A method for preparing a standard sample of hazelnut ultrafine powder for detecting characteristic values ​​of food allergens

Abstract

The invention belongs to the field of food testing and discloses a method for preparing a hazelnut ultrafine powder standard sample for detecting characteristic quantitative values ​​of food allergens. The standard sample adopts hazelnut kernels as raw materials and is ground at low temperature by an ultrafine grinder to obtain hazelnut ultrafine powder. The obtained hazelnut ultrafine powder standard sample retains the characteristic quantitative values ​​of hazelnut component characteristic 2S globulin gene nucleic acid and allergen protein Cora9 to the greatest extent, and the preparation process does not add any auxiliary materials and preservatives. It is purely green production and highly purified, can simultaneously meet the requirements of allergen characteristic 2S globulin gene detection and allergen protein Cora9 detection, and its uniformity and stability meet the requirements of national standard samples, filling the gap of international standard samples of this type, improving the detection level of food allergen safety testing laboratories, and can be used for food allergen hazelnut detection method verification, detection kit evaluation, laboratory detection process quality control, inter-laboratory proficiency verification and comparison, and personnel detection ability assessment.

Description

Technical Field

[0001] The present invention belongs to the field of food detection, and particularly relates to a method for preparing a hazelnut ultrafine powder standard sample for detecting the characteristic quantitative values ​​of food allergens, and specifically relates to a method for preparing a hazelnut ultrafine powder standard sample for detecting the characteristic quantitative values ​​of the allergen hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a9, which are limited in the field of food allergens. Background Art

[0002] Hazelnuts, a restricted allergen in the food allergy field, are rich in protein, oils (mostly unsaturated fatty acids), carotene, vitamins, and minerals, earning them the nickname "King of Nuts." Hazelnuts can be eaten directly or used as an ingredient in various pastries and candies. While highly nutritious, hazelnuts are also one of the most common food allergens. Allergic reactions can cause chest tightness, sore throat, difficulty breathing, nausea, stomach cramps, vomiting, and diarrhea, and in severe cases, death. Approximately 3%-5% of adults and 8% of children worldwide suffer from food allergies, with allergies to nuts accounting for approximately 25% of all known food allergies. To prevent allergic reactions from unknowingly ingesting hazelnuts, my country, the United States, the European Union, and other countries have enacted regulations requiring clear labeling of food packaging containing hazelnuts. The incidence of allergic diseases continues to rise worldwide. Conducting allergen testing and labeling to help consumers avoid allergens is the most effective prevention and control measure. There is no effective treatment for food allergies so far. An important way to prevent food allergies is to strictly avoid consuming allergens, and food allergen detection and analysis is the most critical link.

[0003] Hazelnut ingredient characterization, the 2S globulin gene and the allergen Cor a 9, are the most common tests for hazelnut food allergens. The 2S globulin gene test is a molecular assay targeting the allergen's DNA. High-temperature heating of foods can cause DNA degradation, but the allergen protein remains. Hazelnut allergen protein testing, on the other hand, is an immunological assay targeting the allergen Cor a 9 protein. The allergen protein is heat-stable but susceptible to loss of antigenicity during storage and processing, and is also susceptible to interference from complex components such as preservatives and the food matrix. These two distinct testing methods complement each other in their effectiveness in hazelnut allergen testing. This necessitates that the preparation of standard samples must meet the requirements of both methods, namely, the stability requirements for the characteristic values ​​of the 2S globulin gene and the allergen Cor a 9 protein. At present, there are no standard samples of allergen hazelnut powder at home and abroad. Since the characteristic 2S globulin gene and allergen protein of hazelnut components are easily denatured and aggregated during processing, their linear epitopes and conformational epitopes are changed, which brings difficulties to the detection of the characteristic quantity values ​​of the hazelnut component characteristic 2S globulin gene and allergen protein Cor a 9, and is more likely to cause detection errors, false positive and false negative results. The above reasons make the preparation very difficult, resulting in the lack of hazelnut ultrafine powder standard samples rich in allergen hazelnut component specific 2S globulin gene and hazelnut allergen protein for detection value guarantee at home and abroad, which brings inconvenience to the detection of hazelnut allergen components in my country's food.

[0004] There are various hazelnut flour products and corresponding preparation processes in the prior art. For example, a patent discloses a method for producing hazelnut flour, comprising the following steps: shelling hazelnut kernels, cold-pressing the kernels at low temperature using an oil press, separating the hazelnut oil to obtain a meal cake, crushing the meal cake into a meal cake powder with a particle size of 40-60 mesh, extracting the meal cake powder using supercritical carbon dioxide extraction, and separating the extracted hazelnut oil to obtain hazelnut flour. However, this method uses supercritical carbon dioxide extraction for degreasing, which destroys and degrades the nucleic acid content of the characteristic 2S globulin gene of hazelnut components and interferes with the determination of allergen protein antigenicity, thereby reducing the detection and addition recovery rate. Another example is a patent that discloses a nut flour processing method, comprising the following steps: material selection - rinsing - machine cutting - machine selection - color sorting - freeze-crushing - autoclaving - drying - packaging. The autoclaving step involves placing the freeze-crushed nut flour in a high-pressure device for autoclaving, and the packaging step involves packaging and sealing the dried nut flour after it passes inspection. The high-pressure sterilization in the above-mentioned steps not only destroys non-covalent bonds (such as hydrogen bonds, disulfide bonds, and ionic bonds) in microbial proteins, but also disrupts the higher-order structure of allergenic proteins, leading to protein coagulation and enzyme inactivation, which in turn affects the determination of allergenic protein characteristic values ​​and the recovery rate of additives. Furthermore, while ultrahigh pressure can cause multiple cell damages, such as rupturing bacterial cell membranes and the outflow of chemical components within the bacteria, it also destroys the nucleic acid (DNA and other genetic material) of the nut component characteristic 2S globulin gene, significantly damaging and reducing the characteristic values ​​of the hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9. Nut powder packaging is not vacuum-sealed, which can easily cause unsaturated fatty acids to oxidize and deteriorate, making it unsuitable for long-term storage and unable to ensure the long-term stability of characteristic values ​​for more than two years. For example, a patent discloses a hazelnut nutritional powder and its preparation method, which specifically includes water-grinding and sieving the hazelnut kernels, mixing with ginseng juice, colloid milling, sieving, vacuum concentration, and spray drying at 140-170°C. However, this method significantly destroys the hazelnut component characteristic 2S globulin gene through high-temperature spray drying, thus affecting DNA detection, and the addition of a mixed ginseng colloid matrix can interfere with the immunoassay of allergen characteristic allergen proteins. For example, a publicly published article on the preparation process of hazelnut powder uses hazelnut cake after squeezing as the base material, and adds non-dairy creamer, white sugar, maltodextrin, ethyl maltol, and sodium carboxymethyl cellulose to produce hazelnut powder. However, the addition of complex matrix components in this method can seriously interfere with the molecular detection of allergen DNA and the immunoassay of allergen proteins. Moreover, more importantly, the above-mentioned products do not aim to provide standard samples for the detection of characteristic values ​​of the hazelnut component characteristic 2S globulin gene and the allergen protein Cor a 9. The uniformity and stability of the characteristic values ​​do not meet the requirements of national standard samples. Therefore, it is still necessary to develop a standard sample preparation process for hazelnut ultrafine powder used for the detection of characteristic values ​​of allergen components. Summary of the Invention

[0005] Standard samples can be used by food safety testing laboratories to validate test methods, evaluate test kits, and perform quality control during the testing process. They are of great significance for ensuring the accuracy and validity of laboratory test results. At the same time, the emergence of standard samples will provide reliable support for assessing and evaluating the testing capabilities and result comparison of food allergen testing laboratories and technicians, and is an effective supplement to the methods of assessing and monitoring laboratory capabilities.

[0006] The research on preparing a standard sample of hazelnut ultrafine powder for detecting the characteristic 2S globulin gene of the allergen hazelnut component and the characteristic value of the allergen protein Cor a 9 will provide support for the comprehensive assessment of the level of food allergen detection laboratories in test method validation, test kit evaluation, quality control during the detection process, etc. 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.

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a method for preparing a hazelnut ultrafine powder standard sample for detecting the characteristic values ​​of food allergens. The hazelnut ultrafine powder standard sample is limited in the field of food allergens and is used to detect the characteristic values ​​of the allergen hazelnut component 2S globulin gene and the allergenic protein Cor a 9. The method comprises the following steps:

[0008] (1) Determine the weight of a standard sample of ultrafine hazelnut powder for detecting the characteristic values ​​of the allergen hazelnut component 2S globulin gene and the allergenic protein Cor a 9, which is limited in the field of food allergens, and consider that the loss during the preparation process is 10-20 wt% of the raw material;

[0009] (2) Low temperature grinding: The dried hazelnut kernels were crushed using an ultrafine grinder to obtain hazelnut ultrafine powder with a mass fraction of more than 98%. Different grinding temperatures affected the values ​​of the characteristic 2S globulin gene and the characteristic value of the allergenic hazelnut component in the standard sample.

[0010] (3) Freeze-vacuum drying: The cryogenically ground hazelnut ultrafine powder is freeze-dried; the total bacterial count of hazelnut ultrafine powder standard samples with different moisture contents varies during storage, which affects the long-term stability of the characteristic values ​​of the standard samples;

[0011] (4) Sieving treatment: The ultrafinely crushed hazelnut ultrafine powder is passed through a sterilized metal sieve to obtain hazelnut ultrafine powder with a particle size that meets the detection requirements; according to the quality control needs of the added recovery rate of the food matrix to be tested, the particle size of the hazelnut ultrafine powder standard sample to be crushed is determined. At the same time, it is necessary to consider the determination of common allergenic proteins in GB / T 38163-2019, the food matrix added recovery rate under the specified conditions of liquid chromatography-tandem mass spectrometry standard, and the added recovery rate of hazelnut allergenic protein Cor a 9 characteristic value detection of different particle sizes.

[0012] (5) Dry powder mixing: The sieved hazelnut ultrafine powder is placed in a sterile packaging bag and placed on a dry powder mixer to shake and mix. The sample is further mixed to obtain a hazelnut ultrafine powder standard sample with uniformity, stability and moderate uncertainty, which contains the hazelnut component characteristic 2S globulin gene and allergenic protein Cor a 9 characteristic value for detecting the allergen hazelnut component characteristic within the scope of food allergens;

[0013] (6) Vacuum packaging and storage: The hazelnut ultrafine powder standard sample was packaged into brown glass sample bottles, vacuum-sealed using a freeze vacuum dryer, and then sealed using a three-stage sealing device and placed in the environment for long-term storage.

[0014] The hazelnut allergen 2S globulin gene was detected using the "SN / T 1961.8-2013 Standard for the Detection of Allergens in Exported Foods - Part 8, Real-Time Fluorescence PCR Method," and the hazelnut allergen Cor a 9 protein was detected using the "GB / T 38163-2019 Standard for the Determination of Common Allergens by Liquid Chromatography-Tandem Mass Spectrometry." The homogeneity and stability of the hazelnut components were tested, and the recovery rate of the hazelnut powder after addition to food matrices was evaluated. Finally, a batch of uniform and stable hazelnut ultrafine powder standard samples was prepared for the detection of the allergen hazelnut allergen 2S globulin gene and the allergen Cor a 9 protein. The homogeneity and stability of the hazelnut allergen 2S globulin gene and the allergen Cor a 9 protein in the standard samples met the relevant requirements for national standard samples.

[0015] Furthermore, the temperature of the cryogenic grinding in step (2) is below 0°C, preferably -80 to -20°C.

[0016] Furthermore, the crushing time in step (2) is 5 to 10 minutes.

[0017] Furthermore, after the vacuum drying in step (3), ultrafine hazelnut powder with a moisture content of ≤5% is obtained.

[0018] Furthermore, the diameter of the sterilized metal sieve in step (4) is 100 μm.

[0019] Furthermore, the shaking and mixing time of the dry powder mixer in step (5) is 1 day.

[0020] Preferably, the cover for vacuum sealing in step (6) is a silicone cover.

[0021] Preferably, the three-stage sealing device in step (6) consists of a silicone cover, an aluminum foil cover, and a plastic cover.

[0022] Furthermore, the long-term storage condition in step (6) is an ambient temperature of 0-4°C.

[0023] The key technical points of the present invention are:

[0024] (1) Process research: The low-temperature grinding process of hazelnut kernels, freeze-vacuum drying, and the particle size of ultrafine powder particles and vacuum packaging are very important for the characteristic value, additive recovery rate, uniformity and long-term stability of the allergen hazelnut component characteristic 2S globulin gene and allergen allergen Cor a 9 to avoid changes in characteristic value. If the hazelnut grinding temperature is too high, the allergen hazelnut component characteristic 2S globulin gene and hazelnut allergen Cor a 9 will be degraded and destroyed, resulting in a decrease in content. If the water content of the hazelnut ultrafine powder is too high, it will cause bacteria to grow during the storage of the standard sample and affect the long-term stability of the characteristic value. If the particle size of the hazelnut ultrafine powder is too large, it will affect the additive recovery rate of different food matrix detection. Therefore, it is necessary to consider the low-temperature freezing time and the water content of the hazelnut ultrafine powder, grinding temperature, particle size, low-temperature vacuum packaging and other processes.

[0025] (2) Study on sample uniformity: The characteristic quantities of the developed standard samples are based on the detection of hazelnut component characteristic 2S globulin gene by real-time fluorescence PCR and hazelnut allergy protein Cor a 9 by liquid chromatography-tandem mass spectrometry. The values ​​of characteristic 2S globulin gene and allergy protein Cor a 9 are affected by many factors such as the temperature of the preparation test and the particle size of the sample. Therefore, it is necessary to strictly control the low-temperature freeze-drying temperature and sample particle size of the sample to ensure the uniformity of the characteristic quantity values ​​of the standard samples.

[0026] (3) Sample stability study: The characteristic values ​​of the developed standard samples are based on the detection of hazelnut component characteristic 2S globulin gene by real-time fluorescence PCR and the characteristic value of hazelnut allergy protein Cor a 9 by liquid chromatography-tandem mass spectrometry. The characteristic values ​​of characteristic 2S globulin gene and allergy protein Cor a 9 under long-term storage conditions are affected by factors such as water content and the easy oxidation of unsaturated fatty acids. Therefore, it is necessary to strictly control the low-temperature freeze-drying temperature, water content and low-temperature vacuum packaging of the samples to ensure that the characteristic values ​​of hazelnut component characteristic 2S globulin gene and allergy protein Cor a 9 do not change over time within a certain period of time.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The hazelnut ultrafine powder standard sample obtained by the present invention, which is limited in the field of food allergens and is used to detect the characteristic values ​​of the hazelnut component characteristic 2S globulin gene and the allergy protein Cor a 9, has uniformity, stability and moderate uncertainty. Within a certain period of time, the characteristic values ​​of the hazelnut component characteristic 2S globulin gene and the allergy protein Cor a 9 do not change with time. The uniformity and stability of the sample meet the requirements of national standard samples, filling the gap of the lack of such standard samples in my country. The detection level of food allergen safety testing laboratories is improved, and the sample can be used for food allergen hazelnut detection method verification, detection kit evaluation, laboratory testing process quality control, inter-laboratory proficiency verification and comparison, and personnel testing ability assessment. DETAILED DESCRIPTION

[0029] The following is a further description of the present invention, but not a limitation thereof. 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.

[0030] Example 1

[0031] 1. Sample Preparation

[0032] In this example, each batch prepares 2.5 kg, or 500 bottles, of 5 g / bottle of hazelnut ultrafine powder standard samples for detecting the characteristic values ​​of the allergen hazelnut component 2S globulin gene and the allergenic protein Cor a 9, which are restricted in the field of food allergens. First of all, it is necessary to consider the loss rate of the sample during the grinding process and the low-temperature freeze-drying and dehydration, so the number of samples to be prepared is about 3.2 kg.

[0033] The specific preparation steps are as follows:

[0034] (1) Ultra-low temperature freezing and grinding: The hazelnut kernels that have been ultra-low temperature frozen at -80℃ for 48 hours are crushed in an ultra-fine grinder for 2-3 minutes to obtain hazelnut powder samples; without ultra-low temperature freezing treatment, the characteristic values ​​of the allergen hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9 in the standard sample will be affected;

[0035] (2) Vacuum freeze drying and dehydration treatment: The hazelnut ultrafine powder is subjected to vacuum freeze drying and dehydration treatment, namely: pre-freezing at -80°C for 2 hours, then vacuum freezing at -45 to -55°C at 0.1 mbar for 10 hours, and finally rapid heating and drying at -10 to 30°C for more than 8 hours, with a rapid heating rate of ≥1.0°C / min, to obtain freeze-dried hazelnut ultrafine powder with a moisture content of ≤5%; the total bacterial count of hazelnut ultrafine powder standard samples with different moisture contents varies during storage, which affects the long-term stability of the characteristic values ​​of the standard samples;

[0036] (3) Sieving: The ultrafinely crushed hazelnut ultrafine powder standard sample is passed through a 100 μm sterilized metal sieve to obtain a hazelnut ultrafine powder standard sample with a particle size of less than 100 μm; according to the quality control requirements of the added recovery rate of the food matrix to be tested, the particle size of the hazelnut ultrafine powder standard sample to be crushed is determined, and at the same time, the recovery rate of hazelnut ultrafine powder under the conditions specified in the "GB / T 38163-2019 Standard for the determination of common allergenic proteins by liquid chromatography-tandem mass spectrometry" needs to be considered. The added recovery rate of hazelnut allergenic protein Cor a 9 characteristic value detection of different particle sizes has certain differences;

[0037] (4) Dry powder stirring and mixing: The sieved hazelnut ultrafine powder standard sample is placed in a sterile packaging bag and placed on a dry powder mixer for shaking and mixing for 1 day. The standard sample is further mixed to obtain a hazelnut ultrafine powder standard sample with uniformity, stability and moderate uncertainty in the field of food allergens for detecting the characteristic value of the allergen hazelnut component 2S globulin gene and the allergenic protein Cor a 9;

[0038] (5) Vacuum packaging and storage: The hazelnut ultrafine powder standard sample was packaged into brown glass sample bottles and sealed with silicone caps using a freeze vacuum dryer under vacuum conditions of 0.05-0.2 mbar. Then, a three-level sealing device was used, namely, a silicone cap, an aluminum foil cap, and a plastic cap. The ambient temperature of the sample was kept below 0-4°C for long-term storage.

[0039] Comparative Example 1

[0040] Compared with Example 1, the difference is that the grinding temperature is different. This comparative example adopts the method of grinding at room temperature, and the other operating steps are the same as Example 1.

[0041] Experiments have shown that pre-treatment of hazelnut kernels with ultra-low temperature freezing for 48 hours at temperatures below 0°C increases their hardness and brittleness. Grinding, through the use of physical force, reduces the degradation of the hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9 in the standard sample, making it easier to crush the hazelnut kernels into fine particles without agglomeration. This results in higher values ​​of the hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9 in the standard sample. In contrast, grinding at room temperature affects the values ​​of the allergen hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9 in the standard sample.

[0042] Comparative Example 2

[0043] Compared with Example 1, the difference lies in the drying method and water content. In this comparative example, the water content of the prepared hazelnut ultrafine powder sample is about 15% without vacuum freeze-drying and dehydration treatment. The other operating steps are the same as those in Example 1.

[0044] Experiments have shown that low-temperature vacuum drying, a physical process in which water freezes and expands, causing hazelnut tissue to burst, and ice sublimation causes surface water evaporation, does not destroy or degrade the characteristic hazelnut component 2S globulin gene and allergenic protein Cor a 9 in the standard sample. This allows the hazelnut kernel to be easily crushed into fine particles without agglomeration. The hazelnut ultrafine powder standard sample, which has a moisture content of ≤5% after low-temperature vacuum drying, is stable over the long term, extending its shelf life. In contrast, the hazelnut ultrafine powder sample, which has not been subjected to vacuum freeze drying and dehydration, has a moisture content of approximately 15%, and the characteristic values ​​in long-term stability monitoring show a significant trend change, indicating sample instability.

[0045] Comparative Example 3

[0046] Compared with Example 1, the difference is that the particle size of the hazelnut ultrafine powder sample particles is different. This comparative example is not subjected to sieving treatment, and the other operating steps are the same as Example 1.

[0047] Experiments have shown that the analysis of the characteristic values ​​of hazelnut ultrafine powder standard samples, namely the hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9, requires thorough extraction of nucleic acid DNA or allergenic proteins before testing. This complex testing process and the uneven sample particle size affect the characteristic values. Screening of hazelnut ultrafine powder standard samples with particle sizes less than 100 μm fully releases internal structural substances, facilitating the uniform dispersion of the 2S globulin gene DNA and allergenic proteins. This results in greater uniformity and higher values ​​for the characteristic values ​​of the hazelnut component characteristic 2S globulin and hazelnut allergenic protein Cor a 9. In contrast, unscreened hazelnut powder samples affect the spike recovery rate of the hazelnut allergenic protein Cor a 9 characteristic value.

[0048] Comparative Example 4

[0049] Compared with Example 1, the difference is that the vacuum packaging of the hazelnut ultrafine powder standard sample is different. This comparative example is not vacuum-sealed, and the other operating steps are the same as Example 1.

[0050] Due to the high oil content in hazelnuts, with unsaturated fatty acids accounting for over 90% of the oil, which are easily oxidized and deteriorated, hazelnut powder generally has a short shelf life. After vacuum treatment in a low-temperature vacuum dryer, automatic sealing (silicone cap) can effectively isolate the air, inactivate the enzymes, stop their action, and prevent oxidation. Standard samples of ultrafine hazelnut powder sealed with a three-stage sealing device (silicone cap, aluminum foil cap, plastic cap) have better stability of characteristic values ​​and can ensure the long-term stability of the characteristic values ​​of allergen hazelnut components within two years. In contrast, hazelnut powder samples that are not vacuum-sealed affect long-term stability and stability under transportation conditions.

[0051] Comparative Example 5

[0052] With reference to CN101336735A, the method comprises the following steps: taking shelled hazelnut kernels, cold-pressing the hazelnut kernels with an oil press at low temperature, isolating hazelnut oil, obtaining a meal cake, crushing the meal cake into a meal cake powder with a particle size of 40 to 60 meshes, extracting the meal cake powder by supercritical extraction with carbon dioxide, isolating the extracted hazelnut oil to obtain hazelnut powder, the extraction pressure being 24 to 26 MPa, the extraction temperature being 50 to 60°C, the extraction time being 2 to 3 hours, and the carbon dioxide flow rate being 110 to 130 L / h. This comparative example first isolates part of the hazelnut oil in the hazelnut kernel by the cold-pressing method, then isolates the hazelnut oil in the meal cake by supercritical extraction with carbon dioxide, and the hazelnut powder can be used as a raw material or ingredient to process a variety of foods with unique flavors. The above-mentioned steps of cold-pressing to separate hazelnut oil and extracting meal powder will greatly destroy the higher-order structure of the hazelnut component allergenic protein and the hazelnut component characteristic 2S globulin gene, resulting in a reduction in the characteristic quantitative values ​​of the hazelnut component characteristic 2S globulin gene and the allergenic protein Cor a 9. They are not suitable for preparing hazelnut powder standard samples with the goal of providing accurate characteristic quantitative values.

[0053] Comparative Example 6

[0054] Referring to CN104886663A, the process includes the following steps: material selection - rinsing - machine cutting - machine selection - color sorting - freeze crushing - high pressure sterilization - drying - packaging. Among them, freeze crushing is to put the color-sorted nuts into a freeze crushing device for freeze crushing, using liquid nitrogen as the refrigerant, controlling the freezing temperature at -100°C, and crushing the nuts into nut powder; high pressure sterilization is to put the frozen crushed nut powder into a high pressure device for high pressure sterilization, maintaining the temperature in the high pressure device at 20-40°C, the pressure holding time at 5-15 minutes, and the pressure at 300-500MPa; drying is to collect the high pressure sterilized nut powder and put it into a vacuum freeze dryer, controlling the temperature at -80°C and the vacuum degree at 133×10 －3 mBar, drying time 2 to 4 hours, moisture content in the raw materials ≤ 7.0%; packaging is to package and seal the dried nut powder after it passes the inspection. Using liquid nitrogen as the refrigerant in the above steps will increase the preparation cost; high-pressure sterilization can destroy the non-covalent bonds in the bacterial proteins of microorganisms, such as hydrogen bonds, disulfide bonds and ionic bonds, and also destroy the higher-order structure of allergenic proteins, thereby leading to protein coagulation and enzyme inactivation, which in turn affects the characteristic values ​​of allergenic proteins; in addition, ultra-high pressure can cause multiple cell damages such as rupture of bacterial cell membranes and outflow of chemical components in the bacteria, while also destroying the characteristic values ​​of genetic materials such as nut component DNA, thereby greatly damaging and reducing the characteristic values ​​of hazelnut component characteristic 2S globulin gene and allergenic protein Cor a 9; nut powder is not packaged in a vacuum-sealed manner, which can easily cause unsaturated fatty acids to oxidize and deteriorate, which is not conducive to long-term storage and cannot guarantee the long-term stability of hazelnut component characteristic values ​​within 2 years; more importantly, no screening treatment is performed, and the uniformity of the sample particle size cannot be guaranteed to meet the requirements of national standard samples. Therefore, it is not suitable for preparing hazelnut powder standard samples with the goal of providing accurate characteristic values.

[0055] Comparative Example 7

[0056] Reference CN1706287A relates to a hazelnut nutritional powder and its preparation method, with the goal of providing a hazelnut solid beverage with both medicinal and edible properties. The powder is a spray-dried product of hazelnut juice or ginseng juice, comprising the following steps: grinding the hazelnuts with 2-12 times the amount of water, sieving, vacuum concentrating at 50-55°C to a solids content of 50-60%, spray drying at an inlet air temperature of 140-170°C, and sieving after cooling. The 140-170°C spray drying process in this comparative example significantly degrades the characteristic values ​​of the hazelnut component 2S globulin gene and allergenic proteins, making it unsuitable for preparing a hazelnut ultrafine powder standard sample intended to provide accurate hazelnut characteristic values.

[0057] Example 2

[0058] The hazelnut ultrafine powder standard sample prepared in Example 1 of the present invention and the hazelnut powder samples prepared in Comparative Examples 1, 2, 3, 4, 5, 6 and 7 were used to determine the Ct value of the hazelnut component characteristic 2S globulin gene by real-time fluorescence PCR and the characteristic value of the hazelnut allergenic protein Cor a 9 by liquid chromatography-tandem mass spectrometry, to systematically evaluate their compliance with the national standard sample requirements for uniformity and stability performance, as well as the additive recovery rate of the characteristic value in the food matrix.

[0059] 1. Standard sample uniformity test

[0060] Sampling and homogeneity testing were carried out according to the random sequence 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 into the smallest package for homogeneity testing, with each sample repeated twice. The Ct value of the hazelnut component characteristic 2S globulin gene was detected using the real-time fluorescence PCR method in SN / T 1961.6-2013. The homogeneity of the standard sample was evaluated using the one-way analysis of variance (F test) with the hazelnut component characteristic 2S globulin gene as the representative. 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.

[0061] The results of the statistical analysis of the homogeneity test of the hazelnut ultrafine powder standard sample prepared in Example 1 of the present invention and the hazelnut powder samples prepared in Comparative Examples 5, 6, and 7 are shown in Table 1. The homogeneity test was performed using the Ct value of the hazelnut component characteristic 2S globulin gene as a representative. According to the F test, the F ratio of the hazelnut ultrafine powder standard sample prepared in the present invention was 1.29, which was less than the F critical value F 0.05(14,15) =2.42, and the standard uncertainty of inter-sample uniformity was 0.0159%, indicating that at a 95% confidence level, there were no significant differences between or within bottles, indicating that the standard samples had good uniformity and met their intended use. However, the hazelnut powder samples prepared in Comparative Examples 5, 6, and 7 showed significant differences in both inter-unit variance and intra-unit variance, indicating that the standard samples were non-uniform and failed to meet the performance evaluation requirements for uniformity of national standard samples.

[0062] Table 1 Results of variance analysis of 2S globulin gene homogeneity of hazelnut component characteristics in hazelnut powder samples

[0063]

[0064] 2. Stability test of standard samples

[0065] 1. Long-term stability test

[0066] According to the random sequence repeated measurement method in GB / T 15000.3-2008 "General principles and statistical methods for the determination of national standard samples", samples were taken, and the long-term storage stability was investigated by measuring the characteristic gene of hazelnut component 2S globulin as a representative. The real-time fluorescence PCR method in SN / T 1961.6-2013 was used to detect the characteristic gene of hazelnut component 2S globulin. For the long-term stability test, according to the sampling principle of dense sampling at the beginning and sparse sampling at the end of the time interval, the packaged samples selected by the present invention were stored for a long time at a low temperature of 0 to 4°C. Every 2 months in the first year and every 3 months in the second year, samples were randomly selected for long-term stability experiments. Three standard samples were taken each time, and each sample was tested twice. According to the standard sample stability test method recommended by GB / T 15000.3, a straight line was used as the empirical model for statistical testing. The Ct value of the hazelnut component 2S globulin characteristic gene is less than its t (0.95,n-2) ×s(b1), indicating that during the stability test period, the value of the standard sample did not undergo obvious trend changes, that is, the stability was good.

[0067] The hazelnut ultrafine powder standard sample prepared in Example 1 of the present invention and the hazelnut powder samples prepared in Comparative Examples 2, 4, 5, 6, and 7 were subjected to long-term stability testing. The statistical analysis results are shown in Table 2. The stability test was performed using the hazelnut component 2S globulin characteristic gene as a representative. According to the linear empirical model statistical test, the Ct value of the hazelnut 2S globulin characteristic gene in the hazelnut ultrafine powder standard sample prepared in the present invention is less than t (0.95,n-2) ×s(b1), indicating that the standard sample has good stability and meets the national standard sample stability requirements. However, the Ct values ​​of the hazelnut 2S globulin characteristic gene in the hazelnut powder samples prepared in Comparative Examples 2, 4, 5, 6, and 7 are all greater than t (0.95,n-2) ×s(b1), that is, the value of the sample has undergone an obvious trend change, indicating that the standard sample is unstable and cannot meet the performance evaluation requirements of the national standard sample for stability.

[0068] Table 2 Results of linear empirical model analysis of the long-term stability of hazelnut characteristic 2S globulin gene in hazelnut powder samples

[0069]

[0070] 2. Short-term stability test under transportation conditions

[0071] Following the randomized, repeated measurement method outlined in GB / T 15000.3-2008, "General Principles and Statistical Methods for the Determination of Values ​​of National Standard Samples," samples were collected for short-term stability under transportation conditions, using the hazelnut component characteristic 2S globulin gene as a representative. Considering that the short-term stability of standard samples is primarily influenced by the effects of short-term transportation on characteristic values ​​under temperature fluctuations, this experiment simulated extreme transportation conditions by short-term storage of samples at temperatures between -20°C and 50°C. Stability studies were conducted at -20°C, 0-4°C, 25°C, and 50°C to investigate short-term stability. The hazelnut component characteristic 2S globulin gene was detected using the real-time fluorescence PCR method outlined in SN / T 1961.6-2013. Hazelnut powder samples were stored at -20°C for 7 days, 0-4°C for 7 days, 25°C for 7 days, and around 50°C for 7 days before testing. Three standard samples were tested at each temperature, with two replicates per sample. The mean consistency test was used to verify and evaluate short-term stability. After the transportation conditions, the statistical t-test value of the Ct value of the hazelnut component characteristic 2S globulin gene was less than the critical value t (0.95,10) , indicating that under the transportation conditions of -20℃ and 50℃, the characteristic values ​​of the standard sample did not change significantly within four weeks, and the short-term stability was good.

[0072] The hazelnut ultrafine powder standard sample prepared by the present invention and the hazelnut powder samples prepared by comparative examples 5, 6 and 7 were subjected to short-term stability test results. The statistical analysis results are shown in Table 3. The short-term stability test was performed using the Ct value of the hazelnut component characteristic 2S globulin gene as a representative. According to the consistency T test, the t test value of the hazelnut component characteristic 2S globulin gene of the hazelnut ultrafine powder standard sample prepared by the present invention was less than the critical value t (0.95,10) , indicating that the standard sample has good stability under transportation conditions, and the national standard sample has good performance evaluation requirements for stability under transportation conditions. However, the hazelnut powder samples prepared in Comparative Examples 5, 6, and 7 have t-test values ​​of hazelnut component characteristic 2S globulin gene greater than the critical value t (0.95,10) , indicating that the sample is unstable under transportation conditions and cannot meet the performance evaluation requirements of national standard samples for stability under transportation conditions.

[0073] Table 3 Results of short-term stability consistency test analysis of hazelnut characteristic 2S globulin gene in hazelnut powder samples

[0074]

[0075] 3. Determination of characteristic values ​​of standard samples added to food matrix

[0076] The hazelnut ultrafine powder standard sample prepared by the present invention and the hazelnut powder samples prepared in Comparative Examples 1, 3, 5, 6, and 7 were used to determine the Ct value of the hazelnut component characteristic 2S globulin gene by real-time fluorescence PCR and the characteristic value of the hazelnut allergenic protein Cor a 9 by liquid chromatography-tandem mass spectrometry to systematically evaluate their compliance with the national standard sample requirements for uniformity and stability performance, as well as the detection and addition recovery rate of the characteristic value in the food matrix.

[0077] Randomly sample the hazelnut ultrafine powder standard sample prepared by the present invention, and the hazelnut powder samples prepared by Comparative Examples 1, 3, 5, 6, and 7, 3 each, and add them to the biscuit matrix wheat flour at two addition levels of 40 mg / kg and 20 mg / kg. The hazelnut component characteristic 2S globulin gene Ct value is detected by the real-time fluorescence PCR method in SN / T 1961.6-2013, and the hazelnut allergy protein Cor a 9 characteristic value detection recovery rate is analyzed by liquid chromatography-tandem mass spectrometry according to GB / T 38163-2019 for the determination of common allergenic proteins. Each sample is measured in parallel twice. The Kruskal Wallis test is used to compare the hazelnut component characteristic 2S globulin gene quantity value analysis of the embodiment with comparative examples 5, 6, and 7, and the statistical significance is set at 0.05.

[0078] The results of the Ct value analysis of the hazelnut component characteristic 2S globulin gene in the biscuit matrix at different addition levels of hazelnut powder are shown in Table 4. The results of the recovery rate analysis of the characteristic value of the hazelnut allergy protein Cor a 9 in the biscuit matrix at different addition levels of hazelnut powder are shown in Table 5.

[0079] Table 4 Analysis results of hazelnut characteristic 2S globulin gene in hazelnut powder samples at different addition levels in biscuit matrix

[0080]

[0081] Table 5 Recovery analysis results of hazelnut allergy protein Cor a 9 characteristic value detection in biscuit matrix at different addition levels of hazelnut powder samples

[0082]

[0083] The hazelnut ultrafine powder standard sample developed by the present invention maximizes the hazelnut component characteristic 2S globulin gene value, with a smaller Ct value indicating a higher characteristic gene content, as well as the hazelnut allergy protein Cor a 9 characteristic value. A higher hazelnut allergy protein characteristic value recovery rate indicates a higher allergy protein characteristic value. Furthermore, the preparation process does not add any preservatives, achieving a purely green production process, and the prepared hazelnut ultrafine powder standard sample is highly purified. The hazelnut ultrafine powder standard sample preparation method for detecting allergens provided by the present invention is simple, meets national standard sample performance requirements, and meets expected usage requirements. It has low production costs and has excellent commercial prospects.

[0084] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a standard sample of hazelnut ultrafine powder for detecting characteristic values ​​of food allergens, characterized in that: The method comprises the following steps: (1) Determine the weight of the hazelnut ultrafine powder standard sample to be prepared for the detection of the characteristic values ​​of the hazelnut component 2S globulin gene and the allergenic protein Cor a 9 within the scope of food allergens, and consider that the loss during the preparation process is 10-20wt% of the raw material; (2) Cryogenic grinding: The dried hazelnut kernels are crushed at -80~-20℃ using an ultrafine grinder to obtain hazelnut ultrafine powder with a mass percentage of more than 98%; (3) Freeze vacuum drying: freeze vacuum drying the ultrafine hazelnut powder after cryogenic grinding to obtain ultrafine hazelnut powder with a moisture content of ≤5%; (4) Screening treatment: The ultrafinely crushed hazelnut ultrafine powder is passed through a sterilized metal sieve with a diameter of 100 μm to obtain hazelnut ultrafine powder with a particle size that meets the test requirements; (5) Dry powder mixing: The sieved hazelnut ultrafine powder is placed in a sterile packaging bag and placed on a dry powder mixer and shaken for 1 day. The sample is further mixed to obtain a hazelnut ultrafine powder standard sample with uniformity, stability and moderate uncertainty, which contains the characteristic 2S globulin gene and the characteristic value of the allergen hazelnut component and the allergen protein Cor a 9 for the detection of allergen hazelnut component characteristics within the scope of food allergens; (6) Vacuum packaging and storage: The hazelnut ultrafine powder standard sample was packaged into brown glass sample bottles, vacuum sealed using a freeze vacuum dryer, and then sealed using a three-stage sealing device consisting of a silicone cap, an aluminum foil cap, and a plastic cap, and then stored in an environment of 0-4°C for a long time.

2. The method for preparing a standard sample of hazelnut ultrafine powder for detecting characteristic values ​​of food allergens according to claim 1, wherein the pulverization time in step (2) is 5 to 10 minutes.

3. The method for preparing a standard sample of hazelnut ultrafine powder for detecting characteristic values ​​of food allergens according to claim 1, wherein the cover for vacuum sealing in step (6) is a silicone cover.

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