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WO1998031824A1 - Matieres tinctoriales fluorescentes obtenues a partir de micro-organismes - Google Patents

Matieres tinctoriales fluorescentes obtenues a partir de micro-organismes Download PDF

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Publication number
WO1998031824A1
WO1998031824A1 PCT/AU1998/000023 AU9800023W WO9831824A1 WO 1998031824 A1 WO1998031824 A1 WO 1998031824A1 AU 9800023 W AU9800023 W AU 9800023W WO 9831824 A1 WO9831824 A1 WO 9831824A1
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Prior art keywords
compound
biomolecule
yeast
fungal species
cells
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PCT/AU1998/000023
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English (en)
Inventor
Philip Bell
Jian Shen
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Access Macquarie Ltd
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Macquarie Research Ltd
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Priority to AU55437/98A priority Critical patent/AU5543798A/en
Publication of WO1998031824A1 publication Critical patent/WO1998031824A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • This invention relates generally to compounds produced by microorganisms which can function as fluorescent dyes, methods of producing the compounds, and uses of compounds in scientific applications.
  • the argon-ion laser is the excitation source in many flow cytometers and confocal laser scanning microscopes, as well as in certain laser scanners.
  • the wavelengths used to excite green, yellow, orange and red fluorescent dyes are limited primarily to the laser ' s 488 nm and 514 nm spectral lines, which severely restricts simultaneous multicoloured detection.
  • the commonly used Texas Red® fluorophore has a particularly low fluorescence output that is easily obscured by the more intense fluorescein fluorescence in a double- labelling reaction.
  • Red fluorescent dyes (each with their own disadvantages) are used extensively in many fields of biological study. Most of these, such as Texas
  • Tetramethylrhodamine-isothiocyanate or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their utility for flow cytometry as most cytometers are only capable of excitation at blue wavelengths (488 nm).
  • Phycobilloprotein. phycoerythrin can be excited at 488 nm and does emit in the red wavelengths. This compound, however, has poor stability and a high molecular weight making it unsuitable for cell tracking or labelling of nucleic acid probes.
  • Microorganisms and particularly some fungi, are known to produce pigmented or coloured compounds. Most of the coloured compounds produced by microorganisms do not fluoresce and are not suitable as fluorescent dyes in scientific applications. Many coloured compounds produced by fungi have been used as food dyes or as antimicrobial or antitumour agents. For example, the fungus Monascus purpurem is well known for producing a variety of coloured compounds including orange (Monascorubin and rubropunctatin) and purple (monascorubramine and rubropunctamine).
  • yeast strains of the yeast genus Saccharomyces are used in some of the largest and oldest biotechnology industries, including baking, brewing, distilling and wine making. Improvements in the performance of the yeast strains used in these processes has come about as a result of the development of yeast with novel genotypes.
  • the methods for producing these improved genotypes include genetic engineering, protoplast fusion, and mutation/selection techniques. However, in many situations, traditional techniques involving mating followed by selection are still effective for strain improvement. In these techniques, spores derived from parental strains of Saccharomyces are isolated, germinated and allowed to mate. The hybrids produced from these matings can be screened to identify novel strains that combine desirable traits.
  • the present inventors have produced a new compound derived from fungi that is particularly suitable for use as a non-genetic marker in scientific applications.
  • the present invention consists in a compound produced by a fungal species, the compound binds to biomolecules and is suitable for use as a fluorescent dye for biomolecules.
  • the compound produced by the fungal species when bound to a biomolecule emits fluorescence after excitation at blue wavelengths, preferably emitting in the red wavelengths.
  • the fungal species is an Epicoccum species, preferably Epicoccum nigrum.
  • the blue wavelength is 488 nm.
  • Biomolecules which may be bound by the compound of the invention when used as a fluorescent dye include, for example, proteins, peptides. sugars, nucleic acids, antibodies, cell surface biomolecules. and cells. Due to the compound ' s fluorescent and biomolecule-binding characteristics, it will be appreciated that the compound will have use in any application where detection of a fluorescent dye attached to a biomolecule is required.
  • the present invention consists in a process for producing a compound according to the first aspect of the present invention comprising culturing a fungal species under conditions such that the fungal species produces the compound; and separating the compound from the culture.
  • the fungal species is cultured in the presence of a yeast such that the fungal species produces the compound.
  • the fungal species is preferably an Epicoccum species, more preferably Epicoccum nigrum, and most preferably Epicoccum nigrum PBl.
  • the present inventors have deposited, under the provisions of the Budapest Treaty, a sample of Epicoccum nigrum PBl that produces a compound according to the present invention. The deposit was made at the Australian Government Analytical Laboratories (AGAL) on 15 January 1998 and was given the Accession Number NM98/00507.
  • Epicoccum nigrum PBl is believed by the present inventors to be the first example of a fungal strain that produces a compound suitable as a fluorescent dye for biomolecules.
  • the present inventors have isolated other strains of Epicoccum nigrum and all of these strains produced a similar compound to that of Epicoccum nigrum PBl.
  • the yeast is preferably a Saccharomyces species, more preferably Saccharomyces cerevisiae.
  • a strain that has been found by the present inventors to be particularly suitable is the commercially available strain NCYC 996
  • the compound can be used as a fluorescent dye when in a crude culture extract or when purified by extraction and separation techniques. It will be appreciated that after the compound is produced by the fungus during culture it can be obtained in substantially pure form by a standard purification technique.
  • the compound according to the present invention may also be produced synthetically by chemical means.
  • the knowledge that a new fluorescent compound is produced by fungi may lead to other means of producing the compound apart from culturing the fungi under the required conditions.
  • the present invention has the distinct advantage that it binds to cells and other biomolecules in its fluorescent form so can be used as a means to track the cells or the other biomolecules when labelled with the compound.
  • the present invention consists in use of the compound according to the first aspect of the present invention as a fluorescent dye in scientific techniques for staining, labelling and/or detecting biomolecules.
  • the use of the compound according to the first aspect of the present invention include cell tracking dyes for microscopy, membrane fluidity dyes, conjugation with antibodies, conjugation to nucleic acids, cell surface ligand imaging dyes, conjugation to sugars, cytometric analysis, and confocal microscopy. It will be appreciated, however, that the compound would be suitable for any use where fluorescence in the red wavelengths is required, particularly when excited at 488 nm.
  • the present invention consists in an improved method of fluorescent-labelling a biomolecule comprising causing the compound according to the first aspect of the present invention to bind to the biomolecule such that the biomolecule is fluorescently labelled with the compound.
  • the biomolecule may include, but not limited to. proteins, peptides, sugars, nucleic acids, antibodies, cell surface biomolecules. and cells.
  • the compound may bind directly to the biomolecule due to a chemical or physical association or may bind to the biomolecule via a linking molecule. If the compound is attached to a ligand specific for the biomolecule. for example an antibody or lectin, then the binding of that ligand to the biomolecule will cause the biomolecule to be fluorescently labelled.
  • the present invention consists in a method of detecting a biomolecule in a sample comprising labelling the biomolecule according to the method of the fourth aspect of the present invention: and detecting the biomolecule in the sample by monitoring or detecting its fluorescence.
  • the monitoring or detecting of the fluorescence of the labelled- biomolecule may be by any means known to the art. Such means include, but not limited to. microscopy and cytometry.
  • the compound according to the first aspect of the present invention has several advantages over currently used fluorescent dyes presently available.
  • the compound has been called Beljian Red (BR) by the present inventors and has the following characteristics:
  • the fluorescent form has wide range of absorbances • has strong fluorescent emission after staining but a low fluorescent emission when free in solution. This ensures low background fluorescence levels.
  • the compound has been found by the present inventors to stain fungal, bacterial and mammalian cells
  • the Stokes shift of cells fluorescently labelled with this dye is such that flow cytometers can detect the emission in a separate channel from green- emitting dyes (e.g. fluorescein) making the fluorescent labelling conferred by the dye ideal for two-colour fluorescence imaging in conjunction with fluorescein.
  • green- emitting dyes e.g. fluorescein
  • Figure 1 shows a NMR spectrum of Beljian Red.
  • a fluorescent compound according to the first aspect of the present invention peak 1 is the compound showing benzene ring structures indicating the compound is aromatic, peak 2 is water, and peak 3 is DMSO solvent peak:
  • Figure 2 shows the separation of cells stained using Beljian Red.
  • a fluorescent compound according to the present invention :
  • Figure 3 shows results of mating of haploid strains of S. cerevisiae.
  • Figure 4 shows gating region used for isolation of small clusters of mating cells. Linear gains were used for both SSC (Side scatter) and FSC (Forward scatter) parameters to provide sufficient resolution for size discrimination. Values are in arbitrary units; and
  • Figure 5 shows results of rare mating of an industrial polyploid strain with a laboratory haploid strain of S.
  • Streaks should be ideally 1 cm apart and parallel or arranged in a cross formation. Incubate culture at 20°C for several days (3 - 5) until Epicoccum nigrum fungal filaments are approximately 3-4 mm long and start secreting a pale yellow compound.
  • This raw extract can be either used directly as a stain or purified to homogeneity.
  • the fraction with the highest 508 nm absorbance was selected and the running buffer in the fraction evaporated.
  • the fraction was then further purified on a reversed phase HPLC C18 column using 85% acetonitrile. 0.045% TFA in Milli Q water.
  • the fraction with maximum 508 nm absorbance was selected and further purified on a Mono Q ion-exchange column.
  • the compound after purification, had an absorbance maximum at 508 nm and a second peak absorbance at 386 nm.
  • This compound becomes fluorescent after contact with and possibly modification by the bakers yeast, Saccharomyces cerevisiae.
  • the compound is easy to produce, easy to use. and stable to freeze/thaw and storage.
  • the fluorescent form that forms in contact with yeast emits in the longer orange to red wavelengths.
  • Uses of Dye Strains and culture conditions Studies were performed using three strains of Saccharomyces cerevisiae: X [MATa trpl hisl MAL6T::l ⁇ cZ). SMC19-A ⁇ MAT ⁇ MAL2-8 c MAL3 leul SUC3). and an industrial baking strain Nl (Burns Philp Technology and Research Centre culture collection). Yeast strains were grown on a variety of media. Rich medium was composed of.
  • glucose minimal indicator medium was composed of 20 g glucose. 20 g agar. 5g yeast extract (Oxoid): 10 g peptone (Oxoid) and 3 g KH2PO4 (Sigma- Aldrich. Sydney. Australia). 20 g agar (Oxoid). Two types of minimal media were used: maltose minimal indicator medium was composed of 20 g maltose. 6 g Na 2 HPO 4 . 6g KH 2 PO . 20 g agar. 6.7 g yeast nitrogen base (YNB. Difco. Sydney. Australia) and X- ⁇ - Gal at 40 ⁇ g per litre: glucose minimal indicator medium was composed of 20 g glucose. 20 g agar.
  • CT Cell Tracker
  • CMFDA CT-Green BODIPY.
  • CT-Orange CT-Yellow- Green and CT-SNARF were made up to 10 inM with DMSO from a freshly opened flame sealed ampoule (99.9 atom % DMSO. Sigma. Sydney).
  • Dye stocks were stored frozen at -50°C in single use aliquots and sealed from moisture or light. After defrosting, any unused dye from each aliquot was discarded.
  • Staining for flow cytometry was performed in 1 ml final reaction volumes consisting of 25 ⁇ l of a suspension of overnight yeast culture added to 975 ⁇ l YNB. Thus, during staining the cell density was approximately 10 7 yeast cells/ml.
  • cells were typically incubated at 30°C for 45 min. in darkness with a working dye concentration of 10 ⁇ M. This staining concentration was determined after staining using working dye concentrations ranging from 0.5 ⁇ M to 25 ⁇ M (data not shown). Unbound dye was removed by centrifugally washing (pelleting for 1 min. at 12.000 x g. removal of accessible supernatant with pipette, re-suspension in 1 ml YNB) three times.
  • Aqueous dye stocks of the dye according to the present invention were prepared. Staining reactions were prepared in 150 ⁇ l final reaction volumes by pelleting 25 ⁇ l cells from overnight yeast cultures and resuspending in 50 ⁇ l YNB followed by addition of 100 ⁇ l BR stock. Thus, during staining the cell density was approximately 10 8 yeast cells/ml. Staining reactions were incubated at room temperature (21 - 25°C) for 30 min. Unbound dye was removed by centrifugal washing three times. incubating for 30 min. at 30°C in YNB in new microfuge tubes, followed by three further centrifugal washes. Mating procedure.
  • the FACSCalibur-Sort flow cytometer (Becton Dickinson, Lane Cove, NSW, Australia) was operated using Isoton II (Coulter Electronics Ltd, Brookvale, NSW, Australia) diluted 1:1000 with filtered (0.2 ⁇ m) purified (MilliQ, Millipore, Sydney, Australia) distilled water as the sheath fluid. Coulter ImmunoCheck beads were analysed each day to ensure the cytometer was correctly aligned. The flow rate was adjusted to keep the total data rate below 1000 events per second during analysis or below 300 events per second during sorting. The detection threshold was set in the FSC channel at a level just below that of the lowest yeast cell signals. The excitation light, wavelength 488 nm.
  • Sort regions were defined on an FLl vs FL2 dot-plot. To determine the nature of cells sorted from the defined regions, sorted cells were examined using microscopy and regions modified until sorting accuracy was confirmed. The sorter was always set to single cell mode. For sorting rare mating hybrids, the first round of sorting consisted of collecting 20.000 events in BSA coated (FACSCalibur Users Manual. Becton-Dickinson) 50 ml sterile Falcon tubes (Bacto. Sydney. Australia). Sorted cells are recovered in high volume from catcher tube sorters such as the FACSCalibur. Therefore, cells were concentrated by centrifugation at 4.000 x g for 20 min.
  • the first round of sorting was performed as above, collecting 20.000 cells into 50 ml tubes and re-suspending in 2 ml YNB. Re- suspended cells were loaded into the cytometer for a second round of sorting with 150 cells being collected directly onto both rich medium and glucose minimal indicator medium in triplicate. Plates were incubated at 30°C for 48 h. Comparison between the number of colonies on the rich medium vs the number on the minimal medium indicated the efficiency of hybrid isolation. As a further confirmation. 100 colonies from the rich medium were picked randomly and patched onto maltose minimal agar. On this medium, hybrids were identified by the unique ability to grow and synthesise ⁇ -galactosidase enzyme.
  • strain X has lacZ linked to the MAL6 promoter and integrated into the genome. As a result, lacZ is expressed at high levels on maltose based medium.
  • Microscopy A Nikon Optiphot II epifluorescence microscope (Nikon.
  • PCR fingerprinting To discriminate parent strains from rare mated hybrid strains. PCR fingerprints were obtained using commercially available primers (Yeast Mutilplex PCR primers. Bresatec. Sydney. Australia). Banding patterns were scored visually after agarose gel electrophoresis of PCR products.
  • the compound has exceptionally good characteristics.
  • the fluorescence of cells labelled with the dye is not detected significantly in the FLl channel of Becton-Dickinson FACScan/Calibur type instruments, but is detected in the FL2 and FL3 channels.
  • the compound does not diffuse from stained cells and does not stain neighbouring cells making it ideal for cell tracking. It is likely that the compound will have uses in other scientific applications due to its excellent fluorescent characteristics, low molecular weight, and its moderate water solubility.
  • a compound according to the present invention was used as a fluorescent dye to stain cells which were subsequently separated by flow cytometry.
  • a scan of the separated cells stained by the compound is shown in Figure 2.
  • the fluorescent dye allowed the differentiation of several populations of cells and their sorting by the cytometer.
  • Isometric plots of Figure 2 show the number of cells having particular fluorescence and light scatter characteristics. The number of cells is given on the vertical axis (cell number) and the side scatter (SSC) axis is as indicated.
  • Plots A and B show green fluorescence (FLl).
  • plots C and D show orange fluorescence (FL2) and plots E and F show red fluorescence (FL3). Data were collected using the FACSCalibur instrument at Macquarie University.
  • Plots A. C and E are unstained rehydrated high activity dry yeast (HADY) and plots B.
  • D and F are rehydrated HADY stained in distilled water with 5 ⁇ l per ml of a crude culture extract containing the compound of the present invention.
  • the fluorescent and SSC axes are logarithmic scale, the cell number axis is linear. Note the great increase in fluorescence for stained cells in channels FL2 and FL3. but not in FLl. Rapid Selection of Novel Yeast Hybrids
  • the criteria for selecting the most appropriate dyes were as follows.
  • the two dyes preferably should have different spectral emission properties (e.g. one green, the other one orange) such that labelled cells can be readily discriminated by flow cytometry.
  • Each dye should be bright enough to allow discrimination between parental cell types.
  • the dyes should be retained by the cells of the parent strain for the duration of the mating reaction. In addition they should not rapidly leak from one strain to the other.
  • a range of potentially suitable dyes were tested from the "CellTracker” (CT) dye range of Molecular Probes Inc. and the tracking dye, PKH-26.
  • CT CellTracker
  • PKH-26 the tracking dye according to the present invention.
  • the dye according to the present invention "Beljian Red", (BR).
  • cell clusters could not be precisely distinguished from doublets or mating pairs.
  • the FLl vs FL2 dot-plot was gated by a region defined on the forward scatter (FSC) vs side scatter (SSC) dotplot (Fig. 4) that included the events of lowest light scatter.
  • FSC and SSC amplifiers were set to linear. This low-scatter population was expected to correspond to the smallest clusters which would have included mated pairs. Cell sorting and microscopic observation of the low-scatter gated dual- stained population confirmed that this gating strategy excluded large clusters and included a large proportion of mating pairs.
  • Hybrid strains were identified by being able to form colonies after sub- culturing onto glucose minimal indicator medium, and by expression of lacZ derived from strain X. Using this method of analysis it was found that prior to sorting 33% of the population were hybrids. After one round of sorting the percentage of hybrids within the mixed population had increased to 70% and after two rounds it had increased to 96%. Rare mating of a polyploid industrial strain with a laboratory haploid. Shortly after mixing, two distinct populations representing the unmated parent strains were detectable in the mating reaction (data not shown). Cell sorting and microscopic observation confirmed that one of the populations was fluorescent green (strain Nl) and the other population was fluorescent orange (strain X).
  • the method presented here was developed for the rapid production and isolation of yeast hybrid strains without the need for genetic markers.
  • the percentage of yeast hybrids isolated following mating between two strains with opposite mating types was increased from 33% to 96% after two rounds of sorting.
  • the major advantage of the method is that a population highly enriched for hybrids can be produced without the use of genetic markers. This process is useful for production of new strains since isolates selected from the mating reaction are likely to be hybrids.
  • the present inventors also have demonstrated that this method can be applied to situations where hybrids are only rarely produced such as in 'rare mating'.
  • Rare mating can occur when a diploid or polyploid yeast strain which does not have a mating type spontaneously mutates into a strain with either an a or alpha mating type.
  • the resultant mutant can mate with a haploid or another polyploid strain of opposite mating type to produce a hybrid yeast.
  • heterothallic strains of yeast which generally includes the brewing and baking strains, this is a rare event.
  • the reported frequency of spontaneous ma ting-type switching in heterothallic haploid strains is increased by DNA damaging agents due to a gene conversion process.
  • three rare mated hybrids were identified from 50 isolates sorted from a mating pool of greater than two million cells.
  • the new technique could be used for the isolation of interspecific crosses.
  • Such hybrids may have significant industrial applications since it has been reported that some lager strains (Saccharomyces carlsbergensis) are the result of interspecific hybridisation.
  • Sacharomyces carlsbergensis Sacharomyces carlsbergensis
  • the ability to rapidly and efficiently isolate hybrids between two sexual gametes need not only be applied to yeast. It may be possible to apply the techniqvie to other situations where mating is required between organisms which do not have convenient genetic markers.

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Abstract

L'invention concerne un composé produit à partir d'une espèce de champignon, de préférence du genre Epicoccum, lequel est approprié à une utilisation en tant que marqueur fluorescent d'une biomolécule, peut se fixer sur une biomolécule, et, lorsqu'il est fixé sur une molécule, émet une fluorescence dans des longueurs d'onde rouges après excitation à des longueurs d'onde bleues.
PCT/AU1998/000023 1997-01-21 1998-01-20 Matieres tinctoriales fluorescentes obtenues a partir de micro-organismes Ceased WO1998031824A1 (fr)

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AU55437/98A AU5543798A (en) 1997-01-21 1998-01-20 Fluorescent dyes from microorganisms

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AUPO4701A AUPO470197A0 (en) 1997-01-21 1997-01-21 Fluorescent dyes from microorganisms
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Cited By (5)

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JP2003533446A (ja) * 2000-04-26 2003-11-11 フルオロテクニックス ピーティーワイ リミテッド 蛍光化合物
GB2346971B (en) * 1999-02-19 2004-07-21 Audrey Ann Coop Blood test for infectious prion protein using antibodies raised to eye lens crystallin proteins
WO2011071396A1 (fr) * 2009-12-09 2011-06-16 Auckland Uniservices Limited Compositions fongicides et leurs procédés d'utilisation
DE112010001347T5 (de) 2009-03-24 2012-07-05 Council Of Scientific And Industrial Research Verfahren zur Herstellung von kristallinen und nicht-hygroskopischen phenolreichen Farbfraktionen aus Pflanzen
CN112384618A (zh) * 2018-05-11 2021-02-19 喜力供应链有限公司 杂交有机体的稀有产物的鉴定

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2346971B (en) * 1999-02-19 2004-07-21 Audrey Ann Coop Blood test for infectious prion protein using antibodies raised to eye lens crystallin proteins
JP2003533446A (ja) * 2000-04-26 2003-11-11 フルオロテクニックス ピーティーワイ リミテッド 蛍光化合物
US8114963B2 (en) 2000-04-26 2012-02-14 Flurotechnics Pty Limited Fluorescent compounds
DE112010001347T5 (de) 2009-03-24 2012-07-05 Council Of Scientific And Industrial Research Verfahren zur Herstellung von kristallinen und nicht-hygroskopischen phenolreichen Farbfraktionen aus Pflanzen
US9255109B2 (en) 2009-12-09 2016-02-09 Auckland Uniservices Limited Fungicidal compounds and methods of their use
CN102844319A (zh) * 2009-12-09 2012-12-26 奥克兰联合服务有限公司 杀真菌的化合物和它们的使用方法
WO2011071396A1 (fr) * 2009-12-09 2011-06-16 Auckland Uniservices Limited Compositions fongicides et leurs procédés d'utilisation
CN102844319B (zh) * 2009-12-09 2016-03-02 奥克兰联合服务有限公司 杀真菌的化合物和它们的使用方法
KR101923234B1 (ko) 2009-12-09 2018-11-28 오클랜드 유니서비시즈 리미티드 살진균성 화합물 및 이의 사용 방법
US10477865B2 (en) 2009-12-09 2019-11-19 Auckland Uniservices Limited Fungicidal compounds and methods of their use
CN112384618A (zh) * 2018-05-11 2021-02-19 喜力供应链有限公司 杂交有机体的稀有产物的鉴定
CN112384618B (zh) * 2018-05-11 2024-01-23 喜力供应链有限公司 杂交有机体的稀有产物的鉴定
US12215309B2 (en) 2018-05-11 2025-02-04 Heineken Supply Chain B.V. Identification of rare products of crossing organisms

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