KR20210013995A - Bacillus sp, a microorganism product for treating food waste and a treating method of food waste using it - Google Patents
Bacillus sp, a microorganism product for treating food waste and a treating method of food waste using it Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
Description
본 발명은 염분 내성 및 셀룰라아제, 프로테아제, 아밀라제 활성을 갖는 바실러스(Bacillus) 균주 Bacillus sp를 동정하는 한편, 동정된 균주 Bacillus sp를 이용한 미생물 제제를 제조하고, 상기 미생물 제제를 음식물 쓰레기에 투입하여 음식물 쓰레기를 처리토록 하는 미생물 제제, 미생물 균주 및 이를 이용한 음식물쓰레기 처리방법에 관한 것이다. The present invention identifies Bacillus sp, which has salt tolerance and cellulase, protease, and amylase activity, while preparing a microbial preparation using the identified strain Bacillus sp, and putting the microbial preparation into food waste It relates to a microbial preparation, a microbial strain, and a food waste treatment method using the same.
국내 음식물 쓰레기는 수분 함량과 유기물 함량이 80%이상으로 매우 높고, 염도가 높아서 저장, 수거 및 운송과정에서 쉽게 부패하여 악취와 침출수 등의 악영향으로 주거단지 내에서 쾌적성 저해 요인으로 작용하고 있다.Domestic food waste has a very high moisture content and organic matter content of more than 80%, and has high salinity, so it is easily decayed during storage, collection and transportation, and is acting as a factor that hinders comfort in residential complexes due to adverse effects such as odor and leachate.
따라서, 발생원에서부터 원천적으로 음식물 쓰레기의 적정 처리에 대한 효과적 기술 개발 필요성이 증대되고 있으며, 공동주택에서 발생하는 음식물 쓰레기는 발생량, 성상이 거의 일정하여 생물학적 처리 후 활용이 용이하여 유용한 에너지 자원으로 기대되고 있다.Therefore, the need for effective technology development for the proper treatment of food waste from the source is increasing, and food waste generated in apartment houses is expected to be a useful energy resource because it is easy to use after biological treatment because the amount and nature of food waste is almost constant. have.
이러한, 음식물류폐기물은 2006년 분리배출 이후 정부의 정책에 따라 2008년부터 감량 추세로 매년 평균 2.3%의 감량폭을 기록하였지만, 2014년부터 13,222톤/일로 약 1.9% 증가추세를 나타내고 있으며, 음식물류폐기물은 사료화에 의해 47.34% 처리가 되고. 퇴비화 38.1%, 하수처리 2.6%, 혐기성소화 3.6%로 각각 처리되는 있다.Food waste has been reduced by an average of 2.3% every year since 2008 in accordance with the government's policy after separate discharge in 2006, but has increased by about 1.9% at 13,222 tons/day since 2014. Is treated 47.34% by feed conversion. There are 38.1% of composting, 2.6% of sewage treatment, and 3.6% of anaerobic digestion.
이에따라, 음식물류폐기물 자원화 정책이 퇴비화, 사료화에 집중되어 있는 처리시템을 다양화, 다각화하여 재활용을 활성화시키고 그에 따른 체계적인 관리방안 또한 대두되고 있다.Accordingly, the policy of recycling food waste to resources is diversifying and diversifying treatment systems concentrated on composting and fodder, activating recycling, and systematic management plans are also emerging.
또한, 국내외 폐기물 처리 및 자원화 시설들은 감량화/자원화 방식으로 주로 대형·집중형 형태로 운영되고 있으나 다양한 기술적·사회적·경제적 한계를 내포하고 있으며, 음식물 종량제 추진 등 다양한 정책이 추진되고 있으나 자원순환 인식 및 실천부족으로 효과는 미미한 수준이다.In addition, domestic and overseas waste treatment and recycling facilities are mainly operated in large-scale and concentrated form in a reduction/resource-ization method, but they have various technical, social, and economic limitations, and various policies such as the promotion of a food waste rate system are being promoted. Due to the lack of practice, the effect is insignificant.
이와같은 음식물 쓰레기를 효율적으로 처리하기 위한 한 방편으로 미생물을 이용한 소멸화방식이 적용되고 있으며, 이러한 미생물을 이용한 소멸화 처리 방법은 투입된 음식물을 호기적 조건 및 적합한 공정 하에 짧은 시간 내에 발효시킴으로써 음식물 폐기물의 양을 크게 감소시키고 기존 처리 방법의 문제로 지적된 2차 환경 오염도 최소화하여 현재 음식물 쓰레기 처리를 위한 가장 합리적인 대안이라 할 수 있다.As a way to efficiently treat such food waste, an extinction method using microorganisms is applied, and the extinction treatment method using such microorganisms is a food waste by fermenting the input food in a short time under aerobic conditions and suitable processes. It can be said to be the most reasonable alternative for the current food waste disposal by greatly reducing the amount of food and minimizing the secondary environmental pollution pointed out as a problem of the existing treatment method.
또한, 이러한 미생물을 이용한 소멸화 처리 방법은 생물학적 환경이 적절하게 조절된 장치 내에서 미생물의 대사 활동에 의해 음식물 쓰레기의 유기물을 물과 탄산가스로 분해하여 대기 중으로 휘발시키는 처리공정을 갖는다.In addition, such an extinction treatment method using microorganisms has a treatment process in which organic matter of food waste is decomposed into water and carbon dioxide gas by the metabolic activity of microorganisms in a device in which a biological environment is properly controlled and volatilized into the atmosphere.
현재 국내에서 음식물 쓰레기 처리 방식에 이용되는 미생물로는 고초균인 Bacillus을 비롯한 세군, 효모균, 곰팡이들이 많이 이용된다. Currently, microbes used in the food waste disposal method in Korea include Bacillus, Bacillus bacteria, three groups, yeast, and mold.
그리고, 고초균인 Bacillus은 단백질 분분해 효소를 균체 외부로 분비하여, 열 안정성 및 넓은 pH범위에서 활성을 보유하여 산업적으로 널이 사용되고 있다. In addition, Bacillus, a Bacillus bacillus, secretes proteolytic enzymes to the outside of the cells, retains thermal stability and activity in a wide pH range, and is therefore used industrially.
또한, B. subtilis, B. brevis, B. stearothermophilus 및 B. cereus 등의 균주에서 알칼리성 단백질 분해 효소의 생산이 보고되었으며, 내열성, 내산성 또는 내알칼리성 특성을 지닌 단백질 분해 효소등에 연구가 많이 이루어지고 있다.In addition, production of alkaline proteolytic enzymes has been reported in strains such as B. subtilis, B. brevis, B. stearothermophilus and B. cereus, and many studies are being conducted on proteolytic enzymes having heat resistance, acid resistance, or alkali resistance. .
그러나, 상기와 같은 미생물을 이용한 소멸화 처리 방법은, 장기적으로 음식물쓰레기의 공급에 의해 염분, 온도, pH 등의 환경이 급격히 변화되는 과정이 반복되면, 미생물의 생장 및 분해, 발효가 생리적으로 둔화되고 이로 인해, 음식물쓰레기의 발효, 소멸 시간이 지연되어 음식물쓰레기가 부패하기 쉽다는 단점이 있다. However, in the extinction treatment method using microorganisms as described above, if a process in which the environment such as salt, temperature, and pH is rapidly changed by the supply of food waste in the long term is repeated, the growth and decomposition of microorganisms and fermentation are physiologically slowed. As a result, the fermentation and extinction time of food waste is delayed, and the food waste is liable to perish.
또한, 번식 속도가 느려 접종한 미생물이 쉽게 유실되고 단백질 성분의 분해 능력이 비교적 낮아 암모니아성 및 황화수소성 악취를 유발할 가능성이 높다는 단점이 있다.In addition, there is a disadvantage in that the inoculated microorganisms are easily lost due to the slow propagation rate, and the ability to decompose protein components is relatively low, which is highly likely to cause ammonia and hydrogen sulfide odors.
더하여, 음식물 쓰레기 처리를 위한 단일 미생물 사용의 문제점을 해결하기 위해 대한민국 특허 제10-1161670호에서 음식물 쓰레기 처리용 혼합균주 및 이를 이용한 음식물 쓰레기 처리 방법을 제안하였다. In addition, in order to solve the problem of using a single microorganism for food waste treatment, Korean Patent No. 10-1161670 proposed a mixed strain for food waste treatment and a food waste treatment method using the same.
그러나 이러한 혼합균주는, 음식물 쓰레기 처리시 환경인 높은 온도와 염분도, 낮은 pH에서 미생물의 성장과 활성이 부족한 문제점이 있다.However, these mixed strains have a problem in that the growth and activity of microorganisms are insufficient at high temperature, salinity, and low pH, which are environments when food waste is disposed of.
상기와 같은 종래의 문제점들을 개선하기 위한 본 발명의 목적은, 셀룰라아제, 프로테아제, 아밀라제 효소에 대한 분해활성을 높혀 음식물 쓰레기의 처리시 악취에 대한 문제를 해결하도록 하고활성을 갖도록 하며, 내염성 내산성등의 다양한 특성을 갖는 바실러스 속의 미생물을 분리하도록 하는 미생물 균주, 미생물 제제 및 이를 이용한 음식물쓰레기 처리방법을 제공하는데 있다.An object of the present invention for improving the above-described conventional problems is to increase the decomposition activity of cellulase, protease, and amylase enzymes to solve the problem of odor when disposing of food waste and to have activity, such as salt resistance and acid resistance. It is to provide a microorganism strain, a microorganism preparation, and a food waste treatment method using the same to separate microorganisms in the genus Bacillus having various characteristics.
본 발명은 상기 목적을 달성하기 위하여, 수탁번호 KCTC 18775P로 수탁된 음식물 쓰레기 처리용 Bacillus sp. DP2-9의 미생물 균주를 제공한다.The present invention is to achieve the above object, Bacillus sp. for food waste disposal entrusted with accession number KCTC 18775P. Microbial strains of DP2-9 are provided.
그리고, 본 발명의 미생물 균주를 무균적으로 건조시킨 분말상으로 이루어지는 미생물 제제를 제공한다.And, it provides a microbial preparation consisting of a powdery microbial strain of the present invention aseptically dried.
또한, 본 발명은 수탁번호 KCTC 18775P로 수탁된 미생물 균주 Bacillus sp. DP2-9를 건조한 미생물 제제를 음식물 쓰레기 처리 반응수조에 살포하여 음식물 쓰레기 내의 유기물을 분해시키는 음식물 쓰레기 처리방법을 제공한다.In addition, the present invention is the microbial strain Bacillus sp. entrusted with accession number KCTC 18775P. A food waste treatment method is provided in which a microbial preparation dried DP2-9 is sprayed on a food waste treatment reaction tank to decompose organic matter in the food waste.
이상과 같이 본 발명에 의하면, 생육가능한 온도 및 pH 범위가 매우 넓고, 탄수화물, 단백질, 섬유소, 지방질 모두를 분해하는 효소의 생산력 및 활성도가 높아서 음식물 쓰레기의 소멸화 정도가 매우 높은 효과가 있다.As described above, according to the present invention, the viable temperature and pH range is very wide, and the productivity and activity of enzymes that decompose all carbohydrates, proteins, fibers, and fats are high, so that the degree of extinction of food waste is very high.
또한, 본 발명은 음식물의 용해성 부분 및 비용해성 부분 모두에 대하여 분해력이 우수하고, 고염분 농도하에서도 생육이 가능하며, 음식물로부터 발생하는 취기의 억제력이 우수하며, 내열성, 내산성, 내염기성이어서 우리나라 음식물을 발효 소멸시키는데 매우 유용한 효과가 있다.In addition, the present invention has excellent decomposition power for both soluble and non-soluble parts of food, can grow even under high salt concentration, has excellent suppression power of odors generated from food, and has heat resistance, acid resistance, and alkali resistance. It has a very useful effect in extinguishing fermentation.
도1은 본 발명에 따른 Bacillus sp. DP2-9 생장곡선과 단백질분해효소 활성도를 도시한 그래프이다.
도2는 본 발명에 따른 미생물제제의 제조를 위한 순서도이다.
도3은 본 발명에 따른 균주의 유전자 염기서열이다
도4 및 도5는 각각 본 발명에 따른 균주의 단백질 및 셀룰로스에 대한 활성테스트를 나타낸 사진이다.
도6은 본 발명에 따른 균주의 기탁증 사본이다.1 is a Bacillus sp. It is a graph showing the DP2-9 growth curve and protease activity.
Figure 2 is a flow chart for the production of the microbial preparation according to the present invention.
3 is a gene sequence of the strain according to the present invention
4 and 5 are photographs showing an activity test for protein and cellulose of the strain according to the present invention, respectively.
6 is a copy of the deposit certificate of the strain according to the present invention.
(1). Bacillus속 균주 선발(One). Selection of strains of the genus Bacillus
쓰레기 매립지의 퇴적토를 채취하여, 그 시료를 0.85% 생리식염수로 10-1 ~ 10-6 으로 희석하여 skim-milk을 첨가한 배지(1% skim-milk, 0.5% yeast extracts, 0.1% peptone, 1.5% agar)에 접종하여 30℃에서 7일간 배양하였다. Sediment soil from the landfill was collected, and the sample was diluted with 0.85% physiological saline to 10 -1 to 10 -6 and added skim-milk (1% skim-milk, 0.5% yeast extracts, 0.1% peptone, 1.5). % agar) and incubated for 7 days at 30°C.
배양 후 colony 주변에 생성된 투명환의 크기가 1 cm 이상인 것 즉, 단백질 활성을 띤 균주를 1차적으로 선발하고, 선발된 균주는 3-4회 계대배양(subculture) 후 순수 분리하였다.After cultivation, a strain having a size of 1 cm or more of a transparent ring formed around the colony, that is, a strain with protein activity, was first selected, and the selected strain was subcultured 3-4 times and then purely isolated.
분리된 균주는, 도3과 같이 16s rRNA 유전자 분석하여 유전학적으로 동정을 하였다. The isolated strain was genetically identified by analyzing 16s rRNA genes as shown in FIG. 3.
즉, 단백질 분해 효소 평가에서 선발된 균주를 16s rRNA 유전자 분석 한 결과 아래의 표1에서와 같이 Bacillus sp. 98- 99%와 Lysinibacillus sp. 99% 이내의 유사성을 나타내었다. That is, as a result of analyzing the 16s rRNA gene of the strain selected in the protease evaluation, Bacillus sp. 98-99% and Lysinibacillus sp. It showed a similarity within 99%.
이 중, Bacillus sp. DP2-9 균주는 한국생명공학연구원에 기탁을 하여 KCTC18775P 기탁번호를 받았다. Among them, Bacillus sp. The DP2-9 strain was deposited with the Korea Research Institute of Bioscience and Biotechnology and received the KCTC18775P accession number.
[표 1]. 선발된 균주의 16s rRNA 유전자 분석 유사도[Table 1]. Similarity of 16s rRNA gene analysis of selected strains
(2). 균주 특성(2). Strain characteristics
내염성의 검토를 위하여 음식물 쓰레기의 염도를 고려하여 0, 1, 2, 3, 4, 5, 6, 7, 8% NaCl이 함유된 NB 배지를 사용하여 균주를 배양하였으며, 7일동안 배양상태를 확인하였다.For the examination of salt resistance, the strain was cultured using NB medium containing 0, 1, 2, 3, 4, 5, 6, 7, 8% NaCl in consideration of the salinity of food waste, and the culture was maintained for 7 days. Confirmed.
그리고, 내산성의 확인을 위하여 음식물 쓰레기의 산도를 고려하여 pH 4 ~ 12의 각각 NA 배지 제조하여 균주를 배양하였으며, 이 역시 7일동안 배양상태를 확인하였다.And, in order to confirm the acid resistance, the strains were cultured by preparing each NA medium having a pH of 4 to 12 in consideration of the acidity of the food waste, and the culture condition was also confirmed for 7 days.
또한, 고온성의 확인을 위하여 음식물 쓰레기 처리 중 온도를 에서 배양을 고려하여 10, 15, 20, 25, 30, 35, 40, 45, 50 ℃의 각각 NA 배지에 균주를 접종하여 각각의 온도배양기에 배양하였으며 7일동안 배양상태를 확인하였다.In addition, in order to check the high temperature, the strain was inoculated into each NA medium at 10, 15, 20, 25, 30, 35, 40, 45, 50 ℃ considering the cultivation of the temperature during food waste treatment at each temperature incubator. It was cultured and the culture condition was checked for 7 days.
이상과 같은 조건으로 선발된 9가지 종류의 균주에 대해서 내열성, 내산성, 내염성을 한 결과 다음 표2와 같다. The results of heat resistance, acid resistance, and salt resistance of the nine strains selected under the above conditions are shown in Table 2 below.
즉, 9가지 종류의 균주가 10 ~ 45 ℃ 까지 자라며, Lysinibacillus sp. 균주는 50℃에서 생장을 할 수 있는 것을 확인하였다. That is, 9 kinds of strains grow to 10 ~ 45 ℃, Lysinibacillus sp. It was confirmed that the strain can grow at 50°C.
또한, 일반 바닷물의 염도가 4 %정도로 알려져 있는데 7% 까지 생장이 가능하므로 높은 내염성을 나타내었다. In addition, the salinity of general seawater is known to be about 4%, but it can grow up to 7%, thus exhibiting high salt resistance.
그리고, pH 12에서도 생장이 가능함을 나타내었는데 이는 내알카리성을 나타내고 pH 4.5에서 생장이 가능함을 나타내었다. In addition, it was shown that growth is possible even at
이상과 같은 특성을 갖는 Bacillus sp. 와 Lysinibacillus sp. 은 균주의 생장 범위가 넓으므로 여러 환경조건에서도 활용가능하므로 음식물 처리에 유용하게 활용 할 수 있을것으로 사료된다.Bacillus sp. And Lysinibacillus sp. Since the growth range of silver strain is wide, it can be used in various environmental conditions, so it is thought that it can be usefully used for food treatment.
[표 2]. 선발된 균주의 특성[Table 2]. Characteristics of the selected strain
(3). 분해 효소 활성 테스트(3). Degrading enzyme activity test
분리 균주가 균체외로 방출하는 세포외 효소들 중 셀룰라아제(cellulase), 프로테아제(protease), 아밀라제(amylase)의 활성 검증을 위하여 well diffusion 방법을 사용하였고, 각각의 효소와 특이적으로 반응할 기질 성분이 포함된 고체평판 선별배지를 사용하였다. The well diffusion method was used to verify the activity of cellulase, protease, and amylase among the extracellular enzymes released by the isolated strain to the outside of the cells, and the substrate components that will react specifically with each enzyme The included solid plate selection medium was used.
먼저, 단백질분해효소인 protease 생산능은 skim milk를 기질로 선택하여 2%의 skim milk (Difco, USA)에 1.5%의 agar를 첨가한 skim milk agar 배지에 균체를 접종하고 37℃에서 24시간 반응시킨 후 분해능을 저지원(Clear zone)의 직경으로 조사하였다. First, the protease production ability, a protease, was determined by inoculating the cells in a skim milk agar medium in which 1.5% agar was added to 2% skim milk (Difco, USA) by selecting skim milk as a substrate and reacted at 37°C for 24 hours. After that, the resolution was investigated by the diameter of the clear zone.
그리고, 가수분해효소인 Cellulase 생산능은 1% carboxylmetyl-cellu- lose (CMC, JUNSEI chemical Co. Ltd., Tokyo, Japan)를 함유한 CMC agar 배지에 균체를 접종하고 37℃에서 24시간 반응시킨 후 congo red plate 방법으로 cellulase의 생성능을 확인하였다. In addition, the ability to produce Cellulase, a hydrolase, was obtained after inoculating the cells in CMC agar medium containing 1% carboxylmetyl-cellu-lose (CMC, JUNSEI chemical Co. Ltd., Tokyo, Japan) and reacting at 37°C for 24 hours. The cellulase production ability was confirmed by the congo red plate method.
또한, 전분분해효소인 amylase 생산능은 starch를 기질로 사용하여 1%의 soluble starch (JUNSEI chemical Co. Ltd., Tokyo, Japan)를 함유한 starch agar 배지에 균체를 접종하고 37℃에서 24시간 반응시킨 후 요오드로 염색한 뒤 분해능을 저지원(Clear zone)의 직경으로 조사하였다.In addition, the ability to produce amylase, a starch-degrading enzyme, uses starch as a substrate and inoculates the cells in a starch agar medium containing 1% soluble starch (JUNSEI chemical Co. Ltd., Tokyo, Japan) and reacts at 37°C for 24 hours. After staining with iodine, the resolution was investigated by the diameter of the clear zone.
이상의 조건에서 선발된 9가지 종류의 균주에 대해서 cellulase, protease, amylase의 활성 검증을 실시한 결과 다음 표3과 같다.As a result of verifying the activity of cellulase, protease, and amylase for the nine strains selected under the above conditions, the results are shown in Table 3 below.
또한, 도4의 단백질 분해 활성 및 도5의 가수분해 분해 활성테스트의 그림과 같이 활성을 갖는 것으로 나타난다. In addition, it appears to have activity as shown in the proteolytic activity of Fig. 4 and the hydrolytic decomposition activity test of Fig. 5.
즉, Bacillus cereus와 Bacillus wiedmannii로 동정된 균주는 cellulase, protease, amylase 3가지 효소에 모두 활성을 나타내었고, Bacillus aryabhattai와 Bacillus megaterium로 동정된 균주는 cellulase와 protease는 활성을 나타내었지만 amylase 효소 활성을 나타내지 않았다. That is, Bacillus cereus and Bacillus wiedmannii The identified strains showed activity against all three enzymes: cellulase, protease, and amylase. Bacillus aryabhattai and Bacillus megaterium The identified strains showed cellulase and protease activity, but not amylase enzyme activity.
그리고, Lysinibacillus halotolerans 균주는 protease는 활성을 나타내었지만 cellulase와 amylase 효소 활성을 나타내지 않았다. In addition, Lysinibacillus halotolerans strain showed protease activity, but cellulase and amylase enzyme activities did not.
[표 3]. 선발된 균주의 분해효소 활성 (양성, +; 음성, -)[Table 3]. Degrading enzyme activity of the selected strain (positive, +; negative, -)
(4). Protease 활성 분석(4). Protease activity assay
균주의 효소활성을 정량적으로 평가하기 위하여 단일 집락을 Nutrient broth 배지에 접종하여 37℃에서 24시간 동안 배양하였다. In order to quantitatively evaluate the enzyme activity of the strain, a single colony was inoculated into Nutrient broth medium and cultured at 37°C for 24 hours.
또한, 배양액을 원심분리(15,115 × g, 5분)하여 얻은 상등액은 단백질 농도 및 효소활성 측정을 위한 조효소액으로 사용하였다. In addition, the supernatant obtained by centrifuging the culture solution (15,115 × g, 5 minutes) was used as a crude enzyme solution for measuring protein concentration and enzyme activity.
그리고, 인산염 완충액 (50 mM, pH 7.5)에 casein (Sigma-Aldrich, USA)의 농도가 0.65% (w/v)가 되도록 용해한 것을 기질 용액으로 사용하였다. Then, the casein (Sigma-Aldrich, USA) was dissolved in a phosphate buffer (50 mM, pH 7.5) so that the concentration of 0.65% (w/v) was used as a substrate solution.
1 ml의 기질용액에 200 μl의 조효소액을 혼합하여 37℃ 에서 10분간 반응한 후, 1 ml의 110 mM trichloroacetic acid 를 첨가하여 반응을 종료하였다. 200 μl of the coenzyme solution was mixed with 1 ml of the substrate solution and reacted at 37° C. for 10 minutes, and then 1 ml of 110 mM trichloroacetic acid was added to terminate the reaction.
반응액을 원심분리(15,000 × g, 10분)한 후 상등액 400 μl를 1 ml의 500 mM sodium carbonate (Sigma-Aldrich, USA), 200 μl의 folin & ciocalteu’s phenol reagent (Sigma-Aldrich, USA)와 혼합하여 37℃에서 30분 동안 반응시켜 660 nm에서 흡광도를 측정하였다. After centrifuging the reaction solution (15,000 × g, 10 minutes), 400 μl of the supernatant was added to 1 ml of 500 mM sodium carbonate (Sigma-Aldrich, USA) and 200 μl of folin & ciocalteu's phenol reagent (Sigma-Aldrich, USA). The mixture was reacted at 37° C. for 30 minutes, and absorbance was measured at 660 nm.
효소활성(1 unit)은 반응조건에서 1분간 1 μmole의 tyrosine 을 생성하는 효소의 양으로 정의하였다.Enzyme activity (1 unit) was defined as the amount of enzyme that produced 1 μmole of tyrosine for 1 minute under reaction conditions.
그리고, 균주의 생장은 3시간 마다 600 nm에서 흡광도를 측정하여 생장곡선은 작성하였다.And, the growth of the strain was measured by measuring the absorbance at 600 nm every 3 hours, a growth curve was created.
이상과 같은 조건하에서 균주의 배양특성 및 효소활성 등의 여러 가지 결과를 바탕으로 Bacillus sp. DP2-9 균주을 선발하였다. Based on various results such as the culture characteristics and enzyme activity of the strain under the above conditions, Bacillus sp. The DP2-9 strain was selected.
선발된 균주의 생장속도 및 분석한 결과 Bacillus sp. DP2-9 생장곡선과 단백질분해효소 활성도를 도시한 도1과 같다. The growth rate and analysis result of the selected strains Bacillus sp. Figure 1 shows the DP2-9 growth curve and protease activity.
균주의 12시간 이후부터 생장 속도가 증가하고 이에 따라 Protease 활성도 증가하였다. After 12 hours of the strain, the growth rate increased, and accordingly, the protease activity increased.
특히, 36시간에는 생장율과 단백질 분해 효소 활성가 가장 높았음을 확인 하였다. In particular, it was confirmed that the growth rate and proteolytic enzyme activity were highest at 36 hours.
(5).미생물제제 (5).Microbial preparations
Bacillus속 균주을 대량 배양하여 원심분리 후 균체을 회수 하였다. Bacillus sp. strains were mass-cultured and centrifuged to recover the cells.
이때, Bacillus sp. DP2-9 균주는 내열성, 내산성, 내염성을 갖고 있으며, 단백질 분해능도 탁월하다.At this time, Bacillus sp. The DP2-9 strain has heat resistance, acid resistance, and salt resistance, and is excellent in protein degradation.
Bacillus sp. DP2-9을 Nutrient broth 접종 후 30℃에서 4일 배양 후 회수하였다. Bacillus sp. DP2-9 was recovered after inoculation with Nutrient broth for 4 days at 30°C.
회수한 균체를 이용하여 미생물제제을 제조하였다. A microbial preparation was prepared using the recovered cells.
회수한 균체에 균체를 보호하기 위해 skim milk 첨가하고 미강에 균체를 혼합하고 40~45℃에서 16~24시간 건조하였다. To protect the cells, skim milk was added to the collected cells, and the cells were mixed in the rice bran and dried for 16 to 24 hours at 40 to 45°C.
완성된 미생물제제는 7일동안 생균수 확인하여 균체 함유에 대한 안정성을 평가하였다. The completed microbial preparation was checked for viable cell count for 7 days to evaluate the stability of the cell contents.
그 결과 1일부터 조금씩 감소하는 경향을 보이긴 하지만 5일째부터 균수가 4.2 x 105 CFU/ml 정도를 나타내었다. As a result, although it showed a tendency to decrease gradually from
즉, 평균 4.9 x 105 CFU/ml 균수를 나타내며 적정 생균수를 보였다.That is, the average number of bacteria was 4.9 x 10 5 CFU/ml, and the number of viable cells was appropriate.
[표 4] 미생물제제의 생균수 안정성[Table 4] Stability of the number of viable microorganisms
따라서, 본 발명의 미생물 제제를 이용하여 음식물쓰레기를 처리시 Bacillus sp. DP2-9 균주의 활발한 생육으로 인하여 분해 활성을 높여 음식물 처리 및 악취에 대한 문제를 해결할 수 있는 방안이 될 수 있으며, 우수한 미생물을 확보함으로써 생물자원의 확보 할 수 있을것이라 사료된다.Therefore, when treating food waste by using the microbial preparation of the present invention, Bacillus sp. Due to the active growth of the DP2-9 strain, it can be a solution to the problem of food treatment and odor by increasing the decomposition activity, and it is believed that it will be possible to secure biological resources by securing excellent microorganisms.
이상과 같이 확보된 미생물 균주를 무균적으로 건조시켜 음식물 쓰레기 처리시 투입되는 분말상의 미생물 제제를 완성한다.The microbial strain secured as described above is aseptically dried to complete a powdery microbial preparation that is introduced when disposing of food waste.
그리고, 상기 미생물 제제를 음식물 쓰레기 처리시 음식물 쓰레기가 투입되는 반응수조에 음식물 쓰레기와 동시에 투입하여 음식물 쓰레기 내의 유기물을 분해시키도록 한다.In addition, when the microbial preparation is disposed of the food waste, the organic matter in the food waste is decomposed by simultaneously putting the microbial agent into the reaction tank into which the food waste is added.
이때, 상기 Bacillus sp. DP2-9 균주의 활성 특성에 의해 탄수화물, 단백질, 섬유소, 지방질 모두를 분해하는 효소의 생산력 및 활성도가 높아서 음식물 쓰레기의 소멸화 정도가 매우 높은 효과가 있다.At this time, the Bacillus sp. Due to the active characteristics of the DP2-9 strain, the productivity and activity of enzymes that decompose carbohydrates, proteins, fibers, and fats are high, so the degree of extinction of food waste is very high.
또한, 내열성, 내산성, 내염기성이어서 우리나라 음식물을 발효 소멸시키는데 매우 유용한 효과가 있는 것이다.In addition, it has a very useful effect in fermenting and extinguishing food in Korea because it has heat resistance, acid resistance, and alkali resistance.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also provided. It belongs to the scope of rights
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| CN115786206A (en) * | 2022-12-02 | 2023-03-14 | 森诺技术有限公司 | Self-heat-production high-temperature aerobic strain and application thereof in biological treatment of kitchen waste |
| KR102599313B1 (en) * | 2023-07-21 | 2023-11-07 | 지성산업 주식회사 | Mixed microorganism strain for decomposing food waste |
| CN117229965A (en) * | 2023-09-25 | 2023-12-15 | 南京尚善环境工程技术有限公司 | Preparation method and application of polydopamine nickel-loaded nanocomposite and microbial agent |
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| KR101161670B1 (en) | 2009-12-24 | 2012-07-11 | 강원대학교산학협력단 | Mixed strain for treating food waste and method for treating food waste using the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115786206A (en) * | 2022-12-02 | 2023-03-14 | 森诺技术有限公司 | Self-heat-production high-temperature aerobic strain and application thereof in biological treatment of kitchen waste |
| CN115786206B (en) * | 2022-12-02 | 2023-12-19 | 森诺技术有限公司 | Self-heat-generating high-temperature aerobic strain and application thereof in biological treatment of kitchen waste |
| KR102599313B1 (en) * | 2023-07-21 | 2023-11-07 | 지성산업 주식회사 | Mixed microorganism strain for decomposing food waste |
| CN117229965A (en) * | 2023-09-25 | 2023-12-15 | 南京尚善环境工程技术有限公司 | Preparation method and application of polydopamine nickel-loaded nanocomposite and microbial agent |
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