RU98190U1 - DEVICE FOR MONITORING THE BIOTECHNOLOGICAL PROCESS OF OBTAINING ACETIC ACID BY MEASURING THE CONTENT OF ETHYL ALCOHOL IN THE FERMENTATION MEDIUM CONTAINING ACETIC ACID - Google Patents

Abstract

The utility model relates to the field of biotechnology and the food industry, namely, to a biosensor analytical device that can be used to determine the content of ethyl alcohol in the process of fermentation of obtaining acetic acid. A device for determining ethyl alcohol in the presence of acetic acid contains a measuring cell with a magnetic stirrer and a biosensor for determining ethanol, including a Clark electrode, on which the bioreceptor is placed in the form of cells of the bacterial strain Gluconobacter oxydans BKM B-1280 immobilized on a carrier.

Description

The utility model relates to the field of food biotechnology, namely, the fermentation process for the production of acetic acid. The utility model also relates to the field of biosensor development. In this utility model, a biosensor is used to determine the ethanol content in the presence of acetic acid during the microbiological production of vinegar from food raw materials.

The production of vinegar by an industrial microbiological method is based on the oxidation of ethyl alcohol by acetic acid bacteria. In the production of acetic acid for optimal process performance it is necessary to have information on the content of the amount of produced acetic acid; this information can be obtained by measuring the content of ethyl alcohol. At the initial stage of the process in the fermenter is its maximum amount, which decreases in the process of increasing the content of acetic acid. The decrease in the concentration of ethyl alcohol to a certain level indicates the completion of the process (Becker ME Introduction to biotechnology. M .: Food industry, 1978. S.143-145). This gives reason to judge the phase of the process of producing acetic acid by the content of ethyl alcohol.

To control the fermentation process of obtaining acetic acid, parameters such as the concentration of acetic acid and ethyl alcohol are used (Hekmat D., Vortmeyer D. Measurement, control, and modeling of submerged acetic acid fermentation // Journal of fermentation and bioengineering. 1992. V. 73. No. 1. P. 26-30).

To determine aliphatic alcohols, gas methods are mainly used (Clarkson SP, Onnrod IHL, Sharpe FR Determination of ethanol in beer by direct injection gas chromatography. A comparison of 6 identical systems // J. Inst. Brew. 1995. V. 101. P 191-193) and high performance liquid chromatography (Liden H., Vijayakumar AR, Gorton L., Marko Varga G. Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection // J. Pharm. Biomed. Anal. 1998 . V. 17. P. 1111-1128); spectrophotometry methods are also used (Wilson Scoggins M. Determination of trace quantities of alcohols by ultraviolet spectrophotometry of alkyl nitrobenzoates // Anal. Chem. 1964. V. 36. No. 6. P. 1152 - 1154), refractometry (Williams S. Official Methods of Analysis of the Association of Official Analytical Chemists, 15th ed. Inc. Washington DC: Association of Official Analytical Chemists. 1990. P. 702). However, despite the low detection limits of alcohols, the common disadvantages of these methods are the complexity of the equipment used, in some cases, sample preparation, as well as the duration of the analysis. The results of refractometric determination are influenced by the color and heterogeneity of the solution (Wagner K., Bilitewski U., Schmid R. D. Flow injection analysis of wine - accomplishments and needs // Microchemical Journal. 1992. V. 45. P. 114-120).

Acetic acid content was determined by gas chromatography (Brett J. Savary, Alberto Nuñez. Gas chromatography-mass spectrometry method for determining the methanol and acetic acid contents of pectin using headspace solid-phase microextraction and stable isotope dilution. Journal of Chromatography A. 2003. V. 1017. No. 1-2. P. 151-159). This method is quite lengthy and difficult to monitor the fermentation process in real time.

There are various biosensor approaches for the detection of ethyl alcohol and acetic acid, based on the use of enzymes or microorganism cells. One of the principles of analysis is based on the detection of oxygen, which is a cosubstrate of the oxidation reaction of ethanol, acetic acid. The rate of oxygen absorption by the receptor element of the biosensor is proportional to the concentration of acetic acid (Hikuma M., Kubo T., Yasuda T., Karube I., Suzuki S. Amperometric determination of acetic acid with immobilized Trichosporon brassicae // Analytica Chimica Acta. 1979. V. 109. . P. 33-38) or ethanol (Hikuma M. Microbial electrode sensor for alcohols // Biotechnol. Bioeng. 1979. V. 21. No. 10. P. 1845-1853).

The following are analogues of the proposed microbial sensor for monitoring the concentration of ethyl alcohol. So, biosensors based on cells of microorganisms and an oxygen electrode for the detection of ethyl alcohol are described. One of the first commercially available ethanol sensors included immobilized Trichosporon brassicae cells and an oxygen electrode (Hikuma M. Microbial electrode sensor for alcohols // Biotechnol. Bioeng. 1979. V. 21. No. 10. P. 1845-1853). The response time of the sensor was 6 minutes. A linear relationship between the decrease in current and the concentration of ethanol was observed in the concentration range from 2 to 22.5 mg / L. The difference current signal was reproducible with a relative error of 6%. The stability of the sensor was 3 weeks. The sensor was coated with a gas-permeable membrane and was not sensitive to volatile compounds such as methanol, formic, acetic and propionic acids, as well as to non-volatile substances (carbohydrates and amino acids).

Two types of electrochemical biosensors have been developed for the detection of alcohols based on mutant cells of Hansenula polymorpha methylotrophic yeast (Gonchar MV, Maidan MM, Moroz OM, Woodward JR Sibirny AA Microbial O ₂ - and H ₂ O ₂ -electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements // Biosens. Bioelectron 1998. V. 13. P. 945-952.). Cells were immobilized in a calcium alginate gel and placed on the surface of an O ₂ or H ₂ O ₂ electrode. The biosensor based on the O ₂ electrode contained mutant cells with increased alcohol oxidase activity. The biosensor using an H ₂ O ₂ electrode as a transducer contained catalase deficient mutant cells that produced H ₂ O ₂ during the oxidation of alcohol. Both strains were treated with digitonin, which allowed to significantly reduce sensitivity to glucose and glycerin and increase the selectivity of biosensors. The linear ranges for the determination of ethanol and methanol for a biosensor based on an oxygen electrode were 0.2–1.2 and 0.4–4.0 mM, respectively. A biosensor based on the detection of hydrogen peroxide made it possible to determine ethanol and methanol in the ranges 0.03–0.35 mM and 0.05–1.2 mM, respectively.

An amperometric biosensor based on Candida tropicalis cells containing AOs is presented in (Akyilmaz E., Dinçkaуa E. A new enzyme electrode based on ascorbate oxidase immobilized in gelatin for specific determination of L-ascorbic acid // Talanta. 1999. V. 50 P. 87-93). Cells were immobilized in gelatin with the addition of glutaraldehyde. Ethanol detection was based on recording the respiratory activity of cells using an oxygen sensor. The response of the microbial sensor linearly depended on the ethanol concentration in the range 0.5–7.5 mM; the response time was 2 min.

An amperometric biosensor based on Gluconobacter suboxydans cells developed by Karube et al. (Kitagawa Y., Ameyama M., Nakashima K., Tamiya E. and Karube I. Amperometric Alcohol Sensor Based on an Immobilised Bacteria Cell Membrane // Analyst. 1987. V. 112. P. 1747). The detection range of ethyl alcohol was 5-25 mg / L. The sensor was also sensitive to propanol-1 and butanol-1 and insensitive to glucose. The stability of the sensor was 15 days with a drop in activity over the specified time by 70%.

In contrast to the above prototype, the proposed model uses the Gluconobacter oxydans BKM B-1280 strain belonging to the All-Russian Collection of Microorganisms (IBPM RAS). Another difference is the use of sodium phosphate buffer solution (acetate buffer is used in the prototype model). The strain used to create the utility model is insensitive to high concentrations of acetic acid (400 mM (2.4%) in the measured sample, which is diluted to 20 mM (0.12%) in the measured cuvette; in this case, the change in pH of the measured medium is ~ 1 pH). Strain G. oxydans BKM B-1280 has the ability to oxidize a number of sugars, including glucose. The prototype does not present such characteristics as the sensitivity of the biosensor in relation to the detection of ethanol when changing the pH of the medium caused by acetic acid. With respect to this biosensor, it can be noted that its sensitivity to ethanol is 2.2 (nA / s) / mM, and the lower detection limit is 12.5 μM. Both of these parameters are not statistically significantly changed when measured in a medium containing the indicated concentration of acetic acid.

The problem to which the claimed utility model is directed is to create a device for determining the content of ethyl alcohol in the presence of a high concentration of acetic acid.

The technical result that can be obtained using the proposed utility model is that the proposed biosensor is resistant to samples containing acetic acid and high sensitivity to ethyl alcohol.

The essence of the utility model is that the biosensor for determining ethyl alcohol includes a Clark electrode coupled to a bioreceptor containing cells of the bacterial strain G. oxydans BKM B-1280 immobilized on a carrier. The strain is sensitive to the presence of ethyl alcohol and insensitive to acetic acid (Lusta K., Reshetilov A.N. Physiological and biochemical features of Gluconobacter oxydans and prospects for use in biotechnology and biosensor systems // Prikl. Biochemistry and microbiology. 1998. V. 34. P . 339-353.)

Figure 1 presents a diagram of a device for determining ethyl alcohol in the presence of acetic acid. The proposed device includes the following elements: a biosensor, consisting of a transducer - Clark electrode (1), on which a bioreceptor (2) is placed, which is a cell of the bacteria strain G. oxydans VKM B-1280 immobilized on a carrier, as well as a measuring cell (3) and magnetic stirrer (4).

The main analytical parameter of the biosensor is the calibration dependence. For its construction, various concentrations of ethyl alcohol were added to the measuring cell. Figure 2 presents the calibration dependence of the sensor based on the cells of the strain G. oxydans VKM B-1280 The range of determined concentrations of ethyl alcohol was 0.0125-2.00 mm. Sensor responses were recorded in 30 mM sodium phosphate buffer solution at pH 6.6. The sensitivity in the linear range was 2.2 (nA / s) / mM.

For ethanol (the concentration in the sample was 0.025%), the pH dependences for various buffer solutions were studied (Fig. 3). When using sodium phosphate buffer, the bioreceptor is stable in the pH range of 5-7 when analyzing ethyl alcohol samples (curve 2a). When analyzing samples containing ethyl alcohol (0.025%) and acetic acid (2.4%) (curve 2b), the sensor responses were stable in the pH range of 6–6.6.

The range of investigated pH values for citrate buffer solution (according to McIlvane) was 3.2-7. As can be seen from figure 3, the bioreceptor without loss of activity allowed measurements in the pH range from 3.2 to 6.4. At pH 7, the measurement error exceeded 10%.

When using a buffer solution containing Tris-maleate, the range of the studied pH values was 5-6.1. The optimal pH for this buffer solution is pH 5, with the remaining pH being determined, a noticeable decrease in sensor responses was observed.

The type of buffer solution affected the absolute value of the sensor response. So, when using a citrate buffer solution, the highest sensor response value was obtained. However, when studying the stability of the sensor in nitrate buffer for 6 h, a decrease in sensor responses was observed. Thus, sodium phosphate buffer solution was chosen as the optimal base solution.

In the production of acetic acid for optimal process performance, it is necessary to have information on the content of ethyl alcohol, since its decrease to a certain final level (0.1%) indicates the completion of the process.

An analysis of samples simulating the production of acetic acid at various stages of the process was carried out. The dependence of the sensor responses on the ethyl alcohol content in the acetic acid sample is presented in Table 1. When analyzing the sample containing 9% acetic acid (concentration in the measuring cell 0.11%) and 0.1% ethyl alcohol (0.00125% in the cell), the pH of the buffer solution decreased by one . For the analysis of subsequent samples, the dilution was 800 or more times, which practically had no effect on the pH of the buffer solution.

Table. one. The dependence of sensor responses on the concentration of ethyl alcohol in the model sample. Try sample pH Buffer Dilution pH of samples in a measuring cell The initial pH value of the buffer Sensor Response, nA / s 0.1% ethanol in 9% acetic acid 2.4 80 times 5.5 6.6 0.201 1% ethanol in 9% acetic acid 2.4 800 times 6.6 0.206 5% ethanol in 5% acetic acid 2.5 4000 times 6.6 0.200 10% ethanol 5.0 8,000 times 6.6 0.217

Thus, to evaluate ethanol in the analyzed samples, they must be diluted 80 (final dilution in the cuvette) and more than once. When using such a dilution, the pH of the base buffer solution changes slightly and does not lead to a change in the response value of the biosensor.

The residual ethanol content of samples of food vinegar obtained by the microbiological method was evaluated (Table 2). The correlation coefficient of the data obtained using microbial and enzyme-based alcohol oxidase sensors was 0.98.

Table 2. The residual ethyl alcohol content in apple grape and grape vinegar. Sample Ethanol, mm G. oxydans Alcohol Oxidase Apple Cider Vinegar (Yegorye) 12.0 ± 0.7 11.9 ± 1.1 Apple Cider Vinegar ("Apricot") 10.9 ± 1.0 11.7 ± 0.8 Grape Vinegar (Baltimore) 16.6 ± 1.2 14.9 ± 1.1

The data obtained show the possibility of monitoring the process of acetic acid by both microbial and enzymatic biosensors. An enzymatic biosensor with a higher substrate specificity than a microbial one can be used as a control method for assessing the total alcohol content.

Thus, the possibility of analyzing the ethyl alcohol content in samples with a high content of acetic acid by a microbial sensor based on Gluconobacter oxydans BKM B-1280 was shown. The working pH range of the buffer solution was investigated. A high degree of dilution of the samples does not significantly affect the pH of the buffer.

Claims (1)

A device for determining the content of ethyl alcohol against the background of high concentrations of acetic acid, containing a measuring cell with a magnetic stirrer and a biosensor for determining ethanol, including a Clark electrode, on which the bioreceptor is placed in the form of Gluconobacter oxydans bacteria cells immobilized on a carrier, characterized in that the biosensor contains cells of the bacterial strain Gluconobacter oxydans BKM B-1280.