KR100265540B1 - Mass production method using new Bacillus subtilis S-W8-24 (KFC-11027) and soybean meal - Google Patents
Mass production method using new Bacillus subtilis S-W8-24 (KFC-11027) and soybean meal Download PDFInfo
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Abstract
단백질 가수분해효소,섬유소 가수분해효소를 분비하며 성장이 우수하여 사료 효율을 향상시키는 생균제로 적합한 신균주와 이의 대량 생산방법에 관한 것이다.The present invention relates to a new strain and its mass production method suitable for probiotics that secrete proteinase and fibrinase and improve growth efficiency.
단백질 분해효소,섬유소 분해효소를 동시에 분비하며, 대량 생산이 유리한 신규의 단일균주를 얻고, 이의 배양을 위한 배지에 대하여 검토하였다. 이를위하여 부숙된 토양과 퇴비에서 샘플을 채취하여 배양 선별한 결과 단백질가수분해와 섬유소 가수분해효소를 동시에 분비하며 내열성,내산성이 우수한 신균주 바실러스 서브틸리스 SY8-24(KFCC-11027)를 얻었고 이의 대량생산을 위해 배지들을 검토한 결과 전분, 인산이수소나트륨등 별도의 첨가물이 필요없이 대두막과 당밀만을 원료로한 배지를 사용한 방법을 얻었다. 이들은 돼지,닭등 소화기관이 짧은 가축의 사료효율을 향상을 위한 생균제로써 활용도 가치가 높다.Proteolytic enzymes and fibrinolytic enzymes were secreted at the same time, and a new single strain with favorable mass production was obtained, and the medium for its culture was examined. To this end, samples were collected from aged soil and compost to be cultured and selected. The bacterium Bacillus subtilis SY8-24 (KFCC-11027) was obtained by releasing proteolytic and fibrinase at the same time and having excellent heat and acid resistance. As a result of examining the medium for mass production, a method using only soybean and molasses as a raw material without a separate additive such as starch and sodium dihydrogen phosphate was obtained. They are highly useful as probiotics for improving feed efficiency of livestock with short digestive organs such as pigs and chickens.
Description
본 발명은 신규한 바실러스 SY8-24 및 대두박을 배양원료로 이용한 대량생산 방법에 관한 것으로, 더욱 상세하게는 토양에서 분리한 단백질 가수분해효소, 섬유소 가수분해효소 분비 및 성장이 우수하여 단위가축의 사료효율 향상 목적의 생균제로 적합한 신균주 바실러스 서브틸리스 SY8-24와 대두박을 배양원료로 이용한 경제적이고도 효율적인 대량생산 방법에 관한 것이다.The present invention relates to a mass production method using the novel Bacillus SY8-24 and soybean meal as a culture raw material, more specifically, it is excellent in the secretion and growth of protein hydrolase and fibrinase isolated from the soil unit feed of livestock The present invention relates to an economical and efficient mass production method using the new strain Bacillus subtilis SY8-24 and soybean meal, which are suitable as probiotics for improving efficiency.
일반적으로 균주로부터 효소를 얻고자 할 경우에는 다른 효소를 분해시키는 단백질 가수분해효소 분비가 없거나 저해제를 이용하여 목적하는 효소를 얻으나, 본 발명과 같이 가축 생균제로 이용하고자 균주를 직접생산하는 경우에는 단일 균주가 복합적으로 효소를 분비할 경우 가축 체내에서 효과적으로 작용이 가능하며 대량생산에 유리하다. 또한 현재까지의 바실리스 속 균주배양에 있어 트립톤,염화나트륨,효모 추출물(대한민국 특허 공고 97-590호) 등과 같은 고가의 배양배지를 이용하거나 대두박을 이용할 경우 가용성 전분,인산이수소나트륨,염화칼슘(대한민국 특허공고 92-6397호)을 첨가한 경우가 있다.In general, if you want to obtain an enzyme from the strain, there is no protein hydrolase secretion that breaks down other enzymes, or to obtain the desired enzyme using an inhibitor, but when producing the strain directly to use as livestock probiotics as in the present invention When a single strain secretes enzymes in a complex way, it is possible to work effectively in the livestock body and is advantageous for mass production. Also, in the culture of strains of the genus Bacillus, high-quality culture media such as tryptone, sodium chloride, yeast extract (Korean Patent Publication No. 97-590), or soybean meal are used, and soluble starch, sodium dihydrogen phosphate, and calcium chloride (Korea) Patent Publication No. 92-6397) may be added.
따라서 사료원료 및 첨가제로의 이용이 낮고, 사용에 따른 경제적 이득을 거의 보지 못하고 있는 실정이다.Therefore, the use of feedstock and additives is low, and the situation is hardly seen the economic benefits.
본 연구자들은 상대적으로 이용율이 낮은 사료원료의 이용성을 극대화하여 축산농가들의 경제적 이득을 얻게 하기 위해서는 효소분비력이 우수하고 성장속도가 빨라 돼지, 닭과 같이 소화시간이 짧은 가축의 장내에서 효과적으로 사료원료를 분해사며 펠렛팅과 같은 사료가공시의 열처리에 견딜 수 있는 미생물을 선별하여 값싸게 축산농가들애게 공급하기 위해 매우 저렴한 원료를 이용한 대량생산방법을 강구하였다.In order to maximize the availability of feedstocks with relatively low utilization rates and to obtain economic benefits for livestock farmers, the researchers found that feedstocks with high enzyme secretion and fast growth rate are effective in the gut of livestock with short digestion time such as pigs and chickens. In order to select microorganisms that can withstand the heat treatment during feed processing such as pelleting, we have devised a mass production method using very inexpensive raw materials to supply them to livestock farmers at low cost.
도 1은 엔도 베타 1,4-글루카네이즈(CMCase) 분비를 나타내는 사진1 is a photograph showing endo beta 1,4-glucanase (CMCase) secretion
도 2는 배양원료별 성장곡선2 is a growth curve for each raw material
도 3은 대두박의 첨가량에따른 균주성장곡선3 is a strain growth curve according to the amount of soybean meal added
도 4는 당밀의 첨가효과곡선4 shows the effect curve of molasses
본 발명의 발명자들은 가축 생균제로 널리 이용되고 있는 균주 중 바실러스속 균주가 고온, 낮은 산도 등의 열악한 조긴애서는 포자를 형성하여 내열성 및 내산성이 우수하다는 점을 고려하여 자연에서 얻은 사료들을 곰팡이, 유산균 등도 자라지만 바실러스속 균주들이 잘 자라는 영양 한천배지(nutrient agar; 육즙 0.3%, 펩톤 0.5%, 한천 1.5%)를 이용하여 특히 단위가축에 있어 단백질의 이용성을 높여성장촉진 및 분변내로의 질소배출량을 억제하는 효소인 단백질 가수분해효소와 사료곡물내에 많이 존재하여 단위가축의 소장에서 점성을 높여 영양소 이용효율을 낮추는 물질인 글루칸을 분해하는 효소인 섬유소 가수분해효소의 일종인 엔도 베타 1,4-글루카네이즈 분비력이 우수한 균주를 선벌하기 위해 부숙이 잘 된 흙이나 퇴비등의 샘플을 적정하게 희석한 다음 도말하여 37℃ 배양기에 넣어 배양하였다. 배양된 많은 균주들을 상이한 콜로니들 만을 선별하여 효소역가 측정용 배지에 그어 균주 성장에 따라 환을 나타내는 균주들을 선별하였다. 효소역가 측정용 배지는 단백질 가수분해효소는 2.0%의 탈지분유를 함유한 영양 한천배지를 이용하였으며 엔도 베타1,4-글루카네이즈역가측정을 위하여 카르복시메틸셀룰로스를 1.0% 함유한 영양 한천배지에서 배양 후 0.2% 콩고레드 염색액을 배지에 부어 염색시킨 다음 1몰의 염화나트륨용액으로 씻어내어 환을 나타내는 균주를 선벌하였다.The inventors of the present invention, the bacterium of the genus Bacillus strains widely used as livestock probiotics, such as the high temperature, low acidity, poor coarse eggs in consideration of the excellent heat resistance and acid resistance to form spores, fungi, lactic acid bacteria It also grows back, but Bacillus strains grow nutrient agar (0.3% juicy, 0.5% peptone, 1.5% agar) to increase the availability of protein, especially in unit livestock, to promote growth and nitrogen discharge into feces. Endo beta 1,4-glycol, an enzyme that breaks down glucan, an enzyme that inhibits nutrient utilization by increasing the viscosity in the small intestine of unit livestock because it is present in a large amount of protein hydrolase, which is an inhibitory enzyme, and feed grains. Properly dilute samples such as well-adjusted soil or compost to select strains with excellent lucanise secretion Then, smeared and incubated in a 37 ℃ incubator. Many cultured strains were screened only for different colonies and screened on the enzyme titer medium to select strains that showed a ring as the strain grew. The enzyme titer was used as a nutrient agar medium containing 2.0% skim milk powder, and in a nutrient agar medium containing 1.0% carboxymethylcellulose for the determination of endo beta 1,4-glucanase titer. After incubation, 0.2% Congo red staining solution was poured into the medium, stained, and washed with 1 mol of sodium chloride solution to obtain a strain showing a ring.
또한 이와같이 선별된 균주를 대두박과 당밀을 이용한 대량 배양방법을 획득하였다.In addition, the strain thus selected was obtained a mass culture method using soybean meal and molasses.
본 발명의 효과를 실시예를 통하여 더욱 상세히 기술하면When describing the effects of the present invention in more detail through the examples
실시예 1 : 균주의 분리 및 동정Example 1 Isolation and Identification of Strains
단백질 분해효소 및 섬유소 분해효소 분비력이 우수한 바실러스속 균주를 함유하리라 판단된 부속이 잘 된 흙, 두엄 등에서 샘플을 채취하여 생리식염수로 적당히 희석하여 상등액을 취한 다음 영양 한천배지(nutrient agar)에 도말하여 37℃ 배양기에서 12시간 배양한 다음 콜로니의 모양에 따라 균주를 선별적으로 분리하여37℃ 배양기에서 배양시켰다.Samples are taken from well-equipped soil, manure, etc., which are believed to contain Bacillus strains, which are known to contain protease and fibrinolytic enzymes, and diluted with physiological saline to obtain a supernatant, followed by smearing on nutrient agar. After incubating for 12 hours in a 37 ℃ incubator, strains were selectively isolated according to the shape of colonies and incubated in a 37 ℃ incubator.
이렇게 분리된 균들을 단백질 가수분해효소 및 섬유소 가수분해효소의 일종인 엔도 베타 1,4-글루카네이즈 분비력을 측정하기 위해 각각 2.0%의 탈지분유 및 1.0%의 카르복시메틸셀룰로스 기질로 함유한 영양 한천배지에 선별된 균주를 37℃ 배양기에서 배양시켜 나타나는 환의 크기로 선별하였다. 이때 단백질 가수분해효소는 곧바로 환을 판독할 수 있으며 섬유소 가수분해효소의 일종인 엔도 베타 1,4-글루카네이즈의 경우 전술한 바와 같이 0.2% 콩고레드 염색액을 배지에 부어 염색시킨 다음 1몰의 염화나트륨 용액으로 씻어내어 나타나는 환의 크기로 선별하였다. 이들 중 단백질 가수분해효소, 섬유소 가수분해효소(특히, 엔도 베타 1,4-글루카네이즈) 분비력 및 성장이 가장 우수하며 내열성 및 내산성이 우수한 바실러스속 균주를 얹기위해 영양 한천배지 상에서 특유의 바실러스속 균주의 콜로니 형태, 현미경 검경을 통한 형태 및 포자형성 등을 파악하여 최종적으로 균주를 선발하였다.These isolates were nutrients containing 2.0% skim milk powder and 1.0% carboxymethylcellulose substrate, respectively, to measure the endocrine 1,4-glucanase secretion ability, which is a kind of proteinase and fibrinase. Strains selected on agar medium were selected by culturing in a 37 ℃ incubator to the size of the ring. At this time, the protease can immediately read the ring, and in the case of endo beta 1,4-glucanase, a kind of fibrinase, 0.2% Congo red dye solution was poured into the medium and stained, followed by 1 mole of Rinsed with sodium chloride solution and selected to the size of the ring appearing. Among them, a unique Bacillus on a nutritious agar medium to put on Bacillus strains with the best secretion and growth of protein hydrolase and fibrinase (especially endo beta 1,4-glucanase) and excellent heat resistance and acid resistance The strains were finally selected by identifying colony morphology, morphology and spore formation through microscopic examination of the genus strains.
상술한 바에 따라 최종적으로 선별된 균주는 [표 1]에 나타난 바와 같이 호기성 균이며 그람 양성간균으로 밝혀졌고 상업적으로 시판되는 당테스트 키트를 이용하여 당이용 결과를 판정하여 동정하였다.The strains finally selected as described above were identified as aerobic bacteria and Gram-positive bacillus as shown in [Table 1], and were determined by determining sugar use results using a commercially available sugar test kit.
동정결과 바실러스 서브틸리스(Bacillus subtilis)로 동정되었으며, 이 균주는 1998년 4월 1일 KFCC-11027 로서 한국종균협회(KFCC)에 기탁하였다.As a result, it was identified as Bacillus subtilis, and this strain was deposited to KFCC on April 1, 1998 as KFCC-11027.
[표 1]에서 알수 있는 바와 같이, 본 발명의 바실러스 서브틸리스 8-24는 99.6%의 확률로 바실러스 서브틸리스에 속하는 균주로 판명되었으나,둘시톨 이용등에 있어 통상의 바실러스 서브틸리스의 경우 거의(5%) 이용할 수 없는 것과 달리이용능에 있어 신규한 균주로 판명되었으며,요소를 분해하여 암모니아를 발생시키는 효소인 우레이즈를 분비하지 않아 가축의 호흡기 질병을 유발하는 암모니아를 발생시키지 않기 때문에 가축 생균제용 균주로 적합한 것으로 판명되었다.As can be seen from Table 1, Bacillus subtilis 8-24 of the present invention was found to be a strain belonging to Bacillus subtilis with a probability of 99.6%, but in the case of the usual Bacillus subtilis in the use of dulcitol, etc. Unlike the almost unavailable (5%), it was found to be a novel strain in its capacity, and because it does not secrete urease, an enzyme that breaks down urea and generates ammonia, it does not produce ammonia, which causes respiratory diseases in animals. It has been found to be suitable as a probiotic strain.
실시예 2 : 값싼 배지원료를 이용한 균주 성장 비교 실험Example 2 Strain Growth Comparison Experiment Using Cheap Media Raw Materials
실시예 1에 따라 선별된 바실러스 서브틸리스 SY8-24 균주를 1차적으로 균주의 활성을 높이기 위해 영양 한천배지에서 계대배양한 다음 50ml의 영양 한천배지에 접종한 후 37℃, 150rpm으로 교반배양기에서 배양하였다. 8시간 배양 후 영양 액체배지(펩톤 0.5%, 육즙 0.3%)를 최적 비교배지로하여 대두박, 면실박, 채종박, 미강, 밀기울을 1mm 체로 분쇄하여 거른 것을 증류수 100ml에 각각 1.0%(w/v)씩 첨가하여 멸균시킨 배양액에 균주를 1.0%(v/v)씩 접종한 후 37℃, 150mm으로 교반배양기에서 배양하였다. 배양 후 2시간 간격으로 균수를 측정한 결과 [도 2]에 나타난 바와 같이 대두박 첨가 배지에서 가장 우수한 균주성장을 나타냈는데 최적 배지인 영양 액체배지와 비교해볼 때 정지기에 도달하는 시간은 다소 지연되었으나 10시간 배양시 영양 액체배지의 최고 성장도와 유사한 결과를 나타내어 대두박이 본 발명이 균주 배양시 적합한 배지성분임을 알 수 있다.Bacillus subtilis SY8-24 strains selected according to Example 1 were first subcultured in nutrient agar medium to increase the activity of the strain and then inoculated in 50 ml of nutrient agar medium in a stirred culture at 37 ℃, 150rpm Incubated. After 8 hours of cultivation, nutrient liquid medium (peptone 0.5%, juicy 0.3%) was used as the optimum comparison medium. Soybean meal, cottonseed meal, rapeseed meal, rice bran, and bran were pulverized with 1mm sieve and then filtered by 1.0% (w / v) in 100ml of distilled water. ) Were inoculated with 1.0% (v / v) of the strain into the sterilized culture solution, followed by incubation at 37 ° C. and 150 mm. As a result of measuring the number of bacteria at 2 hour intervals after cultivation, as shown in [Fig. 2], the best strain growth was shown in the soybean meal-added medium. Soybean meal is a suitable medium component for culturing strains by showing results similar to the highest growth of nutrient liquid medium at time culture.
실시예 3 : 대두박 첨가량 결정 실험Example 3: Soybean meal addition amount determination experiment
실시예 2와 동일 조건과 방법으로 접종 및 배양하되, 대두박의 첨가량을 각 0.5%(w/v), 1.0%(w/v) 및 2.0%(w/v)씩 첨가하여 균주성장을 파악한 바 [도 3]에서와 같이 0.5%(w/v) 첨가시 1.0%(w/v) 첨가시에 비해 균주 성장이 낮았으며, 2.0%(w/v) 첨가시 1.0%(w/v) 첨가시와 비교해볼 때 더 나은 균주 성장을 나타내지 않아 경제적 측면을 고려해 보았을 때 대두박 1.0%(w/v) 첨가배지가 적합하다는 결론을 얻었다.Inoculated and cultured under the same conditions and methods as in Example 2, but the strain growth was determined by adding 0.5% (w / v), 1.0% (w / v) and 2.0% (w / v) of soybean meal, respectively. As shown in FIG. 3, when 0.5% (w / v) was added, strain growth was lower than when 1.0% (w / v) was added, and when 1.0% (w / v) was added, 1.0% (w / v) was added. It was concluded that the soybean meal added 1.0% (w / v) medium was suitable in consideration of economic aspects because it did not show better strain growth compared to the city.
실시예 4 : 균체회수를 용이하게 하기 위한 대두박 추출용액 비교 실험Example 4 Comparative Test of Soybean Meal Extract Solution for Facilitating Cell Recovery
별도의 멸균장치가 필요없고 균체회수가 용이하게 하기 위해 1.0%(w/v)에 해당되는 대두박을 1시간 동안 100℃로 끓인 후 4겹의 거즈를 이용하여 거른 용액을배양액으로하여 1.0%(w/v) 대두박을 함유한 배양액에 실시예 2와 동일한 방법으로 균주성장을 비교한 결과 [도 3]에 나타난 바와 같이 균주성장이 다소 낮았으나 거의 유사한 결과를 얻었다.There is no need for a separate sterilizer and boil the soybean meal corresponding to 1.0% (w / v) at 100 ° C for 1 hour in order to facilitate cell recovery. w / v) Strain growth was compared to the culture solution containing soybean meal in the same manner as in Example 2, but as shown in FIG.
실시예 5 : 당밀 첨가에 따른 영향 실험Example 5 Influence Experiment by Molasses Addition
1.0%(w/v) 대두박 함유배지를 기본배지로하여 상대적으로 탄소원인 당밀의 첨가효과를 파악한 결과 도식 4에 나타난 바와 같이 2.0%(w/v) 첨가수준에서 나은 균주성장을 나타낸다는 결과를 얻었는데 이는 대두박에 비해 상대적으로 탄소원인 당밀공급에 따른 탄소대 질소비의 상승으로 균주성장에 적합한 배지조성이 이루어졌기 때문이다. 대두박과 당밀의 탄소대 질소비를 구하기 위해 탄소함량은 Poincelot과 Day(1973, Compost Sci. 14(3):23-25)가 제시한 공식인 [% 탄소(건물중량)=(100-회분,%)/1.8]에 의거하여 구하였으며 질소함량은 AOAC(1990) 방법에 의하여 분석한 바 각각 7.6과 10.5의 결과를 얻었다.As a basic medium of 1.0% (w / v) soybean meal containing medium, the effect of molasses, which is a relatively carbon source, was identified. As shown in Scheme 4, it showed a good strain growth at 2.0% (w / v) addition level. This is because a medium composition suitable for strain growth was achieved due to an increase in the carbon-to-nitrogen ratio due to the supply of molasses, a carbon source, relative to soybean meal. To determine the carbon-to-nitrogen ratio of soybean meal and molasses, the carbon content was determined by the formula given by Poincelot and Day (1973, Compost Sci. 14 (3): 23-25): [% carbon (building weight) = (100- ash, %) / 1.8], and the nitrogen content was analyzed by AOAC (1990), and the results were 7.6 and 10.5, respectively.
상기의 실시예 결과들에서 보는 바와 같이, 본 발명에 따른 단백질 분해효소, 섬유소 분해효소를 동시에 분비하며 성장이 우수한 신균주 바실러스 서브틸리스 SY8-24를 대두박 및 당밀이 각각 1%(w/v)와 2%(w/v)로 함유된 배지에서 배양하였을 때 가장 우수한 균주성장을 나타낸바 값싼 원료를 이용하여 저렴하게 농가들에게 균주를 공급할 수 있게 되었다.As shown in the results of the above examples, the new strain Bacillus subtilis SY8-24, which simultaneously secretes protease and fibrinase according to the present invention and has excellent growth, has 1% soybean meal and molasses (w / v). When grown in a medium containing 2% (w / v) and showed the best strain growth was able to supply strains to farmers cheaply using cheap raw materials.
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