DETECTION AND PERSISTENCE OF SPOILAGE ORGANISMS IN FERMENTATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0210284
• Project No.: NC06864
• Project Start Date: Jan 12, 2007
• Project End Date: Sep 30, 2012

Project Director
Phister, T. G.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Food, Bioprocessing, and Nutrition Sciences.

Non Technical Summary
Wine spoilage microbes, such as Brettanomyces, have been found to persist in wine for long periods. These organisms may not be detected by conventional methods such as plating. However, they may still have a negative impact on the finished product. In fact, previous research has demonstrated that Brettanomyces may produce spoilage compounds even in the absence of a culturable population. In this project, we will develop DNA based methods to detect wine spoilage microbes and to differentiate the living and dead populations. We will further examine the impact the living but nonculturable populations may have on the wine fermentation.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	4010	1040	30%
501	4010	1100	10%
501	4020	1040	40%
501	4020	1102	20%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
4010 - Bacteria; 4020 - Fungi;

Field Of Science
1100 - Bacteriology; 1102 - Mycology; 1040 - Molecular biology;

Keywords

brettanomyces

dekkera

lamp pcr

real-time pcr

ethidium monoazide

wine

fermentation

viable nonculturable

yeast

Goals / Objectives
This project has three objectives. The first objective is the development of detection methods for wine-spoilage organisms. The second objective is the differentiation of living and nonculturable microbes in alcoholic fermentation and the final objective is to determine the impact of nonculturable yeasts and bacteria on alcoholic fermentations.

Project Methods
This project will examine the detection and persistence of spoilage organisms in fermentations. In the first phase of the project we will develop inexpensive detection methods for various spoilage yeasts and bacteria. The method used will be based on LAMP PCR. Primers have been designed to Brettanomyces/Dekkera bruxellensis. This assay will be optimized beginning with the DNA purification. In order to limit cost for the wine industry we will examine the preparation of samples by boiling as this is a cheaper less technically demanding replacement for other DNA isolation procedures. Once the DNA isolation is optimized we will begin optimizing the visualization methods using SYBR green. Finally we will develop methods to quantify B. bruxellensis in wine. Initially the time to fluorescence for each sample will be measured and compared to a standard curve. Concurrent with optimization of the Brettanomyces assay, we will develop a LAMP assay to detect and possibly enumerate other wine-spoilage organisms. One possibility is an assay for lactic acid bacteria capable of producing histamine. Levels of histamine in wine are becoming a greater concern to the industry and are even regulated in Switzerland and a few other European countries. Once the different LAMP primer sets are developed, they will be tested for specificity using wine related yeast and bacteria including both histamine producing and nonproducing LAB strains. Assay conditions for the most promising LAMP primers will be further optimized. Other organisms may also be possible targets including Zygosaccharomyces, Pediococcus, O. oeni, as well as, other spoilage yeast and bacteria. In the second phase of the project, we will try and differentiate viable and nonviable populations of B. bruxellensis and Acetobacter pasteurianus using Quantitative real-time PCR (QPCR) and ethidium monoazide. We will optimize the concentration of EMA required to inhibit the PCR reaction, the amount of light exposure needed to inactivate unbound EMA and the proper length of light exposure to effectively cross link the EMA with the DNA of dead cells. We will next examine the amplification of live cell DNA in a dead cell background to establish the minimum number of live cells that can be detected in a dead cell background. In the final phase of the project we will determine if the nonculturable populations of B. bruxellensis and A. pasteurianus can have an effect on wine quality. The microbes will be grown in a model wine system. The fermentation will be incubated at 25oC with aeration and growth will be monitored using QPCR, QPCR plus EMA, rt-real-time PCR, the Molecular Probes Live/Dead yeast kit and the standard plate count on Malt Extract Broth at both neutral and acidic pH as this helps determine the populations level of injury. The 4-ethylphenol and 4-ethylguaiacol concentrations will be measured using the gas chromatography-mass spectrometry. The direct viable counting methods (QPCR plus EMA and the Molecular Probes Live/Dead yeast kit) may correlate with the level of 4-ethylphenol and 4-ethylguaiacol production by the Brettanomyces sp.

Progress 01/12/07 to 09/30/12

Outputs
OUTPUTS: todate we have published 11 papers with 4 more in preparation and presented 18 posters or papers PARTICIPANTS: Mara Massel- technical Michael Lenordelii M.S. student Victoria Gray M. S. Student Megan Beckner M.S. student Jinjun Gong-Postdoc Steven Gray-Postdoc Melissa Ivey M. S. Student Vanessa Pittet-(visiting PhD Student) Blake Layfield M.S. student TARGET AUDIENCES: In this project we developed knowledge that would help fermented beverage and biofuel producers prevent or control contaminating organsims. We developed at least one assay to detect spoilage microbes and explored the genomes and physiology of dekkera and pediococcus both major contaminants in biofuel fermentations. these studies will lead to better control systems for these spoilage agents. In addition, we trained nine researchers two of which have gone onto work in the food fermentation industry. In addition two other M.S. students ahve gone onto PhD programs still focused in the fermented foods industries. The first Megan Beckner is doing a PhD working on wine in Italy. Blake is working on Brewing at NCSU. Finally Michael Leanordelli is working as an Extension Associate at the University of Missouri. Both postdocs have gone into the biofuel industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have sequenced the genome of the major wine spoilage agent Dekkera bruxellensis which will provide insight into the physiology of this spoilage organism. We have developed assays to detect Dekkera anomola, looked at gene expression for the fermentation contaminating microbe Pediococcus claussenii and determined that flavor compounds produced by lactobacilli can impact Saccharomyces during the course of a fermentation.

Publications

• Gray, Victoria, and T. G. Phister. 2011. The influence of yeasts in the survival of probiotics in yogurt. IFT. New Orleans, LA. (June 2011)-poster
• Beckner, Megan, and T. G. Phister. 2011. The effects of aromatic alcohols on the growth of Saccharomyces cerevisiae during fermentation. ASM. New Orleans, LA. (May 2011)-poster
• Phister, Trevor G. 2011. Cell-cell signaling in microbial interactions. Plant pathology Society of North Carolina, Raleigh, NC. (March 2, 2011)-presentation
• Gray, Steven R., H. Rawsthorne, B. Dirks and T. G. Phister. 2011. Detection and Enumeration of Dekkera anomala in Beer and Cola Using Real-Time PCR. Lett. Appl. Microbiol. 52:352-359
• Rawsthorne, H., T. G. Phister and L. Jaykus. 2009. Development of a fluorescent in situ method for visualization of enteric viruses. Appl. Environ. Microbiol. 75:7822-7
• Rawsthorne, H. and T. G. Phister. 2009. The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide bromide. Lett. Appl. Microbiol. 49: 652-654
• Phister, T. G. 2009. Using Microbial Succession to the Processor's Advantage: Food Fermentation and Biocontrol. In L. Jaykus, H. Wang, and L. Schlesinger (ed.), Foodborne Microbes: Shaping the Host Ecosystem. ASM Press, Washington D. C.
• Gray, Steven, J. Gong, John Sheppard and Trevor G. Phister. 2010. Bioethanol Production by the yeast Kluyveromyces marxianus using five and six carbon sugars. ASM, San Diego, CA. (May 23-27, 2010)-poster
• Phister, Trevor G. 2010. Enhancing Bioethanol Fermentation using Kluyveromyces marxianus. Novozymes, Franklinton NC (July 9, 2010)-Presentation
• Corro-Herrera, V. A., Phister, T., Mendoza-Garcia, P., and Aguilar-Uscanga, M. G. 2010. Acetic acid production byBrettanomyces bruxellensis in continuous culture. Fourth International Congress on Food Science and food Biotechnology in Developing Countries. Veracruz, Mexico
• Beckner, Megan, Melissa Ivey, Mara Massel and Trevor G. Phister. 2010. Interactions between Saccharomyces cerevisiae and Lactobacillus species during fermentation. 110th IFT Annual Meeting. Chicago IL. (July 17-20, 2010)-Poster
• Gray, Victoria, Michael Leonardelli and Trevor G. Phister. 2010. Affect of Saccharomyces on the growth of Dekkera bruxellensis. 110th IFT Annual Meeting. Chicago, IL. (July 17-20, 2010)-Poster
• Massel, Mara and Trevor G. Phister. Affect of amplicon size on the differentiation of viable and nonviable Dekkera bruxellensis using EMA and real-time PCR. 2010. Society for Industrial Microbiology Annual Meeting. San Francisco, CA. (August 1-5, 2010)-Poster
• Trevor Phister. 2009. Functional genomic analysis of Saccharomyces-Lactobacilli Interactions in Bio-ethanol fermentations Genes to Products Agricultural Plant, Microbe, and Biobased Product Research. USDA Bethesda MD
• Aguilar-Uscanga, U. and T. G. Phister. 2008. Use of propidium monoazide (PMA) for live/dead distinction of Brettanomyces bruxellensis. 108th General Meeting American Society for Microbiology. Boston
• H. Rawsthorne, T. G. Phister and L.A. Jaykus. 2008. Fluorescent labeling of mouse norovirus facilitates the study of interactions between enteric viruses and foods. 108th General Meeting American Society for Microbiology. Boston
• J. Blake Layfield, Trevor G. Phister and John Sheppard. 2009. Characterization of hybrid strains of Saccharomyces pastorianus as related to desiccation tolerance and fermentation performance. ASBC, Tucson AZ
• Piskur, J. Z. Ling, M. Marcet-Houben, O. P. Ishchuk, A. Aerts, K. LaButti, A. Copeland, E. Lindquist, K. Barry, C. Compagno, L. Bisson, I. V. Grigoriev, T. Gabaldon and T. Phister. 2012. The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties. Int. J. Food Microbial. 157: 202-209
• Ivey, M. and T. G. Phister. 2011. Detection of Common Microorganisms in Wine: A Review of Molecular Techniques. J. Industrial Microbiol Biotech. 38: 1619-1634
• Beckner, M., M. Ivey, and T. G. Phister. 2011.Contamination in Biofuel Fermentations. Lett Appl. Microbiol. 53: 387-394
• Uscanga, M. G. A., Y. G. Alvarado, T. G. Phister, M. L. Delia and P. Strehaiano. 2011. Modeling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations. Lett Appl. Microbiol. 53:141-149
• Layfield, J. B., T. G. Phister and J. D. Sheppard. 2011. Characterization of Hybrid Strains of Saccharomyces pastorianus for Desiccation Tolerance. J. Am. Soc. Brewing Chem. 69:108-115
• Rawsthorne, H. and T. G. Phister. 2009. Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR. Int. J. Food Microbiol. 131:246-250
• Pettit, Vanessa, T. G. Phister and Barry Ziola. 2012. Pediococcus claussenii genetic expression during growth in beer assessed by transcriptome sequencing (RNA-seq). World Brewing Congress, Portland OR, USA. (July 28-August 1, 2012)-presentation
• Pettit, Vanessa, Mara Massel, Barry Ziola and T. G. Phister. 2011.RNA-seq of Pediococcus claussenii during growth in beer. 10th Symposium on Lactic Acid Bacteria. Egmond aan Zee, Netherlands. (August28-September1, 2011)-presentation
• Gray,S., L. Bisson, J. Piskur, F. Detrich, T. Henick-Kling, S. Baker and T. G. Phister. 2010 Completion of the Dekkera(Brettanomyces) bruxellensis Genome Sequence JGI User Meeting, Walnut Creek, CA
• Phister, T. 2009. Functional genomic analysis of Saccharomyces-Lactobacilli Interactions in Bio-ethanol fermentations 95th Annual ASM Southeastern Branch Conference. Savannah, GA
• Jinjun Gong, John Sheppard, and Trevor G. Phister. 2009. Characterization of a newly discovered Kluyveromyces marxianus strain for its carbohydrate utilization and ethanol production compared to commercial Saccharomyces cerevisiae strains CAT-1 and SuperstartTM. the 27th International Specialized Symposium on Yeast. Paris
• Melissa, Ivey, Mara Massel and Trevor G. Phister. 2009. Effects of lactic acid bacteria on ethanolic fermentations of Saccharomyces cerevisiae the 27th International Specialized Symposium on Yeast. Paris, poster
• Maria Guadalupe Aguilar-Uscanga, Mara Massel and Trevor G. Phister. 2009. The effect of ethanol and sulfite on the growth of Brettanomyces. XII National congress of Biotechnology and Bioengineering and VII International symposium of alcohol and yeast production. Acapulco Mexico

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: During this project period we have worked on both LAMP-PCR and Real-time PCR plus PMA. The LAMP-PCR assays have been found to be inconsistent. When the same sample of Brettanomyces was used for 5 different trials with a plus minus assay two where positive and three where negative. With regard to the detection of the nonviable yeasts. we used ethidium monoazide (EMA) a dye commonly used to differentiate viable and nonviable populations of bacteria in real-time PCR (QPCR) assays to eliminate the nonviable cells from the Z. bailii population. Thus we are able to determine the viable Z. bailii population using QPCR plus EMA. To do this we first, optimized the EMA exposure conditions; EMA concentration of 50 ug/ml with an incubation at 30oC in the dark for 5 minutes. Followed by light exposure on ice, for 5 minutes using a 500W halogen lamp at a distance of 12 cm. Using these optimized conditions, we determined that the assay could detect as few as 12.5 viable Z. bailii cells in the presence of 105 CFU/ml of heat killed-cells. The EMA assay was also more consistent at determining viable cell counts when compared to plating than fluorescent microscopy viable cell counts. Finally, we used the assay to determine the viable population in heat-treated (72oC, 2 min), ethanol-treated and raspberry cranberry juice Z. bailii cultures. When examining Z. bailii cells treated with 70% ethanol the QPCR assay with EMA (1.22 x 102) showed a better correlation with plating (4.5 x 101 cfu/mL) compared to the QPCR assay without EMA (5.31 x 106 cfu/mL) and this was also seen in the other two injured populations. Thus we feel that we have designed an assay which will be useful for the detection of viable spoilage yeasts in various fruit juices. In addition we used EMA-QPCR to remove contaminating DNA found in yeast growth media. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The development of the Z. bailii EMA-QPCR assay will provide beverage producers with a useful assay to rapidly determine the spoilage potential of their product.

Publications

• Rawsthorne, H. and T. G. Phister. 2009. Detection of viable Zygosaccharomyces bailii by utilizing ethidium monoazide bromide and real-time PCR. Int J. Food Microbiol. 131:246-250
• Rawsthorne, H. and T. G. Phister. 2009. The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide bromide. Lett. Appl. Microbiol. 49: 652-654

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We optimized the DNA isolation procedures comparing the use of a kit with boiling. Samples were prepared by boiling and gave a time to fluorescence for 100 cells of 15.12 minutes using LAMP-PCR,staining with sybergreen and detection on a multiwell plate reader. The kit gave a time to fluorescence of 12.7 minutes. Thus the boiling method worked as well as the kit at a much lower cost and time commitment. To determine the best visualization method we first needed to determine the limit of detection for the assay. Using a series of 1 in ten dilutions DNA was isolated from 10 million cells down to 1 cell. Using either extraction method we detected Brettanomyces down to 10 cells when the samples were visualized by gel electrophoresis. We next attempted to apply the use of sybergreen staining and a fluorometer to detect the DNA amplification. Using this method we were able to detect 10 cells. We also detected the fluorescent color change by the naked eye. Using this method we were able to detect down to 100 cells. We are currently trying to optimize this detection method through use of other dyes different reaction volumes and addition of handheld UV lights. We are currently adapting the assay for enumeration of the population in wine. We diluted Brettanomyces in media, wine, and wine plus Saccharomyces. The DNA was isolated by both methods and subjected to the LAMP-PCR assay. To enumerate the yeast we monitored the time to a predetermined fluorescence reading and plotted this for cultures of known population sizes. This generates a standard curve of time versus cell number. Using this method, we can now generate standard curves down to 10 yeasts. Additionally we worked on the detection of viable Brettanomyces using Propridium monoazide (PMA) and real-time PCR. PMA is a dye that is excluded from living cells but can penetrate the cell membranes of dead cells. Once inside it binds to the organisms DNA and blocks amplification by PCR. The goal of this work was to examine the suitability of PMA to selectively remove genomic DNA of dead cells from yeast culture. Incubation time PMA concentration and light exposure were all optimized. These conditions generated a strong correlation (r2=0.994) between CT and CFU/mL of viable cells. To elucidate the relationship between the proportion of viable cells, the DNA yield and the QPCR signals after PMA treatment, mixtures with defined ratios of viable and dead cells were used. An aliquot of B. bruxellensis was subjected to heat treatment resulting in a decrease in culturable cell counts to zero. Heat-killed cells were mixed with the untreated original culture in defined ratios with viable cells representing 0,10,20,30,40 and 50% of the total respectively. Increasing proportions of viable cells led to a substantial increase in the amount of genomic DNA not bound by PMA and to decreasing CT values in QPCR. While using only heat killed cells presented the highest CT value, i.e. greatest signal reduction. A plot of percent viability versus CT values revealed a linear correlation with a R2 value of 0.997. This suggested that the PMA-QPCR assay quantified only the living Brettanomyces cells. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
As this is the first year of the project there is limited impact from the results. We are however hoping that the LAMP assay will be developed and useful for winemakers as an inexpensive method to test for the spoilage yeast Brettanomyces. The PMA assay will also provide a future impact if it is able to develop a better correlation between the Brettanomyces population present in wine and the production of spoilage compounds.

Publications

• M. G. Aguilar-Uscanga and T. G. Phister. Use of propidium monoazide (PMA) for live/dead distinction of Brettanomyces bruxellensis. 108th General Meeting American Society for Microbiology. Boston (June 2008)-poster