Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-09
2002-09-24
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06455256
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to methods for identifying microorganisms present in fruit juices generally and more specifically in wine. More specifically, the invention relates to methods of isolating nucleic acids present in fruit juice in order that they may be successfully amplified and/or detected by molecular amplification and detection methods including that known to the art such as polymerase chain reaction (PCR).
The fermentation of grape juice (must) to produce wine is a complex biological process in which yeasts, most typically
Saccharomyces cerevisiae,
are used to convert the sugar content of grape must to ethanol, water, and carbon dioxide. Not only do the many different strains of
Saccharomyces cerevisiae
differ with respect to their metabolic processes and products, but grape must typically comprises a multiplicity of other microorganisms including other fungi, yeasts, and bacteria which react with the grape must during fermentation to produce various metabolic products including aromatic and flavoring compounds. Such products can influence the sensory character of the resulting fermentation product in both positive and negative ways. Conversely, the presence of unwanted microorganisms can lead to undesirable flavors or spoilage. In some cases contaminated must can produce fermentation products characterized by undesirable commercial attributes, affecting flavor, color, and/or mouthfeel. It is thus of intense interest to the vintner to know whether the fermentation is characterized by such microbial contaminants. Similarly, the presence of contaminating microorganisms in the finished wine product is of concern to the wine producer and there exists a desire to ensure the absence of such contaminants. It is also crucial to know if microbes are present after the fermentation has been completed, as the majority of spoilage occurs during the aging and storage of the finished product.
Alternatively, where it is desired to inoculate grape must with a malolactic fermentation bacterium to carry out malolactic fermentation, it is useful to evaluate the growth of the inoculated strain and determine whether the fermentation medium need be reinoculated. Previously, to determine the presence and identity of microorganisms in the grape must, it has been necessary to culture samples of the grape must to “grow up” cell cultures in different selective media. The presence of microbial contaminants can then be identified morphologically or otherwise. Not only is morphological identification sometimes uncertain, but in the case of Dekkera sp., several days are required to culture a sample to determine its presence.
More recently, advances in molecular biology have made it possible to identify microorganisms by amplifying and/or detecting nucleic acid sequences which are uniquely characteristic of those microorganisms by methods such as polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), RFLPs, direct sequencing (dideoxy method), direct hybridization, and immuno-based assay techniques. In this regard, much recent work has been conducted in the identification of polynucleic acid sequences which are uniquely characteristic of microorganisms present in wine fermentations. For example, ribosomal DNA (rDNA) genes are particularly useful as targets for molecular probes and PCR primers because of their high copy number. Moreover, non-transcribed and transcribed spacer sequences associated with ribosomal genes are usually poorly conserved and thus are useful for the detection and identification of different closely related fungal pathogens. The internal transcribed spacer (ITS) region lies between the 18S and 28S rRNA genes and contains two variable non-coding spacers referred to as ITS1 and ITS2 separated by the 5.85 gene. In addition, the transcriptional units are separated by non-transcribed spacer (NTS) sequences. The ITS and NTS sequences are particularly suitable for the detection of fungal pathogens. PCT International Application WO 99/46405 discloses unique DNA sequences which are useful in the identification of microorganisms involved in fermentations such as
Saccharomyces cerevisiae, Saccharomycodes ludwigii,
Dekkera sp.,
Botrytis cinerea,
Penicillium sp.,
Hanseniaspora guilliermondii, Debaryomyces carsonii, Pichia anomala, Pichia kluyveri,
and
Candida krusei.
See also Bartowsky, et al., Australian J. of Grape and Wine Research 5:39-44 (1999) which describes the use of PCR for specific detection of the malolactic fermentation bacterium
Oenococcus oeni
(
Leuconostoc oenos
) isolated from bacterial culture.
Despite advances in the molecular identification of microorganisms associated with wine, fruit juice, and fruit-juice fermentations, it has not been possible to successfully conduct a nucleic acid amplification/detection method such as PCR amplification directly on a wine sample. While all the reasons for this remain unclear, they may include concentration effects and may also be related to the presence of potentially inhibitory compounds in the wine, among other factors. It thus remains the case that microorganisms present in wine samples must be subjected to a culturing step in order to provide sufficient quantities of cells to serve as a source of nucleic acids for amplification by PCR or other molecular amplification or detection techniques. The requirement that sample cells be amplified by cell-culturing constitutes a significant limitation to the utility of molecular identification methods because of the time delay (typically several days) inherent in cell-culturing. The vintner is therefore unable to apply real-time adjustments to the fermentation to take advantage of the information provided by the molecular identification techniques. In the case of the presence of undesired microorganisms in a wine fermentation, the loss of time in which to address the outbreak may mean the difference between successfully applying remediation techniques to salvage the fermentation and degradation or ruination of the fermentation batch. Real-time identification of potential microbial problems will enable the proactive, rather than reactive, use of one or more targeted treatments such as cooling, filtering, application of clarification and/or fining agents, anti-microbial substances, and reinoculation, thereby increasing their effectiveness. For this reason, there remains a desire in the art for methods for conducting “real-time” PCR or other amplification techniques not requiring an intermediate cell-culturing step to multiply the cell sample quantity.
While the preceding discussion has focused on the need for improved nucleic acid amplification and/or detection methods for application to the wine industry, it is apparent that there exist similar needs for testing of other fruit juices. Such a need is important in the cider industry but is particularly acute given the growth in popularity of “organic” and other “natural” fruit juices and ciders which are sometimes not subject to pasteurization or treated with preservatives.
Attempts have been made to use conventional cellulosic filters to isolate and concentrate organisms within grape must or wine in order to perform PCR or other amplification or nucleic acid detection techniques on the retentate. Such methods have not been successful and the failure of those methods may relate to the retention of inhibitory compounds within the retentate. More recently, methods such as those of DiMichele and Lewis,
ASBC Journal,
51:2, 63-66 (1993) have shown promise in conducting real-time PCR for the identification of organisms involved in the fermentation of beer. According to DiMichele and Lewis,
Lactobacillus brevis, L. casei,
and
L. plantarum
were isolated from water and beer by membrane filtration and membrane dissolution using a dissolvable polycarbonate membrane having a pore size of 0.45 micron. Regions of the 16S rRNA sequences unique to each organism were used to generate species-specific PCR products, which were then visualized by gel
Chunduru Prabha
E. & J. Gallo Winery
Fredman Jeffrey
Marshall Gerstein & Borun.
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