RU2077717C1 - Process of detection of affection of grain by microscopic fungi - Google Patents

Abstract

FIELD: agricultural food processing industry. SUBSTANCE: invention can find use in detection of affection of grain by metabolites of fungi. Examined sample of grain and check sample are formed, they are around and pelletized, they are exposed to radiation in wave range 360-500 nm. Difference of bioelectric potentials is measured and judgement on degree of affection of examined sample of grain by metabolites of fungi is made. EFFECT: improved authenticity of detection of affection of grain by microscopic fungi. 4 cl, 5 dwg, 1 tbl

Description

 The invention relates to food industry and agriculture in the field of determination of harmful substances of microbiological origin and can be used to determine the degree of damage to grain metabolites of mushrooms.

 The greatest danger to Russian grain farming is contamination of grain with fusariotoxins. About 10% of wheat with the presence of Fusarium grains comes from the average annual purchases to grain receiving points of the Russian Federation, and 2.5% of it is completely unsuitable for food purposes. This not only leads to significant economic losses. but, given the irreversible effects of exposure to Fusarium infection of grain products on the human body, creates a serious social problem. In a market economy, when control over the storage conditions of grain and grain products largely leaves the sphere of state bodies, this problem is even more acute. Along with this, there is an increasing need for simple and affordable means of control that can, in practice, ensure compliance with accepted laws and certification rules.

Currently, the following maximum permissible concentrations of microtoxins are established in Russia and the CIS countries:
aflatoxin B1-5 mg / kg for grain and grain products;
comitoxin 1.0 mg / kg for strong and durum wheat; 0.5 mg / kg for the rest of the wheat for food purposes;
T-2 toxin 0.1 mg / kg for grain and grain products;
zearalenone 1.0 mg / kg for grain and grain products.

 In baby food, the content of the listed microtoxins is not completely allowed.

 Currently, most of the known methods for determining the degree of damage to grain by microscopic fungi are time-consuming, expensive and require highly qualified researchers, which makes them unsuitable for the system of production, storage and processing of grain. For this reason, the "Rules for the certification of grain and grain products" as test methods for infection with microscopic fungi, it is recommended to use only visual methods of determination, based on purely subjective estimates and which are also time-consuming and time-consuming. Therefore, for the actual implementation of the above-mentioned rules, a high-precision express method for determining the degree of damage to grain by various types of microscopic fungi is required.

 A known method for determining the degree of damage to grain by microscopic fungi, including grinding the grain, preparing the extract, introducing it into the reaction mixture containing NADI: FMN oxidoreductase, luciferase and their substrates, determining the presence of mushroom metabolites in the extract by the intensity of their glow and judging the degree of damage to the grain by the ratio of the intensities of the bioluminescence of the reaction mixture in the presence of grain extracts and without it (class G01N 33/10, USSR author's certificate N 1557521, 1990).

However, the known method is time-consuming, requires specific scarce reagents for its existence and specialist chemists, and is also quite long (1 1.2 hours) with a sufficiently high measurement error of up to 10%
Since the maximum permissible concentration of microtoxins for various types of microscopic fungi is only 0.1 5 mg / kg of grain, and their content in baby food is generally completely unacceptable, this magnitude of the measurement error is unacceptably high.

 The closest technical solution (prototype) is a method for determining the degree of grain damage by microscopic fungi, including weighing the selected grain sample, preparing it for examination by peeling 38 42 s with a gap between rubberized rolls of 0.3 0.5 mm, grinding 19 32 s with a gap between the abrasive drum and brake pad 2.9 3.2 mm, UV irradiation of the sample, separation of the detected grain with green-yellow fluorescence, weighing it and determining the degree of damage to the batch of grain by the percentage of grains with green-yellow th fluorentsentsiey in the selected sample of grain (Cl. G01N 33/10, 21/33, author's certificate USSR N 1822496, 1993).

 However, the known method is time-consuming, requires special bulky peeling and grinding equipment, and is also quite long (more than 1 hour) and is based on purely subjective estimates, which leads to a rather high (10 12%) measurement error; and such a value for grain products, especially for baby food, as noted above, is often unacceptably high.

 Achievable new technical result of the invention is to increase the accuracy of determination while reducing the duration.

где S(W) амплитудно-частотная характеристика в виде плотности спектральной мощности биотока таблетки в области фликер шума;
W частота;

скорость изменения массы-энергии пространственно-временных диссипативных структур таблетки, формирующих субпространства структуры биотока таблетки при изменении частоты в пределах интервала ΔW;;
ΔW интервал частот, показывающий область устойчивости пространственно-временных диссипативных структур таблетки, формирующих субпространства структуры биотока определенной степени изотропности и определяемый из следующего выражения:
ΔW = W $\genfrac{}{}{0ex}{}{\text{n}}{\text{кр}}$ - W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{кр}}$ ,
где W $\genfrac{}{}{0ex}{}{\text{n}}{\text{кр}}$ и W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{кр}}$ - критические частоты, определяемые пересечением последовательных параллельных прямых, образуемых совокупностью максимального количества точек, включающих минимальные значения точек S(W_i)_min, принадлежащих каждому из интервалов
ΔW^n-(n-1), ΔW^(n-1)-(n-2),...,
и осуществляют построение из спектрограммы относительного изменения массы-энергии пространственно-временных диссипативных структур от времени

а о степени поражения зерна судят по степени смещения по крайней мере двух частот W_i и W_j на наиболее длинных интервалах ΔW, на которых относительное изменение соответствующей скорости массы энергии пространственно-временных диссипативных структур от времени,

на частотах W_i и W_j происходит в противофазе, относительно аналогичных частот

для контрольной пробы с известным содержанием здорового зерна.A new technical result is achieved by the fact that in the method for determining grain damage by microscopic fungi, including the selection of the studied grain sample with microscopic mushrooms and a control sample with known healthy grain content, preparation of samples for research, irradiation, registration of the physical parameter of the samples and judging the degree of grain damage, unlike the prototype, when preparing samples for research, they are ground and tabletted, training is carried out at a wavelength of 360 500 nm, and as a physical This parameter uses the difference in bioelectric potentials, after recording the bioelectric potentials of the tablets, their difference is amplified, converted into the amplitude-frequency characteristic of the spectral power density of the biocurrent, the high-frequency noise flicker regions are filtered out, for example, over 2000 Hz the components of the amplitude-frequency characteristic are laid out in the interval 0 - 2000 Hz in the field of flicker noise, determine the dependence of the amplitude-frequency characteristics on BP names, decipher it by constructing a flicker noise spectrogram described by the following expression:

where S (W) is the frequency response in the form of the spectral power density of the tablet biocurrent in the flicker noise region;
W frequency;

the rate of change in mass-energy of spatio-temporal dissipative structures of the tablet, forming subspaces of the structure of the biocurrent of the tablet when the frequency changes within the interval ΔW ;;
ΔW is the frequency interval showing the stability region of spatiotemporal dissipative tablet structures forming subspaces of the biocurrent structure of a certain degree of isotropy and is determined from the following expression:
ΔW = W $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ - W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ ,
where w $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ and W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ - critical frequencies determined by the intersection of consecutive parallel lines formed by the combination of the maximum number of points, including the minimum values of points S (W _i ) _min , belonging to each of the intervals
ΔW ^{n- (n-1)} , ΔW ^{(n-1) - (n-2)} , ...,
and construct from the spectrogram the relative change in mass-energy of spatio-temporal dissipative structures from time to time

and the degree of damage to the grain is judged by the degree of displacement of at least two frequencies W _i and W _j on the longest intervals ΔW, at which the relative change in the corresponding velocity of the mass of energy of spatio-temporal dissipative structures from time to time,

at frequencies W _i and W _j occurs in antiphase, relative to similar frequencies

for a control sample with known healthy grain content.

 The registration of the difference in bioelectric potentials can be carried out when applying to two additional, installed on the tablet extreme, relative to two removable, DC electrodes from an external energy source.

 FIG. 1 5 explain the proposed method.

 The amplitude-frequency characteristic of the density of the spectral power of the biocurrent and the parameters of the spatiotemporal dissipative structures can be determined by time during the day by recording with an interval of 600 ± 1 s based on the linear averaging of 128 spectra of the amplitude-frequency characteristics of the density of the spectral power of the biocurrent for each of the width intervals no more than 12.5 Hz obtained in real time. The tablet can be installed at the temperature of liquid nitrogen and shielded from the influence of electromagnetic fields.

 A method for determining the degree of damage to grain by microscopic fungi is implemented as follows.

 The main part of the studied samples was type IV wheat - winter red-grain high-glassy wheat from the regions of the North Caucasus, cultivated in the foci of spike fusarium.

 To establish the dose-effect relationship, model mixtures were compiled in which the content of Fusarium grains ranged from 0.5 to 50%. A 100% sample of normal healthy grain and a 100% sample of Fusarium grain were also used as a control. Model mixtures are presented in the table. A test sample was also prepared with an unknown degree of damage by microscopic fungi.

 Samples were prepared for the study by grinding grain in a laboratory mill LZM up to 250 microns for 3 minutes. The homogeneity of the model mixtures was ensured by repeated mixing and sieving of the mixed components.

Two characteristics were used to characterize the analyzed wheat samples:
the content of fusarium grains;
the content of mycotoxins, i.e., deoxynivalenol (DON) and zearalenol (3H).

 The determination of the content of Fusarium grains was carried out in accordance with the "Methodological guidelines for accounting for spike Fusarium and visual determination of wheat grain Fusarium" (approved by the USSR Ministry of Health, USSR State Agro-Industrial Committee and USSR Ministry of Bread and Beverage on July 15, 87).

Then the samples were tabletted by placing the crushed grain in the matrix of the pressing machine under a pressure of 250 kg / cm ² and a size of 10x5x2 mm.

 4 electrodes are mounted on the tablet surface. The conductivity of the ohmic contacts was provided by organosilicon paste.

 A tablet with electrodes is placed in an optically transparent cell equipped with a thermal monitoring system and placed in a shielded chamber with an optically transparent window. The sample is cooled to the temperature of liquid nitrogen and irradiated with an IFK-120 type xenon flash lamp with a frequency of 5 Hz, a pulse duration of 30 μs and a flash electric energy of ≈ 0.1 J. To select luminescence-exciting radiation with a wavelength of 350–500 nm, we used suitably selected SS filters -4 and SZS-22.

 The essence of the invention is in fixing the energy, which is the difference between the excitation energy (360 500 nm) and the luminescence energy (520 700 nm) using spectral analysis of part of this energy, which falls on the low-frequency region.

 Before irradiation, a direct electric current of 0.6 mA from an external current source is passed through two extreme electrodes, and the difference in bioelectric potentials is measured through two middle electrodes.

 The captured bioelectric potential difference is fed to the preamplifier PU-2 with a noise level of 1-2 dB to amplify the signal 200 times. The difference in bioelectric potentials is converted into the amplitude-frequency characteristic (AFC) of the spectral power density of the biocurrent, the high-frequency components of the AFC are filtered out and the latter is laid out in the range of 0 to 2000 Hz in the noise flicker region using the Brule and Kier narrow-band frequency spectrum analyzer BK 2031 (Denmark) connected to the output of the preamplifier PU-2.

где S(W) АЧХ в виде плотности спектральной мощности биотока таблетки в области фликер-шума,
W частота;

скорость изменения массы-энергии пространственно-диссипативных структур биотока пробы в виде таблетки при изменении частоты в пределах интервала ΔW;
ΔW интервал частот, показывающий область устойчивости пространственно-временных диссипативных структур пробы в виде таблетки, формирующих субпространства структуры биотока определенной степени изотропности и определяемый из следующего выражения:
ΔW = W $\genfrac{}{}{0ex}{}{\text{n}}{\text{кр}}$ - W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{кр}}$ ,
где W $\genfrac{}{}{0ex}{}{\text{n}}{\text{кр}}$ и W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{кр}}$ критические частоты, определяемые пересечением последовательных параллельных прямых 1,2,3. и 1', 2', 3',образуемых совокупностью максимального количества точек, включающих значения точек S(W_i)_min, принадлежащих каждому из последовательных интервалов

.Then determine the dependence of the frequency response from time to time. At the same time, the analyzer operates in real time, performing linear averaging of 128 values obtained from a sample in the form of a tablet of information of the AFC spectra in each of the narrow frequency intervals (12.5 Hz) of the spectrum from 0 to 2000 Hz in the flicker noise region for 2.10 ^-2 c. Automatically decipher the frequency response of the investigated processes in the structure of the biocurrent versus time in decibels (dB) by constructing a flicker noise spectrogram (Fig. 1), described by the following expression:

where S (W) AFC in the form of the spectral power density of the tablet biocurrent in the flicker noise region,
W frequency;

the rate of change in mass-energy of the spatially dissipative structures of the sample biocurrent in the form of a tablet with a change in frequency within the interval ΔW;
ΔW is the frequency interval showing the stability region of spatio-temporal dissipative sample structures in the form of tablets, forming subspaces of the biocurrent structure of a certain degree of isotropy and determined from the following expression:
ΔW = W $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ - W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ ,
where w $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ and W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ critical frequencies determined by the intersection of consecutive parallel lines 1,2,3. and 1 ', 2', 3 ', formed by the combination of the maximum number of points, including the values of points S (W _i ) _min , belonging to each of consecutive intervals

.

для каждого интервала

и т. д. определяют как семейства параллельных прямых, принадлежащих соответствующему интервалу.In this case, the value

for each interval

etc. are defined as families of parallel lines belonging to the corresponding interval.

(фиг.3,4,5) относительного изменения массы-энергии пространственно-временных диссипативных структур от времени, для оценки скорости

.Then determine the dependence on time

(Fig.3,4,5) the relative change in mass-energy of spatio-temporal dissipative structures from time to time,

.

Moreover, the values ΔS (W _i ) and ΔS (W _j ) the relative long-term change in the spectral density of the noise power of the biocurrent at frequencies W _i , W _j , characterizing the conditions of the stationary state of the biological object (sample in the form of a tablet), will be determined as shown in Fig.3.

The verifiability of the data obtained by the proposed method on the physiological parameters of a grain sample in the form of a tablet is achieved by rigidly fixing the time of day (± 1c) of measurements, fixing in space the position of the test sample in the form of a tablet and measuring electrodes, and the accuracy of maintaining the temperature (± 0.5 ^o C), which determines the physiological state of the sample, the strength of the bioelectric current and the parameters of its structure. A rigid “linking” to time is due to the dependence of self-organization processes on time and the fact that for non-ergodic systems, to which any biological objects belong, the state average is not equivalent to the time average. For this reason, it is possible to compare the dependence of the parameters of the macrostructure of the tablet resulting from the processes of self-organization on the composition only if the experiment is fixedly fixed.

 Statistical analysis for non-ergodic systems arises only with respect to the coherent time of day.

для соответствующих интервалов частот для максимальной величины силы биотока контрольной и исследуемой проб составляет 1%
Относительная ошибка определения частот W_i, W_j, W_k и т.д. связанных с существованием пространственно-временных диссипативных структур, между которыми происходит перераспределение энергии ΔS(W_i), ΔS(W_j), ΔS(W_k),..., от времени, составляет 3%
Определив, как описано выше, для таблетки пробы зерна с микроскопическими грибами и таблетки контрольной пробы здорового зерна величины

для каждого из интервалов

и величины этих интервалов, частоты W_i, W_j, W_k, на которых фиксируется изменение во времени значений S(W_i), S(W_j), S(W_k), а также относительного изменения величин ΔS(W_i), ΔS(W_j), ΔS(W_k),...,, строят графическую зависимость этих величин от времени, соблюдая условия верифицируемости данных о неэргодичных системах, проводят сравнительный анализ полученных результатов, выбирая для анализа по крайней мере две частоты W_i, W_j, наиболее длительных интервалах ΔW, на которых относительное изменение соответствующей скорости массы-энергии пространственно-временных диссипативных структур от времени

на частотах W_i и W_j происходит в противофазе.Relative error in the determination of quantities

for the corresponding frequency intervals for the maximum value of the biocurrent strength of the control and studied samples is 1%
The relative error in determining the frequencies W _i , W _j , W _k , etc. associated with the existence of spatio-temporal dissipative structures between which there is a redistribution of energy ΔS (W _i ), ΔS (W _j ), ΔS (W _k ), ..., from time to time, is 3%
Having determined, as described above, for a tablet of a sample of grain with microscopic mushrooms and a tablet of a control sample of healthy grain, the values

for each of the intervals

and the values of these intervals, the frequencies W _i , W _j , W _k , on which the change in time of the values S (W _i ), S (W _j ), S (W _k ), as well as the relative change in the values ΔS (W _i ) is recorded , ΔS (W _j ), ΔS (W _k ), ... ,, build a graphical dependence of these values on time, observing the verification conditions of data on nonergodic systems, conduct a comparative analysis of the results, choosing at least two frequencies W _i for analysis , W _j , the longest intervals ΔW, in which the relative change in the corresponding mass-energy velocity is spatially dissipative structures from time to time

at frequencies W _i and W _j occurs in antiphase.

и т.д.The stationary nature of the process of absorption of light energy by the molecular structure of the fungus and its redistribution between heat and luminescence work is manifested in the appearance of new frequencies

etc.

 These frequencies are compared with the reference frequencies of tableted flour prepared from grains with a known degree of infection with fusarium, defined, for example, with respect to 100 grains and, thus, determine the degree of infection. The sensitivity of the proposed method is great.

 This is indicated by a simple calculation. Fixing accuracy S (W) 0.1 dB.

0.1DB 10 ^-9 B 10 ^-8 W / A: at 6.10 ^-4 A, the apparent power is 10 ^-13 W.

The noise flicker region accounts for 0.5.10 ^-13 W, and 5 / 400.10 ^-14 or 10 ^-16 W 10 ^-16 J / s falls on one of the 400 bands in the range of the spectrum under study.

The absorption energy at a wavelength of λ 400 nm is 4.2.10 ^-9 dis.

When irradiated with a xenon lamp operating with a frequency of light pulses of 1 10 Hz, at a frequency of 5 Hz and electric flash energy ≈ 0.1 J, pulse duration ≈ 30 μs at the studied object, the power density ≈ K.10 W / cm ² , the energy density ≈ K 0.3 mJ / cm ² (K transmittance of the filter; K≅1).

 The sensitivity of the method, estimated from a fixed power, is 7 orders of magnitude higher than the amount of energy absorbed by photoexcitation.

 Due to this, it seems possible to fix the degree of infection with fusarium at the level of 1% (Fig. 5).

While in the method according to the prototype the measurement error is at least 10%. The duration of determining the degree of damage to the grain by the proposed method with a known arrangement of frequencies W _i , W _j , etc. for the corresponding control samples with a known amount of healthy grain, carried out once for the subsequent evaluation of samples with an unknown degree of infection by microscopic samples, does not exceed 5 minutes, which is no less than an order of magnitude less in duration than in the prototype.

 The boundaries of the irradiation wavelengths of 360 500 nm are due to the fact that at wavelengths of the irradiated emission spectrum below 360 nm, a luminescence band of less than 500 nm appears (mainly at 420 430 nm), in which the presence of fusarium relatively weakly affects the change in the intensity of the grain spectrum. At a wavelength of the irradiated spectrum above 500 nm, a luminescence band above 700 nm appears, at which the presence of fusarium also relatively weakly affects the change in the intensity of the grain spectrum.

Based on the foregoing, a new achievable technical result of the invention is:
1. Improving the accuracy of determining the degree of damage to grain by at least an order of magnitude.

 2. Reducing the duration of determining the degree of damage to the grain by at least an order of magnitude.

 3. Simplification of the technology for determining the degree of grain damage due to the rejection of the use of bulky grinding equipment.

 Currently, the enterprise GP "NPO Astrophysics" tested the inventive method for determining the defeat of grain by microscopic fungi and issued technological instructions for implementing the method.

Claims (4)

 1. A method for determining grain damage by microscopic fungi, including the isolation of a test grain sample with microscopic fungi and a control sample with a known content of healthy grain, preparation of samples for research, irradiation, registration of the physical parameter of the samples and judgment on the degree of grain damage, characterized in that in preparation of samples for research, grinding the grain of the test and control lots and tabletting of the flour, irradiation of the tablets is carried out by optical radiation with a wavelength of 360 500 nm, and the registration of the physical parameter of the samples is carried out by measuring the bioelectric potentials of the tablets, and the degree of damage to the grain is judged by comparing the measured values.  2. The method according to claim 1, characterized in that the measurement of the difference of the bioelectric potentials of the tablets is carried out when applying to two additional optionally mounted on the tablet extreme relative to two removable DC electrodes from an external energy source.  3. The method according to PP.1 and 2, characterized in that before irradiation, the tablet is placed at a temperature of liquid nitrogen and shielded from the influence of electromagnetic fields. 4. The method according to claims 1 to 3, characterized in that after measuring the difference in the bioelectric potentials of the tablets, they are amplified, converted into the amplitude-frequency characteristic of the density of the spectral power of the biocurrent, high-frequency components of the amplitude-frequency characteristic are filtered out over 2000 Hz, the latter is laid out in the range 0 - 2000 Hz in the field of flicker noise, determine the dependence of the parameters of the amplitude-frequency characteristic on time, decipher it by constructing a spectrogram of the flicker noise described the following expression:

where S (W) is the frequency response in the form of the spectral power density of the tablet biocurrent in the flicker noise region;
W frequency;

the rate of change in mass-energy of the spatiotemporal dissipative structures of the tablet biocurrent with a change in frequency within the interval ΔW;
ΔW frequency interval showing the stability region of spatio-temporal dissipative tablet structures forming subspaces of the biocurrent structure of a certain degree of isotropy and determined from the following expression ΔW = W $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ - W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ ,
where w $\genfrac{}{}{0ex}{}{\text{n}}{\text{cr}}$ and W $\genfrac{}{}{0ex}{}{\text{n-1}}{\text{cr}}$ - critical frequencies determined by the intersection of consecutive parallel lines formed by the combination of the maximum number of points, including the minimum values of points S (W _i ) _{m i n} belonging to each of the intervals ΔW ^{n- (n-1)} , ΔW ^{(n-1) - ( n-2)} , ..., and construct from the spectrogram the speed of the relative change in mass-energy of spatio-temporal dissipative structures from time to time

and the degree of damage to the grain is judged by the degree of displacement of at least two frequencies W _i and W _j at the longest time intervals ΔW, at which the relative change in the corresponding mass-energy velocity of spatio-temporal dissipative structures from time to time

at frequencies W _i and W _j occurs in antiphase relative to similar frequencies

for a control tablet with a known healthy grain content.

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