DD220142A1 - PROCESS FOR DETERMINING NATURAL MATERIALS BY MEANS OF SELECTIVE MICROBIOLOGICAL ELECTRODES - Google Patents

Abstract

The invention relates to a method for the specific determination of natural substances by means of microbiological electrodes. Its aim is to obtain a specificity and speed of determination comparable to those of the enzyme electrodes by using microbiological electrodes. According to the invention, the microorganisms used for the determination of a natural substance in microbiological electrodes are attracted to nutrient solutions with this particular substance. After dosing the substrate to be determined in the measuring cell, the rate of change of the breathing is used as a measure of its concentration. Current current change is measured per time. The method is suitable for use in microbiology, agriculture, food industry and analysis, pharmacy and toxicology, and environmental protection.

Description

Riedel.

Renneberg

.Wise

Scheller,

* f

"Method for the determination of natural products by means of selective microbiological electrodes".

Field of application of the invention

The invention relates to a method for the specific determination of natural products (carbohydrates, organic acids, alcohols, amino acids, etc.) in complex solutions with microbiological sensors containing specific microorganisms.

The method is suitable fpr use in microbiology, agriculture, Lebensraittelindustrie and -Ana-. lytics, pharmacy, toxicology and environmental protection.

Characteristic of the known technical solutions For the determination of natural substrates, such as carbohydrates, alcohols, organic acids and amino acids are in addition to the

Electrochemical sensors in the form of enzyme and microorganism electrodes are increasingly used in conventional spectrometric and chromatographic processes (GUILBAULT, GG, Enzymes raicrob.Techn., 2, 258-264 (1980), SUZUKI, S. and KARUBE, I., Proc. Int. Ferm. Symp., London (Canada), Vol. III, ρ 355-360 (1980), FUKUI and TANAKA, Ann. Rev. Microbiol, 36, 145-172 (1982)). The use of enzyme electrodes is essentially limited to those reactions which lead to the formation or consumption of 0 "and HJDp in conjunction with the polarographic detection. Furthermore, the availability of enzymes, which in some cases requires very extensive preparation and low

ge stability of their use. The cofactor.negenerienjng also presents great difficulties ·, microbiological electrodes can be used wherever O _{2 is} consumed by substrate use. A complex enzyme preparation and a cofactor regeneration eliminated, multi-enzyme reactions can be easily carried out by microorganisms. These advantages of the microbiological electrodes are, however, partially reversed by the disadvantage of the unspecific effect, which is why specific detection of certain substances in complex solutions, eg fermentation solutions, is only possible with microbiological electrodes.

Object of the invention

The invention has the object, o. Disadvantage of the microbiological electrode to eliminate and to obtain the enzyme electrodes comparable specificity and speed of measurement. ,

Explanation of the essence of the invention

The object of the invention is to determine specific natural substances in natural systems and complex solutions by means of microbiological electrodes. According to the invention, the microorganisms used in the microorganism electrodes are cultured by each of the microorganisms used for the analysis of a particular natural product (carbohydrates, organic acids, alcohols, amino acids, etc.) are grown in a Nährraedium, which preferably contains this natural product as C source. This cultivation induces the microorganism uptake system required for the corresponding natural product and does not or only weakly forms other transport systems (whose substrates are not present). The use of the respective specific transport systems of the microorganisms for the substrate analysis

"*" J "

not only allows a high specificity, but also a very rapid measurement by detecting the change in respiratory rate as a result of substrate uptake The microorganisms grown in the above-mentioned manner are immobilized or used for the production of microbiological electrodes After metering the substance to be determined into the measuring cell, the rate of change of the respiration is used as a measure of the substrate concentration.

, This change is measured as current per unit time. The advantage of the method is that natural substances with which the microorganisms used have been cultivated can be determined without great expenditure of time. Another advantage is the possibility of simultaneous substrate determination. Several selective microbiological electrodes are used.

Exemplary embodiments

The invention will be explained in more detail below with reference to exemplary embodiments.

1st example .

Determination of glucose by means attracted by the glucose mediura

Bacillus subtilis cells

In a glucose-containing medium (20 g glucose, 5 g Casamino acids, 5 g Aramoniurachlorid, 20 ml stock solution salts ad 1 1 0.134 M phosphate buffer pH 6.0; stock solution salts: 0.4 % FeCl ₃ .6H ₂ O, 0, 13 % CaCl ₂ , 0.2 % ZnSO ₄ .7H ₂ O ,, 2.46 % MgSO ₄ .7H ₂ O, 3.7 % HCl), B. subtilis cells are grown after 30 to 20 hours of culture on rotary shakers ⁰ C at the beginning of the stationary phase and used to prepare a microbiological electrode according to the manner described in DD-WP G 01 N / 238 412/8. For this purpose, 100 .mu.l of culture solution are centrifuged on a filter plate of 2-5 ram diameter, so that a Zellkon-

ct concentration of about 0.2 mg dry weight per filter achieved. The filter is placed on a polyethylene membrane of an oxygen electrode and closed by a dialysis membrane to the solution side. In a stirred at 30 ⁰ C tempered measuring cell 2.5 ml of 0.1 mol Phosphatpqffer pH 6.8 is introduced. The oxygen concentration is measured in amps by means of a measuring amplifier at an applied voltage of -600 mV or the change in the current intensity after metering of the substrate in A / min, this rate of change is used as a measure of the substrate concentration. After dosing 100 μUl glucose containing solution, the oxygen concentration (measured as current) drops rapidly due to increased respiration. This change in current per unit time (A / min) is proportional to the glucose concentration (Table 1). The response time of the electrode is 2 sec. If maltose solutions are metered, only an extremely small change in the current intensity, ie the oxygen consumption, occurs (Table I). After a substrate determination, the measuring cell is rinsed twice with phosphate buffer.

Table 1 /

Determination of glucose by means of glucose-selective microorganism electrode (B. subtilis) in the glucose medium.

As a check, the rate of change of the current intensity per min. indicated after Maltosedosierung

Substrate Conc. / RaM / 'Amperage change per time ^: ''·; ·. >,.

min

0 0 glucose 0.15 0.14 0.6 0.64 1.2 ⁵ 0.39 ' - 0 0 maltose 0.15 0.02 0.6 0.03 · 1.2 0.04

Concentration in the measuring approach, d. H. at dosage of 100 yul solution this contains 25 times.

2nd example

Maltose determination by means of maltose, B. subtilis

Cells _______

The B.subtilis cells used to prepare a root-selective microorganism electrode are grown in the medium described in Example 1, which contains 20 g of maltose instead of glucose. Cultivation, production of the electrodes and type of measurement correspond to the description in Example 1.

Table 2 demonstrates the dependence of the change in current per minute on maltose and glucose concentration. A comparison with the glucose-attracted cells in Table 1 illustrates the selective effect of culture.

Table 2

Maltosebestitnmung center, ls raaltoseselective microorganism electrode (B. subtilis).

To control the rate of change of the current per min. indicated after glucose dosage

Substrate Conc. / Ϊ́πΜ / 'Change in current intensity per time

Γ ι ι Λ 1

conjures

Maltose O O

0.15 0.25

0.6 '"- O', 81 1.2 , 0.76

Glucose O 'O.

0.15. 0.09

0.6 0.21

, 1,2 \ 0.22

'See Note Table 1

3 »Example . .

Simultaneous determination of maltose and glucose in solutions

by means of selective microorganism electrodes  - * /

A simultaneous determination of glucose and maltose in natural solutions is achieved by the simultaneous use of maltose- and glucose-selective microorganism electrodes in a measuring cell. The selective electrodes are prepared as described in Examples 1 and 2, and introduced into a double measuring cell. Glucose liquor solutions calibrate the electrodes and compensate for the different sensitivities of the electrodes (using different sensitivity settings on the sense amplifiers), i. H. the sensitivity of the glucose-selective electrode is adjusted to the sensitivity of the maltose-selective electrode, since the glucose electrode, as can be seen from the comparison example: 1 and 2, is more sensitive to glucose.

The glucose-selective electrode directly indicates the glucose concentration. The difference in the current intensity of the maltose- and glucose-selective electrode corresponds to the maltose quantity (Table 3) v '. , , '

Table 3 ·

Simultaneous determination of glucose and maltose with the help of selective microorganism electrodes

Test solutions / mmol /

Change in the current intensity per unit time / / ^u / concentrations

/ Ht-lox?

troms

£ Ϊ

Glucose, maltose glucose-maltose difference

selective- maltoseve Elek- Elekelectr. electrode trode glucose electrode

Glucose maltose

0.15 _ 0.06 0.06 O 0.15 - 0.60 - 0.20 0.20 0 0.60 0.15 - 0.15 0.01 0.25 0.24 - 0.6. - 0.6 0.02 0.81 0.79 - 0.16 0.15 .0,15 0.06 0.31 0.25 0.15 .0,61 0.15 0.6 0.07 0.87 0.80 0.17 0.15 0.5 0.15 0.20 0.26 0.06 0.60 0.59 0.6 0.6 0.22 1.00 0.78 0.65 4. Seispiel in the Glutaminsäurebestimmung by means of B. subtilis cells, rden a glutamic acid containing Medium dressed wu

The electrode used for determination of glutamic acid was prepared as described in Example 1, whereby the B.subtilis cells were grown without carbohydrates. Although this achieves an 8-fold increase in sensitivity to glutamic acid compared to cells grown in a glucose diura, the glucose sensitivity is only reduced by 50 % (Table 5), so that a direct determination of glutamic acid with an electrode in the presence of Glucose is not possible. By using 2

Electrodes, a glucose-specific electrode (with cells that are attracted to the glucose medium) that does not respond to glutamic acid, and a so-called glutamic acid-selective electrode (cells carbohydrate-attracted), which has sensitivity to glutamic acid, but still indicates glucose, it is possible to determine by subtraction glutamic acid by means of microbiological electrodes.

Table '4 -

Glutamic acid determination by means of glutamic acid sensitive microorganism electrode,.

For comparison, the change in current intensity after dosing of glutamic acid and glucose is given to an electrode containing glucose-attracted cells.

Substrate Conc. Change in current intensity per time

L min '

ibM '·. Electrode with cells Electrode with

_: glutamic acid cells from Gluko medium semedium

Glucose 0,15 .0,13 0,25

Glutamic acid 1.2, 0.16 ; 0.02

-'Note: Table 1

5th example - ', ...

Determination of glucose by means of immobilized, glucose- attracted S. subtilis cells \ v

The cultivation is carried out as in Example 1. The cells specially cultivated on glucose are separated from the broth by centrifugation, using 100 ml. The residue is mixed with 10 ml of phosphate buffer and stirred. \

5 / μl of the adhesive solution (2.5 g of commercial adhesive, Epasol contact, mixed with 30 ml of ethyl acetate) are placed on a polyethylene film. Directly on the drying adhesive is applied 10 μl of the cell suspension. The thus prepared polyethylene film is stored under vacuum for 20 minutes in a desiccator. Then another 5 μl of the adhesive solution are added to the cells and covered with a dialysis membrane.

The next day, the carrier-fixed microorganism film is used to cover an oxygen electrode. The preparation of the electrode and the type of measurement correspond to those described in Example 1 !. Table 5 gives the current values for glucose and maltose.

table 5 1st measurement B. subtilis cells immobilized, glucose 2nd measurement Glucose train ^: • and Maltosebestimraung means 3rd measurement ; 0.15 mM AuA / min / Maltosezügabe: glucose-selective 4. Measurement    0.12 0.15 mM ", uÄ / min / ⁷ 1st measurement 0.11 .. ^l : '0 2nd measurement 0.13 o, ·:. ' 1 day 3rd measurement 0.12 0 , - 1st measurement 0.11 0 2nd measurement ; 0.12 0 3rd measurement    0.10 ° - 2 day 1st measurement 0.11   , ο · 2nd measurement ' ^: '^; 0.10 · '.- · · · o' ^! · ' 3rd measurement , 0.10 ° - '0 .. 3rd day 6th example , 0.09   : 0 · Maltosebesjcimmung 0.10 O coated , , 0.09 0 6th day 0 immobilized by means of  ". ' B. subtilis cells he, on maltose,

Cultivation is carried out as in Example 2. Specifically on '. ·. '' * '* /. ,

Cultured maltose cells are fixed in accordance with Example 5 on a polyethylene film with the adhesive solution and in the same way, using an oxygen feed, each glucose or maltose solutions measured.

Table 6

Maltose and glucose confirmation by means of 1st measurement , subtilis cells immobilized. maltoses'selective B 2nd measurement Glucose addition: \ -  3rd measurement 0.15 mM / ", uA / min / Maltosezugabe: 1st measurement 0.02 0.15 mM / μA / min / 1 day 2nd measurement 0.01 0.30 3rd measurement 0.03 0.28 1 .Measurement 0.01 0.31 2 day 2nd measurement 0.01 0.29 3rd measurement 0.02 0.30 1st measurement 0.01 0.30 3rd day 2nd measurement O ₁ oil 0.29 3rd measurement 0.01 0.28 0.01 .0,30 6th day 0.01 0.23 0.02 0.23 0.29

Claims (3)

4t invention claim 1) A method for the determination of natural substances (carbohydrates, organic acids, alcohols, amino acids, etc.) in granular mixtures or solutions with microorganism electrodes, characterized in that the microorganisms used for the analysis of a particular substance in microbiological electrodes are grown in nutrient solutions with this natural substance and measuring the rate of change of respiration after dosing the particular substance as MaS for its concentration as a change in current intensity per time. 2) Method according to item 1, characterized in that microorganisms obtained after o · g · specific cultivation are immobilized or fixed in the microbiological electrodes  ; be used. '' 3) Method according to item 1, characterized in that a plurality of selective microbiological electrodes are used for simultaneous Substratbestiramung.