액을 상기 키토산 용액에 떨어뜨리는 단계이고,
상기 유산균이 락토코쿠스락티스(Lactococcus lactis)이고,
상기 제조방법으로 제조된 유산균-키토산 복합체는 디부틸프탈레이트(DBP), 디에틸프탈레이트(DEP), 디(2-에틸헥실)프탈레이트(DEHP), 디이소노닐프탈레이트(DINP) 및 벤질부틸프탈레이트(BBP) 중 어느 하나 이상의 프탈레이트계 화합물을 흡착하는 것을 특징으로 하는 유산균-키토산 복합체의 제조방법. Dissolving chitosan powder in water;
Adding and mixing the lactic acid bacteria solution into the chitosan solution;
Adding a cross-linking agent dropwise to the chitosan solution;
A method for producing a lactic acid bacteria-chitosan complex comprising the step of separating the generated particles from the solution,
The chitosan powder is 0.1 to 5 g/L, the lactic acid bacteria are diluted in saline solution to 50 to 200 g/L, and the volume ratio of the chitosan solution and the lactic acid bacteria solution is in the range of 20 ml: 0.1 to 5 ml,
The dropping step is dropping 5 to 20 ml of 0.05 to 0.5 (w/v)% crosslinker solution into the chitosan solution,
The lactic acid bacterium is Lactococcus lactis,
The lactic acid bacteria-chitosan complex prepared by the above manufacturing method is dibutyl phthalate (DBP), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), and benzylbutyl phthalate (BBP). ) A method for producing a lactic acid bacteria-chitosan complex, characterized in that it adsorbs any one or more phthalate-based compounds.
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| KR102009220B1 (en) * | 2017-05-02 | 2019-08-09 | 포항공과대학교 산학협력단 | Nahochitin-nanochitosan complex body, network structure body comprising thereof and manufacturing methods thereof, respectively |
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| KR102210922B1 (en) * | 2019-03-29 | 2021-02-01 | 가천대학교 산학협력단 | Chitosan nanoparticles adsorbent loading lactic acid bacteria and method of preparing the same |
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