KR102551904B1 - Bacillus licheniformis BS and composition for reducing oder containing the same - Google Patents
Bacillus licheniformis BS and composition for reducing oder containing the same Download PDFInfo
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Abstract
본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 및 이를 포함하는 악취 감소용 조성물에 관한 것으로, 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)는 악취가스 제거능이 우수하다. 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P), 이의 배양액 및 이 배양액의 건조분말을 포함하는 악취 감소용 조성물은 축산분뇨 또는 음식물쓰레기의 발생 현장에 처리하여, 악취의 근본적인 원인물질을 제거시켜 발생되는 악취를 감소시킬 수 있다.The present invention relates to Bacillus licheniformis BS (KACC 92483P) and a composition for reducing odor containing the same, and the Bacillus licheniformis BS (KACC 92483P) of the present invention has excellent odor gas removal ability do. Bacillus licheniformis BS (KACC 92483P) of the present invention, a culture solution thereof, and a composition for reducing odor containing a dry powder of this culture solution are treated at the site of occurrence of livestock manure or food waste, and are the fundamental causative agent of odor By removing it, it is possible to reduce the odor generated.
Description
본 발명은 악취유발물질 제거능이 우수한 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 및 이를 포함하는 악취 감소용 조성물에 관한 것이다.The present invention relates to Bacillus licheniformis BS (KACC 92483P) having excellent odor-causing substances removal ability and a composition for reducing odor containing the same.
축산가축 사육시에 유기성 오염 물질인 축산분뇨가 대량으로 발생되는데, 축산분뇨가 그대로 배출되는 경우 커다란 환경오염을 야기시키는 문제가 있다.Livestock manure, which is an organic contaminant, is generated in large quantities during breeding of livestock, and when the livestock manure is discharged as it is, there is a problem of causing great environmental pollution.
그 중 우선적으로 발생하고 있는 문제점으로 축산악취 발생, 가축의 생산성 격감, 폐사율 증가, 사료효율 저하 등이 나타났다. 또한, 농경지 오염, 하천 오염, 악취로 인한 대기 오염과 주변 민원의 대상으로 증폭되어 왔다. Among them, the occurrence of livestock odor, decrease in productivity of livestock, increase in mortality rate, and decrease in feed efficiency were found to be the problems that occurred first. In addition, it has been amplified as a target of air pollution and civil complaints due to agricultural land pollution, river pollution, and odor.
축산악취는 대부분이 축산가축의 분뇨로 인한 것으로, COD(Chemical Oxygen Demand), BOD(Biological Oxygen Demand), CODMn, SS(Suspended Solids), TN(Total Nitrogen) 등의 농도가 매우 높은 상태를 가진다. 또한, 항생제의 남용으로 인해 유익균의 생장억제, 자연 미생물의 활성인자 생장저해, 발효균주 활성억제, 지방산 휘발성 취기물질(VFC)의 악취 발생증대의 상태를 가진다. Most of the livestock odor is caused by livestock manure, and has a very high concentration of COD (Chemical Oxygen Demand), BOD (Biological Oxygen Demand), CODMn, SS (Suspended Solids), and TN (Total Nitrogen). In addition, due to the abuse of antibiotics, it has a state of inhibiting the growth of beneficial bacteria, inhibiting the growth of active factors of natural microorganisms, inhibiting the activity of fermenting strains, and increasing the odor of fatty acid volatile odorants (VFC).
취기물질로는 암모니아, 메틸머캅탄, H2S, 설픽메틸(sulfic methyl), 디설픽 메틸, 트리메틸아민, CH3COOH, 스티렌 등이 있는데, 대표적인 물질로 암모니아 및 아민류가 축산악취의 95%를 차지한다. Odor substances include ammonia, methyl mercaptan, H 2 S, sulfic methyl, disulfic methyl, trimethylamine, CH 3 COOH, styrene, etc. Representative substances include ammonia and amines, which account for 95% of livestock odor. occupy
한편, 음식물쓰레기 역시 축산분뇨와 마찬가지의 문제를 가지고 있다. 즉, 음식물쓰레기는 유기물의 함량이 풍부하고 중금속이나 농약 등의 인공 독성물질이나 자연독성을 함유하지 않기 때문에 농업용 퇴비 또는 동물의 사료 등으로 재활용할 수 있다는 점에서 다른 일반쓰레기와 큰 차별성을 갖지만, 악취로 인해 장시간의 방치나 보관이 어렵다는 문제가 있다. On the other hand, food waste also has the same problems as livestock manure. In other words, food waste has a great difference from other general waste in that it can be recycled as agricultural compost or animal feed because it is rich in organic matter and does not contain artificial or natural toxins such as heavy metals or pesticides. There is a problem that it is difficult to leave or store for a long time due to the odor.
음식물쓰레기를 퇴비 등으로 재활용하고 있긴 하지만, 음식물쓰레기를 퇴비화하는 경우에는 미생물에 의한 생분해과정인 전처리와 분해산물을 부식질(humus)로 전환하기 위한 후숙과정이 필요하기 때문에 3~4 주에 이르는 장시간의 처리를 요구하고 지속적으로 악취가 발생한다는 문제가 있다. Although food waste is recycled into compost, etc., in the case of composting food waste, a pre-treatment, which is a biodegradation process by microorganisms, and a post-ripening process to convert decomposition products into humus are required, which takes a long time of 3 to 4 weeks. There is a problem that it requires treatment and continuously generates an odor.
이에, 상기와 같은 축산분뇨 및 음식물쓰레기에서 발생되는 악취를 감소시킬 수 있는 여러 연구들이 시행되고 있는 실정이다.Accordingly, several studies that can reduce the odor generated from livestock manure and food waste as described above are being conducted.
본 발명에서는 악취유발물질 제거능이 우수한 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 제공하고자 한다. In the present invention, it is intended to provide Bacillus licheniformis BS (KACC 92483P) having excellent odor-causing substance removal ability.
또한, 본 발명에서는 축산분뇨 및 음식물쓰레기 등에서 유발되는 악취를 효과적으로 감소시킬 수 있는 조성물을 제공하고자 한다.In addition, the present invention is intended to provide a composition capable of effectively reducing odors caused by livestock manure and food waste.
본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 제공한다.The present invention provides Bacillus licheniformis BS (KACC 92483P).
또한, 본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P), 이의 배양액 또는 이 배양액의 건조분말을 포함하는 것을 특징으로 하는 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물을 제공한다.In addition, the present invention provides a composition for reducing the odor of livestock manure or food waste, comprising Bacillus licheniformis BS (KACC 92483P), a culture medium thereof, or a dry powder of the culture medium.
한편, 본 발명의 악취 감소용 조성물은 바람직하게 대나무잎을 더욱 포함하는 것이 좋다.Meanwhile, the composition for reducing odor of the present invention preferably further includes bamboo leaves.
본 발명의 악취 감소용 조성물에 있어서, 상기 배양액은 바람직하게 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 포도당(glucose), 효모 추출물 (Yeast extract) 및 염화나트륨(Nacl)을 포함하는 배지에 배양한 것이 좋다.In the odor reduction composition of the present invention, the culture medium is preferably Bacillus licheniformis BS (KACC 92483P) in a medium containing glucose, yeast extract and sodium chloride (Nacl) It is good to cultivate
본 발명의 악취 감소용 조성물에 있어서, 상기 축산분뇨 또는 음식물쓰레기는, 아민가스(amine gas) 또는 황화수소가스(H2S)를 악취 성분으로 배출하는 것이 좋다.In the odor reduction composition of the present invention, it is preferable to discharge amine gas or hydrogen sulfide gas (H 2 S) as an odor component from the livestock manure or food waste.
또한, 본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 포도당(glucose), 효모 추출물 (Yeast extract) 및 염화나트륨(Nacl)을 포함하는 배지에 1차 배양하여 배양액을 제조하는 제1단계; 및 상기 제1단계 후, 상기 배양액과 대나무잎 건조분말을 혼합하는 제2단계;를 포함하는 것을 특징으로 하는 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물 제조방법을 제공한다. 이때, 상기 제2단계는 바람직하게 포도당을 더욱 혼합하고 2차 배양하는 것이 좋다.In addition, the present invention is Bacillus licheniformis ( Bacillus licheniformis ) BS (KACC 92483P) in a medium containing glucose (glucose), yeast extract (Yeast extract) and sodium chloride (Nacl) by primary culture to prepare a culture medium first step; And after the first step, a second step of mixing the culture solution and dried bamboo leaf powder; provides a method for producing a composition for reducing the odor of livestock manure or food waste, characterized in that it comprises. At this time, the second step is preferably further mixed with glucose and secondary culture.
본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)는 악취유발물질 제거능이 우수하다. Bacillus licheniformis BS (KACC 92483P) of the present invention has an excellent ability to remove odor-causing substances.
또한, 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P), 이의 배양액 및 이 배양액의 건조분말을 포함하는 악취 감소용 조성물은 축산분뇨 또는 음식물쓰레기의 발생 현장에 처리하여, 악취의 근본적인 원인물질을 제거시켜 발생되는 악취를 감소시킬 수 있다.In addition, the odor reduction composition comprising Bacillus licheniformis BS (KACC 92483P), its culture medium, and the dried powder of this culture medium of the present invention is treated at the site of occurrence of livestock manure or food waste, thereby reducing the fundamental odor It is possible to reduce odor by removing the causative substance.
본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 제공한다.The present invention provides Bacillus licheniformis BS (KACC 92483P).
바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)는 양돈농가에서 새롭게 분리한 균주로써, 서열번호 1의 16s rRNA 부분서열을 포함하고 있다. 또한, 악취유발물질 제거능이 우수한 균주이다. Bacillus licheniformis BS (KACC 92483P) is a strain newly isolated from pig farms and contains the 16s rRNA subsequence of SEQ ID NO: 1. In addition, it is a strain with excellent ability to remove odor-causing substances.
또한, 본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P), 이의 배양액 또는 이 배양액의 건조분말을 포함하는 것을 특징으로 하는 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물을 제공한다.In addition, the present invention provides a composition for reducing the odor of livestock manure or food waste, comprising Bacillus licheniformis BS (KACC 92483P), a culture medium thereof, or a dry powder of the culture medium.
하기 실험예에서, 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)의 배양액을 축산분뇨에 처리하였더니, 축산분뇨로부터 발생되는 아민가스(amine gas) 및 황화수소가스(H2S)가 상당하게 감소되는 것을 확인하였다.In the following experimental example, when the culture solution of Bacillus licheniformis BS (KACC 92483P) of the present invention was treated with livestock manure, amine gas and hydrogen sulfide gas (H 2 S) generated from livestock manure was confirmed to be significantly reduced.
한편, 본 발명에서 배양액은 다양한 종류의 배지에 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 균주를 배양한 것을 의미한다. 다만, 하기 실험예에 따르면, 포도당(glucose)을 2~4중량%, 효모 추출물 (Yeast extract)을 1~2중량%, 염화나트륨(Nacl)을 0.01~0.03중량%로 포함하는 배지에 배양시키는 경우 본 발명의 균주가 더욱 잘 배양되어, 조성물이 더욱 우수한 악취 감소 효능을 발휘할 수 있다. On the other hand, in the present invention, the culture medium refers to culturing the Bacillus licheniformis BS (KACC 92483P) strain of the present invention in various types of media. However, according to the following experimental example, when cultured in a medium containing 2 to 4% by weight of glucose, 1 to 2% by weight of yeast extract, and 0.01 to 0.03% by weight of sodium chloride (Nacl) The strain of the present invention is better cultivated, so that the composition can exhibit a better malodor reduction effect.
한편, 본 발명의 악취 감소용 조성물은 바람직하게 대나무잎을 추가로 포함하는 것이 좋은데, 더욱 바람직하게는 대나무잎의 건조분말을 추가로 포함하는 것이 더욱 좋다.On the other hand, the odor reduction composition of the present invention preferably further includes bamboo leaves, more preferably, it is even better to further include dry powder of bamboo leaves.
대나무잎은 사계절 동안 대나무 생육지 주변에서 쉽게 얻을 수 있는 자원확보가 용이한 식물 유래 부산물이다. 본 발명에서 이러한 대나무잎이 악취유발 물질을 제거시키는 효과를 가지고 있으며, 발효시 향기유발 성분을 발생시켜 축사 내외의 악취를 감소시키는 효과를 나타냄을 확인하였다. 또한, 상기 대나무잎을 다양한 형태로 가공한 후 실험한 결과, 건조분말 형태로 가공한 대나무잎을 포함하는 경우 조성물의 악취 감소 효능이 더욱 우수한 것을 확인하였다.Bamboo leaves are plant-derived by-products that can be easily obtained in the vicinity of bamboo habitats during the four seasons and are easy to secure resources. In the present invention, it was confirmed that these bamboo leaves have an effect of removing odor-causing substances and exhibit an effect of reducing odor inside and outside the barn by generating fragrance-inducing components during fermentation. In addition, as a result of experiments after processing the bamboo leaves in various forms, it was confirmed that the odor reduction effect of the composition was more excellent when the dried powdered bamboo leaves were included.
한편, 대나무잎 건조분말은 대나무 잎을 60~80℃에서 0.8~1.2시간 동안 처리하여 건조시킨 후 파쇄하여 제조한 것을 사용하는 것이 좋다. 상기 온도 및 시간 조건으로 제조한 대나무잎 건조분말을 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)가 발효시키는 경우 향기유발 성분이 더욱 많이 발생된다.On the other hand, bamboo leaf dry powder is preferably prepared by treating bamboo leaves at 60 to 80 ° C. for 0.8 to 1.2 hours, drying them, and crushing them. When the dried bamboo leaf powder prepared under the above temperature and time conditions is fermented with Bacillus licheniformis BS (KACC 92483P), more fragrance-inducing components are generated.
한편, 본 발명의 악취 감소용 조성물은 다양한 종류의 악취유발물질에 처리하여 발생되는 악취를 감소시킬 수 있는데, 바람직하게 축산분뇨 또는 음식물쓰레기와 같이 유기물의 함량이 높은 악취유발물질에 처리하는 것이 좋다. 상기와 같은 악취유발물질은 주로 아민류의 가스를 발생시켜 악취를 유발시키는데, 하기 실험예에 따르면, 본 발명의 악취 감소용 조성물은 아민류 가스 발생을 효과적으로 감소시킬 수 있다. On the other hand, the composition for reducing odor of the present invention can reduce odors generated by treatment with various types of odor-causing substances. . The odor-inducing substances as described above mainly generate amine gas to cause odor. According to the following experimental examples, the odor-reducing composition of the present invention can effectively reduce the generation of amine-type gas.
또한, 본 발명은 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P)를 이용한 악취 감소용 조성물의 제조방법을 제공한다. 이하 본 발명의 악취 감소용 조성물의 제조방법에 대하여 구체적으로 설명한다.In addition, the present invention provides a method for preparing a composition for reducing odor using Bacillus licheniformis BS (KACC 92483P). Hereinafter, a method for preparing the composition for reducing odor of the present invention will be described in detail.
<단계 1: 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 균주 배양액을 제조하는 단계><Step 1: Preparing Bacillus licheniformis BS (KACC 92483P) strain culture solution>
본 단계는 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 균주를 배지에 1차로 배양하여 악취유발물질 제거능이 우수한 배양액을 제조하는 단계이다. 본 단계에서 제조된 배양액을 그대로 악취 감소용 조성물로 사용할 수도 있다.This step is a step of preparing a culture solution excellent in removing odor-causing substances by first culturing the Bacillus licheniformis BS (KACC 92483P) strain in a medium. The culture solution prepared in this step may be used as a composition for reducing odor.
한편, 본 단계에서 배지는 균주가 더욱 잘 배양되도록 탄소원인 포도당(glucose) 2~4 중량%, 질소원인 효모 추출물 (Yeast extract) 1~2중량%, 무기염류인 염화나트륨(Nacl) 0.01~0.03중량%를 포함하는 배지를 사용하는 것이 좋다. On the other hand, in this step, the medium contains 2 to 4 wt% of glucose as a carbon source, 1 to 2 wt% of yeast extract as a nitrogen source, and 0.01 to 0.03 wt% of sodium chloride (Nacl) as an inorganic salt so that the strain can be cultured better. It is recommended to use a medium containing %.
본 단계에서 배양은 30~35℃의 온도, 100~150rpm의 교반속도, 0.01~0.1 L/min의 통기량, 6~7의 pH의 조건하에 1~3일 동안 배양하는 것이 좋다. In this step, the culture is preferably cultured for 1 to 3 days under the conditions of a temperature of 30 to 35 ° C, an agitation rate of 100 to 150 rpm, an aeration rate of 0.01 to 0.1 L / min, and a pH of 6 to 7.
하기 실시예에 따르면, 상기와 같은 배지 구성 및 조건으로 배양하는 경우 악취를 더욱 효과적으로 감소시킬 수 있는 배양액을 제조할 수 있다.According to the following examples, a culture solution capable of more effectively reducing odor can be prepared when cultured under the above medium configuration and conditions.
<단계 2: 대나무잎 건조분말과 혼합하여 악취 감소용 조성물을 제조하는 단계><Step 2: Preparing a composition for reducing odor by mixing with dried bamboo leaf powder>
본 단계는 상기 단계 1에서 제조된 배양액에 대나무잎 건조분말을 더욱 첨가하여 악취유발물질 제거능을 더욱 향상시키고, 조성물이 발효될 시 향기유발 성분이 발생되게 하여, 악취를 더욱 효과적으로 감소시킬 수 있는 조성물을 제조하는 단계이다.In this step, dried bamboo leaf powder is further added to the culture solution prepared in step 1 to further improve the ability to remove odor-causing substances, and to generate odor-causing components when the composition is fermented, thereby reducing odor more effectively. It is a step of manufacturing.
본 단계에서 대나무잎 건조분말은 바람직하게 대나무잎을 60~80℃에서 0.8~1.2시간 동안 처리하여 건조시킨 후 파쇄하여 제조한 것을 첨가하는 좋다. 상기 온도 및 시간 조건으로 제조한 대나무잎 건조분말을 바실러스 리체니포미스 (Bacillus licheniformis) BS (KACC 92483P) 균주가 발효시키는 경우 향기유발 성분이 더욱 많이 발생된다.In this step, the dried bamboo leaf powder is preferably prepared by treating bamboo leaves at 60 to 80 ° C. for 0.8 to 1.2 hours, drying them, and then crushing them. When the dried bamboo leaf powder prepared under the above temperature and time conditions is fermented by the Bacillus licheniformis BS (KACC 92483P) strain, more fragrance-inducing components are generated.
본 단계에서 대나무잎의 건조분말은 상기 단계 1의 배양액: 대나무잎 건조분말의 중량 비율이 1:0.1~0.5가 되도록 첨가하는 것이 좋다. 상기 대나무잎 건조분말 중량 비율이 0.1 미만으로 첨가하는 경우에는 상기 단계 1의 배양액과 악취감소능에 큰 차이가 없어 이점이 없으며, 0.5를 초과하여 첨가하는 경우에는 제품의 제형화를 위해 추가로 첨가하는 재료들과 혼합이 어려워져 제품화가 어렵게 된다.In this step, the dry powder of bamboo leaves is preferably added so that the weight ratio of the culture solution of step 1: dry powder of bamboo leaves is 1:0.1 to 0.5. When the bamboo leaf dry powder weight ratio is less than 0.1, there is no significant difference between the culture solution and the odor reduction ability of step 1, so there is no advantage. It is difficult to mix with the materials, making productization difficult.
한편, 본 단계는 대나무잎의 건조분말을 첨가한 후, 포도당을 더욱 첨가하고 2차로 발효시키는 것이 좋다. 이때, 본 발명의 균주가 포도당에 의해 당발효가 더 진행되는데, 대나무잎 건조분말과 반응하며 향기성분을 더욱 많이 발생시켜 악취감소능이 더욱 향상되는 것이다.On the other hand, in this step, after adding the dry powder of bamboo leaves, it is preferable to further add glucose and ferment it secondarily. At this time, the sugar fermentation of the strain of the present invention further proceeds with glucose, and reacts with the dried bamboo leaf powder to generate more fragrance components, further improving the odor reduction ability.
본 단계에서 포도당을 더욱 첨가하는 경우에는 상기 단계 1의 배양액: 포도당의 중량 비율이 1:0.001~0.05가 되도록 첨가하는 것이 좋다. 상기 포도당의 중량 비율이 0.001 미만으로 첨가되는 경우에는 상기 단계 1의 배양액과 악취감소능에 큰 차이가 없어 이점이 없으며, 0.05를 초과하여 첨가하는 경우에는 추가함량에 비례한 악취감소능이 나타나지 않아 경제적으로 적합하지 않다.In the case of further adding glucose in this step, it is preferable to add such that the weight ratio of the culture solution in step 1: glucose is 1:0.001 to 0.05. When the weight ratio of glucose is added to less than 0.001, there is no significant difference in odor reduction ability from the culture solution in step 1, so there is no advantage. Inappropriate.
한편, 본 단계를 통해 제조된 악취 감소용 조성물을 그대로 사용할 수 있으나, 건조시킨 후 분말형태로 가공시켜 사용할 수도 있다. 구체적으로 35℃~40℃의 온도에서 40~48시간 건조한 후 분쇄하여 최종적으로 수분함량이 5~10%가 되도록 가공하여 사용할 수 있는 것이다.On the other hand, the odor reduction composition prepared through this step may be used as it is, but may be used after being dried and processed into a powder form. Specifically, after drying at a temperature of 35 ° C. to 40 ° C. for 40 to 48 hours, it can be ground and finally processed to have a moisture content of 5 to 10%.
이하, 본 발명의 내용을 하기 실시예 및 실험예를 통하여 보다 상세하게 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited only to the following examples and experimental examples, and includes modifications of equivalent technical ideas.
[실시예 1: 본 발명의 바실러스 리체니포미스 BS 분리 및 동정][Example 1: Isolation and identification of Bacillus licheniformis BS of the present invention]
대한민국 양돈농가에서 500여종의 미생물을 분리한 후, 아민가스를 90%이상 제거시킬 수 있는 1종의 균주를 선별하였다.After isolating about 500 types of microorganisms from pig farms in Korea, one strain that can remove 90% or more of amine gas was selected.
선별된 균주의 동정을 위하여 액체배지(TSB)에서 28℃에서 72시간 동안 진탕배양하였다. For the identification of the selected strain, shaking culture was performed at 28° C. for 72 hours in liquid medium (TSB).
배양액에 키트(genomic DNA prep kit)를 사용하여 구입회사에서 제시한 방법을 준수하여 genomic DNA를 추출하였다. 추출된 genomic DNA에 16s rDNA V3 영역은 PCR 프라이머로 NS3 및 ITS4를 사용하였으며, ITS-2 영역은 PCR 프라이머로 ITS86 및 ITS4를 사용하였다.Genomic DNA was extracted using a kit (genomic DNA prep kit) for the culture medium and following the method suggested by the purchasing company. NS3 and ITS4 were used as PCR primers for the 16s rDNA V3 region in the extracted genomic DNA, and ITS86 and ITS4 were used as PCR primers for the ITS-2 region.
PCR을 위한 반응조건은 전체 PCR반응액을 50㎕로 하였고 반응조성물 (10mM Tris-HCl (pH 8.0), 50mM Koam1 Cl, 1.5mM MgCl2, 0.01% gelatin, 100ng primer, 50ng Template DNA, 2.5mM dNTP, 및 2.5unit Taq polymerase(Promega))을 넣어 실시하였다. Reaction conditions for PCR were 50 μl of the total PCR reaction solution, and the reaction composition (10 mM Tris-HCl (pH 8.0), 50 mM Koam1 Cl, 1.5 mM MgCl 2 , 0.01% gelatin, 100 ng primer, 50 ng Template DNA, 2.5 mM dNTP , and 2.5unit Taq polymerase (Promega)) was put in and carried out.
PCR반응은 94℃에서 4분간 초기변성(pre denaturation) 시킨 후 94℃에서 1분간 변성(denaturation), 55℃에서 1분간 부착(annealing), 72℃에서 2분 증폭(extension)하여 총 35 Cycle 실시하였으며 최종 DNA 합성은 72℃에서 10분으로 하였다. PCR reaction was performed at 94℃ for 4 minutes, followed by denaturation at 94℃ for 1 minute, annealing at 55℃ for 1 minute, and amplification at 72℃ for 2 minutes, for a total of 35 cycles. And the final DNA synthesis was 10 minutes at 72 ℃.
PCR 증폭산물은 1.5% 아가로스 겔로 전기영동하여 ETBR(Ethidium bromide) 염색으로 확인 한 후 겔추출 키트(gel extraction kit, QIAGEN)로 PCR산물을 정제하고 염기서열을 분석하여 서열번호 1을 얻을 수 있었다. 서열번호 1을 NCBI의 BLAST의 데이터베이스와 염기서열의 상동성을 조사한 결과, 해당 균주는 바실러스 리체니포미스 속 균주임을 알 수 있었다. The PCR amplification product was electrophoresed on a 1.5% agarose gel, confirmed by ETBR (ethidium bromide) staining, and then the PCR product was purified with a gel extraction kit (QIAGEN) and sequenced to obtain SEQ ID NO: 1. . As a result of examining the homology of SEQ ID NO: 1 with NCBI's BLAST database and nucleotide sequence, it was found that the strain was a strain of the genus Bacillus licheniformis.
본 발명에서 상기 균주를 바실러스 리체니포미스(Bacillus licheniformis) BS라고 명명하였고, 2022년 12월 5일자로 농촌진흥청 국립농업과학원 미생물은행(KACC)에 기탁하여 수탁번호 KACC(92483P)를 부여받았다.In the present invention, the strain was named Bacillus licheniformis BS, and on December 5, 2022, it was deposited with the National Institute of Agricultural Science Microbial Bank (KACC) of the Rural Development Administration and was given accession number KACC (92483P).
[실시예 2: 악취 감소용 조성물 1 제조][Example 2: Preparation of composition 1 for reducing odor]
1) 배양액 제조1) Preparation of culture medium
본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS 균주를 하기 표 1의 조건에서 24시간 동안 배양하였다. Bacillus licheniformis BS strain of the present invention was cultured for 24 hours under the conditions shown in Table 1 below.
질소원: 효모 추출물 1.5중량%(배양액 중량기준)
무기염류: NaCl 0.01중량%(배양액 중량기준)Carbon source: 4.0% by weight of anhydrous glucose (based on the weight of the culture medium)
Nitrogen source: yeast extract 1.5% by weight (based on the weight of the culture medium)
Inorganic salts: NaCl 0.01% by weight (based on the weight of the culture medium)
교반속도: 120rpm
통기량: 0.05L/min
pH(산도): 6.5Temperature: 35℃
Stirring speed: 120 rpm
Aeration rate: 0.05L/min
pH (acidity): 6.5
2) 대나무잎 건조분말 제조2) Manufacture of dried bamboo leaf powder
대나무잎 50g을 70℃에서 24시간동안 건조하여 대나무잎 건조분말을 제조하였다.50 g of bamboo leaves were dried at 70° C. for 24 hours to prepare dried bamboo leaf powder.
3) 악취 감소용 조성물 제조3) Preparation of a composition for reducing odor
상기 1)에 제조된 배양액 1ml에 상기 2)에서 제조된 대나무잎 건조분말 0.1mg을 넣고 혼합하여, 본 발명의 악취 감소용 조성물 1을 제조하였다.0.1 mg of dried bamboo leaf powder prepared in 2) was added to 1 ml of the culture solution prepared in 1) above and mixed to prepare odor reduction composition 1 of the present invention.
[실시예 3: 악취 감소용 조성물 2 제조][Example 3: Preparation of composition 2 for reducing odor]
상기 실시예 2에서 제조한 본 발명의 악취 감소용 조성물 1에 포도당을 상기 조성물 중량을 기준으로 2중량%를 더욱 첨가한 후, 24시간 동안 배양하여 악취 감소용 조성물 2를 제조하였다.After further adding 2% by weight of glucose based on the weight of the composition to the malodor reducing composition 1 of the present invention prepared in Example 2, the composition was cultured for 24 hours to prepare a malodor reducing composition 2.
[실시예 4: 분말 형태의 악취 감소용 조성물 제조][Example 4: Preparation of a composition for reducing odor in powder form]
상기 실시예 2에서 제조한 본 발명의 악취 감소용 조성물 1을 40℃에서 40시간 동안 건조시킨 후 분쇄하여 분말 형태로 제조하였다.After drying the odor reduction composition 1 of the present invention prepared in Example 2 at 40° C. for 40 hours, it was pulverized to prepare a powder form.
[실험예 1: 본 발명 바실러스 리체니포미스 BS 균주의 악취유발물질 제거능 확인 실험][Experimental Example 1: Experiment to confirm odor-causing substance removal ability of Bacillus licheniformis BS strain of the present invention]
본 실험예에서는 본 발명 바실러스 리체니포미스 BS 균주가 악취유발물질을 제거하여 축산분뇨 등에서 발생되는 아민가스(Amine gas)를 효과적으로 감소시킬 수 있는지 확인하고자 했다. In this experimental example, it was attempted to confirm whether the Bacillus licheniformis BS strain of the present invention can effectively reduce amine gas generated from livestock manure by removing odor-causing substances.
1) 실험방법1) Experiment method
상기 표 1의 조건으로 균주를 배양하였다. 플라스크에 배양액 2ml 및 돈분 100g을 첨가한 후, 3일간 30℃에서 배양하였다. 이후 (주)가스텍 감지관을 사용하여 플라스크 내 아민가스 농도를 측정하였다.The strain was cultured under the conditions of Table 1 above. After adding 2 ml of the culture medium and 100 g of pig excretion to the flask, it was cultured at 30°C for 3 days. Afterwards, the concentration of amine gas in the flask was measured using a Gastech detector.
한편, 대조군에서는 TSB 배지 (casein 1.7중량%, soybean 0.3중량%, Dextrose 0.25중량%, NaCl 0.25중량%, Dipotassium phosphate 0.25중량%)에 본 발명의 바실러스 리체니포미스 (Bacillus licheniformis) BS 균주를 배양하여 얻은 배양액을 사용하였다.On the other hand, in the control group, TSB medium (casein 1.7% by weight, soybean 0.3% by weight, Dextrose 0.25% by weight, NaCl 0.25% by weight, Dipotassium phosphate 0.25% by weight) of the present invention Bacillus licheniformis ( Bacillus licheniformis ) By culturing the BS strain The obtained culture medium was used.
2) 실험결과2) Experimental results
상기 표 2에 나타나 있듯이, 표 1의 배지에서 배양된 바실러스 리체니포미스(Bacillus licheniformis) BS 배양액이 더욱 높은 아민가스 제거능을 나타냄을 확인하였다. 상기와 같은 결과는 본 발명의 균주가 더욱 잘 배양되어 더 높은 농도의 균주를 포함하는 배양액이 악취 감소능이 더욱 우수하다는 것을 의미하고, 본 발명의 균주가 악취유발물질을 제거시켜 돈분에서 발생되는 아민가스(Amine gas)를 효과적으로 감소시켰음을 의미한다.As shown in Table 2, it was confirmed that the Bacillus licheniformis BS culture medium cultured in the medium of Table 1 showed a higher amine gas removal ability. The above results mean that the strain of the present invention is cultured better, and the culture solution containing the strain at a higher concentration has a better odor reduction ability, and the strain of the present invention removes odor-causing substances to reduce amines generated from pig manure. This means that amine gas has been effectively reduced.
[실험예 2: 본 발명 악취 감소용 조성물의 악취 감소능 확인 실험][Experimental Example 2: Experiment to confirm malodor reducing ability of the composition for reducing odor of the present invention]
본 실험예에서는 상기 실시예 2 및 3에서 제조한 본 발명의 악취 감소용 조성물이 황화수소가스(H2S)로 인한 악취를 효과적으로 감소시킬 수 있는지 확인하고자 했다.In this experimental example, it was investigated whether the odor reduction composition of the present invention prepared in Examples 2 and 3 can effectively reduce odor caused by hydrogen sulfide gas (H 2 S).
1) 실험방법1) Experiment method
플라스크에 상기 실시예 2 또는 3에서 제조한 '악취 감소용 조성물 1 또는 악취 감소용 조성물 2' 3ml 및 돈분 100g을 첨가한 후, 30℃에서 배양하였다. 이후 (주)가스텍 감지관을 사용하여 플라스크 내 황화수소가스(H2S) 농도를 측정하였다. 3 ml of 'composition for reducing odor 1 or composition 2 for odor reduction' prepared in Example 2 or 3 and 100 g of pig manure were added to the flask, followed by incubation at 30°C. Then, the concentration of hydrogen sulfide gas (H 2 S) in the flask was measured using a gas tech detector.
한편, 대조군으로는 조성물이 처리되지 않은 돈분, 대나무잎 분말, 대나무잎 물 추출물, 대나무잎 에탄올 추출물, 동종의 바실러스 리체니포미스 배양액 + 대나무잎 분말을 설정하여 비교하였다. On the other hand, as a control group, pig manure, bamboo leaf powder, bamboo leaf water extract, bamboo leaf ethanol extract, and Bacillus licheniformis culture solution of the same species + bamboo leaf powder were set and compared as control groups.
2) 실험결과2) Experimental results
++: 1ppm 이하, +: 5ppm 이하, -: 10ppm 이상++: 1 ppm or less, +: 5 ppm or less, -: 10 ppm or more
상기 표 3에 나타나 있듯이, 본 발명 실시예 2의 악취 감소용 조성물 1의 경우에는 본 발명 바실러스 리체니포미스(Bacillus licheniformis) BS 균주를 포함하는 배양액과 대나무잎 건조분말을 포함하여 황화수소가스(H2S) 감소능이 높게 나타남을 확인하였다. 반면, 대나무잎 분말을 사용한 대조군 2, 대나무잎 물추출물을 사용한 대조군 3, 대나무잎 에탄올추출물을 사용한 대조군 4, 본 발명 균주와 동종의 바실러스 리체니포미스(Bacillus licheniformis)와 대나무잎 분말을 혼합하여 구성된 대조군 5 모두 황화수소가스(H2S) 제거능이 본 발명의 실시예 2 및 3보다 낮게 나타남을 확인하였다.As shown in Table 3, in the case of odor reduction composition 1 of Example 2 of the present invention , hydrogen sulfide gas (H 2 S) It was confirmed that the reduction ability was high. On the other hand, control 2 using bamboo leaf powder, control 3 using bamboo leaf water extract, control 4 using bamboo leaf ethanol extract, composed of a mixture of Bacillus licheniformis and bamboo leaf powder of the same species as the strain of the present invention. It was confirmed that all of the control groups 5 had lower hydrogen sulfide gas (H 2 S) removal capabilities than those of Examples 2 and 3 of the present invention.
상기와 같은 결과는, 본 발명의 악취감소용 조성물이 악취유발물질을 제거시켜 돈분에서 발생되는 황화수소가스(H2S)를 효과적으로 감소시켰음을 의미한다.The above results mean that the composition for reducing odor of the present invention effectively reduced hydrogen sulfide gas (H 2 S) generated from pig manure by removing odor-causing substances.
[실험예 3: 본 발명 악취 감소용 조성물의 처리 농도별 악취 감소능 확인 실험][Experimental Example 3: Experiment to confirm malodor reduction ability of the composition for reducing odor of the present invention by treatment concentration]
본 실험예에서는 상기 실시예 4에서 제조한 분말 형태의 악취 감소용 조성물의 처리 농도에 따른 악취 감소능을 확인하고자 했다. In this experimental example, the malodor reducing ability of the composition for reducing malodor in the form of powder prepared in Example 4 was confirmed according to the treatment concentration.
1) 실험방법 1) Experiment method
플라스크에 돈분을 첨가하고, 분말 형태의 악취 감소용 조성물 (실시예4)을 돈분 중량 기준으로 0.01, 0.1, 0.5, 1 중량%를 각각 첨가한 후, 30℃ 항온 인큐베이터에 5일동안 방치하고, 상온에서 23일간 방치한 후, 아민가스 농도를 측정하였다.Pork manure was added to the flask, and after adding 0.01, 0.1, 0.5, and 1% by weight of the composition for reducing odor in powder form (Example 4) based on the weight of pig manure, respectively, it was left in a constant temperature incubator at 30 ° C. for 5 days, After leaving it at room temperature for 23 days, the amine gas concentration was measured.
2) 실험결과2) Experimental results
상기 표 4에 나타나 있듯이, 분말 형태의 악취 감소용 조성물 1을 돈분 전체중량 대비 0.1~0.5 중량% 농도로 처리 하였을 때 무처리 대비 약 90%의 아민가스 농도가 감소하였음을 확인하였다. 반면, 0.01 중량%를 처리할 경우에는 아민가스 농도 감소율이 약 30%로 낮게 나타났으며 1 중량%로 처리할 경우에는 0.5 중량%와 비슷하게 감소하였음을 확인하였다.As shown in Table 4, it was confirmed that when the composition 1 for odor reduction in powder form was treated at a concentration of 0.1 to 0.5% by weight based on the total weight of pork manure, the amine gas concentration was reduced by about 90% compared to untreated. On the other hand, when 0.01% by weight was treated, the reduction rate of amine gas concentration was as low as about 30%, and when treated with 1% by weight, it was confirmed that it decreased similarly to 0.5% by weight.
이를 통해, 본 발명의 분말형태의 악취 감소용 조성물은 돈분 중량대비 0.1~0.5 중량%로 처리하는 것이 가장 적합함을 확인할 수 있었다. Through this, it was confirmed that the composition for reducing odor in powder form of the present invention is most suitable to be treated with 0.1 to 0.5% by weight based on the weight of pork manure.
Claims (7)
축산분뇨 또는 음식물쓰레기의 중량 대비 0.1~0.5중량%로 처리되는 것을 특징으로 하는 분말형태의 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물.
Including dry powder of Bacillus licheniformis BS (KACC 92483P) culture medium and dry powder of bamboo leaves cultured in a medium containing glucose, yeast extract and sodium chloride (Nacl),
A composition for reducing the odor of livestock manure or food waste in powder form, characterized in that it is treated with 0.1 to 0.5% by weight relative to the weight of livestock manure or food waste.
상기 축산분뇨 또는 음식물쓰레기는,
아민가스(amine gas) 또는 황화수소가스(H2S)를 악취 성분으로 배출하는 것을 특징으로 하는 분말형태의 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물.
According to claim 2,
The livestock manure or food waste,
A composition for reducing the odor of livestock manure or food waste in powder form, characterized in that amine gas or hydrogen sulfide gas (H2S) is discharged as an odor component.
상기 제1단계 후, 상기 배양액과 대나무잎 건조분말을 혼합한 후 분말화하는 제2단계;를 포함하여 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물을 제조하며,
상기 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물은 축산분뇨 또는 음식물쓰레기의 중량 대비 0.1~0.5중량%로 처리되는 것을 특징으로 하는 분말 형태의 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물 제조방법.
A first step of preparing a culture medium by primary culturing Bacillus licheniformis BS (KACC 92483P) in a medium containing glucose, yeast extract and sodium chloride; and
After the first step, a second step of mixing the culture solution and dried bamboo leaf powder and then pulverizing them; to prepare a composition for reducing the odor of livestock manure or food waste,
The livestock manure or food waste odor reduction composition is a method for producing a composition for reducing livestock manure or food waste odor in powder form, characterized in that treated with 0.1 to 0.5% by weight relative to the weight of livestock manure or food waste.
상기 제2단계는,
포도당을 더욱 혼합하고 2차 배양하는 것을 특징으로 하는 분말형태의 축산분뇨 또는 음식물쓰레기 악취 감소용 조성물 제조방법.
According to claim 6,
The second step is
A method for producing a composition for reducing the odor of livestock manure or food waste in powder form, characterized in that the glucose is further mixed and secondary cultured.
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| KR20220095082A (en) * | 2020-12-29 | 2022-07-06 | 오헌식 | Livestock complex with organic substance decomposition ability and odor reduction effect |
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