SE462004B - PROCEDURE AND COMPOSITION FOR DETERMINATION OF MICROBIAL POLLUTANTS IN CEREALS USING POLYMERIC DOUBLE PHASE SYSTEMS - Google Patents

Abstract

The invention relates to the use of polymeric two-phase systems for quantitative or semi-quantitative determination of microbial contaminants, especially fungal spores and/or bacterial cells, in grain products; a method for determining the total microbial contaminants in grain products by extracting micro-organisms from a grain sample, mixing the extraction solution with a polymeric two-phase system and allowing the system to separate such that the microbial contaminants are collected in the top-phase and non-microbial contaminants in the bottom-phase, and optically determining the contents of microbial contaminants; a method of determining the contents of fungal spores in a grain sample in corresponding manner but directing also the bacterial contaminants to the bottom-phase; a method of determining the contents of bacterial contaminants in a grain sample by determining both the total microbial contaminants and the fungal contaminants, and subtracting the latter measuring data from the former; and compositions suitable for carrying out the methods.

Description

462 10 15 zo '25 30 35 004 2 The cereal analysis shall i.a. form the basis for determining the price of cereals and how the cereals are to be stored and used. For example, a substandard grain batch must not be stored together with a high-quality grain batch, as the latter can then be microbially contaminated by the former, and there is no time to send samples to a laboratory for analysis.

There is thus a great need for improved methods for grain analysis.

I E.. E! The present invention aims to provide an improved analysis method for quality assessment of cereals.

In particular, the invention aims to provide an analysis method which is fast (can be performed on site), simple (can be performed by unqualified personnel), reliable (gives quantitative or semi-quantitative results) and significantly cheaper than those currently available. available quantitative methods.

The invention also aims to provide compositions and test kits for carrying out the new analysis method.

These and other objects and advantages of the invention will become apparent from the following description and the accompanying drawings. s En. E ..

The stated objects and advantages are achieved according to the invention in that the assay procedure, assay compositions and assay kit have been given the distinctive features set forth in the appended claims. Preferred embodiments of the invention are set out in the subclaims.

The assay method according to the invention is based on optical detection of bacterial cells and mold spores in extraction solutions from grain kernels. The optical detection requires relatively pure microorganism suspensions, free from starch granules and shell residues. In order to enable this, separation in polymeric two-phase systems is used according to the invention.

Polymeric two-phase systems are known per se, for example through Albertsson P Å, 1960, Partition of cell particles and macromolecules, 2nd edition, Uppsala, Sweden, but this technology has not been used - even less adapted - for grain analysis.

The invention comprises two cooperating such systems, which can also be used independently of each other, namely a first system (here called fungal system), which separates organic non-microbial particles and bacterial cells from spores from storage damage fungi, and a second system (here called total system), which separates organic non-microbial particles from bacterial cells and fungal spores. By combining the separation of the two systems with optical detection, the amount of fungal spores and bacterial cells in an extraction liquid from grain kernels can be estimated. '.t. S .vn.

The accompanying drawings show the following: 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 spores.

Figure 2 is a graph corresponding to Figure 1, illustrating the correlation between measured optical absorbance according to the invention as a function of the concentration of bacterial SOITI cells.

Figure 3 is a diagram, between measurement data for fungal spores in cereals obtained by the spectrophotometric method according to the invention resp. with the conventional laboratory method epifluorescence microscopy.

Figure 4 is a diagram corresponding to Figure 3, but refers to the determination of bacterial cells in cereals. which illustrates the correlation. . Some preferred embodiments of the invention will be described in the following examples, in which the present data on concentrations and the like are weight-based, unless otherwise indicated. Bulk solutions (20%) of dextran and DEAE-dextran are prepared by weighing 220 grams of powder together with 780 grams of water. The powder is dissolved by heating in, for example, a water bath or in a microwave oven. The exact concentration of dextran or DEAE-dextran in the stock solutions is determined by measuring the optical rotation (polarimetry) or gravimetrically by evaporating a known amount of solution. The stock solutions are diluted to 20.0%.

Bulk solution of polyethylene glycol 8000 is prepared by dissolving 400 g of Carbowax 6000 in 600 g of water. Other solutions, 0.5 M citrate / phosphate buffer pH 4.8 and 7 and 2 M Li2SO4, are prepared from chemicals of p.a. quality.

The bulk solutions of the polymers, buffer, salt and water are weighed into 12x100 mm glass test tubes. the composition of the two types of systems is given below and is calculated on 2 grams (fungal system) and 3 grams (total system), respectively, which includes 1 g of extraction liquid. yyampsystgmi 4.8% dextran 500 Uppsala), 3.85% polyethylene glycol 8000 Union Carbide, New York), 1.1% diethylaminoethyldextran 500 Fine Chemicals, Uppsala), 26 mM citrate / phosphate buffer pH 4.8, 1 mm Li2so4.

(Pharmacia Fine Chemicals, (Carbowax 6000, (Pharmacia 8.4% dextran 500, 5.8% polyethylene glycol 8000, 25 mM Citrate / phosphate buffer pH 7.0, 50 IHM Li 2 SO 4).

The peak phase volumes in the two systems after separation are about 1.0 ml (fungal system) and 1.5 ml (total system) and the separation time is about 1.5 minutes in the horizontal position and about 6 minutes in the vertical position. c) Egtraktiog of grain samples Grain samples (whole kernels) are extracted with a sterile-filtered aqueous solution containing 0.5% Tween 80, as follows: Some grain kernels are weighed in plastic bags with 1/2 part or 1 part washing liquid. The plastic bags are sealed and placed in an ultrasonic bath for 3 minutes. After the ultrasound treatment is completed, the bags are opened, after which the washing liquid is coarsely filtered through glass wool. s e ss 1 ml of test liquid from Example 1 c) is added to the respective fungal and total systems (prepared according to Example 1b), after which the contents of the systems are mixed well 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 sponge and total system, respectively. After mixing, the test tubes are placed in a horizontal position in a test tube rack until the phases have separated.

The absorbance in the peak phases for each fungal and total system is measured by means of a spectrophotometer (NOVASPEC 40/49) at 400 or 560 nm as follows; The blank for the fungal system is inserted into the cuvette holder in the spectrophotometer, after which it is reset. The absorbance of the fungal system is measured. Correspondingly, the absorbance of the total system is determined.

With knowledge of the grain / sample liquid extraction ratio, peak phase volume and the absorbances in each peak phase, the concentration of fungal spores and bacteria in the grain sample can be determined, as will be explained in more detail below in Example 4.

EK§E2§l_3 E J 1 En! ll.

To improve the separation between interfering organic 462 004 6 10 15 20 25 30 35 non-microbial particles and fungal / bacterial cells in the total system, and to improve the separation between fungal and bacterial cells in the fungal system to thereby increase the precision of the assay, a pre-extraction was performed in the following way. 1 ml of extraction liquid is added to each of a fungal system and a total system (systems 1a and 2a, respectively). In the same way, add 1 ml of sterile filtered water containing 0,5% Tween 80 to the corresponding fungal and total systems (systems 1b and 2b respectively). The systems are mixed well using a test tube shaker for about 10 seconds, after which the tubes are placed horizontally on a test tube rack until the phases are separated.

After phase separation, 0.5 ml of the peak phases from systems 1b and 2b are removed and discarded, after which 0.5 ml of the peak phase from system 1a is transferred to the peak phase in system 1b. In the same way, 0.5 ml of the peak phase in system 2a is transferred to system 2b. Systems 1b and 2b are mixed again on a test tube shaker and allowed to separate in a horizontal test tube rack for 2-3 minutes.

As a blank system, 1 ml of sterile filtered water is added to a fungal and total system, respectively, which are treated and allowed to separate in the same way as the test systems.

The spectrophotometric. the measurement in systems 1b and 2b is performed in the same way as. described for analysis without pre-separation in Example 2 above. fi xsmmLi E lf. _ 1 1 1. Ü 3 In the accompanying drawing figures 1 and 2, the optical relationship between the log absorbance is reported as a function of the (A) log concentration of fungal spores and (B) log bacterial cells in a liquid suspension. The measurements were performed at 560 nm. Penicillium brevi compactum (fungal spores) and Bacillus subtilis (bacterial cells) have been used as type organisms.

In the graphical estimation of the amount of fungal spores and bacterial cells in the washing liquid obtained from the grain extraction, the two absorbance values from the fungal and total system are used as follows: 10 15 20 25 462 ÛÛ4 7 1. The amount of fungal spores is estimated directly by reading the log concentration of spores in the system measured the log absorbance. 2. The amount of bacterial cells is estimated by subtracting the absorbance from the fungal system from the absorbance in the total system, after which log bacterial cells can be determined by means of the log residual absorbance. With knowledge of the peak phase volumes (1.0 ml), the estimated concentrations of bacterial cells and fungal spores and the ratio of grain / washing liquid, the amount of fungal and bacterial cells per gram of grain kernels can be determined. e 's e' v 'grain kernels result in a bacterial cell extraction of the entire mixture of starch granules, shell residues, and fungal spores. In order to quantify the amount of fungal spores and bacterial cells in the extraction liquid by means of optical methods, it is required: 1. That starch granules and shell residues can be separated from the bacterial cells / fungal spores. 2. That bacterial cells can be separated from fungal spores.

Table 1 shows the approximate purification efficiency in the two systems (fungal and total systems) in separation experiments performed on extraction solutions of oat bran, wheat bran and wheat flour, the purification efficiency being expressed as the difference in absorbance before and after separation according to the invention. 10 15 20 25 30 35 40 "4e2 004 8 Iâb§ll_l Havrekli ------- --abs-før- _ abs efter reningseff.

Mushroom system 15.50 0.32> 98% Total system 15.60 0.46> 97% Wheat bran abs_before abs after cleansing effect Mushroom system 17.80 0.25> 99% Total system 17.80 0.32> 98% Wheat flour abs before abs after reningseff. fungal system "šfZš '_"' 8fšš ""> 98% Total system 9.45 0.45> 95% _______________________________________________ Since starch granules and shell residues cause the strongest absorbance in extraction solutions of crushed cereals and flour, as shown in Table 1, a very good separation (> 95%) in the two systems. Separation and spectrophotometric detection of the amount of bacterial cells and fungal spores in extraction liquids from whole grain kernels has been performed on a large number of grains of different quality using the method described in Example 3 above. As a reference method, direct counting of the amount of bacterial cells and fungal spores, after staining with acridine orange, was used in an epifluorescence microscope. The results of 72 comparative measurements are presented graphically in the drawing figures 3 and 4. Figure 3 shows the correlation in measurement data between the two methods regarding fungal spores, while Figure 4 shows the correlation in measurement data for bacterial cells.

As can be seen from Figures 3 and 4, the method according to the invention gives a good agreement with the time-consuming (and not useful for daily practical use) laboratory method epifluorescence microscopy, with a correlation of 0.90 when determining the amount of fungal spores and 0.82 when determining the bacterial concentration. 10 15 20 25 30 35 9 462 004 EzemBel_§ B i I 1! le ln.

The reproducibility of an analysis method can be defined as the reproducibility of the analysis response when several analyzes are performed on one and the same sample (= grade 1 reproducibility), alternatively when several grain samples are taken from a larger batch, extracted and analyzed separately (= reproducibility of grade 2).

Tables 2a-b below show the results of some reproducibility experiments performed with the method according to the invention.

Table 2a. Triple test performed on grain with excellent quality.

(Reproducibility grade 1) Sample no. Mold spores * Bacteria * 1 2,2x1ø5 ___- 2, sx1o7 2 z, 2x1o5 z, sx1o7 3 z, zx1o6 2,6x1o7 = Number expressed in microorganisms / g wet weight Table 2b. Two samples from different batches of cereals of different quality, extracted and analyzed separately.

(Reproducibility grade 2).

Sample round 1 * Sample round 2 * Mold spores o. 93x1o7 1, oox1o7 Bacteria 2.1ox1o8 3, sox1o8 Mold spores o. 71x1o6 z, 2ox1ø6 Bacteria 2, sox1o7 2, sox1o7 Mold spores 4.50x105 Borex1x1xx1 , 35x106 2.70x105 Bacteria 2, s3x1o7 2.9ox1o7 ___________________________________-________________________ = The number expressed in microorganisms / g wet weight 462 004 10 10 15 20 25 30 35 The results from this study show that the reproducibility of the first as well as the second degree is very good. With regard to the second degree of reproducibility, the homogeneity of microbial presence on the cereal kernels naturally plays a decisive role, which may explain the small differences in concentrations recorded in the two cases of analysis.

Since separation in polymeric two-phase systems is a distribution phenomenon, ie differences in the partition coefficient between interfering particles and microorganisms are used, an extraction process has an advantage when it comes to the separation of organic non-microbial particles from the total system. In the same way, you get a more secure value in the fungal system thanks to an increased separation of bacterial cells and other particles from the fungal spores.

The effect of the extraction procedure on the detection of fungal spores in an extraction liquid is presented in Table 3.

Table 3. Significance of extractions for spectrophotometric determination of mold spores in fungal systems.

Sample No. Extraction 1 Extraction 2 Epifluorescence value 1 1.2x1o7 1.1x1o6 9.2x1o6 2 2, sx1o6 1, sx1o6 2, ox1o6 3 1, sx1o6 9, sx1o5 1.2x1o5 4 3, sx1o5 2, sx1o6 2.4x1o6 7, sx1o5 4, sx1o5 4, ox1o6 6 2.2x1o6 1.7x1o5 1.9x1o5 7 4, sx1o7 3.9x1o7 4.1x1o7 a 4.9x1o5 4, ox1o5 3.1x1o5 As shown in the table, an extraction in the fungal system that the concentration of spores in the system approaches the reference value, in this case quantified by, epifluorescence microscopy.

Claims (16)

10 15 20 25 30 462 004 ll B_A_I_E_E_ILK_B_A_ ¥ Use of polymeric two-phase systems for the quantitative or semi-quantitative determination of fungal spores or bacterial cells in cereal products. Method for the qualitative, semi-quantitative or quantitative determination of microbial contaminants in cereal products, characterized in that it comprises the steps of: (a) forming a sample solution by extracting microorganisms from a cereal sample, (b) mixing the sample solution with a polymeric two-phase system, (c) allowing the system to separate so that microbial contaminants accumulate in the upper phase of the system and substantially all non-microbial particles collect in the lower phase of the system, (d) determining the level of microbial contaminants in the upper phase optically. 3. A process according to claim 2, characterized in that the extraction liquid in step (a) consists of particle-free water, which preferably contains a surfactant. A method according to claim 2 or 3, characterized in that the optical determination in step (d) is performed spectrophotometrically, turbidimetrically or visually by comparison with a standard. A method according to any one of the preceding claims, characterized in that the polymeric two-phase system comprises a dye capable of binding to or being taken up by at least some of the microorganisms in the superphase, or changing color upon contact with them. Process according to any one of the preceding claims, characterized in that the microbial contaminants comprise fungal spores or bacterial cells, and that the polymeric two-phase system is chosen so that substantially all fungal spores and bacterial cells in the sample solution collects in the superphase when the two-phase system separates at step (c). Process according to any one of the preceding claims, characterized in that the polymeric two-phase system comprises at least one polyalkylene glycol, preferably a polyethylene glycol having an average molecular weight of 200 - 20,000, in particular 1,000 ~ 10,000, in particular 4,000 - 8,000, which forms the upper phase of the system, and at least one polysaccharide, which is preferably crosslinked and has a higher average molecular weight than the polyalkylene glycol in the superphase, especially at least 40,000, which forms the lower phase of the system. Process according to one of the preceding claims, characterized in that the polysaccharide is crosslinked dextran, starch, cellulose or crosslinked mono-, di- or oligosaccharides. * Process according to any one of the preceding claims, characterized in that the polymeric two-phase system also comprises at least one inorganic salt, preferably an alkali metal salt, which is capable of enhancing the enrichment of the microbial impurities in the over-phase. A method for determining the content of fungal spores in a cereal sample, characterized in that it comprises the steps of (a) forming a sample liquid by extracting microorganisms from a cereal sample, (b) mixing the sample liquid with a polymeric two-phase system, (c) allowing the system separates, so that the upper phase will essentially contain only fungal spores, while other microbial and non-microbial contaminants in the sample liquid accumulate in the sub-phase, and I (d) determine the amount of fungal spores in the upper phase optically. 10 15 20 25 30 35 462 004 13 11. A method according to claim 10, characterized in that the two-phase polymeric system comprises such a system according to claim 1 or 2 and in addition either (i) a positively charged polymer, as in separation. is in the bottom phase, or (ii) a negatively charged polymer which in separation is in the upper phase. Process according to Claim 10 or 11, characterized in that the polymeric two-phase system also comprises an inorganic salt, in particular alkali metal salt, which is capable of enhancing the distribution of a positively charged polymer to the bottom phase. 13. A process according to claim 10, 11 or 12, characterized in that the acidic pH of the polymeric two-phase system is maintained. A method for quantitatively determining the content of microbial contaminants in a cereal sample, characterized in that (a) the content of fungal spores in a first aliquot of the cereal sample is determined by the method according to any one of claims 9 to 13, (b) determines the total content of microbial contaminants in a second aliquot of the cereal sample by the method of any one of claims 2 to 8, and (c) estimates the content of bacterial contaminants by subtracting the value obtained in the determination step (a) from the one in the determination step ( b) the value obtained. Composition suitable for the quantitative determination of contaminants of fungal spores in cereal samples, characterized in that it contains: (a) 0.5 to 15 parts by weight of at least one optionally crosslinked polysaccharide and / or crosslinked mono-, di- or oligosaccharide , (b) 0.5 to 15 parts by weight of at least one polyalkylene glycol, 462 004 14 (C) (d) (6) 5 At least 10'4 parts by weight of at least one charged polymer, a buffer system which gives the composition an acidic pH, and at least one alkali metal salt. Composition suitable for the quantitative determination of the total content of microbial impurities in a cereal sample, characterized in that it contains components (a), (b) and (e) of claim 15, a buffer system which gives neutral to basic pH.