Method for detecting vomitoxin produced by bacillus cereus in raw milk or fermented feed

Abstract

The invention discloses a method for detecting vomitoxin produced by bacillus cereus in raw milk or fermented feed, and relates to the technical field of quality safety detection of raw milk and feed. According to the method provided by the invention, high-accuracy and high-sensitivity detection of the vomitoxin in the raw milk or the fermented feed can be realized, and rapid, accurate and quantitative detection requirements can be met. Raw milk or a fermented feed sample is pretreated through a Captiva EMR-GPD purification column, interference of protein macromolecular substances and polar and non-polar small molecular substances such as pigments and sugar on a target object is effectively removed, the matrix effect caused by impurities in the mass spectrometric detection process is reduced, and the detection sensitivity and accuracy can be greatly improved. The method provided by the invention is strong in specificity, high in qualitative and quantitative detection accuracy and good in sensitivity, and has important significance for guaranteeing the safety of raw milk and feed.

Description

Technical Field

[0001] This invention relates to the field of feed and raw milk quality and safety testing technology, and more specifically, to a method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed. Background Technology

[0002] Bacillus cereus is a spore-forming, Gram-positive facultative aerobic bacterium that can grow at temperatures ranging from 5 to 45°C. It is widely distributed in nature, including soil, air, and water. Bacillus cereus is an opportunistic pathogen, causing food poisoning incidents that account for 1.4% to 12.0% of all food poisoning events globally, ranking fifth in incidence after Salmonella, Campylobacter, norovirus, and Staphylococcus aureus. Enterotoxins and cereulides produced by Bacillus cereus are the root causes of foodborne illnesses, causing diarrhea and vomiting, respectively. The hemolytic enterotoxin Hb1, the non-hemolytic enterotoxin Nhe, and the cytotoxin CytK, which cause diarrhea, are relatively unstable and easily inactivated. Cereulides are dodecapeptide cyclic toxins with strong heat stability, can tolerate extreme acidity and alkalinity, and have extremely strong resistance to pepsin and insulin proteolysis, leading to hepatocellular damage, liver failure, and ultimately death. Vomiting-induced poisoning is more common in Asia and other regions.

[0003] Fermented feed is a new type of feed made from plant-based raw materials through fermentation by beneficial microorganisms. It boasts advantages such as degrading anti-nutritional factors, optimizing nutritional composition, and being rich in active bacteria. It also improves feed digestibility and palatability, and has significant advantages in improving intestinal health and replacing antibiotics. With ongoing research, the application of fermented feed in livestock and poultry farming is expanding, demonstrating broad development potential. However, current fermented feed production processes are relatively rudimentary. If fermentation conditions are not strictly controlled, it can easily provide opportunities for the growth of pathogenic microorganisms such as Bacillus cereus. Contamination of fermented feed with Bacillus cereus can cause poisoning in livestock and poultry, posing a serious threat to the healthy development of animal husbandry. Studies have reported that mastitis and endometritis in dairy cows are related to Bacillus cereus infection. Some strains of Bacillus cereus colonize the mammary glands of dairy cows, causing mastitis. Such infections not only reduce dairy cow productivity but also directly lead to raw milk contamination. In addition, there is a risk that farmed animals may metabolize the toxins into raw milk by consuming fermented feed containing emetic toxins. Furthermore, Bacillus cereus can form biofilms in milking equipment, milk storage tanks, and transportation pipelines and remain there for a long time, increasing the safety risk of contaminating raw milk and other dairy products.

[0004] Currently, there are reports on methods for detecting emetogenic toxins in food, mainly animal testing, biological detection, and chemical methods. However, these methods are mostly semi-qualitative and semi-quantitative, with low accuracy and sensitivity, and the instruments are expensive and the pretreatment procedures are complex. However, there are few reports on highly sensitive and efficient pretreatment methods for detecting emetogenic toxins from Bacillus cereus in raw milk and fermented feed.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting emetogenic toxins produced by Bacillus cereus in raw milk or fermented feed, so as to achieve high accuracy and high sensitivity in detecting emetogenic toxins in fermented feed.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed, comprising the following steps: (1) Extract the raw milk or fermented feed to be tested with acetonitrile to obtain acetonitrile extract; centrifuge the acetonitrile extract, take the supernatant and load it onto the Captiva EMR-GPD purification column, discard the supernatant flowing through the purification column; elute the purification column with elution buffer and collect the elution buffer; filter to obtain the test solution of emetic toxin. (2) The emetogenic toxin test solution was loaded onto the chromatographic column and the test sample was detected by liquid chromatography-mass spectrometry. The chromatographic column was a C18 column; mobile phase A was a 1-3 mM ammonium acetate aqueous solution containing 0.05-0.15% (v / v) formic acid; mobile phase B was methanol; flow rate: 0.3-0.5 mL / min; gradient elution conditions for liquid chromatography were: During the 0-1.5 min period, mobile phase A decreased from 20% to 10%, while mobile phase B increased from 80% to 90%. From 1.51 to 4.0 min, mobile phase A decreased from 10% to 0%, while mobile phase B increased from 90% to 100%. From 4.01 to 6.0 min, mobile phase A was maintained at 0%, and mobile phase B was maintained at 100%. From 6.01 to 7.0 min, mobile phase A was maintained at 20% and mobile phase B was maintained at 80%.

[0008] The present invention has the following beneficial effects: This invention provides a method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed. This method can achieve high accuracy and high sensitivity in detecting emetogenic toxin in raw milk or fermented feed, and can meet the needs of rapid, accurate and quantitative detection.

[0009] Raw milk or fermented feed samples are rich in fats and proteins. Pretreatment of raw milk or fermented feed using Captiva EMR-GPD purification columns helps to effectively remove macromolecules (such as proteins and lipids), and can even eliminate interference from polar and nonpolar small molecules such as pigments and small sugars on the target analytes. This reduces the matrix effect caused by impurities during mass spectrometry detection, mainly by reducing the inhibitory effect on the target compounds, which can significantly improve detection sensitivity and accuracy. The purification process of Captiva EMR-GPD purification columns also has the advantages of simple and rapid operation.

[0010] In summary, the method provided by this invention can effectively separate the analyte from interfering components, enhance the recovery rate and detection effect of the target compound, and improve the detection sensitivity. This invention can meet the requirements of rapid and accurate qualitative and quantitative analysis of emetogenic toxins produced by Bacillus cereus in raw milk or fermented feed within a short time, satisfying the timeliness requirements of practical detection. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 A flowchart of the pretreatment method for raw milk and fermented feed samples provided by the present invention; Figure 2 The following are images showing the pretreatment effects of fermented feed samples provided by this invention: Image A shows the effect of adding homogenants to the fermented feed sample; Image B shows the effect of vortex extraction with added acetonitrile; Image C shows the effect of the supernatant after centrifugation; and Image D shows the effect after purification by the purification column. Figure 3 The following are images showing the pretreatment effect of raw milk samples provided by this invention: Image A shows the effect of adding homogeneous protons to the raw milk sample; Image B shows the effect of vortex extraction with added acetonitrile; Image C shows the effect of the supernatant after centrifugation; and Image D shows the effect after purification by the purification column. Figure 4 Quantitative standard curve and curve equation for emetic toxin using internal standard method; Figure 5 Emetogenic toxins and emetogenic toxins in the test solutions of fermented feed (left) and raw milk (right). 13 Characteristic daughter ion extracted ion chromatogram of C6; Figure 6 Structural formula of emetogenic toxin produced by Bacillus cereus; Figure 7This is a graph showing the elution effect of methanol chromatography. Figure 8 This is a chromatographic elution effect diagram of acetonitrile; Figure 9 Extracted ion chromatogram at the limit of quantification concentration of emetic toxin in raw milk; Figure 10 Extracted ion chromatogram at the detection limit concentration of emetic toxins in raw milk. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] In a first aspect, the present invention provides a method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed, comprising the following steps: (1) Extract the raw milk or fermented feed to be tested with acetonitrile to obtain acetonitrile extract; centrifuge the acetonitrile extract, take the supernatant and load it onto the Captiva EMR-GPD purification column, discard the supernatant flowing through the purification column; elute the purification column with elution buffer and collect the elution buffer; filter to obtain the test solution of emetic toxin; (2) The emetogenic toxin test solution was loaded onto the chromatographic column and detected by liquid chromatography-mass spectrometry. The chromatographic column was a C18 column; mobile phase A was a 1-3 mM ammonium acetate aqueous solution containing 0.05-0.15% (v / v) formic acid; mobile phase B was methanol; flow rate: 0.3-0.5 mL / min; gradient elution conditions for liquid chromatography were: During the 0-1.5 min period, mobile phase A decreased from 20% to 10%, while mobile phase B increased from 80% to 90%. From 1.51 to 4.0 min, mobile phase A decreased from 10% to 0%, while mobile phase B increased from 90% to 100%. From 4.01 to 6.0 min, mobile phase A was maintained at 0%, and mobile phase B was maintained at 100%. From 6.01 to 7.0 min, mobile phase A was maintained at 20% and mobile phase B was maintained at 80%.

[0015] In step (1), the Captiva EMR-GPD purification column used in this invention has the following functions and advantages: it can remove macromolecular substances (such as proteins and lipids) in raw milk or fermented feed, and can even eliminate the interference of polar and non-polar small molecules such as pigments and small molecule sugars on the target, reduce the matrix effect caused by impurities during mass spectrometry detection, mainly reduce the inhibitory effect on the target compound, and thus improve the detection sensitivity.

[0016] The purification procedure of the purification column used in this invention differs from the conventional procedure of the Captiva EMR-GPD. The conventional purification procedure involves passing the supernatant through the purification column and collecting the effluent as the test solution. However, this application employs a method of discarding the supernatant and then eluting the target analyte again after passing the effluent through the purification column, which more effectively removes matrix interference.

[0017] Mass spectrometry in liquid chromatography-mass spectrometry includes, but is not limited to: quadrupole tandem mass spectrometry, time-of-flight mass spectrometry, ion trap mass spectrometry, orbital trap mass spectrometry, etc.

[0018] For example, the Captiva EMR-GPD purification column has a specification of 595 mg and 6 mL. After all the supernatant has passed through the purification column, positive pressure can be applied to expel the residual liquid remaining on the purification column.

[0019] The acetonitrile in step (1) includes analytical grade acetonitrile and chromatographic grade acetonitrile, preferably chromatographic grade acetonitrile; In this invention, acetonitrile can be used to precipitate proteins, dissociate and extract target substances.

[0020] In a preferred embodiment of the present invention, the eluent in step (1) is methanol. Methanol is used as the eluent, and the volume of the eluent is 2-5 mL, preferably 3 mL. After the eluent has completely flowed out, positive pressure can be applied to push out the eluent remaining on the purification column.

[0021] In a preferred embodiment of the present invention, the filtration in step (1) is performed using a filter membrane.

[0022] In a preferred embodiment of the present invention, the filter membrane is selected from regenerated cellulose filter membranes (RC filter membranes) or nylon filter membranes (NYL filter membranes). Nylon filter membranes are preferred.

[0023] In a preferred embodiment of the present invention, in step (1), the amount of acetonitrile added to each 2-5 g of raw milk or fermented feed to be tested is 10-20 mL.

[0024] Fermented feeds are selected from: grain and processing by-product feeds, crop by-product feeds, green fodder feeds, unconventional feed resources, probiotic fermented functional feeds, or feeds fermented with a combination of bacteria and enzymes.

[0025] Feeds made from grains and processing by-products include, but are not limited to, feeds prepared using corn flour, wheat bran, rice bran, soybean meal, distiller's grains, vinegar residues, etc., as fermentation substrates or nutritional supplements.

[0026] Agricultural by-product feeds include, but are not limited to, feeds prepared using corn stalks, wheat stalks, rice stalks, soybean stalks, and sugarcane tops and leaves as fermentation substrates or nutritional supplements.

[0027] Green fodder includes, but is not limited to, feed prepared using whole corn plants, ryegrass, elephant grass, sweet potato vines, and peanut vines as fermentation substrates or nutritional supplements.

[0028] The raw milk is selected from cow's milk, goat's milk, mare's milk, or camel's milk.

[0029] In a preferred embodiment of the present invention, after adding acetonitrile, vortex extraction is performed under the following conditions: vortex rotation speed is 1500~2500 rpm, preferably 1800~2000 rpm; vortex oscillation time is 5~15 min, preferably 8~10 min.

[0030] In one specific implementation, the vortex rotation speed is 1500 rpm, 1800 rpm, 2000 rpm, 2200 rpm, or 2500 rpm; the vortex oscillation time is 5 min, 8 min, 10 min, or 15 min.

[0031] In a preferred embodiment of the present invention, before vortex extraction, a homogenant is added to the raw milk or fermented feed to be tested; in a preferred embodiment of the present invention, one ceramic homogenant is added to 2-5 g of raw milk or fermented feed.

[0032] The addition of homogenants can exert shear force, resulting in better uniformity during sample extraction, which is beneficial for the recovery of the compounds detected in this invention and can help improve the recovery rate of the compounds.

[0033] The centrifugation conditions in step (1) are as follows: centrifugation temperature is 4~10℃, preferably 6~8℃; centrifugation speed is 7000~10000 r / min, preferably 8000~9000 r / min; centrifugation time is 5~15 min, preferably 8~10 min.

[0034] In one specific implementation, the centrifugation temperature is 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, or 10℃; the centrifugation speed is 7000, 7500, 8000, 8500, 9000, 9500, or 10000 r / min; and the centrifugation time can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 min.

[0035] In a preferred embodiment of the present invention, the supernatant liquid extracted is passed through a column in a volume of 2 to 5 mL, preferably 3 mL.

[0036] In a preferred embodiment of the present invention, the liquid chromatography column is a Zorbax Eclipse Plus C18 column, 2.1 × 50 mm, 1.8 µm.

[0037] In a preferred embodiment of the present invention, the sample loading volume during liquid chromatography is 2~10 μL.

[0038] In a preferred embodiment of the present invention, the mass spectrometry conditions are as follows: the mass spectrometer is a quadrupole tandem mass spectrometer, and the conditions of the quadrupole tandem mass spectrometer are as follows: ion source: ESI+; drying gas flow rate: 13~17 L / min; drying gas temperature: 240~260℃; sheath gas flow rate: 10~14 L / min; sheath gas temperature: 300~330℃; nebulizer pressure: 28~32psi; capillary voltage: 2800~3100V (+); nozzle voltage: 480~520V.

[0039] In a preferred embodiment of the present invention, mass spectrometry is used to detect emetogenic toxins and emetogenic toxin- 13 The characteristic ion of C6 (emetic toxin isotope internal standard) was used to qualitatively and quantitatively determine the signal, emetogenic toxin, and emetogenic toxin- 13 A standard curve is plotted using the peak area ratio of C6 quantitative ion pairs as the ordinate and the concentration of the emetogenic toxin standard solution as the abscissa. The peak area ratio of the characteristic signal intensity ions in the raw milk or fermented feed is obtained by mass spectrometry detection. Based on the standard curve, the concentration of emetogenic toxin produced by Bacillus cereus in the raw milk or fermented feed is obtained.

[0040] Characteristic ion pairs refer to the fragmentation of the target analyte parent ion into multiple daughter ions in mass spectrometry. The daughter ions are then screened by signal intensity, and the daughter ions with high signal intensity and high recognition are selected as the parent ion paired with the daughter ion.

[0041] In a preferred embodiment of the present invention, the mass-to-charge ratio of the characteristic ion pair of the emetogenic toxin is 1170.7 > 1125.8 and 1170.7 > 357.2, where 1170.7 > 357.2 is the quantitative ion pair; emetogenic toxin - 13 The mass-to-charge ratios of characteristic C6 ion pairs are 1176.7 > 1159.8 and 1176.7 > 1130.7, with the 1176.7 > 1130.7 pair being the quantitative ion pairs. At this mass-to-charge ratio, good resolution is achieved, and a high-performance standard curve can be constructed.

[0042] The liquid chromatography-mass spectrometry method of this invention uses multiple reaction monitoring (MRM) mode to monitor fragment ions of the target compound, and performs qualitative or quantitative analysis of fragment ions of the target compound based on the compound retention time and the ratio of two pairs of characteristic parent ions > daughter ions.

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1 This embodiment provides a method for detecting emetogenic toxin produced by Bacillus cereus in fermented feed, which includes pretreatment of fermented feed samples and detection by liquid chromatography-mass spectrometry.

[0045] 1. Refer to the flowchart of the pretreatment method for fermented feed samples. Figure 1 As shown, the specific steps include the following: (1) Fermented feed (collected from dairy farms and fermented with lactic acid bacteria in a mixture of cornmeal and wet distiller's grains) was pulverized using a blade grinder; (2) Weigh 2g of the pulverized fermented feed sample into a polytetrafluoroethylene centrifuge tube, add 200 μL of emetogenic toxin (standard purchased from Tianjin Alta Technology Co., Ltd.) standard solution (100 ng / mL) and 200 μL of emetogenic toxin- 13 C6 (standard purchased from Tianjin Alta Technology Co., Ltd.) standard solution (50 ng / mL); (3) Add 1 ceramic homogenant and 10 mL of acetonitrile, and vortex extract at a rotation speed of 2000 rpm for 10 min to obtain a mixed extract; add homogenant to fermented feed sample as a reference. Figure 2 As shown in Figure A, the effect of vortex extraction after adding acetonitrile is illustrated in the figure below. Figure 2 As shown in B; (4) The above mixed extract was centrifuged at low temperature to obtain the supernatant. The centrifugation temperature was 6℃; the centrifugation speed was 8000 r / min; and the centrifugation time was 10 min. The supernatant after centrifugation was taken as a reference. Figure 2 As shown in C; (5) Pass 3 mL of the supernatant through a Captiva EMR-GPD purification column (595 mg, 6 mL). After all the supernatant has passed through the purification column, positive pressure can be applied to push out the residual liquid remaining on the purification column. (6) Elute the purification column with 3 mL of methanol as the eluent and collect the eluent; after the eluent has completely flowed out, positive pressure can be applied to push out the eluent remaining on the purification column; refer to the effect diagram of the eluent after purification by the purification column. Figure 2 As shown in D; (7) The eluent is filtered through a nylon filter membrane to obtain the sample solution to be tested.

[0046] 2. Liquid chromatography-mass spectrometry (LC-MS) detection steps: The conditions for liquid chromatography are: The chromatographic column was a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm). Mobile phase A is a 2 mM ammonium acetate aqueous solution containing 0.1% (volume concentration) formic acid, and mobile phase B is methanol; Flow rate: 0.3 mL / min; Column temperature: 40℃±1℃; Injection volume: 2 μL.

[0047] The gradient elution conditions for liquid chromatography are: During the 0-1.5 min period, mobile phase A decreased from 20% to 10%, while mobile phase B increased from 80% to 90%. From 1.51 to 4.0 min, mobile phase A decreased from 10% to 0%, while mobile phase B increased from 90% to 100%. From 4.01 to 6.0 min, mobile phase A was maintained at 0%, and mobile phase B was maintained at 100%. From 6.01 to 7.0 min, mobile phase A was maintained at 20%, and mobile phase B was maintained at 80%. The mass spectrometer used was an Agilent 6495 quadrupole tandem mass spectrometer. The conditions for quadrupole tandem mass spectrometry were as follows: ion source: ESI+; drying gas flow rate: 15 L / min; drying gas temperature: 250℃; sheath gas flow rate: 12 L / min; sheath gas temperature: 320℃; nebulizer pressure: 30 psi; capillary voltage: 3000V (+); nozzle voltage: 500V.

[0048] Fragment ions of the target compound were monitored using multiple reaction monitoring (MRM) mode, with Bacillus cereus emetogenic toxin and emetogenic toxin- 13 Qualitative or quantitative analysis was performed on the retention time and peak area of ​​the C6 characteristic precursor ion > daughter ion pair. Specifically: The two daughter ions of the emetogenic toxin, m / z=357.2 and m / z=1125.8, were detected by mass spectrometry, along with the emetogenic toxin- 13 The two daughter ions of C6 have m / z values ​​of 1159.8 and 1130.7. A standard curve was plotted with the peak area ratio of m / z = 357.2 and 1130.7 as the ordinate and the concentration of emetogenic toxin in the emetogenic toxin standard solution as the abscissa. The peak area ratio of m / z = 357.2 and 1130.7 in the fermented feed was obtained by mass spectrometry. Based on the standard curve, the concentration of emetogenic toxin produced by Bacillus cereus in the fermented feed was obtained.

[0049] The names of the target compounds in the fermented feed, their chromatographic retention times, and qualitative and quantitative ion information obtained by mass spectrometry are shown in Table 1.

[0050] Table 1. Chromatographic retention time and mass spectrometry qualitative and quantitative ion information of emetogenic toxins.

[0051] Note: Ions marked with "*" are quantitative ions.

[0052] Figure 5 The left and middle images show the emetic toxins and emetic toxins in the test solution of the fermented feed. 13 Extracted ion chromatogram of C6 daughter ions.

[0053] Example 2 This embodiment provides a method for detecting emetogenic toxin produced by Bacillus cereus in raw milk, which includes sample pretreatment and detection by liquid chromatography-mass spectrometry.

[0054] 1. Refer to the flowchart of the pretreatment method for raw milk samples. Figure 1 As shown, the specific steps include the following: (1) The raw milk (milk collected from the milk storage tanks of dairy farms) is mixed evenly; (2) Weigh 5g of raw milk sample into a polytetrafluoroethylene centrifuge tube, add 200 μL of emetogenic toxin (standard purchased from Tianjin Alta Technology Co., Ltd.) standard solution (100 ng / mL) and 100 μL of emetogenic toxin- 13 C6 (standard purchased from Tianjin Alta Technology Co., Ltd.) standard solution (200 ng / mL); (3) Add 1 ceramic homogenizer and 15 mL of acetonitrile, vortex extract at 2000 rpm for 5 min to obtain a mixed extract; add homogenizer to the raw milk sample as a reference. Figure 3 As shown in Figure A, the effect of vortex extraction after adding acetonitrile is illustrated in the figure below. Figure 3 As shown in B; (4) The above mixed extract was centrifuged at low temperature to obtain the supernatant. The centrifugation temperature was 5℃; the centrifugation speed was 9000 r / min; and the centrifugation time was 5 min. The supernatant after centrifugation was taken as a reference. Figure 3 As shown in C; (5) Pass 3 mL of the supernatant through a Captiva EMR-GPD purification column (595 mg, 6 mL). After all the supernatant has passed through the purification column, positive pressure can be applied to push out the residual liquid remaining on the purification column. (6) Elute the purification column with 3 mL of methanol as the eluent and collect the eluent; after the eluent has completely flowed out, positive pressure can be applied to push out the eluent remaining on the purification column; refer to the effect diagram of the eluent after purification by the purification column. Figure 3 As shown in D; (7) The eluent is filtered through a nylon filter membrane to obtain the sample solution to be tested.

[0055] 2. Liquid chromatography-mass spectrometry (LC-MS) detection steps: The conditions for liquid chromatography are: The chromatographic column was a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm). Mobile phase A is a 1 mM ammonium acetate aqueous solution containing 0.1% (volume concentration) formic acid, and mobile phase B is methanol; Flow rate: 0.3 mL / min; Column temperature: 40℃±1℃; Injection volume: 2 μL.

[0056] The gradient elution conditions for liquid chromatography are: During the 0-1.5 min period, mobile phase A decreased from 20% to 10%, while mobile phase B increased from 80% to 90%. From 1.51 to 4.0 min, mobile phase A decreased from 10% to 0%, while mobile phase B increased from 90% to 100%. From 4.01 to 6.0 min, mobile phase A was maintained at 0%, and mobile phase B was maintained at 100%. From 6.01 to 7.0 min, mobile phase A was maintained at 20%, and mobile phase B was maintained at 80%. The mass spectrometer used was an Agilent 6495 quadrupole tandem mass spectrometer. The conditions for quadrupole tandem mass spectrometry were as follows: ion source: ESI+; drying gas flow rate: 15 L / min; drying gas temperature: 250℃; sheath gas flow rate: 12 L / min; sheath gas temperature: 320℃; nebulizer pressure: 30 psi; capillary voltage: 3000V (+); nozzle voltage: 500V.

[0057] Fragment ions of the target compound were monitored using multiple reaction monitoring (MRM) mode, with Bacillus cereus emetogenic toxin and emetogenic toxin- 13 Qualitative or quantitative analysis was performed on the retention time and peak area of ​​the C6 characteristic precursor ion > daughter ion pair. Specifically: The two daughter ions of the emetogenic toxin, m / z=357.2 and m / z=1125.8, were detected by mass spectrometry, along with the emetogenic toxin- 13The two daughter ions of C6 have m / z values ​​of 1159.8 and 1130.7. A standard curve was plotted with the peak area ratio of m / z = 357.2 and 1130.7 as the ordinate and the concentration of emetogenic toxin in the emetogenic toxin standard solution as the abscissa. The peak area ratio of m / z = 357.2 and 1130.7 in the raw milk was obtained by mass spectrometry. Based on the standard curve, the concentration of emetogenic toxin produced by Bacillus cereus in the raw milk was obtained.

[0058] The names of the target compounds in the raw milk, their chromatographic retention times, and qualitative and quantitative ion information obtained by mass spectrometry are shown in Table 1.

[0059] Table 1. Chromatographic retention time and mass spectrometry qualitative and quantitative ion information of emetogenic toxins.

[0060] Note: Ions marked with "*" are quantitative ions.

[0061] Figure 5 The right-middle image shows the emetic toxins and emetic toxin- in the raw milk test solution. 13 Extracted ion chromatogram of C6 daughter ions.

[0062] Experimental Example 1 This experimental example optimized the chromatographic separation method for emetogenic toxin produced by Bacillus cereus. Emetogenic toxin produced by Bacillus cereus is a cyclic polypeptide composed of 12 amino acid units. The molecule contains several basic amino acids (such as arginine and lysine), resulting in an overall basic pH. Its molecular weight is 1153.42, and its structural formula is as follows: Figure 6As shown in the figure. This experimental example compared the retention performance of different chromatographic columns for emetogenic toxins and further optimized the flow composition and elution conditions. By comparing three chromatographic columns—C18 (Zorbarx Eclipse Plus C18, 2.1×50 mm, 1.8 µm), pentafluorophenyl (Poroshell 120 PFP, 100×3.0 mm, 2.7 µm), and phenylhexyl (ZORBAX RRHD EP95, 100×3.0 mm, 2.7 µm)—the results showed that while the pentafluorophenyl and phenylhexyl columns could retain the target analytes, the retention times were long and the peak shapes were broad. The ZORBAX Eclipse Plus C18, a rapid separation column, is resistant to high column pressures. Its packing material, a monolayer of densely bonded porous silica gel with dimethyl-N-octadecylsilane bonding, reduces or eliminates the strong adsorption of basic and highly polar compounds. Simultaneously, secondary end-capping minimizes the activity of the silica surface, significantly improving the retention of basic compounds with poor peak shapes. The Zorbarx Eclipse Plus C18 column achieves good retention and sharp peak shape for emetogenic toxins. Furthermore, this column is tolerant of pH values ​​from 2 to 9, making it suitable for most commonly used mobile phases. This experimental example further compares the elution effects of methanol and acetonitrile on the target analyte. When methanol is used as mobile phase B, the target analyte peak is sharp and symmetrical, with the elution time located in the middle of the elution program. When acetonitrile is used as mobile phase B, the target analyte elutes relatively later, exhibits tailing, and has a wider peak shape. The elution effects of methanol and acetonitrile are comparable. Figure 7 and Figure 8 As shown.

[0063] Experimental Example 2 This experiment optimized the extraction solvent and purification materials for emetogenic toxin produced by Bacillus cereus. Emetogenic toxin is a moderately polar, weakly basic compound, readily soluble in organic solvents such as methanol and acetonitrile. This experiment compared methanol, acetonitrile, 1% formic acid-acetonitrile, and 1% formic acid-methanol as extraction solvents. Emetogenic toxin (at a concentration of 10 μg / kg) was added to fermented feed and raw milk, and the extraction efficiency of different solvents was compared by comparing the target analyte detection intensity. The results showed that formic acid-methanol and methanol extracts were darker in color, and the raw milk sample extract was turbid, which was unfavorable for subsequent solid-phase extraction purification and resulted in lower response intensity. Formic acid-acetonitrile and acetonitrile had the highest extraction efficiency, and the raw milk sample extract was clear. This is because acetonitrile has a stronger ability to denature proteins, causing protein denaturation and precipitation, while its extraction ability for non-polar compounds such as pigments is relatively weak, thus improving the target analyte extraction efficiency and effectively reducing matrix effect interference. After protein precipitation, the main impurities in the sample extract were lipids and pigments. To further purify the sample matrix and reduce matrix effects, this study first investigated the purification and enrichment effect of a Bond Elut HLB solid-phase extraction column (6 mL, 500 mg) on ​​the target analytes. It was found that the HLB solid-phase extraction column effectively adsorbed the target analytes, but methanol was difficult to elute them. Next, the purification effect of a through-feed purification column, Captiva EMR GPD (595 mg, 6 mL), on the target analytes was investigated. The results showed that the target analytes were not detected in the sample receiving solution, indicating that they were adsorbed on the purification column and could not be dissolved and eluted by acetonitrile in the extract. Further, methanol elution of the purification column revealed the detection of the target analytes in the eluent, with external standard recoveries exceeding 70% and internal standard recoveries exceeding 90%. Therefore, Captiva EMR GPD was ultimately selected as the purification column.

[0064] Experimental Example 3 This experimental example validates the linear range, accuracy, precision, and sensitivity of the method.

[0065] 1. Linear range test Precisely measure the emetogenic toxin and its isotopic internal standard emetogenic toxin. 13 C6 was prepared with methanol to form a series of standard working solutions with external standard concentrations of 0.2 μg / L, 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, and 50.0 μg / L, and an internal standard concentration of 1.0 μg / L for each solution. These solutions were then analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) (the HPLC-MS / MS detection conditions were the same as in Example 1). The results showed good linearity within the above concentration range, and the linear correlation coefficient R was [value missing]. 2 >0.999. See the internal standard curve and curve equation. Figure 4 .

[0066] 2. Sensitivity Test The limits of detection (LOD) and quantitation (LOQ) for emetic toxins in fermented feed using this method are 1.0 μg / kg and 5.0 μg / kg, respectively. The LOD and LOQ for emetic toxins in raw milk are also 1.0 μg / kg and 5.0 μg / kg, respectively.

[0067] To test the signal-to-noise ratio at the aforementioned limits of detection and quantitation, this invention selected blank fermented feed (collected from a dairy farm, prepared by fermenting a mixture of cornmeal and wet distiller's grains with lactic acid bacteria) and raw milk (milk collected from milk storage tanks at a dairy farm) as test subjects. 1.0 μg / kg (limit of detection) and 5.0 μg / kg (limit of quantitation) of emetogenic toxin standard were added to the blank fermented feed, respectively. Similarly, 1.0 μg / kg (limit of detection) and 5.0 μg / kg (limit of quantitation) of emetogenic toxin standard were added to the raw milk sample, respectively. Detection was performed using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) according to the method of Example 1.

[0068] The results showed that the signal-to-noise ratios of each matrix were greater than 3 and 10, respectively. Figure 10 The detection limit for raw milk ( Figure 10 The ion chromatogram of emetogenic toxin extracted when the emetogenic toxin content was 1.0 μg / kg. Figure 9 The limit of quantification for raw milk ( Figure 9 The ion chromatogram of emetogenic toxin at a concentration of 5.0 μg / kg.

[0069] 3. Accuracy and precision testing Fermented feed and raw milk samples were selected as test subjects. Three different concentrations (5 μg / kg, 10 μg / kg, and 50 μg / kg) were added to each sample matrix at the limit of quantitation (LOQ), 2 times the LQ, and 10 times the LQ, respectively, and the recovery rate was tested using both internal and external standard methods. Five parallel experiments were performed for the same concentration within each batch. The results showed that the recovery rate of the external standard method was >70% at low, medium, and high spiking levels, and the recovery rate of the internal standard method was >90%, with relative standard deviations <5.0% for both. Therefore, the detection method provided by this invention has extremely high accuracy and precision. The accuracy and precision test results are shown in Table 2.

[0070] Table 2. Results of Accuracy and Precision Tests

[0071] In summary, this invention, through pretreatment using the Captiva EMR-GPD purification column, effectively removes large molecules such as proteins and lipids, eliminates interference from small molecules such as pigments and sugars, and improves detection sensitivity. Furthermore, this invention can rapidly and accurately complete the qualitative and quantitative analysis of Bacillus cereus emetogenic toxins within a short time. This method is highly specific, with high accuracy and sensitivity in both qualitative and quantitative detection, and is of great significance for ensuring the safety of raw milk and feed.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed, characterized in that, It includes the following steps: (1) Extract the raw milk or fermented feed to be tested with acetonitrile to obtain acetonitrile extract; centrifuge the acetonitrile extract, take the supernatant and load it onto the Captiva EMR-GPD purification column, discard the effluent of the supernatant flowing through the purification column; elute the purification column with elution buffer and collect the elution buffer; filter to obtain the test solution of emetic toxin; (2) The test solution containing the emetogenic toxin was loaded onto a chromatographic column and detected by liquid chromatography-mass spectrometry. The chromatographic column is a C18 column; mobile phase A is a 1-3 mM ammonium acetate aqueous solution containing 0.05-0.15% (v / v) formic acid; mobile phase B is methanol; flow rate: 0.3-0.5 mL / min; the gradient elution conditions for the liquid chromatography are: During the 0-1.5 min period, mobile phase A decreased from 20% to 10%, while mobile phase B increased from 80% to 90%. From 1.51 to 4.0 min, mobile phase A decreased from 10% to 0%, while mobile phase B increased from 90% to 100%. From 4.01 to 6.0 min, mobile phase A was maintained at 0%, and mobile phase B was maintained at 100%. From 6.01 to 7.0 min, mobile phase A was maintained at 20% and mobile phase B was maintained at 80%.

2. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 1, characterized in that, The eluent in step (1) is methanol.

3. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 1, characterized in that, The filtration in step (1) is performed using a filter membrane; Preferably, the filter membrane is selected from regenerated cellulose filter membrane or nylon filter membrane.

4. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 3, characterized in that, In step (1), the amount of acetonitrile added to each 2-5 g of raw milk or fermented feed to be tested is 10-20 mL; Preferably, the fermented feed is selected from: grain and processing by-product feed, crop by-product feed, green fodder feed, unconventional feed resource feed, probiotic fermented functional feed or bacteria-enzyme combined fermented feed; Preferably, the raw milk is selected from cow's milk, goat's milk, mare's milk, or camel's milk.

5. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 4, characterized in that, After adding acetonitrile, vortex extraction is performed under the following conditions: vortex speed of 1500~2500 rpm, preferably 1800~2000 rpm; vortex oscillation time of 5~15 min, preferably 8~10 min.

6. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 5, characterized in that, Before vortex extraction, add homogenants to the raw milk or fermented feed to be tested; preferably, add 1 ceramic homogenant to 2-5 g of raw milk or fermented feed. The centrifugation conditions in step (1) are as follows: centrifugation temperature is 4~10℃, preferably 6~8℃; centrifugation speed is 7000~10000r / min, preferably 8000~9000r / min; centrifugation time is 5~15 min, preferably 8~10 min.

7. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 1, characterized in that, The liquid chromatography column was a Zorbax Eclipse Plus C18 column, 2.1 × 50 mm, 1.8 µm.

8. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 1, characterized in that, The sample loading volume for liquid chromatography is 2~10 μL.

9. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 1, characterized in that, The conditions for the mass spectrometry are as follows: The mass spectrometry is a quadrupole tandem mass spectrometer, and the conditions for the quadrupole tandem mass spectrometer are as follows: Ion source: ESI+; Drying gas flow rate: 13~17 L / min; Drying gas temperature: 240~260℃; Sheath gas flow rate: 10~14 L / min; Sheath gas temperature: 300~330℃; Nebulizer pressure: 28~32psi; Capillary voltage: 2800~3100V (+); Nozzle voltage: 480~520V.

10. The method for detecting emetogenic toxin produced by Bacillus cereus in raw milk or fermented feed according to claim 9, characterized in that, The characteristic ions of emetogenic toxin and emetogenic toxin isotope internal standard were detected by mass spectrometry to perform qualitative and quantitative analysis of the signals. The peak area ratio of the quantitative ions of emetogenic toxin and emetogenic toxin isotope internal standard was used as the ordinate, and the concentration of the emetogenic toxin standard solution was used as the abscissa to plot a standard curve. The peak area ratio of the characteristic signal intensity ions of the raw milk or fermented feed was obtained by mass spectrometry detection. Based on the standard curve, the concentration of emetogenic toxin produced by Bacillus cereus in the raw milk or fermented feed was obtained. Preferably, the isotopic internal standard is an emetogenic toxin. 13 C6; the mass-to-charge ratio of the characteristic ion pair of the emetogenic toxin is 1170.7 > 1125.8 and 1170.7 > 357.2, where 1170.7 > 357.2 is a quantitative ion pair; the emetogenic toxin - 13 The mass-to-charge ratios of the characteristic C6 ion pairs are 1176.7 > 1159.8 and 1176.7 > 1130.7, with 1176.7 > 1130.7 being the quantitative ion pairs.

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