KR100805044B1 - Bacillus clausii strain for fats degradation and microbial agent for foodwaste treatment using it - Google Patents
Bacillus clausii strain for fats degradation and microbial agent for foodwaste treatment using it Download PDFInfo
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- KR100805044B1 KR100805044B1 KR1020070087363A KR20070087363A KR100805044B1 KR 100805044 B1 KR100805044 B1 KR 100805044B1 KR 1020070087363 A KR1020070087363 A KR 1020070087363A KR 20070087363 A KR20070087363 A KR 20070087363A KR 100805044 B1 KR100805044 B1 KR 100805044B1
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- food waste
- strain
- fermentation
- bacillus
- extinction
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 33
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 20
- 239000003925 fat Substances 0.000 title abstract description 10
- 241001328122 Bacillus clausii Species 0.000 title abstract description 6
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
Abstract
본 발명은 내염성과 유지분해 능이 있는 바실러스 클라우지 균주를 이용한 음식물쓰레기 발효소멸용 미생물제제에 관한 것이다. 좀 더 구체적으로 본 발명의 균주는 전통적인 한식 식단의 음식물쓰레기로부터 1~4%의 염분농도에서 최고의 활성을 나타내며 지방분해효소 생산능력이 우수한 바실러스 클라우지(Bacillus clausii) SKAL-16(KACC 91320P)를 분리 동정하고, 이 균주를 이용하여 음식물쓰레기 발효소멸시 가장 큰 문제점 중의 하나인 염분과 유지에 의한 미생물의 분해활성 저하를 방지함으로써, 일반 가정 및 사업장에서 발생하는 음식물쓰레기를 처리하는데 뛰어난 효과가 있는 음식물쓰레기 발효소멸용 미생물제제 제조에 관한 것이다. The present invention relates to a microbial preparation for food waste fermentation and extinction using a Bacillus cloudage strain having flame resistance and oil decomposition. More specifically, the strain of the present invention exhibits the highest activity at a salinity of 1 to 4% from food waste of the traditional Korean diet, and has excellent ability to produce lipase, Bacillus clown ( Bacillus clausii ) SKAL-16 (KACC 91320P) is isolated and identified by using this strain to prevent degradation of microorganisms by salinity and fats and fats, which is one of the biggest problems in food waste fermentation and extinction, and thus occurs in general homes and workplaces. The present invention relates to a microbial preparation for food waste fermentation and extinction having an excellent effect in treating food waste.
음식물쓰레기, 지방분해효소, 내염성, 미생물제제, 발효소멸 Food waste, lipolytic enzyme, flame resistance, microbial agent, fermentation extinction
Description
본 발명은 생활 폐기물 중 폐유기성 자원인 음식물쓰레기를 미생물의 발효에 의해 분해소멸시키는 미생물제제에 관한 것이다. 좀 더 구체적으로 내염성과 지방분해효소 생산능력을 가지며 음식물쓰레기 분해활성이 우수한 균주 바실러스 클라우지(Bacillus clausii) SKAL-16(KACC 91320P) 균주를 이용한 음식물쓰레기 발효소멸용 미생물제제 제조에 관한 것이다. The present invention relates to a microbial agent for decomposing and extinguishing food waste, which is a waste organic resource, from living waste by fermentation of microorganisms. More specifically, Bacillus clout ( Bacillus) has excellent flame retardant and lipolytic enzyme production and excellent food waste degrading activity. clausii ) relates to the preparation of microbial products for food waste fermentation and extinction using SKAL-16 (KACC 91320P) strain.
현재 우리나라의 1일 음식물쓰레기의 발생량은 11,520톤/일(국립환경과학원 '2004 폐기물통계)에 이르며 이중 극히 일부가 사료화 또는 퇴비화하여 재활용되고 있으나 대부분이 소각 또는 매립이 주로 이루어졌으며, 소각은 다이옥신과 같은 유독 물질을 유발하여 인체에 치명적인 영향을 끼치고, 또한 환경오염의 심각한 원인을 제공하며, 매립은 수분이 많은 음식물쓰레기가 외부 미생물에 의해 분해가 일어나 고농도의 침출수와 심한 악취가스를 발생하며 이로 인한 2차 환경오염을 일으킨 다. 이로 인해 2005년 1월 이후에는 감량화 및 재활용이 안 되는 음식물쓰레기의 매립이 금지되고 있다. Currently, the daily amount of food waste in Korea reaches 11,520 tons / day (National Institute of Environmental Science '2004 waste statistics), and only a part of it is recycled by feed or compost, but most of it is incinerated or landfilled. It causes the same toxic substances and has a fatal effect on the human body, and also provides a serious cause of environmental pollution.In landfills, watery food waste is decomposed by external microorganisms, generating high concentrations of leachate and odorous gas. It causes secondary environmental pollution. As a result, after January 2005, landfills of food waste that cannot be reduced or recycled are prohibited.
따라서 음식물쓰레기 발생원단위에서 처리시설을 설치하여 발생 즉시 바로 처리된다면 음식물쓰레기의 발생을 근본적으로 해결할 수 있고, 경제적이며, 안전한 방법으로써 발생한 음식물쓰레기를 1차 현장에서 완전 소멸을 유도하여 2차 오염으로 이한 여러 가지 폐해를 줄일 수 있다. Therefore, if a treatment facility is installed at the source of food waste generation and processed immediately, it can fundamentally solve the occurrence of food waste, and induces the complete disappearance of food waste generated in an economical and safe way at the first site, resulting in secondary pollution. This can reduce several hazards.
발효소멸 방법은 최상의 방법으로써, 발효분해 방법과 비슷하지만, 퇴적물의 완전소멸을 유도함으로써, 직접적인 잔존물의 교체를 수개월에 1회 단위로 배출이 가능하다. 이 같은 발효소멸 처리장치는 국내뿐 아니라 일본 등지에서도 활발히 연구가 진행 중에 있으나 기 개발된 처리기술에 대한 신뢰도는 상당히 낮은 편이며, 판매 설치된 처리장치 들마저도 적정한 공정효율을 보이지 못하고 있으며 기존의 발효소멸 장치를 이용한 처리방법은 ①악취를 많이 발생시키고, ②사용하는 미생물제제의 발효소멸 효율이 낮으며, ③소음 및 유지비 등의 문제점으로 인해 사용자들에 의해 기피 되어 왔던 것이 사실이다. 또한, 대부분 업체가 발효소멸 장치라고 주장하지만 소멸화가 아닌 액상분해 배출방식이나 단순건조 방식으로 미생물에 의한 유기물 분해에 의한 실질적인 감량효율을 보이지 못하고 있다. Fermentation and extinction method is the best method, similar to fermentation method, but by inducing complete disappearance of sediment, it is possible to discharge direct residue once every few months. Such fermentation and destruction treatment devices are being actively researched not only in Korea but also in Japan, but the reliability of the developed treatment technology is quite low, and even the installed treatment devices do not show proper process efficiency. It is true that the treatment method using the device has been avoided by the users due to problems such as ① generating a lot of odor, ② low fermentation efficiency of microbial agents used, and ③ noise and maintenance costs. In addition, most companies claim fermentation and extinction device, but it does not show the actual weight loss efficiency due to the decomposition of organic matter by microorganisms in a liquid decomposition discharge method or a simple drying method, not annihilation.
따라서, 발효소멸용 장치 및 공정효율을 높이기 위한 운용기술 개발과 그에 따른 미생물과 발효소멸에 필요한 주요인자 및 분해매체제(담체)를 포함하는 미생물제제, 효소활성이 높은 미생물의 분리 등에 체계적인 연구가 필요하고 특히 우리나라 음식 문화의 특성에 따른 연구가 요구되고 있다.Therefore, systematic researches on the development of fermentation extinction device and operation technology for enhancing process efficiency, microorganisms including microorganisms and major factors necessary for fermentation and extinction, and the separation of microorganisms with high enzyme activity, etc. In particular, research is required according to the characteristics of Korean food culture.
한편, 한국의 음식물쓰레기의 조성은 서구의 음식물쓰레기와 비교하여 된장, 간장, 김치, 젓갈 등의 음식물에 의해 높은 염분과 각종 찌개, 전, 튀김 및 육류 소비 증대에 따른 지방성분 축적으로 인해 미생물의 활동이 어려워질 수 있다. 이와 같은 음식물쓰레기의 특성에 맞춘 미생물 균주의 분리 및 미생물제제의 제형 연구가 무엇보다도 중요하다.On the other hand, the composition of food waste in Korea is higher than that of Western food waste due to the high salinity of foods such as doenjang, soy sauce, kimchi, and salted fish, and the accumulation of fat components due to increased consumption of various stew, pan, fried, and meat. Activities can be difficult. The isolation of microbial strains and the formulation of microbial agents according to the characteristics of such food waste are of paramount importance.
종래기술의 문헌정보Literature Information of the Prior Art
[문헌1] KR 10-0443267 A 2004.07.26.
[문헌2] KR 10-0452125 A 2004.09.30.
[문헌3] KR 10-0503678 A 2005.07.16.
본 발명은 우리나라 근래의 음식물쓰레기 특성에 맞춰 음식물쓰레기로부터 1~4%의 염분농도에서 최고의 활성을 나타내며 지방분해효소 생산능력이 우수한 바실러스 클라우지(Bacillus clausii) SKAL-16(KACC 91320P)를 분리 동정하고, 이 균주를 이용한 음식물쓰레기 발효소멸용 미생물제제를 제공하는데 그 목적이 있다.The present invention shows the best activity at 1 ~ 4% salinity from food waste in accordance with the characteristics of food waste in Korea and Bacillus Cluj ( Bacillus clout) with excellent lipolytic enzyme production capacity. clausii ) SKAL-16 (KACC 91320P) is isolated and identified, and the purpose of the present invention is to provide a microbial agent for food waste fermentation and extinction using this strain.
본 발명은 내염성과 지방분해효소 생산능력이 우수한 세균을 선발하기 위하여 음식물쓰레기로부터 지방분해효소 생산능력이 있는 세균을 순수분리하는 단계, 분리된 세균 중 우수한 균주를 선별하여 동정하는 단계, 동정된 세균의 내염성 및 지방분해효소 생산능력을 측정하는 단계, 가장 우수한 균주를 선발하고 선발된 균주를 이용하여 음식물쓰레기 발효소멸용 미생물제제를 제조하는 단계, 제조된 미생물제제를 이용하여 음식물쓰레기 발효소멸 시험을 하는 단계로 이루어진 것에 특징이 있다. The present invention is the step of separating the bacteria having the ability to produce lipolytic enzymes from food wastes in order to select a bacterium with excellent flame resistance and lipolytic enzyme production, the step of identifying and identifying the excellent strains of the isolated bacteria, identified bacteria Measuring the salt tolerant and lipolytic enzyme production capacity of the selected, the best strains were selected using the selected strain to produce food waste fermentation microbial agent, food waste fermentation and extinction test using the prepared microbial agent It is characterized by consisting of steps.
본 발명에 따른 바실러스 클라우지 SKAL-16은 내염성 및 지방분해 능력이 우수하여 우리나라 음식물쓰레기를 발효소멸시키는데 매우 유용하다. 특히 본 발명의 미생물을 제오라이트에 안정되게 흡착시켜 미생물제제를 제조하여 사용할 경우 분해소멸률이 더욱 향상되어 매우 경제적이다. 이러한 본 발명의 기술로 인하여, 음식물쓰레기를 매우 효율적으로 처리, 처분할 수 있으며 발효소멸 후 잔량은 그린(1 급)퇴비 조건에 적합하여 2차 오염원이 전혀 발생하지 않는다. 따라서 경제적이고 위생적으로 음식물쓰레기를 처리할 수 있어 환경산업상 매우 유용한 발명인 것이다.Bacillus cluj SKAL-16 according to the present invention is excellent in flame resistance and lipolysis ability is very useful for fermentation and extinction of Korean food waste. In particular, when the microorganism of the present invention is stably adsorbed to zeolite to prepare and use a microbial agent, the decomposition and extinction rate is further improved, which is very economical. Due to the technology of the present invention, food waste can be treated and disposed of very efficiently, and the remaining amount after fermentation extinction is suitable for the green (primary) composting conditions, so that no secondary pollution occurs. Therefore, the food waste can be disposed of economically and hygienically, which is a very useful invention for the environmental industry.
본 발명에서 사용된 바실러스 클라우지(Bacillus clausii) SKAL-16은 우리나라의 전통적인 식단에 근거한 음식물쓰레기로부터 분리하였다. 분리에 사용된 최소배지의 조성은 증류수 1L에 폐식용유 5g, 효모추출물 1g, 제2인산칼륨 2g, 염화암모늄 2g, 콩단백추출물 1g이 포함된다. 위의 배지에서 배양한 후 배양액에 1.5%의 agar를 넣은 평판배지를 이용 각각의 균주를 분리하였다. 분리된 균주는 다시 위의 배지상태에서 염분농도 0-10%까지의 각 단계별로 조건을 달리하여 배양을 하였으며 배양된 결과는 흡광광도계(660nm)를 이용하여 O.D값(균밀도)을 측정하였다. 이러한 조건에서 가장 우수한 형질을 나타내는 균주를 분리한 후 수차례 순수분리과정을 통하여 단일 균주 SKAL-16을 선별하였으며, 분자유전학적인 방법으로 16S rRNA partial sequencing을 이용하여 동정한 결과 바실러스 클라우지(Bacillus clausii)로 동정되었다. 본 발명자들은 상기 동정된 균주를 바실러스 클라우지 SKAL-16으로 명명하고 농업생명공학연구원에 기탁하여 2007년 6월 20일자로 수탁번호 KACC 91320P를 부여받았다.Bacillus Cluj used in the present invention ( Bacillus clausii ) SKAL-16 was isolated from food waste based on Korean traditional diet. The composition of the minimum medium used for separation includes 5 g of waste cooking oil, 1 g of yeast extract, 2 g of potassium diphosphate, 2 g of ammonium chloride, and 1 g of soy protein extract in 1 L of distilled water. After culturing in the above medium, each strain was isolated using a plate medium containing 1.5% agar in the culture. The isolated strains were cultured under different conditions for each step up to a salinity concentration of 0-10% in the above medium state. The cultured results were measured by using an absorbance spectrometer (660 nm) to measure the OD value (density density). Under these conditions, the strain showing the best trait was isolated, and a single strain SKAL-16 was selected through several pure separation processes, and identified by 16S rRNA partial sequencing by molecular genetic method. Bacillus clausii Was identified). The present inventors named the identified strain Bacillus Cluj SKAL-16 and deposited it with the Agricultural Biotechnology Research Institute and received the accession number KACC 91320P on June 20, 2007.
호염성 해양세균인 바실러스 클라우지는 최근 학계에 보고된(한국생물공학회지, 20권 3호, p215-220, 2005년) 바로는 고활성의 알칼리성 단백질분해효소만을 생산하는 균주로 연구되어 소개되고 있다. 지방을 분해하는 효소생산능력에 대해서 는 연구되어 진 바가 없다. 그러나 본 발명에서 분리한 바실러스 클라우지 SKAL-16는 10%의 염분농도에서도 지방을 분해할 수 있는 지방분해효소를 다량 생산하는 특징을 보였다.Bacillus Cluj, a basophilic marine bacterium, recently reported to the academic community (Korean Journal of Biotechnology and Bioengineering, Vol. 20 No. 3, p215-220, 2005), has been studied and introduced as a strain that produces only highly active alkaline protease. . The ability to produce enzymes that break down fat has not been studied. However, Bacillus Cluj SKAL-16 isolated from the present invention showed a feature of producing a large amount of lipolytic enzymes capable of breaking down fat even at a salt concentration of 10%.
바실러스 클라우지 SKAL-16이 생산하는 지방분해효소의 활성을 측정하기 위해서 Tributyrin이란 화합물을 사용한다. 지방 분해균은 lipase란 효소를 생성하고 몸 밖으로 분비한다. 이 lipase는 주위에 있는 지방을 분해하고 이 지방분해산물(지방산, 글리세롤 등)은 이 균의 영양분으로 이용된다. lipase의 활성을 추정하기 위해서 지방과 구조가 비슷한 물질과 반응시키는데 이때 tributyrin이라는 화합물을 사용한다. Tributyrin이 분해되면 부틸산(butylic acid)이 생성되는데 HPLC 분석을 통해 부틸산의 농도를 측정하였다. 이 부틸산 농도가 높을수록 많은 tributyrin이 가수분해된 것이다. Tributyrin을 빨리 분해하는 효소는 지방류의 물질을 빨리 분해한다고 볼 수 있다. Tributyrin is used to measure the activity of lipolytic enzymes produced by Bacillus cluj SKAL-16. Lipolytic bacteria produce an enzyme called lipase and secrete it out of the body. This lipase breaks down the surrounding fat, and this fatty acid breakdown product (fatty acid, glycerol, etc.) is used to nourish the bacteria. To estimate lipase activity, a compound called tributyrin is used to react with a substance similar in fat and structure. When tributyrin is decomposed, butyric acid is produced. The concentration of butyric acid was determined by HPLC analysis. The higher the concentration of butyl acid, the more tributyrin hydrolyzed. The enzyme that breaks down tributyrin quickly breaks down fatty substances.
본 발명의 균주가 음식물쓰레기의 발효소멸용 미생물제제로 사용되는 경우, 본 발명의 균주를 분해매체제(무기담체)에 흡착시켜 사용하는 것이 바람직하며, 적절한 무기담체는 제오라이트, 활성규조토, 질석 및 화이트카본 중에서 1) 수분조절능력이 뛰어나고 2) 미생물흡착에 의한 담체역할을 할 수 있고 3) pH 버퍼 능력이 있고 4) 기계적인 마모에 안정적이고 5) 악취물질 흡착효과가 뛰어난 것을 선택하는 것을 포함한다. When the strain of the present invention is used as a microbial agent for fermentation and extinction of food waste, it is preferable to use the strain of the present invention by adsorbing it to a decomposition medium (inorganic carrier), and suitable inorganic carriers are zeolite, activated diatomaceous earth, vermiculite and white. Among the carbons, it includes 1) excellent moisture control ability, 2) carrier function by adsorption of microorganisms, 3) pH buffering ability, 4) stable to mechanical wear, and 5) excellent adsorption of odorous substances. .
본 발명의 음식물쓰레기 발효소멸용 미생물제제 제조방법은, 바실러스 클라우지 SKAL-16을 영양배지에서 호기배양하여 발효균배양액을 제조하는 단계; 상기 발효균배양액을 무기담체에 흡착시키고 건조시켜 발효균건조물을 제조하는 단계; 및 상기 발효균건조물에 부형제 및 발효균 먹이성분을 혼합하는 단계를 포함하여 이루어진다. Microbial preparation method for food waste fermentation and extinction of the present invention comprises the steps of preparing a culture of fermentation bacteria by aerobic culture of Bacillus Cluj SKAL-16 in a nutrient medium; Adsorbing the fermentation broth culture solution to an inorganic carrier and drying to prepare a fermentation bacteria dry matter; And mixing the excipient and the fermentation bacteria food ingredient in the fermentation bacteria dry.
상기 발효균건조물 제조시 사용되는 무기담체는 제오라이트, 활성규조토, 질석 및 화이트카본으로 이루어진 군에서 선택된 하나 이상인 것이 바람직하고, 상기 발효균배양액에 대해 중량비로 0.5 내지 5배인 것이 바람직하다.The inorganic carrier used in the preparation of the fermentation bacteria dry matter is preferably at least one selected from the group consisting of zeolite, activated diatomaceous earth, vermiculite and white carbon, and preferably 0.5 to 5 times by weight relative to the fermentation culture medium.
상기 부형제는 일라이트, 활성탄 및 맥반석으로 이루어진 군에서 선택된 하나 이상인 것이 바람직하고, 상기 발효균건조물에 대해 중량비로 0.1 내지 5배인 것이 바람직하다. 또한, 이때 발효균 먹이성분으로 당밀 및 포도당으로 이루어진 군에서 선택된 하나 이상인 것이 바람직하고, 상기 부형제에 대해 중량비로 0.5 내지 5중량%인 것이 바람직하다.The excipient is preferably one or more selected from the group consisting of elite, activated carbon and elvan, and preferably 0.1 to 5 times by weight relative to the fermentation bacteria dry matter. In addition, it is preferable that at least one selected from the group consisting of molasses and glucose as the fermentation bacteria feed ingredient, it is preferable that the weight ratio of the excipients 0.5 to 5% by weight.
본 발명의 균주 바실러스 클라우지 SKAL-16은 음식물쓰레기에 처리하여 초기에 왕성한 번식 및 안정성을 유도하기 위한 배지성분 및 무기담체를 함유하는 미생물제제 형태로서 음식물쓰레기 발효소멸에 사용되며, 구체적으로는 음식물쓰레기에 본 발명의 균주를 포함하는 미생물제제를 투여하고 교반하면서 적정온도에서 호기적인 조건을 유지하면, 본 발명의 미생물은 발효 초기에 미생물제제 내에 포함된 배지성분을 이용하여 활발하게 번식함으로써 음식물쓰레기 더미 내에서 우점미생물이 된다. 이 바실러스 클라우지 SKAL-16은 내염성 및 유지분해 능력이 우수하므로, 본 발명의 미생물을 포함하는 미생물제제를 사용하면 빠른 시간 내에 음식물쓰레기를 분해소멸시킬 수 있다. The strain Bacillus Cluj SKAL-16 of the present invention is used in fermentation and extinction of food waste as a microbial formulation containing a medium component and an inorganic carrier to induce vigorous propagation and stability at first by treating food waste. If the aerobic conditions are maintained at the appropriate temperature while administering and stirring the microbial agent containing the strain of the present invention to the waste, the microorganism of the present invention by actively breeding using the media components contained in the microbial agent at the beginning of fermentation, food waste Become a dominant microbe in the pile. Since Bacillus Cluj SKAL-16 is excellent in flame resistance and oil and fat decomposition ability, using a microbial agent containing the microorganism of the present invention can decompose and destroy food waste quickly.
이하, 실시 예를 통하여 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail with reference to the following examples.
[실시 예 1] Example 1 바실러스Bacillus 클라우지Cluj SKALSKAL -16 균주의 분리Isolation of -16 Strains
본 발명에서 사용한 바실러스 클라우지 SKAL-16은 전통적인 한식 식단의 음식물쓰레기에서 분리하였다. 본 균주는 채소와 젓갈, 김치 등이 혼합된 음식물쓰레기를 분쇄한 후 생성된 액으로부터 시료를 채취하고, 그 시료를 0.85% 생리식염수로 10-4, 10-5, 10-6, 10-7 등으로 희석하여 지방분해효소 분리용 고체 평판배지에 접종하여 30℃에서 3일간 배양하였다. 배양결과 형성된 미생물 콜로니(colony)를 3~5회 연속적으로 계대배양하여 순수하게 40종 균주를 분리하였고, 그 중 우수한 균주 8종을 선별하여 분자유전학적인 방법으로 16S rRNA partial sequencing을 이용하여 동정한 결과 7종이 Bacillus sp. 이었고 SKAL-16만이 Bacillus clausii로 나타났다. 분리 동정된 균들은 다시 염분농도에 따른 생육특성을 알아보기 위하여 최소배지(폐식용유 5g, 효모추출물 1g, 제2인산칼륨 2g, 염화암모늄 2g, 콩단백추출물 1g)에 염분(NaCl)을 0~10% 첨가한 후 30℃에서 120rpm으로 120시간 배양하였으며 흡광광도계(660nm)를 이용하여 O.D값(균밀도)를 측정한 결과를 표1에 나타내었다. Bacillus Cluj SKAL-16 used in the present invention was isolated from the food waste of the traditional Korean diet. This strain is crushed the food waste mixed with vegetables, salted fish, kimchi, etc., and the sample is collected from the resulting solution, and the sample is 10 -4 , 10 -5 , 10 -6 , 10 -7 with 0.85% saline. The mixture was diluted with and inoculated into a solid plate medium for separating lipase, and incubated at 30 ° C. for 3 days. The cultured microorganism colonies (colony) formed as a result of the culture was subcultured three to five times, and 40 strains were isolated purely. Among them, eight excellent strains were selected and identified by 16S rRNA partial sequencing by molecular genetic method. Results Seven Bacillus sp. Only SKAL-16 Bacillus clausii . To identify the growth characteristics according to the salt concentration, the identified bacteria were added with salt (NaCl) in a minimum medium (5g waste oil, 1g yeast extract, 2g potassium diphosphate, 2g ammonium chloride, 1g soy protein extract). After addition of 10%, the cells were incubated at 120 ° C. for 120 hours at 120 rpm. The results of measuring the OD value (density density) using an absorbance spectrometer (660 nm) are shown in Table 1.
[표1] 염분농도가 분리 동정된 균의 생육에 미치는 영향[Table 1] Effect of salinity on the growth of isolated bacteria
상기 표1에 보이는 바와 같이 염분농도별로 균의 성장을 O.D(optical density)값을 구하여 간접적으로 측정한 결과 SKAL-4와 SKAL-14를 제외한 6가지 균이 염분농도 10%에서도 생장하였다. 대부분이 높은 염분농도에서 성장이 더 빠른 것으로 보아 내염성(haloduric) 균들이며 일반 바닷물의 염도가 4% 정도로 알려져 있는데 염분농도 10%에서도 생장을 보이는 높은 내염성이 있었다. As shown in Table 1 above, indirectly measuring the growth of bacteria by salinity concentration, O.D (optical density) value was obtained. As a result, six bacteria except SKAL-4 and SKAL-14 grew at 10% salinity. Most of them are haloduric bacteria that grow faster at high salinity and are known to have 4% salinity of general seawater.
[실시 예 2] 분리 동정된 균을 이용한 지방분해효소 생산능력 측정Example 2 Measurement of Lipolytic Enzyme Production Capacity Using Isolated Isolates
지방분해효소인 lipase의 활성을 측정하기 위해서 지방과 구조가 유사한 물질인 tributyrin을 0.1M 첨가한 최소배지(폐식용유 5g, 효모추출물 1g, 제2인산칼륨 2g, 염화암모늄 2g, 콩단백추출물 1g)에서 30℃, 120rpm으로 0~8일 동안 배양하면서 2일 간격으로 생산된 부틸산(butyric acid)의 농도를 HPLC를 이용하여 측정한 결과를 표2에 나타내었다.Minimal medium containing 0.1M of tributyrin, a substance similar in structure to fat, to measure the activity of lipase, a lipolytic enzyme (5g waste cooking oil, 1g yeast extract, 2g potassium diphosphate, 2g ammonium chloride, 1g soy protein extract) The concentration of butyric acid produced at 2 days intervals at 30 ° C. and 120 rpm at 0 to 8 days was measured using HPLC.
[표2] 분리 동정된 균의 지방분해효소(lipase) 생성[Table 2] Lipase production of isolated bacteria
상기 표2에 보이는 바와 같이 대부분 지방분해효소 생성능력이 우수하였고 분리 동정된 균 중에서 바실러스 클라우지 KAL-16의 지방분해효소 생산능력이 가장 높게 나타났다.As shown in Table 2, most lipolytic enzyme production ability was excellent, and among the identified bacteria, Bacillus Cluj KAL-16 showed the highest lipolytic enzyme production capacity.
상기의 실시 예 1과 실시 예 2를 통하여 내염성을 갖춘 균주로써 지방분해효 소 생산능력이 가장 우수한 바실러스 클라우지 SKAL-16 균주를 선발하였다.Through Example 1 and Example 2, the bacterium Cluj SKAL-16 strain having the best lipolytic production capacity was selected as a strain having flame resistance.
[실시 예 3] 음식물쓰레기 발효소멸용 미생물제제 제조Example 3 Preparation of Microbial Preparation for Food Waste Fermentation and Extinction
본 발명의 미생물 바실러스 클라우지 SKAL-16의 배양액을 무기담체로서 제오라이트, 활성규조토, 질석 및 화이트카본과 혼합함으로써 본 발명의 미생물을 무기담체에 흡착시켜 발효소멸용 미생물제제를 제조하였다.The microorganism of the present invention was adsorbed to the inorganic carrier by mixing the culture solution of the microorganism Bacillus cluj SKAL-16 of the present invention as zeolite, activated diatomaceous earth, vermiculite and white carbon as an inorganic carrier to prepare a microbial agent for fermentation and extinction.
본 발명의 미생물제제의 제조방법은 바실러스 클라우지 SKAL-16 균주를 Nutrient broth에 접종하여 30℃에서 24시간 동안 호기배양하여 균주 배양액을 제조한 후, 중량비로 2배의 제오라이트, 활성규조토, 질석 및 화이트카본 각각에 균주 배양액을 골고루 균일하게 혼합하여 40℃에서 건조시켜 각각의 균주 건조물을 제조하고, 미생물 균주의 음식물쓰레기 내 초기번식을 촉진하기 위하여, 이 균주 건조물에 당밀, 포도당을 중량비로 각각 0.5%, 1%를 첨가하고 부형제로 일라이트를 50% 첨가하여 발효소멸용 미생물제제를 제조하였다. 이렇게 제조한 미생물제제를 이용하여 이후의 시험을 실시하였다.
The microbial preparation of the present invention is inoculated Bacillus Cluj SKAL-16 strain in Nutrient broth and incubated at 30 ℃ for 24 hours to prepare a strain culture, and then by weight ratio of zeolite, activated diatomaceous earth, vermiculite and Evenly mix the strain culture solution to each white carbon and dry it at 40 ° C. to prepare each strain dry matter, and in order to promote the initial breeding in the food waste of the microorganism strain, molasses and glucose were respectively added in a weight ratio of 0.5 by weight ratio. %, 1% was added and 50% illite was added as an excipient to prepare a microbial preparation for fermentation. Subsequent tests were carried out using the microbial preparation thus prepared.
[실시 예 4] Example 4 무기담체별By weapon 미생물제제 내의 In microbial products 생균수Viable count 및 안정성 측정 And stability measurements
여러 종류의 무기담체를 사용하여 본 발명의 균주를 음식물쓰레기 발효소멸용 미생물제제에 적용시, 최적의 무기담체를 조사하였다. 실시 예 3의 여러 가지 무기담체를 이용하여 제조된 미생물제제 내의 생존하는 생균수를 조사하였으며, 그 안정성 측정을 위하여 40℃ 인큐베이터 내에서 4주간 보관한 후 생균수를 조사하였으며, 그 결과를 하기 표3에 나타내었다. Using various kinds of inorganic carriers, the optimum inorganic carriers were investigated when the strains of the present invention were applied to food waste fermentation and microbial agents. The number of viable cells in microbial preparations prepared using various inorganic carriers of Example 3 was investigated, and the number of viable cells was stored after 4 weeks in a 40 ° C incubator to measure the stability thereof. 3 is shown.
[표3] 각각의 무기담체에 따른 미생물제제 내의 생균수 [Table 3] Number of Viable Cells in Microbial Preparations for Each Inorganic Carrier
상기 표3에 보이는 바와 같이, 무기담체로 제오라이트를 사용한 경우에 많은 생균이 안정되게 유지되는 것이 확인되었다. As shown in Table 3 above, when zeolite was used as the inorganic carrier, it was confirmed that many live bacteria were stably maintained.
[실시 예 5] 음식물쓰레기의 실제 발효소멸 실험Example 5 Fermentation and Extinction Experiment of Food Waste
본 발명의 미생물이 포함된 미생물제제의 음식물쓰레기 발효소멸 효과를 검증하기 위하여 다음과 같은 실험을 하였다. 실시 예 4에서 생균수가 가장 높게 잘 유지된 제오라이트흡착 발효소멸용 미생물제제 15kg를 교반기, 온도조절장치, 탈취장치 및 팬이 부착된 10kg/일 처리용량의 발효소멸장치에 투입하여 발효소멸 여부를 실험하였다. 실험기간은 60일 동안이었으며, 매일 10kg 음식물쓰레기(당사 근처 식당에서 배출된 음식물쓰레기, 평균 함수율 80%)를 투여하여 소멸율, 생균수, 악취발생 여부, pH, 함수율, 온도 등을 조사하였고, 그 결과를 하기 표4에 나타내었다. In order to verify the effect of fermentation and extinction of food waste of the microbial agent containing the microorganism of the present invention, the following experiment was performed. In Example 4, 15 kg of the zeolite adsorption fermentation annihilation microorganisms having the highest viable cell count was added to a fermentor annihilation device of 10 kg / day treatment capacity equipped with a stirrer, a temperature control device, a deodorizer and a fan to test whether or not fermentation disappeared. It was. The experiment period was 60 days, and 10kg food waste (food waste discharged from restaurants near us, average water content of 80%) was administered to check the extinction rate, viable cell count, odor occurrence, pH, water content, temperature, etc. The results are shown in Table 4 below.
[표4] 음식물쓰레기 발효소멸 실험 결과[Table 4] Result of Food Waste Fermentation and Extinction
상기 결과에서, 생균수의 경우 평균 63×106 CFU/g로 매우 안정한 상태를 유지하였으며 악취발생의 원인으로 알려진 암모니아와 황화수소의 농도를 가스검지기 를 이용하여 측정한 결과 감지되지 않았으며, 음식물쓰레기의 최종 감소율은 92%로 나왔다. 투여된 음식물 가운데 소뼈 등의 이물질이 포함되었던 것을 고려한다면 실제 무게감소율은 더 높을 것으로 사료된다. In the above results, the average number of viable cells was 63 × 10 6 CFU / g, which was very stable, and the concentration of ammonia and hydrogen sulfide, which are known to cause odors, was not detected by using a gas detector. The final reduction rate was 92%. Considering that foreign foods such as beef bones were included in the administered food, the actual weight loss rate would be higher.
한편, 음식물쓰레기 발효소멸 후 잔량을 농업과학기술원 인증 퇴비원료 분석지정기관에 의뢰한 결과를 하기 표5에 나타내었다. On the other hand, after the food waste fermentation and extinction, the remaining amount was commissioned by the Agricultural Science and Technology Institute certified compost raw material analysis designated agency is shown in Table 5 below.
[표5] 발효소멸 후 잔량 분석결과[Table 5] Analysis of residual amount after fermentation extinction
위 모든 항목은 그린(1급)퇴비 기준에 적합함.All of the above items meet Green (Class 1) composting criteria.
따라서, 음식물쓰레기에 대한 본 발명의 미생물제제를 이용한 발효소멸에 있어서 활발한 대사과정으로 인해 음식물쓰레기 소멸시간을 단축할 수 있으며 높은 효소활성을 가지므로 소멸 전 기간 동안의 분해활성을 유지하여 발효소멸을 성공적으로 수행할 수 있다. Therefore, in the fermentation annihilation using the microbial agent of the present invention for food waste, the food waste extinction time can be shortened due to the active metabolic process, and has high enzymatic activity, thus maintaining the degradation activity for the entire period of extinction. You can do it successfully.
도 1은 본 발명에 의해 분리된 바실러스 클라우지 SKAL-16 균주(KACC 91320P)의 염농도에 따른 균 성장을 나타낸 그래프이다.1 is a graph showing the bacterial growth according to the salt concentration of Bacillus Cluj SKAL-16 strain (KACC 91320P) isolated by the present invention.
도 2는 본 발명에 의해 분리 동정된 8종 미생물의 배양시간 경과에 따른 부틸산의 생성을 나타낸 그래프이다.Figure 2 is a graph showing the production of butyric acid over the cultivation time of 8 microorganisms isolated and identified by the present invention.
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