Method for measuring content of mono-rhamnolipid and di-rhamnolipid through nuclear magnetic resonance hydrogen spectrum

Abstract

The invention provides a method for determining the content of mono-rhamnolipid and di-rhamnolipid by using a nuclear magnetic resonance hydrogen spectrum, which comprises the following steps: mixing sodium p-hydroxybenzoate as an internal standard substance with a deuterated reagent and a sample to be detected to prepare a sample solution, and scanning to obtain the nuclear magnetic resonance hydrogen spectrum; and substituting the characteristic peak area A internal standard at the delta 6.61-6.69 ppm, the characteristic peak area A single at the delta 4.60-4.63 ppm and the characteristic peak area A double at the delta 4.66-4.69 ppm into a formula for calculation to obtain the concentration of single rhamnolipid in the sample solution or the concentration of double rhamnolipid in the sample solution. The method does not depend on a rhamnolipid standard substance, can directly quantify the content of mono-rhamnolipid and di-rhamnolipid in a sample, has the characteristics of high sensitivity, strong anti-interference capability, good reproducibility and the like, is simple in sample pretreatment method, and breaks through the limitation of a traditional analysis method.

Description

Technical Field

[0001] The present invention belongs to the technical field of rhamnolipid detection, and specifically relates to a method for rapidly and accurately determining the contents of mono- and di-rhamnolipids by proton nuclear magnetic resonance spectroscopy. Background Art

[0002] As a class of excellent biosurfactants, rhamnolipids not only have excellent surface activity and interfacial activity, but also show broad application prospects in many fields such as cosmetics, medicine, agriculture, environmental remediation, and petrochemical industry due to their non-toxic and biodegradable properties. These biosurfactants synthesized by microorganisms exist in the form of mixtures in nature, and their unique performance advantages make them an important research object in the fields of green chemistry and sustainable development.

[0003] From the perspective of molecular structure, rhamnolipids belong to anionic biosurfactants, and their structural characteristics are manifested as 1-2 rhamnose sugars linked to 1-2 fatty acid chains of different lengths (saturated or unsaturated). Due to the changes in the number of rhamnose units, the length and degree of unsaturation of fatty acid chains, the molecular structure of rhamnolipids is very diverse and rich. With the improvement of the sensitivity of analytical methods, the newly discovered molecular structures of rhamnolipids have been increasing. Although more than 60 molecular variants of rhamnolipids are currently known, the rhamnose unit structure part is relatively simple, and rhamnolipids can be clearly divided into two major categories: mono-rhamnolipids and di-rhamnolipids according to the rhamnose unit structure. It should be noted that these two types of rhamnolipids show significantly different physical and chemical properties and application characteristics due to their structural differences, which makes it particularly important to accurately quantify them separately.

[0004] However, the quantitative analysis of rhamnolipids currently faces many technical bottlenecks. Traditional methods such as dry weight method, anthrone-sulfuric acid method, phenol-sulfuric acid method, critical micelle concentration method, and oil diffusion method can only measure the total rhamnolipid content, and the accuracy is poor. The HPLC-UV derivatization method for quantitative analysis of rhamnolipids (CN 115290765 B) is cumbersome to operate, and the efficiency of the derivatization reaction is uncertain; the HPLC-MS / MS method for quantitative analysis of rhamnolipids is accurate, but the equipment is expensive, the sample pretreatment is complex, and the data analysis is difficult; although the HPLC-ELSD instrument can quickly detect and quantify rhamnolipids through rhamnolipid standards, there is currently no commercially available high-purity rhamnolipid sample (>98%), which is the main limiting factor.

[0005] The acquisition of rhamnolipid standards currently faces multiple technical challenges. First, their production relies entirely on the isolation and extraction of microbial fermentation products. However, different strains, under varying process conditions, can produce structurally diverse rhamnolipids, resulting in cumbersome and inefficient extraction and purification procedures, making it difficult to obtain pure products of a single structure. Second, existing commercially available rhamnolipid standards (such as those from AGAE, Inc., USA) are mixtures of multiple structures, exhibiting significant batch-to-batch variability in component ratios, making them incompatible with the stringent consistency requirements for quantitative analysis. Furthermore, rhamnolipid standards exhibit inherently poor stability, susceptibility to moisture absorption during solid storage and decomposition in aqueous solutions, further complicating their preparation and storage. These factors collectively hinder the stable acquisition of high-purity, structurally well-defined rhamnolipid standards, severely hindering the advancement of related research and the standardization of analytical methods. Therefore, the development of a novel analytical method that can rapidly and accurately distinguish and quantify mono- and di-rhamnolipids, independent of standards, has become a critical and pressing need in this area of research. Summary of the Invention

[0006] In response to the above-mentioned problems in the prior art, the present invention provides a method for rapidly and accurately determining the content of mono- and di-rhamnolipids by proton nuclear magnetic resonance spectroscopy. This method selects characteristic peaks and optimizes the selection of deuterated reagents, and then uses an internal standard method to quantitatively determine mono- and di-rhamnolipids. This method does not rely on pure rhamnolipids, is simple, has high detection accuracy, strong anti-interference ability, and good reproducibility.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for determining the content of mono- and di-rhamnolipids by proton nuclear magnetic resonance spectroscopy comprises the following steps:

[0009] (1) Evenly mix the precisely weighed sample to be tested and the precisely weighed internal standard sodium p-hydroxybenzoate in a deuterated reagent to obtain a sample solution;

[0010] (2) scanning the sample solution using a nuclear magnetic resonance spectrometer to obtain a hydrogen nuclear magnetic resonance spectrum;

[0011] (3) In the H NMR spectrum obtained in step (2), the characteristic peak area A at δ 6.61~6.69 ppm is 内标 、Characteristic peak area A at δ4.60~4.63 ppm 单 and the characteristic peak area A at δ 4.66~4.69 ppm 双 Substitute into formula (1) or formula (2) to calculate and obtain the concentration C of monorhamnolipid in the sample solution 单 Or the concentration C of dirhamnolipid in the sample solution 双 :

[0012] (1)

[0013] (2)

[0014] Among them, C 内标 is the molar concentration of the internal standard substance in the sample solution, and the units of C 内标 and C 单 or C 双 are the same.

[0015] Preferably, the molar ratio of the sample to be measured to the internal standard substance is 1:1.

[0016] Preferably, the sample to be measured is rhamnolipid obtained from microbial fermentation.

[0017] To know the mass fraction of mono-rhamnolipid or di-rhamnolipid in the sample to be measured, the method further includes the following steps:

[0018] (4) Substitute the C 单 or C 双 obtained in step (3) into formula (3) or formula (4) to obtain the mass fraction P 单 of mono-rhamnolipid or the mass fraction P 单 of di-rhamnolipid in the sample to be measured.

[0019] (3)

[0020] (4)

[0021] Among them, m 待测 is the mass of the sample to be measured, and V is the volume of the sample solution.

[0022] Rhamnolipids are divided into two major categories: mono-rhamnolipid and di-rhamnolipid according to the number of sugar rings. Their hydrophobic chains are mainly C 10 -C 10 . For rhamnolipids with other chain lengths, on the one hand, their contents are relatively low, and on the other hand, their molar masses differ little from that of C 10 -C 10 . Therefore, using 504 (mono-rhamnolipid) and 650 (di-rhamnolipid) as the molar masses for calculation can achieve both accuracy and convenience in measurement.

[0023] Preferably, the sampling amount of the sample to be measured is 5 - 20 mM.

[0024] Preferably, the deuterated reagent is a mixed reagent of deuterated dimethyl sulfoxide (DMSO-d6) and deuterated methanol (CD3OD). More preferably, the deuterated reagent is a mixed reagent of deuterated dimethyl sulfoxide (DMSO-d6) and deuterated methanol (CD3OD) with a volume ratio of 4:1.

[0025] Preferably, the frequency of the nuclear magnetic resonance spectrometer is above 400 MHz.

[0026] Preferably, the sample to be tested is pretreated, and the pretreatment is selected from the following methods:

[0027] For the aqueous solution of the analyte, first centrifuge to collect the supernatant, then adjust the pH of the supernatant to 1.5 - 2.5, then let it stand at 3 - 5 °C for 10 - 16 h, centrifuge again, collect the solid and dry it to obtain the sample to be tested;

[0028] For the solid analyte, first dissolve the solid in an organic solvent, centrifuge to collect the supernatant, and then remove the organic solvent in the supernatant to obtain the sample to be tested.

[0029] Preferably, the method for adjusting the pH of the supernatant is: adding hydrochloric acid. More preferably, the concentration of the hydrochloric acid is 4 - 8 M.

[0030] Preferably, the organic solvent is ethanol.

[0031] Preferably, the method for removing the organic solvent in the supernatant is: rotary evaporation.

[0032] The beneficial effects of the present invention are as follows:

[0033] (1) Since the properties of deuterated reagents will affect the peak appearance of rhamnolipids. For example, the active hydrogen of rhamnolipids does not peak in CD3OD, and the peak overlap degree of rhamnolipids in DMSO-d6 is relatively high, which is not conducive to the reasonable attribution of peaks. At the same time, the positions of the water peak, solvent peak, etc. of different deuterated reagents are different, which will also interfere with the peaks of rhamnolipids, making it more difficult to study the 1H NMR spectrum of rhamnolipids and difficult to perform quantitative analysis. By selecting sodium p-hydroxybenzoate as the internal standard and selecting the characteristic hydrogen corresponding to the single and double rhamnolipids in the range of δ 4.5 - 5.0 ppm as the characteristic peak, and cooperating with a suitable deuterated reagent, the present invention ensures that the area of the characteristic hydrogen peak is not affected, and uses the peak area of the characteristic hydrogen of rhamnolipids for the quantitative calculation of rhamnolipids. The method is simple and has high accuracy.

[0034] (2) The mixed deuterated reagent of DMSO-d6 and CD3OD selected in the present invention is used for the nuclear magnetic resonance quantitative analysis of rhamnolipids. The introduction of CD3OD successfully eliminates the interference of active hydrogen (δ 4.4 - 4.8 ppm) on the characteristic hydrogen signal (δ 4.5 - 5.0 ppm), and at the same time maintains the complete separation of the characteristic hydrogen signals of single and double rhamnolipids. This mixed deuterated reagent not only avoids the interference of solvent peaks and water peaks, but also significantly improves the spectral resolution.

[0035] (3) In the present invention, without relying on pure rhamnolipid, the contents of mono-rhamnolipid and di-rhamnolipid in the rhamnolipid sample can be quickly quantified by an internal standard (sodium p-hydroxybenzoate) in a nuclear magnetic resonance spectrometer.

[0036] (4) The present invention has a high detection accuracy for rhamnolipids, and can accurately quantify when the proportion of a single structure is more than 5%.

[0037] (5) In the present invention, the rhamnolipid sample only needs simple pretreatment to carry out nuclear magnetic resonance analysis, with strong anti-interference ability and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the appearance diagram of the pure rhamnolipid obtained in Example 1 (I), where the left figure is mono-rhamnolipid and the right figure is di-rhamnolipid.

[0039] Figure 2 It is the LC-MS spectrum of the mono-rhamnolipid and its fragments obtained in Example 1 (II).

[0040] Figure 3 It is the LC-MS spectrum of the di-rhamnolipid and its fragments obtained in Example 1 (II).

[0041] Figure 4 It is the analysis diagram of the hydrogen peak attribution of the mono-rhamnolipid obtained in CD3OD in Example 1 (III).

[0042] Figure 5 It is the analysis diagram of the hydrogen peak attribution of the di-rhamnolipid obtained in CD3OD in Example 1 (III).

[0043] Figure 6 It is the nuclear magnetic resonance hydrogen spectrum of rhamnolipid in D2O in Example 1 (IV).

[0044] Figure 7 It is the nuclear magnetic resonance hydrogen spectrum of rhamnolipid in DMSO-d6 in Example 1 (IV).

[0045] Figure 8 It is the nuclear magnetic resonance hydrogen spectrum of rhamnolipid in a mixed reagent of DMSO-d6 and CD3OD in Example 1 (IV).

[0046] Figure 9 It is the determination diagram of the detection limit (mono:di = 1:20) of the rhamnolipid nuclear magnetic resonance hydrogen spectrum detection method in Example 1 (VII).

[0047] Figure 10 It is the determination diagram of the detection limit (mono:di = 20:1) of the rhamnolipid nuclear magnetic resonance hydrogen spectrum detection method in Example 1 (VII).

[0048] Figure 11It is a comparative graph of the anti-interference results of the rhamnolipid nuclear magnetic resonance hydrogen spectrum detection method in Example 1 (VIII).

[0049] Figure 12 It is a graph showing the influence of the rhamnolipid sample pretreatment method on the peak appearance of nuclear magnetic resonance hydrogen spectrum in Example 1 (IX).

[0050] Figure 13 It is a standard curve graph established by using the HPLC-ELSD detection method with pure mono- and di-rhamnolipids respectively in Comparative Example 1.

[0051] Figure 14 It is an HPLC-ELSD peak appearance graph of a commercially available rhamnolipid standard in Comparative Example 3. Specific implementation mode

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation modes and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0053] In the following examples, the rhamnolipid-producing strain used is Pseudomonas aeruginosa.YM4 strain, and the preservation number of this strain is CCTCC NO: M 2017494.

[0054] Example 1

[0055] (I) Preparation of pure mono- and di-rhamnolipids by column chromatography:

[0056] (1) In a laminar flow hood, inoculate the YM4 strain into LB liquid medium and culture overnight at 37 °C and 200 rpm to obtain a seed solution. Among them, the LB liquid medium includes the following components and mass concentrations: NaCl 10 g / L, peptone 10 g / L, yeast powder 5 g / L.

[0057] (2) In a laminar flow hood, transfer the seed solution to the fermentation medium at an inoculation amount of 2%, and shake and culture at 37 °C and 200 rpm for 60 h to obtain a fermentation broth. Among them, the fermentation medium includes the following components and mass concentrations: soybean oil 50 g / L, sodium nitrate 8 g / L, potassium dihydrogen phosphate 1.9 g / L, disodium hydrogen phosphate dodecahydrate 7.8 g / L, anhydrous calcium chloride 0.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, potassium chloride 1 g / L, sodium chloride 1 g / L, yeast powder 1 g / L, 2 mL / L trace elements (ferrous sulfate heptahydrate 0.5 g / L, copper sulfate pentahydrate 0.3 g / L, zinc sulfate heptahydrate 2.9 g / L, manganese sulfate heptahydrate 1.7 g / L), and adjust the initial pH of the fermentation medium to 7.0 with 2 M sodium hydroxide.

[0058] (3) The fermentation broth was centrifuged to remove the cells, and the supernatant was adjusted to a pH range of 1.5 - 2.5 with 6 M hydrochloric acid and allowed to stand at 4 °C for 12 h.

[0059] (4) The acid-precipitated solution was centrifuged to remove the supernatant, and the precipitate was dissolved in 0.2 M sodium hydroxide to one-tenth of the original volume, and finally the pH of the rhamnolipid solution was controlled at 7.0 - 7.3.

[0060] (5) The solution was freeze-dried, dissolved in ethanol, centrifuged to obtain the supernatant, and the ethanol was removed by rotary evaporation at 55 °C using a rotary evaporator to obtain the crude rhamnolipid.

[0061] (6) The above-mentioned crude rhamnolipid was purified by column chromatography with gradient elution. The process was monitored by spotting plates with a color-developing agent to achieve the separation of mono- and di-rhamnolipids. The eluent was dichloromethane, methanol, and 1% acetic acid, where the ratio of dichloromethane to methanol was from 20:1 to 7:1. The color-developing agent (α-naphthol, sulfuric acid, ethanol, water, 10.5 / 6.5 / 40.5 / 4.5, m / v / v / v) was heated for color development or the alkaline potassium permanganate solution was used for color development. The alkaline potassium permanganate solution was composed of potassium permanganate, potassium carbonate, sodium hydroxide, and water in a ratio of 2.5:20:0.25:40 (m / m / m / v).

[0062] (7) The eluate was collected, concentrated by rotary evaporation, and acetic acid was removed by vacuum pumping. An appropriate volume of pure water was added to dissolve the product, and the pH was adjusted to 7.0 - 7.3 with 0.2 M sodium hydroxide, and it was allowed to stand overnight at low temperature. After freeze-drying the aqueous solution, the pure rhamnolipid in the form of a white powder as shown in Figure 1 was obtained.

[0063] (II) Structure identification of rhamnolipid:

[0064] The pure rhamnolipid obtained in (I) was analyzed and identified by LC-MS, and the results are as shown in Figure 2 , 3 . The samples were Rha-C 10 -C 10 and Rha-Rha-C 10 -C 10 , showing an ion peak corresponding to Rha-C 10 -C 10 +NH4 + at m / z 522.3642, and an ion peak corresponding to Rha-Rha-C 10 -C 10 +NH4 + at m / z 668.4223. In addition, a characteristic peak at m / z 359.2793 was observed in the spectra of both samples, which was confirmed by analysis to be the protonated fragment peak of the fatty acid chain C 10-C 10 +H + 。

[0065] (III) Analyzing the structures of mono- and di-rhamnolipids by nuclear magnetic resonance hydrogen spectroscopy and explaining the characteristic hydrogen signals of the molecular structures of mono- and di-rhamnolipids:

[0066] In nuclear magnetic resonance hydrogen spectroscopy analysis, using CD3OD as the solvent can effectively suppress the signals of active hydrogens such as hydroxyl and carboxyl groups in rhamnolipid molecules through hydrogen-deuterium exchange, significantly simplifying the spectral analysis. Therefore, 20 mM pure rhamnolipid samples were prepared with CD3OD as the solvent and subjected to 500 MHz nuclear magnetic resonance analysis. The results are as Figure 4 、 5 shown. The obtained spectra are clear and characteristic. Through detailed assignment analysis of each characteristic peak in the spectra, the structural characteristics of mono- and di-rhamnolipids were further confirmed. This result provides accurate and reliable nuclear magnetic resonance hydrogen spectroscopy data support for the structural identification of mono-rhamnolipid and di-rhamnolipid.

[0067] Through in-depth comparative analysis of the nuclear magnetic resonance hydrogen spectra of mono- and di-rhamnolipids, it was found that the high structural similarity between them led to significant signal overlap in the δ 0.5 - 4.5 ppm range. Nevertheless, characteristic hydrogen signals due to the structural differences between the two could still be identified. For example, the characteristic peaks of the hydrogen atoms connected to the carbon atoms numbered 26, 32, 34, 36, and 38 in di-rhamnolipid, and the characteristic peak of the hydrogen atom connected to the carbon atom numbered 26 in mono-rhamnolipid.

[0068] Considering the interference from the peaks of hydrogen atoms at other positions of the compound in the δ 3.0 - 4.5 ppm range. Finally, the characteristic hydrogen signals in the interference-free range of δ 4.5 - 5.0 ppm were selected for further analysis and research, namely the characteristic hydrogen of the hydrogen atom connected to the carbon atom numbered 26 (the hemiacetal carbon of the sugar ring) in mono-rhamnolipid, and the characteristic hydrogen of the hydrogen atoms connected to the carbon atoms numbered 26 and 36 (the hemiacetal carbons of the sugar ring) in di-rhamnolipid.

[0069] (IV) Screening deuterated solvents suitable for mixed rhamnolipids:

[0070] Based on the above research results, the present invention preferably selects the characteristic hydrogen signals in the interference-free range of δ 4.5 - 5.0 ppm for further analysis.

[0071] By measuring the relative peak areas of characteristic hydrogens of mono- and di-rhamnolipids, the relative ratio between the two can be accurately determined. However, when CD3OD is used as the solvent, its water peak (δ 4.87 ppm) seriously interferes with the analysis of labile hydrogens in this interval. Therefore, the present invention further investigated the effects of different deuterated solvents (deuterochloroform, heavy water, DMSO-d6, mixed deuterated reagents) on the analysis of characteristic hydrogens of rhamnolipids to optimize the detection conditions of 1H NMR and improve the accuracy and reliability of the analysis.

[0072] (1) Deuterochloroform (CDCl3) has low solubility for rhamnolipids, so CDCl3 is not suitable for the NMR quantitative analysis of rhamnolipids.

[0073] (2) As can be seen from Figure 6 , heavy water (D2O) has poor splitting effect on the labile hydrogen peaks of rhamnolipids, and the solvent peak of heavy water (δ 4.79 ppm) also seriously interferes with the analysis of labile hydrogens. Rhamnolipids have excellent foaming properties in aqueous solution, and foaming problems will also occur during the preparation of samples with heavy water. Therefore, heavy water is not suitable for the NMR quantitative analysis of rhamnolipids.

[0074] (3) As can be seen from Figure 7 , in the DMSO-d6 solvent, although the characteristic hydrogen signals of mono- and di-rhamnolipids in the range of δ 4.5 - 5.0 ppm can be completely separated and are not interfered by solvent peaks or water peaks, the presence of the labile hydrogens of the sugar ring hydroxyl groups (δ 4.4 - 4.8 ppm, showing a broad peak feature) still has a significant impact on the analysis of the characteristic hydrogen signals in this region. Specifically, the broad peak of the hydroxyl labile hydrogen will partially overlap the characteristic hydrogen signals, resulting in deviation in the calculation of the integral area of the characteristic hydrogens, thereby reducing the accuracy of the quantitative analysis.

[0075] (4) As can be seen from Figure 8 , the mixed deuterated reagent prepared by mixing DMSO-d6 and CD3OD in a volume ratio of 4:1 can effectively solve the key problems in the analysis of rhamnolipids. In this mixed solvent system, the introduction of CD3OD successfully eliminates the interference of labile hydrogens (δ 4.4 - 4.8 ppm) on the characteristic hydrogen signals (δ 4.5 - 5.0 ppm), while maintaining the complete separation of the characteristic hydrogen signals of mono- and di-rhamnolipids. It should be noted that this mixed solvent system not only avoids the interference of solvent peaks and water peaks, but also significantly improves the spectral resolution. Therefore, the DMSO-d6 / CD3OD (4:1, v / v) mixed deuterated reagent is proven to be the optimal solvent choice for the NMR quantitative analysis of rhamnolipid mixtures. In this deuterated reagent, the characteristic hydrogen signal of mono-rhamnolipid is located at δ 4.62 ppm, and di-rhamnolipid has two characteristic hydrogen signals, located at δ 4.80 ppm and δ 4.68 ppm respectively.

[0076] (V) Selecting an internal standard suitable for the nuclear magnetic resonance hydrogen spectrum detection method of rhamnolipid:

[0077] This example is used to illustrate the preferred results of the internal standard. Based on the research results in (IV), for the hydrogen signal range (δ 0.5 - 6.0 ppm) of rhamnolipid in the mixed deuterated reagent, the applicability of five candidate internal standards, namely maleic acid, phenol, methyl p - benzoate, sodium benzoate, and sodium p - hydroxybenzoate, was systematically investigated. An ideal internal standard needs to meet key requirements such as the nuclear magnetic resonance signal completely avoiding the analysis range, stable chemical properties, and good solubility in the solvent. The experimental results show (Table 1) that maleic acid is not applicable because its strong acidity easily causes sample precipitation, phenol has a highly toxic safety hazard and needs to be prohibited, methyl p - benzoate has insufficient solubility in this deuterated reagent, the hydrogen spectrum of sodium benzoate shows complex multiplets, resulting in an unattractive peak shape, while sodium p - hydroxybenzoate shows two sets of symmetric doublets at δ 6.6 ppm and δ 7.7 ppm, with no overlap with all the hydrogen peaks of rhamnolipid, and has advantages such as high stability, easy availability, and excellent solubility. The methodological verification shows that when adding 20 mM of this internal standard, the chemical shift deviation (Δδ < 0.01 ppm) and peak shape change (half - peak width difference < 5%) of rhamnolipid both meet the quantitative requirements. Therefore, it is established as the optimal internal standard selection.

[0078] Table 1 Screening results of internal standards for the nuclear magnetic resonance hydrogen spectrum detection of rhamnolipid

[0079]

[0080] (VI) Verification of the repeatability and accuracy of the nuclear magnetic resonance hydrogen spectrum detection method of rhamnolipid:

[0081] Based on the above research results, a DMSO - d6 / CD3OD (4:1, v / v) mixed deuterated reagent containing 20 mM of sodium p - hydroxybenzoate was used as the internal standard system, and stock solutions of 30 mM mono - and di - rhamnolipid were prepared respectively.

[0082] Five parallel samples were prepared by mixing in a volume ratio of 1:1 and analyzed on a 500 MHz nuclear magnetic resonance spectrometer. The measured peak areas are summarized in Table 2. The relative standard deviation of each peak of the five parallel samples is within 2%, indicating that the method has good repeatability.

[0083] Table 2 Verification of the repeatability of the nuclear magnetic resonance hydrogen spectrum detection method of rhamnolipid

[0084]

[0085] Mix the mono- and di-rhamnolipid mother liquors in different ratios (1:5, 2:4, 3:3, 4:2, 5:1), and add the mono- and di-rhamnolipid mother liquors, for a total of 7 rhamnolipid samples, which are analyzed on a 500 MHz nuclear magnetic resonance spectrometer. Set the characteristic peak area at δ 6.6 ppm of sodium p-hydroxybenzoate in each spectrum to 2.00 as the quantitative reference, and set the characteristic peak area A 内标 of sodium p-hydroxybenzoate at δ 4.60 - 4.63 ppm and the characteristic peak area A 单 at δ 4.66 - 4.69 ppm into equations (1) and (2) respectively for calculation to obtain the concentration C 双 of mono-rhamnolipid or the concentration C 单 of di-rhamnolipid in the sample solution: 双 :

[0086] (1)

[0087] (2)

[0088] The calculation results are shown in Table 3. The measured contents of mono- and di-rhamnolipid by this method are highly consistent with the theoretical ratios, and the relative errors are all controlled within 5%, indicating that this method has good accuracy.

[0089] Table 3 Verification of the accuracy of the nuclear magnetic resonance hydrogen spectrum detection method for rhamnolipid

[0090]

[0091] (VII) Experiment for determining the detection limit of the nuclear magnetic resonance hydrogen spectrum detection method for rhamnolipid:

[0092] By preparing mixtures of mono- and di-rhamnolipid with extreme ratios (molar ratios of 1:20 and 20:1 respectively), systematically evaluate the detection sensitivity of the nuclear magnetic resonance quantitative method. When calculating the characteristic hydrogen peak area in the sample, the content of di-rhamnolipid is calculated based on the characteristic hydrogen peak area at δ4.66 - 4.69 ppm. The quantitative analysis results are as shown in Figure 9 and Figure 10 . The measured value of the sample with a high di-rhamnolipid ratio is 20.05:1.00 (theoretical value 20.00:1.00), and the measured value of the sample with a high mono-rhamnolipid ratio is 1.00:20.07 (theoretical value 1.00:20.00). The relative error is within 5%, confirming that this method can accurately detect trace components as low as 5% in the mixture.

[0093] It can be seen that the characteristic hydrogen peak area quantitative method of the present invention has excellent sensitivity and a low detection limit, providing a reliable solution for the trace analysis of rhamnolipid mixtures.

[0094] (8) Evaluation of the anti-interference property of the nuclear magnetic resonance hydrogen spectrum detection method for rhamnolipid:

[0095] The present invention realizes quantitative analysis by identifying the characteristic hydrogen signals on the hemiacetal carbon of the sugar ring in the rhamnolipid structure. To verify the specificity of this method, for the possible interfering substances in the rhamnolipid fermentation system, the fermentation carbon source components (including glycerol, glucose, sucrose) and the structural analog L-rhamnose were selected as the research objects.

[0096] In the experiment, a blank control sample containing only rhamnolipid was prepared using a mixed deuterated reagent system containing sodium p-hydroxybenzoate as an internal standard, and the concentrations of mono- and di-rhamnolipids were both 15 mM. Interference tests were carried out by adding 15 mM of the above interfering substances to the blank sample respectively. Calculated by the internal standard method, the rhamnolipid content in the added interfering substance sample was consistent with that of the blank sample, and the interfering substances had no effect on the rhamnolipid quantification. The specific nuclear magnetic resonance hydrogen spectrum of the experimental sample is as Figure 11 shown. The characteristic hydrogen signal of mono-rhamnolipid in the blank sample is located at δ 4.62 ppm, and the two characteristic hydrogen signals of di-rhamnolipid are located at δ 4.80 ppm and δ 4.68 ppm respectively.

[0097] The test results show that in the presence of glycerol, no interference peak appears near the characteristic hydrogen signal; although sucrose and glucose generate new hydrogen signals near the characteristic hydrogen chemical shift, they have no significant effect on the peak area integration of the characteristic hydrogen; the introduction of L-rhamnose causes the characteristic hydrogen signal at δ 4.80 ppm of di-rhamnolipid to overlap with it, but the characteristic hydrogen signal at δ 4.68 ppm still remains independent and does not affect the calculation. Based on this, when calculating the quantity of di-rhamnolipid, the hydrogen peak area at δ 4.68 ppm can be selected as the quantitative basis to ensure the accuracy of the detection results.

[0098] The method described in the present invention can still maintain high detection specificity in complex matrices, providing a reliable technical solution for the quantitative analysis of rhamnolipid.

[0099] (9) Rhamnolipid sample pretreatment method:

[0100] For liquid samples, first remove the insoluble substances by centrifugation, and then adjust the pH to the range of 1.5 - 2.5 with 6 M hydrochloric acid solution to precipitate rhamnolipid, and the acid-precipitated sample can be obtained after drying. For solid samples, dissolve them with anhydrous ethanol and remove the insoluble substances, and then remove the ethanol by rotary evaporation. Further, the acid-precipitated sample can be redissolved with 0.2 M sodium hydroxide solution and freeze-dried to obtain an alkali-redissolved sample; the alkali-redissolved sample can be further purified by dissolving it with anhydrous ethanol, and the ethanol is removed and freeze-dried to obtain an ethanol-purified sample.

[0101] Using the rhamnolipid fermentation broth in (1) as the raw material, the fermentation broth was removed of cells to obtain a clear solution, and purification treatments such as acid precipitation, alkali re-dissolution, and alcohol dissolution were carried out successively in steps. The rhamnolipid samples obtained in each step were used for analysis. The results of nuclear magnetic resonance hydrogen spectrum analysis ( Figure 12 showed that the samples pretreated by the method of the present invention could maintain stable characteristic hydrogen signals, and no other impurity peaks interfering with the characteristic hydrogen of mono- and di-rhamnolipids appeared in the spectra. This method is simple and efficient in operation: only acid precipitation and drying treatments are required for liquid samples, while for solid samples, only the insoluble substances need to be removed by alcohol dissolution and then ethanol is removed by rotary evaporation to meet the requirements of nuclear magnetic resonance detection. This pretreatment process significantly simplifies the sample pretreatment steps and at the same time ensures the accuracy and reliability of the analysis results.

[0102] The purity changes of rhamnolipid samples at different treatment stages were calculated by the internal standard method, and the results are shown in Table 4. With the increase of pretreatment steps, the purity of rhamnolipid showed a significant increasing trend.

[0103] Table 4 Purity of rhamnolipid samples at each treatment step

[0104]

[0105] Note: All data are from three independent repeated experiments.

[0106] Comparative Example 1: Determination of rhamnolipid by HPLC-ELSD detection method:

[0107] Quantitative analysis of rhamnolipid usually adopts high performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD). The specific operation process is as follows: First, the rhamnolipid sample is diluted to an appropriate multiple with ethanol, filtered through a 0.22 μm organic filter membrane and transferred to a liquid phase vial for standby. The analysis is carried out using an Agilent HPLC-ELSD system (Palo Alto, California, USA) equipped with a C18 chromatographic column (4.6×150 mm, 5 µm; Sepax Technologies, Suzhou, China). The detection conditions are set as follows: ELSD drift tube temperature 103°C, atomizing gas flow rate 2.8 L / min. The mobile phase uses acetonitrile (phase A) and an aqueous solution containing 0.05% formic acid (phase B), and is eluted with a linear gradient of 30% - 100% within 26 minutes, and the injection volume is 20 μL.

[0108] Using the purified mono-rhamnolipid and di-rhamnolipid pure products in Example 1 (1), standard curves of concentration and peak area were established respectively. Taking the concentration (taking the lg logarithm) as the abscissa and the peak area (taking the lg logarithm) as the ordinate, an X-Y scatter plot was drawn and linearly fitted ( Figure 13). The results showed that within the instrument detection limit, the standard curve equation of dirhamnolipid in the concentration range of 0.2 - 0.7 mM was y = 2.1252x + 2.5146 (R² = 0.9997); monorhamnolipid showed a good linear relationship in the concentration range of 0.2 - 1.0 mM, and its standard curve equation was y = 1.9796x + 2.3015 (R² = 0.9992). Based on this, HPLC-ELSD analysis was performed on the 7 rhamnolipid samples prepared in Example 1 (vi), and their contents were calculated through the standard curves of mono- and dirhamnolipid respectively. The analysis results showed (Table 5) that the contents of the two rhamnolipids measured by this method were in good agreement with the theoretical ratio, and their relative errors were all controlled within 5%. It should be noted that the results obtained by this method were highly consistent with the data measured by nuclear magnetic resonance technology in Example 1 (vi) (the relative errors were all controlled within 5%), further confirming the accuracy and reliability of the nuclear magnetic resonance analysis method and providing a reliable technical means for the quantitative analysis of rhamnolipids.

[0109] Table 5 Concentration of rhamnolipid detected by HPLC-ELSD method

[0110]

[0111] Comparative Example 2: Determination of rhamnolipid by anthrone-sulfuric acid method:

[0112] The anthrone-sulfuric acid method is a commonly used method for the rapid quantitative detection of rhamnolipids, and its detection principle is based on the characteristic color reaction of carbohydrate compounds. In this method, concentrated sulfuric acid is used to dehydrate carbohydrates to form furfural derivatives, and these products condense with anthrone reagent to form a blue-green complex with characteristic absorption at 620 nm, and the color development intensity has a good linear relationship with the sugar content. This method uses L-rhamnose as the standard to establish a standard curve, and the measured value is converted into the rhamnolipid concentration through the empirical correction coefficient 3.4. It should be noted that this method responds to all reducing sugars, so interfering substances such as reducing sugars like glucose must be completely removed in the sample pretreatment stage.

[0113] The specific detection process is as follows: accurately prepare anthrone-sulfuric acid reagent (0.2 g anthrone dissolved in 100 mL 75% sulfuric acid), and use it immediately after preparation. Take 200 μL of the sample to be tested in a test tube, slowly add 1 mL of anthrone-sulfuric acid reagent under ice-water bath conditions, vortex and mix well, then place it in a boiling water bath for reaction for 10 minutes. After the reaction is completed, immediately cool it to room temperature in an ice bath, and finally measure the absorbance at a wavelength of 620 nm using an enzyme-labeled instrument. In this study, L-rhamnose was used as the standard to establish a standard curve, and the measured rhamnose mass concentration was converted into the rhamnolipid mass concentration through the empirical correction coefficient 3.4.

[0114] Seven samples from Example 1 (6) were detected for rhamnolipid by the anthrone - sulfuric acid method, and the measurement results are shown in Table 6. The results indicate that the rhamnolipid content measured by the anthrone - sulfuric acid method is generally higher than the true value and shows an obvious regular change: as the content of dirhamnolipid in the sample increases, the relative error of the measurement result increases significantly (from 16.34% to 70.15%). This phenomenon can be attributed to the fact that the dirhamnolipid molecule contains two rhamnose unit structures. Under the condition of the same molar concentration, the number of active sites participating in the color reaction is twice that of monorhamnolipid, resulting in a significant increase in the intensity of the color reaction. It is further confirmed that the 3.4 correction factor used in the traditional anthrone - sulfuric acid method is only an empirical value, and its applicable range has obvious limitations, especially not suitable for the accurate quantitative analysis of rhamnolipid samples with different structural ratios.

[0115] Table 6 Determination of rhamnolipid concentration by anthrone - sulfuric acid method

[0116]

[0117] Comparative Example 3: Error of commercially available standard product:

[0118] In quantitative analysis, the establishment of the standard curve is decisive for the accuracy of the results. Aiming at the problem that commercially available rhamnolipid standard products (such as products of AGAE Company) usually exist in the form of mixtures and the proportion of each structural component is not fixed, this study systematically compared the influence of two commercially available mixed standard products, AGAE - R90 (No. A791255126058, purity 90%) and AGAE - R95Dd (No. A33056058058, purity 95%), on the measurement results. As Figure 14 shown, HPLC - ELSD analysis shows that except for the solvent peak (1.3 min), both standard products contain 3 - 4 rhamnolipid homologues, among which Rha - Rha - C 10 -C 10 (10.6 min) and Rha - C 10 -C 10(13.5 min) is the main component. When the standard curve established based on the total mass concentration versus the total peak area was used to analyze the 7 rhamnolipid samples in Example 1 (Six), the results showed that when using the AGAE-R95Dd standard, the measured values were significantly lower than the theoretical values (relative error -4.63% to -15.08%); while when using the AGAE-R90 standard, the measured values were significantly higher than the theoretical values (relative error +96.13% to +119.91%). This significant deviation was mainly due to the unfixed proportion of each component in the standard, the lack of molecular weight information, and the purity differences between batches. This result fully demonstrates that in the quantitative analysis of rhamnolipids by HPLC-ELSD, it is necessary to establish an exclusive standard curve using a pure product with a clear structure and a single component in order to effectively eliminate the systematic error introduced by the mixed standard, improve the data comparability between different laboratories, and ensure the reliability of the quality control results.

[0119] Table 7 Comparison of quantitative results and error analysis of rhamnolipids detected by HPLC-ELSD using different standards

[0120]

[0121] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should be within the protection scope of the present invention.

Claims

1. A method for determining the contents of mono- and di-rhamnolipids by nuclear magnetic resonance hydrogen spectrum, characterized in that, It includes the following steps: (1) Uniformly mix a precisely weighed sample to be measured and a precisely weighed internal standard, sodium p-hydroxybenzoate, in a deuterated reagent to obtain a sample solution; (2) Scan the sample solution with a nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance hydrogen spectrum; (3)In the nuclear magnetic resonance hydrogen spectrum obtained in step (2), substitute the characteristic peak area A at δ 6.61~6.69 ppm 内标 , the characteristic peak area A at δ 4.60~4.63 ppm 单 and the characteristic peak area A at δ 4.66~4.69 ppm 双 into Equation (1) or Equation (2) for calculation to obtain the concentration C of monorhamnolipid in the sample solution 单 or the concentration C of dirhamnolipid in the sample solution 双 : （1） （2） Among them, C 内标 is the molar concentration of the internal standard substance in the sample solution, and C 内标 and C 单 or C 双 have the same unit.

2. The method according to claim 1, wherein The deuterated reagent is a mixed reagent of deuterated dimethyl sulfoxide and deuterated methanol.

3. The method according to claim 2, wherein The deuterated reagent is a mixed reagent of deuterated dimethyl sulfoxide and deuterated methanol with a volume ratio of 4:

1.

4. The method according to claim 1, characterized in that, The sample to be measured is derived from rhamnolipid obtained by microbial fermentation.

5. The method according to claim 1, wherein The method further includes the following steps: (4) Substitute C obtained in step (3) 单 or C 双 into formula (3) or formula (4) to obtain the mass fraction P of monorhamnolipid 单 or the mass fraction P of dirhamnolipid 单 in the sample to be measured: （3） （4） where m 待测 is the mass of the sample to be measured, and V is the volume of the sample solution.

6. The method according to claim 1, characterized in that, The frequency of the nuclear magnetic resonance spectrometer is above 400 MHz.

7. The method according to claim 1, wherein The sample to be measured is pretreated, and the pretreatment is selected from the following methods: For an aqueous solution of the analyte, first centrifuge to collect the supernatant, then adjust the pH of the supernatant to 1.5 - 2.5, then stand at 3 - 5 °C for 10 - 16 h, centrifuge again, collect the solid and dry it to obtain the sample to be measured; For a solid analyte, first dissolve the solid in an organic solvent, centrifuge to collect the supernatant, and then remove the organic solvent in the supernatant to obtain the sample to be measured.

8. The method according to claim 8, wherein The method for adjusting the pH of the supernatant is: adding hydrochloric acid.

9. The method according to claim 8, characterized in that, The organic solvent is ethanol.

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