Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-03-06
2004-08-03
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S034000
Reexamination Certificate
active
06770453
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable
FIELD OF THE INVENTION
This invention relates to the field of microorganism detection. In particular, this invention provides an improved methodology for detecting chitinous contaminants in samples, e.g. biological samples.
BACKGROUND OF THE INVENTION
A methodology for rapid detection and identification of microorganisms, and other contaminants, has long been a concern to the medical, pharmaceutical and food processing fields, among others. Because of this sustained interest, significant advances over the classical time consuming methods of plate counting, membrane filtration, or multiple tube fermentation procedures have been noted. Such approaches include, differential dye-cell wall binding, mass spectrometry, bacteriophage lysis, computer assisted probability methods, gel ferrography, flow cytometry, and the like. Such methods, however, typically have not achieved industry acceptance particularly in the agricultural industry. This lack of acceptance/industry implementation is due to disadvantages such as laborious and time-consuming laboratory preparation and sample handling, long observation times and nonspecificity with respect to microorganism characterization and subsequent identification, and increased expense in terms of labor and/or instrumentation. Thus, relatively primitive methods are employed for monitoring contaminants, e.g. in agricultural products.
Thus, for example, California tomato industry monitors mold levels in raw product at inspection stations and in processed product in quality control laboratories. At the inspection stations, 23 kg of fruit from each 24 metric ton truckload of processing tomatoes are visually inspected for defects. Tomatoes with visible signs of mold are weighed to obtain a percentage of decayed fruit on a mass basis (PTAB, 1996). At the processor's quality control laboratories, mold is quantified by the Howard mold count (HMC) method (AOAC, 1984), where a small drop of homogenized juice is inspected using a microscope. In the HMC method, two slides of twenty-five fields each are viewed, and the percentage of fields containing mold are recorded. An accurate HMC takes up to thirty minutes to conduct.
Over the last several decades, many attempts have been made to replace manual grading and the Howard mold count with a less subjective and less labor-intensive measurement, but no method has been accurate, rapid, and simple enough to use at inspection stations or in quality control laboratories (Jarvis and Williams (1987). p.599-636. In
Food and Beverage Mycology
, ed. L. R. Beuchat, 2
nd
ed. Van Nostrand Reinhold, N.Y.; Gourama and Bullerman (1995)
Journal of Food Protection
58:1389-1394; and Cousin (1996)
Journal of Food Protection
, 59: 73-81). Despite the difficulties and limitations of the HMC (e.g. Williams (1968)
J. Ass. Pub. Analysts
, 6: 69-84; Jarvis et al. (1983)
J. Appl. Bacteriol
. 55: 325), it remains the universal standard for mold assessment almost ninety years after it was first introduced (Howard (1911)
U.S. Dept. Agr., Bureau of Chemistry, Circular No
. 68).
Chitin is an important structural component in fungal cell walls, but absent from plant tissue. The detection of mold based on the chemical isolation and quantification of N-acetyl-D-glucosamine, a breakdown product of chitin, has been proposed as an alternative mold measurement (Ride and Drysdale (1972)
Physiol. Plant. Pathol
. 2: 7-15; Jarvis (1977)
J. Food Technol
. 12: 581-591; Lin and Cousin (1985)
Journal of Food Protection
, 59: 73-81). Jarvis (1977), supra., found a coefficient of variation (CV) of around 20% for this method. Although the high performance liquid chromatography based isolation method is too slow and labor intensive to be utilized as an industrial replacement for the HMC, a more rapid method that detects chitin could have commercial promise (Cousin (1996)
Journal of Food Protection
, 59: 73-81).
Lectins are naturally occurring proteins or glycoproteins that bind to specific carbohydrates. They are becoming increasingly valuable as molecular probes, including the labeling of cell-surface components in tissue typing (Lis and Sharon (1986)
Ann. Rev. Biochem
. 55: 35-67). Hundreds of lectins from microbial, plant, and animal cells have been identified, but most commercially available lectins are isolated from plant seeds. They are available with various enzymatic and fluorescent labels, and their nomenclature derives from the name of their source.
There are numerous commercially available lectins that bind polymers of N-acetyl-D-glucosamine. Stoddard and Herbertson (1978)
J. Med. Microbiol
. 11: 315-324, utilized fluorescein labeled lectins to detect human pathogenic fungi. Patel (1992) The applications of lectins in food analysis.
Trends in Food Sci
. &
Technol
. 3: 35-39, used fluorescein isothiocyanate (FITC) labeled lectins to observe mold in processed foods. He tested several chitin-binding lectins, and found that a lectin from wheat germ agglutinin (WGA) had the strongest binding to fungal cell walls and the least amount of nonspecific binding to tomato cells. He observed considerable autofluorescent signal, tomato cell tissue that fluoresces at similar wavelengths as the fluorescent probe. Patel et al. (1993) pages 31-41 In
New Techniques in Food and Beverage Microbiology
Eds. Kroll, R. G., Gilmour, A., and Sussman, M. Blackwell Science Inc. Oxford, England, used biotinylated lectins and streptavidin labeled magnetic particles to separate and concentrate mold spores and yeasts in fruit juices.
SUMMARY OF THE INVENTION
This invention provides novel methods for the detection of chitinous contaminants of non-chitinous biological materials. The methods are accurate, highly reproducible, rapid and relatively inexpensive. The methods are well suited to commercial applications, particular in the food and agriculture industry where biological materials (e.g. food products) are regularly screened for contaminants (e.g. insect, mold, fungus, etc.).
In one embodiment this invention provides a method of detecting chitinous material in a processed non-chitinous biological sample. The method involves contacting the biological sample with a probe that is a lectin that binds chitin, contacting the biological sample with a pectinase, and detecting binding of the lectin to a chitin wherein the binding indicates the presence of chitin (and hence a chitinous contaminant) in the biological sample. The chitinous contaminant can be any of a wide variety of contaminants including, but not limited to insects, insect parts, other animals or parts of animals of the phylum arthropoda (e.g. crustacea), nematodes, annelids, molds, fungi, slimes, yeasts, and various other microorganisms, and the like. In particularly preferred embodiments, the detected contaminant is a fungus of phylum Ascomycota, Basidomycota, Chytridiomycota, or Zygomycota, or a member of the phylum Oomycota in the Stramenopila kingdom. Particularly preferred fungi include, but are not limited to Cladosporium spp, Fusarium spp, Stemphylium spp, Alternaria spp, Geotrichum spp, Fusarium spp, Rhizopus spp, Botrytis spp, Phytophthora spp, Pythium spp, or Pythium spp (e.g.
Cladosporium herbarum, Fusarium oxysporum
, and
Stemphylium botryosum, Alternaria alternata, Geotrichum candidum, Fusarium oxysporum, Rhizopus stolonifer, Botrytis cinerea, Phytophthora parasitica, Pythium aphanidermatum, Pythium ultimum
, etc.).
Preferred biological samples include, but are not limited to an agricultural product, a food product, a wood product, a textile, and an animal tissue product. Particularly preferred agricultural products include, but are not limited to fruits, vegetables, grains, forages, silages, juices (vegetable or fruit), a wood, flowers, or seeds. In one embodiment the agricultural product is a tomato, a pepper a grape, an apple, an orange, a lemon, a berry, or a juice or concentrate thereof.
Preferred lectins for use in t
Payne Jennifer J.
Potts Steven J.
Slaughter David C.
Thompson James F.
Gitomer Ralph
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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