PMID- 22819715
OWN - NLM
STAT- MEDLINE
DCOM- 20121210
LR  - 20181201
IS  - 1879-3460 (Electronic)
IS  - 0168-1605 (Linking)
VI  - 158
IP  - 1
DP  - 2012 Aug 1
TI  - Survival rate of wine-related yeasts during alcoholic fermentation assessed by 
      direct live/dead staining combined with fluorescence in situ hybridization.
PG  - 49-57
LID - 10.1016/j.ijfoodmicro.2012.06.020 [doi]
AB  - Real-time detection of microorganisms involved in complex microbial process, such 
      as wine fermentations, and evaluation of their physiological state is crucial to 
      predict whether or not those microbial species will be able to impact the final 
      product. In the present work we used a direct live/dead staining (LDS) procedure 
      combined with fluorescence in situ hybridization (FISH) to simultaneously assess 
      the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora 
      guilliermondii (Hg) during fermentations performed with single and mixed 
      cultures. The population evolution of both yeasts was determined by plating and 
      by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since 
      the FISH method involves the permeabilization of the cell membrane prior to 
      hybridization and that it may influence the free diffusion of PI in and out of 
      the cells, we optimized the concentration of this dye (0.5 mug of PI per 10(6) 
      cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by 
      hemocytometry and flow cytometry. Results showed that the survival rate of Sc 
      during mixed cultures was high throughout the entire process (60% of viable cells 
      at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead 
      cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at 
      the 4th day. However, under single culture fermentation the survival rate and 
      culturability of Hg decreased at a much slower pace, exhibiting at the 7th day 
      (67 g/l of ethanol) 8.7x10(4) CFU/ml and 85% of dead cells. Thus, our work 
      demonstrated that the LDS-FISH method is able to simultaneously assess the 
      viability and identity of these wine-related yeast species during alcoholic 
      fermentation in a fast and reliable way. In order to validate PI-staining as a 
      viability marker during alcoholic fermentation, we evaluated the effect of 
      ethanol on the membrane permeability of Sc and Hg cells, as well as their 
      capacity to recover membrane integrity after being exposed to different levels of 
      ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to 
      recover membrane integrity after ethanol exposure, Hg cells were not. However, 
      under alcoholic fermentation Sc cells didn't recover membrane integrity after the 
      mid-term (4-5 days) of alcoholic fermentation.
CI  - Copyright (c) 2012 Elsevier B.V. All rights reserved.
FAU - Branco, Patricia
AU  - Branco P
AD  - Unidade Bioenergia, LNEG, Estrada do Paco do Lumiar, 22, 1649-038 Lisboa, 
      Portugal.
FAU - Monteiro, Margarida
AU  - Monteiro M
FAU - Moura, Patricia
AU  - Moura P
FAU - Albergaria, Helena
AU  - Albergaria H
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120707
PL  - Netherlands
TA  - Int J Food Microbiol
JT  - International journal of food microbiology
JID - 8412849
RN  - 3K9958V90M (Ethanol)
SB  - IM
MH  - Cell Membrane/physiology
MH  - Ethanol/metabolism
MH  - *Fermentation
MH  - Flow Cytometry
MH  - Hanseniaspora/*growth & development
MH  - In Situ Hybridization, Fluorescence
MH  - Saccharomyces cerevisiae/*growth & development/metabolism
MH  - Staining and Labeling
MH  - Wine
MH  - Yeasts/metabolism
EDAT- 2012/07/24 06:00
MHDA- 2012/12/12 06:00
CRDT- 2012/07/24 06:00
PHST- 2012/04/25 00:00 [received]
PHST- 2012/06/21 00:00 [revised]
PHST- 2012/06/30 00:00 [accepted]
PHST- 2012/07/24 06:00 [entrez]
PHST- 2012/07/24 06:00 [pubmed]
PHST- 2012/12/12 06:00 [medline]
AID - S0168-1605(12)00344-3 [pii]
AID - 10.1016/j.ijfoodmicro.2012.06.020 [doi]
PST - ppublish
SO  - Int J Food Microbiol. 2012 Aug 1;158(1):49-57. doi: 
      10.1016/j.ijfoodmicro.2012.06.020. Epub 2012 Jul 7.