J. Milk Sci. Biotechnol. Vol. 34. No. 1, pp. 9~20(2016) 1, 2 3, 4 Novel Molecular-Based Approaches for Analyzing Microbial Diversity in Raw-Milk Long-

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1 J. Milk Sci. Biotechnol. Vol. 34. No. 1, pp. 9~20(2016) 1, 2 3, 4 Novel Molecular-Based Approaches for Analyzing Microbial Diversity in Raw-Milk Long-Ripened Cheeses: A Review Dong-Hyeon Kim 1, Jung-Whan Chon 1,2, Hyunsook Kim 3, Soo-Kyung Lee 1, Hong-Seok Kim 1, Joo-Yeon Lee 4, Jin-Hyuk Yim 1, Kwang-Young Song 1*, Young-Ji Kim 1, Il-Byung Kang 1, Dana Jeong 1, Jin-Hyeong Park 1, Ho-Seok Jang 1 and Kun-Ho Seo 1 1 Center for One Health, College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea 2 National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA 3 Dept. of Food & Nutrition, College of Human Ecology, Hanyang University, Seoul 04763, Korea 4 Korea Livestock Products HACCP Accreditation Service, Daegu 41256, Korea Abstract Various microflora, including lactic acid bacteria, are important and necessary components of various cheeses and have significant roles in cheese manufacturing and ripening. In general, the starter culture and secondary microflora could affect the physicochemical properties of various cheeses and could contribute to modifications during manufacturing and ripening. Therefore, during cheese manufacturing and ripening, microbial diversity may depend on continuous interactions among microflora and various environmental conditions. The microbial diversity of cheese is very complex and difficult to control using the classical microbiological techniques. However, recent culture-independent methods have been rapidly developed for microflora in cheese, which could be directly detected using DNA (and/or RNA) in combination with culture-dependent methods. Therefore, this review summarizes state-of-the-art molecular methods to analyze microbial communities in order to understand the properties that affect quality and ripening as well as the complex microbial diversity of various raw-milk, long-ripened cheeses. Keywords: cheese, raw milk, long-ripened, lactic acid bacteria, culture-dependent, culture-independent These authors contributed equally to this study. * Corresponding author: Kwang-Young Song, Center for One Health, College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea. Tel: , Fax: , drkysong@gmail.com, (Bottari et al., 2013). 2, 9

2 10 Journal of Milk Science & Biotechnology 제 34 권제 1 호 (2016),., dynamic (Coppola et al., 2000).,, (Neviani et al., 2013).., (Cremonesi et al., 2011). dynamic. (Neviani et al., 2013)., DNA( RNA) (Duthoit et al., 2003).,,, (El Baradei et al., 2007)., (Neviani et al., 2013).. (, Grana Padano Parmigiano Reggiano).. (Neviani et al., 2013). 2,.,. (1) (enumeration), (2), (3), (4) (biotype) (Gatti et al., 2008). MRS (de Man et al., 1960). (Gatti et al., 2003; Fornasari et al., 2006; Neviani et al., 2009).. (species). Staphylococcus thermophilus (Neviani et al., 2013). Fornasari et al.(2006) M17 S. thermophilus. Grana Padano S. thermopilus. (, WAM) fresh Gatti et al.(2003), Parmigiano Reggiano thermophillc lactobacilli streptococci. Neviani et al.(2009) (10 2 cfu/ml ) (, CAM). 24 grated Parmigiano Reggiano (Neviani et al., 2013). CAM Parmigiano Reggiano Lactobacillus rhamnosus ,,. phenotyping genotyping.,... (identification).

3 비살균숙성치즈의미생물균총분석에이용되는새롭게개발된분자생물학적방법 : 총설 11 (species-specific), amplified ribosomal DNA restriction analysis(16s-ardra), 16S ribosomal RNA(rRNA) gene (Chagnaud et al., 2001; Roy et al., 2001; Bouton et al., 2002; Vasquez et al., 2005). ARDRA(tRNAAla-23S rdna-restricion fragment length polymorphism) Mancini et al.(2012), Grana Padano. 16S~23S rdan. 16S rrna (Cremonesi et al., 2011). Bottari et al.(2013) phenylalanyl-trna phenylalanine (phes). real-time PCR Parmigiano Reggiano 29 thermophilic. DNA 600, (Neviani et al., 2013). thermophilic. 10, (subspecies) (Neviani et al., 2013)., typing. DNA fingerprinting, RFLP 16S rrna, restriction enzyme analysis pulsed-field gel electrophoresis(rea-pfge), randomly amplified polymorphic DNA(RAPD), amplified fragment length polymorphism(aflp), repetitive extragenic palindromic- PCR (REP-PCR) (Giraffa et al., 2003; Gala et al., 2008; Lazzi et al., 2009; Bove et al., 2011; Herrero-Fresno et al., 2012; Solieri et al., 2012). (Neviani et al., 2013). RAPD, (Randazzo et al., 2009; Monfredini et al., 2012)., 2 (1), (2), (Tyler et al., 1997). REP-PCR,. REP-PCR, artifacts, (Li et al., 2009). AFLP RAPD (Clerc et al., 1998)., DiCagno et al(2010) RAPD-PCR AFLP D Index Lazzi et al (2009) AFLP. Parmigiano Reggiano Grana Padano (Neviani et al., 2013)., (Rossetti et al., 2009; Bottari et al., 2013)., thermiphilic Lb. helveticus (Andrighetto et al., 2004). Lb. helveticus,, (Gatti et al., 2008; De Dea Lindner et al., 2009). Lb. helveticus Broadbent et al.(2011). Parmigiano Reggiano Grana Padano Lb. helveticus. Parmigiano Reggiano Grana Padano. Parmigiano Reggiano Grana Padano (non-starter LAB, NSLAB) (Zago et al., 2007; Monfredini et al., 2012; Solieri et al., 2012).,, (Giraffa and Neviani, 2001; Santarelli et al., 2008).,

4 12 Journal of Milk Science & Biotechnology 제 34 권제 1 호 (2016) (Neviani et al., 2013)., (Neviani et al., 2013). Gatti et al.(2006) (fluorescence in situ hybridization, FISH) Grana Padano.. (culture-depenpent), (culture-independent) (Neviani et al., 2013).,. in vitro in vitro (Head et al., 1998; Hugenholtz et al., 1998).,,. DNA( RNA) (Neviani et al., 2013)., (Coppola et al., 2008).,,. PCR (Quigley et al., 2011). DNA,. DNA (Rudi et al., 2005)., DNA DNA. DNA EMA(ethidium bromide monoazide) PMA(propidium monoazide) (Schaad et al., 1995; Josefsen et al., 2010). RNA, (Bodrossy et al., 2006). DNA RNA (Neviani et al., 2013)., DNA, PCR. DNA( RNA) (Bonaiti et al., 2006)., Matrix, PCR (Jany and Barbier, 2008)., DNA( RNA) matrix,,, DNA (Duthoit et al., 2003; El Baradei et al., 2007; Parayre et al., 2007). matrix PCR. marker (Juste et al., 2008). target 16S 26S rrna (Florez and Mayo, 2006).., PCR annealing site(dna target). phes RNA polymerase B subunit (Martin-Platero et al., 2009; Zago et al., 2009). PCR. PCR, (probe) (Delbes et al., 2007; Neviani et al., 2013).. PCR-DGGE PCR-TTGE PCR,

5 비살균숙성치즈의미생물균총분석에이용되는새롭게개발된분자생물학적방법 : 총설 13 (Myers et al., 1987; Yoshino et al., 1991). acrylamide DNA, GC,, (melting domain) (discrete region). (Neviani et al., 2013). PCR-DGGE (formamide urea). PCR-TTGE (denaturing gradient), (Jany and Barbier, 2008; Juste et al., 2008). PCR-DG/TTGE (Ogier et al., 2004). database. (amplicon) DG/TTGE acrylamide., (Jany and Barbier, 2008). PCR SSCP-PCR (Delbes et al., 2007). DGGE TTGE DNA, DGGE/TTGE PCR. DNA acrylamdie. 2 (Neviani et al., 2013). (SSCP) (DGGE) (TTGE) discrete, (fingerprint). (band),,,,. data base (Coppola et al., 2008). DNA. terminal-rflp length heterogeneity-pcr (Rademaker et al., 2006; Bottari et al., 2010). LH-PCR 16S ribosomal DNA (Ritchie et al., 2000). PCR,., (Suzuki et al., 1998). T-RFLP PCR (Liu et al., 1997). PCR 5` 3`. acrylamide. (TRFs) database (Dickie et al., 2002). PCR TRF, DNA. TRF (Jany and Barbier, 2008). SSCP-PCR T-RFLP HPLC (Xiao and Oefner, 2001). PCR-DG/TTGE (Jany and Barbier, 2008). DHPLC, (Ercolini et al., 2008; Mounier et al., 2009)., real-tiem PCR (Neviani et al., 2013). PCR PCR PCR. DNA. DNA., DNA DNA. DNA (Juste et al., 2008). DNA RNA, -qpcr (Falentin et al., 2012).

6 14 Journal of Milk Science & Biotechnology 제 34 권제 1 호 (2016) (dynamics) (Juste et al., 2008; Neviani et al., 2013).. (polyphasic) (Delbes et al., 2007; Neviani et al., 2013)., Parmigiano Reggiano Grana Padano., Rossetti et al.(2008) RT-LH-PCR Grana Padano. RT-LH-PCR Grana Padano,,, (Santarelli et al., 2008). Gatti et al.(2008) Parmigiano Reggiano., LH-PCR. PCR-DGGE rrna 12 Parmigiano Reggiano (Gala et al., 2008).,,,. Parmigiano Reggiano Grana Padano (Neviani et al., 2013). Grana Padano Parmigiano Reggiano. (Neviani et al., 2013). Grana Padano Parmigiano Reggiano (Bottari et al., 2010)., (Neviani et al., 2013). Grana Padano Parmigiano Reggiano.. butyric late blowing, vat lysozyme (Coppola et al., 2000)., Grana Padano 8 (Neviani et al., 2013). vat Parmigiano Reggiano Grana Padano (Cremonesi et al., 2011). ( ), 32~34. 2~4 mm, 53~56. (4.0~4.3 SH/50 ), (Neviani et al., 2013)., vat. grana, paste, (Zago et al., 2007). (Neviani et al., 2013). Vat. 2,, 3.,. Grana Padano 9 Parmigiano Reggiano 12,,. 30% 70% (Neviani et al., 2013). Grana Padano Parmigiano Reggiano dynamic (Neviani et al., 2013). ( thermophilic )...,

7 비살균숙성치즈의미생물균총분석에이용되는새롭게개발된분자생물학적방법 : 총설 15, deamination decarboxylation (Gatti et al., 2008).,, - stress., (Neviani et al., 2013).,. (Rossetti et al., 2009: Rasolofo et al., 2010). Grana Padano Parmigiano Reggiano.,, ph (Neviani et al., 2013). 1., (,, ) matrix (Settanni and Moschetti, 2010). Rasolofo et al.(2010), T-RFLP DGGE. Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acetobacger Trichococcus., Coppola et al.(2000) Parmigiano Reggiano mesophilic 10 4 cfu/ml, De Dea Lindner et al.(2008) Neviani et al. (2009) 1 log.. Coppola et al.(2010) mesophilic lactobacilli. Lb. paracasei subsp. paracasei, Lb. paracasei subsp. tolerans. thermophilic Streptococcus thermophilus, Lb. helveticus, Lb. delvrueckii subsp. bulgaricus. Lb. rhamnosus. Neviani et al.(2009) CAM MRS Lb. rhamnosus. 16S rrna St. uberis, Lactococcus lactis subsp. lactis subsp. cremoris. Lb. delbrueckii subsp. lactis, Leuconostoc(Ln.) mesenteroides subsp. mesenteroides Lc. lactis subsp. cremoris Enterococcus genus(faecalis and E. faecium) (Neviani et al., 2013).. Grana Padano Santarelli et al.(2012) fluorescence microscopy %. LH-PCR (Neviani et al., 2013). 2., Grana Padano Parmigiano Reggiano epifluorescence microscopy. MRS (Neviani et al., 2009). WAM MRS, WAM ( ) (Neviani et al., 2013). 24 Grana Padano natural whey starter Rossetti et al.(2008), Lb. helveticus, Lb. delbrueckii subsp. lactis, St. thermophilus, Lb. fermentum. Lb. helveticus, Lb. delbrueckii subsp. lactis St. thermophilus, Lactobacilli. Santarelli et al.(2012). Parmigiano Reggiano LH-PCR FISH(Bottari et al., 2010). LH-PCR electropherogramsd FISH, Lb. helveticus, Lb. delbrueckii ( subsp. lactis), St. thermophilus, Lb. fermentum. Parmigiano Reggiano 3. Lb. helveticus Lb. delbrueckii, Lb. helveticusa, Lb. helveticus Lb. delbrueckii (Bottari et al., 2010).

8 16 Journal of Milk Science & Biotechnology 제 34 권제 1 호 (2016) 3. Grana Padano Parmigiano Reggiano (SLAB) NSLAB., (Beresford et al., 2001)., SLAB, NSLAB (Neviani et al., 2013). Grana Padano Parmigiano Reggiano SLAB. ( 20 ) (Grana Padano 9, Parmigiano Reggiano 12 ) (,, ) (Neviani et al., 2013).,,, ph. SLAB,. SLAB,, (De Dea Lindner et al., 2008). maxtrix. NSLAB., dynamics (Neviani et al., 2013). LH-PCR.. Grana Padano Parmigiano Reggiano,,,., dynamics (colony) matrix..,.,,,,,.,,. Grana Padano Parmigiano Reggiano.., (culture-dependent) (culture-independent) dynamic., ( ) (No. 2015R1A2A2A ) Disclaimer: The views expressed herein do not necessarily reflect those of the US Food and Drug Administration or the US Department of Health and Human Services. 1. Andrighetto, C., Marcazzan, G. and Lombardi, A Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese. Lett. Appl. Microbiol. 38: Beresford, M. R., Andrew, P. W. and Shama, G Listeria monocytogenes adheres to many materials found in food-processing environments. J. Appl. Microbiol. 90: Bodrossy, L., Stralis-Pavese, N., Konrad-Koszler, M., Weilharter, A., Reichenauer, T. G. and Shofer, D mrna-based parallel detection of active methanotroph populations by

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