WO2017084064A1 - Use of electronic olfactory device in on-line detection of cyclohexanecarboxylic acid concentration during dolomycin fermentation - Google Patents

Abstract

Provided is a use of an electronic olfactory device in on-line detection of a cyclohexanecarboxylic acid concentration during dolomycin fermentation. An optimization method for supplementing a precursor during the dolomycin fermentation is determined. Namely, the cyclohexanecarboxylic acid in the fermentation broth is maintained at the optimum concentration by means of on-line detection and feedback of the supplementary addition amount of the precursor substance of cyclohexanecarboxylic acid using the electronic olfactory device, not only satisfying the amount of the precursor required for the bacterial synthesis of doramectin, but also not adversely affecting the metabolic activity of the bacteria. This method makes up for the shortcomings where sampling during the process to perform liquid chromatography or gas chromatography is time-consuming and where it is difficult to achieve on-line detection and timely control.

Description

Application of Electronic Olfactory Device in On-line Detection of Cyclohexanoic Acid Concentration in Doramectin Fermentation Process Technical field

The invention belongs to the field of analytical chemistry, and particularly relates to the application of an electronic olfactometer in the online detection of the concentration of cyclohexanic acid in the fermentation process of doramectin.

Background technique

Doramectin, the trade name "Dectomax", is a new generation of macrolide anti-parasitic drugs developed by Pfizer Inc. in the 1990s. It is composed of mutated avermectin. The Streptomyces strain is biosynthesized in an environment containing cyclohexanoic acid (CHC). Doramectin belongs to avermectins, but has better pharmacokinetic properties and efficacy than avermectin. It has a wide range of antiparasitic, high blood concentration in the body, and slow elimination (duracilin). The half-life is 5.7 days, the avermectin half-life is 4.2 days), the drug effect is maintained for a long time, and there is no allergic reaction. Doramectin has been approved by the US Food and Drug Administration (FDA) as a treatment for parasitic infections such as gastrointestinal tract nematodes, scorpion worms, ocular parasites, ticks, sucking lice, cockroaches, etc. in cattle, sheep, pigs, etc. The veterinarian uses an anthelmintic. Compared with mammals, Musca domestica is 600-700 times more sensitive to avermectin insecticides and has a high selectivity. It is safer at normal dosages.

In the doramectin batch fermentation process, cyclohexanecarboxylic acid is an important precursor in the synthesis of doramectin, which is directly used in the molecular structure synthesis of doramectin. Reports on the effect of cyclohexanic acid on the fermentation of doramectin showed that the precursor had a significant inhibitory effect on cell growth, but insufficient addition of cyclohexanecarboxylic acid would limit the rapid synthesis of the product. The detection of cyclohexanecarboxylic acid residues in the fermentation process is mainly determined by gas chromatography or liquid chromatography. This method takes a long time to process, and it is difficult to achieve rapid detection of large-volume samples in the actual production process. Cyclohexanecarboxylic acid is often used in the fermentation process of racastatin by means of timed detection and batch addition. In order to realize on-line monitoring and feedback control of cyclohexanecarboxylic acid residues and optimize the optimal cyclohexanecarboxylic acid control technology, the present invention firstly proposed and found a loop of doramectin fermentation liquid using an electronic olfactory specific sensing membrane. Online real-time detection and optimization control method for residual concentration of hexanecarboxylic acid.

Summary of the invention

Technical Problem: In order to solve the defects of the prior art, the present invention provides an application of an electronic olfactometer for online detection of cyclohexanoic acid concentration in a doramectin fermentation process.

Technical Solution: The present invention provides an application of an electronic olfactometer in detecting cyclohexanic acid content.

The invention also provides the application of an electronic olfactometer in the online detection of the concentration of cyclohexanic acid in the fermentation process of doramectin.

The application includes the following steps:

(1) Standard curve drawing: different concentrations of cyclohexanoic acid were introduced into the fermenter, and the response of the electronic olfactometer was taken as the abscissa and the concentration of cyclohexanecarboxylic acid was taken as the ordinate. The fitting equation of the value and the concentration of cyclohexanecarboxylic acid in the fermentation broth is a standard curve;

(2) The fermentation tail gas of the doramectin fermentor is introduced into an electronic olfactometer, and the response value of cyclohexanic acid in the fermentation odor in the electronic olfactometer, that is, the concentration of cyclohexanoic acid in the terramycin fermentor is detected.

Preferably, the electronic olfactometer uses a gas sensing membrane TGS2440.

As another preferred, the doramectin fermentation conditions are: a temperature of 19 to 45 ° C; a stirring speed of 50 to 800 rpm; and aeration ratio of 4.5 vvm or less, preferably 4.0 vvm or less.

The invention also provides an application of an electronic olfactor for on-line detection of cyclohexanic acid concentration and control of cyclohexanoic acid content in the fermentation process of doramectin.

Advantageous Effects: The present invention determines a new optimization process for the precursor of the fermentation process of the doramectin fermentation process, that is, the electronic scent on-line detection and feedback control of the amount of the precursor substance cyclohexanecarboxylic acid, the fermentation liquid Cyclohexanecarboxylic acid is maintained at an optimum concentration, which not only satisfies the amount of precursor required for the synthesis of doramectin by the cells, but also does not adversely affect the metabolic activity of the cells. The method makes up for the short time taken by the process sampling for liquid phase or gas chromatography, and it is difficult to realize on-line detection and timely control, and has an important guiding role for industrial fermentation production of doramectin.

DRAWINGS

Figure 1 is a schematic diagram of the application of an electronic olfactometer in the online detection of cyclohexacarboxylic acid concentration in the fermentation of doramectin.

Figure 2 is a specific radar fingerprint of an electronic olfactor of cyclohexanecarboxylic acid;

Figure 3 is a graph showing the response of a specific channel at different concentrations;

Figure 4 is the response of the electronic olfactometer in the case of different ventilation ratios;

Figure 5 is a fitting curve of the electronic olfactometer for detecting the concentration of cyclohexanecarboxylic acid;

Figure 6 is a graph showing the concentration of cyclohexanecarboxylic acid by gas chromatography;

Figure 7 is the online feedback control cyclohexane formic acid addition rate and traditional batch addition process for doramectin The effect of fermentation synthesis.

detailed description

Fermentation conditions of doramectin in the examples of the present invention:

(1) strains and culture medium

Production strain: Streptomyces fuliginea JT-01, preserved in Jiangsu Weiling Biochemical Co., Ltd.

Bevel seed, plate medium:

Starch 0.6%, yeast extract 0.4%, magnesium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium chloride 0.05%, sodium nitrate 0.1%, agar powder 1.5%; pH 7.

Shake flask seed medium:

Starch 2%, soybean cake powder 1%, dipotassium hydrogen phosphate 0.2%, sodium chloride 0.1%, calcium carbonate 0.2%; pH 7.

Fermentation medium:

Starch 15%, soybean cake powder 1.2%, yeast extract 0.7%, dipotassium hydrogen phosphate 0.5%, sodium chloride 0.06%, 10% concentration of sodium cyclohexylate 3%; pH 7.

(2) Instruments and reagents

Instrument: Fermentation tank: Shanghai Guoqiang Biochemical Equipment Co., Ltd. 15L and 50L fermenter; Haoyu Hengping tail gas mass spectrometer and analysis software; 722 UV-visible spectrophotometer; rotary shaker.

(3) Training method

Doramectin fermentation uses tertiary fermentation, which includes secondary seed fermentation.

The deposited spores were uniformly spread on a slant/plate medium with an inoculation needle or a sterile bamboo stick, and cultured at 28 ° C and 40% relative humidity for 5-7 days.

100 ml of seed medium was added to a 500 ml shake flask, and after sterilization, 3-4 ml of the spore suspension was added and cultured at 28 ° C, 240 rpm for 24-48 hours.

100 ml of fermentation medium was added to a 500 ml Erlenmeyer flask, 8 ml of seed solution was added, and culture was carried out for 13 days at 28 ° C, 240 rpm.

(4) Determination method:

Reducing sugar determination: DNS method.

Biomass determination: Off-line measurement Using the wet volume method, 10 mL of the fermentation broth was placed in a centrifuge tube, centrifuged at 3000 rpm for 15 min, and the centrifuged supernatant was poured into a graduated cylinder, and the volume of the fermentation broth was calculated from the volume of the supernatant. Potency determination: using the National Pharmacopoeia method.

On-line determination of pH and DO: On-line measurement was performed using a Mettler Toledo high temperature electrode.

Temperature: Platinum temperature electrode was measured on-line.

Determination of oxygen and carbon dioxide in the intake and exhaust gases: Real-time online collection and analysis of the intake and exhaust gases during the fermentation process using the Hanyu Hengping tail gas mass spectrometer.

Oxygen consumption rate OUR and carbon dioxide production rate CER measurement: OUR and CER calculations were calculated by analyzing data on fermentation tail gas.

Electronic scent gas sensor: The electronic scent contains 16 gas sensors. The sensitive film material of these sensors is the semiconductor oxide SnO ₂ . When the gas to be tested is in contact with the ceramic film, the resistance of the SnO ₂ sensitive film coated on the surface of the ceramic film It varies with the type and concentration of the gas to be tested. When the air passes over the surface of the sensitive membrane, the oxygen in the air and the negative electrons in the sensitive membrane are combined by electron affinity to form a potential barrier. This barrier will cause the resistance of the gas sensor to become larger, generally reaching tens of thousands. Hundreds of thousands ohms. When the gas to be tested passes through the surface of the sensitive film, the free electrons of the reducing gas combine with O ₂ to reduce the barrier, resulting in a decrease in the resistance of the gas sensor. According to Ohm's law

U=I×R

Where U is the voltage value, I is the current value, and R is the resistance value. With the voltage remaining the same, the voltage across the resistor in series with the sensor changes as the resistance changes.

Example 1 to examine the corresponding situation of a series of gas sensing membranes

The sterile medium solution with different concentration of cyclohexanecarboxylic acid was prepared in the fermenter, and the qualitative and quantitative detection of the fermentation process was carried out under aeration and agitation.

Investigate 8 TGS-8 series gas sensing sensors (TGS800, 812, 813, 821, 822, 826, 830, 842) and 7 TGS-2000 series gas sensing sensors (TGS2100, 2180, 2181, 2281) 2440, 2601, 2610) and a TGS4161 sensing membrane for a total of 16 sensing membranes for detection and identification of responses.

The experimental results of each sensor element are shown in Table 1.

The specific radar fingerprint of the electronic olfactory of cyclohexanecarboxylic acid is shown in Fig. 2. The specific channel response curve at different concentrations is shown in Fig. 3. The specific radar fingerprint of the electronic olfactor according to cyclohexanecarboxylic acid and the different concentrations of cyclohexane The response curve of the formic acid concentration was changed. We selected the gas sensing membrane TGS2440. The response intensity of the sensing membrane showed a good consistency in the concentration of cyclohexanecarboxylic acid solution (50 to 4500 ppm).

Table 1 16 sensing membrane response detection and recognition results

concentration	TGS800	TGS812	TGS813	TGS821	TGS822	TGS826	TGS830	TGS842
50.00	0.33	0.30	0.11	0.73	0.50	1.60	0.69	0.74
200.00	0.37	0.38	0.10	1.05	0.63	2.17	0.93	0.95
500.00	0.47	0.45	0.08	1.35	0.76	2.73	1.11	1.14
800.00	0.49	0.53	0.08	1.68	0.88	3.15	1.33	1.31
1000.00	0.55	0.57	0.10	1.90	1.01	3.54	1.49	1.45
2000.00	0.58	0.63	0.09	2.13	1.11	3.83	1.65	1.64
3000.00	0.69	0.74	0.09	2.39	1.32	4.23	1.88	1.84
4500.00	0.76	0.81	0.12	2.57	1.38	4.44	2.04	1.98

concentration	TGS2100	TGS2180	TGS2181	TGS2281	TGS2440	TGS2601	TGS2610	TGS4161
50.00	0.24	1.50	1.00	2.47	0.71	1.74	0.45	0.84
200.00	0.34	1.78	1.15	2.82	1.38	2.99	1.04	1.00
500.00	0.44	2.13	1.30	3.21	2.47	3.87	1.85	1.15
800.00	0.54	2.44	1.48	3.51	3.52	4.22	2.93	1.30
1000.00	0.65	2.73	1.64	3.82	4.10	4.41	3.59	1.44
2000.00	0.73	3.02	1.75	4.04	5.56	4.53	5.43	1.58
3000.00	0.88	3.38	1.99	4.37	6.18	4.73	6.21	1.79
4500.00	1.01	3.64	2.14	4.57	6.47	4.85	6.89	1.91

Example 2 examines the effect of different operating control conditions on the stability of the assay

The fermentation process control conditions simulation experiment was carried out in a 500 ppm concentration cyclohexanecarboxylic acid solution fermentor.

In a 50 liter fermentor, a 30 liter aqueous solution was added, and the pH was adjusted to a neutral condition of 7.0 ± 0.2; the fermenter was a three-layer flat blade paddle stirring system, and the vent pipe had a bottom entry. The normal fermentation process conditions: speed 450 rpm, temperature 27 ° C, tank pressure 0.03 MP, ventilation ratio 1.2 vvm, the exhaust gas is connected to the electronic sniffer sampling port for data acquisition.

The effects of different aeration ratios, temperature, fermentation speed and different tank pressures on the response values of TGS2440 sensing membranes were investigated. The test results show that:

(1) The temperature of the detected electronic olfactometer has hardly changed in the case where the temperature fluctuates from 19 ° C to 45 ° C of the test;

(2) When the stirring speed of the fermenter is 50 to 800 rpm, the response value remains the same;

(3) When the same tank pressure changes within the normal fermentation control range, the response value remains constant;

(4) With the change of ventilation ratio, when the ventilation is not higher than 4.5vvm, there is almost no influence on the detection result, indicating that the diffusion rate of cyclohexanecarboxylic acid in the liquid phase to the bubble and the bubble are below the ventilation ratio. The overflow rate reached equilibrium (see Figure 4); when the ventilation ratio is higher than 4.5vvm, the response value detected by the electronic olfactometer begins to decrease, indicating that the diffusion rate of cyclohexanecarboxylic acid into the bubble in the liquid has begun to be slower than that of the bubble. The rate of overflow; while the normal fermentation process is often at a rate of 4.0 vvm to prevent the power consumption cost caused by excessive ventilation ratio.

Therefore, under normal control conditions, the electronic olfactor sensing membrane can achieve on-line detection of the residual concentration of cyclohexanecarboxylic acid.

Example 3 standard curve drawing

The concentration of cyclohexanecarboxylic acid in the fermenter was controlled at 50 ppm, 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm, 4500 ppm, respectively, and the average value of the response values was obtained by three times of detection, and the response value of the electronic olfactor was used as the abscissa to the concentration of cyclohexanecarboxylic acid. As the ordinate, the fitting equation of the response value of the electronic olfactory detection and the concentration of cyclohexanecarboxylic acid in the fermentation broth is obtained.

y=148.58x ² -329.74x+219.71

Wherein y is the concentration of cyclohexanecarboxylic acid and x is the electronic olfactory response value. The R ² of the fitted equation is 0.995, indicating that the fitting equation is believable.

By comparing the cyclohexanecarboxylic acid concentration calculated by the fitted equation (see Figure 5) with the results of off-line gas chromatography (see Figure 6), the experimental results obtained by the two detection methods are very close, indicating the results calculated by fitting the equation. It is correct that it is feasible to use the electronic scent to detect the content of cyclohexanecarboxylic acid in the fermentation broth.

Example 4

The fermentation tail gas of the doramectin fermentor was introduced into an electronic olfactometer, and the response value of cyclohexanic acid in the fermentation odor in the electronic olfactometer, that is, the concentration of cyclohexanoic acid in the doramectin fermentor was detected. Specifically, as shown in Fig. 1, the detection of the residual concentration of cyclohexanecarboxylic acid in the fermentation broth by using electronic olfactory and the feeding flow control are as follows. The tail gas of the fermenter is directly subjected to water odor detection after the water removal treatment, and the electronic scent is detected by four channels. The device can perform on-line detection on four fermenters at the same time, and the signal obtained by the monitoring is converted into a numerical signal, and is used for feedback control of the feed adjustment acceleration of the feed.

Excellent control of doramectin fermentation by electronic scent on-line detection and feedback control of cyclohexanecarboxylic acid content Chemical:

In the 50 liter fermenter, the on-line detection of the concentration of cyclohexanecarboxylic acid in the fermentation process was carried out by using an electronic olfactory sensor, and the feed rate of the feed pump was controlled to control the concentration of cyclohexanecarboxylic acid to be 200 ppm, 500 ppm, and 800 ppm, respectively. And 1500ppm to investigate the effect of different concentrations on the fermentation synthesis of doramectin, see Figure 7, through the addition of different concentrations of cyclohexanecarboxylic acid in the fermentation process, the optimal addition concentration of cyclohexanecarboxylic acid was 500±40ppm. The titer of doramectin was up to 1886 μg/mL, which was 85.2% higher than that of the intermittent supplemental mode. When the concentration of cyclohexanecarboxylic acid was higher than 800 ppm, the synthesis rate of doramectin was significantly reduced. When the temperature is higher than 1500ppm, the growth of the bacteria is obviously restricted, which means that the addition of too much precursor substance can inhibit the metabolic process of the bacteria.

Claims (6)

1. The application of electronic olfactometer in the detection of cyclohexanic acid content.
2. Application of electronic olfactometer in on-line detection of cyclohexanic acid concentration in doramectin fermentation process.
3. The application of claim 2, comprising the steps of:

   (1) Standard curve drawing: different concentrations of cyclohexanoic acid were introduced into the fermenter, and the response of the electronic olfactometer was taken as the abscissa and the concentration of cyclohexanecarboxylic acid was taken as the ordinate. The fitting equation of the value and the concentration of cyclohexanecarboxylic acid in the fermentation broth is a standard curve;

   (2) The fermentation tail gas of the doramectin fermentor is introduced into an electronic olfactometer, and the response value of cyclohexanic acid in the fermentation odor in the electronic olfactometer, that is, the concentration of cyclohexanoic acid in the terramycin fermentor is detected.
4. The use according to claim 3, characterized in that the electronic olfactometer uses a gas sensing membrane TGS2440.
5. The use according to Claim 3, characterized in that the doramectin fermentation conditions are: a temperature of 19 to 45 ° C; a stirring speed of 50 to 800 rpm; and aeration ratio of 4.5 vvm or less, preferably 4.0 vvm or less.
6. The application of electronic olfactometer in the online detection of cyclohexacarboxylic acid concentration in the fermentation of doramectin and the control of cyclohexanoic acid content.

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