CN116818958B

CN116818958B - Method for measuring content of 5-aminolevulinic acid and glycine in fermentation broth

Info

Publication number: CN116818958B
Application number: CN202311068302.8A
Authority: CN
Inventors: 梁艺超; 华跃飞; 李爽; 张广民; 蔡辉益
Original assignee: Tianjin Bofeide Science & Technology Co ltd; Beijing Challenge Bio Technology Ltd
Current assignee: Tianjin Bofeide Science & Technology Co ltd; Beijing Challenge Bio Technology Ltd
Priority date: 2023-08-24
Filing date: 2023-08-24
Publication date: 2023-11-21
Anticipated expiration: 2043-08-24
Also published as: CN116818958A

Abstract

The invention relates to a method for measuring the content of 5-aminolevulinic acid and glycine in fermentation broth, which uses an HPLC-fluorescence method for measurement, wherein chromatographic conditions and programs are as follows: the mobile phase is: phase A: 0.1% trifluoroacetic acid solution; and B phase: acetonitrile; a gradient elution mode is adopted; chromatographic column temperature: 35 ℃; sample injection volume: 10. Mu.L; excitation wavelength: 398nm; emission wavelength: 480nm; flow rate: 1.4mL/min. The method can simultaneously detect the content of 5-aminolevulinic acid and glycine in the fermentation broth, solves the problems of unseparated peak shape, non-return of peak to baseline, asymmetric peak shape, tail sweeping of peak shape and the like in the spectrum when the conventional HPLC-fluorescence method is adopted for measurement, has good recovery rate and is suitable for production requirements; the method of the invention has short time, only needs 25min, has simple operation and high repeatability and accuracy, and provides powerful guarantee for efficiently producing 5-ALA.

Description

Method for measuring content of 5-aminolevulinic acid and glycine in fermentation broth

Technical Field

The invention relates to the field of feed component detection, in particular to a method for measuring the content of 5-aminolevulinic acid and glycine in fermentation broth.

Background

5-aminolevulinic acid (5-ALA) is a precursor for synthesis of tetrapyrroles and porphyrins in organisms, and is attracting increasing attention as a new generation of photodynamic agents for use as herbicides, pesticides, antimicrobial agents, plant growth promoters and medical diagnostics. However, little research has been done with respect to the fermentative production of 5-ALA, and in particular with respect to the timely and accurate monitoring of the target metabolites during fermentation. At present, the microbial fermentation technology is mainly adopted to produce 5-ALA, and the condensation of succinyl CoA and glycine into 5-ALA under the action of 5-ALA synthetase is a main way for producing 5-ALA by a current fermentation method.

In the current production of 5-ALA by using a microbial fermentation technology, glycine is required to be used as a precursor substance for 5-ALA synthesis, so that the glycine content and the 5-ALA content in fermentation need to be accurately measured in time in fermentation, the conversion rate of glycine and a product in the current stage is calculated, the microbial state is known, fermentation conditions are better controlled, and the product synthesis is promoted. In addition, 5-ALA is very unstable in solution, and temperature, concentration, pH and dissolved oxygen all cause it to condense two molecules to produce other substances. Therefore, it is important to find a rapid, simple, stable detection method.

The main detection methods of the content of 5-ALA and glycine at present are two types:

one is spectrophotometry: the current detection of 5-ALA content is mostly determined by using a chemochromic method, the principle of which is that 5-ALA and acetylacetone can be condensed in acetic acid-sodium acetate buffer solution with pH of 4.6 (water bath at 100 ℃ for 15 min) to generate pyrrole derivatives, and the pyrrole derivatives form orange-red substances in strong acid medium, and the detection is carried out under the wavelength of 553 nm. According to the method, a new standard solution is required to be prepared for drawing a new standard curve each time of measurement, the middle process is required to use perchloric acid, acetylacetone and other toxic strong acid dangerous chemicals, the operation process is complex, the time for each measurement is 2 hours, and in the 5-ala fermentation production, the consumption rate of a precursor substance and the synthesis rate of a synthetic substance are required to be tracked in time, so that the timely control of a production link is realized, and the method cannot be used for timely tracking of product measurement and the content of the precursor substance.

Secondly, detecting the dansyl chloride (DST): there are several reports currently in the literature of simultaneous glycine and 5-ALA determination using the dansyl chloride derivatization method, which exploits the affinity of dansyl chloride (DST) for dominant amine groups to convert the amine groups to dansyl amide groups under basic conditions, thereby allowing nitrogen-containing compounds in the analyte to form dansyl amide derivatives. However, the method has the advantages of longer derivatization time (1 h), complicated operation steps in the process, longer sample elution time, more impurity peaks in detection, more than 2 hours for each measurement, and incapability of timely tracking production.

Glycine and 5-ALA are two primary amine substances, and fluorescamine is a specific derivative of primary amine substances, can specifically generate fluorescence with the primary amine substances, and fluorescamine does not have fluorescence, and can be hydrolyzed into non-fluorescent substances in water, so that no interference is generated to the detection peak shape, the derivative time of fluorescamine and primary amine substances is shorter, the detection time can be obviously reduced, and therefore, the method optimization is carried out by adopting fluorescamine as a derivative reagent. However, because some primary amine-containing substances are generated in the fermentation liquid for producing 5-ALA by thallus fermentation, the primary amine-containing substances react with fluorescamine specifically, other miscellaneous peaks can be generated, the traditional detection method does not consider to separate the miscellaneous peaks, but the detection of 5-ALA and glycine can be influenced by the fact that the miscellaneous peaks are not separated.

Therefore, the prior art cannot meet the production requirement, so that a method with simple operation mode, short derivatization time and short overall detection time is urgently needed, and the method can effectively help grasp and adjust all important links in the production process so as to produce 5-ALA more efficiently.

Disclosure of Invention

In order to solve the problems, the invention provides a method for measuring the content of 5-aminolevulinic acid and glycine in fermentation broth.

The method for measuring the content of 5-aminolevulinic acid and glycine in the fermentation broth provided by the invention adopts an HPLC-fluorescence method for measurement, and comprises the following specific steps:

step 1), taking 5mL of fermentation liquor in a centrifuge tube, and centrifuging at 4000r/min for 5 minutes;

step 2), taking 0.1mL of supernatant in the centrifuge tube, placing the supernatant in a 100mL volumetric flask, fixing the volume, and shaking uniformly;

step 3) taking 100 mu L of the solution in the volumetric flask, putting the solution in a 1.5mL centrifuge tube, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 200 mu L of BBS buffer solution, adding 100 mu L of 0.5% triethylamine solution, oscillating for 1min by using an oscillator, standing for 3-5min, and microfiltering into a sample injection bottle for detection.

Wherein, chromatographic conditions and procedures are set as follows:

the mobile phase is: phase A: 0.1% trifluoroacetic acid solution; and B phase: acetonitrile; a gradient elution mode is adopted;

chromatographic column temperature: 35 ℃; sample injection volume: 10. Mu.L; excitation wavelength: 398nm; emission wavelength: 480nm; flow rate: 1.4mL/min.

Preferably, the BBS buffer is dissolved in 0.46g sodium borate tetradecahydrate and 0.51g boric acid to a volume of 100mL volumetric flask.

Preferably, the AB phase ratio and procedure of the gradient elution mode are shown in the following table:

the beneficial effects of the invention are as follows:

1. the method can simultaneously detect the content of 5-aminolevulinic acid and glycine in the fermentation liquid.

2. The method of the invention is optimized in detail, and solves the problems of unseparated peak shape, non-return of peak to baseline, asymmetric peak shape, tail sweeping of peak shape and the like in the map when the conventional HPLC-fluorescence method is adopted for measurement.

3. The method has good linear relation between 0.1-50ppm of 5-ALA and 0.5-50ppm of glycine, and the obtained result has high accuracy and good repeatability.

4. The method has good recovery rate and is suitable for production requirements.

5. The method provided by the invention can be used for simultaneously detecting the contents of 5-aminolevulinic acid and glycine in the fermentation broth, has the advantages of short time consumption, much faster output result than the average 2 hours of the existing detection technology, simple operation and high repeatability and accuracy, and provides a powerful guarantee for efficiently producing 5-ALA.

Drawings

FIG. 1 is a partial peak shape screenshot of the detection map of example 1;

FIG. 2 is a partial peak shape screenshot of the detection map of example 2.1;

FIG. 3 is a partial peak shape screenshot of the detection map of example 2.2;

FIG. 4 is a partial peak shape screenshot of the detection map of example 2.3;

FIG. 5 is a partial peak shape screenshot of the detection map of example 2.4;

FIG. 6 is a partial peak shape screenshot of the detection map of example 2.5;

FIG. 7 is a partial peak shape screenshot of the detection map of example 2.6;

FIG. 8 is a partial peak shape screenshot of the detection map of example 2.7;

FIG. 9 is a partial peak shape screenshot of the detection map of example 2.8;

FIG. 10 is a partial peak shape screenshot of the detection map of example 2.9;

FIG. 11 is a partial peak shape screenshot of the detection map of example 2.10;

FIG. 12 is a partial peak shape screenshot of the detection map of 2.11 in example 2;

FIG. 13 is a partial peak shape screenshot of the detection map of 2.12 in example 2;

FIG. 14 is a glycine standard curve of example 3;

FIG. 15 is a 5-ALA standard curve for example 3;

fig. 16 is a partial peak shape screenshot of the mixed label detection spectrum of example 3.

Detailed Description

The invention is further illustrated by the following examples.

Example 1: detecting 5-ALA and glycine content in fermentation broth by conventional HPLC-fluorescence method

Preparation of 5-ALA fermentation broth: and (3) after rejuvenating the strain of the 5-ALA production strain, inoculating the strain into a 1L shaking flask to prepare the 5-ALA strain shaking flask, inoculating the 5-ALA seed shaking flask into a 5L fermentation tank after the culture reaches the inoculation requirement, and carrying out induced expression after the culture for a period of time to obtain the 5-ALA fermentation liquor.

step 2), taking 0.1mL of supernatant, placing the supernatant into a 100mL volumetric flask, fixing the volume, and shaking uniformly;

step 3) taking 100 mu L of solution into a 1.5mL centrifuge tube, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 300 mu L of BBS buffer solution (0.46 g sodium borate tetrahydrate and 0.51g boric acid are dissolved and fixed to a 100mL volumetric flask), oscillating for 1min by using an oscillator, standing for 3-5min, and then micro-filtering into a sample injection bottle for detection

Detection conditions for the method of example 1: chromatographic column: diamond C18 (2) 5 μm 250X 4.6mm

Mobile phase: phase A: 0.1% trifluoroacetic acid solution, phase B: acetonitrile; phase A: phase B = 7:3, a step of;

chromatographic column temperature: 40 ℃; sample injection volume: 20. Mu.L; excitation wavelength: 398nm; emission wavelength: 480nm; flow rate: 1.5mL/min.

The spectrum obtained in example 1 is shown in FIG. 1, and it has been confirmed that the peak shape of 1# is glycine peak shape and the peak shape of 3# is 5-ALA peak shape by using an internal standard method for fermentation broth, and that the peak shape of 2# is interference peak shape, and that the interference peak is not separated from the peaks of 1# and 3#, and the measurement result is affected.

As can be seen from FIG. 1, the conventional HPLC-fluorescence method is adopted to detect the 5-ALA and glycine contents in the fermentation broth, and the obtained spectrum has the problems of unseparated peak shape, unsetting peak, base line regression and asymmetric peak shape, so that the problems of peak shape separation, base line regression and the like are required to be optimized.

Example 2: optimized and improved screening experiments for conventional HPLC-fluorescence methods

With the fermentation broth of example 1,

2.1 Reducing the proportion of mobile phase B

The flow phase was adjusted to a: b=8: 2, the rest of the procedure is the same as in example 1.

2.1 As shown in FIG. 2, the peak elution effect was poor by decreasing the proportion of mobile phase B, and the elution ability for two amino acids was reduced due to the decrease of phase B, and the effective elution was not performed.

2.2 Increasing the proportion of mobile phase B

The flow phase was adjusted to a: b=6: 4, the rest of the procedure is the same as in example 1.

2.2 the obtained pattern is shown in fig. 3, increasing the proportion of mobile phase B, from phase a: phase B = 7:3 to phase a: phase B = 6: and 4, the peak shapes are gathered together, and the separation effect is worse, so that the peak shapes can not be effectively separated while the peak shapes can not be maintained by simply adjusting the proportion of the isocratic elution mobile phase, and the peak shapes are separated by taking gradient elution into consideration on the basis of analyzing the influence of other factors on the peak shape effect.

2.3 The flow rate of the mobile phase is reduced to 1.2mL/min, and the sample injection amount is reduced to 10 mu L

The procedure of example 1 was repeated except that the flow rate of the mobile phase was reduced from 1.5mL/min to 1.2mL/min and the sample injection amount was reduced from 20. Mu.L to 10. Mu.L.

The flow velocity of the mobile phase is reduced, the peak-out time is adjusted by changing the flow velocity of the mobile phase, so that the peak shape is separated, the sample injection amount is reduced, and the problems that the peak shape has a front-extending peak and a base line does not fall back are attempted to be solved.

2.3 As shown in FIG. 4, reducing the sample injection amount to 10. Mu.L has a certain help to the fall-back of peak shape, and the sample injection amount of 10. Mu.L can be adopted in the subsequent experiments; at the same time, the flow rate of the mobile phase is reduced to 1.2mL/min, so that the No. 2 peak cannot be eluted, the peak spread is large, and a certain peak distance can be increased.

2.4 The flow rate of the mobile phase is reduced to 1.4mL/min, and the sample injection amount is reduced to 10 mu L

The procedure of example 1 was repeated except that the flow rate of the mobile phase was reduced from 1.5mL/min to 1.4mL/min and the sample injection amount was reduced from 20. Mu.L to 10. Mu.L.

2.4 As shown in FIG. 5, decreasing the flow rate of the mobile phase to 1.4mL/min can elute the peak shape of 2# and combining 2.3 and 2.4 shows that decreasing the flow rate can increase the peak shape spacing, but too low a flow rate can affect the elution effect of the peak shape; the reduction of the sample injection amount can solve the problem that the peak shape falls back to a certain extent, and the subsequent sample injection amount of 10 mu L is adopted.

2.5 Reducing the temperature of the chromatographic column to 30 DEG C

The column temperature was reduced from 40℃to 30℃while the sample loading was reduced from 20. Mu.L to 10. Mu.L, in the same manner as in example 1.

The effect of temperature singles on peak appearance was further examined by varying column temperature.

2.5 As shown in FIG. 6, at a column temperature of 30 ℃, the front peaks # 1 and # 2 were not separated and the overall elution effect was poor, so that the decrease in column temperature had a large effect on the elution of the target.

2.6 Reducing the temperature of the chromatographic column to 35 DEG C

The column temperature was set at 35℃and the remainder was 2.5 as in example 2.

2.6 As shown in FIG. 7, the peak effect is affected by the column temperature as shown in FIG. 6 and FIG. 7, and the elution ability for the target substance is deteriorated as the column temperature is lowered, but the distance between peaks is increased by lowering the column temperature, and the peak-to-peak interval in FIG. 7 is ideal.

2.7 use of other alkaline buffers

The same procedure as in example 1 was repeated except that the BBS buffer was changed to 0.01mol/L phosphate buffer, pH 7.9, and the sample amount was reduced from 20. Mu.L to 10. Mu.L.

2.7 the profile obtained is shown in FIG. 8, and after phosphate buffer was used, the peak shape was disordered and was unfavorable for peak shape separation, so BBS buffer should be used continuously.

2.8 By gradient elution

On the basis of the influence of the single factors on the peak, the gradient elution mode is further optimized, the gradient elution program A is shown in the table 1, the sample injection amount is reduced from 20 mu L to 10 mu L, and the rest is the same as the example 1.

In order to separate the middle peak, the B mobile phase ratio was reduced after 7min, changing the middle peak out time.

2.8 the spectrum obtained is shown in fig. 9, the last peak form is poor in elution effect, but the last peak form is effectively separated from the previous two peaks, and the 2# peak form is not effectively separated, which means that the elution effect is poor due to the reduction of the mobile phase B, and the proportion of the mobile phase B needs to be improved, so that the 2# peak can be eluted, and the elution procedure is optimized again for the problems.

2.9 Improved gradient elution mode

Gradient elution was performed by the same method as in example 2, except that the gradient elution procedure B is as shown in Table 2, which is 2.8.

On the basis of the 2.8 method, the mobile phase B proportion at the beginning and the mobile phase B proportion at the time of 8-20min are increased, the eluting capacity is increased, and the eluting effect of peak shape is improved.

2.9 the pattern obtained is shown in fig. 10, the peak shape is approximately separated, but the 1# and the 2# are still connected together to form a shoulder peak, the two peaks still need to be separated, the peak shape of the 1# and the 2# is further separated by downwards regulating the temperature and the flow rate of the mobile phase, and prolonging the peak outlet time.

2.10 Continuing to improve gradient elution mode

The column temperature of the lower chromatography was 35℃and the flow rate was reduced to 1.4mL/min, with the remainder being 2.9 as in example 2.

2.10 As shown in FIG. 11, after the flow rate of the mobile phase is reduced and the column temperature is adjusted downwards, the peak shape is further separated, and the phenomenon that the peak shape does not fall back still exists between the No. 1 peak and the No. 2 peak is further regulated, so that the No. 1 peak is earlier out, and the No. 2 peak out time is delayed.

2.11 Again improving the gradient elution mode

The gradient elution procedure was adjusted on the basis of 2.10, and the gradient elution procedure C is shown in table 3.

The proportion of the phase B is kept at 35% in the initial 5 minutes, the peak-out time of the phase 1 is quickened, the phase 1 is eluted for 6-9 minutes as the normal elution concentration, the phase B proportion is reduced, the peak-out time of the phase 2 is prolonged, the phase B proportion is kept at the normal state after the phase 2 is out, and the phase 3 is eluted.

2.11 the pattern obtained is shown in fig. 12, the required peak shape is basically obtained, but the peak shape of the No. 1 peak has a tailing problem, so that the peak shape is asymmetric, the peak shape needs to be further optimized, and the addition of a tail sweeping agent triethylamine solution is considered for tail sweeping.

2.12 Improved tail sweeping

On the basis of 2.11, the dosage of BBS solution is reduced from 300 mu L to 200 mu L, and simultaneously 100 mu L of 0.5% triethylamine solution is added in a supplementary way, so that the problem of tailing of the No. 1 peak is solved.

2.12 the obtained pattern is shown in figure 13, and after 100 mu L of 0.5% triethylamine solution is added to replace 100 mu L of BBS solution, the tail sweeping problem of the No. 1 peak is solved, the peak shapes are effectively separated, the symmetry of the peak shapes is good, and the detection method meeting the actual production is obtained.

In summary, the method of the invention is 2.12, and the 5-aminolevulinic acid and glycine contents of the fermentation broth are determined in production practice. The method comprises the following steps:

step 3) taking 100 mu L of the solution in the volumetric flask, putting the solution in a 1.5mL centrifuge tube, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 200 mu L of BBS buffer solution, adding 100 mu L of 0.5% triethylamine solution, oscillating for 1min by using an oscillator, standing for 3-5min, and carrying out microfiltration sample injection on a liquid chromatograph; wherein, chromatographic conditions and procedures are set as follows:

The BBS buffer is dissolved and fixed in a 100mL volumetric flask with 0.46g sodium borate tetradecahydrate and 0.51g boric acid.

The AB phase ratio and procedure for the gradient elution mode are shown in table 3, gradient elution procedure C.

Example 3 glycine, 5-ALA standards and mixed standards of both were tested and standard curves were drawn using the method of the invention.

Test reagent:

reagent (1): precisely weighing 0.126g of 5-ALA hydrochloride in a 100mL volumetric flask, fixing the volume, shaking uniformly to obtain a solution with the concentration of 1000 mug/mL, then taking 10mL of the solution in the 100mL volumetric flask, fixing the volume, shaking uniformly to obtain a 5-ALA standard mother solution with the concentration of 100 mug/mL

Reagent (2): precisely weighing 0.1g of glycine (AR) in a 100mL volumetric flask, fixing the volume, shaking uniformly to obtain a solution with the concentration of 1000 mug/mL, then taking 10mL of the solution in the 100mL volumetric flask, fixing the volume, and shaking uniformly to obtain 100 mug/mL glycine standard mother liquor.

Test protocol:

1. glycine, 5-ALA single standard curve plot: respectively diluting the reagent (1) according to 0.5, 1, 5, 10, 20 and 50ppm to obtain a gradient solution, then carrying out sample treatment, sample injection and measurement according to a method 2.12, and finally drawing a standard curve; reagent (2) was subjected to the same operation, and a standard curve was finally drawn, as shown in tables 4 and 5, and fig. 14 and 15.

2. Glycine, 5-ALA mix test: sequentially adding 1mL of reagent (1) and 1mL of reagent (2) into a 10mL volumetric flask, diluting with ultrapure water, and fixing the volume to obtain a liquid to be detected containing glycine and 5-ALA with the content of 10ppm, and processing according to a 2.12 method to obtain a map (see figure 16), wherein the peak-forming time and peak-forming area of glycine and 5-ALA in the map are consistent with the peak-forming time and area of each single mark, which indicates that the two substances cannot interfere with detection after mixing. As can be seen from FIG. 16, the detection result of the mixture of 5-ALA and glycine standard product shows no impurity peak, and the fact that other substances which react with fluorescamine are generated in the process of producing 5-ALA by fermentation of strains is further explained, so that the content measurement of 5-ALA and glycine in fermentation liquor is influenced, so that an improved detection method is needed to separate the impurity peak, the content of 5-ALA and glycine is obtained more accurately, and the production practice can be guided better.

Fig. 14 shows a glycine standard curve, fig. 15 shows a 5-ALA standard curve, and fig. 16 shows a mixed standard map.

The results of this example show that the method of the invention provides a good linear relationship between 5-ALA and glycine standard solution (R ² =1), glycine has a good linear relationship between 0.5-50ppm (R ² =0.9991）。

Example 4 recovery measurement of the present method

The recovery of the process was determined by adding low, medium and high concentrations of 5ALA and glycine to the fermentation broth for standard sample recovery.

1) The preparation method of the reagent in the test comprises the following steps:

reagent (1): precisely weighing 0.256g of 5-ALA hydrochloride in a 100mL volumetric flask, fixing the volume, shaking uniformly to obtain a solution with the concentration of 2000 mug/mL, then taking 1mL of the solution in a 10mL volumetric flask, fixing the volume, and shaking uniformly to obtain a 5-ALA standard solution with the concentration of 200 mug/mL;

reagent (2): precisely weighing 0.2g of glycine (AR) in a 100mL volumetric flask, fixing the volume, shaking uniformly to obtain a solution with the concentration of 2000 mug/mL, then taking 1mL of the solution in a 10mL volumetric flask, fixing the volume, and shaking uniformly to obtain a glycine standard solution with the concentration of 200 mug/mL;

reagent (3): mixing the reagent (1) and the reagent (2) standard solution together, and uniformly mixing to obtain a standard solution containing 100 mug of 5-ALA and glycine per milliliter for later use;

reagent (4): taking a reagent (3) (1 mL in a 10mL volumetric flask, fixing the volume, and shaking uniformly to obtain a standard solution containing 10 mug of 5-ALA and glycine per milliliter for later use;

reagent (5): taking 5mL of fermentation liquor in a centrifuge tube, centrifuging at 4000r/min for 5min, taking 0.1mL of supernatant in a 100mL volumetric flask, fixing the volume, and shaking uniformly for later use;

2) Low concentration 5-ALA (5 ppm) with glycine (5 ppm) recovery test:

taking reagent (5. Mu.L, reagent (4. Mu.L in a 1.5mL centrifuge tube), shaking and mixing uniformly, adding 100. Mu.L of 0.1% fluorescamine acetone solution, adding 200. Mu.L of BBS buffer solution (0.46 g sodium borate tetradecahydrate and 0.51g boric acid are dissolved and fixed to a volume of 100mL volumetric flask), adding 100. Mu.L of 0.5% triethylamine solution, shaking for 1min by using a shaker, standing for 3-5min, and carrying out microfiltration and sample injection.

Control: the reagent (4) in this method was replaced with 50. Mu.L of ultrapure water, and the remaining steps were the same, and the recovery rate was calculated by performing three operations.

3) Medium concentration 5-ALA (20 ppm) with glycine (20 ppm) recovery test:

taking 80 mu L of reagent (5 mu L and 20 mu L of reagent (3 mu L in a 1.5mL centrifuge tube), shaking and mixing uniformly, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 200 mu LBBS buffer solution (0.46 g sodium borate tetradecyl water and 0.51g boric acid are dissolved and fixed to a 100mL volumetric flask), adding 100 mu L of 0.5% triethylamine solution, shaking for 1min by using a shaker, standing for 3-5min, and carrying out microfiltration sample injection.

4) High concentration 5-ALA (40 ppm) and glycine (40 ppm) recovery test:

taking 60 mu L of reagent (5 mu L and 40 mu L of reagent (3 mu L in a 1.5mL centrifuge tube), shaking and mixing uniformly, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 200 mu LBBS buffer solution (0.46 g sodium borate tetradecyl water and 0.5 g boric acid are dissolved and fixed to a 100mL volumetric flask), adding 100 mu L of 0.5% triethylamine solution, shaking for 1min by using a shaker, standing for 3-5min, and carrying out microfiltration sample injection.

As can be seen from table 7/8/9, the average recovery of glycine in 9 recovery runs was 99.9%, rsd=1.09 (n=9); the average recovery of 5-ALA was 100.7%, rsd=1.1 (n=9). Therefore, the method has good recovery rate and is suitable for production requirements.

Example 5 comparative test with Current detection methods

The same fermentation broth is detected by the method of the invention and the existing common detection method. The specific operation of each detection method is as follows:

spectrophotometry color development: (1) firstly, preparing 1mL of 0, 2, 4, 6 and 8ppm5-ALA standard solution (each time of preparation), and placing 1mL of a sample to be tested into a 15mL centrifuge tube. (2) Then 0.5mL of sodium acetate buffer (1M, PH4.6) and 25 mu L of acetylacetone are added in sequence respectively, the shaking is uniform, the centrifugation is carried out for one minute (4000 r/min), and the boiling water bath is carried out for 15min after the centrifugation. (3) Cooling to room temperature, shaking uniformly, centrifuging for one minute (4000 r/min), adding 1.525mL Ehrrish's reagent (42 mL glacial acetic acid, 8mL70% perchloric acid, 1g dimethylaminobenzaldehyde) into each centrifuge tube after centrifuging, shaking uniformly, developing color for 10min, detecting light absorption value at 553nm wavelength, drawing standard curve, substituting sample detection light absorption value after standard curve is obtained, and calculating

Pre-column derivatization of dansyl chloride (DST): (1) sample treatment: and (3) freezing and centrifuging the fermentation liquor for 5min to remove macromolecular impurities, adding trichloroacetic acid with the same volume of 40% into 0.2mL of supernatant, and centrifuging for 5min (13000 r/min) to obtain a sample to be detected. (2) 100ul of a sample to be detected is taken, 100ul of dansyl chloride solution (28.8 mmol/L) is added, the dansyl chloride solution is led for 1h at 30 ℃ in a dark place, 200 ul of hydrochloric acid solution (0.01 mol/L) is taken for stopping the reaction, 0.9mL of water is added for dilution, and the sample is subjected to microfiltration sample injection measurement on a conventional ultraviolet detector.

The operation of the present invention was the same as that of 2.12 in example 2.

The comparison of the 3 methods is shown in table 10:

comparison by the different methods described above can be seen:

1. the method can detect the content of 5-ALA and glycine in the fermentation broth at the same time, and the time spent by the method is only 25 minutes, so that the method has good timeliness, can meet the timely monitoring in production, and the average detection duration in the prior art is 2 hours;

2. the concentration range for detecting the content of 5-ALA and glycine by the method is wide;

3. the method has the advantages of short pretreatment time, reduced pretreatment steps, no need of protein removal, great optimization of derivatization time and conditions, shortened detection time from 1 hour to 3-5 minutes, and more contribution to the accuracy of test operation.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. A method for measuring the content of 5-aminolevulinic acid and glycine in fermentation broth is characterized by comprising the following specific steps of:

step 2), taking 0.1ml of supernatant in the centrifuge tube, placing the supernatant in a 100ml volumetric flask, and shaking the supernatant uniformly by using ultrapure water to fix the volume;

step 3), taking 100 mu L of the solution in the volumetric flask, putting the solution in a 1.5mL centrifuge tube, adding 100 mu L of 0.1% fluorescamine acetone solution, adding 200 mu L of BBS buffer solution, adding 100 mu L of 0.5% triethylamine solution, oscillating for 1min by using an oscillator, standing for 3-5min, and microfiltering the solution into a sample injection bottle for detection;

wherein, chromatographic conditions and procedures are set as follows:

chromatographic column temperature: 35 ℃; sample injection volume: 10. Mu.L; excitation wavelength: 398nm; emission wavelength: 480nm; flow rate: 1.4mL/min;

the AB phase ratio and procedure for the gradient elution mode are shown in the table below:

。

2. the method for measuring the content of 5-aminolevulinic acid and glycine in fermentation broth according to claim 1, wherein the BBS buffer is dissolved in a volumetric flask of 0.46g sodium borate tetradecahydrate and 0.51g boric acid to a volume of 100 mL.