Food Science and Preservation (한국식품저장유통학회지)

The Korean Society of Food Preservation (한국식품저장유통학회)
권호 표지
DOI QR코드

DOI QR Code

Identification and Characterization of Wild Yeasts Isolated from Korean Domestic Grape Varieties

국산 포도로부터 분리한 야생효모의 동정 및 특성

  • Choi, Sang-Hoon (Department of Food Science and Technology, Kyungpook National University) ;
  • Hong, Young-Ah (Department of Food Science and Technology, Kyungpook National University) ;
  • Choi, Yoon-Jung (Department of Food Science and Technology, Kyungpook National University) ;
  • Park, Heui-Dong (Department of Food Science and Technology, Kyungpook National University)
  • 최상훈 (경북대학교 식품공학부 식품생물공학) ;
  • 홍영아 (경북대학교 식품공학부 식품생물공학) ;
  • 최윤정 (경북대학교 식품공학부 식품생물공학) ;
  • 박희동 (경북대학교 식품공학부 식품생물공학)
  • Received : 2011.03.03
  • Accepted : 2011.08.05
  • Published : 2011.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

Several wild yeasts were isolated from Korean grape varieties before and during spontaneous fermentation. Among them, four strains were isolated based on the alcohol content and flavor production in wine after fermentation of apple juice. In this study, the four yeast strains were identified and characterized. PCR-restriction fragment length polymorphism analysis of ITS I-5.8S-ITS II region with restriction endonuclease Hae III and Hinf I resulted in that all the strains showed a typical pattern of Saccharomyces cerevisiae. Pulse field gel electrophoresis showed three different chromosome patterns with a same band between strains SS89 and SS812. When ITS I-5.8S-ITS II sequences of the four strains were compared with one another, they were similar to those of Saccharomyces cerevisiae CBS 4054 type strain. Identity of the sequences was higher than 97% with those of the type strain. Phylogenetic analysis showed based on the sequences showed they were genetically closed to the type strain. The four identified strains were tested in a medium containing 200 ppm potassium metabisulfite, and the MM10 and WW108 inhibition rates resulted at up to 24 h. The four strains were tested at an incubation temperature of $30^{\circ}C$. The 30% sugar concentration in the medium (w/v) showed the highest growth in 36 h, especially in the case of SS89, which was close to growth 40. The four strains were tested in an 8% ethanol medium (v/v). Alcohol tolerance was initially kept in the incubation process. The strains began to adapt, however, to the exceeded resistance. The four strains showed the lowest inhibition rate at 24 h.

본 연구에서는 국산 포도로부터 분리된 효모들 중 와인 발효 적성이 우수한 균주들을 선정하여 ITS-I-5.8S-ITS II DNA 염기서열 분석과 분자생물학적인 방법을 통하여 동정하였고 발효환경 내성을 알아보았다. 분리 효모들의 ITS I-5.8S-ITS II 영역을 PCR로 증폭한 결과 약 800bp의 DNA가 증폭됨을 확인하였다. 이 DNA를 제한효소 HaeIII로 처리한 경우 모두 약 400bp의 DNA band 확인되었고, Hinf I로 처리한 경우에는 약 350 bp와 300 bp의 DNA band를 나타내었다. 분리된 4 균주의 염색체 DNA를 PFGE로 확인한 결과 MM10, WW108 그리고 SS89(SS812 동일)가 서로 다른 3가지 염색체 패턴을 나타내었다. ITS I-5.8S-ITS II DNA 염기서열을 분석한 결과 모두 S. cerevisiae CBS 4054 표준균주와 97% 이상의 상동성을 나타내어 매우 가까운 근연관계에 있음을 알 수 있었다. 근린결합분석을 이용한 phylogenetic 분석을 통하여 분리 균주인 MM10, SS89, SS812 그리고 WW108 균주는 S. cerevisiae CBS 4054 표준 균주와 계통 유연관계에서 매우 가까운 위치에 있는 것을 확인할 수 있었다. 동정된 4종의 야생효모 중 200 ppm의 아황산 함유 배지에서 MM10과 WW108 균주는 24시간대에서 6% 미만의 생육 저해률을 보였다. 또한 $30^{\circ}C$ 배양온도에서 30% 포도당을 함유하는 YPD 배지에서 36시간대에서 가장 높은 성장을 나타내었으며, 특히 SS89 균주는 660 nm에서의 흡광도가 약 40에 가까운 수치를 나타내었다. 배양온도 $40^{\circ}C$에서는 모든 효모군이 10~15의 수치를 나타내어 낮은 성장을 나타내었다. 알코올(8%, v/v)을 함유하는 YPD 배지에서 배양초기에는 내성이 약하였으나 배양이 진행되면서 적응을 하기 시작하고 24시간대에서 가장 낮은 생육 저해률을 보였다.

Keywords

References

  1. Montville TJ, Matthews KR (2005) Food microbiology: an introduction. ASM Press, Washington, DC, USA, p 223-239
  2. Kurtzman CP, Fell JW (1998) The Yeasts, A Taxonomic Study(fourth edition). Elsevier, Amsterdam, The Netherlands, p 358-371
  3. Attfield PV (1997) Stress tolerance. The key to effective strains of industrial baker's yeast. Nat Biotechnol, 15, 1351-1357 https://doi.org/10.1038/nbt1297-1351
  4. Pigeau G, Inglis D (2005) Upregulation of ALD3 and GPD1 in Saccharomyces cerevisiae during Icewine fermentation. J Appl Microbiol, 99, 112-125 https://doi.org/10.1111/j.1365-2672.2005.02577.x
  5. Erasmus DJ, Merwe GK, Vuuren HJJ (2003) Genome-wide expression analyses: metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Res, 2, 375-399
  6. Jimenez-Martí E, Zuzuarregui A, Gomar-Alba M, Gutierrez D, Gil C, del Olmo M (2011) Molecular response of Saccharomyces cerevisiae wine and laboratory strains to high sugar stress conditions. Int J Food Microbiol, 145, 211-220 https://doi.org/10.1016/j.ijfoodmicro.2010.12.023
  7. Caridi A, Crucitti P, Ramondino D (1999) Winemaking of must at high osmotic strength by thermotolerant yeast. Biotechnol Lett, 21, 617-620 https://doi.org/10.1023/A:1005528613379
  8. Aguilera F, Peinado RA, Millan C, Ortega JM, Mauricio JC (2006) Relationship between ethanol tolerance, H+-ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains. Int J Food Microbiol, 110, 34-42 https://doi.org/10.1016/j.ijfoodmicro.2006.02.002
  9. Zuzuarregui A, Del Olmo M (2004) Expression of stress genes in wine strains with different fermentative behavior. FEMS Yeast Res, 4, 699-710 https://doi.org/10.1016/j.femsyr.2004.01.008
  10. Yook C. Seo MH, Kim DH, Kim JS (2007) Quality improvement of Campbell Early wine by mixing with different fruits. Korean J Food Sci Technol, 39, 390-399.
  11. Guthrie C, Fink RG (1991) Methods in Enzymology: Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, California, USA, 194, p 13
  12. Amberg DC, Burke DJ, Strathern JN (2005) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. COLD SPRING HARBOR LABORATORY PRESS, Cold Spring Harbor, New York, USA, p 119-120
  13. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene, 57, 267-272 https://doi.org/10.1016/0378-1119(87)90131-4
  14. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds.) PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, California, USA, p 315-322
  15. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual(3rd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, Volume 1, Chapter 5, p 5.2-5.17, p 5.55-5.60, p 5.65-5.67, p 5.79-5.82
  16. Clark SM, Lai E, Birren BW, Hood L (1988) A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields. Science, 241, 1203-1205 https://doi.org/10.1126/science.3045968
  17. Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV, the CDC PulseNet Task Force (2001) PulseNet: The Molecular Subtyping Network for Foodborne Bacterial Disease Surveillance, United States. Emerg Infect Dis, 7, 382-389 https://doi.org/10.3201/eid0703.017303
  18. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nuclic Acids Symp Ser, 41, 95-98
  19. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol, 24, 1596-1599 https://doi.org/10.1093/molbev/msm092
  20. Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A (2001) Yeast population dynamics in spontaneous fermentations: comparison between two different wine-producing areas over a period of three years. Antonie Van Leeuwenhoek, 79, 345-352 https://doi.org/10.1023/A:1012027718701
  21. Nisiotou AA, Spiropoulos AE, Nychas GJE (2007) Yeast community structures and dynamics in healthy and Botrytis-affected grape must fermentations. Appl Environ Microbiol, 73, 6705-6713 https://doi.org/10.1128/AEM.01279-07
  22. Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneuf-Pomarede I (2008) Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. Int J Food Microbiol, 125, 197-203 https://doi.org/10.1016/j.ijfoodmicro.2008.04.001
  23. Ough CS, Crowell EA (1987) Use of sulphur dioxide in winemaking. J Food Sci, 52, 386-389 https://doi.org/10.1111/j.1365-2621.1987.tb06620.x
  24. Abe H, Fujita Y, Takaoka Y, Kurita E, Yano S, Tanaka N, Nakayama K (2009) Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase ${\delta}$. J Biosci Bioeng, 108, 199-204 https://doi.org/10.1016/j.jbiosc.2009.03.019

Cited by

  1. Rapid Fermentation of Freeze-Concentrated Ice Apple Wine by a Sugar Tolerant Yeast, Saccharomyces cerevisiae SS89 vol.19, pp.3, 2012, https://doi.org/10.11002/kjfp.2012.19.3.413
  2. Isolation and Characterization of Acidophilic Yeasts Producing Urease from Korean Traditional Nuruk vol.19, pp.2, 2012, https://doi.org/10.11002/kjfp.2012.19.2.308
  3. Leavening Ability of the Isolate Saccharomyces cerevisiae MF10003 in Bakery Dough vol.23, pp.2, 2013, https://doi.org/10.5352/JLS.2013.23.2.222
  4. Isolation of indigenous Lactobacillus plantarum for malolactic fermentation vol.51, pp.2, 2015, https://doi.org/10.7845/kjm.2015.5022