KR101605516B1 - Method for Increasing Viability, Storage Stability, Acid Tolerance or Oxgall Tolerance of Lactic Acid Bacteria - Google Patents
Method for Increasing Viability, Storage Stability, Acid Tolerance or Oxgall Tolerance of Lactic Acid Bacteria Download PDFInfo
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- KR101605516B1 KR101605516B1 KR1020150082813A KR20150082813A KR101605516B1 KR 101605516 B1 KR101605516 B1 KR 101605516B1 KR 1020150082813 A KR1020150082813 A KR 1020150082813A KR 20150082813 A KR20150082813 A KR 20150082813A KR 101605516 B1 KR101605516 B1 KR 101605516B1
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- lactic acid
- acid bacteria
- proline
- resistance
- survival rate
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Abstract
Description
본 발명은 유산균의 생존율, 저장안정성, 내산성 또는 내담즙성을 증가시키는 방법에 관한 것이다.The present invention relates to a method for increasing the survival rate, storage stability, acid resistance or biliary cholestasis of a lactic acid bacterium.
유산균(lactic acid bacteria)은 당류를 에너지원으로 사용하여 유산을 생성하는 세균으로 사람이나 포유동물의 소화관, 구강 및 질 등에서 발견되며, 각종 발효식품 등 자연계에 널리 분포되어 있다. 유산균은 인류가 가장 오랫동안 광범위하게 활용하고 있는 미생물 중 하나로서, 사람이나 동물의 장에 해로운 물질을 생성하지 않으며 장내에서 부패를 방지하는 순기능을 가진 미생물이다. Lactic acid bacteria are bacteria that produce saccharides by using saccharides as an energy source. They are found in digestive tracts, oral cavity and vagina of humans and mammals. They are widely distributed in nature such as various fermented foods. Lactic acid bacteria is one of the microorganisms that humans have used extensively for a long time and is a microorganism that does not produce harmful substances in human or animal fields and has a pure function to prevent corruption in the intestines.
현재 유산균은 12개 속(Lactobacillus, Carnobacterium , Atopobium , Lactococcus , Pediococcus , Tetragenococcus, Leuconostoc , Weisella , Oenococcus , Enterococcus , Streptococcus, Vagococcus)으로 분류되며 일반적으로 우리가 사용하고 있는 유산균은 간균인 락토바실러스 속(Lactobacillus sp.), 구균인 락토코커스 속(Lactococcus sp.), 스트렙토코커스 속(Streptococcus sp.), 류코노스톡 속(Leuconostoc sp.), 그리고 페디오코커스 속(Pedicoccus sp.) 균 등이다(BioWave, 2009, Vol. 11, No. 7, pp. 1-20).Currently, lactic acid bacteria are classified into 12 genera ( Lactobacillus, Carnobacterium , Atopobium , Lactococcus , Pediococcus , Tetragenococcus, Leuconostoc , Weisella , Oenococcus , Enterococcus , Streptococcus and Vagococcus ). Lactobacillus commonly used by us is bacterium Lactobacillus sp .), a strain of Lactococcus ( Lactococcus sp .), Streptococcus sp ., Leuconostoc sp . sp .), and Pedioccus spp. sp .) (BioWave, 2009, Vol.11, No. 7, pp. 1-20).
한편, 프로바이오틱스(Probiotics)란 숙주에게 건강효과를 나타내는 살아있는 미생물 또는 살아있는 미생물이 함유된 식품을 말하며(FAO/WHO 2001), 인체의 장내 미생물을 정상적인 수준으로 유지하고 조절하는 중요한 기능을 가지고 있다. 대표적인 프로바이오틱스로는 유산균과 비피더스균(Bifidobacterium)이 있으며 그 외 효모(Saccharomyces cerevisiae와 Saccharomyces boulardii)와 사상균(Aspergillus oryzae) 등이 있다(BioWave, 2009, Vol. 11 No. 7, pp. 1-20).Probiotics, on the other hand, are foods that contain live microorganisms or live microorganisms that have a health effect on the host (FAO / WHO 2001), and have important functions to maintain and regulate human intestinal microorganisms at normal levels. Typical probiotics include lactic acid bacteria and Bifidobacterium , and yeast ( Saccharomyces cerevisiae and Saccharomyces boulardii ) and Aspergillus oryzae (BioWave, 2009, Vol. 11, No. 7, pp. 1-20).
프로바이오틱스의 대표적인 효능으로는 항균활성, 항생제 관련 설사 개선, 유당불내증 경감, 항암 효과, 혈중 콜레스테롤 저하, 위 내 헬리코박터 파이로리(Helicobacter pylori) 균 억제, 과민성 대장염, 크론병, 궤양성 대장염 경감, 면역 기능 조절 등이 알려져 있다(International Dairy Journal, 2007, Vol. 17, pp. 12621277). 프로바이오틱스는 인체 의약품인 정장제나 유산균 제제, 사료첨가제로서의 생균제 및 건강식품의 일종인 유산균 식품으로 구분할 수 있다. 유산균 식품은 유산간균, 유산구균, 비피더스균 등의 생균을 배양하여 식품에 혼합한 것을 안정적이고 섭취가 용이하도록 분말, 과립, 정제, 캡슐 등으로 만든 것으로 유산균 발효식품, 유산균 발효유, 유산균 음료 이외의 것을 말한다.Antibiotic activity, antibiotic-related diarrhea reduction, anticancer effect, anti-cancer effect, blood cholesterol lowering, Helicobacter pylori fungus inhibition, irritable colitis, Crohn's disease, ulcerative colitis relief, immune function control (International Dairy Journal, 2007, Vol. 17, pp. 12621277). Probiotics can be divided into the formulations of human drugs, lactose, lactic acid bacteria, probiotics as feed additives, and lactic acid bacteria, a kind of health food. Lactic acid bacteria food is made by cultivating live bacteria such as lactic acid bacterium, lactic acid bacterium and bifidus bacteria and mixing them with foodstuffs in powder, granules, tablets, capsules etc. for stable and easy ingestion. Lactic acid bacteria fermented food, lactic acid fermented milk, It says.
상기와 같은 유산균 식품을 제조하는 공정은 크게 유산균 배양, 균체 회수, 동결건조, 분쇄, 제품화 등으로 구분되는데 이 과정에서 유산균은 다양한 물리화학적인 스트레스에 노출된다. 즉, 균체회수 시에는 농축에 따른 삼투압 영향을 받으며, 동결건조 과정에서는 급격한 온도 변화에 따른 세포질 내 얼음결정이 형성되거나 세포 외부에 생성되는 얼음 결정 및 탈수(dehydration) 현상으로 인하여 온도와 삼투압 영향을 동시에 받게 된다. 또한, 분쇄 및 제품화 과정에서 고온, 고압에 노출되거나 공기 중의 수분에 의하여 수화(hydration)될 경우 유산균의 안정성이 떨어지게 되며(International Dairy Journal, 2004, Vol. 14, pp. 835847; Journal of Applied Microbiology, 2005, Vol. 98, pp. 14101417; Microbial Ecology in Health & Disease, 2012, pp. 2-5), 단기간 보관되는 유산균 발효식품, 유산균 발효유, 유산균 음료와 같은 액상 제품뿐만 아니라 장기 보관을 목적으로 분말 형태로 제조되는 제품에 있어서도 산소에 노출될 경우 세포막을 구성하고 있는 지방산이 산화되어 생존율이 감소하게 되는 문제점이 지적되어 왔다(Comprehensive Reviews in Food Science and Food Safety, 2004, Vol. 3, pp. 117-124; Curr Issues Intest Microbiol, 2004, Vol. 5, pp. 1-8; Letters in Applied Microbiology, 1996, Vol. 22, No. 1, pp. 3438).The process for producing the above-mentioned lactic acid bacteria food is largely classified into culturing of lactic acid bacteria, recovery of cells, freeze-drying, crushing, and commercialization. In this process, lactic acid bacteria are exposed to various physicochemical stresses. In other words, the cells are affected by the osmotic pressure due to concentration during the recovery of the cells. During the lyophilization process, the temperature and the osmotic pressure are influenced by the formation of ice crystals in the cytoplasm or the dehydration generated outside the cells due to the rapid temperature change At the same time. In addition, the stability of the lactic acid bacterium is lowered when exposed to high temperature and high pressure or by moisture in the air during pulverization and commercialization (International Dairy Journal, 2004, Vol. 14, pp. 835847; Journal of Applied Microbiology, 2005, Vol. 98, pp. 14101417), liquid foods such as fermented foods of lactic acid bacteria, fermented milk of lactic acid bacteria and drinks of lactic acid bacteria stored for a short period of time, It has been pointed out that even in the case of a product made in the form of a fatty acid, the fatty acid constituting the cell membrane is oxidized and the survival rate is reduced when exposed to oxygen (Comprehensive Reviews in Food Science and Food Safety, 2004, Vol. Curr Issues Intest Microbiol, 2004, Vol. 5, pp. 1-8; Letters in Applied Microbiology, 1996, Vol. 22, No. 1, pp. 3438).
한편, 다른 산업용 미생물과 달리 프로바이오틱스 제품은 생균 자체를 이용하기 때문에 섭취 후 장에 도달하기 전에 인체 내에서 다양한 스트레스에 노출된다. 위장에서는 pH 3 이하로 떨어지는 산성 환경에 노출되고 소장에서는 분비되는 소화효소와 담즙산 등의 영향을 받아 생존율이 크게 감소할 수 있다. 또한 장에 도달해서도 기존 장내 정착한 미생물과 경쟁함과 동시에 각종 유해 성분과 활성산소 등에 의한 성장 저해를 받게 된다(Immunology and Cell Biology, 2000, Vol. 78, pp. 8088; Am J Clin Nutr, 2001, Vol. 73, pp. 393S398S(suppl); Probiotic Bacteria and Enteric Infections, 2011, Chap. 2, pp. 41-63). On the other hand, unlike other industrial microorganisms, probiotics products are exposed to various stresses in the human body before reaching the post-intake stage because they use live bacteria. In the gastrointestinal tract, the exposure to acidic environment falls below pH 3, and in the small intestine, the survival rate may be greatly reduced due to the effects of secreted digestive enzymes and bile acids. In addition, even when it reaches the intestine, it competes with the microorganisms established in the intestinal tract and is inhibited by various harmful components and active oxygen (Immunology and Cell Biology, 2000, Vol. 78, pp. 8088; Am J Clin Nutr, 2001, Vol. 73, pp. 393S398S (suppl); Probiotic Bacteria and Enteric Infections, 2011, Chap.2, pp. 41-63).
상기와 같은 문제점을 해결하고자 유산균을 코팅하는 다양한 방법이 개발되었는데 초기에는 캡슐제를 이용한 장용 코팅제와 젤라틴, 다당류, 검류 등을 이용한 마이크로캡슐화 등이 있었지만 고가의 코팅제를 사용하거나 공정이 추가되는 문제점이 지적되어 왔다. 이를 해결하기 위해 고농도의 유산균이 살아있는 이중 구조의 젤리를 제조하는 방법 또는 단백질과 다당류로 이중 코팅하는 방법을 도입하거나(대한민국 특허출원 제10-2003-0020375, 대한민국 특허출원 제10-2001-0010397) 단백질과 다당류 코팅에 나노 입자를 추가한 삼중 코팅방법이 도입되기도 하였다(대한민국 특허출원 제10-2008-0008267). 그러나 이와 같이 개선된 유산균 코팅 기술 또한 유산균의 표면을 완전히 코팅하지 못하여 여전히 유산균의 내열성, 내산성 및 내담즙성이 충분히 우수하지 못하다는 문제점이 있다. 이러한 문제점을 극복하기 위하여 삼중 코팅에 식용유지를 추가하여 다중코팅 하거나(대한민국 특허출원 제10-2011-0093074), 수용성 폴리머, 히알루론산, 다공성 입자를 가지는 코팅제 및 단백질을 추가하여 4중 코팅하는 방법(대한민국 특허출원 제10-2011-0134486) 등이 경쟁적으로 개발되어 왔다. 하지만 상기의 유산균 코팅 기술은 통상의 방법으로 배양된 유산균체를 회수한 다음 코팅제 조성물을 혼합하여 교반하는 다단계 공정을 수행하기 때문에 생균제로서 무균 조작이 어렵다는 단점이 있고, 특히 산업적인 대량생산에 있어 경제성이 떨어지는 문제점이 있다.Various methods for coating lactic acid bacteria have been developed in order to solve the above problems. Initially, encapsulation using an encapsulating agent and microencapsulation using gelatin, polysaccharide, gum, etc. have been performed. However, Has been pointed out. To solve this problem, a method of producing a double structure jelly in which a high concentration of a lactic acid bacterium is active or a method of double coating a protein with a polysaccharide (Korean Patent Application No. 10-2003-0020375, Korean Patent Application No. 10-2001-0010397) A triple coating method in which nanoparticles are added to a protein and polysaccharide coating has been introduced (Korean Patent Application No. 10-2008-0008267). However, the improved lactic acid bacteria coating technique also fails to completely coat the surface of the lactic acid bacteria, so that the heat resistance, acid resistance and bile resistance of the lactic acid bacteria are not sufficiently excellent. In order to overcome this problem, there has been proposed a method in which a triple coating is further coated with an additional edible oil (Korean Patent Application No. 10-2011-0093074), a method of coating a water-soluble polymer, hyaluronic acid, a coating agent having porous particles, Korean Patent Application No. 10-2011-0134486) have been competitively developed. However, the above-mentioned lactic acid bacteria coating technology has a disadvantage in that aseptic manipulation is difficult as a probiotic agent because the lactic acid bacteria cultured by a conventional method is recovered and then the multi-step process of mixing and stirring the coating composition is carried out. There is a problem of falling.
한편, 프로바이오틱스 미생물은 배양 시점부터 삼투압, pH, 산소 등 다양한 스트레스에 노출될 수 있고 이에 따라 외부환경 변화에 대응하기 위하여 다양한 생리적 반응을 나타낸다는 것이 알려져 있으며, 배양 조건에 따라 이 후 공정인 동결건조 과정에서의 생존율에 차이가 나타난다는 것이 보고되어 있다(Comparative Biochemistry and Physiology Part A, 2001, Vol. 130, pp. 437-460; Journal of Applied Microbiology, 2005, Vol. 99, pp. 13301339; J. Dairy Sci., 2005, Vol. 88, pp. 21-29; Biochemical Engineering Journal, 2010, Vol. 52, pp. 65-70). On the other hand, it is known that probiotic microorganisms can be exposed to various stresses such as osmotic pressure, pH, and oxygen from the time of cultivation, and thus exhibit various physiological responses to cope with changes in the external environment. According to culturing conditions, (Comparative Biochemistry and Physiology Part A, 2001, Vol. 130, pp. 437-460; Journal of Applied Microbiology, 2005, Vol. 99, pp. 13301339; Dairy Sci., 2005, Vol. 88, pp. 21-29, Biochemical Engineering Journal, 2010, Vol.52, pp. 65-70).
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 유산균의 배양 및 보관 과정에서 안정성을 높이기 위해 연구 노력한 결과, 프롤린을 포함한 아미노산을 유산균 배양 중 또는 배양 후 첨가하면 동결건조 후 생존율 및 저장안정성을 현저히 높일 수 있고, 스트레스에 대한 저항성 지표인 내산성, 내담즙성이 크게 증가한다는 것을 규명함으로써 본 발명을 완성하였다. The inventors of the present invention have made efforts to increase the stability of lactic acid bacteria in culture and storage. As a result, it has been found that when amino acids including proline are added during or after lactic acid bacteria culture, the survival rate and storage stability after lyophilization can be remarkably increased, Acidity and brittleness were greatly increased, thereby completing the present invention.
따라서, 본 발명의 목적은 유산균의 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성을 증가시키는 방법을 제공하는데 있다.Accordingly, an object of the present invention is to provide a method for increasing survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of lactic acid bacteria.
본 발명의 다른 목적은 상기 방법에 의해 제조된 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성이 증가된 유산균을 제공하는데 있다. Another object of the present invention is to provide a lactic acid bacterium having increased survival rate, storage stability, acid resistance and biliary cholesteriness after lyophilization produced by the above method.
본 발명의 다른 목적 및 이점은 하기 발명의 상세한 설명 및 청구범위에 의해 보다 명확하게 된다. Other objects and advantages of the present invention will become more apparent from the following description and claims of the present invention.
본 발명의 일 양태에 따르면, 본 발명은 유산균의 배양 중 또는 유산균을 배양한 후 프롤린, 아스파르트산, 세린, 트레오닌, 글루탐산, 라이신 및 발린으로 구성된 군으로부터 선택되는 1종 이상의 아미노산을 첨가하여 유산균의 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성을 증가시키는 방법을 제공한다. According to one aspect of the present invention, there is provided a method for producing a lactic acid bacterium, comprising adding at least one amino acid selected from the group consisting of proline, aspartic acid, serine, threonine, glutamic acid, lysine and valine, A method for increasing the survival rate, storage stability, acid resistance, and bile resistance after lyophilization is provided.
본 발명자들은 유산균의 배양 및 보관 과정에서 안정성을 높이기 위해 연구 노력한 결과, 프롤린을 포함한 아미노산을 유산균 배양 중 또는 배양 후 첨가하면 동결건조 후 생존율 및 저장안정성을 현저히 높일 수 있고, 스트레스에 대한 저항성 지표인 내산성, 내담즙성이 크게 증가한다는 것을 규명하였다. The inventors of the present invention have made efforts to increase the stability of lactic acid bacteria in culture and storage. As a result, it has been found that when amino acids including proline are added during or after lactic acid bacteria culture, the survival rate and storage stability after lyophilization can be remarkably increased, Acidity, and biliary properties were significantly increased.
본 명세서의 용어 “생존율”은 동결건조 후 생존균 수를 동결건조 전 생존균 수로 나눈 값의 백분율을 의미한다.The term " survival rate " as used herein means the percentage of viable cell count after lyophilization divided by viable cell count before lyophilization.
본 발명의 방법을 상세하게 설명하면 다음과 같다:The method of the present invention will be described in detail as follows:
본 발명에 따르면, 우선 유산균을 배양한다. According to the present invention, lactic acid bacteria are first cultured.
본 발명에서 사용하는 유산균은 특별히 한정되지 않으며, 예를 들어, 락토바실러스 속(Lactobacillus sp.), 스트렙토코커스 속(Streptococcus sp.), 락토코커스 속 (Lactococcus sp.), 엔테로코커스 속(Enterococcus sp.), 페디오코커스 속(Pediococcus sp.), 류코노스톡 속(Leuconostoc sp.), 비셀라 속(Weissella sp.) 또는 비피도박테리움 속(Bifidobacterium sp.) 유산균이다. The lactic acid bacteria used in the present invention are not particularly limited and include, for example, Lactobacillus sp., Streptococcus sp., Lactococcus sp., Enterococcus sp. Pediococcus sp., Leuconostoc sp., Weissella sp., Or Bifidobacterium sp. Lactic acid bacteria.
본 발명의 다른 구현예에 따르면, 본 발명에서 사용하는 유산균은 락토바실러스 플랜타럼(L. plantarum), 락토바실러스 애시도필러스(L. acidophilus), 비피도박테리움 비피덤(B. bifidum), 비피도박테리움 롱검(B. longum) 또는 류코노스톡 메센테로이드(L. mesenteroides)이다. According to another embodiment of the present invention, the lactic acid bacteria to be used in the present invention may be selected from the group consisting of L. plantarum , L. acidophilus , Bifidum bifidum , Bifidobacterium longum ( B. longum ) or leucono stokescensenteroid ( L. mesenteroides ).
본 발명의 특정 구현예에 따르면, 본 발명의 유산균은 락토바실러스 플랜타럼 KCTC3108, 락토바실러스 애시도필러스 KCTC3142, 비피도박테리움 비피덤 KCTC3202, 비피도박테리움 롱검 KCTC3128 또는 류코노스톡 메센테로이드 KCTC3100이다. According to a particular embodiment of the present invention, the lactic acid bacteria of the present invention are selected from the group consisting of Lactobacillus plantarum KCTC3108, Lactobacillus acidophilus KCTC3142, Bifidobacterium bifidum KCTC3202, Bifidobacterium longum KCTC3128 or Leuconostomercenteroid KCTC3100.
본 발명에서의 유산균 배양은 종래에 공지된 통상적인 유산균 배양배지 및 배양 조건에서 실시된다.The cultivation of lactic acid bacteria in the present invention is carried out under conventionally known lactic acid bacteria culture medium and culture conditions known in the art.
본 발명에 따르면, 유산균의 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성을 증가시키기 위해 유산균의 배양 중 또는 유산균을 배양한 후 아미노산을 첨가한다. According to the present invention, in order to increase the survival rate, storage stability, acid resistance and bile resistance after lyophilization of lactic acid bacteria, amino acid is added during cultivation of lactic acid bacteria or after cultivation of lactic acid bacteria.
본 발명의 일 구현예에 따르면, 아미노산은 유산균 배양 배지조제 시, 유산균 배양 0시간 내지 배양종료 시점 또는 유산균체 회수 후 동결건조 전에 첨가한다. According to one embodiment of the present invention, the amino acid is added at the time of preparing the culture medium for lactic acid bacteria, at 0 hours after the cultivation of the lactic acid bacteria or at the end of the culture, or before the lyophilization after the recovery of the lactic acid bacteria.
본 발명의 다른 구현예에 따르면, 유산균의 배양 중 유산균의 성장이 정지기에 도달한 시점에 아미노산(예컨대, 프롤린)을 첨가한다. According to another embodiment of the present invention, an amino acid (for example, proline) is added at the time when the growth of the lactic acid bacteria reaches the stationary phase during the culture of the lactic acid bacterium.
본 발명의 일 구현예에 따르면, 유산균의 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성을 증가시키기 위해 첨가되는 아미노산은 프롤린, 아스파르트산, 세린, 트레오닌, 글루탐산, 라이신 및 발린으로 구성된 군으로부터 선택되는 1종 이상의 아미노산이다. According to one embodiment of the present invention, the amino acid added to increase survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of lactic acid bacteria is selected from the group consisting of proline, aspartic acid, serine, threonine, glutamic acid, lysine and valine One or more amino acids selected.
본 발명의 다른 구현예에 따르면, 본 발명에서 이용되는 아미노산은 프롤린이다. According to another embodiment of the present invention, the amino acid used in the present invention is proline.
본 발명의 일 구현예에 따르면, 상기 프롤린의 첨가 농도는 1 g/L 내지 50 g/L, 2 g/L 내지 20 g/L 또는 2 g/L 내지 10 g/L이다. According to one embodiment of the present invention, the added concentration of proline is 1 g / L to 50 g / L, 2 g / L to 20 g / L or 2 g / L to 10 g / L.
본 발명의 일 구현예에 따르면, 아미노산 첨가에 의해 안정성이 증가한 유산균에 동결보호제 또는 코팅제를 첨가하여 피막 형성 유산균을 제조하는 단계를 추가적으로 포함한다. According to an embodiment of the present invention, there is further included a step of adding a cryoprotectant or a coating agent to the lactic acid bacteria having increased stability by addition of an amino acid to prepare a film-forming lactic acid bacterium.
본 발명에서 이용되는 동결보호제는 프롤린(proline), 트리할로오스(trehalose) 또는 글리세린(glycerin)이고, 코팅제는 키토산(chitosan), 말토덱스트린(malto-dextrin), 난소화성 덱스트린(indigestible dextrin), 잔탄검(xanthan gum, XG), 구아검(guar gum, GG), 카르복시메틸셀룰로오스(carboxymethyl cellulose, CMC), 하이드록시에틸셀룰로오스(hydroxyethylcellulose, HEC), 폴리비닐피롤리돈(polyvinylpyrroridone, PVP), 카보폴(carbopol), 소듐알기네이트(sodium alginate), 프로필렌글리콜 알기네이트(propylene glycol alginate), 알지네이트(alginate), 폴리에틸렌글리콜(polyethyleneglycol, PEG), 트리아세틴(triacetin), 프로필렌 글리콜(propylene glycol), 아세틸트리에틸 시트레이트(acetyl triethyl citrate) 또는 트리에틸 시트레이트(triethyl citrate)이다. The cryoprotectant used in the present invention is proline, trehalose or glycerin, and the coating agent is chitosan, malto-dextrin, indigestible dextrin, It is known that xanthan gum (XG), guar gum (GG), carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), polyvinylpyrroridone (PVP) But are not limited to, carbopol, sodium alginate, propylene glycol alginate, alginate, polyethyleneglycol (PEG), triacetin, propylene glycol, acetyl Acetyl triethyl citrate or triethyl citrate.
본 발명에 따르면, 본 발명의 유산균은 아미노산 첨가에 따른 배양 완료 후 동결건조한다. According to the present invention, the lactic acid bacterium of the present invention is freeze-dried after completion of cultivation in accordance with addition of amino acid.
하기 실시예에서 검증된 바와 같이, 본 발명의 방법에 의해 배양 후 동결건조된 유산균은 현저히 증가한 생존율, 저장안정성, 내산성 및 내담즙성을 나타냈다. As verified in the following examples, the lyophilized Lactobacillus after the culture according to the method of the present invention exhibited remarkably increased survival rate, storage stability, acid resistance and bile resistance.
본 발명에 따라 배양 중 또는 배양 후 아미노산을 첨가하여 균체 자체의 극한환경에 대한 저항성을 높이는 방법은 기존 다단계 코팅으로 균체에 물리적 장벽을 구축하는 방법에 비하여 제조 공정이 단순하고 경제적이어서 산업적 이용 가능성이 매우 높다. 한편, 종래 기술에 의해 생산된 다단계 코팅 유산균 제품을 섭취할 경우 장 내에서 유산균 코팅제가 해리되지 않아 장을 통과하여 배출될 가능성이 높고, 해리되더라도 급격히 변화하는 장 내 환경에서 생존율을 담보하기 어려운 문제점이 있다. 반면, 본 발명의 방법에 의해 제조된 유산균은 유산균 자체가 극한환경에 대하여 높은 저항성을 갖고 있기 때문에 장에 도달하여 재수화(rehydration)되는 과정에서 높은 생존율을 보인다.According to the present invention, the method of increasing the resistance of the cells themselves to extreme environments by adding amino acids during or after culturing is simpler and more economical than the method of constructing physical barriers with the conventional multi-stage coating, Very high. On the other hand, when the multistage coated lactobacillus product produced by the prior art is ingested, there is a high possibility that the lactic acid bacterial coating agent is not dissociated in the intestines and is discharged through the intestines. Even if dissociated, the survival rate is difficult to be secured in a rapidly changing intestinal environment . On the other hand, the lactic acid bacterium produced by the method of the present invention has a high survival rate in the process of rehydration when the lactic acid bacterium itself has a high resistance to the extreme environment.
본 발명은 유산균 배양 중 아미노산을 첨가함으로써 균체 내에 아미노산이 고농도로 축적되도록 하며, 균체를 회수한 다음 보호제 또는 코팅제와 혼합하고 동결건조하여 균 자체가 산성, 담즙 및 산소에 대하여 높은 내성을 갖는 개량된 유산균을 제조할 수 있다.The present invention relates to a process for the production of microorganisms capable of accumulating high concentrations of amino acids in microbial cells by adding amino acids during lactic acid bacterial cultivation, recovering the microbial cells, mixing them with a protective agent or a coating agent and lyophilizing the microbial cells so that the microbes are highly resistant to acid, Lactic acid bacteria can be produced.
본 발명의 다른 일 양태에 따르면, 본 발명은 상술한 방법에 의해 제조된 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성이 증가된 유산균을 제공한다. According to another aspect of the present invention, there is provided a lactic acid bacterium having increased survival rate, storage stability, acid resistance, and bile resistance after lyophilization prepared by the above-described method.
상기 방법에 의해 제조된 유산균은 과산화수소 내성 및 과산화수소 분해 활성을 갖는다. The lactic acid bacteria produced by the above method have hydrogen peroxide resistance and hydrogen peroxide decomposing activity.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(ⅰ) 본 발명은 유산균의 배양 중 또는 유산균을 배양한 후 프롤린을 첨가하여 유산균의 동결건조 후 생존율, 저장안정성, 내산성 및 내담즙성을 증가시키는 방법 및 상기 방법에 의해 제조된 유산균을 제공한다. (I) The present invention provides a method for increasing survival rate, storage stability, acid resistance, and bile resistance after lyophilization of lactic acid bacteria by adding proline during culturing or culturing of lactic acid bacteria and lactic acid bacteria produced by the method .
(ⅱ) 본 발명의 방법은 유산균체 내에 프롤린 등 보호제를 고농도로 함유하도록 함으로써 유산균 자체의 동결건조 안정성, 저장안정성 등 외부 환경 스트레스에 대한 안정성을 증가시킬 뿐만 아니라 유산균 섭취 시 장관환경 안정성의 지표인 내산성, 내담즙성을 현저히 향상시킨다. (Ii) The method of the present invention not only increases the stability against external environmental stresses such as freeze-drying stability and storage stability of the lactic acid bacteria itself by including a proline or the like such as proline in the lactic acid bacterium at a high concentration, Acid resistance, and bile resistance.
(ⅲ) 종래 기술이 단백질, 다당류, 다공성 폴리머, 식용유지 등으로 이중 코팅, 삼중 코팅, 사중 코팅과 같은 다단계 코팅을 하여 유산균체 외부에 물리적인 방어벽을 구축하는 복잡한 방법을 사용함으로써 경제성이 떨어지고 산업적인 적용에 한계가 있음에 반하여, 본 발명은 유산균체 자체의 외부 물리화학적인 스트레스에 대한 저항성을 크게 증가시켜 위장관 통과 시 생존력을 증가시키고 장내에서 유산균 고유의 기능성을 발휘할 수 있게 하며 기존 다단계 코팅공정에 따른 원가상승의 문제점을 극복할 수 있도록 하는 경제적인 방법이다. (Iii) The prior art uses a complicated method of building a physical barrier outside the lactic acid bacteria by applying a multi-step coating such as a double coating, a triple coating, or a quadruple coating with protein, polysaccharide, porous polymer, edible oil, The present invention significantly increases the resistance to external physicochemical stress of the lactic acid bacteria itself, thereby increasing the viability during passage through the gastrointestinal tract and allowing the lactic acid bacteria to exert their inherent functionality in the intestines. In addition, This is an economical way to overcome the problem of cost increase caused by
도 1은 락토바실러스 플랜타룸 KCTC3108(Lactobacillus plantarum KCTC3108)의 배양 중 삼투압과 세포내 프롤린의 농도 변화를 확인한 결과이다.
도 2는 고속액체크로마토그래피-증기화 광산란검출기(HPLC-ELSD)로 프롤린 표준품(a)과 배양 중 프롤린을 5 g/L 첨가한 경우(b), 프롤린을 첨가하지 않은 경우(c)의 세포내 프롤린을 분석한 크로마토그램이다.
도 3은 프롤린 첨가 농도에 따른 세포내 프롤린의 농도를 정량화한 결과이다.
도 4는 프롤린 첨가 시점에 따른 세포내 프롤린의 농도를 정량화한 결과이다.
도 5는 다단계 코팅을 이용하는 선행 기술 제조방법에 대한 모식도이다.
도 6은 본 발명의 제조방법에 대한 모식도이다.FIG. 1 shows the results of confirming osmotic pressure and intracellular proline concentration changes during the culture of Lactobacillus plantarum KCTC3108.
FIG. 2 is a graph showing the relationship between proline (a) and proline (b), proline (b) and proline (c) This is the chromatogram of my proline.
FIG. 3 is a result of quantifying intracellular proline concentration according to the concentration of proline.
FIG. 4 shows the results of quantifying intracellular proline concentration according to the time point of addition of proline.
5 is a schematic diagram of a prior art manufacturing method using a multistage coating.
6 is a schematic view of the manufacturing method of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example
실시예 1: 균체 회수 후 아미노산 첨가Example 1: Amino acid addition after cell recovery
생균제 제조에 사용할 유산균의 고농도 배양을 위하여 각각의 균종에 최적화된 배지와 배양조건을 사용하였으며, 이후 균체 회수 및 동결건조 공정부분은 통상적인 방법(원심분리 후 -60℃ 냉동고에서 급속 동결한 다음 0℃에서 45℃ 사이의 조작 조건에서 동결건조 수행)을 적용하였다. 균체 회수 후 첨가한 아미노산 종류에 따라 동결건조 및 보관 과정에서의 안정성에 미치는 영향을 조사하기 위하여 아래와 같은 실험을 진행하였다. 본 발명에서는 락토바실러스 플랜타룸 KCTC 3108(Lactobacillus plantarum KCTC3108) 배양액과 락토바실러스 애시도필러스 KCTC 3142(Lactobacillus acidophilus KCTC3142) 배양액을 농축하고 여기에 20가지 아미노산을 각각 5 g/L가 되도록 첨가하였다. 20가지 아미노산을 종류별로 농축액에 첨가하고 동결건조한 다음 동결건조 시 생존율, 가속 안정성(40℃, 습도 70%), 내산성 및 내담즙성을 확인하였다. In order to cultivate lactic acid bacteria at a high concentration for use in the production of probiotics, medium and culture conditions optimized for each species were used. After the centrifugation, the cells were rapidly frozen in a freezer at -60 ° C, ≪ / RTI > and < RTI ID = 0.0 > 45 C < / RTI > The following experiment was conducted to investigate the effect of amino acid added after cell recovery on stability during freeze drying and storage. In the present invention, the culture medium of Lactobacillus plantarum KCTC3108 and Lactobacillus acidophilus KCTC3142 were concentrated and 20 amino acids were added to the culture medium to have a concentration of 5 g / L, respectively. The 20 amino acids were added to the concentrate by lyophilization, and then the survival rate, accelerated stability (40 ° C, 70% humidity), acid resistance and biliary cholesterol were observed upon freeze drying.
장기간 유통시 생균제의 보관 안정성을 검토하기 위하여 온도 40℃, 습도 70%의 조건에서 4주 동안 가속 시험을 진행하여 가혹한 조건에서 유산균 생균제의 안정성을 평가하였으며, 내산성을 측정하기 위하여 생균제를 pH 2.5의 인공위액 조건에 노출시킨 후, 생균수를 분석하였다. 구체적으로, 인공위액 조건은 식품 붕해도 시험 상의 인공위액 조건(염화나트륨 2.0 g, 묽은 염산 24.0 ㎖/L, pH 1.2)을 사용하였으며, 10% 농도로 생균제 분말을 첨가한 후 위 수축 운동을 고려하여 분당 100회의 왕복운동을 실시하였고, 노출 시간은 위를 통과하는 시간을 고려하여 2시간으로 실시하였다. 인공위액 조건에 노출한 실험군은 추후 pH를 7.0으로 재조정한 후 통상적인 생균수 측정방법에 따라 분석하였으며 비교 실험군으로는 아미노산을 첨가하지 않은 생균제를 사용하였다. In order to evaluate the storage stability of the probiotics during long term circulation, the stability of the probiotics was evaluated under the conditions of 40 ℃ and 70% humidity for 4 weeks. After exposure to artificial gastric conditions, viable counts were analyzed. Specifically, artificial gastric juice conditions were artificial gastric juice conditions (2.0 g of sodium chloride, 24.0 ml / L of diluted hydrochloric acid, pH 1.2) in the food shattering test, and 10% concentration of probiotics powder was added, 100 reciprocating movements per minute were carried out and the exposure time was 2 hours considering the passage time. Experimental groups exposed to artificial gastric conditions were analyzed by the usual method of measuring viable counts after pH was adjusted to 7.0. Probiotics without amino acids were used as comparative test groups.
한편, 담즙산(bile acid)은 간(liver)에서 만들어져 담도를 통하여 소장(small intestine)으로 분비되고 소장 말단의 회장(ileum)에서 다시 95% 흡수되어 다시 간으로 들어가는 장관순환을 한다. 이 과정에서 담즙산은 소장에 정착한 유산균에 영향을 미치기 때문에 유산균이 담즙산에 노출되었을 경우 생존율 차이를 시험관 환경에서 비교하였다. 구체적으로는 담즙산이 첨가되지 않은 배지와 담즙산 0.5%를 여과하여 무균적으로 첨가한 배지를 사용하였으며, 제조된 배지에 각각의 유산균 시료를 1 g씩 접종하고, 2시간 동안 반응시킨 후 통상적인 방법에 따라 생균수를 측정하였다.On the other hand, bile acid is produced in the liver, secreted into the small intestine through the bile duct, absorbed again by the ileum at the end of the small intestine, and circulates intestinally into the liver again. Because bile acid affects the small intestine, the survival rate of lactic acid bacteria exposed to bile acid was compared in vitro. Specifically, a medium in which bile acid was not added and a medium in which 0.5% of bile acid was filtered and aseptically added was used. 1 g of each lactic acid bacterium sample was inoculated into the prepared medium, reacted for 2 hours, And the number of viable cells was measured.
표 1과 표 2에서와 같이 유산균 배양 후 농축액에 각각의 아미노산을 처리한 결과를 분석하면 다음과 같다.As shown in Table 1 and Table 2, the amino acid treatment of the concentrate after culturing the lactic acid bacteria was analyzed as follows.
락토바실러스 플랜타룸 KCTC 3108(Lactobacillus plantarum KCTC3108) 균의 동결건조 후 유산균의 생존율이 가장 높은 것은 프롤린과 아스파르트산(aspartic acid) 처리군이었고, 가속 실험 결과 프롤린, 세린(serine) 처리군에서 가장 우수한 생존율을 보였다. 그리고 내산성과 내담즙산성에 있어 우수한 결과를 보인 아미노산은 프롤린이었으며 그 외 트레오닌(threonine)과 글루탐산(glutamic acid)을 사용할 경우에도 각각 내산성, 내담즙성이 우수함을 확인하였다.The highest survival rate of Lactobacillus plantarum KCTC3108 after lyophilization of Lactobacillus plantarum KCTC3108 was in the group treated with proline and aspartic acid. The results of accelerated experiment showed that the survival rate of Lactobacillus plantarum KCTC3108 was highest in the proline, Respectively. In addition, the amino acid which showed excellent results in acid resistance and bile acid resistance was proline. Furthermore, it was confirmed that acid tolerance and bile resistance were also excellent when threonine and glutamic acid were used.
락토바실러스 애시도필러스 KCTC 3142(Lactobacillus acidophilus KCTC3142)균의 동결건조 후 생존율이 우수한 것은 라이신(lysine)과 프롤린 처리군이었며, 가속시험(40℃, 습도 70%) 결과 프롤린과 발린(valine) 처리군에서 우수한 결과를 보여주었다. 그리고 내산성과 내담즙성은 각각 프롤린과 글루타민(glutamine) 처리군에서 우수한 결과를 보여주었다. Lactobacillus acidophilus KCTC3142 ( Lactobacillus acidophilus KCTC3142) survived after lyophilization. Lecithin (lysine) and proline treatment were the best survival rates. The accelerated test (40 ℃, 70% Treated group showed excellent results. And acid tolerance and biliary properties showed excellent results in proline and glutamine treatment groups, respectively.
(40℃, 습도 70%)Accelerated test
(40 DEG C, 70% humidity)
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
(40℃, 습도 70%)Accelerated test
(40 DEG C, 70% humidity)
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
실시예 2: 균체 회수 후 농도별 첨가Example 2: Addition by concentration after cell recovery
유산균 배양 후 농축액에 20가지 아미노산을 각각 첨가하였을 때 동결건조 생존율, 가속안정성, 내산성 및 내담즙성에 있어 전체적으로 가장 효과적이었던 아미노산이 프롤린이었기 때문에 이의 최적 농도를 확인하고자 락토바실러스 플랜타룸 KCTC 3108 배양액을 농축한 후 농축액 대비 프롤린을 0-50 g/L 농도 범위로 첨가하여 동결건조를 수행하였다. 이후 4주 동안 가속 시험(40℃, 습도 70%) 및 내산성과 내담즙성 시험을 수행한 결과 프롤린 첨가 농도를 5 g/L에서 10 g/L까지 늘려 첨가한 경우 동결건조 생존율, 가속시험 후 생존율이 모두 향상되는 것을 확인할 수 있었으며 내산성도 개선되는 경향을 보였다(표 3).Since 20 amino acids were added to the concentrate after the cultivation of the lactic acid bacteria, the proline was the most effective amino acid in the freeze-drying survival rate, the acceleration stability, the acid resistance and the bile resistance. Therefore, the lactobacillus plantarum KCTC 3108 culture was concentrated After lyophilization, proline was added to the concentrate in the concentration range of 0-50 g / L. After 4 weeks of accelerating test (40 ℃, 70% humidity) and acid tolerance and biliary tests, the concentration of proline was increased from 5 g / L to 10 g / L. Survival rates were all improved and acid resistance was also improved (Table 3).
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
실시예 3: 배양액 농축 후 균주별 프롤린 첨가 영향Example 3: Effect of proline addition on strains after concentration of culture medium
실시예 2에서 락토바실러스 플랜타룸 KCTC 3108 배양 후 농축액에 프롤린을 첨가한 영향이 다른 유산균에도 적용되는지 확인하고자 락토바실러스 플랜타룸 KCTC 3108을 포함한 5종류의 유산균주를 각각 배양하고 이를 회수한 다음 프롤린을 10 g/L 농도가 되도록 첨가하고 실시예 1의 방법에 따라 동결건조를 수행하였다. 실험 결과, 실험에 이용한 모든 균주에서 동결건조 후 생존율, 가속안정성 및 내산성이 개선되었다. 특히 락토바실러스 플랜타룸 KCTC 3108 균주의 경우 프롤린을 10 g/L 첨가한 후 동결건조를 수행할 경우 동결건조 생존율과 가속시험 후 생존율이 가장 높았으며, 내산성도 크게 증가하였다.In order to confirm that the effect of adding proline to the concentrate after the cultivation of Lactobacillus plantarum KCTC 3108 in Example 2 was applied to other lactic acid bacteria, five kinds of lactic acid bacteria including Lactobacillus plantarum KCTC 3108 were cultured and recovered, 10 g / L, and lyophilization was carried out according to the method of Example 1. As a result, the survival rate, accelerated stability and acid resistance after freeze - drying were improved in all strains used in the experiment. In the case of Lactobacillus plantarum KCTC 3108 strain, the freeze - drying survival rate and survival rate after accelerated test were the highest when 10 g / L of proline was added and the acid resistance was greatly increased.
첨가 여부Proline
Whether to add
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
첨가10 g / L
adding
실시예 4: 유산균 세포 삼투압에 따른 프롤린 농도 Example 4: Proline concentration according to osmotic pressure of lactic acid bacteria cells
본 발명에서는 락토바실러스 플랜타룸 KCTC 3108(Lactobacillus plantarum KCTC3108), 락토바실러스 애시도필러스 KCTC 3142(Lactobacillus acidophilus KCTC3142), 비피도박테리움 비피덤 KCTC3202(Bifidobacterium bifidum KCTC3202), 비피도박테리움 롱검 KCTC3128(Bifidobacterium longum KCTC3128), 류코노스톡 메센테로이데스 KCTC3100(Leuconostoc mesenteroides KCTC3100) 배양 중 삼투압의 변화와 세포 내 프롤린의 농도변화를 확인하였다. 락토바실러스 플랜타룸 KCTC3108(Lactobacillus plantarum KCTC3108)의 배양 중 삼투압과 세포내 프롤린의 농도 변화는 도 1에 나타내었다.In the present invention, Lactobacillus plan tarum KCTC 3108 (Lactobacillus plantarum KCTC3108), Lactobacillus ash FIG filler's KCTC 3142 (Lactobacillus acidophilus KCTC3142), Bifidobacterium bipyridinium bonus KCTC3202 (Bifidobacterium bifidum KCTC3202), Bifidobacterium ronggeom KCTC3128 (Bifidobacterium longum KCTC3128), Leuconostoc mesenteroides KCTC3100 ( Leuconostoc mesenteroides KCTC3100) and osmotic pressure and intracellular proline concentration. The changes in osmotic pressure and intracellular proline concentration during the culture of Lactobacillus plantarum KCTC3108 are shown in Fig.
유산균 배양을 진행하면서 유산균이 생성하는 각종 유기산 등에 의하여 삼투압이 증가하는 것을 확인할 수 있었으며, 배양 시간이 경과할수록 세포내 프롤린의 농도 또한 증가하는 것을 확인하였다.It was confirmed that the osmotic pressure was increased by the various organic acids produced by the lactic acid bacteria and the concentration of intracellular proline was increased with the lapse of incubation time.
실시예 5: 배양 중 프롤린 첨가 후 세포내 아미노산 농도 분석Example 5: Analysis of intracellular amino acid concentration after addition of proline during culture
유산균 배양 중 프롤린을 5 g/L 첨가한 다음, 세포내 아미노산 중 특히 프롤린의 농도 변화를 확인하기 위하여 실험을 실시하였다. 배양액을 원심분리한 후 MES 버퍼를 이용하여 세척하고, 세척한 세포는 초음파발생장치(Vibra-Cell-Sonics & Materials, Inc.)를 이용하여 파쇄하였다. 파쇄한 세포 내 아미노산 농도는 고속액체크로마토그래피-증기화 광산란검출기(HPLC-ELSD) 시스템을 이용하여 분석하였다. After adding 5 g / L of proline in the culture of lactic acid bacteria, experiments were conducted to confirm the change of the concentration of proline in the intracellular amino acids. The culture was centrifuged, washed with MES buffer, and the washed cells were disrupted using an ultrasonic generator (Vibra-Cell-Sonics & Materials, Inc.). Amino acid concentrations in the disrupted cells were analyzed using a high performance liquid chromatography-vaporization light scattering detector (HPLC-ELSD) system.
분석을 위해 엑스브리지 아마이드 컬럼(Xbridge amide 3.5μm, 250×4.6㎜(Waters))을 사용하였고 이동상으로 A 용액(0.1% 수용성 포름산(aqueous formic acid))과 B 용액(아세토니트릴(acetonitrile))을 이용하여 기울기(gradient) 분석을 수행하였으며 이동률(flow rate)은 1.5 mL/min로 하였다. 기울기 조건(gradient condition)은 초기 : A 용액 20%, B 용액 80%, 0-5분 : A 용액 25%, B 용액 75%, 5-13분 : A 용액 30%, B 용액 70%, 13-18분 : A 용액 20%, B 용액 80%로 하였으며 컬럼 온도는 35℃로 하였다. 검출기(detector)는 380-ELSD(Agilent Technology)를 사용하였다. 분무기(nebulizer)의 질소 가스 주입 속도는 2 L/min이었고 드리프트 관(drift tube) 온도는 90℃로 하였다.A solution (0.1% aqueous formic acid) and a solution B (acetonitrile) were used as the mobile phase, using Xbridge amide column (Xbridge amide 3.5 μm, 250 × 4.6 mm And the flow rate was 1.5 mL / min. Gradient conditions were as follows: Initial 20% solution A, 80% solution B, 0-5 min: 25% solution A, 75% solution B, 5-13 min: 30% solution A, 70% -18 minutes: 20% of solution A and 80% of solution B were used, and the column temperature was 35 ° C. The detector was 380-ELSD (Agilent Technology). The nitrogen gas injection rate of the nebulizer was 2 L / min and the drift tube temperature was 90 ° C.
도 2는 고속액체크로마토그래피-증기화 광산란검출기(HPLC-ELSD)로 프롤린 표준품과 배양 중 프롤린을 첨가하지 않은 경우, 프롤린을 5 g/L 첨가한 경우의 세포내 프롤린을 분석한 크로마토그램이며, 도 3은 프롤린 첨가농도에 따른 세포내 프롤린의 농도를 정량화한 결과이다. FIG. 2 is a chromatogram showing intracellular proline when proline is added to proline at a concentration of 5 g / L when proline is not added to a proline standard product by high-performance liquid chromatography-vaporization light scattering detector (HPLC-ELSD) FIG. 3 is a result of quantifying intracellular proline concentration according to the concentration of proline.
실시예 6: 배양 중 프롤린 첨가 농도의 영향Example 6: Effect of concentration of proline added during culture
실시예 4에서 배양 후기에 세포내 프롤린 농도가 높아진다는 점, 그리고 실시예 1-3으로부터 배양액을 농축한 후 프롤린을 첨가할 경우 이후 공정에서 유산균의 안정성이 개선된 점에 착안하여 배양 중 프롤린을 첨가하여 세포내 프롤린이 고농도로 축적되도록 유도한 후 동결건조 및 가속 안정성을 확인하였다.Considering that intracellular proline concentration is increased in the late stage of culture in Example 4 and that the stability of the lactic acid bacterium is improved in the subsequent stage when proline is added after concentrating the culture from Example 1-3, To induce intracellular proline accumulation at a high concentration, followed by lyophilization and accelerated stability.
이를 위하여 락토바실러스 플랜타룸 KCTC 3108(Lactobacillus plantarum KCTC3108) 배양 중 세포의 성장단계상 대수증식기(log phase)에 해당하는 배양 8시간째에 프롤린을 0 g/L, 2.5 g/L, 5 g/L, 7.5 g/L, 10 g/L의 농도로 배양액에 첨가하였다. 그리고 18시간 배양 후 각각의 배양액을 원심분리에 의해 농축하고 동결건조를 수행한 후 4주 동안 가속 시험(40℃, 습도 70%) 및 내산성과 내담즙성 시험을 실시하였다(표 5).For this purpose, proline was added at 0 g / L, 2.5 g / L and 5 g / L (1 g / L) at 8 hours after the culture corresponding to the log phase of the growth phase of the Lactobacillus plantarum KCTC3108 , 7.5 g / L, and 10 g / L, respectively. After 18 hours of incubation, each culture was concentrated by centrifugation, lyophilized, and then subjected to an accelerated test (40 ° C, 70% humidity) for 4 weeks and an acid-fast bacilli test (Table 5).
그 결과, 프롤린 농도가 증가할수록 동결건조 생존율이 증가하여 5 g/L에서 가장 높은 것으로 나타났고, 이후 유사한 수준을 보였다. 4주 가속 안정성은 5 g/L 첨가 시 가장 우수하였고 그 이상의 농도에서는 오히려 가속 안정성이 낮아지는 결과를 확인하였다. 또한 배양 중 프롤린 첨가 농도가 증가할수록 내산성에 대한 효과도 증가하는 경향을 보였지만 내담즙성은 소폭 증가하는 것을 확인하였다.As a result, the freeze - dried survival rate increased as the concentration of proline increased, which was the highest at 5 g / L, and then showed a similar level. The 4 - week accelerated stability was the best when added at 5 g / L and the accelerated stability was lowered at the higher concentration. In addition, as the concentration of proline increased during the culture, the effect on acid resistance tended to increase, but the biliary properties were slightly increased.
프롤린 첨가(g/L)During cultivation
Proline addition (g / L)
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
실시예 7: 배양 중 프롤린 첨가 시점의 영향Example 7: Influence of proline addition at the time of culture
실시예 6에서 배양 중 대수기에 프롤린 5 g/L 첨가하였을 때 이후 동결건조 및 가속안정성 향상에 효과가 있었으므로 이를 기초로 하여 배양 중 프롤린 첨가시기에 따른 영향을 조사하였다. 구체적으로 락토바실러스 플랜타룸 KCTC 3108(Lactobacillus plantarum KCTC3108)을 이용하여 배양 중 프롤린을 첨가하는 시점에 따른 영향을 조사하였다. 배양초기에 프롤린을 5 g/L 첨가하거나 세포의 성장단계 중 대수증식기(log phase) 또는 정지기(stationary phase) 도달 시점에 프롤린을 각각 5 g/L씩 첨가하였고 배양 18시간 후 유산균 세포내 존재하는 프롤린의 농도를 확인하였다(도 4).The addition of 5 g / L of proline to the medium during the fermentation period in Example 6 was effective in improving freeze-drying and accelerated stability. Therefore, the effect of proline addition during the culture period was examined based on the results. Specifically, the effect of adding proline during culturing was investigated using Lactobacillus plantarum KCTC 3108 ( Lactobacillus plantarum KCTC 3108). 5 g / L of proline was added at the initial stage of culture or 5 g / L of proline was added at the time of reaching the log phase or stationary phase in the growth stage of the cells. After 18 hours of incubation, (Fig. 4). ≪ tb >< TABLE >
이와 함께 각각의 배양액을 원심분리 농축한 다음 통상적인 방법에 따라 동결건조를 수행한 후 4주 동안 가속 시험(40℃, 습도 70%) 및 내산성과 내담즙성에 대하여 실험을 실시하였다(표 6). 그리고 균의 성장이 정지기에 도달한 시점에 프롤린을 첨가하고 2시간 후에 배양액을 회수하여 동결건조를 수행한 결과, 동결건조 생존율, 가속시험 후 생존율이 현저히 높아지고 내산성 및 내담즙성도 개선되는 것을 확인할 수 있었다.In addition, each culture was centrifuged and then lyophilized according to a conventional method. After 4 weeks of accelerated testing (40 ° C, 70% humidity), acid resistance and biliary cholesterol were tested (Table 6) . When the growth of the bacteria reached the stopping point, proline was added, and after 2 hours, the culture solution was recovered and lyophilized, and the freeze-drying survival rate, the survival rate after the accelerated test, and the acidity and bile resistance were improved there was.
프롤린 첨가(5 g/L)During cultivation
Proline addition (5 g / L)
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
실시예 8: 균주별 배양 중 프롤린 첨가 영향Example 8 Effect of Proline Addition on Cultures by Strain
실시예 7에서 락토바실러스 플랜타룸 KCTC3108(Lactobacillus plantarum KCTC3108) 배양 중 프롤린을 첨가할 경우 동결건조 생존율, 가속시험 후 생존율 및 내산성이 높아지는 것을 확인하였기 때문에 다른 유산균에서도 동일한 효과를 보이는지 확인하기 위하여 실험을 수행하였다. 락토바실러스 플랜타룸 KCTC3108 (Lactobacillus plantarum KCTC3108)을 포함하여 5종류의 균주를 각각 배양하면서 대수기 도달 시점에 프롤린 5 g/L를 첨가하였고 2시간 후 배양액을 회수하여 원심분리 후 통상적인 방법에 따라 동결건조를 수행하였다. 실험 결과, 모든 균주에서 동결건조 및 가속안정성과 내산성이 높아졌으며 일부 균주에서 내담즙성이 개선되었다(표 7). It was confirmed that the addition of proline during the cultivation of Lactobacillus plantarum KCTC3108 in Example 7 increased the freeze-drying survival rate, the survival rate after the accelerated test, and the acid resistance, so that experiments were performed to confirm whether the same effect was observed in other lactic acid bacteria Respectively. Five different strains including Lactobacillus plantarum KCTC3108 were cultivated, and 5 g / L of proline was added at the time of arrival of the larvae. After 2 hours, the culture broth was recovered, centrifuged and frozen according to a conventional method Drying was carried out. As a result, the freeze-drying and accelerated stability and acid resistance of all the strains were improved, and the biliary properties of some strains were improved (Table 7).
첨가 여부Proline
Whether to add
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
첨가5 g / L
adding
실시예 9: 배양 중 프롤린 첨가에 따른 동결건조 후 과산화수소 내성 Example 9: Hydrogen peroxide resistance after lyophilization with addition of proline during culture
실시예 8과 같이 각각 프롤린 미첨가 배양액 및 5 g/L 첨가 배양액으로부터 회수한 균체를 사용하여 동결건조를 거친 제품에 대하여 산소 내성을 확인하기 위해 실험을 실시하였다. 산소에 대한 내성을 확인하기 위한 간접적인 방법으로 과산화수소 내성을 비교하였다. 동결건조 후 각각의 샘플을 과산화수소 0 ppm, 5,000 ppm, 10,000 ppm, 15,000 ppm, 20,000 ppm을 포함하고 있는 0.05 M 인산 버퍼(pH 6.8)에 현탁한 다음 37℃에서 1분 동안 반응시켰다. 그리고 반응액 1 ml을 취하여 2 mg/ml 카탈라아제를 포함하고 있는 버퍼에 현탁하고 10배 단위로 희석한 다음 MRS 배지에 도말한 후 배양하여 생균수를 분석하였다. 생존율(%)은 반응 전 인산 버퍼에 첨가된 균수를 100%로 하였으며 분석결과를 표 8에 나타내었다. 균에 따라 차이를 보이지만 일반적으로 과산화수소 처리 농도가 증가할수록 생존율이 감소하였다. 또한, 프롤린을 첨가한 다음 동결건조를 거친 제품은 배양 중 프롤린을 미첨가한 경우와 비교하여 과산화수소에 대한 내성이 현저히 증가하였다. Experiments were carried out to confirm the oxygen tolerance of the products obtained by lyophilization using the cells recovered from the proline-free culture medium and the 5 g / L-containing culture medium as in Example 8, respectively. Hydrogen peroxide tolerance was compared indirectly to confirm resistance to oxygen. After lyophilization, each sample was suspended in 0.05 M phosphate buffer (pH 6.8) containing 0 ppm, 5,000 ppm, 10,000 ppm, 15,000 ppm and 20,000 ppm of hydrogen peroxide, and reacted at 37 ° C for 1 minute. 1 ml of the reaction solution was suspended in a buffer containing 2 mg / ml catalase, diluted 10-fold, and then plated on MRS medium. The survival rate (%) was determined as 100% for the number of bacteria added to the phosphate buffer before the reaction, and the results are shown in Table 8. In general, the survival rate decreased with increasing hydrogen peroxide concentration. In addition, the product after lyophilization with proline added significantly increased the resistance to hydrogen peroxide compared with the case without proline added during the culture.
프롤린 첨가During cultivation
Proline addition
실시예Example 10: 배양 중 프롤린 첨가에 따른 동결건조 후 과산화수소 분해 활성 10: Hydrogen peroxide decomposition activity after lyophilization with addition of proline during incubation
실시예 9와 같이 각각 프롤린 미첨가 배양액 및 5 g/L 첨가 배양액으로부터 회수한 균체를 사용하여 동결건조를 거친 제품에 대하여 과산화수소 분해능을 비교하였다. 0.4 mM 인산 버퍼(pH 6.9)에 수분이 포화된 페놀용액 2%, 4-아미노안티피린(4-aminoantipyrine) 0.04 mg/ml, 페록시다아제(peroxidase) 0.04 Unit/ml, 과산화수소 300 nmol을 포함하는 반응액에 상기의 유산균 동결건조 시료를 첨가하고 37℃에서 1시간 동안 반응시킨 다음 흡광광도계로 505 nm에서 분석하여 반응 후 잔류 과산화수소 농도를 측정하였다. The hydrogen peroxide decomposition ability of the products obtained by lyophilization was compared using the cells recovered from the proline-free culture medium and the 5 g / L-containing culture medium as in Example 9, respectively. A solution containing 2% of water-saturated phenol solution, 0.04 mg / ml of 4-aminoantipyrine, 0.04 Unit / ml of peroxidase, and 300 nmol of hydrogen peroxide in 0.4 mM phosphate buffer (pH 6.9) The lyophilized lactic acid bacterium was added to the reaction solution, reacted at 37 ° C for 1 hour, and analyzed by a spectrophotometer at 505 nm to measure the concentration of residual hydrogen peroxide.
실험결과, 초기 300 nmol에서 잔류농도를 제외한 분해활성을 보면 배양 중 프롤린을 첨가하지 않고 동결건조한 경우보다 프롤린을 첨가한 다음 동결건조를 거친 제품에서 과산화수소 분해 활성이 현저히 증가하였다.As a result of the experiment, the decomposition activity except for the residual concentration at the initial 300 nmol was significantly increased when the proline was added to the lyophilized product after lyophilization.
(nmol of H2O2/mg of cell per hr)Hydrogen peroxide decomposition activity
(nmol of H 2 O 2 / mg of cell per hr)
실시예 11: 동결보호제 또는 코팅제 첨가Example 11: Addition of cryoprotectant or coating agent
배양 시 프롤린을 첨가하는 배양공정을 이용하여 세포 내 프롤린을 고농도로 축적시킴으로써 균주 자체가 동결건조를 포함한 스트레스 환경에 저항성이 강한 특성을 갖도록 하는 것이 가능하였다. 배양 중 프롤린을 첨가하여 세포내 프롤린의 함유량을 높임으로써 균체 자체의 극한환경에 대한 가속 저장 안정성 및 내산성은 증가하였지만 내담즙성에 미치는 효과는 적었기 때문에 이를 보완할 수 있는 방법을 조사하였다.By accumulating intracellular proline at a high concentration using a culture process in which proline is added during the culture, it was possible to make the strain itself resistant to stress environments including freeze drying. The addition of proline to the intracellular proline increased the storage stability and acid resistance of the cell itself to the extreme environment. However, the effect of intracellular proline on the biliary properties was not significant.
이를 위하여 배양 중 프롤린을 첨가한 후 농축한 배양액에 통상적으로 알려져 있는 동결보호제 또는 코팅제를 추가하고 동결건조를 진행하여 그 효과를 확인하였다(표 10).To this end, proline was added during the culture, and then the freeze-drying or lyophilization was carried out by adding a commonly known cryoprotectant or coating agent to the concentrated culture (Table 10).
추가 실험에 사용된 동결보호제는 프롤린(proline), 트리할로오스(trehalose), 글리세린(glycerin)이고 코팅제는 키토산(chitosan), 말토덱스트린(malto-dextrin), 난소화성 말토덱스트린(indigestible dextrin), 전분(starch), 잔탄검(xanthan gum, XG), 구아검(guar gum, GG), 카르복시메틸셀룰로오스(carboxymethyl cellulose, CMC), 하이드록시에틸셀룰로오스(hydroxyethylcellulose, HEC), 셀룰로오스(cellulose), 폴리비닐피롤리돈(polyvinylpyrroridone, PVP), 카보폴(carbopol), 소듐알기네이트(sodium alginate), 프로필렌글리콜 알기네이트(propylene glycol alginate), 알지네이트(alginate), 폴리에틸렌글리콜(polyethyleneglycol, PEG), 트리아세틴(triacetin), 프로필렌 글리콜(propylene glycol), 아세틸트리에틸 시트레이트(acetyl triethyl citrate), 트리에틸시트레이트(triethyl citrate)이다. The cryoprotectants used in further experiments were proline, trehalose, glycerin and coatings were chitosan, malto-dextrin, indigestible dextrin, Starch, xanthan gum, XG, guar gum, carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), cellulose, polyvinylsulfone Polyvinylpyrrolidone (PVP), carbopol, sodium alginate, propylene glycol alginate, alginate, polyethyleneglycol (PEG), triacetin Propylene glycol, acetyltriethyl citrate, and triethyl citrate. The term " anionic surfactant "
이들 중 프롤린(proline), 트리할로오스(trehalose), 말토덱스트린(malto-dextrin), 난소화성 말토덱스트린(indigestible dextrin) 또는 셀룰로오스(cellulose)를 추가로 첨가한 경우와 동결보호제 및 코팅제를 첨가하지 않은 대조군의 내산성 및 내담즙성 확인 결과를 표 10에 나타냈다. 사용된 각각의 5가지 균주마다 추가된 동결보호제 또는 코팅제 성분에 따른 결과가 조금씩 다르지만 전체적으로 내산성을 향상시키는 경향을 보였다. 또한, 동결보호제 또는 코팅제 처리에 의해 내담즙성이 현저히 증가되는 것을 확인하였다.In the case of addition of proline, trehalose, malto-dextrin, indigestible dextrin or cellulose, and addition of a cryoprotectant and a coating agent, Table 10 shows the results of the acid resistance and the bile resistance test of the control group. For each of the five strains used, the result of the addition of the cryoprotectant or coating composition was slightly different, but the overall acidity tended to improve. In addition, it was confirmed that the bile resistance was significantly increased by treatment with a cryoprotectant or a coating agent.
건조 freezing
dry
(인공위액 pH 2.5)Acid resistance
(Artificial gastric juice pH 2.5)
(0.5% oxgall)My bile
(0.5% oxgall)
(proline)Proline
(proline)
(trehalose)Trihalose
(trehalose)
(malto-dextrin)Maltodextrin
(malto-dextrin)
(indigestible- dextrin)Indigestible maltodextrin
(indigestible-dextrin)
(cellulose)cellulose
(cellulose)
(proline)Proline
(proline)
(trehalose)Trihalose
(trehalose)
(malto-dextrin)Maltodextrin
(malto-dextrin)
(indigestible- dextrin)Indigestible maltodextrin
(indigestible-dextrin)
(cellulose)cellulose
(cellulose)
(proline)Proline
(proline)
(trehalose)Trihalose
(trehalose)
(malto-dextrin)Maltodextrin
(malto-dextrin)
(indigestible- dextrin)Indigestible maltodextrin
(indigestible-dextrin)
(cellulose)cellulose
(cellulose)
(proline)Proline
(proline)
(trehalose)Trihalose
(trehalose)
(malto-dextrin)Maltodextrin
(malto-dextrin)
(indigestible- dextrin)Indigestible maltodextrin
(indigestible-dextrin)
(cellulose)cellulose
(cellulose)
L. mesenteroides KCTC3100
L. mesenteroides KCTC3100
(proline)Proline
(proline)
(trehalose)Trihalose
(trehalose)
(malto-dextrin)Maltodextrin
(malto-dextrin)
(indigestible- dextrin)Indigestible maltodextrin
(indigestible-dextrin)
(cellulose)cellulose
(cellulose)
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
(a) 유산균의 배양 중 프롤린을 첨가하여 배양하는 단계로서, 상기 유산균은 락토바실러스 속(Lactobacillus sp.), 비피도박테리움 속(Bifidobacterium sp.) 및 류코노스톡 속(Leuconostoc sp.) 균주로 구성된 군에서 선택된 유산균이고;
(b) 유산균 배양액을 농축하는 단계; 및
(c) 농축된 배양액에 동결보호제 또는 코팅제를 첨가하는 단계로서 상기 동결보호제는 프롤린(proline) 또는 트리할로오스(trehalose)이고, 코팅제는 말토덱스트린(malto-dextrin), 난소화성 말토덱스트린(indigestible dextrin) 또는 셀룰로오스(cellulose)이다.
A method for increasing survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of a lactic acid bacterium comprising the following steps:
(a) culturing lactic acid bacteria by adding proline in the culture, wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus sp., Bifidobacterium sp., and Leuconostoc sp. A lactic acid bacterium selected from the group consisting of;
(b) concentrating the culture liquid of the lactic acid bacteria; And
(c) adding a cryoprotectant or coating to the concentrated culture, wherein the cryoprotectant is proline or trehalose and the coating agent is malto-dextrin, indigestible maltodextrin, dextrin or cellulose.
The method of claim 1, wherein the lactic acid bacterium is selected from the group consisting of L. plantarum , L. acidophilus , B. bifidum , Bifidobacterium longum ( B longum ) or L. mesenteroides .
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