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1 pissn eissn J. Milk Sci. Biotechnol. 2018;36(3): ARTICLE 이봄이 1 이해창 1 문용일 2 오세종 3 1 삼익유가공, 2 우석대학교동물자원식품학과, 3 전남대학교동물자원학부 Development of a Functional Mixed-Starter Culture for Kefir Fermentation Bomee Lee 1, Hae-Chang Yi 1, Yong-II Moon 2, and Sejong Oh 3 1 Samik Dairy & Food Co. Ltd., 453 Yeoksam-ro, Seoul, Korea 2 Dept. of Animal Source Food, Wooseok University, Wanju, Korea 3 Division of Animal Science, Chonnam National University, Gwangju, Korea Received: September 19, 2018 Revised: September 22, 2018 Accepted: September 26, 2018 *Corresponding author : Sejong Oh Division of Animal Science, Chonnam National University, Gwangju, Korea Tel : Fax : soh@chonnam.ac.kr Copyright 2018 Korean Society of Milk Science and Biotechnology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ORCID Bomee Lee Hae-Chang Yi Yong-II Moon Sejong Oh Abstract Kefir, which originates in the Caucasian mountains, is a cultured milk beverage produced by a combination of acidic and alcoholic fermentation. Kefir products are commonly used as food vehicles to deliver health-promoting materials including kefran and lactic acid bacteria to consumers. The aim of this study was to develop a freeze-dried starter culture without yeast and assess the suitability of kefir-like dairy products for the growth of lactic acid bacteria and the acidification of milk. Pasteurized whole milk (SNF 8.5%) stored at 25 was aseptically inoculated with starter cultures (0.002% w/v); it was kept at 25 until the ph attained a value of 4.6. Ten grams of the kefir-like product sample was diluted with 90 ml of 0.15% peptone water diluent in a milk dilution bottle, followed by uniform mixing for 1 min. Viable cells of Lactobacillus species were enumerated on modified-mrs agar (ph 5.2), with incubation at 37 for 48 h. Viable cells of Lactococcus species were enumerated on M17-lactose agar, with incubation at 32 for 48 h. The ph attained a value of 4.6 after fermentation for 9 h 30 min (Starter 1), 9 h 45 min (Starter 2), and 12 h (Starter 3). The viable cell count of Lactobacillu sp. and Lactococcus sp. was initially CFU/g; it increased significantly to 10 9 CFU/g after 12 h of incubation. During the storage of the kefir-like products at 4 for 1 4 days, the total viable cell numbers were unchanged, but the ph decreased slightly. The consistency of the kefir products increased gradually during the storage. The organoleptic properties of the kefir products fermented using the new starter culture are more desirable than those of commercial kefir. These results suggest that the newly developed starter culture without yeast could be suitable for kefir fermentation. Keywords kefir, starter culture, probiotics, fermentation 서론 요구르트와 Kefir 는코카서스산맥의 Elbrus 산에서시작되었는데, 남쪽지역은요구르트, 북쪽지역은 Kefir 의탄생지로알려져있다 (Fig. 1). 여러종류의미생물이적당한온도의우유에옮겨졌고, 인간은이것을발견했던것으로 Elbrus 산의남쪽경사면에서는 의고온을선호하는미생물이터키유목민의우유통에우연히전달되어우유발효가되었고, 터키인들은이를 "Yogurut" 라고불렀다. Yogurut 는 11세기에들어와서현재의이름인요구르트 (yoghurt) 로바뀌었다 (Bylund, 2003). Kefir는 에서잘자라는미생물에의해 Elbrus 산북쪽경사면에서만들어졌는데, 178 J Milk Sci Biotechnol Vol. 36, No. 3

Kefir grain 은점성이있는노란색혹은유백색의비정형형태이다 (Guzel-Seydim 등, 2000). 발효유제조에사용되는종균 ( 단독혹은혼합유산균 ) 을통상적으로스타터로부르지만, Kefir 의경우에는 Kefir grains 으로부르고있다.

2 (A) Fig. 1. The region where Kefir and Yogurt was first made (Bylund, 2003); A, and a Kefir product in Korea; B. (B) Kefir라는이름은터키언어에서파생된것이다. 이름의첫번째음절인 kef는터키어로즐겁다는것을의미하는데, Kefir 는 yoghurt 와다른유형의독특한미생물을함유하고있다. 요구르트와다르게 Lactococcus 속유산균과 CO 2 를생산하는헤테로발효유산균및효모가바로 Kefir 와요구르트와다른점이다. 특히, 효모는알코올을생성할수있기때문에 Kefir 에존재하는최대알코올함량은약 0.8% 정도로알려져있다. Kefir grain 은점성이있는노란색혹은유백색의비정형형태이다 (Guzel-Seydim 등, 2000). 발효유제조에사용되는종균 ( 단독혹은혼합유산균 ) 을통상적으로스타터로부르지만, Kefir 의경우에는 Kefir grains 으로부르고있다. Kefir grain 은효모, lactic acid bacteria, acetic acid bacteria 및 mycelial fungi 등으로구성되어있다. 우리나라의김치가지역별사용재료별다양한미생물분포를나타나는것과같이 Kefir 도지역별, 국가별각기다양한미생물분포를보인다. Kefir grain 에서가장많은비율을차지하는미생물은 Lactobacillus 속유산균으로전체미생물의 65 80% 정도를보인다 (Wouters 등, 2002). 나머지미생물분포는 lactococci 와 yeasts 등이차지하고있다. Kefir에서분리되는유산균으로는 Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus kefiri, Lactobacillus parakefiri, Lactococcus lactis 및 Leuconostoc mesenteroides 등을들수있다 (Kandler 와 Kunath, 1983; Fujisawa 등, 1988; Pintado 등, 1996; Cogan 등, 1997; Takizawa 등, 1998; Micheli 등, 1999; Assadi 등, 2000; Witthuhn 등, 2004). Kefir에서분리되는효모로는 Kluyveromyces marxianus, Torula kefir, Saccharomyces exiguus 및 Candida lambica 가대표적이다 (Kwak 등, 1996; Pintado 등, 1996; Garrote 등, 1997; Wyder 와 Puhan, 1997; Wyder 등, 1999; Assadi 등, 2000; Witthuhn 등, 2004; ). 또한, 초산을생산하는 Acetobacter aceti 와 A. rasens, 곰팡이종류인 Geotrichum candidum 등도분리되었음이보고되었다 (Pintado 등, 1996). 전통방법으로생산하는 Kefir 는종균의안정적확보및각미생물의함유량을제어할수가없어 Kefir starter 에함유되는미생물종류에따라제품의안정성 (stability) 및안전성 (safety) 에 J Milk Sci Biotechnol Vol. 36, No

Lee et al. 많은영향을미친다. 국내에서는티벳버섯이라는이름으로잘못알려져있는데, Kefir의역사적고찰이나, 다른문헌에근거해서도티벳버섯이라는명칭은그유래를찾을수가없다. 아마도일본이나국내의광신적인 Kefir 신봉자들이만든것으로추정되는데, 이는김치를김치로부르지않고엉뚱한다른이름을지어서부르는것과같은것으로참으로우스꽝스러운일이아닐수없다.

최근 Kefir 가미국타임지가선정한 행복감을주는식품 중하나로국내에그소식이소개되면서관심이높아졌는데, 국내에서는 2017년 10월에매일유업에서발효유로 12종의유산균이함유되어 12시간배양하여제조한 Kefir12 라는제품을처음으로출시하였다 (Fig. 2).

3 Lee et al. 많은영향을미친다. 국내에서는티벳버섯이라는이름으로잘못알려져있는데, Kefir의역사적고찰이나, 다른문헌에근거해서도티벳버섯이라는명칭은그유래를찾을수가없다. 아마도일본이나국내의광신적인 Kefir 신봉자들이만든것으로추정되는데, 이는김치를김치로부르지않고엉뚱한다른이름을지어서부르는것과같은것으로참으로우스꽝스러운일이아닐수없다. Kefir 의세계최대의소비국으로러시아는전체발효유중약 70% 를차지하고있는데, 요구르트와는달리독특한맛과건강효능때문에많이음용하고있다. 일본에서는 Kefir 의우수한효능때문에최근학계나생산업계의관심이증대되고있다. 최근 Kefir 가미국타임지가선정한 행복감을주는식품 중하나로국내에그소식이소개되면서관심이높아졌는데, 국내에서는 2017년 10월에매일유업에서발효유로 12종의유산균이함유되어 12시간배양하여제조한 Kefir12 라는제품을처음으로출시하였다 (Fig. 2). 일본의메이지유업에서 Kefir Yogurt Drink, Minami Nihon 에서 Kefir Yogurt, Kansai Runa에서 Kefir, Ohayo 에서 Kefier 등이발매되었고, 그외에 Kefir 발효물을첨가한 Kefir 응용제품이 5 6종출시되었다. 본연구는효모를함유하지않은유산구균과유산간균으로구성된스타터를개발하기위하여 Kefir 배양시생균수변화및 Kefir 제조특성에대한기초자료를얻고자수행하였다. 재료및방법 1. Kefir의제조살균된우유에제조된스타터를 0.02%(w/w) 를접종하여 25 에서정치배양하면서 Kefir 를제조하였다. 판매중인상업용스타터는 2% 를접종하였다. 배양액의 ph가 4.5에이르면배양을종료시켰으며, 바로냉각하여 Kefir 를완성하였다. 2. 유산균수평가유산균수의측정은 ph를 5.2로조정한 MRS-lactose 배지에 Kefir 시료를도말하여 37 에서 48시간동안혐기성배양기에서배양한후집락을계수하였다. Lactococcus 균수의측정은 M17-lactose 배지에서 30 에서 24시간혐기적조건에서배양한후집락을평가하였다. 3. Kefir 스타터의저장중생균수변화 제조된스타터는산소투과가거의없는알루미늄코팅비닐에진공포장하여저장시료로사용하였다. Fig. 2. Developed-starter culture in this study. 180 J Milk Sci Biotechnol Vol. 36, No. 3

제조된스타터는 20 와 5 에서각각저장한다음 12 주동안저장하면서생균수를평가하였다. 4. ph 및적정산도의측정 ph 는 ph meter(fep20; Mettler Toledo; Greifensee, Switzerland) 를사용하여측정하였으며, 적정산도는시료 9 g 을적정하는데소요되는 0.1N NaOH 의 ml 수로환산하였다. 결과및고찰 1.

4 제조된스타터는 20 와 5 에서각각저장한다음 12 주동안저장하면서생균수를평가하였다. 4. ph 및적정산도의측정 ph 는 ph meter(fep20; Mettler Toledo; Greifensee, Switzerland) 를사용하여측정하였으며, 적정산도는시료 9 g 을적정하는데소요되는 0.1N NaOH 의 ml 수로환산하였다. 결과및고찰 1. Kefir Starter 의저장안정성평가제조된스타터 (Fig. 2) 를 20 와 5 에서각각저장하면서생균수를평가한결과는 Fig. 3에서보는바와같다. 냉동보관의경우, 스타터초기균수의 CFU/g 를 4개월간유지하는것으로나타났으며, 냉장보관의경우 CFU/g 의생균수를유지하는것으로나타났다. Yeast 가첨가되지않아 original Kefir 논란이있지만, yeast 는다른우유제품과발효유제품에서제품변질 ( 탄산가스에의한용기팽창 ) 의주된원인미생물로공정상반드시제어되어야한다. 따라서본개발 starter 에는타유제품에영향을줄수있는 yeast 를첨가하지않고, 유산구균과유산간균만으로조성을완성하였다. Table 1은본연구에서개발한 Kefir starter 의 specification 을나타낸것이다. 개발 starter 는유산균 12 species 를함유하며, L. acidophilus 의경우 2종이함유되어있어실제적으로총 13종류로구성되었다. 본연구에서는동일유산균주들을사용하여각유산균혼합비율을다르게하여 Starter 1, 2, 및 3을완성하였다. 2. Kefir 제품의배양중특성제조후총생균수는스타터의경우 CFU/g 이었으며, 장기간동안다소간의증감이있었으나, 이것은측정오차로파악되었다. 냉동저장 12주후 CFU/g, 냉장저장 12주후에는 CFU/g 로나타났다. 본연구에서완성한스타터 2의경우에는제조직후생균수는 CFU/g 이었으며, 냉동저장 12주후 CFU/g, 냉장저장 12주후에는 CFU/g 로초기균수를유지하는것으로나타났다. 3. Kefir 의제조 살균된우유에제조된스타터를 0.02%(w/w) 를접종하여 25 에서정치배양한결과, ph 에 4.5 에 ( 20 ) (5 ) Fig. 3. Changes of viable cells of Kefir starter during storage. J Milk Sci Biotechnol Vol. 36, No

5 Lee et al. Table 1. The sheet of Kefir starter specification Product characteristics Description Composition Manufacturing methods Recommended dosage Directions Activity Microbiological specifications Packaging Product storage Specifications Powder appearing off-white to light brown in color and composed of lyophilized lactic acid bacteria standardized with food grade carbohydrate carriers. Blend of selected strains for the manufacture of Kefir Bifidobacterium longum, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. lactis biovar diacethylactis, L. acidophilus KCNU, L. brevis BMB6, lactose, and maltodextrin Individual cultures are grown in sterilized media under specific conditions, then concentrated, lyophilized, and standardized. Final powder product is tested and aseptically packaged into sealed containers. 100 units of starter culture to treat 2,000 L of milk. Milk used must be free of residual antibiotics. Sanitize hands and outside of bottle prior to inoculation or rehydration. Directly inoculate vat when bottom is completely covered with milk and agitator is on. If inoculating a full vat, culture must be rehydrated in a separate, sanitized container of warm milk for 20 minutes. Activity is measured by acid development. Acidification time to reach a TA>0.45 in rehydrated skim milk powder and glucose medium with % inoculation and 10% NFMS at 32 is less than 8 hours. Lactic acid bacteria > 1x10 10 CFU/g Yeasts/molds <10 /g Coliforms (<10 /g) Negative by test Listeria (25 g) Negative by test Salmonella (40 g) Negative by test Staph. aureus (<1 /g) Negative by test Sealed in polyethylene plastic bottles. Standard size: 100 U (2,000 L). Custom packaging is available upon request. 18 months shelf life is expected when stored below months shelf life is expected when stored at 4. Transportation with dry ice is recommended to maintain temperature below 5. 도달하는시간은스타터 1의경우 9시간 30분, 스타터 2의경우 9시간 45분이걸렸다. 그러나판매중인상업용스타터는 12시간이소요되었다. Kefir를배양하면서생균수를평가한결과는 Fig. 4 에보는바와같다. 0.02%(w/w) 접종시초기생균수는약 CFU/mL 정도로나타났다. Lactobacillus 의총균수는배양 12시간이지나야약 CFU/mL 에이르렀으며, Lactococcus 속유산균의생균수는배양 8시간에약 CFU/mL 에도달하는것으로확인되었다. 따라서 Kefir 제품의생산을위한적정배양시간은 8시간정도로생각되었다. 4. Kefir 배양중 ph의변화발효유의경우, 기호성이가장좋은 ph는당류를첨가하지않은경우 ph 정도이고, 당류를첨가하는경우 ph 정도이다. Fig. 5에서나타난바와같이, 배양 4시간에 ph 정도로저하되었으며, 배양 12시간에는 ph 정도까지저하되었다. 스타터 1과스타터 2는 Kefir 배양 8시간에 ph 4.7 정도로나타나, 적정배양시간은 8 9시간정도로생각되었다. 그러나산업적생산시에는배양탱크에서이송하는시간이있으므로배양 8시간이내에배양을종료하는것이적절한것으로생각된다. 요구르트의경우, Streptococcus thermophilus 와 Lactobacillus delbrueckii ssp. bulgaricus 의 182 J Milk Sci Biotechnol Vol. 36, No. 3

MRS-pH 5.2 M17-lactose Fig. 4. Changes of viable cells during Kefir fermentation. Fig. 5. ph decline curve during Kefir fermentation.

Starter 를구성하고있는유산균의종류에따라우유배양시간이다소다르며, 풍미물질생성또한다소차이가있는데, 이는 lactic acid 이외에다른 metabololites 를만들기때문이다. 다른우유배양과마찬가지로 Kefir 배양중에유산균들은우유내유당은젖산으로빠르게전환시킨다.

Starter 내의다양한유산균들은서로간 symbiotic relationship 으로우유의 ph가 5 이하로저하되면 acetaldehyde 를생산하기시작하여 ph 4.2 4.0 에이르러서최대함량을보인다 (Tamime 과 Robinson, 1999).

6 MRS-pH 5.2 M17-lactose Fig. 4. Changes of viable cells during Kefir fermentation. Fig. 5. ph decline curve during Kefir fermentation. 혼합된 starter 를사용하는데, 배양초기의 acid production 은주로 S. thermophilus 에의해이루어진다. S. thermophilus 는우유에있는 lactose 를 permease 에의해세포안으로가지고들어와서바로에너지생산에이용하기때문이다. Starter 를구성하고있는유산균의종류에따라우유배양시간이다소다르며, 풍미물질생성또한다소차이가있는데, 이는 lactic acid 이외에다른 metabololites 를만들기때문이다. 다른우유배양과마찬가지로 Kefir 배양중에유산균들은우유내유당은젖산으로빠르게전환시킨다. 또한 Kefir starter 에함유된풍미를생산하는유산균들에의해 diacetyl, acetic acid, propionic acid, ketones, aldehydes, fatty acids 등의물질들이만들어진다. Starter 내의다양한유산균들은서로간 symbiotic relationship 으로우유의 ph가 5 이하로저하되면 acetaldehyde 를생산하기시작하여 ph 에이르러서최대함량을보인다 (Tamime 과 Robinson, 1999). 발효유제품의배양시생산되는향미성분은비휘발성물질, 휘발성물질및 carbonyl 화합물등인데, starter culture 로사용될유산균을선발할때에는 acetaldehyde, diacetyl, ehanol, propane, 2-butanone 등의농도를고려해서결정한다. 특히, 발효유에서는 vicinal ketones 2,3-butanedione과 2,3-pentanedione 이주요한향미성분으로알려져있으며, acetaldehyde 는향미성분중 J Milk Sci Biotechnol Vol. 36, No

7 Lee et al. 에서가장많은양을차지하기때문에요구르트와다른발효유제품에서요구르트의독특한향미를 주는성분이다. 비록, 본연구에서향미성분분석을수행하지않았지만 Kefir 와같은발효유제품의 starter 선정에는이러한향미성분의특성을고려하여선발하는것이좋다. 감사의글 본연구는산업통상자원부와한국산업기술진흥원의 R&D 재발견프로젝트 의지원을받아수행된연구결과 ( 과제번호 : N ) 로이에감사드립니다. 또한 Fig. 1의그림을사용할수있도록허가해준 Aniika Nilsson(Dairy Processing Handbook, Petra Pak) 에게감사드립니다. References Assadi, M. M., Pourahmad, R. and Moazami, N Use of isolated kefir starter cultures in kefir production. World Journal of Microbiology and Biotechnology 16: Bylund, G Dairy processing handbook. Tetra Pak Processing Systems AB. Cogan, T. M., Barbosa, M., Beuvier, E., Bianchi-Salvadore, B., Cocconcelli, P. S., Fernandes, I., Gomez, J., Gomez, R., Kalantzopoulos, G., Ledda, A., Medina, M., Rea, M. C. and Rodriguez, E Characterization of lactic acid bacteria in artisanal dairy products. Journal of Dairy Research 64: Fujisawa, T., Adachi, S., Toba, T., Arihara, K. and Mitsuoko, T Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. International Journal of Systematic Bacteriology 38: Garrote, G. L., Abraham, A. G. and De Antoni, G. L Preservation of kefir grains, a comparative study. Lebensmittel-Wissenschaft und Technologie 30: Guzel-Seydim, Z., Seydim, A. C. and Greene, A. K Organic acids and volatile flavor components evolved during refrigerated storage of kefir. Journal of Dairy Science 83: Kakkar, P., Das, B. and Viswanathan, P. N A modified spectrophotometric assay of superoxide dismutase. Indian J. Biochem Biophys. 21: Kandler, O. and Kunath, P Lactobacillus kefir sp. nov., a component of the microflora of kefir. Systematic and Applied Microbiology 4: Kwak, H. S., Park, S. K. and Kim, D. S Biostabilization of kefir with a nonlactosefermenting yeast. Journal of Dairy Science 79: Maffei Facino, R., Carini, M., Aldini, G., Berti, F. and Rossoni, G Panax ginseng administration in the rat prevents myocardial ischemia-reperfusion damage induced by hyperbaric oxygen: Evidence for an antioxidant intervention. Planta Med. 65: Micheli, L., Uccelletti, D., Palleschi, C. and Crescenzi, V Isolation and characterization of a ropy Lactobacillus strain producing the exopolysaccharide kefiran. Applied Microbiology and Biotechnology 53: Miller, J. H Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, NY, pp Pintado, M. E., Lopes Da Silva, J. A., Fernandes, P. B., Malcata, F. X. and Hogg, T. A. 184 J Milk Sci Biotechnol Vol. 36, No. 3

8 1996. Microbiological and rheological studies on Portuguese Kefir grains. International Journal of Food Science and Technology 31: Prado, M. R., Blandón, L. M., Vandenberghe, L. P. S., Rodrigues, C., Castro, G. R., Thomaz-Soccol, V. and Soccol, C. R Milk kefir: Composition, microbial cultures, biological activities, and related products. Frontiers in Microbiology 6:1-10. Takizawa, S., Kojima, S., Tamura, S., Fujinaga, S., Benno, Y. and Nakase, T The composition of the Lactobacillus flora in kefir grains. Systematic and Applied Microbiology 21: Tamime, A. Y. and Robinson, R. K Yoghurt: Science and technology. Woodhead Publishing. Witthuhn, R. C., Schoeman, T. and Britz, T. J Isolation and characterisation of the microbial population of different South African Kefir grains. International Journal of Dairy Technology 57: Wouters, J. T. M., Ayad, E. H. E., Hugenholtz, J. and Smit, G Microbes from raw milk for fermented dairy products. International Dairy Journal 12: Wyder, M., Keile, L. and Teuber, M Description of Saccharomyces turicensi sp. nov., a new species from kefir. Systematic and Applied Microbiology 22: J Milk Sci Biotechnol Vol. 36, No

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(......).hwp 7 6 4 10 20 30 ph 5 4 3 0 10 20 30 40 50 60 Fermentation time(day) Fig.2-1. Changes of ph in DLMK according to various fermentation temperature 2 4 10 20 30 Acidity (%) 1 0 0 10 20 30 40 50 60 Fermentation