Preparation methods and applications of 13 paralytic shellfish toxin matrix reference materials

Abstract

The present invention discloses a preparation method and application of 13 kinds of paralytic shellfish toxin matrix standard material raw materials, including the following steps: (1) a method for expanding the cultivation of four paralytic shellfish toxin-producing algae, and (2) a step of exposing the four paralytic shellfish toxin-producing algae to mussels. The present invention adopts a method of exposing mussels to a mixture of multiple toxin-producing algae in different proportions to obtain raw materials containing 13 kinds of paralytic shellfish toxin matrix standard materials. By comparing the field exposure method and the indoor combined exposure mussel method of the present invention, the present invention can obtain stable, reliable and repeatable raw materials of a large variety of paralytic shellfish toxin matrix standard materials, which can simultaneously meet the quality control and method verification needs of shellfish paralytic shellfish toxin detection and analysis in different sea areas in China, and promote the effectiveness of monitoring and risk assessment of paralytic shellfish toxins in my country.

Description

Technical Field

[0001] The invention relates to a preparation method and application of 13 paralytic shellfish toxin matrix standard material raw materials, belonging to the technical field of biological toxins. Background Art

[0002] Paralytic shellfish toxins (PSTs) pose serious ecological and food safety risks in my country and globally. Strengthening safety monitoring of these toxins to ensure consumer safety is a global consensus. PST-producing algae in my country's coastal waters are diverse and widely distributed, primarily produced by single-celled dinoflagellates such as Alexandrium catenella, A. pacificum, A. tamarense, A. minutum, and Gymnodinium catenatum. The composition and toxicity of PSTs produced by these different algae vary significantly, significantly affecting the toxin accumulation in bivalve shellfish. The PST contamination situation in my country's coastal waters is complex. The main toxins found in shellfish in the South China Sea are GTX, STX, and NeoSTX; in the Yellow Sea, C and GTX are the main toxins; and in the Bohai Sea, GTX and NeoSTX are the main toxins. In particular, GTX is the predominant PST in shellfish from Qinhuangdao, where red tides are particularly severe. PSTs have temporal and spatial distribution and interspecies differences along my country's coast, which makes comprehensive and effective monitoring and management difficult.

[0003] Currently, effective control of PSTs is lacking, so prevention and forecasting are effective means of mitigating their impact. China has implemented effective preventive measures, including controlled monitoring of water sources or harvesting areas. Monitoring changes in shellfish PSTs at all stages of production is crucial. The European Union, the United States, Canada, and other countries and regions have established strict limits (800 μg STXeq. / kg) and monitor these levels. Due to the complexity of food matrices, matrix effects often interfere with the accuracy and precision of detection methods. Using matrix reference materials for quality control during analytical testing can effectively overcome errors caused by matrix effects. High-quality and reliable reference materials are valuable tools for analytical method development, validation, and instrument calibration.

[0004] Common methods for preparing paralytic shellfish toxin matrix reference materials include using shellfish contaminated with paralytic shellfish toxins from naturally occurring red tides as raw materials, or using scallops fed A. tamarense to prepare four paralytic shellfish toxin matrix reference materials, including GTX1, GTX4, C1, and C2. Current techniques for preparing paralytic shellfish toxin matrix reference materials suffer from unstable raw material sources, a limited number of toxins in the products, and a failure to meet international limits. Furthermore, these techniques do not meet the current situation in my country where multiple PSTs coexist. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for preparing 13 kinds of paralytic shellfish toxin standard material raw materials, filling the gap in the simultaneous preparation of 13 kinds of paralytic shellfish toxin matrix standard raw materials at home and abroad. The raw material source is stable, the operation process is simple, and the results are controllable.

[0006] The present invention provides a method for preparing 13 kinds of paralytic shellfish toxin standard material raw materials, comprising the following steps:

[0007] (A) Expansion cultures of four typical PSP-producing algae from my country's coastal waters: primary and secondary culture, and tertiary semi-continuous culture. The four PSP-producing algae are Alexandrium catenella (GY-H25), G. catenatum (GY-H65), A. tamarense (GY-H31), and A. minutum (GY-H46).

[0008] (B) Mixed exposure of four toxic algae: Each mussel was fed with a certain ratio of four toxic algae with a certain total algal cell count, and the mussels were mixed and exposed for 4 to 8 days at a culture water temperature of 6 to 10°C.

[0009] (C) Preparation of paralytic shellfish toxin matrix standard material: After the exposure, all mussels were collected for dissection and all soft tissues were collected. The homogenized and mixed materials were used to obtain the paralytic shellfish toxin matrix standard material.

[0010] Preferably, the four strains of paralytic shellfish toxin-producing algae in step (A) are chain-shaped Alexandrium (A.catenella, GY-H25), chain-shaped gymnodinium (G.catenatum, GY-H65), Tamarense (A.tamarense, GY-H31) and miniature Alexandrium (A.minutum, GY-H46), all of which can be purchased from Shanghai Guangyu Biotechnology Co., Ltd. These four commercialized toxin-producing algae are easy to purchase and inexpensive, with low cost.

[0011] Preferably, the ratio of the four strains of toxic algae in step (B) is: GY-H25 is 7% to 15%, GY-H65 is 10% to 20%, GY-H31 is 8% to 20%, and GY-H46 is 45% to 65%. The change in the proportion of toxic algae affects the proportion and content of different toxins.

[0012] Preferably, the exposure period in step (B) is 5 days.

[0013] Preferably, in step (B), the total amount of algae fed to each shellfish per day is 3.5×10 4 ~2.6×10 5 cells.

[0014] Preferably, the aquaculture water temperature is 9° C. when the shellfish are mixed and exposed in step (B). The water temperature will affect the accumulation rate of toxins from toxic algae in the mussels.

[0015] The paralytic shellfish toxin matrix standard material raw material prepared by the preparation method of the present invention has toxin components including NEO, dcSTX, dcNEO, GTX1, GTX4, GTX2, GTX3, GTX5, GTX6, dcGTX2, dcGTX3, C1 and C2.

[0016] The present invention also provides the application of 13 paralytic shellfish toxin matrix standard substance raw materials for preparing stable and reliable 13 paralytic shellfish toxin standard substances.

[0017] The present invention also provides the use of 13 paralytic shellfish toxin matrix standard substance raw materials in preparing 13 paralytic shellfish toxin standard substances in simultaneously monitoring paralytic shellfish toxins in multiple sea areas in China.

[0018] The paralytic shellfish toxin matrix standard substance prepared according to the preparation method of the present invention can be used in inter-laboratory proficiency testing activities for paralytic shellfish toxin testing projects. It can also be used for quantitative or qualitative detection of paralytic shellfish toxins, as well as for the validation of detection methods, calibration of test instruments, and quality control of test results.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for simultaneously preparing 13 paralytic shellfish toxin matrix standard material raw materials, filling the gap in the simultaneous preparation of 13 paralytic shellfish toxin matrix standard material raw materials at home and abroad, with a stable source of raw materials, a simple operation process, and controllable results. It can ensure the comprehensiveness of paralytic shellfish toxin detection and is conducive to the effective monitoring of paralytic shellfish toxins in multiple sea areas in China. The matrix standard materials prepared from the 13 paralytic shellfish toxin matrix standard material raw materials of the present invention can be used for inter-laboratory paralytic shellfish toxin test capability verification and for quantitative or qualitative detection of 13 paralytic shellfish toxins. It is suitable for use by various departments such as food, fishery supervision, environmental protection, quality inspection, fishery, and health. It can promote the development of aquaculture, processing, and trade, ensure food safety, and will bring significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of mixed exposure of the present invention.

[0021] Figure 2 This is a process flow chart for preparing the raw materials of the paralytic shellfish toxin matrix standard substance of the present invention.

[0022] Figure 3 In Example 2, the total amount of algae fed to each shellfish per day was 3.5×10 4 cells, and fed with four toxin-producing algae, GY-H25, GY-H65, GY-H31, and GY-H46, at a ratio of 14%, 15%, 15%, and 56%, respectively. The exposure time was 5 days, and the toxin content and types in the mussels after mixed exposure were analyzed.

[0023] Figure 4 The total amount of algae fed to each shellfish per day was 2.6×10 5 cells, and fed with four toxin-producing algae, GY-H25, GY-H65, GY-H31, and GY-H46, at a ratio of 8%, 12%, 18%, and 62%, respectively. The exposure time was 5 days, and the toxin content and types in the mussels after mixed exposure were analyzed.

[0024] Figure 5 (a) to (k) are LC-MS spectra of paralytic shellfish toxins in the paralytic shellfish toxin matrix standard material raw materials of Example 2. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0026] The mussels and materials used in the present invention are commercially available or can be obtained by methods known in the art. For example, species of Alexandrium catenella (GY-H25), G. catenatum (GY-H65), A. tamarense (GY-H31), and A. minutum (GY-H46) can all be purchased from Shanghai Guangyu Biotechnology Co., Ltd. Seawater and mussels were purchased from nearby waters.

[0027] Example 1

[0028] Three-stage expansion culture of four toxic algae:

[0029] Primary culture: Toxin-producing algae were inoculated into a 100-250 mL Erlenmeyer flask sterilized by high temperature and cultured in a light incubator for 6-10 days until the cell density reached 1×10 4 ~1×10 5 The incubator was set up with the following conditions: an intensification of 4000 lx and a light-dark ratio of 12 h:12 h; a temperature of 25.0°C for A. catenella (GY-H25) and A. minutum (GY-H46), and a temperature of 22.0°C for G. catenata (GY-H65) and A. tamarense (GY-H31).

[0030] Secondary culture: expand the culture into a 1L triangular flask sterilized by high temperature according to the ratio of 1:3 to 1:1 (algae solution: culture medium, V:V), and culture in a light incubator for 10 to 12 days until the cell density reaches 5×10 5 ~5×10 6 The incubator was set up with the following conditions: an intensification of 4000 lx and a light-dark ratio of 12 h:12 h; a temperature of 24.0°C for A. catenella (GY-H25) and A. minutum (GY-H46), and a temperature of 22.0°C for G. catenata (GY-H65) and A. tamarense (GY-H31).

[0031] Tertiary culture: Spread the culture into sterilized 5L food-grade PET culture jars at a ratio of 1:5 to 1:1 (algae solution: culture medium, V:V). Place in a light-controlled incubator. Every 3-5 days, pump out 1 / 3 of the lower algae solution and replenish with an equal amount of culture medium for semi-continuous culture. The light-controlled incubator should be set up with continuous illumination of 6000 ± 1000 lx on both sides and a light-dark ratio of 12h:12h. The temperature should be air-conditioned at 23.0°C.

[0032] Example 2

[0033] The total algal cell count of the four toxic algae strains GY-H25, GY-H65, GY-H31 and GY-H46 was 2.1×10 7 600 mussels with a shell height of 7.0 ± 0.5 cm and a shell length of 3.5 ± 0.5 cm were purchased and selected for exposure to toxic algae. The mussels were temporarily cultured indoors for 2 days with continuous ventilation and daily replacement of natural seawater. Each mussel was fed 3.5 × 10 4 Cells were fed with four strains of toxic algae, GY-H25, GY-H65, GY-H31, and GY-H46, at a ratio of 14%, 15%, 15%, and 56%, respectively. The toxic algae were fed at 8:00 and 20:00, maintaining a 1:2 ratio between the two feedings. Exposure lasted for 5 days, with the water temperature at 9°C. After exposure, all mussels were collected, dissected, and all soft tissues were collected. The mixture was homogenized and then subjected to paralytic shellfish toxin extraction. Three replicates of each sample were weighed, each weighing 5.0 ± 0.1 g. Extraction was performed with 1% acetic acid in a boiling water bath for 5 minutes. After cooling, the extract was centrifuged at 4500 rpm for 10 minutes. One mL of the extract was transferred to a 5 μL ammonia solution, vortexed, and centrifuged at 10,000 rpm for 10 minutes. Extract with an activated Supelco ENVI-Carb solid-phase extraction column, filter through a 0.22μm filter membrane into an injection vial, and store frozen at -20°C for analysis. Use high-performance liquid chromatography-mass spectrometry to analyze paralytic shellfish poisoning. Chromatographic column: TSK-Amide-80 column, column length 150mm, inner diameter 2.0mm, particle size 3.0μm, or equivalent performance. Flow rate: 0.35mL / min. Column temperature: 40°C. Injection volume: 5μL. Mobile phase A: water (containing 2mmol / L ammonium formate, 50mmol / L formic acid), B: 95% acetonitrile aqueous solution (containing 2mmol / L ammonium formate, 50mmol / L formic acid). Mass spectrometry reference conditions are as follows:

[0034] Detection method: multiple reaction monitoring (MRM), ion source temperature: 550℃, spray voltage IS: 5000V, -4500V, curtain gas pressure CUR: 20psi, nebulizer gas pressure GS1: 50psi, auxiliary heating gas pressure GS2: 50psi, collision gas CAD: Medium, detection as Figure 5 The PSTs content in shellfish is expressed in μg / kg. The results are shown in Figure 3As shown, after testing, the toxin contents in the paralytic shellfish toxin matrix standard material raw material were: NEO 22.9μg / kg, dcSTX 35.4μg / kg, dcNEO 22.5μg / kg, GTX1 253μg / kg, GTX4 72.8μg / kg, GTX2 159μg / kg, GTX3 62.5μg / kg, GTX5 107μg / kg, GTX6168μg / kg, dcGTX2 75.0μg / kg, dcGTX3 30.4μg / kg, C1 278μg / kg and C2 207μg / kg, and the final paralytic shellfish toxin matrix standard material raw material with a total toxicity of 552μg STXeq / kg was obtained.

[0035] Example 3

[0036] The total algal cell count of the four toxic algae strains GY-H25, GY-H65, GY-H31 and GY-H46 was 1.6×10 8 600 mussels with a shell height of 7.0 ± 0.5 cm and a shell length of 3.5 ± 0.5 cm were purchased and selected for exposure to toxic algae. The mussels were temporarily cultured indoors for 2 days with continuous ventilation and daily replacement of natural seawater. Each mussel was fed a total algae intake of 2.6 × 10 5 cells, and fed four strains of toxic algae GY-H25, GY-H65, GY-H31 and GY-H46 in the ratios of 8%, 12%, 18% and 62% respectively. The toxic algae were fed at 8 o'clock and 20 o'clock respectively, and the feeding ratio was controlled at 1:2. The exposure time was 5 days, and the water temperature was 9°C. After the exposure, all mussels were collected for dissection and all soft tissues were collected. After homogenization and mixing, paralytic shellfish toxins were detected using the extraction method and detection method of Example 2. Figure 4 As shown, after testing, the toxin contents in the paralytic shellfish toxin matrix standard material raw material were: NEO 73.8μg / kg, dcSTX 133μg / kg, dcNEO 79.9μg / kg, GTX1 1494μg / kg, GTX4 394μg / kg, GTX2 750μg / kg, GTX3 228μg / kg, GTX5 278μg / kg, GTX6 564μg / kg, dcGTX2 280μg / kg, dcGTX3108μg / kg, C1 930μg / kg and C2 509μg / kg, and the final paralytic shellfish toxin matrix standard material raw material with a total toxicity of 2692μg STXeq / kg was obtained.

[0037] Example 4

[0038] Take the mussels in Examples 2 and 3, and the mussels contaminated by natural red tide in Qinhuangdao. Carefully remove all the shellfish meat, homogenize the shellfish soft tissue samples, mix the homogenate, and use the extraction method and detection method of Example 2 to detect paralytic shellfish toxins. As shown in Table 1, the toxin components of paralytic shellfish toxins in the mussels of Examples 2 and 3 include NEO, dcSTX, dcNEO, dcGTX2, dcGTX3, GTX1, GTX2, GTX3, GTX4, GTX5, GTX6, C1, and C2, a total of 13 toxins. The toxin components of paralytic shellfish toxins in mussels contaminated by natural red tide in Qinhuangdao include NEO, dcSTX, dcNEO, GTX1, GTX2, GTX3, and GTX4, a total of 7 toxins.

[0039] Table 1 Comparison of toxin types in mixed exposure and field exposure

[0040]

[0041] Note: + indicates positive, - indicates negative

[0042] Example 5

[0043] The toxin components of the mussel paralytic shellfish toxins in Examples 2 and 3 were compared with those obtained from other research institutions using scallops fed with Alexandrium tamarense and mixing a culture of G. catenatum with oysters. As shown in Table 2, the toxin components of the mussel paralytic shellfish toxins in Examples 2 and 3 totaled 13. Four paralytic shellfish toxins, GTX1, GTX4, C1, and C2, were obtained from feeding scallops with A. tamarense, while four paralytic shellfish toxins, GTX1, GTX4, GTX2, and GTX3, were obtained from mixing a culture of G. catenatum with oysters.

[0044] Table 2 Comparison of toxin types in mixed exposure with toxin types in other research institutions

[0045]

[0046] Note: + indicates positive, - indicates negative

[0047] Example 6

[0048] Four laboratories were used to compare the toxin content of the paralytic shellfish toxin matrix standard raw material samples prepared in Examples 2 and 3. The paralytic shellfish toxin matrix standard raw material samples prepared in Examples 2 and 3 were distributed to four laboratories. Each laboratory selected experienced testers to test the samples for paralytic shellfish toxin content according to a standardized test procedure. The results are shown in Table 3. Grubb's method confirmed the absence of outliers in each laboratory. Cochran's method was used to test the accuracy of the data determined by each laboratory. The results showed that all data passed the test and the testing accuracy of each laboratory was consistent.

[0049] Table 3 Statistics and analysis of toxin content in samples tested by four laboratories (μg STX eq / kg)

[0050]

[0051]

Claims

1. 13 methods for preparing paralytic shellfish toxin matrix standard material raw materials, characterized in that: The method comprises the following steps: (A) Four typical offshore paralytic shellfish toxin-producing algae were cultured in stages: the first and second stages were graded cultures, and the third stage was a semi-continuous culture. The four paralytic shellfish toxin-producing algae were Alexandrium catenellae ( A. catenella ), Gymnodinium catenatum ( G. catenatum ), Alexandrium tamarense ( A. tamarense ) and Alexandrium microcarpa ( A. minutum ); (B) Mixed exposure of four toxic algae: Among them, the feed ratio for each mussel is: Alexandrium catenella is 7% to 15%, Gymnodinium catenella is 15%. 10% to 20%, Alexandrium tamarense 8% to 20%, and Alexandrium microcarpa 45% to 65%. The total amount of algae fed to each mussel per day is 3.5×10 4 ~2.6×10 5 cells, Mussels were mixed and exposed for 4 to 8 days at a culture water temperature of 6 to 10°C; (C) Obtaining the raw material of paralytic shellfish toxin matrix standard material: After the exposure is completed, all mussels are collected for dissection and all soft tissues are collected. After homogenization and mixing, the raw material of paralytic shellfish toxin matrix standard material is obtained. The 13 paralytic shellfish toxin matrix standard material raw materials include NEO, dcSTX, dcNEO, GTX1, GTX4, GTX2, GTX3, GTX5, GTX6, dcGTX2, dcGTX3, C1 and C2.

2. The method for preparing 13 paralytic shellfish toxin matrix standard material raw materials according to claim 1, characterized in that: The exposure period in the step (B) is 5 days.

3. The method for preparing 13 paralytic shellfish toxin matrix standard material raw materials according to claim 1, characterized in that: The water temperature during the mixing and exposing of the shellfish in step (B) is 9°C, and the water temperature will affect the accumulation rate of toxins from toxic algae in the mussels.

4. The method for preparing 13 paralytic shellfish toxin matrix standard material raw materials according to claim 1, characterized in that: Each mussel was fed with 3.5×10 4 cells, the feeding ratios of Alexandrium catenella, Gymnodinium catenella, Alexandrium tamarense and Alexandrium microphyllum were 14%, 15%, 15% and 56% respectively, the exposure time was 5 days and the water temperature was 9°C.

5. The method for preparing 13 paralytic shellfish toxin matrix standard material raw materials according to claim 1, characterized in that: The total amount of algae fed to each mussel per day was 2.6×10 5 cells, the feeding ratios of Alexandrium catenella, Gymnodinium catenella, Alexandrium tamarense and Alexandrium microphyllum were 8%, 12%, 18% and 62% respectively, the exposure time was 5 days and the water temperature was 9°C.

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