JP3075605B2 - Method and apparatus for measuring sulfurous acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring sulfurous acid and its compounds. More specifically, sulfites such as sulfurous acid, sodium sulfite, sodium bisulfite, crystalline sodium sulfite, anhydrous sodium sulfite, sodium hyposulfite, anhydrous sulfite, potassium metabisulfite, and the like, and bonded sulfite combined with aldehydes, ketones, sugars, etc. And the like for the measurement of sulfite compounds.

[0002]

2. Description of the Related Art Sulfurous acid and its compounds are widely used as food additives in food processing for the purpose of bleaching, preventing oxidation and preserving foods. However, on the other hand, sulfite compounds may adversely affect the human body depending on their concentration, such as being a causative substance of allergies, and the Food Sanitation Law regulates the use standards for each food. Therefore, when using sulfurous acid and its salts,
It is necessary to pay close attention to the amount added and the content. Current,
Methods for measuring sulfurous acid and its compounds include the Monia-Williams method, distillation / iodine titration, and distillation / colorimetry.
(The above, Food Sanitation Inspection Guideline I, 1973, pp. 404-408, published by the Japan Food Sanitation Association) or the Rankin method (BCRankin
e; Aust. Wine Brewing Spirit Rav., 80, 14 (1962)), etc., but all of these methods require distillation operation, require skill and require a large amount of sample. The operation is complicated and the analysis time is long. Also,
In this distillation operation, there is a problem that the recovery rate of the sulfurous acid is reduced due to the volatilization of the sulfurous acid outside the reaction system or the residual in the reactor. Also, a method using sulphite oxidase derived from chicken liver (HOBeutler; Food Chem., 15, 157 (198
4)) has also been put to practical use, but it has problems in hindering the determination of sulfite due to the coexistence of dye and ascorbic acid, and in the stability of the enzyme itself. In addition, ion chromatography (HJKim, YKKi
m; J. Food Sci., 51 , 1360 (1986)) Gas chromatographic method (Hamano et al .; Food and Essays. 19 , 56 (1978)) Polarographic method
(W. Holak, J. Specchio; J. Assoc. Off.Anal.Chem., 72 , 4
76 (1989)). However, there is a problem that the interference of the coexisting substances cannot be completely eliminated and the apparatus is expensive. As a method using a microorganism, a method for measuring sulfite using a strain belonging to the genus Thiobacillus has been clarified by the present inventors (K. Nakamura, Y. Amano, O. Nakayama;
Appl. Microbiol. Biotechnol., 31 , 351 (1989)). This method has high specificity for sulfurous acid and excellent measurement accuracy,
Although the operation is simple and the specification of bacterial enzymes is good, it shows practically excellent properties, but only free sulfite can be measured, and the amount of total sulfite required for food analysis (binding with the amount of free sulfite) (The total amount of type sulfite) cannot be measured.

[0003]

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems in the method for measuring sulfurous acid.
It is another object of the present invention to provide a quantification method having high quantitation accuracy and excellent stability and an apparatus therefor.

[0004]

Means for Solving the Problems A first measuring method of the present invention utilizes a reaction of oxidizing free sulfurous acid to sulfuric acid using a microorganism belonging to Thiobacillus thiooxydans or Thiobacillus ferrooxidans. In the method for measuring the sulfite content, the total sulfite in the sample is subjected to an acidic treatment and / or an alkaline treatment without taking out the bound sulfite out of the system, so that all the sulfite in the sample is converted into free sulfite and the total This is a method for measuring sulfurous acid, which comprises measuring the sulfurous acid content.

[0005] A second measuring method of the present invention is a method for measuring the sulfite content by utilizing the reaction of oxidizing free sulfurous acid to sulfuric acid using a microorganism belonging to Thiobacillus thiooxydans or Thiobacillus ferrooxidans. In the method, the content of free sulfite in the sample was measured without subjecting the bound sulfite in the sample to acidic and / or alkaline treatment, and the measured value was determined by the first measuring method. This is a method for measuring bound sulfurous acid, characterized in that a value obtained by subtracting from the content is defined as bound sulfurous acid content.

In the first and second measurement methods of the present invention, preferably, the amount of oxygen consumed by the microorganism in the process of oxidizing free sulfurous acid to sulfuric acid is detected, or the microorganism is converted to free sulfurous acid by sulfuric acid. Generated by oxidation to p
The change in H or the amount of hydrogen ions released in the process of oxidizing free sulfurous acid to sulfuric acid by the microorganism is detected. Next, the present invention will be described in more detail.

The microorganism used in the present invention is preferably one of such microorganisms, for example, Thiobacillus thiooxidans, for example, Thiobacillus thiooxidans IFO13724, Thiobacillus thiooxydans JCM3866, JCM3867, JCM3868, Thiobacillus. , Thiooxydans ATCC19703, ATCC21835,
Thiobacillus thiooxydance 11773 (FERM BP-311
9), Thiobacillus thiooxydans 20294 (FERM BP
-3467) or Thiobacillus ferrooxidans (Thioba
cillus ferrooxidans) For example, Thiobacillus ferrooxidans JCM3863, Thiobacillus ferrooxidans IFO 14245, IFO 14246, IFO 14262, Thiobacillus ferrooxidans ATCC 13598, ATCC 13661, A
TCC 14119, ATCC 23270, ATCC 33020 and the like.

As a result of extensively searching for strains having a high growth rate and high sulfite oxidation ability, the present inventors isolated one strain belonging to the genus Thiobacillus from the soil of the former Matsuo mine. This strain had practical properties such as a high growth rate, excellent sulfite oxidation ability, and extremely excellent stability. The bacteriological properties of this strain are described below. A. Morphological characteristics (1) Cell shape and size: Bacillus, 0.4 to 0.8 in size
μm × 0.9-2.2μm (2) Cell polyplasia: No (3) Motility: Yes, monopolar flagella (4) Gram staining: Negative (5) Acid-fast: No B. Cultural properties (1) Meat juice Does not grow on agar, broth liquid, broth gelatin stab, litmus milk culture. (2) Thiobacillus medium (ATCC Catalog of BACTERIA
& BACTERIOPHAGES 17thedition, 1989) and Thiosul
phate agar No. 2 medium (ed.
Grows at the University of Tokyo Press (1977). It grows slowly and forms clear, pale yellow microcolonies. C. Physiological properties (1) Reduction of nitrate:-(2) Denitrification reaction:-(3) MR test:-(4) VP test:-(5) Preparation of indole:-(6) Preparation of hydrogen sulfide :-(7) Hydrolysis of starch:-(8) Utilization of citric acid:-(9) Utilization of inorganic nitrogen source: + (10) Formation of pigment: ± (11) Urease:-(12) Oxidase: + (13) Catalase:-(14) Range of growth: Growing in the pH range of 1 to 6. The growth temperature is 10 ℃ ~ 37 ℃. (15) Attitude to oxygen: absolute aerobic (16) OF test: oxidizable D. Generation of acids and gases from the following saccharides E. Other physiological properties (1) Oxidation of sulfur: + (2) Oxidation of iron ions:-(3) Oxidation of thiosulfate: + (4) Inorganic nutrition: Absolute chemoinorganic nutrition (5) Sulfate Production: + F. Chemotaxonomic properties (1) GC content of DNA: 51 mol% (2) Ubiquinone type: Ubiquinone Q-8 (3) Cell fatty acid composition: Contains 3-hydroxytetradecanoic acid.

Based on the above mycological properties, the taxonomic status of the strain was determined by the Bergey's Manual of Systematic Bacteriology, Vol. 3 (1989) (Bergey's
s Manual of Systematic Bacteriology Volume 3 (198
9)), the strain was identified as a new strain belonging to Thiobacillus thiooxydans and named as Thiobacillus thiooxydans 11773. This strain has been internationally deposited as FERM BP-3119 with the Research Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.

Furthermore, the present inventors isolated one strain belonging to the genus Thiobacillus from the soil of Noji Onsen. This strain had practical properties such as a high growth rate, excellent sulfite oxidation ability, and extremely excellent stability. The bacteriological properties of this strain are described below. A. Morphological features (1) Cell shape and size: 0.5 to 0.7 bacilli
μm × 1.0-2.2μm (2) Cell polyplasia: No (3) Motility: Yes, monopolar flagella (4) Gram staining: Negative (5) Acid-fast: No B. Cultural properties (1) Meat juice Does not grow on agar, broth liquid, broth gelatin stab, litmus milk culture. (2) Thiobacillus medium (ATCC Catalog of BACTERIA
& BACTERIOPHAGES 17thedition, 1989) and Thiosul
phate agar No. 2 medium (ed.
Grows at the University of Tokyo Press (1977). It grows slowly and forms clear, pale yellow microcolonies. C. Physiological properties (1) Reduction of nitrate:-(2) Denitrification reaction:-(3) MR test:-(4) VP test:-(5) Preparation of indole:-(6) Preparation of hydrogen sulfide :-(7) Hydrolysis of starch:-(8) Utilization of citric acid:-(9) Utilization of inorganic nitrogen source: + (10) Formation of pigment: ± (11) Urease:-(12) Oxidase: + (13) Catalase:-(14) Range of growth: Growing in the range of pH 1.0 to 5.5. The growth temperature is 10 ℃ ~ 37 ℃. (15) Attitude to oxygen: absolute aerobic (16) OF test: oxidizable D. Generation of acids and gases from the following saccharides E. Other physiological properties (1) Oxidation of sulfur: + (2) Oxidation of iron ions:-(3) Oxidation of thiosulfate: + (4) Inorganic nutrition: Absolute chemoinorganic nutrition (5) Sulfate Production: + F. Chemotaxonomic properties (1) GC content of DNA: 51 mol% (2) Ubiquinone type: Ubiquinone Q-8 (3) Cell fatty acid composition: Contains 3-hydroxytetradecanoic acid.

Based on the above mycological properties, the taxonomic status of the strain was determined by the Bergey's Manual of Systematic Bacteriology, Vol. 3, (1989) (Bergey's Manual).
s Manual of Systematic Bacteriology Volume 3 (198
9)), the strain was identified as a new strain belonging to Thiobacillus thiooxydans and named as Thiobacillus thiooxydans 20294. This strain has been deposited internationally with the Institute of Microbial Industry and Technology of the Agency of Industrial Science and Technology as FERM BP-3467.

Cultivation of the microorganism belonging to Thiobacillus thiooxydans is performed by using a sulfur-containing Silverman 9K medium (Silverman. MP et al .: J. Bacteriol., 77 , 642).
(1959)), Thiobacillus medium (ATCC Catalog of BACTE
RIA & BACTERIOPHAGES 17th edition.1989), Thiosul
phate agar No. 2 medium (ed.
The method can be carried out at the University of Tokyo Press (1977), etc., or in a medium obtained by appropriately modifying these.

[0013] The culture temperature is preferably 10 ° C to 37 ° C,
More desirably, the temperature is 25 ° C to 33 ° C. PH of culture time
Is suitably 1 to 6, and more preferably, pH 3 to 5
It is. The culture period is desirably 2 days to 14 days, more desirably 3 days to 10 days. Culture is liquid culture,
Any of solid culture may be used, but it is necessary to carry out aerobically.

After completion of the culture, when these microorganisms belonging to the genus Thiobacillus are used for measuring sulfite, the microorganisms,
The crushed material, a fractionated component from the crushed material and a crudely purified enzyme may be used. For example, a sulfur residue in a culture may be separated by filtration with a filter paper or the like, and only the cells may be collected and used, or crushed, fractionated, etc. And can be used. Desirably, microbial cells are immobilized between membranes such as an acetylcellulose membrane filter (pore size 0.45 micron or less), and the cells are not eluted and the stability of sulfite oxidizing ability is high and reproducible results are obtained. Can be

The principle of the oxidation of free sulfurous acid to sulfuric acid by Thiobacillus thiooxydans is described in the Abstracts of the Society of Wine Technology (No. 2) (Amano: 1988). Sulfuric acid is converted to sulfuric acid by the action of sulfite dehydrogenase in the presence of C (Fe ³⁺ ) and H ₂ O. On the other hand, cytochrome C (Fe ²⁺ ) and H ⁺ generated by this reaction are again converted to cytochrome C by the action of cytochrome C oxidase in the presence of oxygen.
(Fe ³⁺ ) and H ₂ O.

The above series of reactions is as shown in FIG. In the absence of iron ions, Thiobacillus ferrooxidans oxidizes sulfurous acid to sulfuric acid by the same reaction as Thiobacillus thiooxidans (Journal of the Fermentation Engineering Society, Vol. 67, No. 3, p. 173, 1989). However, according to these principles, all free sulfite in a sample can be measured, but generally no bound sulfurous acid bound with aldehydes, ketones, saccharides and the like.

However, if the sample is adjusted to pH 1-3, preferably pH
Adjust to H1.5-3, 70-110 ° C, preferably 75-105
The acid treatment is carried out for 2 minutes to 25 minutes, preferably 5 minutes to 20 minutes under the condition of ° C., or the sample is adjusted to pH 10 to 14, preferably pH 11 to 13, and the sample is adjusted to 15 ° C. to 70 ° C., preferably Is 20 ℃
2 minutes to 20 minutes, preferably 5 minutes to 15 minutes
It can be released by subjecting it to alkaline treatment for a minute. Therefore, the total amount of sulfurous acid in the sample can be measured by subjecting the sample to these treatments to convert the bound sulfurous acid into free sulfurous acid and then measuring according to the above principle. In addition, the acid treatment and / or the alkaline treatment described above is performed in the system of the apparatus without taking it out of the system, and then measured with a microorganism as free sulfurous acid while maintaining the same system, as in the conventional method. The total amount of sulfurous acid can be measured without lowering the recovery rate due to volatilization or residue. Furthermore, in the above measurement, the sample was treated under acidic conditions and / or
Alternatively, by subtracting the value obtained by measuring only free sulfite from the measured value of total sulfite without performing the treatment under alkaline conditions, the value of only bound sulfite can be accurately obtained.

In the above-described principle of oxidizing the sulfurous acid of the microorganism used in the present invention to sulfuric acid, the amount of free sulfurous acid in the sample, the amount of oxygen consumed in the oxidation and the amount of released hydrogen ions have a high correlation. Therefore, these amounts can be detected by using a commonly used oxygen electrode or pH electrode to detect the amount of change as a potential difference, or by detecting the pH change of the solution caused by the conversion of sulfurous acid to sulfuric acid by using the pH electrode. The amount of free sulfite in the sample can be assayed.

FIG. 2 shows an example of a circuit diagram of a measuring device using the acid treatment method according to the present invention. This measuring device includes a sample injector 1, a switching valve 2 for switching between a sample buffer and a sample buffer, a heating tank 5 for mixing and heating the sample and the buffer, and a sample and buffer in the heating tank 5. Pump 4, a cooling tank 6 for cooling the sample heat-treated in the heating tank 5, a reaction tank 7 equipped with a microorganism-immobilized oxygen electrode or a pH electrode, and a constant temperature Thermostat 8 to keep the temperature and current-voltage converter
10, mV meter 11, computer 12, CRT13
And the printer 14. Sample injector 1
The sample is sent to the heating tank 5 by the pump 4 into which a more constant amount of sample is introduced. The buffer 3 is sent to the heating tank 5 by operating the switching valve 2. The sample mixed and heated in the heating tank 5 enters the cooling tank 6 and is cooled. Next, the sample is sent to a reaction tank 7 maintained at a constant temperature in a thermostat 8, and a change in potential is measured by a microorganism-immobilized oxygen electrode or a pH electrode 9. The measured signal is taken into a computer 12 via a current-voltage converter 10 and an mV meter 11, and is subjected to arithmetic processing, and output to a CRT (display) 13 and a printer 14. These series of operations can be automatically controlled by programming a computer.

FIG. 3 shows an example of a circuit diagram of a measuring apparatus using the alkaline pretreatment method. The measuring apparatus includes a sample injector 15, a switching valve 16 for switching between a sample and an alkaline solution, a heating tank 19 for mixing the sample and the alkaline solution and performing an alkali treatment, and a sample and an alkali in the heating tank 19. Pump 18 for sending liquid and heating tank 19
Cooling tank 20 for cooling the sample alkali-treated with
A mixer 21 for mixing the sample and the acid buffer sent from the cooling bath 20, a pump 23 for sending an acid buffer to the mixer 21, and a microorganism-immobilized oxygen electrode or pH electrode. It comprises a mounted reaction tank 25, a constant temperature tank 24 for keeping the reaction tank 25 at a constant temperature, a current / voltage changer 27, an mV meter 28, a computer 29, a CRT 30, and a printer 31. . The sample introduced from the sample injector 15 enters the heating tank 19 by the pump 18, and then the switching valve 16 is operated to put the alkaline liquid 17 into the heating tank 19. Here, the alkali-treated sample is cooled in a cooling tank 20, and then sent to a mixer 21,
The acid buffer 22 is added thereto using a pump 23 and mixed. The mixed sample is placed in a reaction tank maintained at a constant temperature in a thermostat 24.
25, and a change in potential is measured by a microorganism-immobilized oxygen electrode or a pH electrode 26. The measured signal is a current-to-voltage converter 27, mV
The data is input to a computer 29 via a meter 28 and output to a CRT 30 and a printer 31 which have been processed. These series of operations can be automatically controlled by programming a computer.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0022]

[Example 1] (1) Culture method of microorganisms Thiobacillus thiooxidans 11773 (FERM BP-311)
9) 200m containing 50ml of sterile medium containing the following composition
The flask was inoculated into a one-liter Erlenmeyer flask, and cultured with shaking at 30 ° C. for 4 days. 2 ml of this culture was mixed with 100 ml of the same medium.
Aseptically inoculate a ml Erlenmeyer flask, 30 ° C
For 10 days with shaking. After completion of the culture, the culture solution was filtered using No. 5c filter paper made by Toyo Filter Paper to remove sulfur powder. The amount of cells in the obtained filtrate is 0.2 g dry cells / L
It was a culture solution. Medium composition Sulfur powder 1.0% K ₂ HPO ₄ 0.05% Ca (NO ₃ ) ₂・ 4H ₂ O 0.001% (NH ₄ ) ₂ SO ₄ 0.3% KCl 0.01% MgSO ₄・ 7H ₂ O 0.05% PH 6.5 to be mixed (2) Culture of microorganisms belonging to Thiobacillus thiooxydans and Thiobacillus ferrooxidans and comparison of oxygen absorption capacity A 300 ml Erlenmeyer flask containing 100 ml of the medium described in the above (1) was prepared as follows. Strain as dry cell mass
The cells were inoculated to a concentration of 10 mg / L, and cultured with shaking at 30 ° C. for 4 days. After completion of the culture, the culture solution was filtered with a No. 5c filter paper made by Toyo Filter Paper to remove sulfur powder. For the obtained cells, the cell weight was measured as a measure of the growth rate, and the oxygen absorption capacity was measured as an index of the sulfite oxidation capacity. The oxygen absorption capacity was measured using an oxygen absorption meter (manufactured by Rank Broth Co.) with sodium sulfite as a substrate. Table 1 shows the results.

[0023]

[Table 1]

(3) Method of immobilizing microorganisms The culture filtrate obtained in the above (2) was centrifuged at 11,000 rpm for 15 minutes and collected. The collected cells were suspended in a small amount of cold distilled water and centrifuged again to wash the cells twice to obtain washed cells. The washed cells were suspended in a 0.1 M sodium citrate-NaOH buffer (pH 6.0) so that the concentration of the dried cells was 5 g / L.

As shown in FIG. 4, 50 μL of this suspension was
16 mm diameter acetyl cellulose membrane filter:
A 16 mm diameter double-sided tape 33 made by cutting a circle of 8 mm in diameter from the center into a 0.45 μm pore (made of Millipore) 32 was added to the center of the doughnut-shaped material, and then added to the center of the filter. Immobilized. Further, a membrane filter having a diameter of 16 mm was overlapped on the upper surface of the double-sided tape to form a sandwich-fixed microorganism-immobilized membrane. This immobilized membrane was stable at 4 ° C. for 3 months. (4) Assembly of the apparatus and an example of measuring a standard substance at the oxygen electrode The microorganism-immobilized membrane 37 prepared in (3) was attached to the tip of an oxygen electrode (manufactured by Electrochemical Instruments) 35 as shown in FIG. Buffer tank 36 was filled with 0.1 M sodium citrate-NaOH buffer (p
H5.0) and covered with a gas permeable membrane (Fluoropore filter manufactured by Sumitomo Electric Industries) 38 to prepare a microorganism-immobilized oxygen electrode.

This microbial electrode was mounted on the measuring apparatus of the acidic treatment method shown in FIG. 2, and a standard solution of sodium sulfite adjusted to 10 ppm to 40 ppm was measured. As a result of measuring the current decrease value at the microorganism-immobilized oxygen electrode when each sample was injected, a high correlation was found between the sodium sulfite concentration and the current decrease amount as shown in FIG. (5) Measurement conditions of bound sulfurous acid Table 2 shows the results of examining the treatment conditions under acidic conditions as a glutaraldehyde sulfite-bonded product and a standard substance, and Table 3 shows the results of studying the treatment conditions under alkaline conditions as well.

[0027]

[Table 2]

[0028]

[Table 3]

From the above results, in the acid treatment, p
70 ° C. to 11 under conditions of H1 to 3, preferably pH 1.5 to 3.
The treatment may be performed at 0 ° C., desirably 75 ° C. to 105 ° C. for 2 minutes to 25 minutes, desirably 5 minutes to 20 minutes. In the treatment under alkaline conditions, the temperature may be 15 ° C. under the conditions of pH 10 to 14, desirably pH 11 to 13.
It has been found that the treatment may be performed at a temperature of up to 70 ° C, preferably 20 ° C to 50 ° C for 2 minutes to 20 minutes, preferably 5 to 15 minutes. (6) Measurement example of acidic and alkaline treatment of bound sulfurous acid The microorganism-immobilized oxygen electrode used in the above (4) was attached to the apparatus shown in FIGS. 2 and 3, and an aqueous solution of glutaraldehyde sulfite was used. It was measured as a sample. The results are shown in Tables 4 and 5. At the same time, Table 6 shows the results of the same sample measured using the Rankine method.

[0030]

[Table 4]

[0031]

[Table 5]

[0032]

[Table 6]

As described above, the present apparatus was able to measure bound sulfurous acid accurately and quickly. Also, a higher value was obtained in the recovery rate than in the Rankine method. (7) Comparison between a measurement example in food using an alkaline pretreatment device and a conventional method Using a commercially available wine as a sample, the amount of total sulfurous acid and the amount of free sulfurous acid in red wine were measured using the alkaline pretreatment device shown in FIG. Tables 7 and 8 show the results of comparison with the values measured by the Rankine method.

[0034]

[Table 7]

[0035]

[Table 8]

(8) Measurement Example Using Thiobacillus ferrooxidans [0036] Thiobacillus ferrooxidans IFO 14246 strain was cultured according to the method (1) to obtain 0.08 g of dried cells / L-culture solution. The cells were used to prepare a microorganism-immobilized membrane according to the above (3). This was attached to the tip of an oxygen electrode (manufactured by Electrochemical Instruments) 35 as shown in FIG.
Was filled with a 0.1 M sodium citrate-NaOll buffer solution (pH 5.0), and then covered with a gas-permeable membrane (Fluoropore filter manufactured by Sumitomo Electric Industries) 38 to prepare a microorganism-immobilized oxygen electrode.

This microbial electrode was attached to the measuring device of the acidic treatment method shown in FIG. 2, and a standard solution of sodium sulfite adjusted to 10 ppm to 40 ppm was measured. As a result of measuring the current decrease value at the microorganism-immobilized oxygen electrode when each sample was injected, a high correlation was found between the sodium sulfite concentration and the current decrease amount as shown in FIG. In addition, Thiobacillus ferrooxidans IFO 14246 strain had advantages such as good growth and a small amount of culture solution.

[0038]

Example 2 (a) Example of measuring a standard substance at a pH electrode in a turbidity reaction tank A pH electrode was attached to a reaction tank of a measuring device (FIGS. 2 and 3).
The cells obtained in the above (1) were suspended in 1 mM sodium citrate-NaOH buffer (pH 6.0) so as to have a concentration of 1 g / L as a dry cell mass, put in a reaction tank, and equilibrated. And sodium sulfite was injected into the mixture so as to be 0 to 100 ppm. As a result of performing the reaction at 30 ° C. for 20 minutes under each sodium sulfite concentration, a high correlation was observed between the decrease in pH before and after the reaction and the sodium sulfite concentration. FIG. 8 shows the result. (B) Example of measuring sulfurous acid in a reaction tank using a pH electrode equipped with an immobilized cell membrane A microorganism was measured using a pH electrode (Beckman, flat glass pulp 39533) instead of the oxygen electrode shown in FIG. -A pH electrode type biosensor was prepared. This biosensor was mounted on the device shown in FIG. 2 for measurement.

1) Principle of measuring sulfurous acid by pH change (Fig. 9) In the process of oxidizing sulfurous acid to sulfuric acid by Thiobacillus thiooxydans, hydrogen ions are generated in an amount proportional to the amount of oxidized sulfurous acid. By measuring the changing pH (△ pH), the concentration of sulfite in the sample can be determined. 2) Type, concentration, and measurement time of buffer solution When a 2.5 mM sodium phosphate buffer solution (including 0.1 M NaCl) was used, a large change in pH (△ pH) and a highly reproducible measured value were obtained. The longer the measurement time (ie, the reaction time), the greater the change in pH, but the change in pH per unit time gradually decreased after 10 minutes from the start of measurement.
The measurement time was set to 10 minutes because the pH change with time was linear and a sufficient pH change was obtained.
NaCl has the effect of reducing variations in measured values and increasing reproducibility. If the buffer concentration is too high, the pH change will be small, making it unsuitable for measurement. If it is 1 mM or less, the reproducibility becomes insufficient. 3) Influence of initial pH (Fig.10) Good initial pH of buffer solution when it is between 4.5 and 6.0
Showed a change. When the initial pH is lower than 4.5, 6.0
If it is higher than this, the change in pH (） pH) becomes small, making it unsuitable for measurement. In FIG. 10, the measurement condition is a measurement time of 10 minutes,
The temperature is 30 ° C. In addition, 2.5 mM sodium phosphate buffer is 0.
It contains 1M NaCl. 4) Measurement temperature (Fig.11) The measurement temperature has almost no effect on the pH change (△ pH), and good measurement is possible in the temperature range of 15 to 35 ° C. Figure
In 11, the measurement conditions are a measurement time of 10 minutes and an initial pH of 5.0. The 2.5 mM sodium phosphate buffer contains 0.1 M NaCl. 5) Amount of immobilized cells (Fig. 12) Using the same method as in the example, a microorganism-immobilized membrane was prepared in which the amount of cells to be immobilized was changed, and the resulting pH change was measured. 0.5 to 1.8 mg dry ceil per membrane
Was appropriate. In FIG. 2, the measurement condition is a measurement time of 10
Min, temperature 30 ° C, initial pH 5.0. The 2.5 mM sodium phosphate buffer contains 0.1 M NaCl. 6) Test line (Fig. 13) Based on the above results, a microorganism-immobilized membrane containing 0.6 mg of dry cells was attached to the pH electrode to obtain a microorganism / pH electrode type biosensor. A 2.5 mM sodium phosphate buffer (pH 5.0) was used for the measurement. 0-50 ppm sulfurous acid (SO ₂ )
Was introduced into the measurement cell of the biosensor, and measurement was performed at 30 ° C. for 10 minutes. When the pH change (△ pH) was stapled with respect to the concentration of sulfurous acid in the standard sample, a linear relationship was obtained as shown in the figure, and it became clear that the concentration of sulfurous acid could be measured by the biosensor.

In FIG. 13, the measurement condition is the measurement time.
10 minutes, temperature 30 ° C, initial pH 5.0, bacterial mass: 0.6 mg dry weight / microbial membrane. In addition, 2.5 mM sodium phosphate buffer is 0.1 M
It contains NaCl. As a result of the measurement of sulfurous acid in a commercially available white wine, the results showed good agreement with the values measured by the improved Rankine method. Compared to the method using an oxygen electrode, the method using a pH electrode is 1) independent of the oxygen concentration in the sample, 2) the fluctuation of measured values due to temperature changes is small, 3) inexpensive, and 4) miniaturized. It has advantages such as being possible.

[0041]

Example 3 (1) Culture method of microorganisms Thiobacillus thiooxidans 20294 (FERM BP-346)
7) 200m containing 50ml of sterile medium containing the following composition
The flask was inoculated into a one-liter Erlenmeyer flask, and cultured with shaking at 30 ° C. for 4 days. 2 ml of this culture was mixed with 100 ml of the same medium.
Aseptically inoculate a ml Erlenmeyer flask, 30 ° C
For 10 days with shaking. After completion of the culture, the culture solution was filtered using No. 5C filter paper made by Toyo Filter Paper to remove sulfur powder. The amount of cells in the obtained filtrate was 0.4 g dry cell / L culture solution. Medium composition Sulfur powder 1.0% K ₂ HPO ₄ 0.05% Ca (NO ₃ ) ₂・ 4H ₂ O 0.001% (NH ₄ ) ₂ SO ₄ 0.3% KCl 0.01% MgSO ₄・ 7H ₂ O 0.05% PH 6.5 to be mixed (2) Culture of microorganisms belonging to Thiobacillus thiooxydans and Thiobacillus ferrooxidans and comparison of oxygen absorption capacity A 300 ml Erlenmeyer flask containing 100 ml of the medium described in the above (1) was prepared as follows. Strain as dry cell mass
The cells were inoculated to a concentration of 1.0 mg / L, and cultured with shaking at 30 ° C. for 4 days. After completion of the culture, the culture solution was filtered through No. 5C filter paper made by Toyo Filter Paper to remove sulfur powder. For the obtained cells, the cell weight was measured as a measure of the growth rate, and the oxygen absorption capacity was measured as an index of the sulfite oxidation capacity. The oxygen absorption capacity was measured using an oxygen absorption meter (manufactured by Rank Broth Co.) with sodium sulfite as a substrate. The result is shown in Table 1
Shown below the stock.

[0042]

Example 4 Comparison of Storage Stability of Microorganisms belonging to Thiobacillus thiooxydans 10 mg / L of each of the strains described in Table 9 in a 300 ml Erlenmeyer flask containing 100 ml of the medium described in Example 1 as a dry cell amount. And cultured with shaking at 30 ° C. for 4 days. After completion of the culture, the culture solution was filtered through No. 5C filter paper made by Toyo Filter Paper to remove sulfur powder.

The obtained culture filtrate was centrifuged at 11,000 rpm for 15 minutes and collected. The collected cells were suspended in a small amount of cold distilled water and centrifuged again to wash the cells twice to obtain washed cells. The washed cells were suspended in a 0.1 M sodium citrate-NaOH buffer (pH 6.0) to a concentration of 5 g / L as a dry cell weight. After storing this suspension in a thermostat at 30 ° C. for one week, the enzyme absorption ability when sodium sulfite was used as a substrate was measured in the same manner as in Example 3.

Table 9 shows the results.

[0045]

[Table 9]

The Thiobacillus thiooxydans of the present invention 2
The 0294 strain maintained high activity even after storage at 30 ° C. for one week.

[0047]

The present invention provides a method for measuring the content of sulfurous acid and its compounds in a sample simply and with high precision, and has a high stability and can be used as a practical measuring method.

[Brief description of the drawings]

FIG. 1 is a diagram showing the mechanism by which microorganisms oxidize free sulfurous acid to sulfuric acid.

FIG. 2 is a diagram showing a measuring apparatus using an acid treatment method and an alkaline treatment method.

FIG. 3 is a view showing a measuring apparatus using an acid treatment method and an alkaline treatment method.

FIG. 4 is a diagram showing a microorganism-immobilized membrane.

FIG. 5 is a diagram showing an oxygen electrode equipped with a microorganism-immobilized membrane.

FIG. 6 is a graph showing the relationship between the concentration of sodium sulfite and the amount of current decrease.

FIG. 7 is a graph showing the relationship between sodium sulfite concentration and current decrease.

FIG. 8 is a graph showing the relationship between the concentration of sodium sulfite and the decrease in pH before and after the reaction.

FIG. 9 is a diagram showing a mechanism for generating hydrogen ions by sulfite oxidation of Thiobacillus thiooxydans.

FIG. 10 is a graph showing the effect of initial pH on ΔpH.

FIG. 11 is a diagram showing the effect of temperature on ΔpH.

FIG. 12 is a graph showing the influence of the amount of immobilized cells on ΔpH.

FIG. 13 is a diagram showing a calibration curve for SO ₂ quantification.

──────────────────────────────────────────────────続 き Continuing from the front page Microorganism accession number FERM BP-3467 (72) Inventor Hirofumi Akano 2-46-28, Ariwaki-cho, Handa-shi, Aichi (72) Inventor Yoshiya Kawamura 132, Kowatari, Kochino-cho, Konan-shi, Aichi-ken 58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/00-1/25 BIOSIS (DIALOG)

Claims (7)

(57) [Claims] 1. A method for measuring the sulfite content using a reaction of oxidizing free sulfurous acid to sulfuric acid using a microorganism belonging to Thiobacillus thiooxydans or Thiobacillus ferrooxidans, comprising the steps of: A method for measuring sulfurous acid, wherein all of the sulfurous acid in a sample is converted into free sulfurous acid and the total sulfurous acid content is measured by treating under acidic and / or alkaline conditions without taking out the bound sulfurous acid out of the system. . 2. A method for measuring the sulfurous acid content by utilizing a reaction of oxidizing free sulfurous acid to sulfuric acid using a microorganism belonging to Thiobacillus thiooxydans or Thiobacillus ferrooxidans, comprising the steps of: Without subjecting to acidic and / or alkaline treatment, the content of free sulfite in the sample was measured, and the value obtained by subtracting the measured value from the total sulfite content in the sample determined by the method according to claim 1 was used. A method for measuring bound sulfite, wherein the content of bound sulfite is determined. 3. A method for measuring a sulfite content using a microorganism having the ability to oxidize sulfite, which belongs to thiobacillus thiooxydans or thiobacillus ferrooxidans, according to claim 1 or 2, wherein the microorganism removes free sulfite from sulfuric acid. Detects the amount of oxygen consumed in the process of oxidizing to sulfuric acid, or changes in pH caused by microorganisms oxidizing free sulfurous acid to sulfuric acid, or released during the process of microorganisms oxidizing free sulfurous acid to sulfuric acid A method for measuring sulfurous acid, comprising detecting an amount of hydrogen ions. 4. Thiobacillus thiooxydans is a new strain, Thiobacillus thiooxydans 11773 (FERM BP-311).
The measurement method according to any one of claims 1 to 3, which is 9). 5. Thiobacillus thiooxydans is a new strain, Thiobacillus thiooxydans 20294 (FERM BP-346).
The measurement method according to any one of claims 1 to 3, which is 7). 6. A sample injector 1, a switching valve 2 for switching between a sample and a buffer, a heating tank 5 for mixing and heating the sample and the buffer, and a sample and a buffer in the heating tank 5. A pump 4 for sending the liquid, a cooling tank 6 for cooling the sample heated in the heating tank 5, and a Thiobacillus
Thiooxidans or Thiobacillus ferrooxidans
A reaction vessel 7 equipped with a microorganism-immobilized oxygen electrode or a pH electrode belonging to the same type, and a thermostat 8 for maintaining the reaction vessel 7 at a constant temperature.
And a current-voltage converter 10, an mV meter 11, a computer 12, a CRT 13, and a printer 14. 7. A sample injector 15, a switching valve 16 for switching between a sample and an alkaline solution, a heating tank 19 for mixing the sample and the alkaline solution and performing an alkali treatment, and A pump 18 for sending liquid,
A cooling tank 20 for cooling the alkali-treated sample in the heating tank 19, a mixer 21 for mixing the sample and the acid buffer sent from the cooling tank 20, and an acid buffer solution in the mixer 21 23 to send thiobacillus thiooxydans
Or a reaction vessel 25 equipped with a microorganism-immobilized oxygen electrode or a pH electrode belonging to Thiobacillus ferrooxidans, a thermostatic chamber 24 for maintaining the reaction vessel 25 at a constant temperature, a current-voltage converter 27, and an mV meter 28. And computer 29 and CR
Sulfurous acid measuring device consisting of T30 and printer 31.