Method for detecting urea in halophilic bacteria fermentation liquor

Abstract

The invention relates to the technical field of halophilic bacteria fermentation, in particular to a method for detecting urea in halophilic bacteria fermentation liquor. The method is suitable for a rich high-salt environment for fermentation culture of halophilic bacteria, can be used for detecting relatively high urea content in fermentation liquor, improves the accuracy and the stability of a detection result, greatly shortens the detection time and obviously improves the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of halophilic bacteria fermentation, and particularly relates to a method for detecting urea in a halophilic bacteria fermentation broth. Background Art

[0002] Like other organisms, microorganisms need to continuously absorb nutrients from the outside world, undergo a series of biochemical reactions, obtain energy, form new cell substances, and at the same time discharge waste. There are a wide variety of microorganisms, and their absorption, requirements, and utilization of nutrients are also different. When preparing a culture medium, there must be sufficient carbon sources, nitrogen sources, water, and inorganic salts. In addition, some microorganisms with poor synthetic ability need to add appropriate growth auxiliary substances to grow normally.

[0003] Nitrogen sources are mainly used to form cell substances of bacteria and synthesize nitrogen-containing metabolites, accounting for about 5%-13% in the dry cell matter. When the nitrogen element required by microorganisms is limited and the carbon-nitrogen ratio is too high, the growth of microorganisms is slow, the fermentation time is long, and the fermentation production cost will be greatly increased; when the nitrogen source is too high and the carbon-nitrogen ratio is too low, the energy required for the growth of microorganisms cannot be satisfied, the fermentation speed is slow, and nitrogen will be discharged in the form of ammonia gas, resulting in loss of organic nutrients and bad smell. Commonly used nitrogen sources can be divided into organic nitrogen sources and inorganic nitrogen sources. As the simplest organic nitrogen source, urea is widely used in the biological fermentation industry due to its low price and fast absorption rate. As the main nitrogen source in the microbial fermentation process, the content of urea in the fermentation broth directly affects the growth and reproduction of microorganisms, especially the microbial fermentation culture using urea as the sole nitrogen source. Therefore, if the monitoring of urea in the fermentation medium can be achieved quickly and conveniently, and the timely monitoring and regulation intervention of the carbon-nitrogen ratio in the fermentation process can be carried out, it has great practical significance for the optimization of the fermentation process.

[0004] Urea, also known as carbamide, has the chemical formula CH4N2O or CO(NH2)2. At present, there are many methods for the determination of urea at home and abroad, but they are mainly applied in fields such as clinical diagnosis and environmental monitoring. Generally speaking, there are mainly the following several: direct colorimetry, indirect colorimetry, chromatography, mid-infrared spectroscopy, etc. There is no detection method for urea in microbial fermentation media or fermentation broths. The fermentation medium or fermentation broth often has a relatively complex matrix environment, with characteristics such as high sugar, high salt, and rich mineral salts. The complex components make the accurate detection of urea very difficult.

[0005] At present, the more commonly used direct colorimetric methods include diacetyl monoxime method, o-phthalaldehyde method, and p-dimethylaminobenzaldehyde method. However, the nitrite-Griess reagent method can only be qualitative but not quantitative at this stage. The o-phthalaldehyde method has low detection sensitivity and it is not easy to detect trace amounts of urea in the solution. The indirect colorimetric method detects the products after the enzymatic hydrolysis of urea and then converts them into the urea content. The essence of the urease used in this method is protein, which has the highest activity at 37°C, and its activity will also change significantly with the changes of temperature, water quality, etc. Therefore, this method has certain limitations. For example, copper ions will inactivate the urease, thus seriously affecting the detection accuracy. Chromatographic analysis methods have the characteristics of high speed, high efficiency, and high sensitivity. There is also a method using high performance liquid chromatography-fluorescence detector to detect urea, but the correlation coefficient of this method is low, the processing process is complex, the detection cycle is long, and the instrument cost is high. Special detection instruments and equipment such as a dedicated fluorescence detector are required, and such detectors are not common in related laboratories. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the prior art to a certain extent.

[0007] As the "next-generation industrial biotechnology" (Next-generation industrial biotechnology, NGIB), halophilic bacteria have the characteristics of open fermentation. Due to their halophilic fermentation characteristics, it is possible to avoid abnormal fermentation caused by contamination of some environmental microorganisms. Their open fermentation characteristics determine that the fermentation medium contains a relatively high concentration of inorganic salts. The production process of polyhydroxy alkanoates (PHA) using halophilic bacteria mainly includes two stages: the growth and reproduction of the bacterial strain in the first stage and the accumulation of PHA under nitrogen source limitation in the second stage. The growth of the bacterial cells in the first stage seriously affects the yield of PHA in the second stage. Therefore, it is very necessary to detect and precisely control urea in the PHA fermentation biochemical process.

[0008] For the existing diacetyl monoxime spectrophotometric method for urea detection (such as the national standard method, GB / T18204.2-2014), the principle is based on the reaction of urea with diacetyl monoxime and antipyrine to present yellow, and there is a maximum absorption peak at a wavelength of 460 nm. The detection range of urea content by this method is within 0.001 - 0.015 mg. The detection process requires boiling water bath heating for 45 - 55 min, which takes a relatively long time, and the absorbance of the colored solution decreases with the extension of time. This national standard method is mainly applied to the detection of swimming pool water, with a narrow linear range, a long reaction time, and a relatively low correlation coefficient of the standard curve. Moreover, for the detection of solutions with high urea concentration, dilution is required before detection, and repeated measurements are needed, which is time-consuming and laborious.

[0009] The inventors found that, mainly due to the high content of inorganic salts and the large number of component types in the halophilic bacteria fermentation broth, if the above national standard method is directly applied for detection, the detection results of urea content are not accurate and stable, and the method is complicated and time-consuming.

[0010] To this end, the present invention provides a method for detecting urea in halophilic bacteria fermentation broth, which is suitable for the rich high-salt environment of halophilic bacteria fermentation culture, can detect the higher urea content in the fermentation broth, improve the accuracy and stability of the detection results, greatly shorten the detection time, and significantly improve the detection efficiency.

[0011] The method for detecting urea in the halophilic bacteria fermentation broth of the present invention adopts spectrophotometry.

[0012] A method for detecting urea in a halophilic bacteria fermentation broth, comprising preparing a standard curve and detecting the halophilic bacteria fermentation broth to be detected;

[0013] Wherein, the method for preparing the standard curve comprises:

[0014] A urea standard working solution is prepared with a first solvent; the first solvent is a halophilic bacteria fermentation medium that does not contain urea;

[0015] First, take the urea standard solution, add 1%-2% diacetyl monoxime-isopropanol solution respectively, and mix well; then add 2%-4% antipyrine solution, and mix well; boil in a boiling water bath for 10-20 minutes; then use the first solvent to make the volume constant so that the concentration of urea in the series solution is 0-0.5 mg / mL; use the first solvent as a control to measure the absorbance value of each series solution; use the urea content (mg) as a control absorbance value to obtain a standard curve;

[0016] The method for detecting the halophilic bacteria fermentation broth to be detected comprises: taking the supernatant of the halophilic bacteria fermentation broth to be detected, and performing detection according to the same method as the method for preparing the standard curve.

[0017] The method of the present invention has no particular limitation on the type of halophilic bacteria.

[0018] According to an embodiment of the present invention, the halophilic bacteria are halophilic bacteria strains that can ferment and produce PHA. Generally, these strains are all known.

[0019] According to a specific embodiment of the present invention, the halophilic bacteria are Halomonas, for example, Halomonas bluephagenesis WZY254, WZY278, TDUH, TD40 and other TD01 derivatives, as shown in the document CN115651874A.

[0020] In the present invention, the halophilic bacteria fermentation broth is obtained by fermenting the halophilic bacteria fermentation medium (containing urea) with halophilic bacteria. The addition amount of urea can be adjusted as needed, and the present invention does not make specific limitations. In some specific embodiments, the addition amount of urea in the halophilic bacteria fermentation medium is 0.5 - 10 g / L, such as 1 - 10 g / L or 2 - 5 g / L.

[0021] The method of the present invention is particularly applicable to the halophilic bacteria fermentation medium with urea as the sole nitrogen source.

[0022] The method of the present invention has a good detection effect on the urea content in the halophilic bacteria fermentation broth. Especially for the halophilic bacteria fermentation medium with a relatively high salt content (such as a concentration of 5 - 60 g / L), good accuracy and stability can still be obtained.

[0023] According to an embodiment of the present invention, the halophilic bacteria fermentation medium is a medium capable of fermenting and producing PHA.

[0024] According to an embodiment of the present invention, the halophilic bacteria fermentation medium includes: 1 - 10 g / L of urea, 5 - 25 g / L of glucose, 5 - 60 g / L of sodium chloride, 0.2 - 2 g / L of magnesium sulfate, 2 - 10 g / L of potassium dihydrogen phosphate, 0.1 - 2 g / L of ammonium ferric citrate, and 0.01 - 0.1 g / L of calcium chloride.

[0025] According to an embodiment of the present invention, the halophilic bacteria fermentation medium includes: 2 - 5 g / L of urea, 10 - 25 g / L of glucose, 15 - 30 g / L of sodium chloride, 0.2 - 2 g / L of magnesium sulfate, 2 - 8 g / L of potassium dihydrogen phosphate, 0.1 - 2 g / L of ammonium ferric citrate, 0.01 - 0.1 g / L of calcium chloride, 0.0002 - 0.001 g / L of zinc sulfate heptahydrate, 0.0001 - 0.001 g / L of manganese chloride tetrahydrate, 0.001 - 0.005 g / L of boric acid, 0.0001 - 0.001 g / L of cobalt chloride hexahydrate, 0.0001 - 0.002 g / L of copper sulfate pentahydrate, and 0.0002 - 0.005 g / L of nickel chloride hexahydrate.

[0026] According to a specific embodiment of the present invention, the halophilic bacteria fermentation medium includes: 4.8 g / L of urea, 25 g / L of glucose, 30 g / L of sodium chloride, 1 g / L of magnesium sulfate, 5.2 g / L of potassium dihydrogen phosphate, 0.7 g / L of ammonium ferric citrate, 0.1 g / L of calcium chloride, 0.001 g / L of zinc sulfate heptahydrate, 0.0003 g / L of manganese chloride tetrahydrate, 0.003 g / L of boric acid, 0.001 g / L of cobalt chloride hexahydrate, 0.0002 g / L of copper sulfate pentahydrate, and 0.0001 g / L of nickel chloride hexahydrate.

[0027] According to an embodiment of the present invention, the halophilic bacteria fermentation medium without urea is obtained by removing urea from the halophilic bacteria fermentation medium.

[0028] According to an embodiment of the present invention, the first solvent is preferably the supernatant of the halophilic bacteria fermentation medium without urea.

[0029] According to an embodiment of the present invention, the supernatant of the halophilic bacteria fermentation medium without urea and the supernatant of the to-be-detected halophilic bacteria fermentation broth can both be obtained by centrifugation, for example, centrifuging at 12000 rpm for 1 - 2 min.

[0030] In view of the high inorganic salt content and complex composition in the halophilic bacteria fermentation medium and fermentation broth, the method of the present invention uses the halophilic bacteria fermentation medium without urea or its supernatant to replace pure water for preparing the standard curve, making the environment of the standard curve more consistent with that of the to-be-detected sample, eliminating the interference of inorganic salt metal ions in the microbial fermentation medium on the detection, and greatly improving the accuracy of the detection result.

[0031] According to an embodiment of the present invention, the urea standard working solution can be obtained by diluting the urea standard stock solution, and the first solvent used is the supernatant of the halophilic bacteria fermentation medium without urea. In some specific examples, the concentration of urea in the urea standard working solution is 0 - 5 g / L, such as 0, 0.5 g / L, 1 g / L, 2.5 g / L, 4 g / L, 5 g / L.

[0032] According to an embodiment of the present invention, in the method for preparing the standard curve, after volume fixing, the concentrations of urea in the series of solutions are 0, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0033] According to an embodiment of the present invention, in the method for preparing the standard curve, after volume fixing, the volume of the series of solutions is 10 mL.

[0034] In the aforementioned national standard method, the concentration range of the diluted urea standard working solution is 0 - 0.0006 mg / mL, specifically 0, 0.00004 mg / mL, 0.00012 mg / mL, 0.0002 mg / mL, 0.00028 mg / mL, 0.00036 mg / mL, 0.00044 mg / mL, 0.00052 mg / mL, 0.0006 mg / mL. That is, the highest concentration of the diluted urea standard working solution in the aforementioned national standard method is 0.015 mg / 25 mL.

[0035] The inventor of the present invention unexpectedly found that when the concentration of urea in the series of solutions was increased compared with the aforementioned national standard method (the final constant volume was 10 mL), the color development degree after the reaction was significantly improved, making the measurement data more stable, the curve linearity better, and thus the accuracy of the detection result higher.

[0036] In addition, the boiling water bath time of the aforementioned national standard method is 45 - 55 min, while the method of the present invention only requires 10 - 20 min, and the reaction time is less than 1 / 2 of the original, greatly improving the detection efficiency.

[0037] According to the embodiment of the present invention, the time of the boiling water bath can be 10 min, 15 min or 20 min.

[0038] According to the embodiment of the present invention, after the boiling water bath, there is also a step of cooling, and volume constant is carried out after cooling.

[0039] In particular, in the method of the present invention, urea, diacetyl monoxime and antipyrine are first reacted, and then volume constant is carried out. In this way, the accuracy and stability of urea detection can be significantly improved. Maintaining a reaction system with a relatively high concentration helps to accelerate the reaction efficiency and reduces the reaction time to a certain extent.

[0040] According to the embodiment of the present invention, the concentration of the diacetyl monoxime - isopropanol solution is 1% or 2%.

[0041] According to the embodiment of the present invention, the diacetyl monoxime - isopropanol solution is prepared from diacetyl monoxime and 10% acetic acid and isopropanol with a volume ratio of 1:1.

[0042] The inventor of the present invention unexpectedly found that by adding isopropanol, the accuracy and stability of urea detection can be significantly improved. Isopropanol can act as a color development stabilizer, and the color development effect after the reaction is better, and the standard curve drawn is more linear.

[0043] In the aforementioned national standard method, a 0.2% diacetyl monoxime solution is used. The present invention found that by increasing the concentration of diacetyl monoxime to 1% - 2%, the accuracy and stability of urea detection can be significantly improved.

[0044] According to the specific embodiment of the present invention, the preparation method of the diacetyl monoxime - isopropanol solution includes: dissolving 1.0 - 2.0 g of diacetyl monoxime in 50 mL of 10% acetic acid, and then adding 50 mL of isopropanol and mixing evenly.

[0045] The CAS number of diacetyl monoxime is 57 - 71 - 6, and the molecular formula is CH3COC(NOH)CH3.

[0046] According to the embodiment of the present invention, the concentration of the antipyrine solution is 2%, 3% or 4%.

[0047] In the present invention, the antipyrine solution can be prepared by existing methods.

[0048] According to a specific embodiment of the present invention, the method for preparing the antipyrine solution comprises: dissolving 4.0 g - 8.0 g of antipyrine in 1 + 1 sulfuric acid and diluting with a mixed acid to 200 mL.

[0049] Antipyrine is 1,5 - dimethyl - 2 - phenyl - 3 - pyrazolinone, with a CAS number of 60 - 80 - 0 and a molecular formula of C6H5NN(CH3)C(CH3):CHC:O.

[0050] According to an embodiment of the present invention, in the method for preparing the standard curve, the weight ratio of urea, diacetyl monoxime, and antipyrine is (0 - 5):(0.008 - 0.012):(0.03 - 0.05), for example, (0 - 5):0.01:0.04.

[0051] According to an embodiment of the present invention, in the method for preparing the standard curve, the volume ratio of the series of standard solutions, the diacetyl monoxime - isopropanol solution, and the antipyrine solution is 1:1:2.

[0052] According to an embodiment of the present invention, the absorbance value is measured at a wavelength of 440 - 480 nm, and the preferred wavelength is 460 nm.

[0053] According to an embodiment of the present invention, the steps of preparing the standard curve and the steps of detecting the test halophilic bacteria fermentation broth can use brown stoppered test tubes or brown stoppered colorimetric tubes.

[0054] According to an embodiment of the present invention, a 1 cm cuvette is used to measure the absorbance value.

[0055] According to an embodiment of the present invention, in the method for detecting the test halophilic bacteria fermentation broth, it further includes a step of using the first solvent as a control.

[0056] According to an embodiment of the present invention, the method for detecting the test halophilic bacteria fermentation broth comprises: taking the supernatant of the test halophilic bacteria fermentation broth, adding a 1% - 2% diacetyl monoxime - isopropanol solution, and mixing evenly; then adding a 2% - 4% antipyrine solution and mixing evenly; performing a boiling water bath for 10 - 20 min; then making up the volume with the first solvent; using the first solvent as a control to measure the absorbance value; and obtaining the urea content in the test halophilic bacteria fermentation broth according to the measured absorbance value and the standard curve.

[0057] According to an embodiment of the present invention, the standard curve has the absorbance value as the ordinate and the urea content (mg) as the abscissa.

[0058] According to an embodiment of the present invention, the method for detecting urea in the halophilic bacteria fermentation broth comprises:

[0059] 1) Preparation of standard curve

[0060] Prepare a standard working solution of urea with the supernatant of the halophilic bacteria fermentation medium without urea;

[0061] Use the supernatant of the halophilic bacteria fermentation medium without urea to perform gradient dilution on the standard working solution of urea to obtain a series of standard solutions with urea concentrations of 0, 0.5 g / L, 1 g / L, 2.5 g / L, 4 g / L, and 5 g / L;

[0062] Respectively take 1 mL of the series of standard solutions (the urea contents are 0 mg, 0.5 mg, 1 mg, 2.5 mg, 4 mg, and 5 mg), add 1 mL of a 1% diacetyl monoxime - isopropanol solution to each, and mix well; then add 2 mL of a 2% antipyrine solution and mix well; perform a boiling water bath for 10 - 20 min; then make up the volume to 10 mL with the supernatant of the halophilic bacteria fermentation medium without urea and mix well; using the supernatant of the halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm cuvette to measure the absorbance value; make a standard curve with the urea content (mg) against the absorbance value;

[0063] 2) Detection of the halophilic bacteria fermentation broth to be measured

[0064] Take 1 mL of the supernatant of the halophilic bacteria fermentation broth to be measured, add 1 mL of a 1% diacetyl monoxime - isopropanol solution and mix well; then add 2 mL of a 2% antipyrine solution and mix well; perform a boiling water bath for 10 - 20 min; then make up the volume to 10 mL with the supernatant of the halophilic bacteria fermentation medium without urea and mix well; using the supernatant of the halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm cuvette to measure the absorbance value; according to the measured absorbance value and the standard curve, obtain the urea content in the halophilic bacteria fermentation broth to be measured.

[0065] According to a specific embodiment of the present invention, the regression equation of the standard curve is y = 0.2444x - 0.0115, R 2 = 0.9994; where x is the urea content (mg) and y is the absorbance value (at 460 nm).

[0066] According to a specific embodiment of the present invention, the recovery rate of the spiked recovery determination in the halophilic bacteria fermentation broth is: 97.33% - 103.11%.

[0067] The method of the present invention is applicable to the detection of urea content in the fermentation broth of halophilic bacteria at the initial stage of fermentation (generally from inoculation to 2 - 4 h), the early stage (generally from inoculation to 4 - 6 h), and the later stage of the aforementioned first stage (generally from inoculation to 6 - 10 h), and the detectable range of urea content is 0.2 - 5 g / L.

[0068] When the urea content in the fermentation broth of the halophilic bacteria to be detected is too high, the halophilic bacteria fermentation medium without urea or its supernatant can be appropriately diluted and then detected.

[0069] The present invention also provides the application of the above method for detecting urea in the fermentation broth of halophilic bacteria in the process of halophilic bacteria fermentation (such as the fermentation of halophilic bacteria to produce PHA). The detection method of the present invention can accurately detect and precisely control the urea content in the process of halophilic bacteria fermentation.

[0070] The full text of GB / T 18204.2 - 2014 is incorporated herein by reference.

[0071] The absorbance of the solution after color development by the aforementioned national standard method decreases with the extension of time, and the stability of the method of the present invention is enhanced compared with it.

[0072] It should be noted that terms such as "first" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features.

[0073] The beneficial effects of the present invention at least include:

[0074] The detection method of the present invention is applicable to the detection of urea content in the fermentation broth of halophilic bacteria with high inorganic salt content and complex component types, excluding the influence of fermentation broth impurities, and has high detection accuracy, good stability, short detection time and high efficiency. The method of the present invention improves the detection range of urea content and can be used for the detection of the fermentation broth of halophilic bacteria with high urea content. In addition, the method of the present invention can detect multiple samples simultaneously and can also provide a reference basis for the nitrogen source regulation in the process of halophilic bacteria fermentation. Description of the Drawings

[0075] Figure 1 It is the standard curve prepared in Example 1 of the present invention.

[0076] Figure 2 It is the standard curve prepared in Comparative Example 1 of the present invention. Detailed Embodiments

[0077] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified for the manufacturers, they are all conventional products that can be obtained through regular channels.

[0078] The following halophilic bacterium is the halophilic bacterium strain Halomonas bluephagenesis WZY254 that can ferment and produce PHA.

[0079] The concentration of the following diacetyl monoxime - isopropanol solution is 1%. Preparation method: Dissolve 1.0 g of diacetyl monoxime in 50 mL of acetic acid with a concentration of 10%, and then add 50 mL of isopropanol and mix well.

[0080] The concentration of the following antipyrine solution is 2%. Preparation method: Dissolve 4.0 g of antipyrine in 1 + 1 sulfuric acid and dilute it to 200 mL with the mixed acid.

[0081] The formula of the following halophilic bacterium fermentation medium: urea 4.8 g / L, glucose 25 g / L, sodium chloride 30 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 5.2 g / L, ammonium ferric citrate 0.7 g / L, calcium chloride 0.1 g / L, zinc sulfate heptahydrate 0.001 g / L, manganese chloride tetrahydrate 0.0003 g / L, boric acid 0.003 g / L, cobalt chloride hexahydrate 0.001 g / L, copper sulfate pentahydrate 0.0002 g / L, nickel chloride hexahydrate 0.0001 g / L.

[0082] The preparation method of the halophilic bacterium fermentation medium: First, make ammonium ferric citrate, calcium chloride and concentrated hydrochloric acid into an aqueous solution; then mix the remaining components with this aqueous solution and make up the volume.

[0083] Remove urea from the aforementioned halophilic bacterium fermentation medium, and it is the halophilic bacterium fermentation medium without urea. Further centrifuge (12000 rpm, 2 min) to obtain the supernatant of the halophilic bacterium fermentation medium without urea.

[0084] Example 1 Detection of Urea in Halophilic Bacterium Fermentation Broth

[0085] 1) Preparation of standard curve

[0086] Using the supernatant of the aforementioned halophilic bacteria fermentation medium without urea, prepare a urea standard working solution; then use the supernatant of the aforementioned halophilic bacteria fermentation medium without urea to serially dilute the urea standard working solution into a series of standard solutions with urea concentrations of 0, 0.5 g / L, 1 g / L, 2.5 g / L, 4 g / L, and 5 g / L. Take 6 brown stoppered test tubes of 25 mL, add 1 mL of each of the above series of standard solutions to each tube respectively. Finally, the urea content in each tube is 0 mg, 0.5 mg, 1 mg, 2.5 mg, 4 mg, and 5 mg respectively. Add 1 mL of 1% diacetyl monoxime - isopropanol solution to each tube, mix well, then add 2 mL of 2% antipyrine solution, mix well, and place in a boiling water bath for 15 min. After cooling in the boiling water bath, use the supernatant of the aforementioned halophilic bacteria fermentation medium without urea to make up the volume to 10 mL, and shake each tube well; using the supernatant of the aforementioned halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm cuvette to measure the absorbance value of each tube. Taking the absorbance value of each tube as the ordinate and the urea content (mg) as the abscissa, prepare a standard curve.

[0087] After boiling the water bath of test tubes with different urea concentrations, as the urea content increases, the absorbance value of its ultraviolet - visible absorption light increases significantly, and its maximum absorption value is at 460 nm. By preparing a standard curve for the absorbance value at 460 nm and different urea contents, there is a linear relationship between the absorbance value and the urea content in the range of 0.5 mg - 5 mg, as Figure 1 shown. The regression equation is y = 0.2444x - 0.0115, R 2 = 0.9994, where x is the urea content (mg) and y is the absorbance value.

[0088] 2) Detect the halophilic bacteria fermentation broth to be tested

[0089] Inoculate the aforementioned halophilic bacteria fermentation medium with halophilic bacteria, and the inoculation amount is 10%. Ferment at 37 °C and pH 8.5.

[0090] At the initial stage of fermentation (i.e., 4 h after inoculation), take the halophilic bacteria fermentation broth and detect it by HPLC method. The measured urea content is about 3.25 g / L (high concentration). Add a urea standard solution to this fermentation broth to make the theoretical urea content in the spiked fermentation broth 3.75 g / L; then centrifuge at 12000 rpm for 2 min, take the supernatant as the test sample 1.

[0091] At the early stage of fermentation (i.e., 6 h after inoculation), take the halophilic bacteria fermentation broth and detect it by HPLC method. The measured urea content is about 1.0 g / L (medium concentration). Add a urea standard solution to this fermentation broth to make the theoretical urea content in the spiked fermentation broth 2.25 g / L; then centrifuge at 12000 rpm for 2 min, take the supernatant as the test sample 2.

[0092] At the late stage of the first stage of the aforementioned fermentation (i.e., 8 h after inoculation), take the halophilic bacteria fermentation broth and detect it by HPLC method. The measured urea content is about 0.05 g / L (low concentration). Add urea standard solution to this fermentation broth so that the theoretical urea content in the spiked fermentation broth is 0.75 g / L; then centrifuge at 12000 rpm for 2 min, take the supernatant as the test sample 3.

[0093] Take 1 mL of the above-mentioned test samples 1 - 3 respectively and place them in 3 brown stoppered test tubes of 25 mL. Add 1 mL of 1% diacetyl monoxime - isopropanol solution to each tube and mix well; then add 2 mL of 2% antipyrine solution to each tube and mix well; heat in a boiling water bath for 15 min; cool; then make up the volume to 10 mL with the supernatant of the halophilic bacteria fermentation medium without urea, and shake well; use the supernatant of the halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm colorimetric cell to measure the absorbance value; according to the measured absorbance value and the standard curve, calculate the urea content in the test samples 1 - 3. The results are shown in Table 1 below.

[0094] Comparative Example 1

[0095] 1) Preparation of the standard curve

[0096] Accurately weigh 0.5 g of urea (standard substance) and dissolve it in deionized water, make up the volume to 100 mL, mix well and let it stand for 2 min to obtain a urea standard working solution with a urea concentration of 5 g / L. Respectively transfer 0 μL, 100 μL, 200 μL, 500 μL, 800 μL, 1000 μL of this urea standard working solution into brown stoppered test tubes, and add 1000 μL, 900 μL, 800 μL, 500 μL, 200 μL, 0 μL of deionized water respectively, add 1 mL of 1% diacetyl monoxime - isopropanol, mix well, add 2 mL of 2% antipyrine solution, mix well, heat in a boiling water bath for 15 min, cool, and make up the volume to 10 mL with deionized water.

[0097] Use deionized water as a control, at a wavelength of 460 nm, use a 1 cm colorimetric cell to measure the absorbance value of each tube. Take the absorbance value of each tube as the ordinate and the urea content (mg) as the abscissa to prepare the standard curve.

[0098] After boiling the test tubes with different urea concentrations in a water bath, as the urea content increases, the absorbance value of its ultraviolet - visible absorption light increases significantly, and its maximum absorption value is at 460 nm. By making a standard curve of the absorbance value at 460 nm and different urea contents, there is a linear relationship between the absorbance value and the urea content in the range of 0.5 mg - 5 mg, as Figure 2 shown. The regression equation is y = 0.1627x - 0.0153, R 2= 0.9982, where x is the urea content (mg) and y is the absorbance value.

[0099] 2) Detect the fermentation broth of the halophilic bacteria to be tested

[0100] Take the samples 1 - 3 to be tested in Example 1 for detection. The difference from Example 1 is only that after boiling water bath and then cooling, it is made up to 10 mL with deionized water and shaken well; using deionized water as the control, at a wavelength of 460 nm, with a 1 cm colorimetric cell, measure the absorbance value; according to the measured absorbance value and the standard curve of this Comparative Example 1, calculate the urea content in the samples 1 - 3 to be tested. The results are shown in Table 1 below.

[0101] Table 1

[0102]

[0103] Recovery rate is used to represent accuracy, that is, it represents the degree of closeness between the measured value and the actual value.

[0104] Recovery rate = measured value / theoretical value × 100%

[0105] In Example 1, the supernatant of the halophilic bacteria fermentation medium without urea was used to prepare the urea standard use solution and a series of standard solutions, and the correlation coefficient of its standard curve was R 2 = 0.9994; while in Comparative Example 1, deionized water was used, and the correlation coefficient R of its standard curve 2 = 0.9982. It can be seen that the linearity of Example 1 is better. By detecting the fermentation broth of halophilic bacteria with high, medium, and low concentrations of urea respectively, the recovery rate of the method in Example 1 is closer to 100%, and it is more accurate.

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for detecting urea in a halophilic bacteria fermentation broth, characterized in that, Including preparing a standard curve and detecting the fermentation broth of the halophilic bacteria to be measured; Among them, the method for preparing the standard curve includes: Preparing a standard urea solution for use with a first solvent; the first solvent is a halophilic bacteria fermentation medium without urea; First, take the standard urea solution for use, add a diacetyl monoxime-isopropanol solution with a concentration of 1%-2% respectively, and mix well; then add an antipyrine solution with a concentration of 2%-4%, and mix well; perform a boiling water bath for 10-20 minutes; then make up the volume with the first solvent so that the urea concentration in the series of solutions is 0-0.5 mg / mL; using the first solvent as a control, measure the absorbance values of each series of solutions; prepare a standard curve with the urea content against the absorbance value; The method for detecting the fermentation broth of the halophilic bacteria to be measured includes: taking the supernatant of the fermentation broth of the halophilic bacteria to be measured and performing detection according to the same method as that for preparing the standard curve.

2. The detection method of urea in the halophilic bacteria fermentation broth according to claim 1, characterized in that The first solvent is the supernatant of the halophilic bacteria fermentation medium without urea; and / or, The method for detecting the fermentation broth of the halophilic bacteria to be measured includes: taking the supernatant of the fermentation broth of the halophilic bacteria to be measured, adding a diacetyl monoxime-isopropanol solution with a concentration of 1%-2%, and mix well; then add an antipyrine solution with a concentration of 2%-4%, and mix well; perform a boiling water bath for 10-20 minutes; then make up the volume with the first solvent; using the first solvent as a control, measure the absorbance value; according to the measured absorbance value and the standard curve, obtain the urea content in the fermentation broth of the halophilic bacteria to be measured.

3. The method for detecting urea in the halophilic bacteria fermentation broth according to claim 1 or 2, characterized in that, The halophilic bacteria fermentation broth is obtained by fermenting the halophilic bacteria with the halophilic bacteria fermentation medium; Optionally, the halophilic bacteria are halophilic bacteria strains capable of fermenting and producing PHA; Optionally, the halophilic bacteria are Halomonas; Optionally, in the halophilic bacteria fermentation medium, the salt concentration is 5-60 g / L; Optionally, the halophilic bacteria fermentation medium is a medium capable of fermenting and producing PHA; Optionally, the halophilic bacteria fermentation medium includes: 1-10 g / L of urea, 5-25 g / L of glucose, 5-60 g / L of sodium chloride, 0.2-2 g / L of magnesium sulfate, 2-10 g / L of potassium dihydrogen phosphate, 0.1-2 g / L of ammonium ferric citrate, 0.01-0.1 g / L of calcium chloride; Optionally, the halophilic bacteria fermentation medium includes: 2-5 g / L of urea, 10-25 g / L of glucose, 15-30 g / L of sodium chloride, 0.2-2 g / L of magnesium sulfate, 2-8 g / L of potassium dihydrogen phosphate, 0.1-2 g / L of ammonium ferric citrate, 0.01-0.1 g / L of calcium chloride, 0.0002-0.001 g / L of zinc sulfate heptahydrate, 0.0001-0.001 g / L of manganese chloride tetrahydrate, 0.001-0.005 g / L of boric acid, 0.0001-0.001 g / L of cobalt chloride hexahydrate, 0.0001-0.002 g / L of copper sulfate pentahydrate, 0.0002-0.005 g / L of nickel chloride hexahydrate.

4. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-3, characterized in that, In the method for preparing the standard curve, after volume fixation, the concentrations of urea in the series of solutions are 0, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.4 mg / mL, and 0.5 mg / mL; Optionally, in the method for preparing the standard curve, after volume fixation, the volume of the series of solutions is 10 mL.

5. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-4, characterized in that, The diacetyl monoxime - isopropanol solution is prepared from diacetyl monoxime and 10% acetic acid and isopropanol with a volume ratio of 1:

1.

6. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-5, characterized in that, In the method for preparing the standard curve, the weight ratio of urea, diacetyl monoxime, and antipyrine is (0 - 5):(0.008 - 0.012):(0.03 - 0.05), optionally (0 - 5):0.01:0.

04.

7. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-6, characterized in that, In the method for preparing the standard curve, the volume ratio of the series of standard solutions, the diacetyl monoxime - isopropanol solution, and the antipyrine solution is 1:1:

2.

8. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-7, characterized in that, Measure the absorbance value at a wavelength of 440 - 480 nm, preferably at a wavelength of 460 nm; and / or, Use a 1 cm cuvette to measure the absorbance value.

9. The method for detecting urea in the halophilic bacteria fermentation broth according to any one of claims 1-8, characterized in that, Including: 1) Prepare the standard curve Prepare a urea standard working solution with the supernatant of the halophilic bacteria fermentation medium without urea; Use the supernatant of the halophilic bacteria fermentation medium without urea to perform gradient dilution on the urea standard working solution to obtain a series of standard solutions with urea concentrations of 0, 0.5 g / L, 1 g / L, 2.5 g / L, 4 g / L, and 5 g / L respectively; Take 1 mL of each of the series of standard solutions, add 1 mL of a 1% diacetyl monoxime - isopropanol solution to each, and mix well; then add 2 mL of a 2% antipyrine solution and mix well; perform a boiling water bath for 10 - 20 min; then use the supernatant of the halophilic bacteria fermentation medium without urea to fix the volume to 10 mL and mix well; using the supernatant of the halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm cuvette to measure the absorbance value; prepare a standard curve with the urea content against the absorbance value; 2) Detect the fermentation broth of the halophilic bacteria to be tested Take 1 mL of the supernatant of the fermentation broth of the halophilic bacteria to be tested, add 1 mL of a 1% diacetyl monoxime - isopropanol solution, and mix well; then add 2 mL of a 2% antipyrine solution and mix well; perform a boiling water bath for 10 - 20 min; then use the supernatant of the halophilic bacteria fermentation medium without urea to fix the volume to 10 mL and mix well; using the supernatant of the halophilic bacteria fermentation medium without urea as a control, at a wavelength of 460 nm, use a 1 cm cuvette to measure the absorbance value; according to the measured absorbance value and the standard curve, obtain the urea content in the fermentation broth of the halophilic bacteria to be tested.

10. Application of the method for detecting urea in the fermentation broth of halophilic bacteria according to any one of claims 1 - 9 in the process of halophilic bacteria fermentation; optionally, in the process of halophilic bacteria fermentation for producing PHA.

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