Preparation method of volatile basic nitrogen standard substance in food

Abstract

The invention discloses a preparation method of a volatile basic nitrogen standard substance in food, which comprises the following steps: (1) cutting off tendons, tendons, bones, skins and fat from fresh livestock meat, poultry meat or / and fish meat, and crushing pure meat to obtain minced meat; (2) weighing a certain amount of meat paste, adding a calculated amount of ammonium sulfate aqueous solution into the meat paste, mixing, and continuously crushing and stirring until the mixture is uniform; (3) carrying out freeze drying on the standard-added and uniformly-mixed meat paste to obtain meat powder blocks; (4) crushing the meat meal blocks, and collecting freeze-dried powder; sieving the freeze-dried powder by using a 60-mesh sieve; (5) detecting the moisture content of the sieved freeze-dried powder, wherein the moisture content is less than 5%; and (6) sub-packaging the freeze-dried powder, sealing the freeze-dried powder into independent packaging units, and performing uniformity test to obtain the volatile basic nitrogen standard substance. The standard substance disclosed by the invention has the advantages of uniform content of volatile basic nitrogen, good short-term and long-term stability and accurate content.

Description

Technical Field

[0001] This invention belongs to the field of detection of volatile basic nitrogen in food, and particularly relates to a method for preparing a standard substance of volatile basic nitrogen in food. Background Technology

[0002] Total Volatile Basic Nitrogen (TVB-N) refers to ammonia and amines, alkaline nitrogenous substances produced during the spoilage of animal-based foods due to the decomposition of proteins by enzymes and bacteria. These substances are volatile; higher TVB-N levels indicate greater degradation of amino acids, particularly methionine and tyrosine, thus significantly impacting nutritional value.

[0003] Volatile basic nitrogen is an important physicochemical indicator for evaluating the freshness of foods made primarily from meat and fish. There is a clear correlation between volatile basic nitrogen and spoilage of animal-derived foods; it is a crucial indicator in food safety inspection standards, particularly for meat and fish.

[0004] The National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products (GB2707-2016) stipulates that the maximum limit for volatile basic nitrogen in fresh (frozen) livestock and poultry products is 15 mg / 100g. The National Food Safety Standard for Fresh and Frozen Aquatic Animal Products (GB 2733-2015) stipulates that the maximum limit for volatile basic nitrogen in marine fish and shrimp is 30 mg / 100g, in sea crab is 25 mg / 100g, in freshwater fish and shrimp is 20 mg / 100g, and in frozen shellfish is 15 mg / 100g. The National Food Safety Standard for Aquatic Animal Products (GB10136-2015) stipulates that the maximum limit for volatile basic nitrogen in pickled raw aquatic animal products is 25 mg / 100g, and in pre-processed aquatic animal products (excluding dried and salted products) is 30 mg / 100g.

[0005] Volatile basic nitrogen is the most important physicochemical indicator of food freshness and a routine inspection item in national supervision and spot checks. There is a clear correlation between volatile basic nitrogen and spoilage of animal-derived foods, making it an important indicator in food hygiene inspection standards.

[0006] Currently, most methods for detecting volatile basic nitrogen in meat are traditional chemical detection methods or high performance liquid chromatography. GB 5009.228-2016 Determination of Volatile Basic Nitrogen in Food specifies that the methods for determining volatile basic nitrogen include semi-micro nitrogen determination method, micro-diffusion method and automatic Kjeldahl nitrogen analyzer method. Patent documents CN114264630A and CN103712948A disclose a method for establishing a database of volatile basic nitrogen in mutton based on near-infrared spectroscopy. The method includes: collecting several fresh mutton samples, dividing them into a modeling set and a test set, and collecting the near-infrared spectrum of each sample; determining the volatile basic nitrogen content in each sample according to the chemical detection method specified in national standards; establishing a correspondence between the near-infrared spectrum of any sample and the volatile basic nitrogen content; using the correspondence between the near-infrared spectrum of each sample in the modeling set and the volatile basic nitrogen content, establishing a predictive model for the volatile basic nitrogen content of the sample; using the correspondence between the near-infrared spectrum of each sample in the test set and the volatile basic nitrogen content to correct the accuracy of the predictive model, obtaining a corrected predictive model; and saving the correspondence between the near-infrared spectrum of the samples and the volatile basic nitrogen content included in the corrected predictive model to the database.

[0007] Standard reference materials (SRPs) are substances or materials with one or more sufficiently homogeneous characteristic values ​​that have been determined. As "measuring tools" in the analytical measurement industry, they play an indispensable role in calibrating measuring instruments and devices, evaluating analytical methods, measuring the characteristic values ​​of substances or materials, assessing the operational skills of analysts, and controlling product quality during the production process. As "chemical weights" in relative measurements, SRPs play a crucial role in verifying and ensuring the accuracy of test results.

[0008] Behind the research on the nutritional composition and safety of livestock, poultry, and fish lies the support of efficient and accurate detection technologies. The development and application of reference materials provide strong assurance for quality and safety testing and analysis throughout the entire production process of livestock, poultry, and fish products. Stable reference materials for the analysis of volatile basic nitrogen components are essential key chemical reagents for supporting detection methods.

[0009] Since fresh meat and fish do not contain volatile basic nitrogen, they can be used directly as blank standard material candidates without the addition of ammonium sulfate. The volatile basic nitrogen in meat and fish products measured by the ISO 19615:2026 standard "Determination of total volatile basic nitrogen in meat and fish products" (semi-micro nitrogen determination method) can simulate the determination of total volatile basic nitrogen content in real meat, poultry, and fish products using the semi-micro nitrogen determination method, and the freshness of meat and fish products can be evaluated based on the test results. Summary of the Invention

[0010] The purpose of this invention is to provide a method for preparing volatile basic nitrogen standard substances in food, which facilitates the establishment of chemical traceability for the measurement of livestock, poultry, and fish products, provides quality assurance for laboratory analysis and testing, and provides technical support for the quality assurance of livestock, poultry, and fish products.

[0011] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention discloses a method for preparing a volatile basic nitrogen standard substance for food, comprising the following steps: (1) Remove tendons, bones, skin and fat from fresh meat, poultry and / or fish, and then grind the pure meat to obtain meat paste; (2) Weigh a certain amount of minced meat, add a calculated amount of ammonium sulfate aqueous solution to it, mix and continue to grind and stir until uniform; (3) Freeze-dry the spiked and mixed meat paste to obtain meat powder blocks; (4) Crush the meat powder blocks and collect the freeze-dried powder; sieve the freeze-dried powder using a 60-mesh sieve; (5) The moisture content of the sieved freeze-dried powder should be tested and found to be less than 5%; (6) The freeze-dried powder is divided into individual packaging units and sealed. The volatile basic nitrogen standard substance is obtained by passing the uniformity test.

[0012] Each individual package is numbered, and the number of units to be sampled is determined based on the number of individual package units. A random number of units are then selected for homogeneity testing. If the homogeneity test fails, the preparation process needs to be adjusted and optimized, and the standard material needs to be re-developed until the homogeneity test is passed.

[0013] Homogeneity testing: In accordance with the requirements of JJF 1343-2022 "Assignment of Standard Reference Materials and Evaluation of Homogeneity and Stability", when the total number of units N>500, the number of units sampled is >=15. Each sample bottle undergoes two repeatability tests, and homogeneity is tested by comparing within-group and between-group variances using one-way ANOVA. After the homogeneity test of the standard samples is passed, short-term and long-term stability tests are conducted. The short-term stability test lasts for one week to examine the stability of the standard samples under transportation conditions. Long-term stability testing involves monitoring samples stored at room temperature (25℃) protected from light for 12 months, following a principle of initial close monitoring followed by later close monitoring, to determine a reliable shelf life and optimal storage conditions.

[0014] The meat is one of pork, beef, or mutton; the poultry is chicken or duck; and the fish is cod or tilapia. This study uses one of the top-consuming fish products, both domestically and internationally, from both saltwater and freshwater sources as the research substrate, with cod representing saltwater fish and tilapia representing freshwater fish. The volatile basic nitrogen standard substance in the food is one of the freeze-dried powders made from pork, beef, mutton, chicken, duck, or fish.

[0015] Preferably, the concentration of the ammonium sulfate aqueous solution is set according to the content of volatile basic nitrogen. Based on the amount of nitrogen to be prepared, the concentration of the ammonium sulfate aqueous solution is calculated according to the material balance rule. The preferred concentration is 0.920 to 23.3 g / L.

[0016] Preferably, the freezing temperature of the vacuum freeze dryer is -35 to -65°C, the pre-vacuum degree is 0.1 to 0.5 mbar, the first stage of the freeze drying is set with a vacuum degree of 0.4 to 1.0 mbar, the second stage is set with a vacuum degree of 0.4 to 1.0 mbar, and the total freeze drying time is 24 to 36 hours.

[0017] In step (4), the high-speed pulverizer operates at a rotation speed of 25,000 to 32,000 r / min.

[0018] Compared with the prior art, the present invention has the following technical advantages: This invention provides a standard reference for the analysis of volatile basic nitrogen components in food. The volatile basic nitrogen content was determined using a semi-micro nitrogen determination method. Uniformity was assessed using one-way ANOVA, and the results showed that F < F<0.05. 0.05（r－1,n－r） The stability was assessed using linear regression trend analysis and the t-test, and the results showed that |b1| < t. （0.95,n－2） ×s (b1) Through collaborative determination by 14 laboratories, experimental results have demonstrated that the standard substance of this invention has the advantages of uniform volatile basic nitrogen content, good short-term and long-term stability, and accurate content, and can be used as a reference standard substance for the analysis of volatile basic nitrogen components in food.

[0019] This invention will promote the healthy development of my country's livestock and aquaculture industries, provide strong guarantees for improving the quality of livestock, poultry, and aquatic products, and offer strong support for enterprises to expand overseas. Simultaneously, it also provides technical support for various countries to evaluate and verify newly developed international standard methods. This standard reference material mainly plays the following roles in the quality assurance system for livestock, poultry, and aquatic products: verifying, evaluating, and identifying new technologies and methods; establishing chemical traceability of measurements; and controlling experimental processes. Attached Figure Description

[0020] Figure 1The Mandel statistic h is for 14 laboratories at sample level A.

[0021] Figure 2 The Mandel statistic k is for 14 laboratories at sample level A.

[0022] Figure 3 The Mandel statistic h is for 14 laboratories at sample level B.

[0023] Figure 4 The Mandel statistic k is for 14 laboratories at sample level B.

[0024] Figure 5 The Mandel statistic h is for 14 laboratories at the sample level C.

[0025] Figure 6 The Mandel statistic k is for 14 laboratories at the sample level C.

[0026] Figure 7 The Mandel statistic h is for 14 laboratories at the sample level D.

[0027] Figure 8 The Mandel statistic k is for 14 laboratories at the sample level D.

[0028] Figure 9 The Mandel statistic h is for 14 laboratories at the sample level E.

[0029] Figure 10 The Mandel statistic k is for 14 laboratories at the sample level E.

[0030] Figure 11 The Mandel statistic h is for 14 laboratories at the sample level F.

[0031] Figure 12 The Mandel statistic k is the sample level F for 14 laboratories.

[0032] Figure 13 The Mandel statistic h is used for 14 laboratories at the sample level G.

[0033] Figure 14 The Mandel statistic k is the sample level G for 14 laboratories. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1 A method for preparing a volatile basic nitrogen standard substance for food includes the following steps: 1. Component ratio Fresh chicken was selected, and the volatile basic nitrogen content was set at 15 mg per 100g of chicken. Based on 5200g of freeze-dried chicken powder, 780mg of nitrogen needs to be added. According to the principle of chemical mass, the amount of ammonium sulfate required is calculated to be 3678.69mg.

[0036] 2. Preparation process (1) Remove tendons, bones, skin and fat from fresh chicken, cut the remaining pure meat into small pieces, put them into a high-speed chopper for grinding, set the machine speed to 3700r / min, grind 2000-3000g of material each time, and grind for about 20 minutes to get chicken paste. (2) Weigh 26kg of chicken paste, add 2000mL of ammonium sulfate aqueous solution with a concentration of 1.84g / L, mix and put it into a high-speed chopper for crushing. Set the machine speed to 3800r / min, crush 2000~3000g of material each time, and crush and stir for about 30min until uniform. (3) Place the spiked and mixed chicken paste into a freeze-drying tray and send it into a vacuum freeze dryer for freeze-drying to obtain meat powder blocks: Set the parameters of the vacuum freeze dryer: the pre-vacuum degree is 0.3 mbar, the first stage of freeze-drying is set to 0.7 mbar, the second stage is set to 0.7 mbar, the freezing temperature is shown in Table 1, and the total freeze-drying time is about 24 hours. Table 1 Freeze-drying temperature parameters for Example 1 (4) Crush the meat powder blocks using a high-speed grinder at a speed of 28,000 r / min, using 500-1000 g per batch for 1 minute each time, and collect the freeze-dried powder. Send the freeze-dried powder into a sieve and sieve it using a 60-mesh sieve. If there is too much material on the sieve, it needs to be crushed again and sieved. (5) The sieved freeze-dried powder shall be tested for moisture content in accordance with GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food. The moisture content shall be less than 5%. (6) After the moisture content test is qualified, the freeze-dried powder is repackaged and sealed into individual packages. Each individual packaged sample is numbered and the uniformity test, stability test and value determination are completed. After the test is passed, the standard substance for the analysis of volatile basic nitrogen components in food is obtained.

[0037] Test Experiment Example 1 1. Homogeneity testing and evaluation of standard reference materials for volatile basic nitrogen composition analysis: According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the total number of standard reference materials developed in this study is 500 bottles. Fifteen bottles were randomly selected for homogeneity testing (sampling ratio 3 / 100). Each bottle sample underwent two repeatability tests in a random order. One-way ANOVA was used to evaluate the homogeneity test results. The minimum sampling amount for the homogeneity test of volatile basic nitrogen was 15g. Stability monitoring was conducted after the initial homogeneity test was passed.

[0038] Table 2. Homogeneity test data of standard reference materials for volatile basic nitrogen components in food. One-way ANOVA was used to test the homogeneity within and between bottles. The homogeneity analysis data for the standard substances are shown in Table 2. From the homogeneity test data in Table 2, it can be seen that at a significance level of α=0.05, with r⁻¹=15⁻¹=14 and nr=30⁻¹⁵=15, F⁻¹... 0.05（14,15） Under the condition of F = 2.42, the determination results of volatile basic nitrogen in food conform to the formula: F A <F 0.05 (14,15). Based on this, it can be determined that there are no significant differences within and between groups of the developed standard material, indicating that the standard material is homogeneous and meets the requirements of national standard materials.

[0039] Based on the fact that the within-group variance is less than the between-group variance, the repeatability variance of the detection method is used to assess the uncertainty introduced by sample homogeneity.

[0040] Uncertainty introduced by homogeneity: U bb = = 1.56 mg / 100 g (In the formula: number of repeated measurements n=2, S1) 2 S2 is the sum of squares between groups. 2 (The sum of squares within the group).

[0041] 2. Stability monitoring and evaluation of analytical reference materials for volatile basic nitrogen components The stability of a reference material refers to its property of maintaining its characteristic values ​​within a specified range under certain time intervals and environmental conditions. Short-term and long-term stability tests were conducted on the developed reference materials. Short-term stability tests were performed at four temperature gradients: -20℃, 0℃, 25℃, and 45℃, for one week to examine the stability of the reference material under transportation conditions. Long-term stability tests were conducted by monitoring samples stored at room temperature (25℃) protected from light to determine a reliable shelf life and optimal storage conditions.

[0042] A. Short-term stability study of standard reference material Short-term stability monitoring data and statistical results are shown in Table 3. |b1|<t (0.95,3) ×s (b1) This indicates that the developed standard material sample has good stability under extreme temperatures of -20 to 45°C within a one-week monitoring period, meeting the relevant requirements in JJF1343-2022.

[0043] Table 3. Results of short-term stability testing and statistical evaluation of standard reference materials for volatile basic nitrogen components in food. B. Long-term stability study of standard materials Long-term stability monitoring data and statistical results are shown in Tables 4 and 5. |b1|<t (0.95,4) ×s (b1) All compounds met the requirements. This indicates that the components in the standard reference do not exhibit long-term variation trends, and the developed standard reference sample has good stability within a 12-month monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0044] Table 4. Long-term stability test data of standard reference materials for the analysis of volatile basic nitrogen components in food. Table 5. Linear fitting and statistical parameters of the long-term stability results of the analytical standard references for volatile basic nitrogen components in food. Long-term stability assessment primarily examines the effective time of the reference material under estimated environmental conditions in order to predict its shelf life. As shown in Tables 4 and 5, the estimated environmental conditions are room temperature, protection from light, and dryness. During the 12-month monitoring period, the assessment result of the volatile basic nitrogen content of the reference material is |b1| < t. (0.95,4) ×s (b1) This indicates that the target components of the standard material show no trend of change during the estimated period.

[0045] Uncertainty about long-term stability: US =s (b1) ×t=0.04821×12=0.579 mg / 100 g (In the formula: sb1 is the standard deviation of the slope, and t is the expected shelf life.) 3. Constant value In accordance with the requirements of ISO 5725-1 to ISO 5725-4, multiple laboratories collaborated to determine the values ​​of standard reference materials for the analysis of volatile basic nitrogen components in food. The 14 participating laboratories possessed the relevant qualifications and the conditions for determining volatile basic nitrogen values. Each laboratory implemented strict quality control during its analytical process, employing a semi-micro nitrogen determination method to measure the standard reference materials.

[0046] Table 6. Results of volatile basic nitrogen content determination by each collaborating laboratory Uncertainty of the value of a standard reference = 0.456 mg / 100 g; (In the formula: This represents the average value across all laboratories. (This represents the overall average, where m is the number of laboratories.) (1) Assignment of standard reference material values Based on the uncertainties obtained above, calculate the combined standard uncertainty: U = = = 1.73 mg / 100 g Generally, the coverage factor k=2, determined by the confidence probability and degrees of freedom, resulting in expanded uncertainty: U CRM =k×U=2×1.73=3.46 mg / 100 g In summary, the characteristic value of the volatile basic nitrogen content standard reference material in the food sample of this experiment is expressed as (15.0 ± 3.46) mg / 100 g. (2) Evaluation of the recovery rate of the preparation of standard reference materials The assigned values ​​of the standard substances were compared with the theoretical content of volatile basic nitrogen in the standard substance matrix to verify the effectiveness of the preparation process and the accuracy of the assigned values. The results are shown in Table 7. The recovery rate of the standard substance preparation was 100%, and its prepared values ​​matched the theoretical values, indicating that the preparation route of the standard substance was accurate and feasible.

[0047] Table 7. Recovery rates of standard reference materials Therefore, this invention selects fresh chicken meat as the standard material matrix and optimizes the preparation process to form a stable and reliable technical route, thus preparing a standard material for the analysis of volatile basic nitrogen components in chicken meat.

[0048] The homogeneity and stability of the standard reference materials were determined using the semi-micro nitrogen determination method, and one-way ANOVA and [other methods were employed]. t Statistical evaluation of the test method showed good homogeneity and stability. After collaborative value determination and uncertainty calculation by 14 laboratories, the characteristic value of volatile basic nitrogen was expressed as (15.0±3.46) mg / 100 g. Furthermore, the calculated value of volatile basic nitrogen prepared was in good agreement with the theoretical value. The prepared standard material has advantages such as uniform and stable volatile basic nitrogen content and accurate characteristic values, and can be used as a reference standard for the analysis of volatile basic nitrogen components in chicken meat.

[0049] Example 2 A method for preparing a volatile basic nitrogen standard substance for food includes the following steps: 1. Component ratio Fresh cod was selected, and the volatile basic nitrogen content was calculated to be 190 mg per 100g of fish meat. Based on 5200g of freeze-dried fish meat powder, 9880mg of nitrogen needs to be added. According to the chemical mass principle, the amount of ammonium sulfate to be added is calculated to be 46596.79mg.

[0050] 2. Preparation process (1) Remove the bones and skin from the fresh cod, cut the remaining pure fish meat into small pieces, put them into a high-speed chopper for grinding, set the machine speed to 3600 r / min, grind 2000-3000g of material each time, and grind for about 15 minutes to get meat paste. (2) Weigh 31.2 kg of fish paste, add 2000 mL of ammonium sulfate aqueous solution with a concentration of 23.3 g / L, mix and put it into a high-speed chopper for crushing. Set the machine speed to 3800 r / min, crush 2000-3000 g of material each time, and crush for about 25 min. (3) Place the spiked and mixed fish paste into a freeze-drying tray and send it into a vacuum freeze dryer for freeze-drying to obtain fish powder blocks: Set the parameters of the vacuum freeze dryer: the pre-vacuum degree is 0.2 mbar, the first stage of freeze-drying is set to 0.8 mbar, the second stage is set to 0.8 mbar, the freezing temperature is shown in Table 8, and the total freeze-drying time is about 28 hours. Table 8 Freeze-drying temperature parameters for Example 2 (4) Crush the fish meat powder blocks using a high-speed crusher at a speed of 27,000 r / min, 500-1000 g per batch, and crush for 1 minute each time. Collect the freeze-dried powder. Send the freeze-dried powder into a sieve and sieve it using a 60-mesh sieve. If there is a lot of material on the sieve, it needs to be crushed again and sieved. (5) The sieved freeze-dried powder shall be tested for moisture content in accordance with GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food. The moisture content shall be less than 5%. (6) After the moisture content test is qualified, the freeze-dried powder is repackaged and sealed into individual packages. Each individual packaged sample is numbered and the uniformity test, stability test and value determination are completed. After the test is passed, the standard substance for the analysis of volatile basic nitrogen components in food is obtained.

[0051] Test Experiment Example 2 1. Homogeneity testing and evaluation of standard reference materials for volatile basic nitrogen composition analysis: According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the total number of standard reference materials developed in this study is 500 bottles. Fifteen bottles were randomly selected for homogeneity testing (sampling ratio 3 / 100). Each bottle sample underwent two repeatability tests in a random order. One-way ANOVA was used to evaluate the homogeneity test results. The minimum sampling amount for the homogeneity test of volatile basic nitrogen was 15g. Stability monitoring was conducted after the initial homogeneity test was passed.

[0052] Table 9. Homogeneity test data of standard reference materials for volatile basic nitrogen components in food. One-way ANOVA was used to test the homogeneity within and between bottles. The homogeneity analysis data for the standard substances are shown in Table 2. From the homogeneity test data in Table 2, it can be seen that at a significance level of α=0.05, with r⁻¹=15⁻¹=14 and nr=30⁻¹⁵=15, F⁻¹... 0.05（14,15） Under the condition of F = 2.42, the determination results of volatile basic nitrogen in food conform to the formula: F A <F 0.05 (14,15). Based on this, it can be determined that there are no significant differences within and between groups of the developed standard material, indicating that the standard material is homogeneous and meets the requirements of national standard materials.

[0053] Based on the fact that the within-group variance is less than the between-group variance, the repeatability variance of the detection method is used to assess the uncertainty introduced by sample homogeneity.

[0054] Uncertainty introduced by homogeneity: U bb = =2.45 mg / 100 g (In the formula: number of repeated measurements n=2, S1) 2 S2 is the sum of squares between groups. 2 (The sum of squares within the group).

[0055] 2. Stability monitoring and evaluation of analytical reference materials for volatile basic nitrogen components The stability of a reference material refers to its property of maintaining its characteristic values ​​within a specified range under certain time intervals and environmental conditions. Short-term and long-term stability tests were conducted on the developed reference materials. Short-term stability tests were performed at four temperature gradients: -20℃, 0℃, 25℃, and 45℃, for one week to examine the stability of the reference material under transportation conditions. Long-term stability tests were conducted by monitoring samples stored at room temperature (25℃) protected from light to determine a reliable shelf life and optimal storage conditions.

[0056] A. Short-term stability study of standard reference material Short-term stability monitoring data and statistical results are shown in Table 10. |b1|<t (0.95,3) ×s (b1) This indicates that the developed standard material sample has good stability under extreme temperatures of -20 to 45°C within a one-week monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0057] Table 10. Results of Short-Term Stability Testing and Statistical Evaluation of Standard Reference Materials for Volatile Basic Nitrogen Components in Food B. Long-term stability study of standard materials Long-term stability monitoring data and statistical results are shown in Tables 11 and 12. |b1|<t (0.95,4) ×s (b1) All compounds met the requirements. This indicates that the components in the standard reference do not exhibit long-term variation trends, and the developed standard reference sample has good stability within a 12-month monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0058] Table 11 Long-term stability test data of standard reference materials for the analysis of volatile basic nitrogen components in food. Table 12 Linear fitting and statistical parameters of long-term stability results of standard reference materials for volatile basic nitrogen components in food. Long-term stability assessment primarily examines the effective time of the reference material under estimated environmental conditions in order to predict its shelf life. As shown in Tables 11 and 12, the estimated environmental conditions are room temperature, protection from light, and dryness. During the 12-month monitoring period, the assessment result of the volatile basic nitrogen content of the reference material is |b1| < t. (0.95,4) ×s (b1) This indicates that the target components of the standard material show no trend of change during the estimated period.

[0059] Uncertainty about long-term stability: U S =s (b1) ×t=0.2063×12=2.48 mg / 100 g (In the formula: sb1 is the standard deviation of the slope, and t is the expected shelf life.) 3. Constant value In accordance with the requirements of ISO 5725-1 to ISO 5725-4, multiple laboratories collaborated to determine the values ​​of standard reference materials for the analysis of volatile basic nitrogen components in food. The 14 participating laboratories possessed the relevant qualifications and the conditions for determining volatile basic nitrogen values. Each laboratory implemented strict quality control during its analytical process, employing a semi-micro nitrogen determination method to measure the standard reference materials.

[0060] Table 13 Results of volatile basic nitrogen composition determination by each collaborating laboratory Uncertainty of the value of a standard reference = 2.34 mg / 100 g; (In the formula: This represents the average value across all laboratories. (This represents the overall average, where m is the number of laboratories.) (5) Assignment of standard reference values Based on the uncertainties obtained above, calculate the combined standard uncertainty: U = = = 4.20 mg / 100 g Generally, the coverage factor k=2, determined by the confidence probability and degrees of freedom, resulting in expanded uncertainty: U CRM =k×U=2×4.20=8.40mg / 100g In summary, the characteristic value of the analytical standard reference for volatile basic nitrogen components in the food in this experimental example is expressed as (194±8.40) mg / 100 g. (6) Evaluation of the recovery rate of the preparation of standard substances The assigned values ​​of the standard substances were compared with the theoretical content of volatile basic nitrogen in the standard substance matrix to verify the effectiveness of the preparation process and the accuracy of the standard substance assignment. The results are shown in Table 14. The recovery rate of the standard substance preparation was 100%, and its prepared value basically matched the theoretical value, indicating that the preparation route of the standard substance was accurate and feasible.

[0061] Table 14 Recovery rates of standard reference materials Therefore, this invention selects fresh cod as the standard material matrix and optimizes the preparation process to form a stable and reliable technical route, thus preparing a standard material for the analysis of volatile basic nitrogen components in cod.

[0062] The homogeneity and stability of the standard reference materials were determined using the semi-micro nitrogen determination method, and one-way ANOVA and [other methods were employed]. t Statistical evaluation of the test method showed good homogeneity and stability. After collaborative value determination and uncertainty calculation by 14 laboratories, the characteristic value of volatile basic nitrogen was expressed as (194±8.40) mg / 100 g. Furthermore, the calculated value of the prepared volatile basic nitrogen basically matched the theoretical value. The prepared standard material has advantages such as uniform and stable volatile basic nitrogen content and accurate characteristic values, and can be used as a reference standard for the analysis of volatile basic nitrogen components in cod.

[0063] Interlaboratory validation 1. From April to June 2024, the Shanghai Institute of Quality Inspection led a team of 14 laboratories at home and abroad to conduct interlaboratory comparisons, using volatile basic nitrogen standard materials to examine the universality and precision (repeatability and reproducibility) of the new method, and thus confirm the effectiveness of the method.

[0064] The standard material samples include seven matrix samples: A. pork, B. chicken, C. duck, D. tilapia, E. beef, F. mutton, and G. cod. Each sample was tested in six parallel experiments.

[0065] Table 15 Raw data for the analysis of volatile basic nitrogen content 1 Table 16 Raw data for volatile basic nitrogen content analysis 2 Table 17 Raw data for volatile basic nitrogen content analysis 3 Table 18 Raw data for the analysis of volatile basic nitrogen content 4 Table 19 shows the average values ​​of volatile basic nitrogen content data for the standard material samples including A pork, B chicken, C duck, D tilapia, E beef, F mutton, and G cod. Table 20 shows the standard deviation values.

[0066] Table 19. Average values ​​of volatile basic nitrogen content data analysis Table 20 Standard deviation of volatile basic nitrogen content data analysis 2. Data consistency and outlier tests (1) Mandel Test Figures 1 to 14 The Mandel statistic h and Mandel statistic k are given for 14 laboratories at sample levels A, B, C, D, E, F, and G. The horizontal red and green lines in the figure represent the critical values ​​of Mandel statistic h and k at significance levels of 1% and 5%, respectively.

[0067] from Figures 1 to 14 It can be seen that outliers appeared in laboratory 2 at sample levels A and B; outliers appeared in laboratory 1 at sample level F, in laboratory 2 at sample levels C, D, and E, and in laboratory 8 at sample level E; outliers appeared in laboratory 2 at sample level G, in laboratory 11 at sample levels A, B, and G, and in laboratory 9 at sample levels D, E, and F; and outliers appeared in laboratory 2 at sample level E, in laboratory 7 at sample level A, in laboratory 8 at sample levels D and E, and in laboratory 11 at sample level E.

[0068] The Mandel chart suggests that further analysis of the data is needed using the Cochran and Gtubbs tests.

[0069] (2) Cochran test Table 21 Cochran's test statistic C Note: If the calculated Cochran test statistic C is greater than the 5% confidence level critical value but less than or equal to the 1% confidence level critical value, then the corresponding item being tested is a discrepancy value.

[0070] If the calculated Cochran test statistic C is greater than the 1% confidence level critical value, then the corresponding tested item is an outlier.

[0071] Table 21 shows that the calculated Cochran statistic C for levels B, D, E, and G are 0.247, 0.235, 0.256, and 0.241, respectively, exceeding the critical value of 0.232. This result is basically consistent with the intuitive result obtained from the Mandel plot.

[0072] (3) Grubbs test The results of the Grubbs test, calculated after the Cochran test, are shown in Table 8. Table 22 Grubbs test results for volatile basic nitrogen mass fraction determined by 14 laboratories. For the Grubbs test on one-sided outliers, if the calculated Grubbs test statistic G is greater than the 5% confidence level but less than or equal to the 1% confidence level, then the corresponding item being tested is an outlier. If the test statistic G is greater than the 1% confidence level, then the corresponding item being tested is an outlier.

[0073] For the Grubbs test on two-tailed outliers, an outlier is considered to be an outlier if the calculated Grubbs test statistic G is less than the 5% confidence level. Similarly, an outlier is considered to be an outlier if the test statistic G is less than the 1% confidence level.

[0074] Table 22 shows that there are no outliers in all the existing data from the 14 laboratories, and all of them are valid data that have passed the mathematical statistics test.

[0075] 3. Calculation and analysis of mean and standard deviation Through statistical tests of data consistency and outliers, the average (m) and repeatability standard deviation (S) of the volatile basic nitrogen mass fraction determined by the semi-micro nitrogen determination method in meat and fish from 14 laboratories were analyzed. r ) and reproducibility standard deviation (S R The calculations were performed, and the results are shown in Table 23.

[0076] Table 23 Calculation results of mean and standard deviation of data from 14 laboratories Examine the standard deviation S in Table 23 r S R lgS r lgS R The relationship with the average value m reveals lgS r and lgs R There is a strong positive correlation between lgS and m. Data fitting results confirm this conjecture, showing the following linear relationship: lgS r =0.6903lgm-1.187, R 2=0.9628; lgS R =0.6946lgm-0.6768, R 2 =0.9004; Repeatability standard deviation S r =0.013m+0.2462, R 2 =0.9743; Repeatability limit r=0.0364m+0.6894; Relinearity standard deviation S R =0.0421m+0.8674, R 2 =0.9380; the linear limit R = 0.1179m + 2.4287.

[0077] International interlaboratory comparisons of seven representative meat matrix samples showed that the analytical data of volatile basic nitrogen components in food were accurate.

Claims

1. A method for preparing a standard reference for volatile basic nitrogen in food, characterized in that, Includes the following steps: (1) Remove tendons, bones, skin and fat from fresh meat, poultry and / or fish, and then grind the pure meat to obtain meat paste; (2) Weigh a certain amount of minced meat, add a calculated amount of ammonium sulfate aqueous solution to it, mix and continue to grind and stir until uniform; (3) Freeze-dry the spiked and mixed meat paste to obtain meat powder blocks; (4) Crush the meat powder blocks and collect the freeze-dried powder; sieve the freeze-dried powder using a 60-mesh sieve; (5) The moisture content of the sieved freeze-dried powder should be tested and found to be less than 5%; (6) The freeze-dried powder is divided into individual packaging units and sealed. The volatile basic nitrogen standard substance is obtained by passing the uniformity test.

2. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The homogeneity test shall be conducted in accordance with the requirements of JJF 1343-2022 "Assessment of Standard Reference Materials and Evaluation of Homogeneity and Stability", when the total number of units N>500, the number of units sampled shall be >=15.

3. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The meat is one of pork, beef or mutton; the poultry is chicken or duck; the fish is cod or tilapia.

4. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The volatile basic nitrogen standard substance in the food is one of the freeze-dried powders made from pork, beef, mutton, chicken, duck, or fish.

5. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The concentration of the ammonium sulfate aqueous solution is set based on the content of volatile basic nitrogen. The concentration of the ammonium sulfate aqueous solution is calculated by working backwards from the amount of nitrogen to be prepared, according to the material balance rules.

6. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The concentration of the ammonium sulfate aqueous solution is 0.920–23.3 g / L.

7. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, The freezing temperature of the vacuum freeze dryer is -35 to -65°C, the pre-vacuum degree is 0.1 to 0.5 mbar, the first stage of the freeze drying is set with a vacuum degree of 0.4 to 1.0 mbar, the second stage is set with a vacuum degree of 0.4 to 1.0 mbar, and the total freeze drying time is 24 to 36 hours.

8. The method for preparing the standard reference material for volatile basic nitrogen in food according to claim 1, characterized in that, In step (4), the high-speed pulverizer operates at a rotation speed of 25,000 to 32,000 r / min.

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