Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N25/00—Investigating or analyzing materials by the use of thermal means
  • G01N25/14—Investigating or analyzing materials by the use of thermal means by using distillation, extraction, sublimation, condensation, freezing, or crystallisation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/10—Starch-containing substances, e.g. dough

Definitions

• a method and a composition for the- determination of micro ⁇ bial contamination in rain using polymeric two- phase systems A method and a composition for the- determination of micro ⁇ bial contamination in rain using polymeric two- phase systems.
• the present invention relates to analysis of grain products. More particularly the invention relates methods and compositions for analysing grain products as regards the presence of micro-organisms.
• organoleptic method of analysis has several severe drawbacks, i.a. the following:
• organoleptic methods which are presently available are epifluorescence microscopy and simi ⁇ lar microscopical quantification methods,, as well as conven ⁇ tional microbial cultivation methods which require specially trained personnel and therefore have to be carried out in laboratories. They are consequently very time consuming and are not any practically useful alternatives to the organo- leptic method.
• the grain analysis should i.a. form the basis for pri cing the grain and determining how the grain should be store and used. For example, a poor-quality grain batch must no be stored together with a high-quality grain batch since th latter might be microbially contaminated by the former, an in such situations there is not sufficient time availabl for sending samples to a laboratory for analysis.
• the present invention aims at providing an improve method of analysis for determining the quality of grain.
• the invention aims at providing a method o analysis which is rapid (can be carried out on-site), simpl (can be carried out by unqualified personnel), reliabl (provides quantitative or semi-quantitative test results) and considerably cheaper than the presently available quan titative methods.
• the invention also aims -at providing compositions an test kits for carrying out the novel method of analysis.
• the analysis method according to the. invention is base on optical detection of bacterial cells and mould funga spores in extraction solutions from grain nuclei.
• the optica detection requires comparatively clean suspensions of micro organisms, which are free from starch granules and grai shell residues.
• the inventio makes use of separation in polymeric two-phase systems.
• the invention comprises two cooperating systems of this type, ' which also can be used independently of each other, viz. a first system (herein called fungal system), which sep ⁇ arates organic non-microbial particles and bacterial cells from the spores of infecting fungi, and a second system (herein called total system), which separates organic non- microbial particles from bacterial cells and fungal spores.
• a first system herein called fungal system
• total system which separates organic non- microbial particles from bacterial cells and fungal spores.
• Figure 1 is a diagram illustrating the correlation between the optical absorbance measured according to the invention as a function of the concentration of fungal spo ⁇ res.
• Figure 2 is a diagram corresponding to Figure 1 which illustrates the correlation between measured optical absor ⁇ saye according to the invention as a function of the con ⁇ centration of bacterial cells.
• Figure 3 is a diagram illustrating the correlation between measured data for fungal spores in grain obtained with the spectrophotometrical process according to the inven ⁇ tion and the conventional laboratory method epifluorescense microscopy respectively.
• Figure 4 is a diagram corresponding to Figure 3 but relates to the determination of bacterial cells in grain. Description of preferred embodiments
• Bulk solutions '(20%) of dextran and DEAE-dextran are prepared by combining 220 g of powder with 780 g of water. The powder is dissolved by heating on e.g. a water bath or in a microwave oven. The exact concentration of dextran or DEAE- dextran in the main solutions is determined by measuring optical rotation (polarimetrically) or gravimetrically by evaporating a known amount of solution. The main solutions are diluted to a concentration of 20.0%.
• a bulk solution of polyethyleneglycol 8000 is prepared by dissolving 400 g of Carbowax 6000 i 600 g of water.
• the remaining solutions 0.5 M citrate/phosphate buffer pH 4.8 and 7 as well as 2 M Li2S ⁇ , are prepared from chemi ⁇ cals of p.a. quality.
• the composition of the phase systems The bulk solutions of the polymers, buffers, salts and water are dosed into 12x100 mm test tubes of glass. The composition of the two types of systems are indicated below and are calculated on the basis of 2 g (fungal system) and 3 g (total system) respectively, 1 g of extraction liquid being included therein.
• Grain samples (whole grain nuclei) are extracted using sterile filtered aqueous solution containing 0.5% Tween 80, as follows:
• One part of grain is charged into plastic bags togethe with 1/2 part or 1 part of washing liquid.
• the plastic bags are sealed and put in an ultrasound bath for 3 minutes. Afte the ultrasound treatment has been terminated, the bags ar opened and the washing liquid is rough-filtered through glass-wool.
• Example lc Analysis without extraction step 1 ml of sample liquid from Example lc) is added to the respective fungal and total system (prepared according to Example lb) . The content of the systems is then thoroughly mixed by means of a test tube shaker for about 10 seconds. As a blank system for the optical measurement 1 ml of sterile filtered water is added to a fungal system and a total system respectively. After mixing the test tubes are put in a hori ⁇ zontal position in a test tube holder until the phases have separated.
• the absorbance in the top-phases for the fungal system and total system respectively is measured by means of a spectrophotometer (NOVASPEC 40/49) at 400 or 560 nm as fol ⁇ lows:
• the blank sample for the fungal system is put in the tube holder of the spectrophotometer which is then set to zero.
• the absorbance in the fungal system is measured.
• the absorbance for the total system is determined in a correspon ding manner.
• the concentrations of fungal spores and bacteri in the grain sample can be determined, as will be explaine further in Example 4 below.
• Example 3 Analysis with pre-extraction
• a pre-extraction wa carried out in the following manner.
• 0.5 ml of the top-phases fro systems lb and and 2b are removed and discarded.
• 0.5 ml o the top-phase from system la is then transferred to the to phase in system lb.
• 0.5 ml of the top-phase system 2a is transferred to system 2b.
• FIGS. 1 and 2 of the drawings show th optical relation between log aborbance as a function of (A) log concentration of fungal spores and (B) log bacteria cells in a liquid suspension ⁇ .
• the measurements have bee carried out at 560 nm.
• Penicilliu brevi compactum (funga spores) and Bacillus subtilis (bacterial cells) have bee used as standard organisms.
• the amount of fungal spores is estimated directly b reading the log concentration of spores for the lo absorbance measured in the system. '
• the amount of bacterial cells is estimated by subtrac- ting the absorbance in the fungal system from the absor ⁇ saye in the total system, and then log bacterial cells can be determined by means of the log residual absor ⁇ saye.
• the amount of fungal and bacterial cells respectively per gram of grains can be determined.
• That starch granules and shell residues can be separated from the bacterial cells/fungal spores.
• That bacterial cells can be separated from fungal spo ⁇ res.
• Table 1 reports the approximate cleaning efficiency in the two systems (fungal and total system) in separation tests carried out on extraction solutions of oat bran, wheat bran and wheat meal, the cleaning efficiency being expressed as the difference in absorbance before and after separation according to the invention.
• Tables 2a-b below report the results of some reproduci ⁇ bility tests carried out by means of the method according to the invention.
• Test run 1 " Test run 2 '
• Table 3 illustrates the effect of the extraction metho as regards detection of fungal spores in an extractio liquid. Table 3. The importance of extractions for spectrophoto metrical determination of mould spores in funga systems.
• the effect of an extractio in the fungal system is that the concentration of the spore in the system approaches the reference value, in this cas quantified by epifluorescense microscopy.

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• Analytical Chemistry (AREA)
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• General Health & Medical Sciences (AREA)
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• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Physics & Mathematics (AREA)
• Food Science & Technology (AREA)
• Pathology (AREA)
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• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
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• Bioinformatics & Cheminformatics (AREA)
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• Genetics & Genomics (AREA)
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• Medicinal Chemistry (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Citations (3)

• 1988
  • 1988-09-12 SE SE8803195A patent/SE462004B/sv not_active IP Right Cessation
• 1989
  • 1989-09-12 WO PCT/SE1989/000487 patent/WO1990002948A1/en unknown
  • 1989-09-12 AU AU42208/89A patent/AU4220889A/en not_active Abandoned

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