Support for the UC-Davis Wine Microbiology Culture Collection

AVF Proj. ID: 124 / /Cat.: ,
Primary Investigator(s): David A. Millsby

The primary objective of this proposal was to increase both the diversity and total number of wine related bacteria in the culture collection. We have used 2 methods to accomplish this goal. First we have obtained interesting isolates from other collections (see Appendix A). From these collections we have been able to obtain some unusual isolates that will be of use for further research. For example, 2 isolates of the acid tolerant Lactobacillus suebicus are being used for a study of malo-lactic fermentations by a masters student, Cameron Parry. These strains may be a commercial alternative to Oenococcus. We also now have in the collection 2 strains of Allicyclobacillus, a spoilage organism from apple juice. These organisms have not been found in grape juice but we may now be able to look for this type of spoilage organism.

Our second method of enhancing the bacterial collection has been to isolate interesting wine-related bacteria directly from wine and must. We worked with a winery that does not use inoculation for malo-lactic fermentaion to obtain isolates of Oenococcus from 5 wine varieties; Zinfandel, Cabernet Sauvignon, Chardonnay, Merlot, and Carignane. We have purified over 600 putative Oenococcus isolates from these wines and are in the process of determining how many different strains they represent. We have also isolated more then 300 other bacteria from these samples and are attempting to determine the genus and species as well as the genetic variety of these organisms. An additional request was made to several wineries to provide us with examples of wines that they felt had microbial spoilage organisms present. We have isolated approximately 20 bacterial spoilage organisms from a variety of such wines at this point. We are currently working in conjunction with the Wine Microbiology class (VEN 128) to obtain more isolates that will be added to the collection.

Over the duration of the granting period we have added 68 well characterized strains to the collection from various collections around the world. We have added an additional 20 spoilage isolates from California. Over the next year we should be able to analyze and characterize many of the other isolates we have collected. We can not as yet put a number on those isolates we will add to the collection. Overall, we have made substantial progress in bringing the bacterial portion of the culture collection up to the level where it can be of substantial use for instructional, research, and industry purposes.

PDF: Support for the UC-Davis Wine Microbiology Culture Collection

Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae

AVF Proj. ID: 3 / /Cat.: ,
Primary Investigator(s): Susan E. Ebeler, Linda Bissonby

The goal of this project in the 2000-01 funding cycle was to further define the conditions under which phenolic compounds stimulate or inhibit fermentations. Phenolic extracts prepared from Cabernet Sauvignon grape skins were found to be stimulatory to yeast fermentation, particularly accelerating the rate of late fermentation. In contrast, seed phenolic extracts were inhibitory, again with the stronger effect being observed later in fermentation. Purified phenolic compounds were also evaluated for their effects. As reported last year, the effect of the individual phenolic compounds was influenced by media composition. In addition we discovered that there is significant strain variation in response to phenolic compounds.

Rapid Identification of Wine Spoilage Organisms

AVF Proj. ID: 9851 / /Cat.: ,
Primary Investigator(s): Christian Butzkeby

The scope of this project was to systematically analyze all enologically important organisms in the UC Davis wine microorganism collection for correctness of identification, and to create new differential media and a rapid method for identification of spoilage organisms in commercial wine production. During the first part of this project, the MEDI gas chromatography system has been completely set up including all necessary hardware and software upgrades. A variety of wine yeasts was selected and cultured under highly reproducible conditions followed by extraction and fatty acid profiling. The profiling has started on the genus level and the system was tested with Saccharomyces and Kloeckera, Pichia and Schizosaccharomyces strains. Due to the lack of wine-related yeast entries in the MIDI databases, the establishment of a special series of profiles based on cultures from the Department of Viticulture and Enology’s wine yeast collection was initiated. At this point, the system is able to produce well resolved fatty acids peaks that are recognized by the identification software, while the optimization of the cultivation and extraction techniques continues. The project has been part of the on-going long-term research program “Analysis of Saccharomyces during Normal and Problem Fermentations”. In addition to metabolic fatty acid profiling of wine yeast, the MEDI-based identifications will serve as a validation tool for concurrent PCR-based identifications

Effects of Brettanomyces in Winemaking: Chemical, Microbiological and Sensoriale

AVF Proj. ID: 9715 / /Cat.: ,
Primary Investigator(s): Ken Fugelsang, Bruce Zoeckleinby

Historically, winemakers have viewed Brettanomyces sp. as producing spoilage in wines. Despite traditionally negative connotations surrounding the yeast, some are now questioning whether or not subtle “Brett” character, in some cases, may play a positive role in flavor and bouquet development. In addition to potentially contributing to complexity, limited Brettanomyces activity may play a role in accelerated aging in young red wines. Of the nine species of Brettanomyces isolated from wine and juice, B. intermedius is the most frequently identified. This suggests the existence of several, to many, strains that may be involved in the winemaking process. Prior to this study, a comprehensive evaluation of inter- and intrastrain variability has not been undertaken. The objective of this two-year project is to study inter- and intrastrain differences between ten genetically characterized strains of the single species, Brettanomyces intermedius, in aging red wine (Pinot Noir). The project includes comparison of population dynamics as well as chemical and sensorial changes brought about during the growth phase of the respective strains. The goals of the first year were to characterize population changes often strains of B. intermedius in Pinot Noir wine incubated at 20°C and begin GC/MS identification and quantification of primary metabolites. Presently, we are in week 45 of the growth studies inoculated at, <10 CFU/ml. We have observed that three of the ten strains reached maximum cell density at 41 days post-inoculation. Five strains took 60 to 140 days to complete their growth cycle. Final population densities, in these cases, paralleled those of the early group. Two of the ten strains grow very slowly. At present (day 195), both are approaching stationary phase. Depending upon strain and replication, cell density ranges from 28,000 to 50,000 CFU/ml. CG/MS analysis of weekly samples is approximately 60%complete. In every case, 4-ethyl phenol was not detected until cell density reached relatively high numbers. These preliminary results appear to contradict the belief that 4-ethyl phenol can be used as a monitoring tool for population density estimates in the earliest stages of growth. Work also continues on monitoring ieto-glucosidase activity in inoculated wines. Initial results suggest potentially significant enzyme activity in model systems. Follow-up assays are currently being carried out in experimental lots. Upon completion of the growth cycles, each lot was cold-clarified, bottled and stored for upcoming sensory examination. Year two objectives: At four, and nine months post-bottling, each lot will be examined sensorially for differences. Initial sensory comparison of each lot will be carried out using Duo-Trio paired comparison tests. Where differences are noted, descriptive analysis will be performed using a twelve member trained panel.

Control of Current and Emerging Problems of Microbiological Spoilage of Wine

AVF Proj. ID: 9107 / /Cat.: ,
Primary Investigator(s): R.E. Kunkee, M.R. Vilas, K.C. Fugelsang, C.J. Mullerby

Reports have been received from California winemakers in recent years of new types of microbial spoilage problems in both stored and bottled wines. The responsible organisms, not normally associated with wine, are represented by species of the following yeast genera: Zvaosaccharomvces. Pichia. Hansenula, Kloeckera. and Debarvomvces. and_ by species of the following bacterial genera: Acetobacter. Bacillus. and especially vigorous strains of Lactobacillus. There is also continual concern over the apparent ubiquitous presence of Brettanomyces and Dekkera.