CN115201347A - Method for detecting fermentation-derived sialic acid in food - Google Patents
Method for detecting fermentation-derived sialic acid in food Download PDFInfo
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- CN115201347A CN115201347A CN202110393709.2A CN202110393709A CN115201347A CN 115201347 A CN115201347 A CN 115201347A CN 202110393709 A CN202110393709 A CN 202110393709A CN 115201347 A CN115201347 A CN 115201347A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention provides a method for detecting fermentation source sialic acid in food, and belongs to the technical field of analysis in the field of food adulteration detection. The method comprises the following steps: 1) Separating and extracting sialic acid in a food sample to be detected; 2) Determining the carbon stable isotope ratio of sialic acid in the food sample to be detected; 3) Determining the distribution range of the carbon stable isotope ratio of the natural sialic acid; 4) Comparing the carbon stable isotope ratio of sialic acid in the food sample to be detected obtained in the step 2) with the distribution range obtained in the step 3): and if the carbon stable isotope ratio of the sialic acid in the food sample to be detected obtained in the step 2) minus 0.6 per thousand is larger than the maximum value of the distribution range, determining that the sample contains the sialic acid derived from fermentation. The method is mainly used for detecting the fermentation source sialic acid in food, and is simple, rapid in analysis and low in cost.
Description
Technical Field
The invention belongs to the technical field of analysis in the field of food adulteration detection, and particularly relates to a method for detecting sialic acid derived from fermentation in food.
Background
Sialic acid (sialic acid) is an acidic amino sugar containing 9 carbon atoms and having a pyranose structure, commonly known as C-acetylneuraminic acid, also known as cubilose acid, and systematically named 5-amino-3, 5-dideoxy-D-glycerol-D-galactononulose. Since the different linkers at carbon position 5 constitute different types of sialic acid, the two most predominant sialic acids are 5-acetylphthalylamino-3, 5-dideoxy-D-glycerol-D-galactononulose, namely C-acetylneuraminic acid (Ceu 5Ac, ceuAc) and 3-deoxy-D-glycerol-D-galactononulose (KDC).
Sialic acid is one of the components of human milk oligosaccharide in breast milk, and is also a characteristic component of the traditional high-end tonic food cubilose. Studies show that sialic acid plays a very important role in interaction of cells, virus infection and glycoprotein stability, for example, sialic acid plays an important physiological function in a body when being connected with proteins and lipids, sialic acid cholesterol can inhibit the occurrence of senile dementia to a certain extent, sialic acid is greatly related to cerebral ischemia, parkinson's disease and nerve wound healing, and sialic acid derivatives can be used as important anti-adhesion medicaments for effectively preventing excessive leukocyte aggregation and treating diseases such as rheumatoid arthritis, septic shock and the like. In addition, there are studies showing that sialic acid plays an important role in the biological evolution process.
The main food source of the C-acetylneuraminic acid/the cubilose acid/the sialic acid is milk products, eggs and meat products, but the content is low, the substance with the highest sialic acid content in nature is the cubilose (the content is 5-12 percent), therefore, the sialic acid is also called the cubilose acid, and the sialic acid is the only explicit nutrient component in cubilose classification and is also the main index of cubilose classification standard. In addition to cubilose, natural sialic acid is extremely difficult to obtain, and the initial preparation is to extract from food materials such as whey, egg yolk and the like with relatively high sialic acid content, such as JuCeja and the like, which successfully extract sialic acid from egg yolk membrane and frenum of poultry eggs, whitehouse and shimata cli extract sialic acid from cow milk whey and casein, respectively, and alpine peak and the like extract sialic acid from pig blood, but these methods are difficult to prepare on a large scale and with high efficiency due to limitations of raw materials and yield.
In 2017, the examination and passing of sialic acid as a safety evaluation material of a new food material by an organization expert of the evaluation institution according to the regulations of food safety act of the national institutes of health and family planning committee (now the national health committee) means that the mass production and the legal use of sialic acid are officially licensed. It is known that food is not only valuable in sensory flavor and nutritional function, but also in raw material, production process and production place, and is widely used in food or cosmetics due to industrial production of sialic acid derived from fermentation, which is easily confused with natural sialic acid in food, and food sold with natural sialic acid is also easily lost in promotional advantage due to addition of fermented sialic acid.
Disclosure of Invention
The invention provides a simple and efficient method for detecting fermentation-derived sialic acid in food, and provides an effective analysis means for solving the technical problem that natural sialic acid and fermentation-derived sialic acid are difficult to distinguish.
The invention provides a method for detecting fermentation source sialic acid in food, which comprises the following steps:
1) Separating and extracting sialic acid in a food sample to be detected;
2) Determining the carbon stable isotope ratio of sialic acid in the food sample to be detected;
3) Determining the distribution range of the carbon stable isotope ratio of the natural sialic acid;
4) Comparing the carbon stable isotope ratio of sialic acid in the food sample to be detected obtained in the step 2) with the distribution range obtained in the step 3):
if the carbon stable isotope ratio of the sialic acid in the food sample to be detected obtained in the step 2) minus 0.6 per mill is larger than the maximum value of the distribution range, determining that the sample contains the sialic acid from fermentation;
and if the carbon stable isotope ratio of the sialic acid in the food sample to be detected obtained in the step 2) minus 0.6 per thousand is less than or equal to the maximum value of the distribution range, determining that the sample does not contain the sialic acid from fermentation.
Specifically, natural sialic acid in food and sialic acid from fermentation have different isotope characteristics, but since standard deviation of carbon isotope ratio measured by a stable isotope ratio mass spectrometer is required to be better than 0.3 per thousand, 95% confidence interval of the carbon isotope ratio in a sample is determined as +/-0.6, and when the determined value-0.6 per thousand is larger than the distribution range of the natural sialic acid in the bird's nest, the sample to be tested is considered to contain the fermented sialic acid.
Further, in the step 1), the separation and extraction is to extract sialic acid of a food sample to be detected by adopting a preparation liquid phase technology after pretreatment is carried out according to a GB/T30636-2014 method.
Further, in the step 1), the preparation liquid phase technology is specifically to inject 2mL of pretreated solution into a preparation liquid chromatograph, collect an effluent liquid containing sialic acid, remove an organic solvent, freeze-dry to obtain solid powder, and store the solid powder for testing. Specifically, 2mL of the pretreated solution is injected into a preparative liquid chromatograph, the sialic acid-containing effluent is collected, the organic solvent is removed, and the solid powder is obtained by freeze drying and stored for testing.
Further, in the step 1), the food samples to be detected comprise dry edible bird's nest and rock candy edible bird's nest.
Further, in the step 2), a stable isotope ratio mass spectrometer is adopted to determine the nitrogen stable isotope ratio.
Further, in step 3), the natural sialic acid is derived from real edible bird's nest or real edible bird's nest products.
Further, the real edible bird's nest or the real edible bird's nest product is classified according to edible bird's nest quality grade, and the real edible bird's nest collected from edible bird's nest product manufacturers meets the specification of 3.1 in GH/T1092-2014. The real edible bird's nest or the real edible bird's nest product refers to the real edible bird's nest without exogenous sialic acid added. The real cubilose samples are classified according to cubilose quality grades, and the real cubilose without additives collected from cubilose product manufacturers conforms to the specification of 3.1 in GH/T1092-2014.
Further, in the step 3), a sample containing a plurality of natural sialic acids is selected, and carbon stable isotope ratio measurement is carried out, so that the distribution range of the carbon stable isotope ratio of the natural sialic acids is obtained.
The invention has the following advantages:
the method for detecting the fermentation source sialic acid in the food is obtained by comparing the distribution range of the sialic acid nitrogen stable isotope ratio of a sample to be detected with the distribution range of the carbon stable isotope ratio of natural sialic acid, is simple, quick in analysis and low in cost, is suitable for analyzing and confirming the sialic acid source in the food, and is beneficial to research and application of a stable isotope ratio mass spectrometry technology in the field of food adulteration detection.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The embodiments and features of the embodiments of the present invention may be combined with each other without conflict.
Example 1 validation of stable isotope ratio Mass spectrometer method for determining sialic acid carbon stable isotope ratio in bird's nest preparation
1) Taking a commercial sialic acid sample from fermentation source, one dry cubilose and one rock candy cubilose product (No. 1#, no. 2# and No. 3 #), as research objects.
2) The sialic acid content of the dried bird's nest and rock candy bird's nest products was measured according to the method in GB/T30636-2014 and was 119.51g/kg and 1.83g/kg, respectively.
3) Soaking dried nidus Collocaliae with 10 times of water, and adding commercially available fermentation source sialic acid according to 20%, 40%, 60% and 80% of sialic acid content in dried nidus Collocaliae and crystal sugar nidus Collocaliae to obtain simulated sample with serial numbers of 2-1#, 2-2#, 2-3#, and 2-4#; the serial numbers of the rock candy cubilose simulation samples are 3-1#, 3-2#, 3-3# and 3-4#.
4) Taking 0.1g of commercial fermentation source sialic acid sample, diluting with 5mL of purified water, taking 10g of each of a dried cubilose foaming sample and dried cubilose simulation samples (2-1 #, 2-2#, 2-3# and 2-4 #), and respectively pre-treating 60g of each of a rock candy cubilose sample and rock candy cubilose simulation samples (3-1 #, 3-2#, 3-3# and 3-4 #). The pretreatment comprises the steps of pretreating a sample according to a GB/T30636-2014 method to obtain a sample solution, injecting 2mL of the sample solution after pretreatment into a liquid chromatograph, collecting an effluent liquid containing sialic acid, removing an organic solvent, freeze-drying to obtain solid powder, and storing the solid powder to be detected.
2. Testing
5) Direct determination of commercial fermentations without pretreatment using stable isotope ratio mass spectrometry systemCarbon stable isotope ratio (delta) of Source sialic acid 13 C) The results of 5 measurements are shown in Table 1.
TABLE 1 commercial fermentation-derived sialic acid delta 13 C direct measurement (esi)
| Number of measurements | 1 | 2 | 3 | 4 | 5 | Mean value of | SD |
| δ 13 C(‰) | -13.05 | -12.81 | -12.69 | -12.77 | -12.95 | -12.85 | 0.11 |
Table 1 shows sialic acid delta 13 The stability of the C value measurement accords with the analysis requirement (SD less than 0.3 per mill) of stable isotope characteristics in the food field, and saliva can be realizedStability analysis of acid carbon stable isotope characteristics.
6) The sialic acid samples collected after pretreatment of commercial fermentation-derived sialic acid, dry cubilose foam samples and rock candy cubilose samples were measured by using a stable isotope standard mass spectrometry system, and the measurement results are shown in table 2 after 3 times of separation and collection.
TABLE 2 Delta of commercially available sialic acid after pretreatment 13 C measured value (‰)
The results in Table 2 show that the re-determination of sialic acid after treatment had an average value of-12.88%, which differed only by 0.03% from the average value of-12.85% determined directly in Table 1, and was negligible, indicating that the pretreatment step of the samples did not alter the carbon isotope characteristics of sialic acid, and that the re-determination of sialic acid delta after pretreatment of the three samples resulted in a negligible difference 13 The standard deviation of the C value is 0.11 per thousand, 0.15 per thousand and 0.14 per thousand respectively, and the SD meets the requirement of being better than 0.3 per thousand.
7) Determination of sialic acid delta of simulated sample after pretreatment by using stable isotope standard mass spectrum system 13 C values, results are shown in Table 3.
TABLE 3 simulation of sialic acid delta in samples 13 C determination ([ sic ])
| Sample series | δ 13 C(‰) | Sample series | δ 13 C(‰) |
| 2-1# | -24.41 | 3-1# | -24.92 |
| 2-2# | -21.35 | 3-2# | -22.05 |
| 2-3# | -18.69 | 3-3# | -18.15 |
| 2-4# | -15.52 | 3-4# | -15.65 |
The data in Table 3 show that dried and rock candy bird's nests have a negative trend after addition of commercially available sialic acid, and statistical analysis shows that delta in the simulated samples 13 The C value is in a significant negative correlation with the addition of the commercial sialic acid, and the correlation coefficient R 2 All are above 0.99, which shows that the method can accurately determine the delta of sialic acid in the cubilose and products thereof 13 The C value, in turn, indicates the source of sialic acid in the food product.
Example 2 a method for detecting fermentatively derived sialic acid in dry bird's nest comprising the steps of:
1) 24 dry cubilose samples (containing sialic acid as natural sialic acid) with a cubilose tracing code of a Chinese cubilose tracing management service platform are collected from cubilose product production enterprises;
2) 3 samples of fermentation-derived sialic acid were purchased from the market as validation samples;
3) 2 dry cubilose samples are purchased from the market and used as samples to be detected;
4) Sialic acid delta was determined for each sample according to the method of example 1 13 C value;
5) The delta of natural sialic acid in 24 dry cubilose is counted 13 The C value distribution range is-29.23 to-26.56 per mill, and the delta of 3 fermented sialic acid samples 13 The C values are-12.97 per mill, -12.88 per mill and-11.53 per mill respectively, so that the fermented sialic acid has a delta which is obviously different from the natural sialic acid in the dry bird's nest 13 C value distribution characteristics;
6) Delta comparison of Dry cubilose and commercial sialic acid 13 The C value distribution range, in combination with the correlation obtained in example 1, when the amount of fermented sialic acid added to the dried bird's nest product was 4.5% or more, the carbon-stable isotope distribution characteristics (. Delta. Sup.5% or more) were calculated from the minimum value (-12.97% o.) of fermented sialic acid and the maximum value (-26.56% o.) of dried bird's nest product 13 C measured value-0.6 = -26.55 ‰) does not conform to distribution range (greater than-26.56 ‰) of natural sialic acid of dry nidus Collocaliae, and dry nidus Collocaliae product can be judged to contain sialic acid component from fermentation;
7) Delta of 2 commercially available samples of dried bird's nest 13 The measured values of C are-26.78 ‰ and-27.43% 13 The results of the C measured value of-0.6 per mill are all smaller than the maximum value of the distribution range of the dry cubilose, so that the sialic acid of 2 dry cubilose samples is all natural sources, and the sialic acid of fermentation sources is not detected.
Example 3 a method for detecting sialic acid sources in a bird's nest preparation, comprising the steps of:
1) Collecting 17 rock candy cubilose products (containing sialic acid as natural sialic acid) from cubilose product manufacturers;
2) 5 rock candy cubilose products are purchased from the market and used as samples to be detected;
3) Sialic acid delta was determined for each sample according to the method of example 1 13 C value;
4) According to statistics, the delta of sialic acid in 17 rock candy cubilose products 13 The distribution range of the C value is-28.61 per mill to-27.47 per mill;
5) In example 2, 24 dry bird's nests are distributed in the range of-29.23 ‰ -26.56 ‰, since the dry bird's nest is the raw material of crystal sugar bird's nestDelta of rock candy bird's nest in example 3 13 The distribution range of the C value is within the range of the dry bird's nest in the example 2, so that-29.23 per thousand to-26.56 per thousand are taken as the distribution range of the rock candy bird's nest in the example;
6) Sialic acid delta in commercial rock candy bird's nest products 13 The results of the C value are shown in Table 4:
TABLE 4 Delta sialic acid in commercially available rock candy bird's nest 13 C measured value (esi)
| Sample numbering | ss-1 | ss-2 | ss-3 | ss-4 | ss-5 |
| δ 13 C(‰) | -27.01 | -28.85 | -28.18 | -26.83 | -25.43 |
As can be seen from Table 4, sialic acid. Delta. Was found in 4 samples other than ss-5 sample 13 The C values are all less than 26.56 per thousand, so that the 4 products can be judged not to contain the sialic acid of fermentation source; sialic acid delta in ss-5 samples 13 The C measurement was-25.43% o, and the SD measured by the method was 0.3% o25.43-0.6 = -26.03 ‰, greater than maximum value of dry nidus Collocaliae/Crystal sugar nidus Collocaliae distribution range (-26.56 ‰), so in this example, it can be determined that the product contains exogenous fermentation-derived sialic acid.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for detecting fermentation-derived sialic acid in a food product, comprising the steps of:
1) Separating and extracting sialic acid in a food sample to be detected;
2) Determining the carbon stable isotope ratio of sialic acid in the food sample to be detected;
3) Determining the distribution range of the carbon stable isotope ratio of the natural sialic acid;
4) Comparing the carbon stable isotope ratio of sialic acid in the food sample to be detected obtained in the step 2) with the distribution range obtained in the step 3):
if the carbon stable isotope ratio of the sialic acid in the food sample to be detected obtained in the step 2) minus 0.6 per mill is larger than the maximum value of the distribution range, determining that the sample contains the sialic acid from fermentation;
and if the carbon stable isotope ratio of the sialic acid in the food sample to be detected obtained in the step 2) minus 0.6 per thousand is less than or equal to the maximum value of the distribution range, determining that the sample does not contain the sialic acid from fermentation.
2. The method of claim 1,
in the step 1), the separation and extraction is to pre-treat a food sample to be detected according to a GB/T30636-2014 method, and extract sialic acid in the food sample to be detected by adopting a preparation liquid phase technology.
3. The method of claim 2,
in the step 1), the liquid phase preparation technology specifically comprises the steps of injecting the pretreated solution into a liquid chromatograph, collecting an effluent liquid containing sialic acid, removing an organic solvent, and freeze-drying to obtain solid powder.
4. The method of claim 1,
in the step 1), the food samples to be detected comprise dry edible bird's nest and rock candy edible bird's nest.
5. The method of claim 1,
in the step 2), a stable isotope ratio mass spectrometer is adopted to measure the nitrogen stable isotope ratio.
6. The method of claim 1,
in the step 3), the natural sialic acid is derived from real cubilose or a real cubilose product.
7. The method of claim 1,
the real cubilose or the real cubilose product is classified according to cubilose quality grades, and the real cubilose without additives collected from cubilose product manufacturers meets the regulation of 3.1 in GH/T1092-2014.
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