Korean J. Microbiol. Biotechnol. (2014), 42(1), 1 10 pissn 1598-642X eissn 2234-7305 Korean Journal of Microbiology and Biotechnology Review 전통발효식품용종균의안전성평가필요성 정도원, 이종훈 * 경기대학교식품생물공학과 Received: January 28, 2014 / Revised: February 3, 2014 / Accepted: February 6, 2014 Safety Assessment of Starters for Traditional Korean Fermented Foods Do-Won Jeong and Jong-Hoon Lee* Department of Food Science and Biotechnology, Kyonggi University, Suwon 443-760, Republic of Korea Most microorganisms have been used for foods for such a long period of time with no question posed for their safety. However, the progress of food processing technology has activated international food trades, and the consumers and authorities of import countries have come to question the safety of microorganisms used in foods. At present, the most widely known safety standards are Generally Recognized as Safe (GRAS) status from the US Food and Drug Administration (FDA) and Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA). GRAS status is not for the safety of microorganisms themselves but for the permissibility of strains or cultures in specific food uses. QPS provides a qualified generic approval to a defined taxonomic unit. The increase of commercialized traditional fermented foods in Korea has spurred the starter development for traditional food fermentations. However, starter development in Korea has been carried out based on the technological properties of microorganisms with no research on developing a standardized tool for safety assessment. In the globalization of traditional Korean fermented foods, technological properties as well as safety of future starters should be guaranteed, and establishment of the safety assessment regulation for microorganisms used for foods is necessary. Keywords: Starter, traditional fermented foods, safety assessment 발효식품과종균 인간의발효식품섭취는유사이전으로거슬러올라간다. 이집트피라미드에서발견된발효빵, 그리스신화에나오는포도주, 서양의주요발효식품인치즈와요구르트, 동양의장류, 수산발효식품등은인류의문명사와그역사를같이한다. 식품발효에대한과학적토대는현미경의발명과 1665 년 Robert Hooke 와 Antonie van Leeuwenhoek 에의한미생물존재확인으로부터시작되었고, 1857 년 Louis Pasteur 가미생물에의해발효가일어남을증명하면서확립되었다. 식품의발효는식품소재에존재하는미생물에의한자연발효에의존하다가, 미생물첨가에의한식품발효가시작된역사는 100 년정도에불과하다. 서구에서발생한산업혁명은도시 *Corresponding author Tel: +82-31-249-9656, Fax: +82-31-253-1165 E-mail: jhl@kgu.ac.kr 2014, The Korean Society for Microbiology and Biotechnology 인구의증가를초래하였고, 대량생산의필요성은공산품뿐만아니라식품에서도발생하였다. 다행히도 1850년경의미생물학은발효산업을지원할수있는과학적수준에도달해있었기때문에미생물을이용한발효식품의대량생산이시작되었다 [4]. Pasteur가정립한미생물이발효를진행시킨다는기본개념은식품발효를담당하는미생물의탐색으로연결되었고, Christian Hansen이효모를분리하여양조산업에이용하면서종균발효가시작되었다. 종균 (starter) 은미생물이가지고있는대사능력을이용하기위하여식품발효에의도적으로첨가하는미생물을의미하고, 종균첨가제 (starter culture) 는발효식품의빠른제조를위하여종균을배양하여증식시킨첨가용균체를지칭하며, 식품소재에첨가한종균첨가제는발효식품의품질향상및균일화, 식품안전성확보, 저장기간연장등을가능하게하였다 [18]. 자연발효로시작한식품발효는성공적으로제조된발효식품의일부를다음발효식품의제조에첨가하는 back slopping으로발전하여품질향상이달성되었다. Back March 2014 Vol. 42 No. 1
2 Jeong and Lee slopping 은지금까지도발효관련미생물상 (microbial ecology) 과미생물의천이에대한정확한지식이확립되지않은경우사용되고있고, 소규모생산및저비용으로품질균일화를달성할수있는발효법이다 [17]. 한편대량생산에필요한균일하고, 높은관능적품질은종균의사용으로달성되고있다 [27]. 현재, 단일균주를배양한 single-strain culture 와복수의균주를배양한 multiple-strain culture 가제조되어종균첨가제로식품발효에이용되고있다. 종균첨가제의산업적활용을위해서는첨가제의안전성, 첨가제첨가에따른기술적효용성, 첨가제의유지및생산에따르는비용대비얻을수있는경제성이확보되어야하고, 추가적으로첨가제에포함된종균의건강기능성이입증된다면산업적유용성의향상이예상된다 [3]. 식품발효용미생물의안전성평가필요성 유산균은채소, 곡류, 사람과동물의장관을포함하는다양한환경에서검출되는발효미생물로치즈, 요구르트와같은발효유제품, 김치나피클등과같은발효침채류, 발효소시지, 빵의제조등, 수많은발효식품들의종균으로이용된다 [5]. 또한의약품인정장제제와건강보조식품, 가축사료의발효제및영양개선제로사용되고있고, 최근에는백신운반수단으로주목을받고있다. 이외에도 dextran 이나 pullulan 등의고분자화합물, 생분해성플라스틱원료인유산, nisin 과같은 bacteriocin, 식품보존제, 기타유기산, 비타민, 풍미성분등의정밀화학제품, 화장품원료의발효생산에이용되고있고 [15], 우리나라에서도식품발효용종균및 probiotics Table 1. Overview of antibiotic resistances reported in the food-associated lactic acid bacteria [29]. Foods Species Resistance Raw meat products Poultry Lb. reuteri G4 cat Raw ground pork Lb. reuteri 100-63 erm (T) Lb. plantarum catc2r Cm Raw ground pork and beef Lb. sakei, Lb. curvatus, Lb. plantarum, Lb. brevis, Leuco. mesenteroides Tetracycline (69%); chloramphenicol (3%); methicillin (85%) Fermented products Raw milk soft cheese Lc. lactis strain K214 Str-tet (S)-cat Greek cheese Lb. acidophilus ACA-DC 243 Penicillin Yoghurt starter cultures S. thermophilus and Lb. delbruekii ssp. bulgaricus Neomycin, polymyxin B Nigerian fermented foods and beverages Lb. pentosus, Lb.acidophilus, Lb. casei, Lb. brevis, Lb. plantarum, Lb. jensenii Tetracycline (42.5%) Erythromycin (17.5%) Ampicillin (47.5%) cloxacillin (80%); penicillin (77.5%); Fermented dry sausages Lactobacillus species Tetracycline Gentamicin (79%) Penicillin G (64%) Kanamycin (79%) Turkish yoghurts S. thermophiles Vancomycin (65%) European probiotic products Lb. acidophilus, Lb. rhamnosus, Lb. casei, Lb. johnsonnii, Lb. plantarum, Lb. reuteri, Lb. delbreukii ssp. bulgaricus Tetracycline (26%) Penicillin G (23%) Erythromycin (16%) Chloramphenicol (11%) Others Maize silage Lb. plantarum 5057 tet (M) Gene names and abbreviations: cat: chloramphenicol acetylase gene; erm: erythromycin resistance gene; Cm: Chloramphenicol; tet: tetracycline resistance gene; str: streptomycin adenylase gene; Lb: Lactobacillus; Lc: Lactococcus; Leuco: Leuconosstoc; S: Streptococcus.
Starter Safety for Fermented Foods 3 로산업화하려는연구가활발히진행되고있다. 뿐만아니라, 오랜기간의사용이력으로안전성이증명되었다고볼수있다. 이러한맥락에서미국 Food and Drug Administration (FDA) 는유산균을 Generally Recognized as Safe (GRAS) 규격으로오랜기간인정하고있었다. 그러나유산균이항생물질내성을유발할수있는 plasmid 와 transposon을보유하고있다는연구보고가지속적으로발표되면서, 이들의사용이력이더이상안전성에대한근거가되지못하기시작했고, 유산균의항생물질내성에대한연구들은계속증가하고있다 [7, 29, 33, 34]. 특히, 발효유제품의숙성및풍미형성에관여하는것으로알려져발효산업용종균및 probiotics로오랜기간이용되어왔던 Enterococcus 속의경우에는더이상 GRAS로인정되지않는다. Enterococci 는높은항생물질내성유전자획득능력을가지고있을뿐만아니라병원의감염환자로부터빈번히검출되고있어기회감염성균으로도인식되고있다. 아직 enterococci의섭취에의한감염환자발생은보고된바없고, 시제품으로출시된 enterococci 함유 probiotics로부터발견된위해인자에대한보고가없었지만, 식품에서분리되는 enterococci의안전성문제는계속적으로논란이되고있어, 현재식품에사용하기위해서는안전성평가가필요한미생물로분류되고있다 [13, 14, 30]. 이러한항생물질내성의문제는 enterococci 뿐만아니라 Lactococcus, Lactobacillus, Leuconostoc, Pediococcus 속유산균및 bifidobacteria에서도나타나고있어식품미생물분야에서의큰위해요인으로등장하였다. Table 1은 enterococci를제외한유산균에서나타난항생물질내성에대한보고를 Mathur and Singh [29] 가정리한표이다. 유산균은발효식품에다수존재하고있기때문에이들이전이가능한항생물질내성유전자를보유하게된다면발효식품이항생물질확산의매개체가될수있다. 또한종균으로식품발효에사용하거나 probiotics로섭취하게된다면, 이들이인간의장 (gut) 에정착하여항생물질내성유전자를지속적으로위해균및기회감염성균에게전달할수있고, 최종적으로항생물질로는더이상감염균을조절할수없는상황에도달하게될것이다. 따라서유럽에서는식품발효용미생물및식품용미생물제제에대한항생물질내성문제가심각히거론되고있다. 최근에는, 식품발효용미생물의안전성문제중항생물질내성외에도독소생성 (toxin production), 용혈작용 (hemolysis), biogenic amine 생성, biofilm 형성등의여러위해인자들에대한안전성평가가필요한것으로보고되고있다 [32]. 식품용미생물안전성평가시스템 대량생산에의해식품이제조되고있는현대사회에서소비자와생산자에게가장중요한문제는식품의안전성이다. 안전한식품에대한요구증가는발효식품제조에첨가하는종균의안전성평가에대한중요성을부각시켰다. 그러나 2000 년초반까지유럽연합 (EU) 에는식품에첨가하는미생물에대한안전성평가기준이존재하지않았고, 미국 FDA 가사용하고있는 GRAS 가대표적기준으로적용되고있었다. 2002 년 EU 의 European Food Safety Authority (EFSA) 는 GRAS 기준과비슷한개념과목적을위하여미생물안전성평가시스템을제안하였다. 식품에첨가하는생물제제의안전을담당하고있는 EFSA 는 EU 각국에서진행하고있는식품용미생물안전성평가시스템을간소화하기위하여포괄적으로적용할수있는 Qualified Presumption of Safety (QPS) 개념을도입하였다 [8, 28]. 식품에첨가하는미생물제제를용도에따라개별적으로안전성을평가하는 GRAS 시스템과는달리, QPS 시스템은 EFSA 전문가집단의식품에첨가되는미생물단위 ( 분류학적단위, taxonomic unit) 에대한안전성평가이다. 미국의 GRAS 시스템미국에서식품첨가물은연방식품의약품화장품법 (Federal Food, Drug and Cosmetic Act, FFDCA) 에따라규제되고있다. FFDCA 201(s) 및 409 조항에따르면식품첨가물은식품의특성에영향을미칠수있는성분으로의도적으로첨가한물질로정의하고있으며, 신규물질의식품사용을위해서는 FDA 의사전승인 (premarket approval) 을받아야한다. 그러나 GRAS 로인정된식품첨가물의사용에는사전승인을면제하고있다. GRAS 는미국 FDA 가오랜사용이력및전문가들의평가를통하여일반적으로안전하다고판단되는식품첨가물을관리하는규격 (status) 이다. FFDCA 에의해 GRAS 시스템이시행되기시작한 1958 년이전부터사용한물질에대해서는사전승인 (prior sanction) 물질로간주해안전성평가없이 GRAS 로인정되었지만, 현재는안전성에대한과학적근거자료에대한전문가들의심사를통하여인정된다. 미생물및미생물에서유래한성분을식품에첨가하려는경우, 식품첨가물과동일한절차를거쳐 GRAS 로승인된다. GRAS 규격은식품에첨가하는원료자체에대한안전성을제공하는것이아닌, 특정용도에서의안전성을의미한다. 따라서항상용도를지정해야한다. GRAS notification program 은식품원료의안전성을증명하는과정으로, GRAS 로인증된식품원료는 GRAS notice inventory 에등재되어 FDA homepage (http://www.fda.gov) 에공개된다. 1997 년이전에는 GRAS 인증의심사가 FDA 및제조업체의안전성평가전문가들에의해서진행되었지만, 오랜기간에걸쳐진행되는단점을보완하기위해, 1997 년이후에는생산자또는판매자가 GRAS 여부를판단하여 FDA March 2014 Vol. 42 No. 1
4 Jeong and Lee 에심사를청구하면독성, 영양, 분자생물학분야전문가패널의안전성검증을통해 GRAS 규격을획득한다. 다만 1958 년이전에안전하게사용되었다는역사적사용근거가있는물질에대해서는사전승인물질로인정해위해성이없는물질로간주하고있다. Table 2에는미국연방법 (CFR, Title 21, Part 131, http://www.gpo.gov/) 에명기된사전승인물질중, 유산균관련내용을정리하였다. 지금까지 490건의식품원료심사가 FDA에요청되었고, 그중 329건이 GRAS 규격을획득하였다 (2014년 1월기준 ). Table 3에는 GRAS로인정받은 22건의 bacteria 목록을정리하였다. 예를들어 GRN number 49를부여받은 Bifidobacterium lactis Bb1와 Streptococcus thermophilus Th4는유아식에사용할수있으며, GRN number 171을부여받은 Lactobacillus acidophilus, Lactococcus lactis, Pediococcus acidilactici는육가공제품의유해균저해를위해서사용할수있다. 유럽의 QPS 시스템유럽에서식품제조를위해첨가되는미생물은 1997 년부터 Novel Food Regulation (NFR) 의적용을받았다. 전통적으로식품제조에사용되었던미생물은오랫동안안전하게섭취된사용이력에근거규제없이사용되었으나, 새롭게발견되는균주나유전자조작미생물, 사료첨가제를통해식품에유입될수있는미생물은관련법령에의해엄격히규제되고있었다. 특히사료첨가제로사용되는미생물은식품제조에사용되는미생물과동일하거나매우유사한균주가많지만식품과달리엄격한규제를받고있어형평성에대한논란이있었다. 낙농제품생산및축산업의대형화에따른항생제사용의증가로환경시료로부터항생제내성균의분리가증가하고있으며, 식품에서분리되는미생물에서도항생제내성이증가하고있는추세이다. 그러나, NFR 에서는항생 제내성을갖는미생물의사료첨가제사용을제한하지만식품발효에는별다른규제없이사용되었다. 이러한문제의해결을위해 EFSA는 EU의 Scientific Committee on Animal Nutrition (SCAN), Scientific Committee on Food (SCF), Scientific Committee on Plants (SCP) 의전문가그룹을 2002년과 2003년에소집하여식품이나사료에사용될미생물의안전성평가를위한 QPS 개념을제안하였다 [8, 28]. QPS는특정용도로식품에첨가하는미생물의균주 (starin) 수준 (level) 에서의안전성을의미하는미국의 GRAS 와는달리, 미생물단위의일반적인안전성을정의한다. 따라서, QPS 시스템을통하여안전하다고평가된미생물단위 ( 종또는속 ) 은사용용도를제한하지않는다. QPS에의해안전성이확보된미생물단위는 QPS list에등록되고, 이목록은매년업데이트된다 (http://www.efsa.eu.int/). QPS list에등록된미생물단위는일반적인안전성이입증되었기때문에개별적인안전성평가가필요없고, 균주수준에서의특이적안전성문제가없다면식품에사용가능하다. 한편등록되지않은미생물단위에대해서는평가가필요한모든안전성이검증되어야한다. EFSA의과학위원회는 2005년식품에첨가하는미생물의안전성평가에 QPS를적용하도록제안하였고 [9], 2007년에는 bifidobacteria, 유산균, Bacillus 속및일부효모에대한안전성을검토하여 QPS list를작성하였다 [10]. 2008 년부터는 EFSA의 Biological Hazards (BIOHAZ) 패널이안전성을검토하여 QPS list를업데이트한다 [2, 11, 12]. Table 4에 2013년에업데이트된 QPS 미생물목록을속 (genus) 수준에서정리하였다. QPS 규격획득을위한안전성평가는분류 (taxonomy), 정량적정보의양 (familiarity), 병원성 (pathogenicity), 적용상황 (end use) 의 4가지항목을중심으로진행된다 [9]. 안전성에논란이없거나, 위해요소가있더라도제거가능한미생물 Table 2. Food microbial substances of prior sanction in 21 CFR 131. Regulation in 21 CFR Food Substance 131.112 Cultured milk Lactobacillus acidophilus 131.160 Sour cream lactic acid producing bacteria Lactobacillus bulgaricus and Streptococcus thermophiles 133.128 Cottage cheese lactic acid producing bacteria Lactobacillus bulgaricus and Streptococcus thermophiles 131.200 Yogurt lactic acid producing bacteria Lactobacillus bulgaricus and Streptococcus thermophiles 131.203 Low fat yogurt lactic acid producing bacteria Lactobacillus bulgaricus and Streptococcus thermophiles 131.206 Non-fat yogurt lactic acid producing bacteria Lactobacillus bulgaricus and Streptococcus thermophiles 21 CFR 131: Code of Federal Regulations (CFR), Title 21, Part 131 (http://www.gpo.gov/)
Starter Safety for Fermented Foods 5 Table 3. Bacteria in the GRAS notice inventory. GRN No. Strain Use 49 Bifidobacterium lactis strain Bb12 Ingredient in infant formula Streptococcus thermophilus strain Th4 159 Carnobacterium maltaromaticum strain CB1 Inhibitor of Listeria monocytogenes in ready-to-meat products 171 Lactobacillus acidophilus Lactobacillus lactis Pediococcus acidilactici Antimicrobial agent in meat and poultry products 231 Lactobacillus casei subsp. rhamnosus strain GG Ingredient in infant formula 240 Lactobacillus paracasei subsp. paracasei Bacillus coagulans LA-1 Propionibacterium freudenreichii subsp. hermanii mixtures of these microorganisms Antimicrobial agent in meat and poultry products 254 Lactobacillus reuteri strain DSM 17938 Ingredient in processed dairy and vegetable products 268 Bifidobacterium longum strain BB536 Ingredient in various foods 281 Lactobacillus rhamnosus strain HN001 produced in a milk-based medium Ingredient in infant formula 288 Lactobacillus rhamnosus strain HN001 Ingredient in various foods 305 Carnobacterium maltaromaticum strain CB1 Inhibitor of Listeria monocytogenes in various foods (viable and heat-treated) 357 Lactobacillus acidophilus NCFM Ingredient in various foods 377 Bifidobacterium animalis subsp. lactis strain Bf-6 Ingredient in food 378 Streptococcus thermophilus Bacillus coagulans Lactobacillus acidophilus Lactobacillus paracasei subsp. paracasei Lactobacillus plantarum Lactobacillus sakei Lactobacillus bulgaricus Proprionibacterium freudenreichii subsp. shermanii or mixtures of these strains 399 Bacillus coagulans strain GBI-30, 6086 spores Ingredient in baked goods 410 Lactobacillus reuteri strain DSM 17938 Ingredient in infant formula 415 Heat-killed Propionibacterium freudenreichii ET-3 culture Ingredient in specific foods Antimicrobial agents in various food including meat and poultry products excepting infant formula and infant foods 429 Lactobacillus casei strain Shirota Ingredient in fermented dairy products 440 Lactobacillus reuteri strain NCIMB 30242 Ingredient in beverages and various foods 445 Bifidobacterium animalis subsp. lactis strains HN019, Bi-07, Bl-04 and B420 Ingredients in ready-to-eat foods 453 Bifidobacterium breve M-16V Ingredient in baked goods 454 Bifidobacterium breve M-16V Ingredient in infant formulas 455 Bifidobacterium breve M-16V Ingredient in exempt term powdered amino acid-based formulas March 2014 Vol. 42 No. 1
6 Jeong and Lee Table 4. Microorganisms of QPS status in the 2013 updated list. Phylum Family Genus Species a Actinobacteria Bifidobacteriaceae Bifidobacterium 5 Corynebacteriaceae Corynebacterium 1 Propionibacteriaceae Propionibacterium 2 Firmicutes Bacillaceae Bacillus 12 Geobacillus 1 Lactobacillaceae Lactobacillus 36 Pediococcus 3 Leuconostocaceae Leuconostoc 4 Oenococcus 1 Streptococacceae Lactococcus 1 Streptococcus 1 Proteobacteria Acetobacteraceae Gluconacetobacter 1 Ascomycota Saccharomycetaceae Debaryomyces 1 Hanseniaspora 1 Kluyveromyces 2 Komagataella 1 Lindnera 1 Ogataea 1 Saccharomyces 3 Wickerhamomyces 1 Schizosaccharomycetaceae Schizosaccharomyces 1 Basidiomycota Cystofilobasidiaceae Xanthophyllomyces 1 Total number of species 81 a Numbers of QPS status species in the listed genus. Fig. 1. A generalized scheme for assessing the suitability for QPS status of microorganisms. 단위는 QPS 규격획득이가능하고, 4 가지요건에근거한 QPS 규격평가진행은 Fig. 1과같다. 분류 (Taxonomy): 미생물동정은 QPS 규격획득을위한위해도평가의첫단계이다. 미생물은유산균등과같이일반적인분류가아닌, 속 (genus) 또는종 (genus) 과같이학술적분류단위에의해분류되어야하고, 가능한식품용으로적합하지않은균주를제거할수있는분류단위의적용을원칙으로한다. 심사청구자는가능한최신의방법을적용하여가장최근의명명법을동정에사용해야한다. 미생물이분류방법론상의변경으로재분류되더라도 QPS 지위를상실하는것은아니고, 이력상의정보변경으로간주한다. 돌연변이및선발을통해개량된특성을갖는산업용균주의경우에도분류학적위치는변하지않는다. 그러나유전자재조합을통해개량된특성을갖는균주는유전자재조합관련법규에따른다. 정량적정보의양 (Familiarity, Body of knowledge): QPS 시스템의도입시점에는 familiarity 라는용어를사
Starter Safety for Fermented Foods 7 용하였으나, 혼동을일으킬우려가있어 body of knowledge 로수정하였다. 이것은미생물의산업적이용을포함하는역사적사용이력, 주요분포생태계, 질병관련성, 안전성등의과학적평가가가능한모든정보를의미한다. QPS 인증을위해제출한자료는미생물이사람, 가축, 환경등에악영향을미치지않음에대한판단이가능해야한다. 제출한자료가완벽한안전성을증명하지못하더라도, 위해성에대한합리적인증거제시만으로도 QPS 규격획득이가능하다. 병원성 (Pathogenicity): QPS 규격획득을위해서는미생물의사용이력및과학적근거자료를토대로병원성이없음을제시해야한다. 따라서일반적인질병에관여하는미생물은 QPS 인증이불가하지만, 병원성이일부균주에서만한정적으로나타나고발병기작이잘알려져있다면병원성균주만을제외하는제한규정을첨부하여 QPS 의판단대상이될수있다. 예를들어 Bacillus subtilis 중에서위해성을나타낼가능성이있는균주, 곰팡이독소를생산하는곰팡이균주, 위해성이있는 enterococci 를 QPS list 에서제외하는조건으로이들을 QPS 로인증할수있다. 적용상황 (End use): 생물제제의사용에대한적용상황또한 body of knowledge 의일부로써안전성평가에영향을미친다. 미생물의안전성평가에있어모든측면에서의안전성이확보되는것이최선일수있지만, 실용적측면에서조건부사용이따르는경우, end use 는 QPS 인증에큰영향을줄수있다. 예를들어곰팡이의경우, 식물보호의목적으로사용함에있어안전성이검증되었다하더라도곰팡이가가지고있는능력을모든분야로확대하여이용하는점에서는안전성을확대해석하는것에는문제점이있는것으로판단하고있다. 대한민국식품위생법우리나라식품공전에는곰팡이사용을위한종국에대한정의를명시하고있지만, 종균에대한정의및관리가명확하지는않다. 현재식품의발효를위해사용되는종균은식품위생법에정의된식품첨가물 [ 식품에의도적비의도적으로혼입된물질 ( 오염물질제외 )] 에해당하지만, 아직까지요구르트제조에사용하는유산균외에사용허가된것이없으며, 유산균의경우에도균주수준에서의특이적사항에대한제시가없고, 사용허가를위한기준조건이없다. 건강기능성식품법에는 probiotics 로허가된유산균품목이있다 (Table 5). 그러나허가된유산균에대한최신정보를통한업데이트가진행되지않을뿐아니라, 허가품목중, Enterococcus 의경우, 병원성관련문제점이제시되고있고 [13, 14, 30], 캐나다에서는 Natural Health Products Regulations 에의해사용이금지되었지만, 우리나라에서는여전히 probiotics 로승인되어있다. 따라서식품용도로사용되는미생물에대한안전성평가및허가기준에대한검토가필요하다. 우리나라전통발효식품미생물연구 우리나라에는김치, 된장, 젓갈과같은다양한전통발효식품과전통주가있으며, 자연발효기반으로, 지역과가정에따라특징적인제조법으로만들어지고있다. 가정에서만들어지던전통발효식품은 1970 년대에들어각종산업체증가에따른단체급식물량이발생하면서산업화가시작되었고, 경제성장에따른주거환경의변화, 가공식품산업의발달, 여성의사회참여증가등의사회적변화에의해산업규모가지속적으로성장하고있다. 전통발효식품의상업적생산과관련한문제점해결을위하여 1980 년이후다양한물리적, 화학적처리및공정개발이시도되었지만, 품질의변화가발생하는경우가많아, 종균의사용은가장바람직한해결책의하나로주목받고있다. 전통발효식품미생물연구는다양한소재를대상으로진행되었지만, 우리식생활과가장밀접한김치와된장에대한연구가주를이루고있다. 2013 년 10 월기준국가과학기술정보센터 (NDSL, http://ndsl.kr) 의데이터베이스에서검색되는국내에서보고된전통발효식품관련미생물논문은김치 209 건, 된장 84 건, 고추장 70 건, 젓갈 51 건, 청국장 45 건, 막걸리 21 건으로김치에대한연구가가장많이진행되었다. 이러한경향은김치의우리식생활과의관련성뿐만아니라, 상품화및세계화에가장적합한특성때문으로추정된다. Table 5. Approved probiotics in the Health Functional Food Code of Korea. Genus Species Lactobacillus Lb. acidophilus, Lb. casei, Lb. gasseri, Lb. delbrueckii ssp. bulgaricus, Lb. helveticus, Lb. fermentum, Lb. paracasei, Lb. plantarum, Lb. reuteri, Lb. rahmnosus, Lb. salivarius Lactococcus Lc. lactis Enterococcus E. faecium, E. faecalis Streptococcus S. thermophilus Bifidobacterium B. bifidum, B. breve, B. longum, B. animalis ssp. lactis March 2014 Vol. 42 No. 1
8 Jeong and Lee 김치의경우, 미생물뿐만아니라다양한측면에서의품질균일화및유통기간연장을위한가장많은연구가진행되었다. 선택배지를이용한배양법을통하여발효에관여하는미생물상을분석하는미생물연구가진행되다가, 2000 년이후부터는 metagenome 을분석하는배양비의존적미생물군집분석이본격적으로도입되면서발효과정중의미생물천이에대한높은과학수준의지식이확보되었다 [31]. 또한종균개발에대한연구도꾸준히진행되고있고 [20], Leuconostoc 속균주가상품김치에첨가되고있다. 장류관련미생물연구는김치보다는활발하지않지만, 김치의경우와마찬가지로배양법에의한미생물상분석이최근들어배양비의존적미생물군집분석으로발전하면서곰팡이와 Bacillus 속외에도 Enterococcus 속, Staphylococcus 속, Tetragenococcus halophilus 등의새로운 bacteria 의발효관련성이보고되고있다 [23-26]. 개량식된장의제조에서는코지의사용으로품질균일화가달성되었지만, 전통식담금에서는미생물천이에대한충분한지견이얻어지지않았다. 따라서전통식된장의대량생산및품질표준화를위한지속적인연구가필요한시점에있다. 젓갈은 6 개월이상의숙성기간이소요되기때문에숙성기간단축을위한속성발효가주된연구목표이다. 최근까지숙성과정중의미생물상천이및고염발효에대한정확한지식이확립되지않아, 단순히내염성및단백질분해활성이높은균주를선발하여종균제로첨가하려는시도가진행되었고 [1, 6, 19], 20% 이상의고염상태에서숙성이진행되기때문에미생물에의한발효가아닌원재료에서유래하는효소가숙성에주된역할을한다는견해도있다. 최근의배양법및배양비의존적방법을이용한미생물상의분석을통하여미생물군집의천이가밝혀지고있고, Staphylococcus 속이주발효미생물이라는결과들이도출되고있어, 향후종균을이용한속성발효의가능성이높아지고있다 [16, 21, 22]. 그러나최근의건강을우선시하는음식의섭취가증가하고, 젓갈이비위생적으로제조되고있다는소비자들의인식이늘어나면서염도가높은젓갈의소비가감소하고있다. 또한장기간의숙성으로인해자금회수가늦어지는경제적문제점을가지고있어젓갈에대한연구가활발하게진행되고있지는않다. 그러나젓갈은한국인의밥상에빈번하게등장하는부식중의하나이고, 김치의부재료로첨가되고있어향후지속적인종균화를통하여경제성을향상시킬필요성이높은전통발효식품중의하나이다. 전통발효식품의산업화관련미생물연구는 bacteria 의경우, 유산균과 Bacillus 를중심으로진행되었고, 효모와곰팡이에대한연구가꾸준히뒤를따르고있다. 2013 년 10 월기준으로국가과학기술정보센터데이터베이스에서종균을키워드로검색되는국내발표논문은총 502 건이고, 유산균종 균 128 건, Bacillus 종균 52 건, 효모종균 50 건, Aspergillus 종균 17 건등으로이미산업적활용성및안전성이알려진미생물들을대상으로한연구가보고되었고, 그외의미생물의종균화에대한연구는보고된바없다. 제 언 우리나라에서는김치발효와 probiotics 관련의유산균과장류발효에관여하는것으로알려진 Bacillus 속을중심으로종균개발연구가진행되고있어, 다른미생물은종균화대상으로주목받지못하고있다. 그러나 metagenome 분석에기반을둔미생물상분석이전통발효식품에적용되면서기존연구에서알려지지않았던다양한미생물의존재가전통발효식품의발효에관여하는것으로알려지기시작했다. 한예로, 고염에서숙성되는젓갈이 coagulase-negative staphylococci (CNS) 에의한발효에의존한다는보고들의계속적증가를들수있다 [16, 21, 22]. 따라서우리전통발효식품의안전성을입증을위하여전통발효식품에서검출되는새로운우점미생물에대한안전성평가가필요하다. 유럽에서는육류및소시지발효에 CNS 가종균으로사용되고있어, 간접적으로안전성이확보되었다고할수있다. 그러나종균발효를시작한유럽에서식품에첨가하는미생물의안전성을평가하는 QPS 시스템이만들어져시행되고있다는점을고려하면우리나라에서도식품에사용되는미생물안전성평가의필요성이제기되어야할시점으로생각한다. 한식의세계화가활발하게진행되고있는현시점에서김치를비롯한전통발효식품의세계화를위한노력이다양한측면에서진행되고있다. 전통발효식품에대한우수성을알리기위하여건강기능성에대한다양한논문들이보고되고있지만, 안전성과관련한문제들은드물게보고되고있다. 안전성문제는자칫잘못하면그동안의세계화노력을물거품으로만드는가능성을가지고있는민감한사항이다. 그러나, 전통발효식품의산업화및대규모제조의증가에따른품질균일화및속성발효를위한종균개발및종균제첨가의지속적증가가예상되고있어, 종균의안전성은지속적인전통발효식품의발전에필요한전제조건이다. 한편, 우리나라에는발효용종균에대한안전성기준규격없어, 안전성평가없이기능성만으로선발된종균의사용은국내뿐만아니라세계화에걸림돌이될것으로예상된다. 국제적인식품거래가활발해지면서소비자와수입국의정부당국들은안전에대해서더욱민감하게반응하고있다. 전통발효식품의세계화를위해서는향후적용될종균에대한기능적측면뿐만아니라안전성의확보가필요하고, 안전성평가기준의마련이필요한시점에도달해있다.
Starter Safety for Fermented Foods 9 요 약 식품발효용으로사용하는미생물은오랜기간의사용이력에근거하여안전성에대한의문이제기되지않고사용되어왔다. 한편, 가공식품산업의발달로국제적인식품거래가활발해지면서수입국의소비자와정부당국은식품에첨가하는미생물의안전에대해서더욱민감하게반응하고있다. 현재, 식품에첨가하는미생물에대한안전성평가기준은미국 Food and Drug Administration (FDA) 가사용하고있는 Generally Recognized as Safe (GRAS) 와유럽연합 European Food Safety Agency (EFSA) 의 Qualified Presumption of Safety (QPS) 가대표적으로적용되고있다. GRAS는식품에첨가하는미생물자체에대한안전성이아닌특정용도에서의첨가에따른안전성을의미하고, QPS는식품에첨가가능한미생물단위 ( 분류학적단위, taxonomic unit) 에대한안전성평가이다. 우리나라에서는전통발효식품의상업적생산이증가하면서종균적용이추진되고있지만, 식품발효용종균에대한안전성기준규격없어안전성평가없이기능성만으로종균개발이진행되고있다. 전통발효식품의세계화를위해서는향후적용될종균에대한기능적측면뿐만아니라안전성의확보가필요하고, 식품용미생물에대한안전성평가기준의마련이필요한시점에도달해있다. Acknowledgements This project was conducted by the generous financial support of the Youlchon Foundation (Nongshim Corporation and its affiliated companies) in Korea. References 1. Ahn Y-S, Kim C-J, Choi S-H. 1990. Production of protease by the extreme halophile, Halobacterioum sp. J. Korean Agric. Chem. Soc. 33: 247-251. 2. Bourdichon F, Casaregola S, Farrokh C, Frisvad JC, Gerds ML, Hammes WP, et al. 2012. Food fermentations: microorganisms with technological beneficial use. Int. J. Food Microbiol. 154: 87-97. 3. Buckenhuskes HJ. 1993. Selection criteria for lactic acid bacteria to be used as starter cultures for various food commodities. FEMS Microbiol. Rev. 12: 253-272. 4. Caplice E, Fitzgerald GF. 1999. Food fermentations: role of microorganisms in food production and preservation. Int. J. Food Microbiol. 50: 131-149. 5. Carr FJ, Chill D, Maida N. 2002. The lactic acid bacteria: a literature survey. Crit. Rev. Microbiol. 28: 281-370. 6. Cha Y-J, Lee K-H, Lee E-H, Kim J-S, Joo D-S. 1990. Studies on the processing of rapid fermented anchovy prepared with low salt contents by adapted microorganism (Processing of low salt fermented anchovy with proteolytic bacteria and quality stability during storage). J. Korean Agric. Chem. Soc. 33: 330-336. 7. Charteris WP, Kelly PM, Morelli L, Collins JK. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61: 1636-1643. 8. EFSA. 2004. Scientific colloquium summary report: Qualified Presumption of Safety of microorganisms in food and feed. http://www.efsa.europa.eu/. 9. EFSA. 2005. Opinion of the scientific committee on a request from EFSA related to a generic approach to the safety assessment by EFSA of microorganisms used in food/feed and the production of food/feed additives. EFSA J. 226: 1-12. 10. EFSA. 2007. Opinion of the scientific committee on introduction of a qualified presumption of safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA J. 587: 1-16. 11. EFSA. 2008. Scientific opinion of the panel on biological hazards on the maintenance of the list of QPS microorganisms intentionally added to food or feed. EFSA J. 923: 1-48. 12. EFSA. 2009. Scientific opinion of the panel on biological hazards on the maintenance of the list of QPS microorganisms intentionally added to food or feed. EFSA J. 7: 1-93. 13. Franz CMAP, Stiles ME, Schleifer KH, Holzapfel WH. 2003. Enterococci in foods-a conundrum for food safety. Int. J. Food Microbiol. 88: 105-122. 14. Franz CMAP, Huch M, Abriouel H, Holzapfel W, Galvez A. 2011. Enterococci as probiotics and their implications in food safety. Int. J. Food Microbiol. 151: 125-140. 15. Giraffa G, Chanishvili N, Widyastuti Y. 2010. Importance of lactobacilli in food and feed biotechnology. Res. Microbiol. 161: 480-487. 16. Guan L, Cho KH, Lee JH. 2011. Analysis of the cultivable bacterial community in jeotgal, a Korean salted and fermented seafood, and identification of its dominant bacteria. Food Microbiol. 28: 101-113. 17. Holzapfel W. 1997. Use of starter cultures in fermentation on a household scale. Food Control 8: 241-258. 18. Holzapfel WH. 2002. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int. J. Food Microbiol. 75: 197-212. 19. Jung YJ, Park DH. 2004. Physiology and growth properties of halophilic bacteria isolated from jeotgal (salted seafood). Korean J. Microbiol. 40: 263-268. 20. Jung JY, Lee SH, Lee HJ, Seo HY, Park WS, Jeon CO. 2012. Effects of Leuconostoc mesenteroides starter cultures on microbial communities and metabolites during kimchi fermentation. Int. J. Food Microbiol. 153: 378-387. 21. Jung J, Choi S, Jeon CO, Park W. 2013. Pyrosequencingbased analysis of the bacterial community in Korean traditional seafood, ojingeo jeotgal. J. Microbiol. Biotechnol. 23: 1428-1433. March 2014 Vol. 42 No. 1
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