Method for determining stable isotope ratio of non-exchangeable hydrogen in grain starch

Abstract

The invention provides a method for determining the ratio of non-exchangeable hydrogen stable isotopes in grain starch, which is suitable for food stable isotope analysis. The method comprises the following steps: 1) crushing a sample; 2) removing fibers, proteins and moisture; 3) gelatinizing the sample by using a reagent containing exchangeable hydrogen to realize isotope exchange balance; 4) dissolving a starch standard substance and gelatinizing; 5) drying; (6) determining the stable hydrogen isotope ratio of the sample to the standard substance by using a high-temperature cracking / element analysis-stable isotope ratio mass spectrum; and 7) correcting according to the standard curve to obtain the isotope ratio of the non-exchangeable hydrogen in the starch. The method can be used for accurately measuring the ratio of the non-exchangeable hydrogen in the grain starch and eliminating the interference of moisture on hydrogen isotope characteristics, and is suitable for research and application of traceability of grain production places.

Description

Technical Field

[0001] This invention belongs to the field of stable isotope analysis and testing, and relates to a method for determining the stable isotope ratio of non-exchangeable hydrogen in grain starch, so as to analyze the isotope ratio of non-exchangeable hydrogen in grain starch in agricultural product traceability research. Background Technology

[0002] Stable isotope technology is currently an effective tool for tracing food from different sources and implementing geographical indication protection. It has broad application prospects in the field of agricultural product origin traceability, and there are many reports on the research and application of isotope techniques such as carbon, hydrogen, oxygen, nitrogen, and sulfur. Since the differences in hydrogen and oxygen isotopes in organic matter mainly originate from atmospheric precipitation and plant physiological activities, natural fractionation characteristics are more obvious, especially for hydrogen isotopes. Therefore, it is a current research hotspot. For example, Liu Hongyan et al. found significant differences in the δ2H values ​​of whole wheat flour and different extracts, and the δ2H value of defatted wheat was significantly positively correlated with the δ2H value of soil water at 0–20 cm depth during the maturity period; Syed Abdul Wadood et al. revealed that the processing process has a significant impact on the δ2H of wheat; F. Longobardi et al. extracted starch and protein from lentils and measured the δ13C, δ2H, and δ18O in starch, indicating that the stable hydrogen isotopes of protein and starch can be used as characteristic components for identifying origin traceability.

[0003] However, Harold C Urey discovered that, in addition to natural fractionation characteristics, stable isotopes can also undergo atomic exchange in some loosely bonded groups, leading to isotopic changes until exchange equilibrium is reached. The hydrogen atom on the hydroxyl group is a prime example. It is well known that although grains are dried after harvesting before transportation, trade, and production, a large amount of moisture remains (e.g., the quality index for rice in GB / T 1354-2018). Because water and starch have different hydrogen isotopic characteristics, even though water can be removed when measuring the hydrogen isotope ratio of starch, the hydrogen isotope characteristics of the starch hydroxyl groups are still affected by moisture. In the field of origin traceability, the moisture content of different origins has different hydrogen isotope ratios. Therefore, the hydrogen isotope characteristics of starch will be affected by the natural fractionation characteristics during growth and the influence of product moisture on exchangeable hydrogen. If the influence of moisture on exchangeable hydrogen isotopes is eliminated during the measurement process, the measurement results will have higher application value in origin traceability, but this has not been considered in many current studies.

[0004] Aiman ​​Abrahim and Simon Kelly of the International Atomic Energy Agency (Abrahim, Aiman; Kelly, Simon. Determination of carbon bound non-exchangeable (CBNE) hydrogen isotoperatios in starch by Microwave Assisted Hydrolysis (MAH) and GC-CrAg / HTC-IRMS[J]. EGU23, the 25th EGU General Assembly, held 23-28 April, 2023 in Vienna, Austria and Online. Online at https: / / egu23.eu / , id.EGU-16747) attempted to replace the exchangeable hydrogens in starch through derivatization and then use GC-CrAg / HTC-IRMS to determine the hydrogen isotope ratios of the starch derivatives, thereby obtaining the isotopic characteristics of the non-exchangeable hydrogens in starch. However, this method involves derivatization reactions, and the pretreatment conditions are harsh and difficult to control.

[0005] Therefore, how to eliminate the interference of exchangeable hydrogen and achieve the optimal non-exchangeable hydrogen isotope ratio (δD) in starch is crucial. CBNE The determination of ) is not only an innovation in methodology, but also lays the theoretical foundation for the research and application of origin traceability. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention develops a method for determining non-exchangeable hydrogen sites to measure the isotopic ratio of non-exchangeable hydrogen sites in grain starch, comprising the following steps:

[0007] 1) Add a reagent containing exchangeable hydrogen to starch to gelatinize it;

[0008] 2) Dissolve the starch standard in the same proportion and gelatinize;

[0009] 3) Drying;

[0010] 4) The stable hydrogen isotope ratio δD of the dried samples and standards was determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry. 测 ;

[0011] 5) Construct a standard curve based on the given value of non-exchangeable hydrogen in starch standard and the measured value of hydrogen isotope, and calibrate the isotope ratio of non-exchangeable hydrogen in grain starch.

[0012] Exemplarily, in this invention, the original exchangeable hydrogen in starch is replaced by a reagent containing exchangeable hydrogen to achieve hydrogen isotope exchange equilibrium. The key to this process is selecting suitable reagents that can undergo isotope exchange reactions with the hydroxyl hydrogen in starch, thereby eliminating the interference of the original exchangeable hydrogen on the isotope ratio determination. Exemplarily, the reagents include methanol, ethanol, water, etc., which not only have good hydrogen isotope exchange properties but are also readily available and easy to process. By directly adding these reagents to the starch sample, the exchangeable hydrogen in the starch undergoes isotope exchange with the hydrogen atoms in the reagent until equilibrium is reached. This method ensures that the hydrogen isotope ratio obtained in the subsequent high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry determination primarily reflects the isotopic characteristics of non-exchangeable hydrogen. Optionally, the method of this invention may not include the step of processing starch standards, as this can be performed in other scenarios.

[0013] Specifically, during the operation, reagents such as methanol, ethanol, acetic acid, and water can be added to the starch sample in a certain proportion. The selection of reagents should take into account their chemical properties and reactivity with the hydroxyl hydrogens in starch. For example, methanol and ethanol can effectively undergo isotope exchange with the hydroxyl hydrogens in starch.

[0014] After adding the reagent, the sample usually needs to be gelatinized under certain temperature conditions, such as at a temperature above 60°C, for example, 85°C (e.g., in a water bath), for a certain period of time, so that the starch and reagent can come into full contact and reach hydrogen isotope exchange equilibrium. The gelatinization temperature is adjusted according to the solvent.

[0015] For water-soluble samples, such as wheat and rice, water is used as a solvent during gelatinization. Since water boils at 100°C, the gelatinization temperature can be set close to or slightly above the boiling point of water to promote the swelling and hydration of starch granules. It is generally recommended to set the gelatinization temperature between 90°C and 100°C to ensure sufficient dissolution of starch granules and isotope exchange.

[0016] For organic solvent processing methods, such as methanol or ethanol, their boiling points are significantly lower than water, at 64.7°C and 78.5°C, respectively. Therefore, the gelatinization temperature needs to be adjusted accordingly to accommodate the characteristics of these organic solvents.

[0017] When using methanol or ethanol as a solvent, the gelatinization temperature can be appropriately reduced to between 60°C and 70°C. This avoids the organic solvent from evaporating too quickly, while still promoting the expansion and dissolution of starch granules, thus ensuring sufficient isotope exchange.

[0018] By adjusting the gelatinization temperature according to different solvents, optimal gelatinization results can be ensured in various sample processing methods. This guarantees accurate determination of the isotope ratios of non-exchangeable hydrogen in the sample, regardless of whether water or organic solvent treatment is used, thus improving the applicability and reliability of the method.

[0019] Gelatinization is a physical and chemical process involving structural changes in starch granules under the influence of heat and solvents. It is an important step in food science and isotope analysis, especially when isotope exchange reactions are required.

[0020] Starch is typically composed of two polysaccharide components: amylose and amylopectin. Amylose is a long chain of glucose molecules linked by α-1,4-glycosidic bonds; amylopectin has a branched structure in which glucose molecules are linked by α-1,6-glycosidic bonds. Starch granules have a semi-crystalline structure, with amylose mainly distributed in the amorphous region and amylopectin mainly in the crystalline region. When starch is suspended in water and heated, water molecules begin to penetrate the starch granules. Heating causes the starch granules to swell, and water molecules enter the amorphous region. At this point, the starch granules begin to absorb water and swell, causing some hydrogen bonds to break. With continued heating, the starch granules swell further. The interaction between water molecules and starch molecules strengthens, gradually destroying the semi-crystalline structure of the starch granules. Amylose molecules dissolve from the granules, forming a viscous solution, while amylopectin partially dissociates within the granules, forming a colloidal suspension. During gelatinization, the starch granules absorb water to the maximum extent, resulting in a significant increase in viscosity. At this point, the structure of the starch granules is completely destroyed, forming a homogeneous paste. This stage, known as the gelatinization peak stage, is typically reached around 85°C. As the temperature continues to rise, all starch granules undergo complete hydration and expansion, forming a homogeneous paste. At this point, new hydrogen bonds reform between starch molecular chains, leading to a further increase in solution viscosity and ultimately the formation of a gel structure. This process not only disrupts the original structure of the starch granules but also fully exposes the exchangeable hydrogen atoms, facilitating subsequent isotope exchange reactions. During gelatinization, hydrogen atoms in the starch (especially those on the hydroxyl groups) exchange isotopes with hydrogen atoms in external water. This exchange process requires specific time and temperature conditions to ensure isotopic equilibrium is reached. Gelatinization ensures that the exchangeable hydrogen in the starch reaches complete isotopic equilibrium with the hydrogen atoms of the external reagent, thereby eliminating the influence of external water on the hydrogen isotope ratio of the starch.

[0021] In this invention, gelatinization is a key step to ensure that the exchangeable hydrogen isotopes in the starch sample reach equilibrium, thereby eliminating the influence of external moisture.

[0022] For example, the gelatinization may include the following specific steps:

[0023] 1) Dissolve the starch sample in a certain ratio (e.g., 1:40) in acetic acid, methanol, ethanol, or water containing exchangeable hydrogen, and heat it in a water bath at 85°C. This temperature is sufficient to allow the starch granules to fully swell and hydrate, reaching a gelatinized state.

[0024] 2) Keep it in a water bath at 85°C for a certain period of time (e.g., 6 hours) to ensure that the exchangeable hydrogen in the starch is fully exchanged with the hydrogen atoms in the solvent to achieve isotopic equilibrium.

[0025] 3) The gelatinized starch sample was dried at 105°C to constant weight to remove all moisture or solvent. The dried sample no longer contains exchangeable hydrogen, and only non-exchangeable hydrogen retains its original isotopic characteristics.

[0026] After gelatinization and drying, the hydrogen isotope ratio of the starch sample was determined using a high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer.

[0027] Through the above gelatinization steps, the present invention can effectively eliminate the influence of moisture on the hydrogen isotope ratio of starch, ensuring accurate and reliable measurement results and providing a scientific basis for the research on the traceability of agricultural products.

[0028] In the method of the present invention, starch standards are treated with the same reagents as the sample and in the same or similar proportions to make them have the same or similar compositional characteristics as the sample solution. The stable hydrogen isotope ratio of starch is determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry. The method is characterized by the consistency of the exchangeable hydrogen isotope characteristics in the starch sample and starch standards, which can then be eliminated by data correction.

[0029] For example, in step 1), the original exchangeable hydrogen in starch is replaced with a reagent containing exchangeable hydrogen. The mass of this reagent is 4-100 times that of the sample or standard, preferably 40 times, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 times.

[0030] Taking 40 times as an example, in this invention, the ratio of 1:40 refers to the weight ratio of starch sample to reagent (methanol or ethanol or water), that is, 1 gram of starch sample corresponds to 40 grams of reagent.

[0031] The gelatinization process requires sufficient solvent to come into full contact with the starch granules to ensure complete swelling and hydration. A 1:40 ratio provides enough solvent to penetrate evenly into the starch granules, promoting a smooth gelatinization process.

[0032] Stable isotope analysis requires the exchangeable hydrogen atoms in starch to fully exchange with hydrogen atoms in external reagents to achieve isotopic equilibrium. A 1:40 ratio ensures that the number of hydrogen atoms in the reagent is far greater than that in the starch, providing sufficient opportunities for isotope exchange and ensuring the thoroughness and uniformity of the exchange process.

[0033] For example, a weighed starch sample (e.g., 0.1 g) is dissolved in 40 times its weight of a reagent (e.g., 4 g of methanol or ethanol, acetic acid, or water). This ratio ensures that the starch particles can be sufficiently diffused and suspended in the reagent, facilitating subsequent heating and gelatinization.

[0034] Heating is performed in a water bath at 85°C. The high temperature causes the starch granules to absorb the solvent and swell, eventually forming a uniform paste. An excess of solvent ensures thorough contact between the starch granules and the reagent, guaranteeing that each granule is heated evenly.

[0035] During heating, exchangeable hydrogen atoms in the starch undergo isotopic exchange with hydrogen atoms in the reagent. The excess solvent provides a large number of reagent hydrogen atoms, ensuring that all exchangeable hydrogen atoms in the starch have the opportunity to exchange with hydrogen atoms in the reagent, achieving isotopic equilibrium.

[0036] An excess of solvent helps ensure the uniformity of isotope exchange. If the amount of reagent is insufficient, some hydrogen atoms in the starch may not be able to fully exchange with hydrogen atoms in the reagent, leading to inaccurate results. A sufficient amount of reagent covers all starch granules, ensuring the uniformity of the exchange process.

[0037] In isotope analysis, accurate determination of isotope ratios depends on complete isotope exchange. An excess of solvent ensures thorough isotope exchange by providing sufficient reagent, thereby improving the accuracy and reliability of the determination results.

[0038] The excess solvent was not chosen arbitrarily, but was the result of experimental verification. Researchers compared the effects of different ratios (such as 1:4, 1:10, 1:20, 1:40, etc.) and found that an excess solvent could provide sufficient isotope exchange while ensuring gelatinization, thus obtaining the best measurement results.

[0039] In practical applications, an excess of solvent has been proven to be the optimal ratio for various types of starch (such as corn starch, wheat starch, and soybean starch). This ratio is suitable not only for small sample processing under laboratory conditions but also for the analysis and testing of large-scale samples.

[0040] When treating samples with an excess of solvent (e.g., a 1:40 ratio), standards are also treated at the same ratio. This ensures that samples and standards are subjected to identical conditions during gelatinization and isotope exchange, avoiding discrepancies in measurement results due to different treatment conditions.

[0041] A standard curve can be constructed by using standards treated with an excess solvent (e.g., a 1:40 ratio) to correct the measurement results of the samples. The construction of the standard curve is based on the known non-exchangeable hydrogen isotope ratio (δDCBNE) of the standards, ensuring the accuracy and reliability of the final measurement results.

[0042] For example, in a specific implementation of the present invention, the 1:40 ratio is applied as follows:

[0043] First, the grain sample (such as corn, wheat, soybean) is ground into a uniform powder using a mill.

[0044] Add 10 times the amount of water and adjust the pH to 9 with NaOH solution. Vortex for 10 minutes, then filter through gauze to remove residue. Centrifuge to collect the precipitate, and dry to obtain the starch sample.

[0045] Weigh a certain amount of starch sample (e.g., 0.5 g) and add pure methanol (i.e., 20 g of water) in excess of the solvent.

[0046] The dissolved starch sample was heated in a water bath at 85°C for 6 hours to ensure complete gelatinization and isotope exchange.

[0047] The gelatinized starch sample was dried at 105°C to constant weight.

[0048] The hydrogen isotope ratios of the dried samples and standards were determined using a high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer.

[0049] A standard curve was constructed based on the known δDCBNE values ​​and measured values ​​of the reference materials. The measured results of the samples were substituted into the standard curve to calibrate and obtain the isotopic ratio of non-exchangeable hydrogen in starch. Excess solvent plays a crucial role in the gelatinization and isotopic exchange process of this invention. It not only ensures sufficient gelatinization of starch granules but also provides a sufficient amount of reagent to achieve complete isotopic exchange, thereby improving the accuracy and reliability of the measurement results. Through this optimized ratio treatment, the stable isotopic ratio of non-exchangeable hydrogen in grain starch can be accurately determined, providing a scientific basis for the traceability of agricultural products.

[0050] Besides the 1:40 ratio, other ratios can be used in the implementation of this invention for the gelatinization and isotope exchange of starch samples. The following is a description of some other ratios:

[0051] 1:10 scale

[0052] The 1:10 ratio refers to the weight ratio of starch sample to reagent (such as water), that is, 1 gram of starch sample corresponds to 10 grams of reagent.

[0053] A 1:10 ratio is typically used for small-scale experimental studies or samples requiring rapid processing. At this ratio, the reagent volume is relatively small, and the gelatinization process may not be as thorough as at a 1:40 ratio, but a certain degree of isotope exchange can still be achieved. The advantages are ease of operation and speed, making it suitable for situations where experimental conditions are not strictly demanding. However, due to the relatively small reagent volume, uneven gelatinization and incomplete isotope exchange may occur, affecting the accuracy of the measurement results.

[0054] 1:20 scale

[0055] A 1:20 ratio means that 20 grams of reagent (such as water) are needed for every 1 gram of starch sample.

[0056] A 1:20 ratio is common in laboratories and is suitable for general isotope analysis experiments. At this ratio, the reagent volume is moderate, providing sufficient reagent for isotope exchange while ensuring proper gelatinization.

[0057] A 1:20 ratio results in better isotope exchange and more thorough gelatinization compared to a 1:10 ratio. However, it may be slightly less effective than a 1:40 ratio because the smaller reagent volume may result in some starch granules not being fully gelatinized and undergoing isotope exchange.

[0058] In addition to the ratios mentioned above, other ratios, such as 1:30 or 1:50, can be selected according to specific experimental requirements. Different ratios will have varying degrees of impact on the gelatinization process and isotope exchange efficiency. In practical applications, selecting an appropriate ratio requires comprehensive consideration of factors such as the experimental objective, sample properties, and equipment conditions.

[0059] When selecting the ratio of starch gelatinization to isotope exchange, it is necessary to make a reasonable choice based on the specific experimental requirements and conditions. The following are some factors to consider when selecting the ratio:

[0060] Experimental Objective: If the experiment requires a high degree of isotope exchange, it is recommended to choose a larger reagent ratio, such as 1:40 or higher. If the experimental objective is to quickly screen samples or perform preliminary analysis, a smaller ratio may be more suitable.

[0061] Sample properties: Different sources and types of starch samples may require different amounts of reagents. Some starch samples may gelatinize and undergo isotopic exchange more easily, while others may require more reagents to achieve the same effect.

[0062] Laboratory equipment: The availability of equipment in the laboratory also affects the selection of proportions. Some laboratories may only provide a limited quantity of reagents, requiring the selection of proportions within this constraint.

[0063] Previous validation: It is recommended to conduct some small-scale validation experiments before the formal experiment to test the effect at different ratios in order to determine the most suitable ratio.

[0064] In this invention, the high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer measures the stable hydrogen isotope ratio of each starch. This value is the average value of the isotopic characteristics of the numerous hydrogen atoms in the starch, including non-exchangeable hydrogen and exchangeable hydrogen.

[0065] In this invention, the grain samples are starchy products such as cereals (wheat, rice, sorghum, corn, millet, etc.), legumes (soybeans, peas, etc.), and tubers (sweet potatoes, potatoes).

[0066] Furthermore, in the method of this invention, the condition parameters of the high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer are adjusted as follows: the helium carrier gas flow rate is 100 mL / min, the helium purge gas flow rate is set to 175 mL / min; the column oven program is set to a constant temperature of 80℃; and the pyrolysis module is kept at a constant temperature above 1400℃.

[0067] Optionally, confirm that the working environment, airtightness, and vacuum degree of the ion chamber of the stable isotope ratio mass spectrometer meet the analytical requirements, and then check the precision and linearity of the instrument in measuring δD in H2, adjusting the ion source parameter values ​​if necessary.

[0068] Specifically, the present invention provides a pretreatment method for determining the isotope ratio of non-exchangeable hydrogen in grain starch, characterized by comprising the following steps:

[0069] 1) Use physical methods to crush grain samples;

[0070] 2) Add 10 times the amount of water to the grain sample powder, adjust the pH to 9 with NaOH solution, vortex for 10 minutes, and filter with gauze to remove residue;

[0071] 3) Centrifuge at 10000 r / min for 20 min, collect the precipitate, dry it and pass it through a 120 mesh sieve to obtain the starch sample;

[0072] 4) Take starch sample and starch standard, add methanol or ethanol or water at a ratio of 1:40, and gelatinize under water bath conditions at 85℃;

[0073] 5) The gelatinized starch samples were dried at 105℃ to constant weight;

[0074] 6) The hydrogen isotope ratios of starch samples and starch standards were determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry, respectively.

[0075] 7) Construct a standard curve based on the given value of non-exchangeable hydrogen in starch standard and the measured value of hydrogen isotope, and then correct the isotope ratio of non-exchangeable hydrogen in starch in grain samples.

[0076] The operational scheme provided by this invention offers a rapid and stable method for the isotopic analysis of non-exchangeable hydrogen in grain starch and its application in the field of origin traceability. Furthermore, this invention is simpler to operate, has a high safety factor, and is suitable for the analysis and testing of large batches of samples, which is beneficial to promoting further research and application of hydrogen isotope technology in the field of agricultural product origin traceability. This invention provides a method for determining the isotopic ratio of non-exchangeable hydrogen sites in grain starch. This method can eliminate the influence of isotopic exchangeable hydrogen in moisture and starch in grain products, and thus can accurately and stably analyze the isotopic ratio of non-exchangeable hydrogen in grain starch using high-temperature pyrolysis / elemental analysis-stable isotopic ratio mass spectrometry. Detailed Implementation

[0077] In this invention, the various embodiments and features within them are not mutually exclusive, but can be combined according to specific circumstances. This means that, where applicable, different embodiments or features can be flexibly selected and combined to meet specific needs or achieve specific goals. This combinable nature makes the invention more flexible and customizable, adaptable to different practical application scenarios and requirements. For example, different solvents can be selected, gelatinization time and temperature adjusted, or different measurement methods and instrument conditions can be used when processing samples. This flexibility in combination makes the invention more widely applicable and operable in practical applications, better meeting user needs.

[0078] Experimental Example 1 Verification of the instability of starch with exchangeable hydrogen

[0079] Select a starch sample (δD direct measurement result is -32.1‰), dissolve it in water with different hydrogen isotope characteristics at a ratio of 1:40, bathe in water at 85℃ for 10h, let stand for 24h, dry and measure the hydrogen isotope ratio (δD) of starch. The results are shown in Table 1.

[0080] Table 1. δD (‰) determination results of starch after isotopic exchange in water.

[0081] δD value of water (‰) δD value (‰) of starch n Standard deviation -3.1 -27.7 3 0.8 -46.1 -33.3 3 1.3 -142.3 -46.1 3 0.9

[0082] As shown in the table above, the results of direct starch determination differ from those obtained after dissolving / gelatinizing / drying in water. This indicates that exchangeable hydrogen does indeed exist in starch and its composition changes due to the presence of external exchangeable hydrogen. In fact, the molecular structure of starch reveals that approximately 40% of its hydrogen atoms are bonded to the oxygen atoms of the hydroxyl groups, while only about 60% are directly bonded to carbon atoms. This 40% of hydroxyl hydrogen is the reason for hydrogen atom exchange. Table 1 shows that the starch determination results change linearly with variations in the δD value of the water involved in the exchange, indicating that hydrogen isotope exchange varies depending on the external water source, but also demonstrating a predictable pattern. Therefore, based on the theory of natural fractionation of hydrogen isotopes in atmospheric precipitation and Rayleigh fractionation of isotopes in plant transpiration, the hydrogen isotope characteristics of grains are widely used in current agricultural product traceability research. However, only by eliminating the interference of exchangeable hydrogen, especially the influence of differences in the water hydrogen isotope characteristics of grains from different origins, can it be scientifically used for origin traceability research.

[0083] Experimental Example 2 Research on isotopic determination methods for exchangeable hydrogen in starch

[0084] Three treated starch samples from Experiment 2 were reconstituted with water containing δD = -46.1‰ at a ratio of 1:40, bathed in an 85℃ water bath for 10 hours, allowed to stand for 24 hours, dried, and then the hydrogen isotope ratio (δD) of the starch was measured. The results are shown in Table 2.

[0085] Table 2. δD determination results (‰) of starch after excluding differences in exchangeable hydrogen.

[0086] δD value (‰) of starch δD value (‰) of starch after reprocessing -27.7 -32.4 -33.3 -32.5 -46.1 -33.5

[0087] As shown in the table above, although the exchangeable hydrogen isotope characteristics of the three starch samples are different, after being reconstituted with the same water, heated and dried, the hydrogen isotope determination results of the starch are consistent, that is, the original differences in exchangeable hydrogen isotopes have been eliminated.

[0088] Experimental Example 3 Method stability study

[0089] a) Take about 10g of corn, wheat and soybeans respectively and grind them into powder using a grinder;

[0090] b) Weigh 5.00g of corn, wheat and soybean flour samples respectively, add 50g of water, adjust the pH to about 9 with 1N NaOH solution, vortex for 10min, and filter with gauze to remove residue;

[0091] c) Centrifuge at 10000 r / min for 20 min, collect the precipitate, dry it and pass it through a 120 mesh to obtain the starch sample;

[0092] d) Weigh 0.50g of the rice, wheat and soybean starch samples respectively, add 20g of purified water, gelatinize in a water bath at 85℃ for 6h, mix well, and then take 5mL to dry at 105℃ to constant weight.

[0093] e) The hydrogen isotope ratios of each sample were determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry, and the results are shown in Table 3.

[0094] Table 3. Starch δD determination results (‰) after various sample treatments

[0095] Sample type Repeat -1 Repeat -2 Repeat -3 average value Standard deviation corn -54.0 -53.7 -57.5 -55.1 2.1 wheat -70.2 -69.4 -74.6 -71.4 2.8 soybeans -81.1 -85.4 -83.4 -83.3 2.2

[0096] As shown in the table above, this method studies the repeatability of the pretreatment process. The standard deviation of the measurement results after each sample was treated three times was less than 3‰, which meets the repeatability requirements for hydrogen isotope determination.

[0097] Example 1

[0098] a) Take two starch laboratory standards and determine the given ratio (δD) of their non-exchangeable hydrogen isotopes. CBNE The values ​​were -51.2‰ and -107.5‰, respectively.

[0099] b) Weigh 0.1g of each and add them to the purified water in Experiment 3 at a ratio of 1:40, then incubate in a water bath at 85°C for 6 hours;

[0100] c) Dry the starch solution to constant weight at 105℃;

[0101] d) The hydrogen isotope ratios of the two starch standards after treatment were determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry. The results were -63.7‰ and -98.1‰, respectively. Based on this, the standard curve δD can be obtained. CBNE =1.6366δD 测定值 +53.053.

[0102] e) Substituting the mean values ​​from Experiment 3 into the standard curve, it can be seen that the isotope ratios of non-exchangeable hydrogen in starch in corn, wheat, and soybean samples are -37.0‰, -63.8‰, and -83.3‰, respectively.

[0103] Therefore, this method can be used to determine the δD of non-exchangeable hydrogen in starch in various grain samples. CBNE value.

[0104] Example 2

[0105] a) Take two starch laboratory standards with given ratios (δDCBNE) of non-exchangeable hydrogen isotopes of -51.2‰ and -94.3‰, respectively.

[0106] b) Take 0.1 g of each standard and add pure methanol at a ratio of 1:40. After mixing the starch sample with methanol, heat in a water bath at 60 degrees Celsius for 6 hours to ensure that the starch is fully gelatinized and exchanged with isotopes.

[0107] c) Dry the mixed solution at 105°C to constant weight to remove moisture and methanol.

[0108] d) The hydrogen isotope ratios of the two starch standards after treatment were determined using a high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer.

[0109] e) Plot a standard curve based on the measurement results, i.e., the relationship between δDCBNE and the measured value of δD.

[0110] f) Take a sample and repeat steps b)-d) three times. Substitute the sample's measurement results into the standard curve to obtain the corresponding δDCBNE value for the sample. The standard deviation of the calculated results is less than 2‰, which meets the repeatability requirements for hydrogen isotope determination.

[0111] Example 3

[0112] a) Take two starch laboratory standards with given ratios (δDCBNE) of non-exchangeable hydrogen isotopes of -63.8‰ and -107.5‰, respectively.

[0113] b) Take 0.1 g of each standard and add pure ethanol at a ratio of 1:40. After mixing the starch sample with ethanol, heat in a water bath at 60 degrees Celsius for 6 hours to ensure that the starch is fully gelatinized and exchanged with isotopes.

[0114] c) Dry the mixed solution at 105°C to constant weight to remove moisture and ethanol.

[0115] d) The hydrogen isotope ratios of the two starch standards after treatment were determined using a high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometer.

[0116] e) Plot a standard curve based on the measurement results, i.e., the relationship between δDCBNE and the measured value of δD.

[0117] f) Take a sample and repeat steps b)-d) three times. Substitute the sample's measurement results into the standard curve to obtain the corresponding δDCBNE value for the sample. The standard deviation of the calculated results is less than 2‰, which meets the repeatability requirements for hydrogen isotope determination.

[0118] The above description represents only some preferred embodiments of the present invention and is not intended to limit the invention. Various modifications, equivalent substitutions, and improvements can be made according to the principles and spirit of the present invention, and these changes should still be considered within the scope of protection of the present invention.

[0119] For example, parameters such as solvent selection, gelatinization temperature, and gelatinization time during the gelatinization process can be adjusted for specific sample types or experimental needs. Furthermore, different sample pretreatment methods or analytical techniques can be tried to improve the accuracy and repeatability of the determination. These changes are all based on this invention and aim to achieve the same goal: accurately determining the isotopic ratio of non-exchangeable hydrogen in grain starch for applications such as grain origin traceability.

[0120] Therefore, the scope of protection of this invention should include all technically equivalent embodiments, as well as various modifications, substitutions, and improvements made under the principles and spirit of this invention. This broad scope of protection ensures the flexibility and practicality of this invention in the technical field, enabling it to adapt to ever-changing needs and application scenarios.

Claims

1. A method for determining the stable isotope ratio of non-exchangeable hydrogen in grain starch, characterized in that, Includes one, two, three, four, or five of the following steps: 1) Gelatinize starch by adding a reagent containing exchangeable hydrogen; 2) Dissolve the starch standard in the same proportion and gelatinize; 3) Drying; 4) The stable hydrogen isotope ratio δD of the dried samples and standards was determined by high-temperature pyrolysis / elemental analysis-stable isotope ratio mass spectrometry. 测 ; 5) Construct a standard curve based on the given value of non-exchangeable hydrogen in starch standard and the measured value of hydrogen isotope, and calibrate the isotope ratio of non-exchangeable hydrogen in grain starch.

2. The method according to claim 1, characterized in that, The reagent containing exchangeable hydrogen is ethanol, methanol, acetic acid, or water.

3. The method according to claim 1, characterized in that, In step 1), a reagent containing exchangeable hydrogen is added to the sample. The mass of the reagent containing exchangeable hydrogen is 4-100 times the mass of starch.

4. The method according to claim 1, 1) dissolving the starch sample in a certain proportion (e.g., 1:40) in methanol, ethanol, acetic acid, or water containing exchangeable hydrogen, and heating it in a water bath at 40-95°C; 2) Keep in a water bath for a certain period of time to ensure that the exchangeable hydrogen in the starch is fully exchanged with the hydrogen atoms in the solvent to achieve isotopic equilibrium.

5. The method according to claim 4, wherein heating is performed in a water bath at 85°C.

6. The method of claim 1, wherein drying is performed by: The gelatinized starch sample is dried to constant weight to remove all moisture or solvent. The dried sample no longer contains exchangeable hydrogen, and only non-exchangeable hydrogen retains its original isotopic characteristics.

7. The method according to claim 4, wherein the gelatinized starch sample is dried at 105°C.

8. The method according to claim 1, wherein in step 1), the exchangeable hydrogen in the starch is replaced with a reagent containing exchangeable hydrogen, the mass of which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 times that of the sample.

9. The method according to claim 1, excluding the step of processing the starch standard.

10. The method according to any one of claims 1-9, wherein the grain sample is cereal (wheat, rice, sorghum, corn, millet), legume (soybean, pea) and tuber (sweet potato, potato).