CN110672756B - Method for detecting content of 2' -fucosyllactose in milk powder - Google Patents
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Abstract
The invention discloses a method for detecting the content of 2' -fucosyllactose in milk powder, and belongs to the technical field of food detection. The method comprises the following steps: (1) sample pretreatment: dissolving a milk powder sample by adopting a precipitator, centrifuging, diluting and coating a membrane; (2) quantitatively detecting the content of the 2' -fucosyllactose in the pretreated sample by adopting high-efficiency anion exchange chromatography; the precipitant is one of methanol, ethanol, acetonitrile and buffer solution. The method for detecting the 2' -fucosyllactose has the advantages of standard recovery rate of 96.6-103.6%, RSD < 3%, accurate detection result, high sensitivity, no interference and good application value to industrial batch detection.
Description
Technical Field
The invention relates to a method for detecting the content of 2' -fucosyllactose in milk powder, belonging to the technical field of food detection.
Background
Breast feeding is always preferred, the formation of healthy intestinal environment of human bodies is promoted in the early life, in recent years, the health efficacy of some breast milk is gradually emphasized, and the market trend is to add effective components close to breast milk in infant formula milk to obtain corresponding functional effects. The breast milk oligosaccharide is the third main component of human breast milk, which is second to lactose and fat, has very high biological activity, not only has good anti-infection effect on intestinal pathogenic microorganisms, but also can maintain intestinal microecological balance. Fucosyl oligosaccharides, a common breast milk oligosaccharide, select bifidobacteria and pathogen receptor soluble analogs as growth stimulating factors to protect infants from enteric pathogen infection and toxin binding. Among them, 2' -fucosyllactose is the most abundant fucosyllactose, and each liter of breast milk contains about 2.4g, which accounts for more than 30% of total breast milk oligosaccharides, and is widely popularized in the infant nutrition market at present.
Since most of the milk powders contain lactose at present and the content accounts for more than 50% of the total amount of carbohydrates, while 2 '-fucosyllactose is formed by connecting one molecule of lactose with one molecule of fucose through alpha-1, 2 glycosidic bond, the structure and the property are extremely similar to those of lactose, and great difficulty is brought to the separation and the detection of the 2' -fucosyllactose. Moreover, the content of lactose in the infant milk powder is far higher than that of 2' -fucosyllactose, so that the detection difficulty is further increased, and great trouble is brought to the monitoring of the infant milk powder product. Therefore, few reports are made on the detection method of 2' -fucosyllactose at present. Thurl et al (1996) established the determination of 2' -fucosyllactose in human milk, but the samples need centrifugal ultrafiltration, then separated by gel permeation chromatography, and finally analyzed by ion exchange chromatography, the whole process is complicated in operation, high in cost, large in error, and low in feasibility of batch detection of the existing samples. Bao et al (2012) need to reduce lactooligosaccharide in advance by performing separation and determination through graphite carbon high performance liquid chromatography-tandem mass spectrometry, and the method also has the problems of complex operation process, time consumption and low detection sensitivity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the method for detecting the 2 '-fucosyllactose in the milk powder, and the method has the advantages of simple operation process, short time, low cost, high detection accuracy and sensitivity and great breakthrough and application value for quantitative detection means of the 2' -fucosyllactose by optimizing detection conditions such as pretreatment reagents, analytical columns, elution conditions and the like.
The invention provides a pretreatment method for detecting the content of 2' -fucosyllactose in milk powder, which comprises the following steps: (1) weighing a certain amount of milk powder sample; (2) solubilizing the sample with a precipitating agent to remove proteins; (3) centrifuging and taking a supernatant; (4) diluting with ultrapure water; (5) film coating; the precipitant is one of methanol, ethanol, acetonitrile and buffer solution; .
In one embodiment of the present invention, the buffer solution is one of a sodium acetate-acetic acid (NaAc-HAc) buffer solution and an acetic acid (HAc) buffer solution.
In one embodiment of the present invention, the concentration of the ethanol solution is 70 to 80% (v/v).
In one embodiment of the invention, the NaAc-HAc buffer solution has a pH of 4 to 5 and a concentration of 20 to 50 mmol/L.
In one embodiment of the present invention, the HAc buffer solution has a pH of 4 to 5 and a concentration of 0.1 to 0.2% (v/v).
In one embodiment of the invention, the concentration of the milk powder sample dissolved by the precipitant is 5-10 mg/mL.
In one embodiment of the present invention, the centrifugation speed is 8000-.
In one embodiment of the present invention, the dilution factor is 20 to 100 times.
In one embodiment of the invention, the membrane is a 0.22 μm or 0.45 μm nylon microporous membrane.
The second purpose of the invention is to provide a method for detecting 2' -fucosyllactose in milk powder, which comprises the following steps: (1) the pretreatment method is applied to carry out pretreatment on the milk powder; (2) and (3) carrying out high-efficiency anion exchange chromatography quantitative detection.
In one embodiment of the present invention, the analytical column for high performance anion exchange chromatography is a Dionex CarboPac carbohydrate analytical column, including PAl, PAl0, PA20, PAl00, PA200, and MA 1. Preferably, a Dionex CarboPac PA20 anion exchange column is used.
In one embodiment of the present invention, the eluent in the elution is: 0.25mol/L NaOH is used as the solution A, 1mol/L NaAc is used as the solution B, and ultrapure water is used as the solution C.
In one embodiment of the present invention, the elution conditions are one of seven of the following:
condition 1: 0-40min, 10% B + 90% C;
condition 2: 0-40min, 5% B + 95% C;
condition 3: 0-40min, 50% A + 50% C;
condition 4: 0-40min, 20% A + 80% C;
condition 5: 0-40min, 10% A + 90% C;
condition 6: 0-15min, 10% A + 90% C; 15-25min, 20% A + 80% C; mixing the solution A, the solution B and the solution C in any proportion for 25-40 min;
condition 7: 0-20min, 10% A + 90% C; 20-40min, 20% A + 80% C;
the solution A is 0.25mol/L NaOH, the solution B is 1mol/L NaAc, and the solution C is ultrapure water.
In one embodiment of the present invention, the elution conditions are: 0-15min, 10% A + 90% C; 15-25min, 20% A + 80% C; 25-27min, 30-24% of A, 0-36% of B and 70-40% of C; 27-35 min, 24% of A, 36% of B and 40% of C; 35-37min, 24-10% of A, 36-0% of B and 40-90% of C; 37-50 min, 10% A + 90% C.
In one embodiment of the present invention, the detection conditions are: adopting an ampere detector to detect by adopting a four-potential pulse ampere method, wherein the reference electrode is Ag/AgCl; using a Dionex CarboPac PA20 anion exchange column; leaching conditions are as follows: gradient elution is adopted, and 0.25M NaOH and 1M NaAC are used as eluent to carry out gradient elution, wherein the flow rate is 0.5 mL/min; column temperature: 30 ℃; sample introduction volume: 10 mu L of the solution; the exchange column is an analytical column (3X 150mm) or a guard column (3X 30 mm).
In one embodiment of the invention, the content of 2' -fucosyllactose is quantitatively detected by an external standard method.
The third purpose of the invention is to provide the application of the pretreatment method in the detection of products containing 2' -fucosyllactose and derivatives thereof.
The fourth purpose of the invention is to provide an application of the detection method in quality monitoring of infant nutrition.
The invention has the beneficial effects that:
(1) the sample pretreatment process avoids steps of derivatization, reduction, separation and purification and the like, is simple to operate, saves reagent cost, and has great feasibility for batch detection;
(2) compared with a pretreatment method, the pretreatment method adopted by the invention has the advantages of high detection sensitivity, high separation degree and the like, greatly reduces the interference of lactose, improves the quantitative limit of 2 '-fucosyllactose, reduces the quantitative limit to 100 mu g/g, and has important significance for the detection accuracy of low-content 2' -fucosyllactose;
(3) the method for detecting the 2' -fucosyllactose has the advantages of standard recovery rate of 96.6-103.6%, RSD < 3%, accurate detection result, high sensitivity, no interference and good application value to industrial batch detection.
Drawings
FIG. 1 ion chromatogram of a common formula (blank).
FIG. 2 shows the ion chromatogram of a conventional formula milk powder (added at 500. mu.g/g milk powder).
FIG. 3 shows the ion chromatogram of a conventional formula milk powder (added at 800. mu.g/g milk powder).
FIG. 4 shows the ion chromatogram of a conventional formula milk powder (added at 1000. mu.g/g milk powder).
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
Example 1: pretreatment of ethanol solution
Ethanol solutions with different concentrations are adopted for pretreatment and detection in the following way:
(1) sample pretreatment: accurately weighing 0.5g milk powder sample, dissolving with 50mL 80% ethanol solution in advance, centrifuging at 10000rpm for 10min, collecting supernatant, diluting with ultrapure water 20 times, and filtering with 0.22 μm nylon microporous membrane.
(2) Quantitative detection by high performance anion exchange chromatography (with pulsed amperometric detector):
the analysis conditions were: the ampere detector adopts a four-potential pulse ampere method for detection, the reference electrode is Ag/AgCl, a Dionex CarboPac PA20 anion exchange column, and gradient elution and leaching conditions are adopted: performing gradient elution by using 0.25M NaOH (solution A), 1M NaAc (solution B) and ultrapure water (solution C) as leacheate (0-15min, 10% A + 90% C, 15-25min, 20% A + 80% C, 25-27min, 30-24% A + 0-36% B + 70-40% C, 27-35 min, 24% A + 36% B + 40% C, 35-37min, 24-10% A + 36-0% B + 40-90% C, 37-50 min, 10% A + 90% C) and the flow rate of 0.5 mL/min; column temperature: 30 ℃; sample introduction volume: 10 mu L of the solution; the exchange column is an analytical column (3X 150mm) or a guard column (3X 30 mm).
The ethanol concentrations (v/v) were 60%, 70%, 80%, 90%, and 100% (v/v), respectively, and the respective measurements are shown in Table 1. Among these, ethanol concentration had no significant effect on some parameters (e.g., sample resolution, detection sensitivity, retention time accuracy, etc.) (data not shown).
TABLE 1 comparison of the results of the sample determination analysis under the pretreatment method of ethanol solutions of different concentrations
Note:1the state of the sample after precipitation is directly observed by naked eyes;2judging the film passing condition according to the speed and the damage condition of the film passing;3the measurement value RSD was calculated by continuously measuring the peak areas of 6 groups of samples.
As can be seen from Table 1, the concentration of the ethanol solution reaches more than 70%, the method has a good impurity removal effect on milk powder samples, and the obtained solution is clear and transparent and is easy to pass through a membrane; but the concentration is more than 90%, the accuracy of sample detection is greatly reduced, and RSD is more than 5%, probably because the concentration of ethanol is too high, the sample is easy to volatilize and unstable in the determination process. Therefore, the pretreatment of the milk powder sample by using 70-80% ethanol solution is suitable.
Example 2: NaAc-HAc buffer pretreatment
Compared with example 1, the difference is only that: the samples were pretreated with NaAc-HAc buffer solution having a pH of 4.7 at concentrations of 10, 20, 30, 40 and 50mM, respectively, and the pretreatment and detection were carried out in the same manner as in example 1, and the respective detection conditions are shown in Table 2. Among these, NaAc-HAc buffer concentration had no significant effect on some parameters (e.g., measurement accuracy, detection sensitivity, retention time accuracy, etc.) (data not shown).
TABLE 2 comparison of sample assay results under NaAc-HAc buffer pretreatment method at different concentrations
Note:1the state of the sample after precipitation is directly observed by naked eyes;2judging the film passing condition according to the speed and the damage condition of the film passing;3the degree of separation was calculated as the difference between the retention time of the maximum lactose interference peak and the target peak of 2' -fucosyllactose.
As can be seen from Table 2, the NaAc-HAc buffer solution with low concentration (10mM) is not ideal for the precipitation of protein in milk powder, is difficult to pass through the membrane and brings certain difficulty to detection; as the concentration of NaAc-HAc is increased (more than 20 mM), the milk powder sample is easier to pass through the membrane, which indicates that the impurity removal is better. In addition, from the results of the degrees of separation, the degrees of separation become lower as the concentration of the buffer increases, probably because the concentration of NaAc contained in the sample is higher and NaAc has elution ability as an elution solution, thereby causing the migration of the objective peak and reducing the degrees of separation. Therefore, it is appropriate to pretreat the milk powder sample with 20mM NaAc-HAc buffer (pH 4.7).
Example 3: HAc buffer pretreatment
Compared with example 1, the difference is only that: the pretreatment of the sample was carried out using HAc buffer solution of pH 4.7, and other pretreatment and detection methods were the same as in example 1. Preliminary experiments show that after the milk powder is dissolved by 0.15 percent HAC solution, the pH value is about 4.7, and the protein isoelectric principle can be utilized to well precipitate the foreign protein. Therefore, the ethanol solution precipitation method, the NaAc-HAc solution precipitation method and the HAc solution precipitation method are compared to determine a better method for pretreating milk powder samples to improve the content detection of 2' -fucosyllactose in milk powder. The detection conditions after pretreatment with methanol and acetonitrile solution are similar to those of ethanol, and comparison is not shown. Wherein, the concentration of the ethanol solution is 80 percent (v/v), the concentration of the NaAc-HAc buffer solution is 20mM, and the concentration of the HAc solution is 0.15 percent. The other procedures were the same as in examples 1 and 2. The detection of each pretreatment method is shown in Table 3.
TABLE 3 comparison of the results of the sample determination analysis under different pretreatment methods
Note:1the quantitative limit LOQ refers to the lowest detected content with the standard addition recovery rate in the range of 95-105%;2the retention time RSD is calculated by continuously measuring the retention time of 6 groups of samples;3the quantitative time RSD is calculated by continuously measuring the peak areas of 6 groups of samples;4the precision RSD is obtained by measuring the calculated content of 6 groups of samples;5the separation degree is the maximum lactose interference peak and 2' -rockCalculating the difference of the retention time of the objective peaks of the fucosyllactose;6the target peak area is measured at the same sample loading concentration (5 mg/mL);7the target peak height is measured at the same sample loading concentration (5 mg/mL);8the specificity is that fructo-oligosaccharide, galacto-oligosaccharide or isomerized lactose oligosaccharide is added into a sample to prepare a negative control, and the interference is judged.
As can be seen from Table 3, the ethanol solution precipitation method, 0.2mM NaAc-HAc and 0.15% HAc solution precipitation method, these 3 pretreatment methods, for the detection of 2' -fucosyllactose has very good repeatability and precision. However, the ethanol solution precipitation method has a significantly low target peak height value compared with the 0.2mM NaAc-HAc or 0.15% HAc solution precipitation method, which indicates that the method has a lower response value under the same loading concentration, thus resulting in a limit of only 500. mu.g/g; the NaAc-HAc solution precipitation method has low separation degree due to the strong elution capability of NaAc and is greatly interfered by lactose, so that the limit of quantitation is only 300 mug/g. In conclusion, after the milk powder sample is pretreated by the 0.15% HAc solution precipitation method, the quantitative limit can reach 100 mu g/g, and the method has better separation degree, repeatability and precision, is a pretreatment method for well improving the detection of the 2 '-fucosyllactose in the milk powder, and has important significance for the detection of low-content 2' -fucosyllactose.
Example 4: selection of analytical columns
Dionex CarboPac PA20 and PA200 were selected for analytical testing based on the structural features of 2' -fucosyllactose, and the testing results are shown in Table 4.
TABLE 4 comparison of the detection of PA200 and PA20 analytical columns
Note:1retention time may measure the likelihood of separation of the peaks of interest, with smaller values indicating lower likelihood;2the standard adding condition is that the detection possibility of a target peak can be preliminarily judged by adding a 2' -fucosyllactose standard substance and observing the peak area change condition, the higher the standard adding condition isObviously, the greater the detection probability;3the separation is judged by the number of peaks in the spectrum, and the larger the number, the better the separation of the analysis column to the sample.
As can be seen from Table 4, when the milk powder is separated and detected by a Dionex CarboPac PA20 column, the retention time is longer, the labeling condition is obvious, the separation condition is good, and the possibility of separating the 2' -fucosyllactose is extremely high. And the preliminary experiments of the PA20 column show that the interference detection of the lactose peak is only large, and the separation degree of other peaks is good, so a Dionex CarboPac PA20 column is selected for further optimization, and the separation of the lactose interference peak and the target peak of 2' -fucosyllactose is mainly focused. In addition, the inventors have also investigated other analytical columns, such as: PAl, PAl0, PAl00 and MA1, found that PAl and MA1 were unable to separate lactose and 2' -fucosyllactose, while PAl0, PAl00 were less efficient at separation and column efficiency than PA20 and PA 200.
Example 5: selection of elution conditions
And (3) performing gradient elution by using 0.25M NaOH (solution A) and 1M NaAc (solution B) as eluent, wherein the elution strength of the solution B is greater than that of the solution A, controlling the elution capacity by adjusting the proportion of the solution A, the solution B and ultrapure water (solution C), and further controlling the retention time of different sugars so as to accurately separate and detect the 2' -fucosyllactose.
Firstly, the milk powder sample is pretreated and tested on a machine according to the embodiment 2, and then the test optimization is carried out according to the following elution conditions:
condition 1: 0-40min, 10% B + 90% C;
condition 2: 0-40min, 5% B + 95% C;
condition 3: 0-40min, 50% A + 50% C;
condition 4: 0-40min, 20% A + 80% C;
condition 5: 0-40min, 10% A + 90% C;
condition 6: 0-15min, 10% A + 90% C; 15-40min, 20% A + 80% C;
condition 7: 0-20min, 10% A + 90% C; 20-40min, 20% A + 80% C;
the separation under each elution condition is shown in Table 5, and the retention time, the peak shape and the degree of separation are evaluated.
TABLE 5 comparison of the detection conditions under different elution conditions
Note:1retention time may measure the likelihood of separation of the peaks of interest, with smaller values indicating lower likelihood;2the peak shape condition is illustrated by observing the overlapping, crossing, separating, tailing and other conditions of a target peak and a nearby lactose peak in a map;3the degree of separation was calculated as the difference between the maximum interfering lactose peak and the retention time of the desired 2' -fucosyllactose peak.
As can be seen from table 5, even in the case of the low concentration of the solution B (condition 1 and condition 2), the lactose peak with the largest side interference and the target peak cannot be separated well, and the retention time is short, and it is difficult to adjust the concentration of the solution B for optimization, therefore, the solution a with weak elution ability is selected for the elution separation detection. When the elution concentration was 50% a (condition 3), the lactose peak and the desired peak could not be separated yet; when the concentration is further reduced to 20% A (condition 4), the peak shapes and the separation degrees of the lactose interference peak and the target peak are better; when the elution concentration was further reduced and 10% a was set (condition 5), although the retention time and the separation degree of both peaks were improved, the elution ability was weakened and the tailing of the lactose peak was severe; therefore, the gradient was varied and elution conditions were set to 0-15min, 10% a + 90% C; 15-40min, 20% a + 80% C (condition 6), a further improvement in resolution compared to condition 4; but in condition 7, the target peak is at the corner of the gradient change, which affects the accurate determination of the peak. In summary, the elution conditions were defined as: 0-15min, 10% A + 90% C; 15-40min, 20% A + 80% C (Condition 6). In addition, 0-15min, 10% A + 90% C; 15-25min, 20% A + 80% C; the liquid A, the liquid B and the liquid C can be compounded in any proportion for 25-40min, and the detection method is not influenced.
Example 6: accuracy and reproducibility of the method
By the method of example 1 and using 0-15min, 10% a + 90% C; the samples were tested 15-40min, 20% A + 80% C elution conditions, retention time and peak area were recorded, and content and its RSD were calculated, with the results shown in Table 6. It can be seen that the method has accurate detection result, high sensitivity and no interference.
TABLE 6 results of sample measurement and analysis
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for detecting the content of 2' -fucosyllactose in milk powder comprises the following steps:
(1) sample pretreatment: dissolving a milk powder sample by adopting a precipitator, centrifuging, diluting and coating a membrane;
(2) quantitatively detecting the content of the 2' -fucosyllactose in the pretreated sample by adopting high-efficiency anion exchange chromatography; the precipitant is HAc buffer solution;
the analytical column of the high-efficiency anion exchange chromatography is a Dionex CarboPac saccharide analytical column, specifically PA20 or PA 200; eluting with eluent by high-efficiency anion exchange chromatography; the leacheate is as follows: 0.25mol/L NaOH is used as solution A, 1mol/L NaAc is used as solution B, and ultrapure water is used as solution C; the elution condition is 0-15min, 10% A + 90% C; 15-25min, 20% A + 80% C; and (3) compounding the solution A, the solution B and the solution C in any proportion for 25-40 min.
2. The method according to claim 1, wherein the HAc buffer solution has a pH of 4 to 5 and a concentration of 0.1 to 0.2% v/v.
3. The method according to any one of claims 1 to 2, wherein the amperometric detector is used for four-potential pulse amperometric detection, and the reference electrode is Ag/AgCl; using a Dionex CarboPac PA20 anion exchange column; leaching conditions are as follows: gradient elution is adopted, and 0.25M NaOH and 1M NaAC are used as eluent to carry out gradient elution, wherein the flow rate is 0.5 mL/min; column temperature: 30 ℃; sample introduction volume: 10 mu L of the solution; the exchange column is an analytical column of 3X 150mm or a guard column of 3X 30 mm.
4. Use of the method of any one of claims 1-3 for quality monitoring of infant nutrition.
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