Source: CORNELL UNIVERSITY submitted to NRP
CONTROLLING MICROBIAL SPOILAGE IN BEER AND WINE BY NATURAL REMOVAL OF IRON
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1020778
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Feb 1, 2020
Project End Date
Sep 30, 2022
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Food Science
Non Technical Summary
Microbial food spoilage causes 48 million illnesses and costs $77.7 billion dollars per year in the U.S. Microbial spoilage in beer and wine can sometimes have negative health impacts, but impacts are more often reflected as economic losses (entire batches of beer or wine are ruined due to exploding bottles or unpleasant aroma and flavors). One approach to control microbial spoilage is to limit an essential nutrient, such as iron, thus preventing microbial spoilage organisms from growing. This proposal describes a research project aimed at using a natural product (yeast) to remove iron from wine and beer, making the products more microbially stable and less likely to have spoilage issues. This research could not only provide a method for controlling microbial spoilage, but do so in a way that aligns well with the current public interest in "natural" approaches in food/beverage production.The wine and brewing industries in New York, and generally in the United States, are growing along with increased public consumption of wine and beer. The work outlined in this proposal has potential value to winemakers and brewers within the state of New York and throughout the country. By publishing the results of this work in scientific journals, and presenting it at conferences/symposia, the knowledge will be a shared resource available to anybody, and these results will be easily translatable into a commercial product by the large yeast supply companies that would only need to make a small change to their workflow.
Animal Health Component
(N/A)
Research Effort Categories
Basic
15%
Applied
(N/A)
Developmental
85%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50140201060100%
Goals / Objectives
Microbial food spoilage causes 48 million illnesses and costs $77.7 billion dollars per year in the U.S. Microbial spoilage in beer and wine can sometimes have negative health impacts, but impacts are more often reflected as economic losses (entire batches of beer or wine are ruined due to exploding bottles or unpleasant aroma and flavors). One approach to control microbial spoilage is to limit an essential nutrient, such as iron, thus preventing microbial spoilage organisms from growing. This proposal describes a research project aimed at using a natural product (yeast) to remove iron from wine and beer, making the products more microbially stable and less likely to have spoilage issues. This research could not only provide a method for controlling microbial spoilage, but do so in a way that aligns well with the current public interest in "natural" approaches in food/beverage production.The wine and brewing industries in New York, and generally in the United States, are growing along with increased public consumption of wine and beer. The work outlined in this proposal has potential value to winemakers and brewers within the state of New York and throughout the country. By publishing the results of this work in scientific journals, and presenting it at conferences/symposia, the knowledge will be a shared resource available to anybody, and these results will be easily translatable into a commercial product by the large yeast supply companies that would only need to make a small change to their workflow.The major goals of this project are as follows:Identify and optimize growth conditions required to starve yeast cells for ironExamine and optimize the use of iron-starved yeast to remove iron from multiple types of solutions: prepared solutions of iron, wine, and beerExamine the ability of iron-depleted wine and beer to support growth of common spoilage organismsExamine the ability of encapsulated, iron-starved yeast to reduce iron in wine and beer
Project Methods
The methods listed below are associated with each of the major goals of the project.Identify and optimize growth conditions required to starve yeast cells for iron: To begin we will prepare standard yeast (S. cerevisiae) growth media that lacks iron and examine time required until cells cease growth due to iron starvation. Growth will be assessed using plating for viable colony forming units. Iron starvation will be confirmed in multiple ways. First, the total number of cells in a starved culture should be less than in a non-starved, control culture. Second, a number of genes are known to turn on in response to iron starvation; most of them produce proteins that bind and import iron. We will measure activation of these genes by measuring levels of fluorescently-tagged proteins produced in iron-starved conditions vs. control conditions. Growth conditions will be optimized to minimize the media supply costs and minimize the time required to produce iron-starved yeast cells.Examine and optimize the use of iron-starved yeast to remove iron from solution: After preparing iron-starved yeast cells, these cells will be placed into control solutions containing different quantities of iron, and a variety of different beer and wine samples. Iron levels in each solution will be measured both before yeast addition, and at multiple time-points, to determine the speed and efficiency of iron scavenging. Iron measurements will be performed using a flame emission spectrometer available in the department. Optimization parameters include the relationship between cell number and total iron removed, and amount of time required for maximum iron removal.Examine the ability of iron-depleted wine and beer to support growth of common spoilage organisms: Using the optimized protocol developed in #3, we will prepare a variety of iron-depleted beer and wine (including non-depleted controls). To these samples we will add common wine and beer spoilage microorganisms and examine their ability to grow/survive. Growth and survival will be assessed by plating for viable colony forming units. In addition to growth and survival, we will also assess whether or not these spoilage organisms are able to cause typical sensory defects; for example, Brettanomyces spoilage in wine can produce band-aid/barnyard aromas.Examine the ability of encapsulated, iron-starved yeast to reduce iron in wine and beer: To be most useful as a microbial spoilage control in the wine and beer industries, the iron-starved yeast cells would be encapsulated. This encapsulation prevents growth of the iron-starved yeast and makes removal very easy because the encapsulated form is larger and heavier. Encapsulated yeasts are routinely prepared and used in the wine industry as a way to re-start slow/stopped fermentations, so major yeast suppliers already have the mechanism in place to produce these cells. We will encapsulate the iron-starved yeast cells and repeat the optimized experiments performed in #1-3 to validate the results in encapsulated yeasts.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:During this reporting period, the graduate student involved with this research has reported results at a number of internal (Cornell Recent Advances in Viticulture and Enology) and external conferences (American Society for Enology and Viticulture - Eastern Section; she won the best poster award at this conference. I have also reported the research results to students in my Wine Microbiology class. A manuscript describing the results is in preparation. Changes/Problems:We were able to demonstrate that we couldn't reliably remove iron from liquid using iron-starved yeast cells even in the extremely clean/sterile conditions of the laboratory. A great deal of trouble-shooting went into measuring iron at the low levels found in our matrix; eventually we were able to work with a USDA-funded lab in Ithaca to get these iron measurements. It was also difficult to iron-starve cells, even in sterile glassware because there is still trace amounts of metals around and cells need vanishingly small amounts to grow. However, iron starved yeast were no better at removing iron from wine or from model wine solutions. Together these results suggested that this is not going to be a viable approach at industrial scale. However, we decided to continue along the goal of dealing with spoilage issues in wine by looking at fructose as a cause of stuck/sluggish fermentations (no official reporting requirements exist for stuck/sluggish fermentations in the wine industry, but discussions with extension associates or people in the wine industry indicate that this is a significant financial burden). The graduate student doing the work completed a set of nice experiments demonstrating that fructose does not cause stuck/sluggish fermentations, contrary to popular belief. This should save NY winemakers (and all winemakers) money by not having to purchase strains/tools to try and remediate fructose issues in wine. What opportunities for training and professional development has the project provided?The graduate student involved was able to continue developing technical skills, while also developing presentation skills (she has presented the work in both oral presentations and poster presentations). How have the results been disseminated to communities of interest?The manuscript summarizing this work is currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?-Complete some final experiments (testing whether added fructose or glucose to a completed fermentation is consumed; comparing fermentation performance in pure fructose or glucose model wines) -Complete and submit the manuscript resulting from this work (target journal: Journal of the American Society of Enology and Viticulure) -Prepare a version of the above article to be published via Cornell's Viticulture and Enology Extension website also in the Appellation Cornell publication

Impacts
What was accomplished under these goals? We completed all of these goals and were able to demonstrate that we couldn't reliably remove iron from liquid using iron-starved yeast cells even in the extremely clean/sterile conditions of the laboratory. This suggested that this is not going to be a viable approach at industrial scale. However, we decided to continue along the goal of dealing with spoilage issues in wine by looking at fructose as a cause of stuck/sluggish fermentations (no official reporting requirements exist for stuck/sluggish fermentations in the wine industry, but discussions with extension associates or people in the wine industry indicate that this is a significant financial burden). The graduate student doing the work completed a set of nice experiments demonstrating that fructose does not cause stuck/sluggish fermentations, contrary to popular belief. This should save New York (NY) winemakers (and all winemakers) money by not having to purchase strains/tools to try and remediate fructose issues in wine.

Publications


    Progress 02/01/20 to 09/30/20

    Outputs
    Target Audience: Nothing Reported Changes/Problems:A major part of goal #1 includes being able to accurately measure iron concentrations in laboratory model solutions and also in actual food/beverage matrices. While we initially planned on using the atomic absorption spectrometer in Anna Katharine Mansfield's lab (Department of Food Science, Cornell University, Geneva, NY), initial discussions and tests highlighted that this instrument didn't have the appropriate sensitivity (0-70 ppb). A trial was also performed with a facility in Cornell University's geology department, though their analysis took over 4 months, was fairly expensive, and also demonstrated that their instrument lacked the required sensitivity. We contacted a few commercial vendors, including Dairy One in Ithaca, NY, but no vendor was able to supply the sensitivity required. Eventually we discovered that the USDA Holley Center facility had an instrument that could perform the measurements required (managed by Ray Glahn). After multiple rounds of testing, we discovered that the commercially available yeast growth medium lacking iron actually had plenty of iron. So we have had to modify our strategy for iron limitation by using a chelator (BPS). Progress was interrupted by the 3 month laboratory shut-down associated with Covid-19, along with the continued difficulties associated with decreased lab occupancy. So while none of the goals have yet been accomplished, I am hopeful that we have ironed out some of the major issues associated with Goal #1, and as covid restrictions begin to decrease we can concomitantly increase productivity and begin to complete these goals. What opportunities for training and professional development has the project provided?The graduate student involved in this project has learned quite a bit about assay sensitivity, the importance of validating that methods work as expected, how atomic absorption spectroscopy works, and that you can't necessarily trust product vendors without independently confirming their product claims. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?-Use a chelator (BPS) to produce iron-limited yeast growth media to prepare a population of iron-starved yeast cells. -Apply iron-starved yeast cells to model and actual wine solutions to test both a) quantities of iron removed, and b) whether or not this treatment produces a matrix that is inhospitable to potential spoilage organisms.

    Impacts
    What was accomplished under these goals? A major part of goal #1 includes being able to accurately measure iron concentrations in laboratory model solutions and also in actual food/beverage matrices. While we initially planned on using the atomic absorption spectrometer in Anna Katharine Mansfield's lab (Department of Food Science, Cornell University, Geneva, NY), initial discussions and tests highlighted that this instrument didn't have the appropriate sensitivity (0-70 ppb). A trial was also performed with a facility in Cornell University's geology department, though their analysis took over 4 months, was fairly expensive, and also demonstrated that their instrument lacked the required sensitivity. We contacted a few commercial vendors, including Dairy One in Ithaca, NY, but no vendor was able to supply the sensitivity required. Eventually we discovered that the USDA Holley Center facility had an instrument that could perform the measurements required (managed by Ray Glahn). After multiple rounds of testing, we discovered that the commercially available yeast growth medium lacking iron actually had plenty of iron. So we have had to modify our strategy for iron limitation by using a chelator (BPS). Progress was interrupted by the 3 month laboratory shut-down associated with Covid-19, along with the continued difficulties associated with decreased lab occupancy. So while none of the goals have yet been accomplished, I am hopeful that we have ironed out some of the major issues associated with Goal #1, and as covid restrictions begin to decrease we can concomitantly increase productivity and begin to complete these goals.

    Publications