A method for preparing national standard samples of defatted and dehydrated cashew powder for detecting cashew characteristic substances in food allergies

Abstract

The invention belongs to the field of food detection, and discloses a method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting cashew characteristic substances in food allergies. The method comprises the following steps: using cashew kernels as a base material, grinding the cashew kernels to prepare cashew coarse powder; performing n-hexane defatting treatment to obtain loose defatted cashew powder; and then pulverizing the cashew kernels with a grinder, freeze-drying in a vacuum, and screening the cashew powder through at least 50 meshes to prepare a defatted and dehydrated cashew powder standard sample with a water content of ≤5%; packaging at least 500 bottles per batch, and randomly sampling to carry out uniformity and stability inspection and evaluation. The content of the characteristic substances is not affected by the damage and degradation of the preparation process, and the uniformity and stability meet the requirements of the national standard sample. The method can be used for detecting allergic cashew characteristic substances in various food matrices, filling the gap of the national standard sample at home and abroad, and can be used for laboratory quality control activities such as method verification, detection kit performance evaluation, detection efficiency evaluation, and capability verification for detecting food allergenic cashew characteristic substances.

Description

Technical Field

[0001] The invention belongs to the field of food detection, and relates to a method for preparing a national standard sample of defatted and dehydrated cashew nut powder for detecting food allergic cashew nut characteristic substances, and is used for laboratory quality control activities for detecting allergic cashew nut characteristic substances in various food matrices. Background Art

[0002] Cashew (Anacardium occidentale L.) is one of the four major dried fruits in the world and is widely used in various foods. It is one of the main factors causing food allergies, accounting for 20% of allergies caused by nuts. Cashew food allergens seriously endanger the health of allergic people. Allergic patients will experience a variety of symptoms within the first two hours after ingesting cashews. Signs of allergic reactions include itchy skin, wheezing, hoarseness, and tightness in the chest and throat. Moderate to severe cashew allergies can cause swelling of the lips, tongue, and throat, making it difficult to breathe and speak. If the intestines are involved, patients may experience diarrhea and stomach pain. Allergic reactions can cause complete obstruction of the airway and cause a dangerous drop in blood pressure, even life-threatening. In order to prevent cashew allergy patients from ingesting cashews without knowing it and causing allergic reactions, the United States and the European Union have both issued relevant regulations, requiring that food packaging containing ingredients such as cashews must be clearly labeled. In recent years, my country has also implemented the "General Principles for Pre-packaged Food Labeling", which requires that if cashews are used as ingredients, or cashew foods or their products may be introduced during processing, easily recognizable names should be used in the ingredient list, or a reminder should be placed nearby. There is still no specific treatment for food allergies. An important way to prevent food allergies is to strictly prevent the consumption of allergens, and food allergen detection and analysis is the most critical link.

[0003] Cashew allergen characteristic substances (such as allergic protein AnaO3) are the most common detection items for food allergies. However, my country currently lacks national standard samples of cashew powder for the detection of cashew characteristic substances for food allergies. Cashews usually have a high oil content. Cashew powder that has not been defatted is very easy to agglomerate into blocks and has large and uneven particle sizes, which will affect the loss rate of raw material preparation and the detection efficiency when adding food matrix to standard samples; high-temperature heating treatment will cause changes in cashew allergen characteristic substances (such as allergic protein AnaO3), which requires that when preparing standard samples, the preparation method must ensure the stability of the characteristic value of allergic protein AnaO3. The above reasons make the preparation very difficult, resulting in the lack of national standard samples of matrices for the detection of allergen cashew characteristic substances at home and abroad, which has brought inconvenience to the detection of cashew allergen substances in my country's food.

[0004] Standard samples play an important role in ensuring the comparability, accuracy and consistency of laboratory measurement results. Food matrix standard samples can control the entire detection process and play an increasingly indispensable role in food safety testing. The development of food matrix standard samples has become a research hotspot in recent years. The research on the preparation of a national standard sample of defatted and dehydrated cashew powder for the detection of cashew allergen characteristic substances will provide support for food allergen testing laboratories to conduct method validation, test kit evaluation, quality control during the test process, and other comprehensive assessments of the laboratory level. It is of great significance to improve my country's food allergen detection capabilities, protect people's health, and maintain the good international reputation of my country's food industry. Summary of the invention

[0005] In order to fill the gap in the prior art, the present invention relates to a method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances, which is used for laboratory quality control activities for detecting allergy cashew characteristic substances in various food matrices.

[0006] The present invention aims to provide a method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting cashew characteristic substances of food allergy. The content of food allergy cashew characteristic substances (such as characteristic value of allergic protein AnaO3) in the national standard sample of defatted and dehydrated cashew powder is not affected by the defatting and dehydration preparation process. The defatting treatment can greatly reduce the loss rate of raw materials. The particle size reaches at least 50 meshes to ensure the detection efficiency of adding food matrix. The freeze-drying and dehydration make its water content ≤5% to maintain long-term stability. The sample uniformity and stability meet the requirements of national standard samples. The sample can be used for quality control activities of detection laboratories adding various food matrices. The method comprises the following steps:

[0007] (1) Determine the mass of the standard sample and the mass of the raw material: the national standard sample of defatted and dehydrated cashew nut powder prepared in each batch is at least 1500g (3g / bottle, 500 bottles), the mass of fat removed by the defatting process is about 30%, and the mass loss of dehydration in the preparation process is about 2%. It is estimated that the mass of the cashew kernel raw material after shelling and peeling is at least 2200g;

[0008] (2) Grinding coarse powder: Take the shelled and peeled cashew kernels and grind them in a grinder for 5 to 10 minutes to obtain the whole cashew powder sample;

[0009] (3) Hexane defatting: add hexane to the cashew nut powder sample at a volume ratio of 3:1, stir evenly with a glass rod and let it stand for 30 minutes, then discard the yellow oily liquid leached from the upper layer; take the precipitate, add hexane again at a volume ratio of 2:1, stir evenly and let it stand for 30 minutes, then discard the yellow oily liquid leached from the upper layer; repeat the above operation twice until the upper liquid is colorless; place the cashew nut powder sample after hexane defatting in a ventilated place and evaporate naturally for 24 hours. The cashew nut powder sample that has not been defatted can only have 10% of the raw materials that can pass through at least 50 mesh sieves due to the high oil content, and about 90% of the raw materials are lost, which will result in the disadvantage of wasting raw materials. In addition, the defatting treatment will not cause degradation and damage to the nucleic acid characteristic value and allergic protein characteristic value of food allergic cashew substances (such as the characteristic value of allergic protein AnaO3), and the content value can still be kept stable;

[0010] (4) Freeze vacuum drying: freeze vacuum drying the defatted cashew powder sample, pre-freeze at -80°C for at least 24 hours, then freeze vacuum freeze at -45 to -55°C at 0.1 mbar for 8 to 10 hours, and finally rapidly heat dry at -10°C to 30°C for 4 to 6 hours, with a rapid heating rate of ≥1.0°C / min, to obtain a defatted and dehydrated cashew powder sample with a moisture content of ≤5%; the colony counts of defatted cashew powder samples with different moisture contents during the storage period are somewhat different, which affects the long-term stability of the standard sample;

[0011] (5) Grind thoroughly and prepare cashew powder by 50 mesh sieving: Grind the defatted and dehydrated cashew powder sample thoroughly and pass it through a 50 mesh sterilized metal sieve to obtain a defatted and dehydrated cashew powder sample with a particle size of at least 50 mesh. The detection efficiency of cashew powder standard samples with different particle sizes added to the food matrix has certain differences;

[0012] (6) Packaging and storage: The defatted and dehydrated cashew powder standard sample is packaged into brown glass sample bottles (3 g / bottle, 500 bottles) and sealed with a three-level sealing device (silicone cover, aluminum foil cover, plastic cover). The ambient temperature of the sample should be maintained at 0-4°C for long-term storage to meet the shelf life of 2-3 years.

[0013] Real-time fluorescence PCR and ELISA were used to detect the nucleic acid value and protein content of the allergenic cashew characteristic substance allergen protein AnaO3, and the performance of the defatted and dehydrated cashew powder standard sample was evaluated. Finally, a uniform and stable defatted and dehydrated cashew powder national standard sample for detecting food allergic cashew characteristic substances was prepared, and the uniformity, stability and joint determination results met the requirements of the national standard sample.

[0014] The key technical points of the present invention are:

[0015] 1. Research on key preparation processes: Cashew powder degreasing process, dehydration process and particle size are very important for the characteristic values ​​of food allergic cashew characteristic substances, detection efficiency of adding food matrix, uniformity of standard samples and long-term stability. Cashew powder without degreasing will have a raw material loss rate of about 90% through 50 mesh screening due to high oil content, and the degreasing treatment will not cause degradation and damage to the nucleic acid characteristic values ​​and allergic protein characteristic values ​​of food allergic cashew characteristic substances, and the content value can still be kept stable; high-temperature drying and dehydration of cashew powder will cause the characteristic values ​​of allergic cashew characteristic substances to be degraded and damaged, and the content will be reduced; excessive water content of cashew powder will cause microbial growth during the storage of standard samples and affect the long-term stability of standard samples; excessive particle size of cashew powder particles will affect the detection efficiency of adding different food matrices. Therefore, it is necessary to consider key preparation process research such as degreasing, dehydration, and particle size.

[0016] 2. Homogeneity evaluation of standard samples: Homogeneity of standard samples refers to the uniformity of the spatial distribution of characteristic values. It is an evaluation of the overall spatial distribution of standard samples and one of the important quality indicators of standard samples. Randomly select 15 bottles of 500 bottles of standard samples in a large batch that are packaged in independent packages for homogeneity testing (m = 15, n = 2); use real-time fluorescence PCR and ELISA to detect the nucleic acid value and protein content of food allergen cashew characteristic substances (such as allergic protein AnaO3) respectively; use variance analysis (F test) to perform difference analysis and evaluate the homogeneity results of standard samples.

[0017] 3. Evaluation of the stability of standard samples: Under suitable environmental conditions, the characteristic values ​​of standard samples should not change significantly with the extension of storage time. Randomly select samples from a large batch of 500 bottles of standard samples packaged in independent packages and place them at a low temperature of 0-4°C for long-term storage. At the time interval of every 2 months in the first year and every 3 months in the second year, randomly select 2 bottles of standard samples at each time point (m=2, n=2). Real-time fluorescence PCR and ELISA methods are used to detect the nucleic acid value and protein content value of food allergen cashew characteristic substances (such as allergic protein AnaO3), respectively. The linear empirical model is used to analyze the long-term stability of standard samples. For short-term stability testing, the synchronous design method is used to conduct short-term stability experiments under transportation conditions in the range of -20-50°C (select -20°C for 7 days, 0-4°C for 7 days and 14 days, 25°C for 7 days, and 50°C for 7 days). Three bottles of standard samples are tested under each temperature condition, and each bottle is tested twice (m=3, n=2). Linear regression variance analysis is used for short-term stability.

[0018] The beneficial effects of the present invention are as follows: the content of food allergy cashew characteristic substances (such as the characteristic value of the allergic protein AnaO3) in the defatted and dehydrated cashew nut powder national standard sample obtained by the present invention is not affected by the defatting and dehydration preparation process, the particle size can reach at least 50 meshes, the water content is ≤5%, the sample uniformity and stability meet the requirements of the national standard sample, and can be added to various food matrices for laboratory quality control activities such as verification of food allergen cashew characteristic substance detection methods, evaluation of detection kits, evaluation of detection efficiency, capability verification, and assessment of personnel detection capabilities, thereby improving the detection level of food allergen safety detection laboratories. DETAILED DESCRIPTION

[0019] The following is a further description of the present invention, but not a limitation of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0020] Example:

[0021] 1. Preparation of standard sample of defatted and dehydrated cashew powder

[0022] The national standard sample of defatted and dehydrated cashew powder prepared in each batch is at least 1500g (3g / bottle, 500 bottles / batch). The oil loss after defatting is about 30%. Considering the dehydration loss during the preparation process of about 2%, the mass of the cashew kernel raw material after shelling and peeling is at least 2200g.

[0023] The specific preparation steps are as follows:

[0024] (1) Grinding coarse powder: taking at least 2200 g of shelled and peeled cashew kernel raw material, grinding it in a grinder for 5 to 10 min, and harvesting all cashew nut coarse powder samples;

[0025] (2) Hexane defatting: According to the volume ratio of 3:1, add hexane to the cashew nut coarse powder sample, stir evenly with a glass rod and let it stand for 30 minutes, then discard the yellow oily liquid leached from the upper layer; take the precipitate, add hexane again according to the volume ratio of 2:1, stir evenly and let it stand for 30 minutes, then discard the yellow oily liquid leached from the upper layer; repeat the above operation twice until the upper liquid is colorless; place the cashew nut powder sample after hexane defatting in a ventilated place and evaporate naturally for 24 hours. The raw material loss rate of the cashew nut powder sample that has not been defatted is about 90% due to the high oil content, and the defatting treatment will not cause degradation and damage to the nucleic acid characteristic value and the allergic protein characteristic value of the food allergy cashew nut characteristic substance, and the content value can still be kept stable.

[0026] (3) Freeze vacuum drying: freeze vacuum drying the defatted cashew powder sample, pre-freeze at -80°C for at least 24 hours, then freeze vacuum freeze at 0.1 mbar at -45 to -55°C for 8 to 10 hours, and finally rapidly heat dry at -10°C to 30°C for 4 to 6 hours, with a rapid heating rate of ≥1.0°C / min, to obtain a cashew freeze-dried powder sample with a moisture content of ≤5%; the colony counts of defatted cashew powder samples with different moisture contents during the storage period are somewhat different, which affects the long-term stability of the standard sample;

[0027] (4) Grind thoroughly and prepare cashew powder by sieving through at least 50 mesh: Grind the defatted and dehydrated cashew powder sample thoroughly and pass it through a sterilized metal sieve of at least 50 mesh to obtain a defatted and dehydrated cashew powder sample with a particle size of at least 50 mesh. The detection efficiency of cashew powder standard samples with different particle sizes added to the food matrix has certain differences;

[0028] (5) Packaging and storage: The defatted and dehydrated cashew powder standard sample is packaged into brown glass sample bottles (3 g / bottle, 500 bottles) and sealed with a three-level sealing device (silicone cover, aluminum foil cover, plastic cover). The ambient temperature of the sample should be maintained at 0-4°C for long-term storage to meet the shelf life of 2-3 years.

[0029] Comparative Example 1:

[0030] Referring to the example, the difference is that the raw material loss rate of defatted and non-defatted cashew powder after screening with at least 50 mesh is different, and the nucleic acid characteristic value and protein content value of the allergic protein AnaO3 characteristic of the food allergy cashew characteristic substance will not be degraded and destroyed by the defatting process and the content value can still be kept stable.

[0031] Experiments have proved that based on the principle of like-miscible organic solvent degreasing, the oil in cashew nuts is a non-polar molecule. Using the non-polar molecule n-hexane as a solvent, a miscible degreasing effect can be achieved on the oil in cashew nuts, while maintaining the stability of the nucleic acid and protein characteristic values ​​of cashew nut allergenic substances (such as the allergic protein AnaO3).

[0032] The defatted cashew powder sample prepared by the defatting treatment of the present invention and the cashew powder sample prepared without defatting treatment in comparative example 1 were respectively subjected to determination of cashew characteristic substances (such as allergic protein AnaO3), and the nucleic acid characteristic value of the allergic protein AnaO3 was determined by the fluorescence qPCR method and the protein characteristic value of the allergic protein AnaO3 was determined by the ELISA method. The results of the Mann Whitney test statistical analysis are shown in Table 1; the raw material loss rate after at least 50 mesh screening was calculated, and the results of the Mann Whitney test statistical analysis are shown in Table 2. The Ct value of the cashew allergic protein AnaO3 was determined by qPCR for statistical difference analysis. There was no significant difference in the Ct value and protein content value of the allergic protein AnaO3 in the defatted cashew powder sample prepared by the present invention and the cashew powder sample without defatting treatment, indicating that under a confidence probability of 95%, the nucleic acid characteristic value and protein characteristic value of the allergic protein AnaO3, a food allergic cashew characteristic substance, would not be degraded and destroyed by the defatting process, but the characteristic content value would be increased. However, compared with Comparative Example 1, the difference in the raw material loss rate of the defatted cashew powder and the non-defatted cashew powder through at least 50 mesh sieving is extremely significant, indicating that the defatting preparation process of the present invention can greatly improve the utilization rate of the cashew powder raw material, reducing the loss rate from 90.46% to 29.55%.

[0033] Table 1 Statistical difference analysis results of the characteristic values ​​of allergic cashew characteristic substances and allergic protein AnaO3 in defatted cashew powder samples

[0034]

[0035] Table 2 Analysis results of the difference in raw material loss rate of defatted cashew powder samples after 50 mesh sieving

[0036]

[0037] Comparative Example 2:

[0038] With reference to the example, the difference lies in that there is a difference in the effects of low-temperature (-80°C pre-freezing for at least 24 hours, then 0.1 mbar vacuum freezing at -45 to -55°C for 8 to 10 hours, and finally rapid heating and drying at -10°C to 30°C for 4 to 6 hours, with a rapid heating rate ≥1.0°C / min) vacuum drying and dehydration treatment and high-temperature (102°C ± 3°C, 4 hours) drying and dehydration treatment on the characteristic quantity value of the allergic protein AnaO3, a characteristic substance of food allergy cashew nut.

[0039] Experiments have shown that low-temperature vacuum drying is a physical process in which water freezing and expansion causes cashew tissue to burst, and ice sublimation causes surface water to evaporate. It cannot destroy or degrade the characteristic values ​​of allergic cashew characteristic substances in standard samples, such as the allergic protein AnaO3.

[0040] The defatted cashew powder sample of the present invention was subjected to low-temperature vacuum drying and dehydration treatment, and the defatted cashew powder sample was subjected to high-temperature drying and dehydration treatment (102°C ± 3°C, 4h) in Comparative Example 2, and the allergic cashew characteristic substances (such as allergic protein AnaO3) were determined respectively, and the nucleic acid characteristic value of the allergic protein AnaO3 was determined by the fluorescent qPCR method and the protein characteristic value of the allergic protein AnaO3 was determined by the ELISA method, and the results of the Mann Whitney test statistical analysis are shown in Table 3. The Ct value and protein content value of the cashew allergic protein AnaO3 were used for statistical difference analysis. The Ct value and protein content value of the allergic protein AnaO3 in the defatted and dehydrated cashew powder sample prepared by the present invention and the cashew powder sample treated with high-temperature drying and dehydration were extremely significantly different (p<0.0001), indicating that the nucleic acid characteristic value and protein characteristic value of the food allergic cashew characteristic substance allergic protein AnaO3 will be degraded and destroyed by the high-temperature drying and dehydration process, which will affect the reduction of its characteristic substance content value.

[0041] Table 3 Results of statistical difference analysis of the characteristic values ​​of allergy protein AnaO3 in cashew powder samples with different dehydration treatments

[0042]

[0043] Comparative Example 3:

[0044] Referring to the example, the difference is that the different water contents of the cashew nut powder standard samples will result in differences in the total colony counts of the cashew nut powder standard samples.

[0045] The experiment proved that the total colony count of the standard sample of dehydrated cashew powder (water content of 4.8%) after low-temperature vacuum drying was randomly sampled and counted after different storage periods (storage at 0-4℃ for 1 month, 3 months, 6 months, and 12 months). After experiencing different storage periods, the results showed that the total colony count of the cashew powder samples that had not been dehydrated was higher than that of the dehydrated cashew powder samples and showed a gradually increasing trend. Therefore, the lack of dehydration will affect the validity period of the standard sample.

[0046] Table 4 Analysis results of total colony count in cashew nut powder standard samples

[0047]

[0048] Comparative Example 4:

[0049] Referring to the example, the difference is that the cashew powder that has not been defatted is in a state of coarse blocks of oil adhesion, and the particle size is about 10 mm. The different particle sizes of the cashew powder samples will affect the detection efficiency of the added food matrix.

[0050] Experiments have shown that the analysis process of the characteristic substance value (allergic protein AnaO3) of cashew powder samples requires sufficient extraction of nucleic acid DNA or allergic protein before testing the value. The detection process is complicated, and the sample particle size is too large, which affects the detection efficiency of the characteristic value analysis added to the food matrix (such as pepper and salt seasoning). The defatted cashew powder sample can be sieved into a uniform fine powder with a particle size of at least 50 mesh, which will fully release the internal structural substances, which is more conducive to the full release of nucleic acids and proteins of the allergic protein AnaO3, and promote the detection of the characteristic value of the characteristic substance of allergic cashews with better detection efficiency.

[0051] The defatted and dehydrated cashew powder sample of the present invention and the cashew powder sample of comparative example 4 which was not defatted were respectively subjected to determination of cashew characteristic substances (such as allergic protein AnaO3), and the nucleic acid characteristic value of the allergic protein AnaO3 was determined by the fluorescence qPCR method and the protein characteristic value of the allergic protein AnaO3 was determined by the ELISA method, and the results of the Mann Whitney test statistical analysis are shown in Table 5. The Ct value and protein content value of the cashew allergic protein AnaO3 were used for statistical difference analysis, and the Ct value and protein content value of the allergic protein AnaO3 in the defatted and dehydrated cashew powder sample prepared by the present invention and the cashew powder sample which was not defatted were extremely significantly different (p<0.0001), indicating that the nucleic acid characteristic value and protein characteristic value of the allergic protein AnaO3, a food allergic cashew characteristic substance, would be affected by the difference in particle size of the cashew powder which was not defatted, and the detection efficiency of adding food matrix would be reduced.

[0052] Table 5 Detection efficiency analysis results of cashew powder samples with different particle sizes added to the salt and pepper seasoning matrix

[0053]

[0054] The defatted and dehydrated cashew powder standard sample prepared by the present invention and the cashew powder sample prepared by the comparative example are respectively used to detect the nucleic acid amount value and protein content value of the food allergen cashew characteristic substance (such as: allergic protein AnaO3) by real-time fluorescence PCR method and ELISA method, so as to systematically evaluate whether it meets the national standard sample requirements for uniformity performance and stability performance.

[0055] 2. Standard sample uniformity test

[0056] Sampling and uniformity testing were carried out according to the random order repeated measurement method in GB / T 15000.3-2008 "General Principles and Statistical Methods for Determining the Value of National Standard Samples". Fifteen samples were randomly selected from the samples packaged in the smallest package for uniformity testing, and each sample was repeated twice. The Ct value of the cashew nut ingredient characteristic allergic protein AnaO3 gene was detected by real-time fluorescence PCR, and the protein content of the cashew nut allergic protein AnaO3 was detected by ELISA. The uniformity of the standard sample was evaluated by single-factor analysis of variance (F test). When the F ratio is less than the critical value F 0.05(14，15) There is no significant difference between the variance between units and the variance within units, indicating that the standard sample is sufficiently uniform; F ratio> critical value F 0.05(14，15) , there is a significant difference between the variance between units and the variance within units, indicating that the standard sample is not uniform.

[0057] The defatted and dehydrated cashew powder standard sample prepared by the present invention and the cashew powder sample prepared by comparative example 1 were subjected to statistical analysis of uniformity test. The Ct value of the cashew material characteristic allergy protein AnaO3 gene was detected by real-time fluorescence PCR method and the content value of the cashew allergy protein AnaO3 protein was detected by ELISA method, and the uniformity evaluation results were shown in Table 6. According to the F test, the defatted and dehydrated cashew powder standard sample prepared by the present invention, the F values ​​based on the Ct value of the allergy protein AnaO3 gene and the F values ​​based on the content value of the allergy protein AnaO3 protein were 1.81 and 1.78 respectively, both of which were less than the F critical value F 0.05(14,15) =2.42, and the standard uncertainty of uniformity between samples is 0.243% and 0.253%, indicating that there is no significant difference between bottles and within bottles at a 95% confidence probability, and the uniformity of the standard sample is good, meeting the requirements of the national standard sample. However, the F values ​​of the cashew powder samples prepared in Comparative Example 1 are 3.38 and 2.98, both of which are greater than the critical value F 0.05(14,15) =2.42, indicating that the cashew allergy characteristic substances in the cashew powder samples used for testing are uneven and cannot meet the performance evaluation requirements of the national standard samples for the uniformity of characteristic values.

[0058] Table 6 Results of variance analysis for uniformity test of allergic cashew characteristic substances in cashew powder samples

[0059]

[0060] 3. Long-term stability test of standard samples

[0061] According to the random order repeated measurement method in GB / T 15000.3-2008 "General Principles and Statistical Methods for Determining the Value of National Standard Samples", samples were taken for long-term stability testing. The cashew characteristic substance allergy protein AnaO3 gene was detected by real-time fluorescence qPCR, and the cashew characteristic substance allergy protein AnaO3 protein content was detected by ELISA. For the long-term stability test, samples were randomly selected from 500 bottles of large batches of standard samples packaged in independent packages and placed at a low temperature of 0-4℃ for long-term storage. At every 2-month time interval in the first year and every 3-month time interval in the second year, 3 bottles of standard samples (m=3, n=2) were randomly selected at each time point. The long-term stability of the standard samples was analyzed using a linear empirical model.

[0062] The defatted and dehydrated cashew powder standard sample prepared by the present invention and the cashew powder sample prepared by the comparative example were subjected to a long-term stability test. The results of the linear empirical model statistical test analysis under a 95% confidence probability are shown in Table 7. The Ct value of the allergic protein AnaO3 gene and the content value of the allergic protein AnaO3 in the defatted and dehydrated cashew powder standard sample are 0.0064 and 0.0052, respectively, both of which are less than t(0.95,n-2)×s(b1), indicating that the standard sample has good stability within 24 months, and the Ct value of the allergic protein AnaO3 gene and the content value of the allergic protein AnaO3 of the allergic cashew characteristic substance have not undergone obvious trend changes, which meets the stability performance requirements of the national standard sample. However, the cashew powder sample prepared in comparative example 3 has undergone a trend change, indicating that the standard sample is unstable and cannot meet the performance evaluation requirements of the national standard sample for the stability of the characteristic value.

[0063] Table 7 ANOVA results of long-term stability monitoring of allergic cashew characteristic substances in cashew powder samples

[0064]

[0065] The defatted and dehydrated cashew powder national standard sample developed by the present invention has a food allergy cashew characteristic substance content (such as the characteristic value of the allergy protein AnaO3) that is not affected by the defatted and dehydrated preparation process, and the sample uniformity and stability meet the requirements of the national standard sample, and can be added to various food matrices for laboratory quality control activities such as method verification, detection kit evaluation, detection efficiency evaluation, capability verification, and personnel detection capability assessment for detection of food raw cashew characteristic substances. The production cost is low and has a good commercial prospect.

[0066] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances, characterized in that: The method comprises the following steps: (1) Determine the mass of the standard sample and the mass of the raw material: Considering that the mass of fat removed by defatting is 30%, and the mass loss of dehydration during the preparation process is 2%, calculate the required mass of the raw material of the cashew kernel after shelling and peeling; (2) Grinding coarse powder: The cashew kernel raw material after shelling and peeling in step (1) is crushed by a pulverizer to obtain a whole cashew nut coarse powder sample; (3) n-hexane defatting: add n-hexane to the cashew nut coarse powder sample in step (2) at a volume ratio of 3:1, stir evenly with a glass rod, let stand for 30 minutes, and then discard the yellow grease liquid leached from the upper layer; take the precipitate, add n-hexane again at a volume ratio of 2:1, stir evenly, let stand for 30 minutes, and then discard the yellow grease liquid leached from the upper layer; repeat the above operation twice until the upper liquid is colorless; place the cashew nut powder sample after n-hexane defatting in a ventilated place to evaporate naturally; (4) Freeze-vacuum drying: freeze-drying the defatted cashew nut powder sample to obtain a defatted and dehydrated cashew nut powder sample with a moisture content of ≤5%; (5) Grinding thoroughly and sieving to prepare cashew nut fine powder: Grind the defatted and dehydrated cashew nut powder sample thoroughly and pass it through a sterilized metal sieve to obtain a defatted and dehydrated cashew nut fine powder sample; (6) Packaging and storage: The defatted and dehydrated cashew powder standard sample is packaged into brown glass sample bottles, 3 g / bottle, sealed with a three-level sealing device and placed in an environment for long-term storage to meet the shelf life of 2-3 years.

2. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances as claimed in claim 1, characterized in that: The natural volatilization time in step (3) is 24 hours.

3. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances as claimed in claim 1, characterized in that: The freeze-vacuum drying conditions in step (4) are as follows: pre-freezing at -80°C for at least 24 hours, followed by freeze-vacuum freezing at -45 to -55°C at 0.1 mbar for 8 to 10 hours, and finally rapid heating and drying at -10°C to 30°C for 4 to 6 hours, with the rapid heating rate being ≥1.0°C / min.

4. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances as claimed in claim 1, characterized in that: The sterilized metal sieve in step (5) is 50 mesh.

5. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances as claimed in claim 1, characterized in that: The three-stage sealing device in step (6) is composed of a silicone cover, an aluminum foil cover, and a plastic cover.

6. A method for preparing a national standard sample of defatted and dehydrated cashew powder for detecting food allergy cashew characteristic substances as claimed in claim 1, characterized in that: The long-term storage condition in step (6) is an ambient temperature of 0-4°C.

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