KR101192765B1 - Functional foods containing vegetable-origin lactic acid bacteria and method thereof - Google Patents
Functional foods containing vegetable-origin lactic acid bacteria and method thereof Download PDFInfo
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- KR101192765B1 KR101192765B1 KR1020100060760A KR20100060760A KR101192765B1 KR 101192765 B1 KR101192765 B1 KR 101192765B1 KR 1020100060760 A KR1020100060760 A KR 1020100060760A KR 20100060760 A KR20100060760 A KR 20100060760A KR 101192765 B1 KR101192765 B1 KR 101192765B1
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- lactic acid
- acid bacteria
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- lactobacillus
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- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 description 1
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Abstract
본 발명은 식물성유산균을 포함하는 기능성식품 및 그의 제조방법에 관한 것으로써, 보다 상세하게는 배양탱크에 정제수를 넣는 단계; 상기 배양탱크에 배양배지를 넣고, 120℃~122℃에서 14분~16분간 멸균하는 단계; 상기 멸균후 배양탱크의 온도가 41℃~43℃로 떨어지면 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499) 1wt%~2wt%를 접종하는 단계; 상기 락토바실러스 퍼멘텀이 접종된 배양탱크의 pH를 5.45~5.50로, 온도를 41.5℃~42℃로, RPM을 39~41로, 압력을 0.49kgf/cm2~0.51kgf/cm2로 유지하는 단계;를 포함하는 것을 특징으로 하는 식물성유산균을 배양하는 방법과 상기 방법에 의해 배양된 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499)을 49.2wt%~49.4wt% 함유하는 것을 특징으로 하는 식물성유산균을 포함하는 기능성식품 등에 관한 것이다.The present invention relates to a functional food containing a vegetable lactic acid bacteria and a method for producing the same, and more specifically, the step of adding purified water to the culture tank; Putting the culture medium into the culture tank, sterilizing for 14 minutes to 16 minutes at 120 ℃ ~ 122 ℃; Inoculating 1 wt% to 2 wt% of Lactobacillus fermentum (KCCM 10499) when the temperature of the culture tank drops to 41 ° C. to 43 ° C. after sterilization; The pH of the culture tank inoculated with the Lactobacillus fermentum is maintained at 5.45 to 5.50, the temperature at 41.5 ° C to 42 ° C, the RPM to 39 to 41, and the pressure to 0.49 kgf / cm 2 to 0.51 kgf / cm 2 . A method for culturing phyto-lactic acid bacteria, characterized in that it comprises a; and Lacticobacillus fermentum (KCCM 10499) cultured by the method containing 49.2wt% ~ 49.4wt% It relates to a functional food containing.
Description
본 발명은 식물성유산균을 포함하는 기능성식품 및 그의 제조방법에 관한 것이다. 상기 식물성유산균은 락토바실러스 퍼멘텀(Lactobacillus Fermentum; KCCM 10499)이다.The present invention relates to a functional food comprising a plant lactic acid bacteria and a method for producing the same. The phytolactic acid bacteria is Lactobacillus Fermentum (KCCM 10499).
유산균은 자연계에 널리 존재하며 탄수화물을 혐기적으로 이용하여 유산을 생산한다. 유산균이 발견되는 자연환경은 다양한데, 사람이나 동물의 장내에 존재할 뿐 아니라, 다양한 채소와 과일에서도 발견되며 요구르트, 우리나라의 김치나 독일의 사우어크라우트(sauerkraut)와 같은 발효식품에서 그 발효과정에 중요한 역할을 담당한다.
Lactobacillus is widely present in nature and uses carbohydrates anaerobicly to produce lactic acid. The natural environment in which lactobacillus is found is diverse, not only in the gut of humans or animals, but also in various vegetables and fruits, and plays an important role in the fermentation process in fermented foods such as yogurt, kimchi in Korea, and sauerkraut in Germany. In charge of.
이러한 유산균으로는 스트렙토코커스 속(Streptococcus sp.), 페디오코커스 속(Pediococcus sp.), 류코노스톡 속(Leuconostoc sp.), 락토바실러스 속(Lactobacillus sp.), 스포로락토바실러스 속(Sporolactobacilus sp.), 비피도박테리움 속(Bifidobacterium sp.) 등이 있다.
Such lactic acid bacteria include Streptococcus sp. , Pediococcus sp. , Leuconostoc sp. , Lactobacillus sp. , Sporolactobacilli sp. ), and the like Bifidobacterium (Bifidobacterium sp.).
이들 유산균은 장내로 유입된 후 장내 상피세포에 착생하게 되어 유해 미생물의 장 정착을 방지하고 항균 물질을 분비함으로써 유해 미생물의 생육을 억제하고 설사와 변비를 개선할 뿐 만 아니라, 면역활성 증진, 항암 작용, 혈청 콜레스테롤 저하 등의 작용을 수행한다.
These lactic acid bacteria enter the intestine and enter the intestinal epithelial cells, preventing the intestinal settlement of harmful microorganisms and secreting antimicrobial substances, thereby inhibiting the growth of harmful microorganisms and improving diarrhea and constipation. Action, such as lowering serum cholesterol.
또한, 일반적으로 이들 유산균은 직접 혹은 간접적으로 식품에 첨가되어 이들의 대사산물인 유산에 의해 식품의 저장성을 향상시키며 식품의 향미와 조직을 개선한다고 알려져 있다. 따라서 유산균은 동서양을 막론하고 유제품, 육제품, 침채류 및 각종 젓갈류의 가공에 유용한 보조수단뿐만 아니라 생균제(probiotics)로도 이용되고 있다.
In general, these lactic acid bacteria are known to be added directly or indirectly to food to improve the shelf life of the food by their metabolites lactic acid and improve the flavor and texture of the food. Therefore, lactic acid bacteria are used as probiotics as well as auxiliary means useful for processing dairy products, meat products, sesame and various salted fish, regardless of East and West.
최근 채소나 과일과 같은 식물, 혹은 김치와 같은 과채류를 발효한 식품에서 분리해 낸 식물유래 유산균들의 기능성이 밝혀지면서 새로운 기능성 소재로 떠오르고 있다. 이러한 식물 유래 유산균들에는 류코노스톡 속, 페디오코커스 속, 락토바실러스 속 중 락토바실러스 플란타룸(Lactobacillus plantarum)이나 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 브레비스(Lactobacillus brevis) 등이 포함된다. 식물 유래 유산균들은 낮은 pH나 높은 염 농도와 같은 척박한 환경에서도 강한 생존력을 보이며, 배양이 용이하다는 장점을 지니고 있다.
Recently, as the functionalities of plant-derived lactic acid bacteria isolated from fermented foods such as vegetables and fruits, or fruit and vegetable such as kimchi, are emerging as a new functional material. These plant-derived lactic acid bacteria include Lactobacillus plantarum , Lactobacillus paracasei , Lactobacillus brevis , etc. do. Plant-derived lactic acid bacteria have strong viability even in harsh environments such as low pH and high salt concentration, and have the advantage of easy cultivation.
우리가 시중에서 접하는 유산균이나 요구르트 균은 대부분 동물유래에서 분리된 동물성 유산균(우유발효 유산균)이며 배지 영양분으로 탄수화물뿐만 아니라 단백질, 지방 등을 사용한다. 이들 유산균의 발효 양상은 단백질과 지방등을 이용함으로써 시간이 지나면서 부패균수가 증가하여 식용으로 사용할 수 없다.
Most of the lactic acid bacteria and yogurt bacteria that we encounter on the market are animal lactic acid bacteria (milk fermented lactic acid bacteria) isolated from animal origin and use protein, fat, etc. as well as carbohydrates as medium nutrients. The fermentation pattern of these lactic acid bacteria can not be used for food by increasing the number of decaying bacteria over time by using proteins and fats.
그러나 식물성 유산균은 식물유래 식품(김치, 콩발효식품) 등에서 분리되었으며, 배양액 성분을 포도당등 대부분의 탄수화물을 사용함으로써 이체나 환경에 해로운 물질과 균주에 저항하는 물질을 분비하며, 부패균주와 병원성 미생물의 생육을 억제하는 기능을 가지고 있다.
However, vegetable lactic acid bacteria were separated from plant-derived foods (kimchi and soybean fermented foods), and by using most carbohydrates such as glucose in culture medium, they secrete substances that are resistant to variants and environmentally harmful substances and strains. Has the function of suppressing the growth of.
식물성유산균의 유래 및 종류를 구체적으로 살펴보면 야채절임류(containing table salt)에서 유래된 L. plantrum, L. brevis, Leuc. mesenteroides, L. casei, L. fermentum, Ped. pentosaceus 등 이 있고, 야채절임류(not containing table salt)에서 유래된 L. plantrum, L. brevis, L. casei, L. fermentum, B. coagulans 등 이 있으며, 차엽절임에서 유래된 L. plantrum, L.brevis 등 이 있고, 대두발효식품에서 유래된 Tetragenococcus halophilus, L. fermentum 등 이 있으며, 곡류발효식품에서 유래된 L. sakei, Leuc. mesenteroides, L. plantrum, Leuc. lactis Leuc. citreum, Ped. pentosaceus, L. brevis, L. casei 등 이 있다.
Specifically, the origin and types of phyto- lactic acid bacteria are described in L. plantrum, L. brevis, Leuc. mesenteroides, L. casei, L. fermentum, Ped. pentosaceus , L. plantrum, L. brevis, L. casei, L. fermentum, B. coagulans derived from not containing table salt, and L. plantrum, L. brevis , Tetragenococcus halophilus and L. fermentum derived from soybean fermented food, and L. sakei, Leuc. mesenteroides, L. plantrum, Leuc. lactis Leuc. citreum, Ped. pentosaceus, L. brevis, L. casei, etc.
본 발명의 목적은 장(腸) 기능의 활성능력, 면역기능의 향상 등 생리기능성이 검증된 식물성유산균 제품개발 및 상품화하는 데 있다.
It is an object of the present invention to develop and commercialize phyto-lactic acid bacteria products that have been tested for their physiological functions, such as the activity of intestinal function and the improvement of immune function.
일본 및 유럽 등 세계적으로 유산균은 기능성 식품군에서 가장 각광받고 있는 소재로, 단순한 식품차원을 넘어 질병의 치료 등 의학적으로 작용 할 수 있는 방향으로 다양한 연구개발이 이루어지고 있으나 국내적으로는 창의적이고 순발력 있는 기술개발과 소비자에 대한 신뢰제고 등이 아직은 부족한 상태이므로 본 발명을 하기에 이르렀다.
Lactobacillus is one of the most prominent materials in the functional food group in Japan and Europe, and various research and development is being carried out in the direction of medical action such as the treatment of diseases beyond the simple food level. Since the development of technology and the enhancement of trust in the consumer are still insufficient, the present invention has been achieved.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.
The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .
상기 종래기술의 문제점을 해결하기 위해 과제를 해결하기 위해 본 발명에서는, 배양탱크에 정제수를 넣는 단계; 상기 배양탱크에 배양배지를 넣고, 120℃~122℃에서 14분~16분간 멸균하는 단계; 상기 멸균후 배양탱크의 온도가 41℃~43℃로 떨어지면 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499) 1wt%~2wt%를 접종하는 단계; 상기 락토바실러스 퍼멘텀이 접종된 배양탱크의 pH를 5.45~5.50로, 온도를 41.5℃~42℃로, RPM을 39~41로, 압력을 0.49kgf/cm2~0.51kgf/cm2로 유지하는 단계;를 포함하는 것을 특징으로 하는 식물성유산균을 배양하는 방법을 제공한다.In the present invention to solve the problem to solve the problems of the prior art, the step of putting purified water in the culture tank; Putting the culture medium into the culture tank, sterilizing for 14 minutes to 16 minutes at 120 ℃ ~ 122 ℃; Inoculating 1 wt% to 2 wt% of Lactobacillus fermentum (KCCM 10499) when the temperature of the culture tank drops to 41 ° C. to 43 ° C. after sterilization; The pH of the culture tank inoculated with the Lactobacillus fermentum is maintained at 5.45 to 5.50, the temperature at 41.5 ° C to 42 ° C, the RPM to 39 to 41, and the pressure to 0.49 kgf / cm 2 to 0.51 kgf / cm 2 . It provides a method for culturing phyto-lactic acid bacteria, characterized in that it comprises a.
상기 배양배지의 조성은 글루코오즈(glucose)가 24g/L~26g/L이고, 효모추출물(yeast ex.)이 39g/L~41g/L이며, 제2인산칼륨(K2HPO4)이 2g/L~4g/L이고, 소디움아세테이트(sodium acetate)가 3.4g/L~3.6g/L이며, 마그네슘 설페이트(MgSO4)가 0.6g/L~0.8g/L인 것을 특징으로 할 수 있다.The culture medium is composed of glucose (g) of 24g / L ~ 26g / L, yeast extract (yeast ex.) Of 39g / L ~ 41g / L, dibasic potassium phosphate (K 2 HPO 4 ) 2g / L ~ 4g / L, sodium acetate (sodium acetate) is 3.4g / L ~ 3.6g / L, magnesium sulfate (MgSO 4 ) It can be characterized in that the 0.6g / L ~ 0.8g / L.
그리고 본 발명은 상기 방법에 의해 배양된 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499)을 49.2wt%~49.4wt% 함유하고, 사과분말을 7.1wt%~7.3wt% 함유하며, 석류농축액분말을 0.4wt%~0.6wt% 함유하고, 레몬과즙분말을 0.5wt%~0.7wt% 함유하며, 올리고당을 24.0wt%~24.2wt%함유하고, 비타민C를 1.4wt%~1.6wt% 함유하며, 탈지분유를 4.7wt%~4.9wt%함유하고, 식이섬유를 11.9wt%~12.1wt% 함유하는 것을 특징으로 하는 식물성유산균을 포함하는 기능성식품을 제공한다. And the present invention contains Lactobacillus fermentum (KCCM 10499) cultured by the above method, 49.2wt% ~ 49.4wt%, apple powder 7.1wt% ~ 7.3wt%, pomegranate concentrate powder 0.4 wt% ~ 0.6wt%, lemon and juice powder 0.5wt% ~ 0.7wt%, oligosaccharide 24.0wt% ~ 24.2wt%, vitamin C 1.4wt% ~ 1.6wt%, skim milk powder It provides 4.7wt% ~ 4.9wt%, and provides a functional food containing phyto-lactic acid bacteria, characterized in that it contains 11.9wt% ~ 12.1wt% dietary fiber.
이하 본 발명에서 A, B, C라 함은 하기 표 2에 상세하게 기재한 것을 의미한다.
Hereinafter, in the present invention, A, B, and C means those described in detail in Table 2 below.
첫째, 본 발명을 통해 본배양 공정 확립하였다. 보다 구체적으로는 종배양을 거쳐 본배양 공정으로 17시간을 배양한 결과, Lag phase, Log phase, Stationary phase에서 모두 안정적인 결과를 나타내었다.
First, the main culture process was established through the present invention. More specifically, after 17 hours of incubation in the main culture after the species culture, the results were stable in the Lag phase, Log phase, Stationary phase.
둘째, 본 발명을 통해 제조공법 개선의 유리한 효과를 얻을 수 있었다. 보다 구체적으로는 제조공법 개선으로 제조한 유산균 개체수는 기존공정으로 생산한 경우, 액체배양의 경우, 8×108 CFU/g에서 4×109 CFU/g으로 향상되었으며, 동결건조 후는 4.3×1010 CFU/g에서 2×1011 CFU/g을 나타내어 약 75%의 생산성 향상을 나타내었다.
Second, the present invention was able to obtain an advantageous effect of the manufacturing method improvement. More specifically, the lactic acid bacteria population produced by the improvement of the manufacturing method was improved from 8 × 10 8 CFU / g to 4 × 10 9 CFU / g in the case of liquid culture, and 4.3 × after lyophilization. 10 indicated a 10 CFU / g 2 × 10 11 CFU / g are shown in the productivity improvement of about 75%.
셋째, 본 발명을 통해 최적 레시피 확립할 수 있었다. 보다 구체적으로는 식물성유산균이 가지고 있는 기능성과 식이섬유 및 올리고당 등의 prebiotics를 첨가시켜 기능을 더욱 향상시킨 기호성이 우수한 최적의 레시피를 개발하였다
Third, an optimal recipe could be established through the present invention. More specifically, we developed an optimal recipe with excellent palatability by adding the functionality of plant lactic acid bacteria and prebiotics such as dietary fiber and oligosaccharide.
넷째, 본 발명을 통해 내열성을 측정한 결과 동물유래의 일반유산균인 Lactobacillus acidophilus에 비해 제조공법이 개선된 식물성유산균 ‘C’인 Lactobacillus fermentum 유산균의 내열성이 거의 2배 이상 높은 것으로 확인되었다.
Fourth, the heat resistance of the Lactobacillus fermentum lactic acid bacteria of the plant Lactobacillus fermentum lactic acid bacteria improved production method compared to the animal-derived lactic acid bacteria Lactobacillus acidophilus was almost two times higher.
다섯째, 본 발명을 통해 내산성 측정한 결과 체내 위액의 pH와 유사한 정도로 조정한 액에 제조공법이 개선된 식물성유산균 ‘C’와 일반유산균인 Lactobacillus acidophilus를 각각 pH 3과 2에 첨가할 경우 어느 정도의 산에 저항할 수 있는지 측정을 하였다. 그 결과, 식물성유산균인 Lactobacillus fermentum 균주는 각각 pH 3.0과 2.0에서 101 과 102의 균수의 저하가 있었지만 생존율은 매우 높은 상태로 존재하였다. 그러나 Lactobacillus acidophilus 의 경우는 pH 3.0과 2.0 각각에 대해 약 105 정도씩의 균수의 저하가 있었으며 낮은 생존율을 나타내었다. 따라서 식물성유산균은 코팅 등의 보호제를 사용하지 않고도 산에 견딜 수 있는 강력한 내산을 나타내어 공정개선에도 불구하고 내산의 기능성은 그대로 보유하고 있는 것으로 나타났다.
Fifth, some amount of the addition of the present invention, the acid resistance measurement as a result Lactobacillus acidophilus the manufacturing process is improved vegetable lactic acid bacteria 'C' and the common lactic acid bacteria in a liquid adjusted so similar to the pH of the body of gastric juice through the
여섯째, 본 발명을 통해 혈당강하의 효능 검증을 수행한 결과 농도별, 시간별 혈당 측정실험 결과, 식물성 유산균이 일반 마우스에서 혈당강하 효과가 큰 것을 볼 수 있었으며, 이것은 일반적인 평범한 상태에서의 혈당 강하효과를 확인 한 것이라 볼 수 있다. Sixth, as a result of verifying the efficacy of hypoglycemic effect through the present invention, the results of blood glucose measurement by concentration and time, it was found that the plant lactobacillus effect on the hypoglycemic effect in ordinary mice, which has a general hypoglycemic effect It can be confirmed.
급성 고혈당 모델(Fed D-Glucose model)에 있어서 D-Glucose가 혈당 상승에 작용할 때 제조공법이 개선된 식물성유산균 B와 C가 일정부분 제어 하는 것을 볼 수 있었으며, 이것은 급성으로 고혈당을 유발된 모델에서의 혈당 제어 효과를 입증한 것이라 볼 수 있다. In the Fed D-Glucose model, when D-Glucose acts on the blood sugar level, the production process improved the control of plant lactobacillus B and C. It can be seen that the effect of controlling blood sugar.
당뇨 모델(Diabetes model)로서 Streptozotocin(STZ)이 유발한 당뇨 마우스에서 식물성유산균 A, B, C가 혈당 강하 효과를 보임을 확인할 수 있었다. 이것은 당뇨가 유발되어 혈당 제어 능력을 잃어버린 모델에서의 혈당 제어 효과를 입증한 것이라 볼 수 있다. 따라서 일반 사람뿐만 아니라 당뇨환자에 이르기까지 식물성유산균의 혈당 강하효과를 기대할 수 있음을 시사하였다. Phytolactic acid bacteria A, B, and C showed hypoglycemic effect in diabetic mice induced by Streptozotocin (STZ) as a diabetes model. This proved to be effective in controlling blood glucose in a model in which diabetes induced loss of blood sugar control. Therefore, the hypoglycemic effect of phyto-lactic acid bacteria could be expected from diabetic patients as well as general people.
또한 농도별, 날짜별 실험(Normal model)에서 식물성 유산균의 장기간 투여시에도 혈당강하 효과가 큰 것을 볼 수 있었다. 하지만 고농도의 유산균보다 저농도의 유산균에서 혈당 강하효과가 큰 것을 확인하였다. 따라서, 장기간의 유산균 투여시에는 낮은 농도로 꾸준히 유산균을 섭취하였을 때 혈당 강하효과가 탁월함을 알 수 있었다.
In addition, it was found that the hypoglycemic effect was great even in the long-term administration of vegetal lactic acid bacteria in the concentration and date experiments (Normal model). However, it was confirmed that the blood glucose lowering effect was higher in the low concentration of lactic acid bacteria than the high concentration of lactic acid bacteria. Therefore, when the lactic acid bacteria were administered for a long time, it was found that the blood sugar lowering effect was excellent when the lactic acid bacteria were steadily consumed at a low concentration.
일곱째, 본 발명을 통해 장통과 시간 측정 분석결과 다음과 같은 사항을 확인할 수 있었다. 장통과 시간을 측정하는 목적에 있어서 장통과 시간이 빠르다는 것은 장의 활동이 활발하다는 것과 배변 활동이 원활하다는 것을 의미하는데, 식물성유산균은 장활동을 활발히 해줌으로써 배변활동을 좋게 하여 변비예방이나 변비에 효과를 나타낼 수 있는 것으로 판단할 수 있었다. Seventh, through the present invention it was confirmed that the following items as a result of analysis of pain and time measurement. For the purpose of measuring intestinal pain and time, the fast intestinal time and time means that the intestinal activity is active and the bowel movements are smooth. Phytolactic acid bacteria improve bowel activity by promoting bowel activity to prevent constipation and constipation. It could be judged to be effective.
저농도에서는 3일째에 효과가 가장 좋았고, 고농도에서는 장시간 투여할수록 점차 장의 활동이 좋아지는 것을 확일 할 수 있었다. 따라서, 식물성 유산균을 꾸준히 섭취하였을 경우 최소 3일째부터 점차 장의 운동이 활발해짐을 확인할 수 있었다.
At low concentrations, the effect was best at day 3, and at high concentrations, long-term administration gradually improved the intestinal activity. Therefore, when the vegetable lactic acid bacteria were steadily ingested it was confirmed that the bowel movement gradually became active from at least three days.
여덟째, 본 발명을 통해 식물성유산균의 면역력 향상 기능 분석결과 다음과 같은 사항을 확인할 수 있었다. 베타 엔돌핀(β-endorphin)이 분비된 다는 것은 면역역 향상에 도움이 된다는 것을 의미한다. 측정실험 결과, 식물성 유산균의 1회 투여로는 베타 엔돌핀의 분비가 증가되지 않는 것으로 보아 면역력 향상에 대한 효과가 있다고 기대하기 어려워 1회 이상 투여하기로 결정하였다.Eighth, through the present invention it was confirmed that the following items as a result of the analysis of the immune function of the plant lactic acid bacteria. The release of beta endorphins (β-endorphin) means that it helps to boost the immune system. As a result of the measurement experiment, it was determined that the beta endorphin secretion was not increased by one administration of the plant lactic acid bacteria, so it was difficult to expect an effect on the improvement of immunity.
농도별, 날짜별 β-endorphin 발현정도 측정 실험에서는 1회의 식물성 유산균 투여시에 β-endorphin의 분비 증가를 볼 수 없었지만, 7일 이상 장기간 식물성 유산균 투여시 확실히 β-endorphin의 분비 증가를 확인할 수 있었다. 이로써, 식물성 유산균의 장시간 투여가 면역력의 증강에 효과가 있음을 기대할 수 있었다.Β-endorphin expression level by concentration and date did not show increased secretion of β-endorphin after one administration of botanical lactic acid bacteria. . As a result, long-term administration of the plant lactic acid bacteria could be expected to be effective in enhancing the immunity.
시상하부에서의 POMC 유전자 발현에서 제조공법에 따른 시료 A, B, C 식물성유산균 모두에서 시상하부의 POMC 유전자가 두드러지게 증가하는 것으로 미루어 보아, 항스트레스, 쾌감, 면역력 증진, 통증 억제 등의 가능성을 가짐을 알 수 있었다.POMC gene expression in the hypothalamus significantly increases the hypothalamic POMC gene in all of the sample A, B, and C-lactic acid bacteria according to the manufacturing method. It was found.
뇌하수체에서의 POMC 유전자 발현에서 제조공법에 따른 시료 A, B, C 식물성유산균 모두에서 뇌하수체의 POMC 유전자가 두드러지게 증가하는 것으로 미루어 보아, 항스트레스, 쾌감, 면역력 증진, 통증 억제 등의 가능성을 가짐을 알 수 있었다.
The expression of POMC gene in pituitary gland showed that POMC gene of pituitary gland was significantly increased in all samples A, B and C phyto-lactic acid bacteria according to the manufacturing method. Could know.
아홉째, 본 발명을 통해 장내 점착능 측정 실험 결과 다음과 같은 사항을 확인할 수 있었다. 유산균의 점착능이 높다는 것은 그만큼 장내부에 잘 부착될 수 있다는 것을 의미한다. 유산균이 장내부에 잘 부착되면, 그만큼 장 내부에 자리하고 있는 시간이 길어지면서 유산균의 여러 유용한 역할을 잘 해나갈 수 있다는 것을 의미한다. Ninth, through the present invention, the results of the intestinal adhesion measurement experiments were able to confirm the following. The high adhesion of lactic acid bacteria means that it can be adhered well to the intestines. If lactic acid bacteria are well adhered to the intestine, it means that the longer the time spent in the intestines can play various useful roles of lactic acid bacteria.
이 실험을 통해 세가지 균주의 점착능이 우수함을 확인 할 수 있었고, 그중에서 C의 점착능이 아주 우수함을 확인 할 수 있었다.
Through this experiment, it could be confirmed that the adhesion of the three strains was excellent, and among them, the adhesion of C was very good.
열째, 시제품을 생산하여, 당해 시제품의 관능검사 결과 다음과 같은 사항을 확인할 수 있었다. 최적레시피에 의한 시제품을 스틱형 분말제품으로 제조하였으며, 시제품을 가지고 대학생을 대상으로 하여 관능검사를 실시하였다. 시제품을 관능검사하기 위해 대학을 방문하여 남학생 8명, 여학생 52명을 포함하여 총 60명을 대상으로 식물성유산균의 간단한 설명 후에 12문항의 설문을 실시하여 그 결과를 선호 인원 및 백분율로 나타내었다. 그 중 중요한 결과로서 ‘유산균 제품 구매의 주된 요인은?’ ‘장이 안 좋아서’가 약 87%이었으며, 시제품의 맛과 양에서는 각각 약 70%, 60% 선호도로 좋은 결과를 나타내었다.
Tenth, a prototype was produced, and the following items were confirmed by the sensory test of the prototype. Prototype according to the optimum recipe was prepared as a stick-type powder product, and the sensory test was carried out for college students with the prototype. To test the sensory test of the prototype, a total of 60 students, including 8 male students and 52 female students, were surveyed with 12 questions after brief explanation of the phyto-lactic acid bacteria. Among the important results, 'What are the main factors for the purchase of lactic acid bacteria products?' About 87% were 'not good at the market', and the taste and quantity of the prototype showed good results with about 70% and 60% preference, respectively.
나아가 본 발명을 통해 체내 유해균의 활동과 성장의 억제, 장기능 개선(설사와 변비 개선), 항체생산을 촉진하는 면역 증강작용, 발암물질을 감소시키는 항암 작용, 혈청콜레스트롤 저하 작용, 비타민 합성작용 등의 유리한 효과를 얻을 수 있다.
Furthermore, the present invention inhibits the activity and growth of harmful bacteria in the body, improves intestinal function (improvement of diarrhea and constipation), immune enhancer to promote antibody production, anticancer action to reduce carcinogens, serum cholesterol levels, vitamin synthesis, etc. The advantageous effect of can be obtained.
도 1은 종균 배양의 공정도(The method of seed culture for main culture)에 관한 것이다.
도 2는 본 발명인 발효 배양 공정도( Main culture processing of lactic acid bacteria)에 관한 것이다.
도 3은 The graph of OD value에 관한 것이다.
도 4는 배양 후 배양액의 원심분리 공정도(The processing of cell separation)에 관한 것이다.
도 5는 동결건조 과정(The processing of lyophilization)에 관한 것이다.
도 6은 원말제품의 제조 공정도(The manufacturing process of lactic acid bacteria powder)에 관한 것이다.
도 7은 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정한 결과(Blood glucose level test of vegetable lactic acid bacteria)이다.
도 8은 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정한 결과(Blood glucose level test of vegetable lactic acid bacteria)이다.
도 9는 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정한 결과(Blood glucose level test of vegetable lactic acid bacteria)이다.
도 10은 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정한 결과(Blood glucose level test of vegetable lactic acid bacteria)이다. 위쪽은 1×=1×108, 아래의 왼쪽은 5×= 2×108, 아래의 오른쪽은 10×=1×109 CFU/g의 유산균 개체수를 나타낸다.
도 11은 The result of gastointestinal transit time (1×=1×108CFU/g)에 관한 것이다.
도 12는 The result of gastointestinal transit time(5×= 2×108)에 관한 것이다.
도 13은 The result of gastointestinal transit time(10×=1×109 )에 관한 것이다.
도 14는 The result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1×109 CFU/g에 관한 것이다.
도 15는 The result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1×108 CFU/g에 관한 것이다.
도 16은 The result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 2×108 CFU/g에 관한 것이다.
도 17은 The result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1×109 CFU/g에 관한 것이다.
도 18은 시상하부에서의 POMC 유전자 발현(The result of POMC gene expression in the hypothalamus of vegetable lactic acid bacteria at each concentration)에 관한 것이다.
도 19는 뇌하수체에서의 POMC 유전자 발현(The result of POMC gene expression in the pituitary gland of vegetable lactic acid bacteria at each concentration)에 관한 것이다.
도 20은 Colonic mucin-binding assay kit에 관한 것이다.Figure 1 relates to the process of seed culture (The method of seed culture for main culture).
Figure 2 relates to a fermentation culture process (main culture processing of lactic acid bacteria) of the present invention.
3 relates to the graph of OD value.
4 relates to the processing of cell separation of the culture medium after the culture.
5 relates to the processing of lyophilization.
6 relates to the manufacturing process of the raw product (The manufacturing process of lactic acid bacteria powder).
Figure 7 is a blood glucose measurement results (Blood glucose level test of vegetable lactic acid bacteria) using the Accumulus Performa blood glucose meter.
8 is a blood glucose measurement result (Blood glucose level test of vegetable lactic acid bacteria) using the Accuform Performa blood glucose meter.
9 is a blood glucose level (Blood glucose level test of vegetable lactic acid bacteria) using an Accuform Performa blood glucose meter.
10 is a blood glucose measurement result (Blood glucose level test of vegetable lactic acid bacteria) using the Accuform Performa blood glucose meter. The top shows 1 × = 1 × 10 8 , the bottom left shows 5 × = 2 × 10 8 , and the bottom right shows 10 × = 1 × 10 9 CFU / g of lactic acid bacteria population.
Figure 11 relates to The result of gastointestinal transit time (1 × = 1 × 10 8 CFU / g).
12 relates to The result of gastointestinal transit time (5 × = 2 × 10 8 ).
13 relates to The result of gastointestinal transit time (10 × = 1 × 10 9 ).
14 relates to The result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1 × 10 9 CFU / g.
15 relates to the result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1 × 10 8 CFU / g.
Figure 16 relates to the result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 2 × 10 8 CFU / g.
17 relates to the result of plasma β-endorphin of vegetable lactic acid bacteria at the concentration of 1 × 10 9 CFU / g.
FIG. 18 relates to the result of POMC gene expression in the hypothalamus of vegetable lactic acid bacteria at each concentration.
19 relates to the result of POMC gene expression in the pituitary gland of vegetable lactic acid bacteria at each concentration.
20 relates to a colonic mucin-binding assay kit.
본 발명은 배양탱크에 정제수를 넣는 단계; 상기 배양탱크에 배양배지를 넣고, 120℃~122℃에서 14분~16분간 멸균하는 단계; 상기 멸균후 배양탱크의 온도가 41℃~43℃로 떨어지면 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499) 1wt%~2wt%를 접종하는 단계; 상기 락토바실러스 퍼멘텀이 접종된 배양탱크의 pH를 5.45~5.50로, 온도를 41.5℃~42℃로, RPM을 39~41로, 압력을 0.49kgf/cm2~0.51kgf/cm2로 유지하는 단계;를 포함하는 것을 특징으로 하는 식물성유산균을 배양하는 방법에 관한 것이다.The present invention comprises the steps of putting purified water in the culture tank; Putting the culture medium into the culture tank, sterilizing for 14 minutes to 16 minutes at 120 ℃ ~ 122 ℃;
상기 배양배지의 조성은 글루코오즈(glucose)가 24g/L~26g/L이고, 효모추출물(yeast ex.)이 39g/L~41g/L이며, 제2인산칼륨(K2HPO4)이 2g/L~4g/L이고, 소디움아세테이트(sodium acetate)가 3.4g/L~3.6g/L이며, 마그네슘 설페이트(MgSO4)가 0.6g/L~0.8g/L인 것을 특징으로 할 수 있다.The culture medium is composed of glucose (g) of 24g / L ~ 26g / L, yeast extract (yeast ex.) Of 39g / L ~ 41g / L, dibasic potassium phosphate (K 2 HPO 4 ) 2g / L ~ 4g / L, sodium acetate (sodium acetate) is 3.4g / L ~ 3.6g / L, magnesium sulfate (MgSO 4 ) It can be characterized in that the 0.6g / L ~ 0.8g / L.
그리고 본 발명은 상기 방법에 의해 배양된 락토바실러스 퍼멘텀(Lactobacillus fermentum; KCCM 10499)을 49.2wt%~49.4wt% 함유하고, 사과분말을 7.1wt%~7.3wt% 함유하며, 석류농축액분말을 0.4wt%~0.6wt% 함유하고, 레몬과즙분말을 0.5wt%~0.7wt% 함유하며, 올리고당을 24.0wt%~24.2wt%함유하고, 비타민C를 1.4wt%~1.6wt% 함유하며, 탈지분유를 4.7wt%~4.9wt%함유하고, 식이섬유를 11.9wt%~12.1wt% 함유하는 것을 특징으로 하는 식물성유산균을 포함하는 기능성식품에 관한 것이다.
And the present invention contains Lactobacillus fermentum (KCCM 10499) cultured by the above method, 49.2wt% ~ 49.4wt%, apple powder 7.1wt% ~ 7.3wt%, pomegranate concentrate powder 0.4 wt% ~ 0.6wt%, lemon and juice powder 0.5wt% ~ 0.7wt%, oligosaccharide 24.0wt% ~ 24.2wt%, vitamin C 1.4wt% ~ 1.6wt%, skim milk powder It contains 4.7wt% to 4.9wt%, and relates to a functional food comprising phyto-lactic acid bacteria, characterized in that it contains 11.9wt% to 12.1wt% dietary fiber.
나아가 본 발명은 식물성유산균 배양공정 개선에 의한 기능성 향상 즉, 식물성유산균의 기능성을 최대한 확보하기 위한 배양공정 개발 및 Jar-fermenter에 의한 배양공법 개발 후 scale-up하여 제조된 식물성유산균의 in vitro기능성 검증에 관한 것이다.
Furthermore, the present invention is to verify the in vitro functionalities of phyto-lactic acid bacteria prepared by scale-up after the development of cultivation process and cultivation method by Jar-fermenter to improve the functionality by phyto-lactic acid bacteria culture process improvement. It is about.
또한 본 발명은 배양공정 및 제조공정 확립 즉, 지속적인 배양공법 개발로 수율 향상 및 기능성 보존 유지 및 동결건조 방법의 최신공법 개발로 생존율 향상방법 개발 및 생리기능성 효능 검증에 관한 것이다.
In addition, the present invention relates to the establishment of a cultivation process and manufacturing process, that is, the development of a method of improving survival rate and verifying physiological functional efficacy by developing a new method of improving yield and maintaining functional preservation and lyophilization by developing a continuous culture method.
이하에서 첨부된 도면을 참조한 실시예에 의거하여 구체적으로 설명한다.
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
[실시예 1] 균주의 준비Example 1 Preparation of Strains
본 연구개발에 사용된 식물성유산균 Lactobacillus fermentum 은 발명의 명칭이 ‘내산성 유산균을 함유하는 발효음료(특허등록번호 : 10-0435168, 2004년 5월 31일)’인 특허발명의 균주이다. 상기 특허균주의 기탁번호는 KCCM10499이다.
Phytolactic acid bacteria Lactobacillus fermentum used in this research and development is a strain of the invention of the invention named 'fermented beverage containing acid resistant lactic acid bacteria (patent registration number: 10-0435168, 31 May 2004). The accession number of the patent strain is KCCM10499.
식물성유산균 Lactobacillus fermentum 의 특징은 내열성, 내산성, 내담즙성이 매우 우수하고, 장 까지 살아서 도달하는 생존율이 높아 장 기능 개선효과가 탁월하며, 암모니아(NH3) 성분을 분해하는 특성이 있어서 장 기능 저하로 생기는 악취제거 효과가 탁월하다. Phytolactic acid bacteria Lactobacillus fermentum is characterized by excellent heat resistance, acid resistance, and bile resistance, high survival rate to reach the intestine and excellent intestinal function improvement effect, and degrading ammonia (NH 3 ) component. Excellent odor removal effect.
또한 혈당강하, 지질대사 개선(저밀도 콜레스트롤 및 중성지질 저하 효과, 고밀도 콜레스트롤 상승효과), 활성산소 억제로 노화 지연 효과 등이 있다.
In addition, hypoglycemia, improved lipid metabolism (low density cholesterol and neutral lipid lowering effect, high density cholesterol level synergistic effect), and active oxygen inhibition delay the aging effect.
[실시예 2] 최적배양공정 기술개발 Example 2 Development of Optimal Culture Process
[실시예 2-1] 종균 배양Example 2-1 spawn culture
종균 배양의 공정도(The method of seed culture for main culture)는 도 1과 같다.The method of seed culture for the main culture is shown in FIG.
상기 종균 배양의 배지조성은 글루코오즈(glucose) 0.25g/L, 효모추출물(yeast ex.) 0.4g/L, 제2인산칼륨(K2HPO4) 0.03g/L, 소디움아세테이트(sodium acetate) 0.035g/L, 마그네슘 설페이트(MgSO4) 0.007g/L 이다.
The medium composition of the seed culture is glucose 0.25g / L, yeast extract 0.4g / L, dibasic potassium phosphate (K 2 HPO 4 ) 0.03g / L, sodium acetate 0.035 g / L and magnesium sulfate (MgSO 4 ) 0.007 g / L.
[실시예 2-2] 발효 배양Example 2-2 Fermentation Culture
본 발명의 발효 배양 공정도( Main culture processing of lactic acid bacteria)는 도 2와 같다.
Fermentation culture process diagram of the present invention (Main culture processing of lactic acid bacteria) is shown in FIG.
본 발명의 발효 배양 공정으로 17시간을 배양한 결과 Lag phase, Log phase , Stationary phase에서 모두 안정적인 결과를 보였다. 하기 표 1은 Culture processing data에 관한 것이다(표 1 및 도 3 참조).
17 hours of incubation with the fermentation culture process of the present invention showed stable results in both Lag phase, Log phase and Stationary phase. Table 1 below relates to Culture processing data (see Table 1 and FIG. 3).
제조공정 중 조건에 따른 유산균 개체수의 차이는 표 2에 나타낸 바와 같이 'A', 'B', 'C'의 공정으로 생산한 경우, 배양액의 식물성유산균 개체수는 각각 8×108 CFU/g, 1×109 CFU/g, 4×109 CFU/g 이었으며, 동결건조 후는 각각 4.3×1010 CFU/g, 8×1010 CFU/g, 2×1011 CFU/g를 나타내었다. 특히 'C' 공정의 경우는 1011 CFU/g을 나타내어 생산성에 있어서 기존 공정인 'A' 공정과 일부 공정이 개선된 'B' 공정에 비해 각각 75%, 33%의 생산성 향상을 나타내었다.
The difference in the lactic acid bacteria population according to the conditions of the manufacturing process is produced in the 'A', 'B', 'C' process as shown in Table 2, the phyto-lactic acid bacteria population of the culture medium is 8 × 10 8 CFU / g, respectively, 1 × 10 9 CFU / g, 4 × 10 9 CFU / g, and after lyophilization, 4.3 × 10 10 CFU / g, 8 × 10 10 CFU / g, and 2 × 10 11 CFU / g, respectively. In particular, the 'C' process showed 10 11 CFU / g, which showed a 75% and 33% improvement in productivity compared to the 'A' process, which was improved from the existing process 'A' and some processes, respectively.
제조공정 중 조건에 따른 유산균 개체수의 차이는 하기 표 2에 나타낸 바와 같이 'A', 'B', 'C'의 공정으로 생산한 경우, 배양액의 식물성유산균 개체수는 각각 8×108 CFU/g, 1×109 CFU/g, 4×109 CFU/g 이었으며, 동결건조 후는 각각 4.3×1010 CFU/g, 8×1010 CFU/g, 2×1011 CFU/g를 나타내었다. 특히 'C' 공정의 경우는 1011 CFU/g을 나타내어 생산성에 있어서 기존 공정인 'A' 공정과 일부 공정이 개선된 'B' 공정에 비해 각각 75%, 33%의 생산성 향상을 나타내었다. The difference in the lactic acid bacteria population according to the conditions of the manufacturing process is produced in the process of 'A', 'B', 'C' as shown in Table 2, the phyto-lactic acid bacteria population of the culture medium is 8 × 10 8 CFU / g, respectively , 1 × 10 9 CFU / g, 4 × 10 9 CFU / g, and after lyophilization, 4.3 × 10 10 CFU / g, 8 × 10 10 CFU / g, and 2 × 10 11 CFU / g, respectively. In particular, the 'C' process showed 10 11 CFU / g, which showed a 75% and 33% improvement in productivity compared to the 'A' process, which was improved from the existing process 'A' and some processes, respectively.
표 2는 Process improving experiment에 관한 것이다.Table 2 relates to the process improving experiment.
또한 ‘A’공정, ‘B’공정 및 ‘C’공정의 배지조성은 글루코오즈(glucose) 25g/L, 효모추출물(yeast ex.) 40g/L, 제2인산칼륨(K2HPO4) 3g/L, 소디움아세테이트(sodium acetate) 3.5g/L, 마그네슘 설페이트(MgSO4) 0.7g/L 이다.
In addition, medium composition of 'A' process, 'B' process and 'C' process consists of 25g / L glucose, 40g / L yeast extract, 3g potassium diphosphate (K 2 HPO 4 ) / L, sodium acetate (3.5 g / L), magnesium sulfate (MgSO 4 ) 0.7 g / L.
ProcessCondition
Process
[실시예 2-3] 배양 후 배양액의 원심분리(C/S) [Example 2-3] Centrifugation (C / S) of the culture solution after the culture
본 발명의 배양 후 배양액의 원심분리 공정(The processing of cell separation)은 다음 도 4와 같다.
The processing of cell separation of the culture solution after the culture of the present invention is as follows.
[실시예 2-4] 동결건조 Example 2-4 Freeze Drying
본 발명의 동결건조 과정(The processing of lyophilization)은 도 5와 같다.
The processing of lyophilization of the present invention is shown in FIG. 5.
[실시예 2-5] 식물성유산균 원말제품 제조Example 2-5 Preparation of Vegetable Lactic Acid Bacteria Powder
본 발명의 원말제품의 제조 공정(The manufacturing process of lactic acid bacteria powder)은 도 6과 같다.
The manufacturing process of the raw product of the present invention (the manufacturing process of lactic acid bacteria powder) is shown in FIG.
[실시예 3] 제품의 최적레시피 기술개발 Example 3 Development of Optimal Recipe Technology for Products
[실시예 3-1] 식물성유산균 이용 최적레시피 개발 Example 3-1 Development of Optimal Recipe Using Vegetable Lactic Acid Bacteria
식물성유산균이 가지고 있는 기능성과 식이섬유 및 올리고당 등의 prebiotics의 기능을 첨가시켜 기호성이 우수한 최적의 레시피를 개발하였다(표 3, 표 4 참조). 관능적인 측면에서 매우 우수한 결과를 나타냄은 표 8에 나타내었다. 하기 표 3은 식물성유산균 이용 최적레시피(Optimum recipe)에 관한 것이다. The optimum recipe with excellent palatability was developed by adding the functionality of plant lactobacillus and prebiotics such as dietary fiber and oligosaccharide (see Table 3 and Table 4). Very good results in terms of sensuality are shown in Table 8. Table 3 below relates to an optimal recipe using phytolactic acid bacteria (Optimum recipe).
하기 표의 사과분말, 석류농축액분말, 레몬과즙분말은 (주)건우에프피(http://www.geonwoofp.com/)에서 구매하여 사용하였다.
Apple powder, pomegranate concentrate powder, and lemon juice powder in the following table were purchased from Kunwoo Fp Co., Ltd. (http://www.geonwoofp.com/) and used.
(%)Compounding ratio
(%)
(1x109cfu/g)Lactobacillus
(1x10 9 cfu / g)
[실시예 3-2] 제품의 유산균수 측정실험[Example 3-2] Lactic Acid Bacteria Measurement Experiment
유산균수 측정은 식품의약품안전청의 식품공전 및 건강기능식품공전에 준하여 측정하며 측정방법은 다음과 같다.
Lactobacillus count is measured in accordance with the Food Code and Health Functional Food Code of the Korea Food and Drug Administration.
1) 측정방법1) Measuring method
BCP 첨가 평판측정용 한천배지(Plate Count Agar with Brom Cresol Purple)
Plate Count Agar with Brom Cresol Purple
Yeast Extract 2.5gYeast Extract 2.5g
Peptone 5.0gPeptone 5.0g
Dextrose 1.0gDextrose 1.0g
Tween 80 1.0gTween 80 1.0g
L - Cysteine 0.1gL-Cysteine 0.1 g
Agar 15.0g
Agar 15.0 g
위의 성분을 증류수 1,000ml에 녹여 pH 6.8-7.0으로 조정한 후 BCP(Brom Cresol purple)을 0.004-0.006%가 되도록 가하고 121℃에서 15분간 멸균한다.
After dissolving the above components in 1,000ml of distilled water to adjust the pH to 6.8-7.0, BCP (Brom Cresol purple) is added to 0.004-0.006% and sterilized for 15 minutes at 121 ℃.
2) 검체의 채취 및 시험용액의 조제2) Sampling and preparation of test solution
일반적인 주의사항으로는 검체를 무균적으로 취급하되, 본 실험에 사용한 분말상 검체는 검체를 멸균유리봉과 멸균스파테르 등으로 잘 혼합한 후 그 일정양(10g)을 멸균용기에 취해 9배양(90g)의 멸균생리식염수(0.85%)와 혼합한 것을 시험용액으로 하였다.
As a general precaution, treat the sample aseptically, but the powdered sample used in this experiment should be mixed well with a sterile glass rod and a sterile spatter, and then put a certain amount (10 g) into a sterile container and incubated 9 times (90 g). The test solution was mixed with sterile physiological saline (0.85%).
3) 희석배수3) dilution factor
조제된 시험원액(10-1)에 대해서는 멸균생리 식염수를 이용하여 예상되는 균수에 따라 희석액을 10배(10-2), 100배(10-3), 1,000배(10-4), 10,000(10-5), 100,000배(10-6) 등으로 만들어 사용 한다.
For the prepared test stock (10 -1 ), dilute the
4) 측정 방법4) How to measure
상기 ‘2) 검체의 채취 및 시험용액의 조제'에서 조제된 시험용액 1ml와 각 단계 희석액 1ml씩을 멸균 페트리디쉬(petri dish 지름 9-10cm, 높이 1.5cm) 2매 이상을 무균적으로 취하여 약 43-45℃로 유지한 상기 1)의 BCP 첨가 평판측정용 한천배지 약 15ml를 무균적으로 분주하고 petri dish 뚜껑에 부착하지 않도록 주의 하면서 조용히 회전하여 좌우로 기울이면서 검체와 배지를 잘 섞고 냉각 응고 시킨다.1 ml of the test solution prepared in '2) Sampling and Preparation of the Test Solution' and 1 ml of each dilution of each step are aseptically taken with at least two sterile Petri dishes (9-10 cm in diameter and 1.5 cm in height). Aseptic dispensing about 15ml of agar plate for measuring BCP added to -45 ℃ maintained at -45 ℃ and carefully rotating and tilting from side to side while being careful not to adhere to the petri dish lid. .
냉각 응고시킨 petri dish는 거꾸로 하여 35-37℃에서 1~3일 배양 후 발생한 황색의 집락(colony)을 유산균의 집락으로 계측한다.The frozen solidified petri dish is inverted and the colony of yellow color generated after 1 to 3 days of incubation at 35-37 ° C is measured as a colony of lactic acid bacteria.
집락수의 계산은 1평판당(petri dish) 30~300개의 집락을 생성한 평판을 택하여 집락수를 계산하는 것을 원칙으로 한다.The number of colonies is calculated based on the number of colonies that produce 30 to 300 colonies per petri dish.
전 평판에 300개 이상 집락이 발생한 경우 300에 가까운 평판에 대하여 밀집평판 측정법에 따라 안지름 9cm의 페트리디쉬인 경우에는 1cm2 내의 평균 집락수에 65를 곱하여 그 평판의 집락수로 계산한다.If the former is the inner diameter of 9cm petri dish, depending on density measurements for flat close to flat at 300, if more than 300 colonies in the flat has occurred within 1cm 2 The average number of colonies is multiplied by 65 to calculate the number of colonies on the plate.
전 평판에 30개 이하의 집락만을 얻었을 경우에는 가장 희석배수가 얕은 것을 측정한다.
If less than 30 colonies are obtained on the entire plate, the lowest dilution factor is measured.
[실시예 3-3] 유산균 측정 실험결과Example 3-3 Results of Lactic Acid Bacteria Measurement
최적 레시피의 유산균수를 측정한 결과를 표 4에 나타내었다. 제품의 유산균수는 1x108 CFU/g 이상을 원칙으로 하고 있다.
Table 4 shows the results of measuring the number of lactic acid bacteria of the optimum recipe. The number of lactic acid bacteria in the product is 1x10 8 CFU / g or more in principle.
[실시예 4] 안정성을 측정하기 위한 내열성 및 내산성 실험Example 4 Heat resistance and acid resistance experiments for measuring stability
[실시예 4-1] 내열성 실험결과[Example 4-1] Heat Resistance Test Results
일반유산균에 비해 식물성유산균인 Lactobacillus fermentum 유산균의 내열성이 거의 2배 이상 높은 것으로 나타났다. 특히, 90℃의 온도에서 측정 시 일반 유산균은 2분 이상 방치 시 모두 사멸해 버리기 때문에 시간을 짧게 하여 비교치를 구하였다(표 5 참조). 하기 표 5는 내열성 실험결과(Heat resistance of vegetable lactic acid bacteria from manufactured product)에 관한 것이다.
The heat resistance of Lactobacillus fermentum lactic acid bacterium, which is a vegetable lactic acid bacterium, was almost two times higher than that of general lactic acid bacteria. In particular, when measured at a temperature of 90 ℃ general lactic acid bacteria are killed when left for more than 2 minutes, so the time was shortened to obtain a comparison value (see Table 5). Table 5 relates to a heat resistance test result (Heat resistance of vegetable lactic acid bacteria from manufactured product).
(×107cfu/g)Early lactic acid bacteria count
(× 10 7 cfu / g)
(×107cfu/g)90 ℃, after 2 minutes
(× 10 7 cfu / g)
(식물성유산균) Lac. fermentum
(Vegetable lactic acid bacteria)
(일반유산균) Lac. acidophilus
(General Lactobacillus)
[실시예 4-2] 내산성 실험결과Example 4-2 Acid Resistance Test Results
염산을 이용하여 체내 pH와 유사한 정도로 조정한 액에 식물성유산균과 일반유산균을 각각 pH 3과 2에 첨가할 경우 어느 정도의 산에 저항할 수 있는지 측정을 하였다. 그 결과, 식물성유산균인 Lactobacillus fermentum 균주는 각각 pH 3.0과 pH 2.0에서 101 과 102의 균수의 저하가 있었지만 생존율은 매우 높은 상태로 존재하였다. 그러나 일반 유산균인 Lactobacillus acidophilus 의 경우는 pH 3.0과 2.0 각각에 대해 약 105 정도씩의 균수의 저하가 있었으며 낮은 생존율을 나타내었다.
Using hydrochloric acid, it was measured how much acid resistance could be achieved by adding lactic acid bacteria and general lactic acid bacteria to
따라서 식물성유산균은 코팅 등 보호제를 사용하지 않고도 산에 견딜 수 있는 강력한 내산을 나타내어 공정개선에도 불구하고 내산의 기능성은 그대로 보유하고 있는 것으로 나타났다(표 6 참조). 하기 표 6은 내산성 실험결과(The result of acid resistance of vegetable lactic acid bacteria from manufactured product)에 관한 것이다.
Therefore, phyto-lactic acid bacteria showed strong acid resistance to acid without using a protective agent such as coating, and despite the improvement of the process, the acid resistance was retained as it is (see Table 6). Table 6 below relates to the results of the acid resistance test (the result of acid resistance of vegetable lactic acid bacteria from manufactured product).
(cfu/g)Initial bacterial count
(cfu / g)
(cfu/g)1 hour later
(cfu / g)
(cfu/g)2 hours later
(cfu / g)
fermentum
(식물성유산균) Lac.
fermentum
(Vegetable lactic acid bacteria)
acidophilus
(일반유산균) Lac.
acidophilus
(General Lactobacillus)
[실시예 5] 생리기능성의 효능 검증Example 5 Verification of efficacy of physiological function
[실시예 5-1] 농도별, 시간별 혈당 측정실험Example 5-1 Blood glucose measurement experiment by concentration and time
각 마우스 모델별로 농도별, 시간별, 날짜별 혈액 채취를 통한 혈당 수치 분석을 통하여 혈당 저하 기능을 분석하여 다음 도 7 내지 도 10과 같은 결과를 얻었다.
The blood glucose lowering function was analyzed by analyzing blood glucose levels by blood concentration by concentration, time, and date for each mouse model, thereby obtaining the results as shown in FIGS. 7 to 10.
1) Normal model (일반 모델)1) Normal model
가) 실험 방법A) Experiment method
일반사료를 먹이고 있는 normal mice에 A, B, C를 1×=1×108, 5×=2×108, 10×=1×109 CFU/g의 농도로 구강투여 후 시간별 30, 60, 120 분으로 꼬리정맥에서 혈액을 채취하여 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정하였다.30, 60 hourly after oral administration of A, B and C in normal mice fed normal diet at concentrations of 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 CFU / g Blood was collected from the tail vein for 120 minutes and blood glucose was measured using an Accuforma Perma glucose meter.
나) 결과B) result
Normal : 혈당이 잠시 상승하다가 원위치 됨Normal: Blood sugar rises for a while and then returns
Normal+Saline : 혈당이 잠시 상승하다가 원위치 됨Normal + Saline: Blood sugar rises for a while and then returns
Normal+A1× : 혈당이 잠시 상승하다가 2시간 후 급격히 하강Normal + A1 ×: Blood sugar rises briefly and then drops rapidly after 2 hours
Normal+A5× : 혈당이 1시간동안 변화 없다가 이후 급격히 하강Normal + A5 ×: Blood sugar does not change for 1 hour, then drops rapidly
Normal+A10× : 30분까지 급격히 상승 후 2시간까지 급격히 하강Normal + A10 ×: Sharply rises up to 30 minutes and then descends rapidly up to 2 hours
Normal+B1× : 1시간동안 거의 변화가 없다가 이후 급격히 하강Normal + B1 ×: Almost no change for 1 hour, then descends sharply
Normal+B5× : 혈당이 완만하게 하강을 2시간까지 계속 하강함Normal + B5 ×: Blood sugar drops slowly until 2 hours
Normal+B10× : 30분까지 급격히 상승 후 2시간까지 급격히 하강Normal + B10 ×: Sharply rises up to 30 minutes and then descends rapidly up to 2 hours
Normal+C1× : 1시간 동안 완만하게 하강 후 2시간까지 하강Normal + C1 ×: Slowly descend for 1 hour and then descend for 2 hours
Normal+C5× : 1시간동안 거의 변화가 없다가 이후 급격히 하강Normal + C5 ×: Almost no change for 1 hour, then descends sharply
Normal+C10× : 30분까지 급격히 상승 후 2시간까지 급격히 하강
Normal + C10 ×: Sharply rises up to 30 minutes and then descends rapidly up to 2 hours
다) 결론C) Conclusion
위의 실험 결과로 식물성 유산균이 일반 마우스에서 혈당강하 효과가 큰 것을 볼 수 있다. 이것은 일반 평범한 상태에서의 혈당 강하효과를 확인 한 것이라 볼 수 있다(도 7 참조). As a result of the above experiment, it can be seen that the lactic acid bacterium has a large hypoglycemic effect in normal mice. This can be seen to confirm the hypoglycemic effect in the normal state (see Figure 7).
도 7은 Blood glucose level test of vegetable lactic acid bacteria에 관한 것이고, 도 7내의 A, B, C는 표 5의 공정개선실험에 있어서의 A, B, C를 의미한다.
Figure 7 relates to the blood glucose level test of vegetable lactic acid bacteria, A, B, C in Figure 7 means A, B, C in the process improvement experiment of Table 5.
2) Fed D-Glucose model (급성 고혈당 모델)2) Fed D-Glucose model
가) 실험 방법A) Experiment method
16시간 금식 후 D-glucose (2g/kg)를 구강 투여한 mice에 A, B, C를 1×=1×108, 5×=2×108, 10×=1×109 CFU/g의 농도로 구강투여 30분 후에 꼬리정맥에서 혈액을 채취하여 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정하였다.
After 16 hours of fasting, A, B, and C were 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 CFU / g in mice orally administered D-glucose (2g / kg). Blood was collected from the
나) 결과B) result
D-Glucose : 투여 후 30분까지 급격히 상승하다 이후 급격히 하강D-Glucose: It rises sharply until 30 minutes after administration and then drops rapidly
D-Glucose+Saline : 투여 후 30분까지 급격히 상승 후 급격히 하강D-Glucose + Saline: It rises sharply until 30 minutes after administration and then drops rapidly
D-Glucose+A1× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + A1 ×: rises to 30 minutes after administration and then descends
D-Glucose+A5× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + A5 ×: rises to 30 minutes after administration and then descends
D-Glucose+A10× : 투여 후 30분까지 급격히 상승 후 급격히 하강D-Glucose + A10 ×: Sharply rises to 30 minutes after administration and then drops rapidly
D-Glucose+B1× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + B1 ×: Raises up to 30 minutes after administration and then descends
D-Glucose+B5× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + B5 ×: rises to 30 minutes after administration and then descends
D-Glucose+B10× : 투여 후 30분까지 급격히 상승 후 급격히 하강D-Glucose + B10 ×: Sharply rises up to 30 minutes after administration and then descends rapidly
D-Glucose+C1× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + C1 ×: Rise up to 30 minutes after administration and then descend
D-Glucose+C5× : 투여 후 30분까지 상승하다 이후 하강D-Glucose + C5 ×: Rise up to 30 minutes after administration and then descend
D-Glucose+C10× : 투여 후 30분까지 급격히 상승 후 급격히 하강
D-Glucose + C10 ×: Raises rapidly up to 30 minutes after administration and then drops rapidly
다) 결론C) Conclusion
D-Glucose가 혈당 상승에 작용할 때 식물성유산균 B와 C가 일정부분 제어 하는 것을 볼 수 있다. 이것은 급성으로 고혈당을 유발된 모델에서의 혈당 제어 효과를 입증한 것이라 볼 수 있다(도 8참조). 도 8내의 A, B, C는 표 5의 공정개선실험에 있어서의 A, B, C를 의미한다.
Phytolactic acid bacteria B and C control some of the effects of D-Glucose on blood sugar levels. This can be seen to demonstrate the blood sugar control effect in a model that induced acute hyperglycemia (see Fig. 8). A, B, and C in FIG. 8 mean A, B, and C in the process improvement experiment of Table 5. FIG.
3) Diabetes model (당뇨 모델)3) Diabetes model
가) 실험 방법A) Experiment method
Streptozotocin (STZ)으로 마우스에 복강 주사를 하고 일주일간 당뇨를 유도하여 식물성 유산균 A, B, C를 1×=1×108, 5×=2×108, 10×= 1×109 CFU/g의 농도로 구강투여 후 시간별 (30, 60, 120 min)로 꼬리정맥에서 혈액을 채취하여 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정하였다.
Intraperitoneal injection of mice with Streptozotocin (STZ) followed by induction of diabetes for 1 week, resulting in 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 CFU / After oral administration at a concentration of g, blood was collected from the tail vein at hourly intervals (30, 60, 120 min) and blood glucose was measured using an Accuform Performa glucose meter.
나) 결과B) result
Diabetes : 당뇨를 유도한 상태로 시간 경과 후 약간의 혈당이 하락Diabetes: A slight drop in blood sugar after a period of time with diabetes
Diabetes+Saline : 당뇨를 유도, 시간이 경과 후 약간의 혈당 하락Diabetes + Saline: Induces diabetes, slightly decreases blood sugar over time
Diabetes+A1× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음 Diabetes + A1 ×: Significant drop in blood sugar
Diabetes+A5× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음Diabetes + A5 ×: Significant drop in blood sugar
Diabetes+A10× : 혈당이 상승 하다가 크게 하락하였음 Diabetes + A10 ×: Blood sugar rises and then drops significantly
Diabetes+B1× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음Diabetes + B1 ×: Significant drop in blood sugar
Diabetes+B5× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음Diabetes + B5 ×: Significant drop in blood sugar
Diabetes+B10× : 혈당이 상승 하다가 크게 하락하였음Diabetes + B10 ×: Blood sugar rises and then drops significantly
Diabetes+C1× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음Diabetes + C1 ×: Significant drop in blood sugar
Diabetes+C5× : 혈당이 크게 하락 하는 것을 확연히 볼 수 있음Diabetes + C5 ×: Significant drop in blood sugar
Diabetes+C10× : 혈당이 상승 하다가 크게 하락하였음
Diabetes + C10 ×: Blood sugar rises and then drops significantly
다) 결론C) Conclusion
Streptozotocin(STZ)이 유발한 당뇨 마우스에서 식물성유산균 A, B, C가 혈당 강하 효과를 보임을 확인할 수 있었다. 이것은 당뇨가 유발되어 혈당 제어 능력을 잃어버린 모델에서의 혈당 제어 효과를 입증한 것이라 볼 수 있다. 따라서 일반 사람뿐만 아니라 당뇨환자에 이르기까지 식물성 유산균의 혈당 강하효과를 기대할 수 있음을 시사하였다(도 9 참조). 도 9내의 A, B, C는 표 5의 공정개선실험에 있어서의 A, B, C를 의미한다.
Phytolactic acid bacteria A, B, C showed hypoglycemic effect in Streptozotocin (STZ) -induced diabetic mice. This proved to be effective in controlling blood glucose in a model in which diabetes induced loss of blood sugar control. Therefore, it was suggested that the hypoglycemic effect of the plant lactic acid bacteria can be expected from diabetic patients as well as the general person (see Fig. 9). A, B and C in FIG. 9 mean A, B and C in the process improvement experiment of Table 5. FIG.
4) 농도별, 날짜별 실험 - Normal model 4) Experiment by concentration and date-Normal model
가) 실험 방법A) Experiment method
일반사료를 먹이고 있는 mice에 A, B, C를 1×=1×108, 5×= 2×108, 10×=1×109 CFU/g의 농도로 구강투여 후 구강투여 후 날짜별 1, 3, 7, 10, 14일로 꼬리정맥에서 혈액을 채취하여 아큐첵 퍼포마 혈당계를 이용하여 혈당을 측정하였다.
A, B, and C were fed orally at the concentrations of 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 CFU / g in mice fed normal feed, followed by date after oral administration. Blood was collected from the tail vein at 1, 3, 7, 10, and 14 days, and blood glucose was measured using an Accuform Perma glucose meter.
나) 결과B) result
Normal : 혈당이 잠시 상승하다가 원위치 됨Normal: Blood sugar rises for a while and then returns
Normal+Saline : 혈당이 잠시 상승하다가 원위치 됨Normal + Saline: Blood sugar rises for a while and then returns
Normal+A1× : 혈당이 잠시 상승하다가 3일째부터 감소하기 시작 하여 정상범위로 돌아옴Normal + A1 ×: Blood sugar rises briefly, then decreases from day 3 and returns to normal range
Normal+A5× : 혈당이 3일째 잠시 상승했다가 평균수준으로 돌아 옴Normal + A5 ×: Blood sugar rises for 3 days and then returns to average level
Normal+A10× : 혈당이 7일째부터 하강하기 시작하여 평균으로 돌아옴Normal + A10 ×: Blood sugar begins to fall on
Normal+B1× : 혈당이 대조군에 비해 낮은 상태를 유지함.Normal + B1 ×: Blood sugar level is lower than that of the control group.
Normal+B5× : 혈당이 대조군에 비해 낮은 상태를 유지함.Normal + B5 ×: Blood sugar level is lower than that of the control group.
Normal+B10× : 혈당이 초반에 상승했다가 점차 하강하기 시작하여 정상범위로 돌아옴.Normal + B10 ×: Blood sugar rises at the beginning and then gradually descends to return to normal range.
Normal+C1× : 혈당이 대조군에 비해 낮은 상태를 유지함.Normal + C1 ×: Blood sugar level is lower than control.
Normal+C5× : 혈당이 대조군에 비해 낮은 상태를 유지함.Normal + C5 ×: Blood sugar level is lower than control.
Normal+C10× : 혈당이 3초반에 상승했다가 점차 하강하기 시작하여 정상범위로 돌아옴.
Normal + C10 ×: Blood sugar rises in the early 3 seconds, then begins to descend gradually and returns to normal range.
다) 결론C) Conclusion
위의 실험 결과로 식물성 유산균의 장기간 투여시에도 혈당강하 효과가 큰 것을 볼 수 있다. 하지만 고농도의 유산균보다 저농도의 유산균에서 혈당 강하효과가 큰 것을 확인 하였다. 따라서, 장기간의 유산균 투여시에는 낮은 농도로 꾸준히 유산균을 섭취하였을 때 혈당 강하효과가 탁월함을 알 수 있다(도 10참조).
As a result of the above experiment, it can be seen that the hypoglycemic effect is great even in the long-term administration of vegetable lactic acid bacteria. However, it was confirmed that blood glucose lowering effect was higher in low concentration of lactic acid bacteria than high concentration of lactic acid bacteria. Therefore, when the lactic acid bacteria are administered for a long time, it can be seen that the blood glucose lowering effect is excellent when the lactic acid bacteria are continuously ingested at a low concentration (see FIG. 10).
[실시예 5-2] 장통과 시간 측정방법Example 5-2 Method of Measuring Passage Time
1)농도별, 날짜별 장통과 시간 측정실험 1) Measurement of longevity and time by concentration and date
가) 실험 방법A) Experiment method
각각의 유산균을 생리식염수에 녹여 A, B, C를 1×=1×108, 5×= 2×108, 10×=1×109 CFU/g의 농도로 총 14일에 걸쳐 매일 한 번씩 경구 투여하고, 1, 3, 7, 10, 14일에는 Marker인 Carmine red를 0.5%의 농도로 각 유산균 시료에 첨가하여 투여하였다. Each lactobacillus was dissolved in physiological saline, and A, B, and C were added daily at a concentration of 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 CFU / g for a total of 14 days. Orally administered once, 1, 3, 7, 10, 14 days carmine red was added to each lactic acid bacteria sample at a concentration of 0.5%.
도 11, 도 12, 도 13의 그래프에 나타내었듯이 14일 동안에 장통과 시간을 측정하였으며, 이는 급여시작 시간과 marker가 변 중에 처음 나타나기까지의 시간 간격으로 하였다.
As shown in the graphs of FIGS. 11, 12, and 13, the length of intestinal pain was measured for 14 days, which was the time between the start of salary and the time interval between the first appearance of the marker.
나) 결과B) result
① 1×=1×108 CFU/g (도 11 참조)① 1 × = 1 × 10 8 CFU / g (see Fig. 11)
Saline : 일반적인 장통과 시간Saline: common pain and time
A : 식물성유산균을 투입 했을 때 3일째에 일반적인 장통과 시간보다 2배정도 시간이 단축되었다가 점차 평균정도로 돌아가는 것을 확인A: When phyto-lactic acid bacteria were added, the time was shortened by about twice as much as the general intestinal pain time on the third day, and then gradually returned to the average level.
B : 식물성유산균을 투입 했을 때 3일째에 일반적인 장통과 시간보다 2배정도 시간이 단축되었다가 점차 평균정도로 돌아가는 것을 확인B: When the plant lactic acid bacteria were added, the time was shortened by about twice as much as the general intestinal pain time on the third day, and then gradually returned to the average level.
C : 식물성유산균을 투입 했을 때 시간이 지날수록 일반적인 장통과 시간보다 단축되는 것을 확인
C: When vegetable lactic acid bacteria are added, it is confirmed that the time is shorter than the general intestinal pain and time.
② 5×=2×108 CFU/g (도 12 참조)② 5 × = 2 × 10 8 CFU / g (see Fig. 12)
Saline : 일반적인 장통과 시간Saline: common pain and time
A : 식물성유산균을 투입 했을 때 3일째에 일반적인 장통과 시간보다 2배정도 시간이 단축되었다가 점차 평균정도로 돌아가는 것을 확인 A: When phyto-lactic acid bacteria were added, the time was shortened by about twice as much as the general intestinal pain time on the third day, and then gradually returned to the average level.
B : 식물성유산균을 투입 했을 때 꾸준하게 평균시간을 유지하는 것을 확인B: Confirm that the average time is maintained steadily when phyto-lactic acid bacteria are added
C : 식물성유산균을 투입 했을 때 꾸준하게 평균시간을 유지하는 것을 확인
C: Confirm that the average time is maintained steadily when phyto-lactic acid bacteria are added
③ 10×=1×109 CFU/g (도 13 참조)③ 10 × = 1 × 10 9 CFU / g (see FIG. 13)
Saline : 일반적인 장통과 시간Saline: common pain and time
A : 식물성유산균을 투입 했을 때 7일째부터 일반적인 장통과 시간보다 시간이 유의성있게 단축되는 것을 확인 A: When phyto-lactic acid bacteria were added, the time was significantly shorter than the general intestinal pain time from the 7th day.
B : 식물성유산균을 투입 했을 때 7일째부터 일반적인 장통과 시간보다 시간이 유의성있게 단축되는 것을 확인B: When phyto-lactic acid bacteria were added, the time was significantly shorter than the general intestinal pain time from
C : 식물성유산균을 투입 했을 때 7일째부터 일반적인 장통과 시간보다 시간이 유의성있게 단축되는 것을 확인.
C: When phyto-lactic acid bacteria were added, the time was significantly shorter than the general intestinal pain time from the 7th day.
다) 결론C) Conclusion
장통과 시간을 측정함에 있어서 장통과 시간이 빠르다는 것은 장의 활동이 활발하다는 것과 배변 활동이 원활하다는 것을 의미하며, 이 실험결과로 미루어 장활동을 활발히 해줌으로써 배변활동을 좋게 하여 변비예방이나 변비에 효과를 나타낼 수 있다는 것을 판단할 수 있다(도 11, 도 12, 도 13 참조).
In the measurement of intestinal pain and time, the fast intestine and time means that the intestinal activity is active and the bowel movement is smooth.The result of this experiment is that the bowel activity is improved by improving the bowel activity to prevent constipation or constipation. It can be determined that the effect can be obtained (see FIGS. 11, 12, and 13).
이 결과들로 보아 저농도에서는 3일째에 효과가 가장 좋고, 고농도에서는 장시간 투여할수록 점차 장의 활동이 좋아지는 것을 확일 할 수 있었다. 따라서, 식물성 유산균을 꾸준히 섭취하였을 경우 최소 3일째부터 점차 장의 운동이 활발해짐을 확인할 수 있다.
From these results, it was confirmed that the effect was the best at day 3 at low concentrations, and the intestinal activity gradually improved with prolonged administration at high concentrations. Therefore, when the vegetable lactic acid bacteria are ingested steadily, the bowel movement gradually becomes active from at least three days.
[실시예 5-3] 베타 엔돌핀(β-endorphin) 측정실험Example 5-3 Beta Endorphin (β-endorphin) Measurement Experiment
1) 농도별, 1회 투여 90분 후 β-endorphin 발현 측정실험(1회 투여 90분 후 β-endorphin 발현정도 측정 실험)
1) β-endorphin expression measurement test after 90 minutes of single administration by concentration (Test of β-endorphin expression level after 90 minutes of single administration)
가) 실험 방법A) Experiment method
각각의 유산균을 생리식염수에 녹여 위의 농도로 경구 투여하고, 90분 후에 안와 정맥에서 혈액을 채취하여, 원심분리기를 이용해 혈청을 분리하였다. 이렇게 얻어진 혈청으로 베타 엔돌핀 키트를 이용하여 각 혈청 속의 β-endorphin 수치를 측정하였다.
Each lactic acid bacterium was dissolved in physiological saline and orally administered at the above concentration. After 90 minutes, blood was collected from the orbital vein, and serum was separated using a centrifuge. The beta endorphin kit was used to measure β-endorphin levels in each serum.
나) 결과B) result
식물성 유산균 A, B, C 각각은 1회 투여 후에는 대조군에 비교할 때β-endorphin의 수치가 증가하지 않는 것으로 확인 되었다.
Plant Lactobacillus A, B, and C each did not increase the β-endorphin levels after one administration compared to the control group.
다) 결론C) Conclusion
β-endorphin이 분비된 다는 것은 면역역 향상에 도움이 된다는 것을 의미한다. 본 결과로 식물성 유산균의 1회 투여로는 베타 엔돌핀의 분비가 증가되지 않는 것으로 보아 면역력 향상에 대한 효과가 있다고 기대하기 어렵다(도 14 참조).
The secretion of β-endorphin suggests that it helps to boost the immune system. As a result, it is difficult to expect that there is no increase in the secretion of beta endorphin by one administration of the plant lactic acid bacteria, which is effective in improving immunity (see FIG. 14).
2) 농도별, 날짜별 β-endorphin 발현정도 측정 실험2) Experiment of measuring the expression level of β-endorphin by concentration and date
가) 실험 방법A) Experiment method
각각의 유산균을 생리식염수에 녹여 위의 A, B, C를 1×=1×108, 5×=2×108, 10×=1×109의 농도로 경구 투여하고, 1, 7, 14일째 투여 90분 후에 안와 정맥에서 혈액을 채취하여, 원심분리기를 이용해 혈청을 분리하였다. 이렇게 얻어진 혈청으로 β-endorphin 키트를 이용하여 각 혈청 속의 β-endorphin 수치를 측정하였다.
Each lactobacillus was dissolved in physiological saline, and A, B, and C were orally administered at concentrations of 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 , and 1, 7, 90 minutes after the 14th day, blood was collected from the orbital vein, and serum was separated using a centrifuge. The serum thus obtained was measured for β-endorphin levels in each serum using the β-endorphin kit.
나) 결과B) result
A, B, C 유산균 모두에서 아래와 같은 결과를 확인할 수 있었다. The following results were confirmed in all of A, B, C lactic acid bacteria.
1×=1×108 : 투여 7일째부터 약간의 상승을 보이며, 14일째에는 두드러지는 분비 상승을 볼 수 있었음 (도 15 참조)1 × = 1 × 10 8 : Slight increase was observed from
5×=2×108 : 투여 7일째부터 약간의 상승을 보이며, 14일째에는 두드러지는 분비 상승을 볼 수 있었음 (도 16 참조) 5 × = 2 × 10 8 : Slight increase was observed from
10×=1×109 : 투여 7일째부터 약간의 상승을 보이며, 14일째에도 상승정도가 계속 유지됨을 볼 수 있었음 (도 17 참조)
10 × = 1 × 10 9 : A slight increase was observed from the 7th day of administration, and the increase was maintained even on the 14th day (see FIG. 17).
다) 결론C) Conclusion
도 15, 도 16, 도 17에서 볼 수 있듯이 1회의 식물성 유산균 투여시에는 β-endorphin의 분비 증가를 볼 수 없었지만, 7일 이상 장기간 식물성 유산균 투여시 확실히 β-endorphin의 분비 증가를 확인할 수 있었다. 이로써, 식물성 유산균의 장시간 투여가 면역력의 증강에 효과가 있음을 기대할 수 있다.
As shown in FIGS. 15, 16, and 17, the increase in secretion of β-endorphin was not observed in one administration of vegetable lactic acid bacteria, but the increase in secretion of β-endorphin was surely observed in the administration of vegetable lactic acid bacteria for a long time over 7 days. Thus, long-term administration of the plant lactic acid bacteria can be expected to be effective in enhancing the immunity.
[실시예 5-4] POMC 유전자 발현 측정 실험(농도별, 날짜별 POMC 유전자 발현 측정실험) [Example 5-4] POMC gene expression measurement experiment (concentration, POMC gene expression measurement experiment by date)
1) 시상하부에서의 POMC 유전자 발현 관찰1) Observation of POMC Gene Expression in Hypothalamus
가) 실험 방법 A) Experiment method
각각의 유산균을 생리식염수에 녹여 A, B, C를 1×=1×108, 5×= 2×108, 10×=1×109의 농도로 구강 투여하고 90분 후에 마우스의 뇌를 축출하여 뇌의 시상하부 부위를 선별하여 mRNA를 추출하였다. 그리고 real time RT-PCR 방법을 이용하여 시상하부에서의 POMC 유전자 수치를 분석하여 데이터를 산출하여 그 결과를 도 18에 나타내었다. Each lactobacillus was dissolved in physiological saline, and A, B, and C were orally administered at a concentration of 1 × = 1 × 10 8 , 5 × = 2 × 10 8 , 10 × = 1 × 10 9 , and 90 minutes after The hypothalamus of the brain was removed and mRNA was extracted. In addition, the real time RT-PCR method was used to analyze POMC gene values in the hypothalamus to calculate data, and the results are shown in FIG. 18.
나) 결과B) result
세가지 그룹 모두에서 베타 엔도르핀의 전구체인 POMC 유전자가 시상하부 부위에서 두드러지게 증가한 것을 확인 할 수 있었다. In all three groups, the POMC gene, a precursor of beta endorphin, was markedly increased in the hypothalamus region.
다) 결론C) Conclusion
세가지 식물성 유산균 모두에서 시상하부의 POMC 유전자가 두드러지게 증가하는 것으로 미루어 보아, 항스트레스, 쾌감, 면역력 증진, 통증 억제 등의 가능성을 가짐을 알 수 있다(도 18 참조).
Considering that the hypothalamic POMC gene is significantly increased in all three vegetable lactic acid bacteria, it can be seen that it has the possibility of antistress, pleasure, immunity enhancement, pain suppression, etc. (see FIG. 18).
2) 뇌하수체에서의 POMC 유전자 발현 관찰2) POMC gene expression in the pituitary gland
가) 실험 방법 A) Experiment method
각각의 유산균을 생리식염수에 녹여 A, B, C를 1×= (1×108), 2× = (2×108), 10×= (1×109)의 농도로 구강 투여하고 90분 후에 마우스의 뇌를 적출하여 뇌의 뇌하수체 부위를 선별하여 mRNA를 추출하였다. 그리고 real time RT-PCR 방법을 이용하여 뇌하수체에서의 POMC 유전자 수치를 분석하여 그 결과를 도 19에 나타내었다.Each lactobacillus was dissolved in physiological saline, and A, B, and C were orally administered at concentrations of 1 × = (1 × 10 8 ), 2 × = (2 × 10 8 ), and 10 × = (1 × 10 9 ). Minutes later, the brains of mice were extracted, the pituitary parts of the brain were selected, and mRNA was extracted. And the result of analyzing the POMC gene levels in the pituitary gland using a real time RT-PCR method is shown in Figure 19.
나) 결과B) result
세가지 그룹 모두에서 베타 엔도르핀의 전구체인 POMC 유전자가 뇌하수체 부위에서 두드러지게 증가한 것을 확인 할 수 있었다.
In all three groups, the POMC gene, a precursor of beta endorphin, increased significantly in the pituitary gland.
다) 결론C) Conclusion
세가지 식물성 유산균 모두에서 뇌하수체의 POMC 유전자가 두드러지게 증가하는 것으로 미루어 보아, 항스트레스, 쾌감, 면역력 증진, 통증 억제 등의 가능성을 가짐을 알 수 있다(도 19 참조).
Considering the marked increase in the pituitary gland's POMC gene in all three plant lactic acid bacteria, it can be seen that it has the potential of antistress, pleasure, immunity enhancement, pain suppression, etc. (see FIG. 19).
[실시예 5-5] 장내 점착능 측정 실험Example 5-5 Intestinal Adhesion Measurement Experiment
1) 실험 방법1) Experiment Method
김 등의 방법(Korean J Food SCI Technol. Vol. 36, N0. 6, pp. 959~967, 2004)에 의해 Biotinylated surface lectin-like protein(SLP)의 조제는 Biotin protein labeling kit (Roche, cat# 11 418 165 001)를 사용하여 방법대로 조제하였다. 희석(elution)된 4개 그룹의 값을 측정하여 평균값을 비교하였고, 그래프의 수치는 Fraction 1, 2, 3, 4의 4개 그룹 값의 평균값을 나타내었다(2회 각각 실험을 수행하여 결과 값의 평균을 나타내었음).
Preparation of biotinylated surface lectin-like protein (SLP) by the method of Kim et al. (Korean J Food SCI Technol.Vol. 36, N0.6, pp. 959 ~ 967, 2004) was performed by Biotin protein labeling kit (Roche, cat #). 11 418 165 001). The mean values were compared by measuring the values of the four diluted groups, and the numerical values of the graphs represent the average values of the four groups of
2) 결과2) results
동일 조건하에서 비교한 A, B, C 세 균주의 장내 점착능은 상대적으로 C가 가장 우수하며 A와 B는 동일함을 알 수 있고, A와 C 균주를 비교한 결과 통계적으로 유의한(p<0.05) 차이를 보였다 (1way ANOVA, post test: Turkey). 그러나 A vs B, B vs C 는 통계적으로 유의하지 않았다.
Intestinal cohesion of three strains A, B, and C under the same conditions showed that C was the best and that A and B were the same, and A and C strains were statistically significant (p < 0.05) (1way ANOVA, post test: Turkey). However, A vs B and B vs C were not statistically significant.
3) 결론 3) Conclusion
유산균의 점착능이 높다는 것은 그만큼 장내부에 잘 부착될 수 있다는 것을 의미한다. 유산균이 장내부에 잘 부착되면, 그만큼 장 내부에 자리하고 있는 시간이 길어지면서 유산균의 여러 유용한 역할을 잘 해나갈 수 있다는 것을 의미한다. 이 실험을 통해 세가지 균주의 점착능이 우수함을 확인 할 수 있었고, 그중에서 C의 점착능이 아주 우수함을 확인 할 수 있었다(표 7 및 도 20 참조). 하기 표 7은 The OD value of colonic mucin-binding assay에 관한 것이다.
The high adhesion of lactic acid bacteria means that it can be adhered well to the intestines. If lactic acid bacteria are well adhered to the intestine, it means that the longer the time spent in the intestines can play various useful roles of lactic acid bacteria. Through this experiment, it could be confirmed that the adhesion of the three strains was excellent, of which the adhesion of C was very excellent (see Table 7 and Figure 20). Table 7 below relates to The OD value of colonic mucin-binding assay.
[실시예 6] 시제품의 기호성 평가(관능검사)Example 6 Evaluation of Palatability of Prototype (Sensory Test)
시제품을 관능검사하기 위해 인근의 대학을 방문하여 남학생 8명, 여학생 52명을 포함하여 총 60명을 대상으로 간단한 식물성유산균의 설명 후에 12문항의 설문을 실시하여 그 결과를 선호 인원 및 백분율로 나타내어 하기 표 8 및 표 9에 나타내었다.
To test the sensory test of the prototype, a brief questionnaire was given to 60 people including 8 male students and 52 female students, followed by a 12-question questionnaire. Table 8 and Table 9 below.
(86.7%)52 people
(86.7%)
(10%)6 people
(10%)
(3.3%)2 people
(3.3%)
(6.7%)4 people
(6.7%)
(80%)48 people
(80%)
(11.6%)7 people
(11.6%)
(1.7%)1 person
(1.7%)
(31.7%)19 people
(31.7%)
(63.3%)38 people
(63.3%)
(5%)3 people
(5%)
가격 최대 상한선은 어느 정도로 생각? 'Intestinal Lactic Acid Bacteria'
To what extent do you think the price upper limit is?
(6.7%)4 people
(6.7%)
(20%)12 people
(20%)
(26.7%)16 people
(26.7%)
(31.6%)19 people
(31.6%)
(11.7%)7 people
(11.7%)
(3.3%)2 people
(3.3%)
(5%)3 people
(5%)
(63.3%)38 people
(63.3%)
(28.3%)17 people
(28.3%)
(3.4%)2 people
(3.4%)
(60%)36 people
(60%)
(33.3%)23 persons
(33.3%)
(1.7%)1 person
(1.7%)
디자인에 대한 생각은?'Neighboring Vegetable Lactobacillus' Package (for Packaging)
What do you think about design?
(5.0%)3 people
(5.0%)
(60%)36 people
(60%)
(33.3%)20 people
(33.3%)
(1.7%)1 person
(1.7%)
(16.7%)10 people
(16.7%)
(15%)9 persons
(15%)
(16.7%)10 people
(16.7%)
(16.6%)10 people
(16.6%)
(35%)21 people
(35%)
(3.3%)2 people
(3.3%)
(20%)12 people
(20%)
(16.7%)10 people
(16.7%)
(13.3%)8 people
(13.3%)
(46.7%)28 people
(46.7%)
(23.3%)14 people
(23.3%)
(53.3%)32 persons
(53.3%)
(15%)9 persons
(15%)
(8.3%)5 people
(8.3%)
(60%)36 people
(60%)
(5%)3 people
(5%)
(5%)3 people
(5%)
(5%)3 people
(5%)
(25%)15 people
(25%)
제품을 주변인 에게 추천해줄 의향은?'Intestinal Lactic Acid Bacteria'
How would you recommend the product to those around you?
(3.3%)2 people
(3.3%)
(45%)27 people
(45%)
(35%)21 people
(35%)
(15%)9 persons
(15%)
(1.7%)1 person
(1.7%)
중요한 결과로서 ‘유산균 제품 구매의 주된 요인은?’ 장이 안좋아서서 약 87%이었으며, 본 시제품은 맛과 양에서 각각 약 70%, 60% 선호를 나타내어 좋은 결과를 나타내었다.
As an important result, 'what are the main factors for the purchase of lactic acid bacteria products?' The intestine was poor, about 87%, and the prototype showed good results with about 70% and 60% preference in taste and quantity, respectively.
이상에서 첨부된 도면을 참조한 실시예에 의거하여 구체적으로 설명하였다. 그러나 이는 본 발명을 예시하기 위한 것으로 본 발명의 권리범위를 이에 한정하고자 하는 것은 아니고, 이들을 대체할 수 있는 다양한 균등물이 존재한다. 또한 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 안 되고, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.
On the basis of the embodiments with reference to the accompanying drawings it was described in detail. However, this is not intended to limit the scope of the present invention to this intended to illustrate the present invention, there are various equivalents that can replace them. In addition, the terms or words used in the specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.
식물성유산균울 이용하여 생리기능성 측면에서 면역력향상, 혈당강하, 장기능 활성 등이 탁월한 제품을 생산할 수 있으므로 산업상이용가능성이 있다. 이에 일본 및 유럽등 세계적으로 유산균은 기능성 식품군에서 가장 각광받고 있는 소재로, 단순한 식품차원을 넘어 질병의 치료등 의학적으로 작용 할 수 있는 방향으로 다양한 연구개발이 이루어지고 있으나 국내적으로는 창의적이고 순발력 있는 기술개발과 소비자에 대한 신뢰제고 등이 아직은 부족한 상태이므로 본 발명을 하기에 이르렀다.Phytolactic acid bacteria can be used in the industry because it can produce products with excellent immunity improvement, hypoglycemia, and intestinal function in terms of physiological function. As a result, lactic acid bacteria are the most popular materials in the functional food group in Japan and Europe. The present invention has reached the present state because there is still a lack of technology development and the improvement of trust for the consumer.
즉 본 발명을 통해 최적의 배양공정 및 제품의 최적의 레시피를 통해 생산성 향상 및 기능성 향상을 기대 할 수 있고, 경제적으로는 수율의 향상으로 동일의 비용으로 생산성이 50%이상 향상됨으로 경제적으로 비용을 절감 할 수 있으며, 나아가 생리기능성 향상으로 제품의 고급화 및 신뢰성을 제고 할 수 있으므로 산업상이용가능성이 우수하다고 할 것이다.In other words, through the present invention can be expected to improve productivity and functionality through the optimal recipe of the optimal cultivation process and products, economically improved cost by improving the yield at the same cost more than 50% at the same cost economically It can be reduced, and furthermore, it can be said that industrial availability is excellent because it can improve product quality and reliability by improving physiological function.
나아가 본 발명은 건강기능성 식품분야, 일반식품분야, 축산사료분야, 수산사료분야, 친환경농업분야, 환경개선분야에 적용 할 수 있으며, 본 발명을 토대로 한 식물성유산균을 함유하는 기능석식품은 편의점 등에서 판매될 예정이며, 마케팅이 적절히 수반된다면 경제적 효과는 물론이고 산업적으로도 고용창출의 효과가 매우 클것으로 예측된다. 나아가 궁극적으로는 국민건강 향상에 도움을 줄 수도 있으므로 산업상이용가능성이 매우 우수하다고 할 수 있다.
Furthermore, the present invention can be applied to health functional food field, general food field, livestock feed field, fishery feed field, environment-friendly agriculture field, environmental improvement field, and functional stone food containing vegetable lactic acid bacteria based on the present invention in convenience stores, etc. It is expected to be sold and, if properly accompanied by marketing, it is expected that the effect of job creation will be very high not only economically but also industrially. Furthermore, industrial availability is very good because it can ultimately help to improve public health.
Claims (4)
(1) adding purified water to the culture tank; Putting the culture medium into the culture tank, sterilizing for 14 minutes to 16 minutes at 120 ℃ ~ 122 ℃; Inoculating 1 wt% to 2 wt% of Lactobacillus fermentum ( Lactobacillus fermentum , KCCM 10499) when the temperature of the culture tank drops to 41 ° C. to 43 ° C. after sterilization; And maintaining the pH of the culture tank inoculated with the Lactobacillus pertumtum at 5.45 to 5.50, the temperature at 41.5 ° C. to 42 ° C., the RPM to 39 to 41, and the pressure at 0.49 kgf / cm 2 to 0.51 kgf / cm 2 . Making; It contains 49.2wt% to 49.4wt% of Lactobacillus fermentum incubated with (2), 7.1wt% to 7.3wt% of apple powder, and (3) 0.4wt% to 0.6wt% of pomegranate concentrate powder. , (4) 0.5wt% ~ 0.7wt% lemon and juice powder, (5) 24.0wt% ~ 24.2wt% oligosaccharide, (6) 1.4wt% ~ 1.6wt% vitamin C, ( 7) A functional food containing Lactobacillus fermentum, characterized in that it contains 4.7 wt% to 4.9 wt% of skim milk powder, and (8) contains 11.9 wt% to 12.1 wt% of dietary fiber.
According to claim 3, wherein the functionality is anti-stress, pleasure, immunity enhancement and pain suppression, characterized in that any one or more selected from the group consisting of, functional food containing Lactobacillus fermentum.
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