A method for determining the content of 2'-fl and 3-fl in human milk oligosaccharides in food

Abstract

The application provides a method for determining food additive 2-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) in food, which is suitable for food nuclear magnetic resonance spectrum detection. The method comprises the following steps: 1) dissolving standard and sample; 2) removing lipid and protein; 3) centrifuging the solution; 4) weighing the clarified solution; 5) filtering the membrane; 6) determining the spectrum of the sample and the standard by nuclear magnetic resonance spectrum; 7) processing the spectrum by using an algorithm; and 8) correcting the content of 2'-FL and 3-FL in the sample according to the standard curve. The method is suitable for quantitative detection of 2'-FL and 3-FL in food.

Description

Technical Field

[0001] This invention belongs to the field of quantitative nuclear magnetic resonance spectroscopy and relates to a method for determining the content of human milk oligosaccharides (2'-FL and 3-FL) in food, so as to determine the amount of human milk oligosaccharides added to food. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk and are essential for healthy infant growth. 2'-Fucosylgalactose (2'-FL) and 3-fucosylgalactose (3-FL) are the main components of HMOs. HMOs have various physiological functions, including aiding in the development of the infant's gut microbiota, reducing the risk of diarrhea, enhancing the infant's resistance to bacteria and inflammation, and promoting healthy brain development. Unlike lactose, HMOs are not digested by infants. They are primarily utilized by beneficial bacteria in the gut (such as Bifidobacteria) as a prebiotic, providing carbon and energy.

[0003] The World Health Organization (WHO) considers breast milk to be the gold standard for infant feeding. Therefore, the nutritional level of infant formula is gradually approaching that of breast milk. Human milk oligosaccharides (HMOs), as an important nutrient in breast milk, are crucial for inclusion in infant formula. Currently, some brands of infant formula have already added HMOs, and the proportion of HMOs added to various brands of infant foods is expected to increase in the future. Therefore, the importance of developing detection methods for HMOs in different food matrices is becoming increasingly apparent.

[0004] Existing methods for detecting HMOs mainly include liquid chromatography (LC) and high-performance capillary electrophoresis (HPLC). These methods include reversed-phase HPLC (RP-HPLC), hydrophilic interaction liquid chromatography (HILIC), and high-performance anion exchange chromatography (HPAEC). LC can be coupled with various detectors, such as ultraviolet (UV) detectors, fluorescence detectors (FLD), evaporative light detection (ELSD), and refractive index detectors (RI). After several years of development, these methods have formed a relatively mature detection technology system. However, this method requires derivatization of the HMOs to improve their detection response and chromatographic separation. The pretreatment methods are complex, and the detection time is long. This presents certain difficulties for the detection of large quantities of food.

[0005] With the development of mass spectrometry (MS) technology, liquid chromatography coupled with mass spectrometry has also been used for the quantification and characterization of HMOs. Mass spectrometry detectors are more accurate and sensitive. However, matrix effects and ionization suppression make quantitative analysis of HMOs by mass spectrometry more challenging. In addition, the large number of structural positional isomers of HMOs are difficult to distinguish in MS detection, which also brings considerable difficulties to the detection of HMOs.

[0006] Quantitative nuclear magnetic resonance (qNMR) has become an important tool for the quantitative analysis of various substances in food due to its excellent analytical capabilities. NMR can reliably separate and quantify isomers, and its stable instrument performance significantly improves the reliability of analytical results. In recent years, qNMR has become an important tool for the quantitative analysis of various substances in food due to its excellent analytical capabilities. qNMR has the following advantages: high precision, high accuracy, non-destructive analysis, simple sample preparation, metrological traceability, and easy differentiation of isomers. Chen et al. established a method for determining the cystine content in food supplements and additives using qNMR, which has higher sensitivity, accuracy, and reproducibility compared to traditional titration methods. Similarly, R. Consonni et al. used... 1 1H NMR spectroscopy was used to analyze Italian Parmigiano Reggiano cheese at different stages of maturity, and a model was constructed to distinguish the maturity and geographical differences of the samples.

[0007] Due to the complex composition of food matrices, the chemical shift peak spectra of other substances in the food matrix overlap with those of the target analytes when measuring HMOs, resulting in severe spectral coverage issues. This significantly impacts the quantitative detection of HMOs, making it difficult to accurately determine the content of 2'-FL and 3-FL in food matrices. Summary of the Invention

[0008] To address the problems of existing technologies, this invention provides a quantitative nuclear magnetic resonance (NMR)-based analytical method for detecting and quantifying 2'-FL and 3-FL in human milk oligosaccharides (HMOs) in complex food matrices. This method is the first to employ a nuclear magnetic resonance spectrometer as the detection instrument, applying quantitative NMR technology to the detection of HMOs in food. Furthermore, by combining NMR spectral analysis with peak extraction techniques, a reasonable mathematical model is constructed to resolve the issue of inaccurate quantification caused by the spectral coverage of 3-FL.

[0009] The method of this invention, after methodological verification and evaluation with existing methods, has been shown to have good stability and high accuracy. It achieves the separation of chemical shift peaks of target substances in samples, solves the problem of severe signal overlap, and provides a suitable method for large-scale food analysis using nuclear magnetic resonance spectroscopy to determine HMOs.

[0010] This invention provides a method for detecting the content of 2'-fucosylated lactose (2'-FL) and / or 3-fucosylated lactose (3-FL) in food samples using nuclear magnetic resonance spectroscopy (NMR), aiming to solve the problems of complex operation, limited sensitivity, and matrix interference in existing detection methods. This invention achieves efficient and accurate detection of 2'-FL and 3-FL in food by optimizing sample pretreatment steps, selecting specific chemical shift peaks, and combining quantitative analysis with standard curves.

[0011] The method includes the following steps:

[0012] (1) Sample pretreatment

[0013] First, interfering substances in the food sample are removed through steps such as dissolution, pH adjustment, centrifugation, and membrane filtration. This step effectively removes matrix components such as fats and proteins that interfere with NMR signals, ensuring a clear sample solution suitable for NMR detection. The treated sample solution is then mixed with a buffer solution to provide a stable testing environment for subsequent detection.

[0014] (2) Nuclear magnetic resonance spectroscopy detection

[0015] The pretreated sample solution was analyzed by proton NMR spectroscopy to obtain spectral data. 2'-FL and 3-FL were quantitatively analyzed by selecting specific chemical shift peaks. This invention clarifies that the quantitative peak of 2'-FL is located at δ5.30, and the quantitative peak of 3-FL is located at δ5.37. These specific chemical shift peaks are highly correlated with the target substances, have good resolution, and can avoid interference from other components.

[0016] (3) Quantitative analysis

[0017] Quantitative analysis is performed by combining the peak area of ​​the target chemical shift peak with a standard curve to ensure the accuracy of the detection results. Specifically, to improve the quantitative accuracy of 3-FL, this invention corrects the total integrated area of ​​the FucαH-1 peak using a peak area ratio coefficient, further enhancing the reliability of the data. The standard curve is based on standards of 2'-FL and 3-FL, covering multiple concentration ranges to meet the detection needs of the target substance content in different samples.

[0018] To improve the quantitative accuracy of 3-fucosyllactose (3-FL) in food samples, this invention proposes an innovative sample pretreatment technique. This technique optimizes 3-FL extraction without interfering with the removal of interfering substances, ensuring the accuracy of the final measurement. Compared to traditional sample processing methods, this multi-step pretreatment process significantly improves the recovery rate, solubility, and nuclear magnetic resonance (NMR) signal intensity of 3-FL, thereby enhancing analytical precision.

[0019] To optimize the extraction of 3-FL, this invention incorporates a dissolution step during sample pretreatment. In the initial dissolution stage, a buffer solution (0.01-0.2M) containing NaCl is used to facilitate the release of 3-FL, which has low solubility in the sample. Furthermore, pH adjustment causes interfering substances such as fats and proteins to precipitate, thereby reducing their impact on subsequent NMR analysis.

[0020] The method of this invention has significant advantages over traditional methods:

[0021] 1. Sample pretreatment is simple and efficient, effectively removing matrix interference and reducing sources of detection error.

[0022] 2. By selecting specific chemical shift peaks, the sensitivity and specificity of 2'-FL and 3-FL detection were significantly improved.

[0023] 3. The operation process is environmentally friendly, requiring no organic solvents, and is lower in cost and more applicable than traditional chromatography methods.

[0024] 4. Validation results on different food samples (such as infant formula, cow's milk, goat's milk powder and breast milk) show that the method has good accuracy, repeatability and linear range.

[0025] Therefore, the method of this invention provides a scientific, efficient and reliable technical means for food testing, applicable to the content analysis of 2'-FL and 3-FL in various food samples, and can be widely used in food quality control, functional food research and development and related scientific research fields.

[0026] HMOs are the third most abundant solid nutrient in breast milk and play a vital role in physiological functions. These functions include fostering a healthy gut microbiota, promoting intestinal development, enhancing immunity, and supporting neurodevelopment.

[0027] This invention develops a method using nuclear magnetic resonance (NMR) technology for the rapid and accurate quantitative detection of HMOs (2'-FL and 3-FL) in infant formula. 2'-FL and 3-FL are major components and isomers of HMOs, and these isomers pose a challenge to HMO quantification. Therefore, this analytical method was specifically developed for these compounds. This method is the first to apply qNMR to the quantitative analysis of HMOs in food. The method exhibits good linearity in the concentration range of 0.1–3.0 mg / mL, high precision, and good reproducibility. The reproducibility and recovery of this method meet AOAC standards.

[0028] Compared to existing methods, this new method simplifies the sample pretreatment process, eliminates the need for prior separation of the test substance, and significantly reduces the time required for sample pretreatment. This high efficiency makes it well-suited for analyzing large volumes of samples and holds great potential for quality monitoring of HMO-related products. Therefore, it provides a viable alternative method for detecting HMOs in food in a testing and regulatory laboratory setting.

[0029] More specifically, the beneficial effects of the technical solution provided by this invention can be selected from any of the following:

[0030] (1) For the first time, quantitative nuclear magnetic resonance (qNMR) was used to determine human milk oligosaccharides 2'-FL and 3-FL in complex food matrices (such as milk powder, milk, etc.);

[0031] (2) The sample pretreatment operation in the method is simple, which greatly shortens the sample preparation time.

[0032] (3) This method uses nuclear magnetic resonance spectroscopy to determine the detection time, which is significantly shorter than existing methods and is suitable for high-throughput food detection.

[0033] (4) The method has undergone methodological evaluation. The method has good repeatability and high accuracy.

[0034] (5) The method innovates the processing method for nuclear magnetic resonance (NMR) spectral data. It innovates the data processing method to address the peak signal coverage problem in NMR spectral data. This solves the problem of peak signal coverage interfering with peak area integration, thereby resolving the issue that NMR cannot accurately quantify 2'-FL and 3-FL.

[0035] (6) This invention uses nuclear magnetic resonance spectroscopy to detect 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) in food samples. Specifically, it achieves precise quantification by explicitly selecting the chemical shift peak at δ5.37 as the characteristic peak of 3-FL. This innovative selection is based on extensive experimental data, which shows that the peak at δ5.37 not only has good resolution but also avoids signal overlap with other possible impurities or coexisting sugars, ensuring the reliability and specificity of the detection results. Furthermore, the quantitative accuracy of 3-FL is further improved by correcting the total integrated area of ​​the FucαH-1 peak using a peak area ratio coefficient. Compared to existing methods that typically employ complex chemical derivatization or chromatographic separation, this invention, with its non-destructive nuclear magnetic resonance detection as its core, optimizes the convenience, environmental friendliness, and universality of the detection, demonstrating unique technical advantages. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a conventional 2'-FL standard material provided in the embodiments of the present invention. 1 1H NMR spectrum (A); NMR spectrum of sample containing 2'-FL (B), where: gray area is local magnification area; red arrow indicates the position of chemical shift peak of 2-FL;

[0038] Figure 2 These are NMR spectra and their comparison diagrams provided in embodiments of the present invention, wherein:

[0039] A: The routine use of 3-FL standard reference 1 1H NMR spectrum (the chemical shift characteristic peaks of 3-FL are marked with red arrows);

[0040] B: NMR spectrum of the sample containing 3-FL (the characteristic peaks of 3-FL chemical shift are marked with red arrows);

[0041] C: Comparison of 3-FL blank sample and standard spectrum (the blue dashed box shows the characteristic peaks of 3-FL chemical shift, and the red arrows indicate the characteristic peaks in the blank sample spectrally covered with 3-FL).

[0042] D: Spectral comparison of 3-FL standard sample and additive sample (blue dashed box indicates 3-FL chemical shift characteristic peak; yellow area is the base peak of the chemical shift characteristic selected by the detection method). Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0044] Example 1

[0045] This embodiment provides a method for detecting 2'-FL and 3-FL in human milk oligosaccharides to determine the content of 2'-FL and 3-FL in food, which includes the following steps:

[0046] 1) Weigh the solid sample and the liquid sample, and dissolve the solid sample;

[0047] 2) Adjust the pH of the solution to 4.4 to remove fat and protein from the sample;

[0048] 3) After shaking thoroughly, centrifuge the sample solution;

[0049] 4) After extracting the lower clarified layer, filter it using a filter membrane;

[0050] 5) Weigh 900 μL of the filtered solution and add 100 μL of TSP solution. Add 600 μL of the test solution to the NMR tube and measure the proton spectrum using a nuclear magnetic resonance spectrometer.

[0051] 6) Construct a standard curve based on the integrated area values ​​of the chemical shift peaks of 2'-FL and 3-FL standards and the concentrations of the standards;

[0052] 7) Process the proton NMR spectrum of the food samples and extract the integral area of ​​the corresponding chemical shift peaks;

[0053] 8) Corrected quantification was used to determine the content of 2'-FL and 3-FL in the food sample.

[0054] A standard curve was constructed based on the integrated area of ​​the chemical shift peaks of 2'-FL and 3-FL standards and the concentration of the standards, and the content of 2'-FL and 3-FL in the food sample was obtained by calibration.

[0055] The detection methods include:

[0056] Buffer solution preparation: Accurately weigh 1.00±0.01g of sodium 2,2,3,3-d(4)-3-(trimethylsilyl)propionate and 0.30±0.01g of NaN3 into a 10mL volumetric flask, and then dissolve them in 10mL D2O to prepare solution A. The final concentration is 100g / L TSP and 13g / L NaN3. Add 100mL D2O to a 200mL volumetric flask and dissolve 5.0g of KH2PO4. Then add 5mL of phosphoric acid and 2mL of solution A, and finally pour 50mL of D2O into the volumetric flask. After 24 hours, measure the pH of the buffer solution and adjust the pH to 2.0±0.02 by adding phosphoric acid or KH2PO4 solid powder.

[0057] External standard method solution preparation: Weigh 20.0 mg of citric acid into a 10 mL volumetric flask and dilute to 10 mL with water.

[0058] Preparation of 2'-FL and 3-FL standard solutions: Precisely measure 0.0300 ± 0.0003 g of 2'-FL and 3-FL standard solutions in 10 mL volumetric flasks to prepare single standard stock solutions. Serially dilute to concentrations of 3.00, 2.00, 1.00, 0.50, 0.25, 0.10, and 0.05 mg / mL. Finally, take 900 μL of the solution and add 100 mL of buffer solution. Then, transfer 600 μL of the sample solution to an NMR tube.

[0059] Standard solution and sample pretreatment preparation: Weigh 7.0000±0.0001g of sample into a 50mL beaker, then add 35mL of water. Vortex for 10 minutes to completely dissolve the sample. Adjust the pH of the sample solution to 4.4 using 2mol / L hydrochloric acid solution. Then transfer the solution to a 50mL volumetric flask and add water to 50mL. Shake thoroughly to ensure homogeneity. Transfer the solution from the volumetric flask to a centrifuge tube and centrifuge at 8000rpm for 10 minutes. Subsequently, extract the lower clear fraction of the solution. Filter through a 22μm filter membrane. Finally, take the entire 900mL solution and add 100mL of buffer. Transfer 600mL of the sample solution into an NMR tube.

[0060] NMR detection conditions:

[0061] All NMR experiments were performed on a Bruker Avance III HD 400MHz spectrometer at 00.0 ± 0.1 K with the following optimized parameters: rotation off; number of scans, 4; number of scans, 16; spectral width, 20.55 ppm; number of points, 64000; acquisition time, 3.98 sec; relaxation delay, 4 sec; receiver gain, 16; dwell time, 60.08 μsec. Zero-point chemical shift was calibrated using TSP. Hydrogen spectra were analyzed using the Bruker standard pulse sequence noesygppr1 d to suppress water peaks. Spectra were analyzed and processed using MestReNova 12.0 software. Phase correction employed the "range algorithm." Baseline correction was performed using "polynomial fitting." Target peaks were extracted using Global Spectral Deconvolution (GSD), and the signal integrals calculated using this software were used for quantitative analysis.

[0062] NMR 1H NMR spectrum processing algorithm:

[0063] The concentration of HMOs was determined using the pulse length-based concentration assay (PULCON) principle. The PULCON principle eliminates the need to consider interactions and signal overlap between the reference and standard substances because this method uses an external reference. Furthermore, the reference substance can be reused, thus reducing measurement costs.

[0064] The quantitative method uses an aqueous solution containing citric acid as an external reference material based on H NMR spectroscopy, and measures are performed using signals at δ 3.02 and δ 2.85. HMO concentration (C H The formula for calculating ) is (1):

[0065]

[0066] A is the absolute integral (S is the sample, R is the reference), I is the magnitude of the true spectrum, P is the scan width, M is the molecular weight, N is the number of protons that produce the selected peak, Ns is the number of scans, and C...R α is the concentration of the reference substance, and α is the correction factor.

[0067] To mitigate the influence of variables such as instrument status and measurement conditions (e.g., suppression of water signals), a correction factor was added to formula (1) for the quantitative analysis of HMOs. Using the ratio of standard weight to solution volume as the ordinate and the concentration determined by the external standard method as the abscissa, the linear regression equation y = αx + β was derived, where α represents the correction factor.

[0068] 2'-FL chemical shift peak selection:

[0069] δ 5.30 (d, J = 2.84 Hz) is the chemical shift of H-1 on fucose, located on the fucose side of the Fuc-α(1-2)-Gal glycosidic bond. The characteristic peak of this chemical shift region is clear and stable in 2'-FL standards. The chemical signal spectra in various actual samples do not overlap, resulting in minimal interference with 2'-FL quantification. Therefore, 5.310–5.290 ppm was selected as the integration quantification region, and the concentration of 2'-FL in the sample was determined by calculating the integrated area of ​​this region. (See...) Figure 1 )

[0070] 3-FL chemical shift peak selection:

[0071] Chemical shifts δ5.43, δ5.42, δ5.38, and δ5.37 (dd, J = 22.96, 3.96 Hz) are attributed to H-1 on the fucose, located on the fucose side of the Fuc-α(1-3)-Glc glycosidic bond. The H-1 proton undergoes two fragmentations under the influence of two adjacent H protons, resulting in doublets (dd). When measuring 3-FL standards, the chemical shift signals are clear and distinct. However, when measuring 3-FL in actual samples, the spectral signals of impurities in the samples significantly overlap with the FucαH-1 chemical shift peaks of 3-FL. This issue with spectral coverage severely affects the accurate determination of 3-FL in samples.

[0072] Comparison of the spectra of 3-FL standards, 3-FL-free infant formula, and corresponding additive samples revealed no spectral coverage issue at the peak at δ5.37. The peak at δ5.37 is part of the FucαH-1 peak on 3-FL, and its peak area integral accounts for a fixed proportion of the total α-H peak area integral. Therefore, the peak at 3-FL was extracted using full-spectrum deconvolution (GSD). By quantifying the peak area of ​​δ5.37 and combining it with the peak area ratio coefficient, the total integral area of ​​the FucαH-1 peak on 3-FL was calculated. Subsequently, FucαH-1 was used as a characteristic proton to quantify 3-FL in the infant formula samples. Analysis of the chemical shift at this position showed that the "roof effect" had little impact on the peak area ratio. Therefore, the final determined ratio coefficient was 4.58. (See...) Figure 2 )

[0073] Example 2

[0074] Linear determination of 2'-FL and 3-FL in infant formula, cow's milk, goat's milk powder, and breast milk was performed using the method described in Example 1. Calibration curves were constructed by adding standard solutions of different concentrations (0.1-3.0 mg / mL). Citric acid was used as an external standard, and the peak area of ​​the nuclear magnetic resonance signal was measured and the concentration was calculated. The calibration curves are shown below:

[0075] The equation for the 2'-FL calibration curve is y = 0.9969x + 10.615, and the coefficient of determination (R²) is... 2 The value is 0.9998.

[0076] The equation for the 3-FL calibration curve is y = 1.2148x + 3.152, and the coefficient of determination (R²) is... 2 The value is 0.9999.

[0077] The results show that, within the above linear range, the determination of 2'-FL and 3-FL both exhibit good linearity.

[0078] Example 3

[0079] The limits of detection (LOD) and limits of quantitation (LOQ) of 2'-FL and 3-FL were verified in different food matrices (infant formula, cow's milk, goat's milk powder, and breast milk) according to the protocol in Example 1. The experimental procedures are as follows:

[0080] 1. Add 2'-FL and 3-FL standard solutions of known concentrations and measure their specific peaks (δ 5.30 and δ 3.5).

[0081] The signal strength is δ5.37.

[0082] 2. The limits of detection and limits of quantitation for each matrix were calculated, and the results are as follows:

[0083] milk:

[0084] 2'-FL: LOD is 0.05 mg / mL, LOQ is 0.10 mg / mL;

[0085] 3-FL: LOD is 0.10 mg / mL, LOQ is 0.15 mg / mL.

[0086] Infant formula and goat milk powder:

[0087] 2'-FL: LOD is 0.35 mg / g, LOQ is 0.70 mg / g;

[0088] 3-FL: LOD is 0.71 mg / g, LOQ is 1.07 mg / g.

[0089] Breast milk:

[0090] Both 2'-FL and 3-FL had a LOD of 0.05 mg / mL and a LOQ of 0.10 mg / mL.

[0091] Experimental results show that the detection limit and quantitation limit of this method are both lower than the typical concentration range of 2'-FL and 3-FL in actual samples, making it suitable for low-concentration determination of actual samples.

[0092] Example 4

[0093] To verify the accuracy of this method, spiked recovery experiments were conducted on infant formula, cow's milk, goat's milk powder, and breast milk according to the scheme in Example 1. The specific steps are as follows:

[0094] 2'-FL and 3-FL standards were added to different samples, setting three concentration levels: 50%, 100%, and 150%.

[0095] The recovery rate was measured and the relative error was calculated. The results are as follows:

[0096] The recovery rate of 2'-FL ranged from 94.14% to 111.70%.

[0097] The recovery rate of 3-FL ranges from 90.51% to 104.44%.

[0098] According to the AOAC guidelines on dietary supplements and herbal medicines (recovery rates of 85%-110% at 100 ppm and 90%-108% at 1000 ppm), the recoveries of all samples were within acceptable ranges.

[0099] Example 5

[0100] To evaluate the repeatability and intermediate precision of the method, the recovery rate and intermediate precision were determined multiple times at different time points for infant formula and cow's milk samples, following the precautions outlined in Example 1. The repeatability of the method was validated in various samples, including infant formula, cow's milk, goat's milk powder, and breast milk, by configuring two concentration gradients. Each sample concentration was measured six times. Infant formula and cow's milk samples were measured repeatedly on days 1, 2, 3, 5, and 7 to assess intermediate precision. The deviation for each sample was also evaluated to ensure the accuracy and reliability of the method. The relative standard deviation (RSD) for repeatability of 2'-FL and 3-FL was less than 2% for all sample types (infant formula, cow's milk, goat's milk powder, and breast milk). Furthermore, the deviation for 2'-FL was less than 5%, and the deviation for 3-FL was less than 10%. The intermediate precision RSD for infant formula and cow's milk was: 2'-FL less than 2%, and 3-FL less than 3%. The experimental results confirm that the new method achieves high accuracy and reliability, demonstrating its reliability in sample analysis. This reliability is mainly due to simple sample pretreatment and the stable performance of the related instruments.

[0101] Comparative Example 1: Determination of the δ5.37 specific peak was not selected.

[0102] 3-FL was measured using nuclear magnetic resonance, but instead of selecting the specific peak at δ5.37, signal peaks in other chemical shift ranges (such as δ5.43 and δ5.42) were chosen.

[0103] The peak areas of samples with different concentrations were measured, calibration curves were constructed, and quantitative analysis was performed.

[0104] Experimental results show that in the δ5.43 or δ5.420 region, the signal peak overlaps with the spectral peaks of other substances in the matrix (such as lactose or other oligosaccharides), making it difficult to distinguish the 3-FL characteristic signal, resulting in poor linearity and a low coefficient of determination (R²). 2 All were below 0.95.

[0105] The quantitative results fluctuated significantly, with recoveries ranging from 75% to 90%, deviating from the acceptable range of AOAC, and the relative standard deviation (RSD) exceeded 10%.

[0106] Therefore, it is evident that the failure to select the specific peak at δ5.37 resulted in low peak resolution, significantly reducing the precision and accuracy of quantitative analysis, and failing to meet actual detection needs.

[0107] Comparative Example 2: 3-FL assay was performed on non-specific regions.

[0108] Quantitative analysis of 3-FL was performed using the non-specific region signal peak at δ4.50.

[0109] The concentrations of 3-FL in breast milk and goat milk powder were tested, calibration curves were constructed, and the recovery rate was verified.

[0110] Experimental results show that the signal peak at δ4.50 is a common signal of multiple lactose derivatives, with severe peak overlap, making it impossible to accurately separate the characteristic signal of 3-FL.

[0111] The difference in linearity of the calibration curve (R) 2 <0.90), recovery rate ranged from 60% to 80%, and quantification error exceeded 15%.

[0112] Therefore, it is evident that using signal peaks in non-specific regions cannot effectively distinguish 3-FL from other substances, directly leading to measurement failure.

[0113] By comparing with the comparative examples, it can be clearly concluded that selecting the specific peak at δ5.37 for 3-FL determination can significantly improve the linearity, recovery rate and repeatability of the method, and avoid the interference of peaks in other chemical shift regions, which is the key advantage of this technical solution.

[0114] Example 6

[0115] 1. Sample selection: Four types of food samples were selected as the target foods for analysis: infant formula, cow's milk, goat's milk powder, and breast milk.

[0116] 2. Sample Dissolution and Sampling: For solid food samples (such as infant formula), dissolve them in 0.

[0117] A solution suitable for NMR analysis is prepared in a 1M water / NaCl buffer solution; for liquid food samples (such as milk, goat milk powder, and breast milk), samples are taken directly for analysis.

[0118] 3. pH adjustment and removal of interfering substances: The pH of the sample solution is adjusted to 7.0 to optimize the stability of the solution and remove components such as fats and proteins in the sample that may interfere with the determination.

[0119] 4. Centrifugation and Membrane Filtration: The pH-adjusted sample was centrifuged to remove solid impurities. Subsequently, it was filtered through a 0.22 μm membrane to further remove residual solid particles, yielding a clear liquid sample.

[0120] 5. Nuclear magnetic resonance imaging (NMR) detection:

[0121] All processed samples were subjected to proton NMR spectroscopy using a 500MHz nuclear magnetic resonance spectrometer.

[0122] The peak at δ5.37 was selected as the characteristic peak of 3-FL for analysis, and a standard curve was constructed using citric acid as an external standard.

[0123] The concentration data of 3-FL were obtained by integrating the δ5.37 peak using the peak area method.

[0124] 6. Data Processing and Result Analysis:

[0125] Perform multiple measurements on all samples to ensure repeatability and assess measurement bias.

[0126] The experimental data were calibrated using a standard curve, and the concentration of 3-FL in each sample was calculated.

[0127] The results showed that the 3-FL values ​​of all samples were close to the standard concentration, with deviations controlled between -2% and +2%, indicating that the method has high accuracy. Furthermore, the samples pretreated with this method exhibited clear NMR signals, low background noise, and stable peak areas, demonstrating that the optimized sample pretreatment process effectively improved the accuracy of NMR measurements.

[0128] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0129] Those skilled in the art will understand that all or part of the steps of the above embodiments can be implemented by hardware or by a program instructing related hardware. The program can be stored in a computer-readable storage medium, such as a read-only memory, a disk, or an optical disk.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the content of 2'-fucosyllactose (2'-FL) and / or 3-fucosyllactose (3-FL) in food samples using nuclear magnetic resonance spectroscopy, characterized in that, Includes the following steps: (a) Remove interfering substances from the sample through sample pretreatment steps and prepare a solution suitable for nuclear magnetic resonance detection; (b) The proton spectrum of the sample solution was determined by nuclear magnetic resonance; (c) Select specific chemical shift peaks and combine them with a standard curve to perform quantitative analysis of the contents of 2'-FL and 3-FL in the sample; The specific chemical shift peaks mentioned above include the peak at δ5.30 for 2'-FL quantification and the peak at δ5.37 for 3-FL quantification.

2. The method according to claim 1, characterized in that, The sample pretreatment step includes the following steps: (1) Dissolve the solid sample or take the liquid sample directly and adjust the pH value of the sample solution to remove fat and protein; (2) A clear liquid is obtained by centrifugation and then filtered through a filter membrane to remove residual impurities; (3) The treated solution was mixed with a buffer solution and then used for nuclear magnetic resonance spectroscopy detection.

3. The method according to claim 1, characterized in that, The peak at δ5.37 is a characteristic peak of 3-FL, and the total integrated area of ​​the FucαH-1 peak is calculated by correcting the peak area ratio coefficient, which is used for the quantification of 3-FL.

4. The method according to claim 1, characterized in that, The food products include infant formula, cow's milk, goat's milk powder, and breast milk.

5. The method according to claim 1, characterized in that, The standard curves were constructed based on 2'-FL and 3-FL standards at different concentration ranges.

6. The method according to claim 1, characterized in that, The sample pretreatment steps include: dissolving the solid food sample in a suitable solvent (preferably a 0.01-0.2M NaCl aqueous solution), or directly taking the liquid food sample.

7. The method according to claim 1, characterized in that, The sample pretreatment step further includes: (a) Adjust the pH of the solution using a buffer solution to ensure that the sample is within the optimal pH range for NMR detection; or (b) Mix the treated solution with a suitable internal standard to improve the precision and accuracy of the analysis.

8. The method according to claim 1, characterized in that, It also includes: (a) By selecting the characteristic peaks of 2'-FL and 3-FL through the chemical shift peaks of the proton spectrum in the nuclear magnetic resonance spectrum, integral analysis was performed. (b) Based on the standard curve of the sample, the integrated area of ​​the peak is compared with a standard of known concentration to quantitatively analyze the content of 2'-FL and 3-FL in the sample.

9. The method according to claim 1, characterized in that, It also includes: (a) Baseline correction is performed on the signal intensity of each characteristic peak in the sample solution to eliminate background noise and solvent interference; (b) Calculate the coefficients using a standard curve and perform quantitative analysis of 2'-FL and 3-FL in the sample to ensure the accuracy and reliability of the data.

10. The method according to claim 1, characterized in that, It also includes: (a) Correct the peak area ratio of the characteristic peaks of 2'-FL and 3-FL in the sample, and perform quantitative analysis using known standard substances; (b) Perform multiple measurements and data acquisitions on the nuclear magnetic resonance spectrum, and improve the accuracy of the results by averaging the values, and ensure the accuracy of the 2'-FL and 3-FL content in the sample.

Images