Method for identifying filter residues doped with penicillin in nucleotide residues

Abstract

The invention relates to the technical field of feed quality safety detection, and provides a method for identifying penicillin-doped filter residues in nucleotide residues, and the method comprises the following steps: carrying out crushing pretreatment on a nucleotide residue sample; carrying out ion mobility spectrometry detection on the crushed and pretreated nucleotide residue sample; according to the method, an ion mobility spectrometry detection result is analyzed, and whether penicillin filter residues are doped into the nucleotide residues or not is judged by identifying whether preset characteristic volatile compounds exist in the detection result or not, so that rapid doping identification of the penicillin filter residues in the nucleotide residues of the protein feed raw material is realized; the method has the advantages of no pretreatment process, no need of chemical reagents, short detection time, high sensitivity and accurate result, and can quickly and effectively meet the detection purpose of identifying the penicillin-doped filter residues in the nucleotide residues.

Description

Technical Field

[0001] The invention belongs to the technical field of feed quality safety detection, and in particular relates to a method for identifying nucleotide residues mixed with penicillin filter residues. Background Art

[0002] Nucleotide residue is the solid residue remaining after the production of disodium 5'-inosinate and disodium 5'-guanylate by Corynebacterium glutamicum using a culture medium composed of plant-derived ingredients such as sucrose, molasses, starch, or its hydrolyzate, and ammonium salts (or other minerals). It is high in amino acids and crude protein, making it a novel single-cell protein feed ingredient widely used in livestock and poultry farming. Penicillin residue is the solid waste remaining after microbial fermentation to produce penicillin. It is high in crude protein and other ingredients. The physical properties and general chemical composition of penicillin residue are similar to those of nucleotide residue. There have been media reports that penicillin residue is being used as a substitute for nucleotide residue by unscrupulous farmers. Penicillin residue contains trace amounts of penicillin and its metabolites, which have not been evaluated for safety. Direct use of penicillin residue in feed can not only lead to antibiotic resistance in livestock and poultry, but can also cause antibiotics to accumulate in the animals and be transferred through the food chain, ultimately affecting human health. my country explicitly prohibits the addition of penicillin residue to feed.

[0003] Currently, existing detection technologies, both domestically and internationally, primarily focus on detecting parent penicillin. While these technologies can accurately measure penicillin content in protein feed ingredients, they are unable to identify the cause of the excess content—in other words, whether it is due to the addition of parent penicillin or the addition of penicillin residue. This makes it difficult to determine the cause during supervision and law enforcement. Furthermore, traditional wet chemical detection methods require complex pretreatment, are time-consuming, costly, and environmentally unfriendly. Therefore, agricultural administrative departments are in need of identification technology in their efforts to regulate penicillin residue in feed. Summary of the Invention

[0004] In response to the defects in the existing technology, the present invention provides a method for identifying penicillin filter residues added to nucleotide residues, aiming to solve the problems that the detection of penicillin filter residues provided by the existing technology mainly focuses on detecting penicillin prototype drugs, cannot identify the causes of drug exceeding the standard, and has long detection time, high costs and is environmentally unfriendly.

[0005] The technical solution provided by the present invention is: a method for identifying nucleotide residues mixed with penicillin filter residues, the method comprising the following steps: (1) Pulverize and pretreat the nucleotide residue sample; (2) Ion mobility spectrometry was performed on the nucleotide residue samples after crushing pretreatment; (3) Analyze the ion mobility spectrometry test results and determine whether the nucleotide residue is adulterated with penicillin residue by identifying whether the preset characteristic volatile compounds are present in the test results.

[0006] As an improved scheme, in step (3), the preset characteristic volatile compounds include the following: benzaldehyde, 2-methyl-1-pentanol, tetrahydrothiophene, 1-penten-3-ol, 2-methylpropanol, cis-2-penten-1-ol, (E)-2-hexen-1-ol, n-hexanol, propyl acetate, 3-methyl-3-buten-1-ol, trans-2-octenal, butyl lactate, 2-methylpentanal, pentanol, 2-methylpentanoic acid, propionic acid, ethylene glycol dimethyl ether, 2-methylpropanol, isopentanol, and 3-methylbutanal.

[0007] As an improved solution, the step of crushing the nucleotide residue sample for pretreatment specifically includes the following steps: The nucleotide residue mixed with penicillin filter residue is crushed by a cyclone mill, wherein the particle size after crushing reaches 40 meshes.

[0008] As an improved solution, the ion mobility spectrometry detection performed on the nucleotide residue sample after crushing pretreatment is headspace gas phase ion mobility spectrometry detection.

[0009] As an improved solution, the specific steps for implementing the headspace operation are as follows: Accurately weigh 2.0 g of sample and place it in a 20 ml headspace glass injection bottle; Incubate at 80 °C for 20 min in an autosampler (speed 500 r·min-1); The headspace injection needle temperature was 85°C, without splitting, and the injection volume was 500 μL.

[0010] As an improved solution, the gas phase conditions for headspace gas ion mobility spectrometry detection are: FS-SE-54-CB-1 column, column temperature 60 °C, carrier gas nitrogen; Carrier gas flow rate program: initial flow rate 2 mL·min-1, linearly increased to 10 mL·min-1 within 2 min, linearly increased to 100 mL·min-1 within 8 min, linearly increased to 150 mL·min-1 within 10 min, and running time 10 min.

[0011] As an improved solution, the ion mobility spectrometry conditions for headspace gas ion mobility spectrometry detection are: The drift tube length was 9.8 cm; the linear voltage in the tube was 400 V·cm-1; the drift tube temperature was 45°C; the drift gas was high-purity nitrogen with a drift gas flow rate of 150.0 mL·min-1; and the detector temperature was 45°C.

[0012] In an embodiment of the present invention, a nucleotide residue sample is subjected to crushing pretreatment; the crushed pretreated nucleotide residue sample is subjected to ion mobility spectrometry detection; the ion mobility spectrometry detection results are analyzed, and by identifying whether a preset characteristic volatile compound is present in the detection result, it is determined whether the nucleotide residue is adulterated with penicillin filter residue, thereby achieving rapid identification of penicillin filter residue in nucleotide residue of protein feed raw material. In addition, the present invention uses a small amount of identification sample, does not require a pretreatment process, does not require chemical reagents, has a short detection time, high sensitivity, and accurate results, and can quickly and effectively meet the detection purpose of identifying penicillin filter residue adulterated in nucleotide residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0014] Figure 1 These are the fingerprints of volatile compounds of nucleotide residue (A), penicillin filter (B), and nucleotide residue mixture (C, mixed with 10% penicillin filter residue). DETAILED DESCRIPTION

[0015] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0016] The method for identifying nucleotide residues mixed with penicillin filter residues provided by the present invention comprises the following steps: (1) Pulverize and pretreat the nucleotide residue sample; (2) Ion mobility spectrometry was performed on the nucleotide residue samples after crushing pretreatment; (3) Analyze the ion mobility spectrometry test results and determine whether the nucleotide residue is adulterated with penicillin residue by identifying whether the preset characteristic volatile compounds are present in the test results.

[0017] In this step, by identifying volatile compounds, there are characteristic volatile compounds, e.g. Figure 1 The results showed that penicillin residue markers (characteristic volatile compounds) were detected in the nucleotide residue, indicating that penicillin residue was present.

[0018] The preset characteristic volatile compounds include the following: benzaldehyde, 2-methyl-1-pentanol, tetrahydrothiophene, 1-penten-3-ol, 2-methylpropanol, cis-2-penten-1-ol, (E)-2-hexen-1-ol, n-hexanol, propyl acetate, 3-methyl-3-buten-1-ol, trans-2-octenal, butyl lactate, 2-methylvaleraldehyde, pentanol, 2-methylvaleric acid, propionic acid, ethylene glycol dimethyl ether, 2-methylpropanol, isopentanol, and 3-methylbutanal. The information of the characteristic volatile compounds is as follows: In this step, the characteristic volatile compounds are used as qualitative identification markers for penicillin filter residues. Detection of the qualitative identification markers in nucleotide residues indicates that the filter residues contain penicillin.

[0019] In an embodiment of the present invention, the above step (1) is specifically implemented as follows: The nucleotide residue mixed with penicillin filter residue (10%, m / m) was crushed by a cyclone mill, wherein the particle size after crushing reached 40 mesh.

[0020] As a specific embodiment of the present invention, the ion mobility spectrometry detection performed on the nucleotide residue sample after crushing pretreatment is a headspace gas phase ion mobility spectrometry detection, and its specific implementation conditions are: (1) Headspace conditions: ① Accurately weigh 2.0 g of sample and place it in a 20 ml headspace glass injection bottle; ② Incubate in an autosampler at 80°C for 20 min (speed 500 r·min-1); ③The headspace injection needle temperature was 85°C, without split flow, and the injection volume was 500 μL.

[0021] (2) Gas phase conditions: ①FS-SE-54-CB-1 column (1500 mm × 0.53 mm), column temperature 60°C, carrier gas nitrogen (purity >99.999%); ②Carrier gas flow rate program: initial flow rate 2 mL·min-1, linearly increased to 10 mL·min-1 within 2 minutes, linearly increased to 100 mL·min-1 within 8 minutes, linearly increased to 150 mL·min-1 within 10 minutes, and running time 10 minutes.

[0022] (3) Ion mobility spectrometry conditions: ①Drift tube length 9.8 cm; ② Linear voltage in the tube: 400 V·cm-1; ③ The temperature of the drift tube is 45°C, the drift gas is high-purity nitrogen (purity >99.999%), and the drift gas flow rate is 150.0 mL min-1; ④Detector temperature 45℃.

[0023] The present invention uses characteristic volatile compounds as markers for penicillin filter residue. After pre-treatment, the sample is tested using headspace gas ion mobility spectrometry. By comparing the type information of the volatile compounds, the presence of 20 markers in the nucleotide residue is determined, thereby identifying the presence of penicillin filter residue. The present invention uses a small amount of sample, requires no pre-treatment process, and does not require chemical reagents. The detection time is short, the sensitivity is high, and the results are accurate, meeting the purpose of detecting penicillin filter residue spiked in nucleotide residue.

[0024] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for identifying nucleotide residues mixed with penicillin residues, characterized in that: The method comprises the following steps: (1) Pulverize and pretreat the nucleotide residue sample; (2) Ion mobility spectrometry was performed on the nucleotide residue samples after crushing pretreatment; (3) Analyze the ion mobility spectrometry test results and determine whether the nucleotide residue is adulterated with penicillin residue by identifying whether the preset characteristic volatile compounds are present in the test results.

2. The method for identifying nucleotide residues mixed with penicillin residues according to claim 1, characterized in that: In step (3), the preset characteristic volatile compounds include the following: benzaldehyde, 2-methyl-1-pentanol, tetrahydrothiophene, 1-penten-3-ol, 2-methylpropanol, cis-2-penten-1-ol, (E)-2-hexen-1-ol, n-hexanol, propyl acetate, 3-methyl-3-buten-1-ol, trans-2-octenal, butyl lactate, 2-methylpentanal, pentanol, 2-methylpentanoic acid, propionic acid, ethylene glycol dimethyl ether, 2-methylpropanol, isopentanol, and 3-methylbutanal.

3. The method for identifying nucleotide residues mixed with penicillin residues according to claim 1, characterized in that: The step of crushing and pre-treating the nucleotide residue sample specifically comprises the following steps: The nucleotide residue mixed with penicillin filter residue is crushed by a cyclone mill, wherein the particle size after crushing reaches 40 meshes.

4. The method for identifying nucleotide residues mixed with penicillin residues according to claim 1, characterized in that: The ion mobility spectrometry detection performed on the nucleotide residue sample after crushing pretreatment is a headspace gas phase ion mobility spectrometry detection.

5. The method for identifying nucleotide residues mixed with penicillin residues according to claim 4, characterized in that: The specific steps for implementing headspace operation are: Accurately weigh 2.0 g of sample and place it in a 20 ml headspace glass injection bottle; Incubate at 80 °C for 20 min in an automatic sampler (speed 500 r·min-1); The headspace injection needle temperature was 85°C, without splitting, and the injection volume was 500 μL.

6. The method for identifying nucleotide residues mixed with penicillin residues according to claim 4, characterized in that: The gas phase conditions for headspace gas ion mobility spectrometry detection are: FS-SE-54-CB-1 column, column temperature 60 °C, carrier gas nitrogen; Carrier gas flow rate program: initial flow rate 2 mL·min-1, linearly increased to 10 mL·min-1 within 2 min, linearly increased to 100 mL·min-1 within 8 min, linearly increased to 150 mL·min-1 within 10 min, and running time 10 min.

7. The method for identifying nucleotide residues mixed with penicillin residues according to claim 4, characterized in that: The ion mobility spectrometry conditions for headspace gas ion mobility spectrometry detection are: The drift tube length was 9.8 cm; the linear voltage in the tube was 400 V·cm-1; the temperature of the drift tube was 45°C; the drift gas was high-purity nitrogen with a drift gas flow rate of 150.0 mL·min-1; and the detector temperature was 45°C.

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