Apple juice matrix standard sample containing patulin and preparation method thereof

Abstract

The invention relates to a patulin-containing apple juice matrix standard sample and a preparation method thereof, and belongs to the field of preparation of samples for testing. The matrix standard sample is prepared through a series of steps of bacterial suspension preparation, cleaning, inoculated culture, crushing and juicing, screen filtration, adsorption treatment, centrifugal purification, heating sterilization, freeze concentration, packaging and the like, and the matrix standard sample prepared through the method has the advantages of being good in uniformity and high in stability after being subjected to strict uniformity and stability test.

Description

Technical Field

[0001] The invention relates to an apple juice matrix standard sample containing patulin and a preparation method thereof, belonging to the field of preparation of test samples. Background Art

[0002] Patulin is a secondary metabolite produced primarily by certain Penicillium, Aspergillus, and Mycosporium fungi, exhibiting acute toxicity, chronic toxicity, and cytotoxicity. Penicillium expansum is a major toxin-producing fungus, responsible for blue mold in apples, causing the fruit to soften and rot. Because patulin is readily soluble in water and resistant to acid and heat, it easily remains in products after traditional processing methods. In the production of apples and their products, patulin has become a significant factor affecting the development of the apple industry and endangering human health. Therefore, controlling patulin in apple products is of great practical significance.

[0003] Given the potential risks posed by patulin toxicity in food, several countries and institutions have issued standards for patulin levels. China's national standard stipulates a maximum limit of 50 μg / kg for PAT in fruit juice beverages, excluding apples, hawthorn fruits, and their products (excluding fruit leather). Both the United States and the Codex Alimentarius Commission set a maximum PAT limit of 50 μg / kg for apples and apple products. The European Union has even stricter PAT limits: 50 μg / kg for apple juice and alcoholic beverages, 25 μg / kg for fresh apples, and 10 μg / kg for infant foods. my country is the world's largest apple producer and the largest producer, processor, and exporter of apple juice concentrate. Currently, patulin standard samples are primarily imported. However, due to the diverse sample sources, it is difficult to ensure consistent quality across all standards, severely hindering testing and research. Therefore, developing domestically produced patulin standard samples is crucial.

[0004] When preparing an apple juice matrix standard sample containing patulin, it is difficult to obtain a standard sample with qualified uniformity and stability due to various factors, such as unclear metabolic patterns of the target ingredient, uncertain impact of the preparation process on the uniformity and stability of the target substance, and the large number of components in the apple juice matrix standard sample that may affect each other. Summary of the Invention

[0005] The present invention aims to address the problems existing in the prior art and provide a method for preparing an apple juice matrix standard sample containing patulin. The method comprises a series of steps including preparing a bacterial suspension, washing, inoculating and culturing, crushing and juicing, sieve filtration, adsorption treatment, centrifugal purification, temperature-raising sterilization, freeze concentration, and packaging. The matrix standard sample prepared by this method has undergone rigorous homogeneity and stability testing and has the advantages of good uniformity and high stability.

[0006] Another object of the present invention is to provide an apple juice matrix standard sample containing patulin, which has the advantages of good uniformity and high stability.

[0007] To solve the above technical problems, the present invention adopts a technical solution: a method for preparing an apple juice matrix standard sample containing patulin, comprising the following steps:

[0008] Step 1, culturing Penicillium expansum to obtain a bacterial suspension;

[0009] Step 2: Inoculate the bacterial suspension onto clean apples and incubate at 20-24°C for 2-4 days.

[0010] Step 3: crushing and squeezing the cultured apples to obtain apple juice;

[0011] Step 4: Add 2-10g / L activated carbon to the apple juice for adsorption treatment and stir continuously;

[0012] Step 5: Centrifuge to collect apple juice;

[0013] Step 6: Heat the apple juice to 75-85°C and keep it for 25-35 minutes;

[0014] Step 7: After the apple juice returns to room temperature, freeze-concentrate it to a Brix of 18-30 to obtain concentrated apple juice;

[0015] Step 8: Package and store the concentrated apple juice.

[0016] In a preferred embodiment, step 1 is specifically as follows: inoculating Penicillium expansum on a PDA plate, culturing it in a constant temperature incubator at 26-30°C for 6-8 days; scraping spores, and preparing a bacterial suspension with sterile physiological saline, wherein the concentration of the bacterial suspension is (1-5)×10 7 CFU / mL;

[0017] Step 2 involves cleaning the apple surface with sterile water. Two symmetrical wounds with a diameter of 2-3 mm and a depth of 5-7 mm are then inoculated into the equatorial region of each apple. The bacterial suspension is then inoculated into the wounds, with 8-12 μL of the suspension inoculated into each wound. The incubator temperature is set at 22°C and the incubation period is 3 days. This inoculation method provides a more even distribution of lesions, controlling the growth of patulin and improving uniformity. It also better reflects the actual disease status within the apple, demonstrating the antagonistic expression of intact apples against Penicillium expansum infection, similar to authentic samples.

[0018] In a preferred embodiment, step three specifically comprises crushing and squeezing the cultured apples, skimming off the foam on the surface, and separating the apple juice from the residue;

[0019] Step 4 specifically includes collecting all the apple juice, adding activated carbon adsorbent, and stirring at 600-1400 r / min for 20-40 minutes.

[0020] Step 5 specifically comprises clarifying the apple juice after the adsorbent treatment using a centrifuge, setting the centrifuge parameters to 3000-5000 r / min, the centrifugation time to 0.5-1.5 hours, skimming off the surface foam, and collecting the apple juice;

[0021] Step 6 involves slowly heating the apple juice to 80°C and maintaining it for 30 minutes. Heating to 80°C is to sterilize the juice by overheating, thereby ensuring the stability of patulin in subsequent processes.

[0022] Step seven involves freezing and concentrating the apple juice after it has cooled to room temperature to remove some of the water. The freeze concentrator parameters are a coolant temperature of -6 to -18°C and a circulation flow rate of 20 to 40 mL / min. Freeze concentration removes some of the water from the apple juice to maintain a Brix within a certain range, significantly improving the sample's uniformity and stability.

[0023] In a preferred embodiment, in step 1, the concentration of the bacterial suspension is 4×10 7 CFU / mL.

[0024] In the step 4, the amount of activated carbon adsorbent added is 4 g / L.

[0025] In the step 7, the apple juice is concentrated to a Brix of 23.7.

[0026] The apple juice matrix standard sample of the present invention has a shelf life of 12 months when stored at -18°C and can maintain its validity for 7 days during short-term transportation at 4-50°C. The standard value of patulin in the standard sample is 31.0 μg / kg.

[0027] The method for preparing the aforementioned apple juice matrix standard sample containing patulin specifically comprises the following steps:

[0028] Step 1: Inoculate Penicillium expansum onto a PDA plate using the streak method and incubate in a 28°C isothermal incubator for approximately 7 days. Specifically, use an inoculating loop to pick the activated strain, inoculate it onto a PDA slant, and incubate it at 28°C for 7 days. Scrape off the spores and prepare a bacterial suspension with sterile saline.

[0029] Step 2: Clean the apple surface with sterile water, make two symmetrical wounds with a diameter of 2-3 mm and a depth of 5-7 mm at the equator of each apple, and inoculate the bacterial suspension back into the wounds of the apple, inoculating 10 μL of the bacterial suspension into each wound. Set the incubator temperature to 22°C and incubate for 3 days.

[0030] Step 3: Squeeze the cultured apples through a squeezer to separate the apple juice and residue;

[0031] Step 4: Collect all the apple juice, add activated carbon adsorbent, and stir with an overhead stirrer at 1000 rpm for 30 min;

[0032] Step 5: Clarify the apple juice after adsorbent treatment using a centrifuge at 4000 rpm for 1 hour. Skim off the surface foam and collect the apple juice.

[0033] Step 6: slowly heat the apple juice to 80°C and maintain for 30 minutes;

[0034] Step 7: After the apple juice has slowly cooled to room temperature, use a freeze concentrator to freeze concentrate the apple juice to remove some of the water. The freeze concentrator parameters are a coolant temperature of -11°C and a circulation flow rate of 30 mL / min.

[0035] Step 8: Pour the concentrated apple juice into a sterile juice tank and seal it with negative pressure.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention provides a method for preparing an apple juice matrix standard sample containing patulin. Combined with the production process of apple juice, the method is made through a series of steps including preparing bacterial suspension, cleaning, inoculation and culturing, crushing and squeezing juice, screen filtration, adsorption treatment, centrifugal purification, temperature sterilization, freeze concentration, and packaging. The matrix standard sample prepared by the present invention has undergone a strict uniformity test and has the advantages of good uniformity and high stability. Tests have shown that this standard product can ensure product quality regardless of whether it is transported under frozen or high temperature conditions, meets the transportation requirements of different regions, has a long shelf life, and is an effective and reliable physical standard sample that can be used for quality control in related detection and analysis fields. It can also be used as a means to certify and assess the analytical capabilities of laboratories or technicians participating in the test. It has significant economic value and market competitiveness.

[0038] 2. Zhang Xin. Degradation Mechanism of Patulin in Batch Culture of Penicillium expansum [D]. Northwest Agriculture and Forestry University, 2008. This paper discloses the removal of patulin from juice by activated carbon adsorption. However, during the preparation of matrix standard samples, the removal of target substances is rarely considered, and adsorption of target substances is detrimental to obtaining a uniform and stable matrix standard sample. In contrast, the present invention achieves a uniform apple juice matrix standard sample containing patulin through adsorption with an appropriate concentration of activated carbon. Furthermore, activated carbon adsorption removes some free patulin, increasing the uniformity and stability of the remaining patulin.

[0039] The water content of apple juice is partially removed by freeze concentration to adjust the Brix degree, thereby obtaining a matrix standard sample with better uniformity and stability, while freezing ensures that the original substance of the apple juice is not lost.

[0040] The stab inoculation method can achieve more uniform lesion distribution, control the growth of patulin, and help improve uniformity. It can also better reflect the actual disease situation in the apple body, showing the antagonistic expression of intact apples to Penicillium expansum infection, which is close to the real sample.

[0041] The present invention screened multiple toxin-producing strains of Penicillium expansum obtained from a culture collection and controlled toxin production conditions (wound location and size, inoculum volume). Based on the expected toxin production concentration in apples and the varying toxin production rates of different strains, the appropriate strain (ACCC 36188) was selected to achieve controllable toxin production concentration. This also facilitates the operability and uniformity of multiple sample batch replication. The use of ACCC 36188 was primarily intended to simulate real-world contamination conditions.

[0042] 3. The apple juice matrix standard sample containing patulin of the present invention will be mainly used in laboratory proficiency testing, internal quality control, method validation and other activities; it will help laboratory quality control and ensure the accuracy of quantitative test results; it will help strengthen the quality and safety control of apple juice production and improve the pesticide residue detection level of inspection and testing institutions.

[0043] 4. Homogeneity is a fundamental property of reference materials. Obtaining homogeneous, physical reference materials is challenging due to numerous factors, including difficulty in preparing them, high capital investment, long preparation times, and unclear metabolic patterns of the target components. However, the present invention demonstrates that, under specified process conditions, the prepared samples can achieve excellent homogeneity.

[0044] 5. Stability is also a basic property of standard substances. Standard samples involve storage and transportation processes from preparation to use, which may cause changes in the stability of standard samples. Therefore, two aspects of stability assessment need to be carried out. One is to select the temperature for short-term stability assessment based on the form of sample packaging and transportation. It is usually carried out under different temperature conditions to examine the influence of temperature on the characteristic values ​​of standard substances; the second is to regularly test the characteristic values ​​of standard substances over a longer period under specified storage conditions to examine their ability to remain within the specified range. The short-term stability and long-term stability of the standard samples prepared according to the present invention meet the requirements. The validity period is 12 months under a storage environment of -18°C, and it remains valid for 7 days under short-term transportation at 4-50°C. Sample A of Example 1 of the present invention was tested by 9 laboratories, and the standard value was determined to be 31.0μg / kg.

[0045] 6. Since patulin is a toxin produced by the metabolism of Penicillium expansum, the present invention utilizes apple juice to co-culture the strain primarily to obtain authentic samples. This method ensures authentic samples, prioritizing the accuracy and reliability of analytical methods. Standard samples prepared using additives are primarily used to improve analytical accuracy and eliminate interfering substances. The present invention significantly impacts technical performance, primarily in two key areas. First, authentically prepared standard samples outperform additively prepared standard samples in terms of stability. Because they are prepared according to strict standards and procedures, they exhibit superior long-term stability, as demonstrated by long-term stability monitoring data. This is crucial for standard samples requiring long-term storage and use, as well as for commercialization. Second, authentically prepared standard samples also demonstrate advantages as quality control samples. This is because spiked samples for quality control require the addition of solvent based on the actual weight of the liquid being weighed, which inevitably results in inconsistencies associated with manual dosing. Authentically prepared standard samples not only simplify the sample preparation and testing process but also reduce sample size, further improving efficiency and accuracy, as demonstrated by the repeatability and precision of parallel testing experiments. According to the national food safety standard GB5009.185-2016 for the determination of patulin in foods, the precision of the two detection methods was 15%, while the RSD provided in the sample uniformity table of Group A of the present invention was less than 5%.

[0046] 7. In the prior art, the purpose of adjusting the Brix is ​​generally to improve the consistency of the product, and sugars are often added to increase the consistency. In the prior art related to standard samples of target chemical substances, there are no reports of methods for ensuring that the Brix is ​​within a certain low range. In the present invention, the Brix of apple juice is ensured to be within a certain low range by concentrating and removing some water. No sugars are introduced to change the Brix, preventing the introduction of sugars that interfere and affect the stability of the target substance in the standard sample. The present invention also improves the uniformity and stability of the target substance matrix sample.

[0047] The present invention found that low-Brix samples had insufficient uniformity, and high-Brix samples had poor long-term stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The figure is a high performance liquid chromatogram of the apple juice matrix standard sample containing patulin of the present invention. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0050] Example 1

[0051] Preparation of apple juice matrix standard samples containing patulin under different process parameters:

[0052] Group A: A method for preparing a standard sample of apple juice containing patulin, the specific steps of which are as follows:

[0053] Penicillium expansum standard strain ACCC 36188 (purchased from the China Agricultural Microbial Culture Collection Center) was inoculated on a PDA plate and incubated in a 28°C constant temperature isoincubator for 7 days. Specifically, the activated strain was picked with an inoculation loop and inoculated on a PDA slant and incubated at 28°C for 7 days. Spores were scraped and adjusted to a concentration of 4×10 7 The bacterial suspension with a concentration of CFU / mL is ready for use.

[0054] Red Fuji apples from Aksu, Xinjiang, were purchased from a local wholesale market. Undamaged apples of similar maturity and size from the same batch were selected for inoculation. After washing with sterile water and drying, the apples were surface-wiped with 75% alcohol. Using a sterilized pipette tip, two symmetrical wounds with a diameter of 2-3 mm and a depth of 5-7 mm were pricked at the equator of each apple. Each wound was inoculated with 10 μL of the bacterial suspension. After drying, the suspension was placed in an incubator at 22°C for 3 days. Apples with lesions 3 cm in diameter were selected and squeezed using a small juicer (5.5 kW motor, 1.5 tons / hour processing capacity). Surface foam was skimmed off, and the juice was filtered through a mesh to separate the juice from the residue.

[0055] Collect all the apple juice, add 4g / L of activated carbon (80-mesh activated carbon), and stir with an overhead stirrer at 1000 rpm for 30 minutes. Clarify the treated apple juice using a centrifuge at 4000 rpm for 1 hour. Skim off any surface foam and collect the juice. Slowly heat the juice to 80°C and hold for 30 minutes. After the apple juice was slowly cooled to room temperature, it was freeze-concentrated to 23.7°Brix using a freeze concentrator (Brix determination process: use a plastic dropper to draw up the apple juice sample, slowly inject the apple juice in the dropper into the measuring cell of the refractometer, ensure that the liquid covers the prism surface before measurement, and convert the measured refractive index value into a Brix value). The freeze concentrator parameters are a coolant temperature of -11°C and a circulation flow rate of 30 mL / min. The concentrated apple juice is poured into a sterile juice tank. The obtained apple juice matrix standard sample containing penicillin is packaged under negative pressure in PET bottles and stored frozen at -18°C.

[0056] Group B: Different from Group A, the concentration of bacterial suspension in Group B was 0.5×10 7 CFU / mL.

[0057] Group C: Different from Group A, the addition amount of activated carbon adsorbent in Group C was 1 g / L.

[0058] Group D: Different from Group A, the amount of activated carbon adsorbent added in Group D was 15 g / L.

[0059] Group E: Unlike Group A, the apple juice in Group E was concentrated to a °Brix of 10.

[0060] Group F: Unlike Group A, Group F apple juice is concentrated to a Brix of 50.

[0061] like Figure 1 1 is the spectrum corresponding to the apple juice matrix standard sample containing patulin (Group A) obtained by the preparation method of this example. The retention time of 20.815 min corresponds to patulin.

[0062] Uniformity test

[0063] Each of the above groups of samples was tested for homogeneity. The test method employed was the second method of high-performance liquid chromatography, as specified in GB 5009.185-2016, "National Food Safety Standard - Determination of Patulin in Foods." A one-way analysis of variance (ANOVA) statistical method was used, with the F test used to test homogeneity across the three levels. Thirty bags were randomly sampled from each of the above groups of samples, and each sample was tested as an independent subsample. Each subsample was also tested in triplicate, with the average of the replicate measurements taken as the experimental result. All samples were tested in a random order under repeatable conditions.

[0064] The test results are shown in Tables 1 to 6. In Tables 1 to 6, under different concentration conditions, the samples were restored to the mass before concentration for measurement. Specifically, water was added for restoration before measurement (the purpose is to enable direct comparison between groups and improve the accuracy of homogeneity judgment; and the apple juice standard matrix sample also needs to be restored in the actual application of patulin content detection in apples and apple products. This measurement method can simulate the homogeneity of the apple juice standard matrix sample in actual application to the greatest extent). The pH of the samples in Groups A, E, and F after restoration was 3.7-3.8. Each group was stored in the concentrated state, and each group needed to be restored when determining the homogeneity and stability.

[0065] Table 1. Measurement results of uniformity evaluation of group A samples

[0066]

[0067] Table 2. Measurement results of uniformity evaluation of group B samples

[0068]

[0069]

[0070] Table 3. Uniformity evaluation measurement results of group C samples

[0071]

[0072]

[0073] Table 4. Measurement results of uniformity evaluation of group D samples

[0074]

[0075]

[0076] Table 5. Uniformity evaluation measurement results of group E samples

[0077]

[0078] Table 6. Measurement results of uniformity evaluation of Group F samples

[0079]

[0080]

[0081] The results for Group B samples were below the limit of quantification, indicating that the low concentration of the bacterial suspension prevented the strain from producing toxins within a certain period of time. This is related to the strain's growth curve and does not meet the requirements for use as a standard sample. Groups C and D demonstrate that the amount of activated carbon adsorbent added significantly affects the uniformity of this standard sample. Both too low and too high an amount of activated carbon adsorbent can lead to sample inhomogeneity, which is related to the activated carbon's effectiveness in removing patulin. Furthermore, varying amounts of activated carbon adsorbent also affect the characteristic values ​​of the standard sample (especially significantly increasing the uncertainty caused by inhomogeneity during the determination of characteristic values). Groups E and F demonstrate that the Brix of concentrated apple juice significantly affects the uniformity of this standard sample. Excessively low Brix levels can lead to sample inhomogeneity, and Group F's uniformity is inferior to that of Group A.

[0082] The uniformity test of the samples showed that the test results of the samples in group B were lower than the limit of quantitation. Since the concentration of the bacterial suspension was low, the production of patulin was slow, so it was not valuable as a standard sample. The data of group A were subjected to a one-way ANOVA, and the following ANOVA results were obtained: Under the condition of a confidence probability of 0.95, degrees of freedom f1 = 14, and f2 = 30, the F test table showed that F 0.05(14，30) =2.04, the F value in group A and group F samples < critical value F 0.05(14，30) , indicating that there is no significant difference between the two groups of samples within and between the groups. The samples are uniform and meet the requirements of quality control samples. Moreover, the F values ​​of the samples in groups C, D and E are all greater than the critical value F 0.05(14，30) , indicating that the sample is inhomogeneous.

[0083] Stability test

[0084] The samples that meet the uniformity requirements are tested for stability, and samples from Group A and Group F are selected for the experiment. According to JJF 1343-2022 "Value Determination, Uniformity and Stability Evaluation of Standard Substances", the statistical method for stability testing is determined to be the t-analysis test method. The characteristic value change curve over time is used to determine whether the sample characteristic value has a unidirectional change trend, and the linear fitting model is used to evaluate the stability of the standard sample. Stability testing includes two aspects: long-term stability (storage stability) and short-term stability (transportation stability). Sampling is carried out according to the principle of dense sampling first and sparse sampling later. A total of 6 sampling time points are set for short-term stability, stored at 4°C and 50°C, and a total of 6 sampling time points are set for long-term stability testing, stored at -18°C. At each sampling time point, 3 samples from Group A and Group F are randomly selected, and each sample is repeated twice. The average value of the three sub-samples is taken to analyze the content of patulin in the samples of Group A and Group F.

[0085] The test results are shown in Tables 7 to 10.

[0086] Table 7. Short-term stability results of patulin in group A samples

[0087]

[0088]

[0089] Table 8. Short-term stability results of patulin in samples of Group F

[0090]

[0091]

[0092] Table 9 Long-term stability results of patulin in group A samples

[0093]

[0094]

[0095] Table 10 Long-term stability results of patulin in group F samples

[0096]

[0097] It can be seen from Tables 7 to 10 that the samples of group A prepared by the method of the present invention have |β1| less than t in both the short-term stability and the 12-month long-term stability tests. 0.95，n-2 ×s(β1), the content of patulin in the matrix standard sample tends to be stable, and the storage environment of -18℃ can be determined as the storage environment of the matrix standard sample, with a validity period of 12 months. The samples in group F showed stability in the short term, but became unstable within the 12 months of long-term stability. The product quality was lower than that of the samples in group A, indicating that the long-term stability of samples with high brix was poor.

[0098] Fixed value

[0099] The samples in Group A were assigned values ​​according to the requirements of JJF 1343-2022, "Assignment of Values ​​and Evaluation of Homogeneity and Stability of Reference Materials." This matrix standard sample was assigned values ​​using a collaborative approach involving nine laboratories, all of which were accredited. The test results were tested for normal distribution, followed by a Grubbs test to determine if suspicious values ​​were present within each laboratory, and a Cochrane test to determine if data from different laboratories were of comparable accuracy. Only when these requirements were met could the data be considered representative and included in the statistical analysis of the assigned values.

[0100] The test results are shown in Table 11.

[0101] Table 11 Inter-laboratory determination data of prepared Group A standard samples

[0102]

[0103]

[0104] The Shapiro-Wilk test was used for normality testing. From the table, we found W(n, p) = 0.788 (where n = 6 and p = 0.95). Since the W values ​​for the data from each laboratory were all greater than 0.788, we can assume that the data from each laboratory are normally distributed.

[0105] The Grubbs test method is used to test whether the test results of each laboratory group have suspicious values. From the Grubbs critical value table, we can know that λ (0.05,6) =1.887. The results show that the absolute value of the maximum residual value of the detection results is less than λ (0.05,6) *S, * represents the multiplication sign ×, indicating that there are no abnormal values ​​or suspicious values ​​among the fixed-value results of each laboratory. Therefore, all data are retained and can be included in the fixed-value result statistics.

[0106] The Cochran test is used to determine whether the results of the values ​​determined by different laboratories are of equal precision. The Cochran test requires that C is less than or equal to C (α,m,n) , indicating that the average values ​​of each group of data are of equal precision, otherwise it is determined to be an outlier and the data in this group should be removed when calculating the fixed value result. (0.05,9,5) =0.3285. Table 11 shows that the C value of the Cochrane test is 0.1609, which is less than the critical value. Therefore, the data between the laboratory test result groups are of equal precision. All data should be retained and can be included in the fixed value result statistics.

[0107] According to JJF 1343-2022, "Assignment of Values, Uniformity, and Stability Evaluation of Reference Materials," after testing each laboratory's assigned values ​​for suspicious values ​​and equal precision, the average values ​​of the assigned values ​​from each laboratory must be tested for significant differences and normality of the data distribution. Calculations revealed no significant differences between the average values ​​of the nine laboratories and met normality tests, satisfying statistical requirements. Therefore, the standard value of the apple juice-based standard sample containing patulin prepared by Group A was the mean of the average results from each laboratory, resulting in a standard value of 31.0 μg / kg for the apple juice-based standard sample containing patulin prepared by Group A.

[0108] Uncertainty

[0109] According to JJF 1343-2022 "Assessment of the value, uniformity and stability of reference materials", the uncertainty of the value of reference materials consists of three parts: the uncertainty caused by the inhomogeneity of the reference material, U bb , uncertainty U caused by instability of standard substances stsUncertainty U caused by the certification process of the reference material char The combined uncertainty U can be determined by calculating the uncertainties of each part (y) At a 95% confidence level, the expanded uncertainty U is twice the combined uncertainty:

[0110]

[0111] U = 2 × U (y)

[0112] Uncertainty introduced by homogeneity

[0113] According to JJF 1343-2022 "Certification and Evaluation of Homogeneity and Stability of Reference Materials", the within-bottle variance of Group A samples is less than the between-bottle variance of the samples Therefore, the standard deviation s of between-bottle homogeneity can be estimated bb which is equivalent to the uncertainty component U caused by between-bottle inhomogeneity bb However, since U bb < s2, indicating that the estimated between-bottle standard deviation is less than the impact of the repeatability standard deviation on the between-bottle standard deviation. At this time, the uncertainty caused by the inhomogeneity of the reference material can be calculated using the following formula

[0114]

[0115] Where:

[0116] MS within -- Measurement repeatability variance in between-bottle homogeneity evaluation

[0117] n-- Number of measurements within the group

[0118] v MSwithin -- Degrees of freedom of MS within

[0119] After calculation, the uncertainty component U caused by homogeneity finally obtained bb is 0.39953 μg / kg

[0120] Uncertainty introduced by stability​​​​​​​​

[0122] Uncertainty introduced by fixed value

[0123] The method used to determine the value of Group A samples is to use a variety of methods with confirmed accuracy and to determine the value by multiple laboratories. Each laboratory only provides a series of observations. According to the requirements of the standard, in principle, the standard deviation of the mean value under this determination mode is the uncertainty of the determination U char .

[0124]

[0125] The s value is the standard deviation of the mean of the nine laboratories’ determination results, and the p value is the total number of laboratories participating in the determination, which is 9. After calculation, the standard uncertainty U introduced by the joint determination of the laboratories is finally obtained. char =0.276502μg / kg.

[0126] Therefore, the total uncertainty can be calculated as U (y) The final determined characteristic value of patulin in the apple juice matrix standard sample of Group A is 31.0±2.61 μg / kg (k=2).

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a standard sample of apple juice matrix containing patulin, characterized in that: The following steps are involved: Step 1, culturing Penicillium expansum to obtain a bacterial suspension; Step 2: Inoculate the bacterial suspension onto clean apples and incubate at 20-24°C for 2-4 days. Step 3: crushing and squeezing the cultured apples to obtain apple juice; Step 4: Add 2-10g / L activated carbon to the apple juice for adsorption treatment and stir continuously; Step 5: Centrifuge to collect apple juice; Step 6: Heat the apple juice to 75-85°C and keep it for 25-35 minutes; Step 7: After the apple juice returns to room temperature, freeze-concentrate it to a Brix of 18-30 to obtain concentrated apple juice; Step 8: Package and store the concentrated apple juice.

2. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 1, characterized in that: Step 1 is to inoculate Penicillium expansum on a PDA plate and culture it in a constant temperature incubator at 26-30℃ for 6-8 days; scrape the spores and prepare a bacterial suspension with sterile saline. The concentration of the bacterial suspension is (1-5)×10 7 CFU / mL; Step 2 specifically involves cleaning the apple surface with sterile water, making two symmetrical wounds with a diameter of 2-3 mm and a depth of 5-7 mm at the equator of each apple, and inoculating the bacterial suspension back into the wounds of the apple. 8-12 µL of bacterial suspension is inoculated into each wound, and the incubator temperature is set to 22°C for 3 days.

3. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 1, characterized in that: Step three specifically includes crushing and squeezing the cultured apples, skimming off the foam on the surface, and separating the apple juice and residue; Step 4 specifically includes collecting all the apple juice, adding activated carbon adsorbent, and stirring at 600-1400 r / min for 20-40 minutes.

4. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 1, characterized in that: Step 5 specifically comprises clarifying the apple juice after the adsorbent treatment using a centrifuge, setting the centrifuge parameters to 3000-5000 r / min, the centrifugation time to 0.5-1.5 hours, skimming off the surface foam, and collecting the apple juice; Step six specifically includes slowly heating the apple juice to 80° C. and maintaining the temperature for 30 minutes.

5. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 1, characterized in that: Step seven specifically includes: after the apple juice is cooled to room temperature, using a freeze concentrator to freeze concentrate the apple juice to remove some of the water; the freeze concentrator parameters are a coolant temperature of -6 to -18°C and a circulation flow rate of 20 to 40 mL / min.

6. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 2, characterized in that: In step 1, the concentration of the bacterial suspension was 4×10 7 CFU / mL.

7. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 2, characterized in that: In the step 4, the amount of activated carbon adsorbent added is 4 g / L.

8. The method for preparing a standard sample of apple juice matrix containing patulin according to claim 2, characterized in that: In the step 7, the apple juice is concentrated to a Brix of 23.

7.

9. An apple juice matrix standard sample containing patulin obtained by the preparation method according to any one of claims 1 to 8.

10. The apple juice matrix standard sample containing patulin according to claim 9, characterized in that: The standard sample is valid for 12 months when stored at -18°C, and remains valid for 7 days during short-term transportation at 4-50°C. The standard value of patulin is 31.0 μg / kg.

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