A method for extracting and purifying nitrosamines in food

Abstract

The invention discloses a method for extracting and purifying nitrosamines in food, comprising the following steps: adding 10-15% of an extractant, fully dispersing by ultrasonic, then washing with water, filtering, and obtaining a filtrate; extracting and extracting the filtrate using an extractor, and ending the extraction for standby use; finally subjecting the product of step three to gel purification treatment. The present invention is to mix and disperse the raw material to be extracted with water uniformly, so that the extractant and the meat raw material are fully mixed, and the chitosan and sodium alginate in the extractant are matched, so that the ammonium acetate solution, acetonitrile and nitrosamine are dissolved and extracted, and the nitrosamines in the food are further extracted by the extractor, so as to improve the extraction efficiency of the nitrosamines, and the gel purification treatment is carried out by a gel permeation chromatography column, and the extraction efficiency of the nitrosamines is further improved, and the extracted nitrosamines are purified at the same time, so as to improve the extraction and purification effect of the nitrosamines.

Description

Technical Field

[0001] The invention relates to the technical field of nitrosamine detection, and in particular to a method for extracting and purifying nitrosamines in food. Background Art

[0002] Nitrosamines are compounds in which the nitrogen atom of nitroso-NO is connected to the nitrogen atom of amino group. They are strong carcinogens, one of the most important chemical carcinogens, and one of the four major food contaminants. Nitrosamines are found in food, cosmetics, beer, and cigarettes. Smoked foods contain a large amount of nitrosamines. The incidence of certain digestive system tumors, such as esophageal cancer, is related to the amount of nitrosamines ingested in the diet. When smoked foods are taken together with alcohol, the harm of nitrosamines to human health will increase exponentially. Aromatic nitrosamines are more important. They are generated by treating diazonium salts with excessive alkali and are stable in alkaline media.

[0003] Ham, bacon, sausage and other types of pickled foods are very popular foods among Chinese people. Due to the raw materials and production process, these foods are prone to produce nitrosamine carcinogens, which seriously affect people's health. Therefore, the detection of nitrosamines in food has always been a key detection project in the field of food safety testing. According to the current national food safety standard GB5009.26 method, it is extracted by steam distillation and then concentrated by liquid-liquid extraction. The process is very cumbersome and it takes 3 to 5 hours to prepare a sample. In addition, the purification process requires a large amount of dichloromethane, a drug-making reagent that is strictly controlled by the state, which brings great regulatory and environmental pressure to users, resulting in time-consuming, troublesome and laborious nitrosamine detection.

[0004] Based on this, the present invention provides a method for extracting and purifying nitrosamines in food. Summary of the invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a method for extracting and purifying nitrosamines in food to solve the problems raised in the above background technology.

[0006] The present invention solves the technical problem by adopting the following technical solution:

[0007] The present invention provides a method for extracting and purifying nitrosamines in food, comprising the following steps:

[0008] Step 1: Crush the food meat, then add 3-6 times of water and stir to mix;

[0009] Step 2: Add 10-15% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash with water and filter to obtain a filtrate;

[0010] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0011] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0012] Preferably, the power of the ultrasonic dispersion is 350-400 W, and the ultrasonic time is 20-30 min.

[0013] Preferably, the preparation method of the extractant is:

[0014] S01: adding 5-10 parts of chitosan to 10-15 parts of sodium alginate solution, and then adding 1-5 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0015] S02: Add 5-10% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0016] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0017] The inventors of the present invention found that the extraction rate of nitrosamines was significantly reduced when no extractant was added. At the same time, when anhydrous magnesium sulfate was not added to the extractant, the second extractant was not added to the extractant, and ammonium acetate solution was not added to the second extractant, the extraction rate of the product showed a trend of deterioration. The extractant prepared by the method of the present invention can significantly improve the extraction efficiency of nitrosamines. Although other methods can improve the extraction rate, the improvement effect is not as significant as that of the method of the present invention.

[0018] Preferably, the mass fraction of the sodium alginate solution is 10-15%.

[0019] Preferably, the mass fraction of the ammonium acetate solution is 3-5%.

[0020] Preferably, the temperature of the S03 stirring reaction is 55-65°C, the stirring time is 10-20min, and the stirring speed is 1000-1500r / min.

[0021] Preferably, the specific operation steps of the extraction are:

[0022] The filtrate is passed through an extractor with a filler content of 500 mg. The column of the extractor can accommodate 8 to 12 mL of solution. 5 to 8 mL of 5 to 10% ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

[0023] Preferably, the steps of the gel purification treatment are:

[0024] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0025] Preferably, the mass fraction of the methanol aqueous solution is 95-97%.

[0026] Preferably, the flow rate of the mobile phase is 4.5-5.0 mL / min.

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

[0028] The invention mixes and disperses the raw material to be extracted with water uniformly, so that the extractant and the meat raw material are fully mixed. The chitosan and sodium alginate in the extractant are matched, so that the ammonium acetate solution, acetonitrile and nitrosamine are dissolved and extracted. The nitrosamine in the food is further extracted by the extractor, so as to improve the extraction efficiency of the nitrosamine. The gel purification treatment is carried out by washing and purification treatment through a gel permeation chromatography column, so as to further improve the extraction efficiency of the nitrosamine. At the same time, the extracted nitrosamine is purified, so as to improve the extraction and purification effect of the nitrosamine. In addition, the extraction method of the invention can realize rapid extraction, save extraction time and improve extraction efficiency. DETAILED DESCRIPTION

[0029] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0031] Step 1: Crush the food meat, then add 3-6 times of water and stir to mix;

[0032] Step 2: Add 10-15% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash with water and filter to obtain a filtrate;

[0033] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0034] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0035] The power of ultrasonic dispersion in this embodiment is 350-400W, and the ultrasonic time is 20-30min.

[0036] The preparation method of the extractant of the present embodiment is:

[0037] S01: adding 5-10 parts of chitosan to 10-15 parts of sodium alginate solution, and then adding 1-5 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0038] S02: Add 5-10% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0039] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0040] The mass fraction of the sodium alginate solution in this embodiment is 10-15%.

[0041] The mass fraction of the ammonium acetate solution in the present embodiment is 3-5%.

[0042] The temperature of the S03 stirring reaction in this embodiment is 55-65°C, the stirring time is 10-20min, and the stirring speed is 1000-1500r / min.

[0043] The specific operation steps of the extraction in this embodiment are:

[0044] The filtrate is passed through an extractor with a filler content of 500 mg. The column of the extractor can accommodate 8 to 12 mL of solution. 5 to 8 mL of 5 to 10% ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

[0045] The steps of gel purification in this embodiment are as follows:

[0046] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0047] The mass fraction of the methanol aqueous solution in this embodiment is 95-97%.

[0048] The flow rate of the mobile phase in this embodiment is 4.5-5.0 mL / min.

[0049] Example 1.

[0050] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0051] Step 1: Crush the food meat, then add 3 times the amount of water and stir to mix;

[0052] Step 2: Add 10% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash it with water and filter it to obtain a filtrate;

[0053] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0054] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0055] The power of ultrasonic dispersion in this embodiment is 350W, and the ultrasonic time is 20min.

[0056] The preparation method of the extractant of the present embodiment is:

[0057] S01: adding 5 parts of chitosan to 10 parts of sodium alginate solution, and then adding 1 part of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0058] S02: Add 5% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0059] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0060] The mass fraction of the sodium alginate solution in this embodiment is 10%.

[0061] The mass fraction of the ammonium acetate solution in the present embodiment is 3%.

[0062] The temperature of the S03 stirring reaction in this embodiment is 55° C., the stirring time is 10 min, and the stirring speed is 1000 r / min.

[0063] The specific operation steps of the extraction in this embodiment are:

[0064] The filtrate is passed through an extractor with a filler content of 500 mg and a column that can accommodate 8 mL of solution. 5 mL of 5% by mass ammoniated methanol is used to activate and elute the extractor, and the eluent is collected at last.

[0065] The steps of gel purification in this embodiment are as follows:

[0066] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0067] The mass fraction of the methanol aqueous solution in this embodiment is 95%.

[0068] The flow rate of the mobile phase in this example is 4.5 mL / min.

[0069] Example 2.

[0070] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0071] Step 1: Crush the food meat, then add 6 times the amount of water and stir to mix;

[0072] Step 2: Add 15% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash with water and filter to obtain a filtrate;

[0073] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0074] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0075] The power of ultrasonic dispersion in this embodiment is 400W, and the ultrasonic time is 30min.

[0076] The preparation method of the extractant of the present embodiment is:

[0077] S01: adding 10 parts of chitosan to 15 parts of sodium alginate solution, and then adding 5 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0078] S02: Add 10% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0079] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0080] The mass fraction of the sodium alginate solution in this embodiment is 15%.

[0081] The mass fraction of the ammonium acetate solution in the present embodiment is 5%.

[0082] The temperature of the S03 stirring reaction in this embodiment is 65° C., the stirring time is 20 min, and the stirring speed is 1500 r / min.

[0083] The specific operation steps of the extraction in this embodiment are:

[0084] The filtrate is passed through an extractor with a filler content of 500 mg and a column that can accommodate 12 mL of solution. 8 mL of 10% by mass ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

[0085] The steps of gel purification in this embodiment are as follows:

[0086] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0087] The mass fraction of the methanol aqueous solution in this embodiment is 97%.

[0088] The flow rate of the mobile phase in this example is 5.0 mL / min.

[0089] Example 3.

[0090] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0091] Step 1: Crush the food meat, then add 4.5 times the amount of water and stir to mix;

[0092] Step 2: Add 12.5% ​​of the extractant to the product of step 1, disperse it thoroughly by ultrasonication, then wash with water and filter to obtain a filtrate;

[0093] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0094] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0095] The power of ultrasonic dispersion in this embodiment is 375W, and the ultrasonic time is 25min.

[0096] The preparation method of the extractant of the present embodiment is:

[0097] S01: adding 7.5 parts of chitosan to 12.5 parts of sodium alginate solution, and then adding 3 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0098] S02: Add 7.5% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0099] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0100] The mass fraction of the sodium alginate solution in this embodiment is 12.5%.

[0101] The mass fraction of the ammonium acetate solution in the present embodiment is 4%.

[0102] The temperature of the S03 stirring reaction in this embodiment is 60° C., the stirring time is 15 min, and the stirring speed is 1250 r / min.

[0103] The specific operation steps of the extraction in this embodiment are:

[0104] The filtrate is passed through an extractor with a filler content of 500 mg and a column that can accommodate 10 mL of solution. 6.5 mL of 7.5% by mass ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

[0105] The steps of gel purification in this embodiment are as follows:

[0106] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0107] The mass fraction of the methanol aqueous solution in this embodiment is 96%.

[0108] The flow rate of the mobile phase in this example is 4.7 mL / min.

[0109] Example 4.

[0110] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0111] Step 1: Crush the food meat, then add 4 times the amount of water and stir to mix;

[0112] Step 2: Add 12% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash with water and filter to obtain a filtrate;

[0113] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0114] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0115] The power of ultrasonic dispersion in this embodiment is 360W, and the ultrasonic time is 22 minutes.

[0116] The preparation method of the extractant of the present embodiment is:

[0117] S01: adding 6 parts of chitosan to 12 parts of sodium alginate solution, and then adding 2 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0118] S02: Add 6% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0119] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0120] The mass fraction of the sodium alginate solution in this embodiment is 12%.

[0121] The mass fraction of the ammonium acetate solution in the present embodiment is 4%.

[0122] The temperature of the S03 stirring reaction in this embodiment is 58° C., the stirring time is 12 min, and the stirring speed is 1100 r / min.

[0123] The specific operation steps of the extraction in this embodiment are:

[0124] The filtrate is passed through an extractor with a filler content of 500 mg and a column that can accommodate 9 mL of solution. 6 mL of 6% by mass ammoniated methanol is used to activate and elute the extractor, and the eluent is collected at last.

[0125] The steps of gel purification in this embodiment are as follows:

[0126] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0127] The mass fraction of the methanol aqueous solution in this embodiment is 96%.

[0128] The flow rate of the mobile phase in this example is 4.6 mL / min.

[0129] Example 5.

[0130] A method for extracting and purifying nitrosamines in food of this embodiment comprises the following steps:

[0131] Step 1: Crush the food meat, then add 5 times the amount of water and stir to mix;

[0132] Step 2: Add 14% of the extractant to the product of step 1, disperse it thoroughly by ultrasonication, then wash with water and filter to obtain a filtrate;

[0133] Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed;

[0134] Step 4: Finally, the product of step 3 is subjected to gel purification treatment.

[0135] The power of ultrasonic dispersion in this embodiment is 390W, and the ultrasonic time is 28min.

[0136] The preparation method of the extractant of the present embodiment is:

[0137] S01: adding 9 parts of chitosan to 14 parts of sodium alginate solution, and then adding 1-5 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant;

[0138] S02: Add 8% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant;

[0139] S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

[0140] The mass fraction of the sodium alginate solution in this embodiment is 14%.

[0141] The mass fraction of the ammonium acetate solution in the present embodiment is 4%.

[0142] The temperature of the S03 stirring reaction in this embodiment is 62° C., the stirring time is 18 min, and the stirring speed is 1450 r / min.

[0143] The specific operation steps of the extraction in this embodiment are:

[0144] The filtrate is passed through an extractor with a filler content of 500 mg and a column that can accommodate 11 mL of solution. 7 mL of 9% by mass ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

[0145] The steps of gel purification in this embodiment are as follows:

[0146] The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

[0147] The mass fraction of the methanol aqueous solution in this embodiment is 96%.

[0148] The flow rate of the mobile phase in this example is 4.9 mL / min.

[0149] Comparative Example 1.

[0150] The difference from Example 3 is that no extractant was added.

[0151] Comparative Example 2.

[0152] The difference from Example 3 is that anhydrous magnesium sulfate is not added to the extractant.

[0153] Comparative Example 3.

[0154] The difference from Example 3 is that no second extractant is added to the extractant.

[0155] Comparative Example 4.

[0156] The difference from Example 3 is that no ammonium acetate solution is added to the second extractant.

[0157] Comparative Example 5.

[0158] The difference from Example 3 is that the method of step 1 is not used.

[0159] The performance measurement results of Examples 1-5 and Comparative Examples 1-5 are as follows

[0160]

[0161] It can be concluded from Examples 1-5 and Comparative Examples 1-5 that the product of Example 3 of the present invention has an excellent nitrosamine extraction rate, which can be as high as 99.8%;

[0162] It can be seen from Comparative Examples 1-4 that the extraction rate of nitrosamines is significantly reduced without adding an extractant. At the same time, without adding anhydrous magnesium sulfate to the extractant, without adding a second extractant to the extractant, and without adding an ammonium acetate solution to the second extractant, the extraction rate of the product shows a trend of deterioration. The extractant prepared by the method of the present invention can significantly improve the extraction efficiency of nitrosamines. Although other methods can improve the extraction rate, the improvement effect is not as significant as that of the method of the present invention.

[0163] The innovation of the present invention is that chitosan and sodium alginate in the extractant are combined, so that ammonium acetate solution, acetonitrile and nitrosamine are easily dissolved and extracted, and nitrosamine in food is further extracted by an extractor, so as to improve the extraction efficiency of nitrosamine, and gel purification treatment is carried out by washing and purification treatment through a gel permeation chromatography column, so as to further improve the extraction efficiency of nitrosamine, and at the same time, the extracted nitrosamine is purified, so as to improve the extraction and purification effect of nitrosamine, and in addition, the extraction method of the present invention can realize rapid extraction, save extraction time and improve extraction efficiency.

[0164] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0165] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for extracting and purifying nitrosamines in food, characterized in that: The following steps are involved: Step 1: Crush the food meat, then add 3-6 times of water and stir to mix; Step 2: Add 10-15% of the extractant to the product of step 1, disperse it sufficiently by ultrasonication, then wash with water and filter to obtain a filtrate; Step 3: extracting the filtrate with an extractor, and setting aside the extraction after the extraction is completed; Step 4: Finally, the product of step 3 is subjected to gel purification treatment; The preparation method of the extractant is: S01: adding 5-10 parts of chitosan to 10-15 parts of sodium alginate solution, and then adding 1-5 parts of anhydrous magnesium sulfate, stirring and mixing thoroughly to obtain a first extractant; S02: Add 5-10% acetonitrile to the ammonium acetate solution, stir and disperse evenly to obtain a second extractant; S03: The first extractant and the second extractant are mixed in a weight ratio of 5:1, stirred for reaction, and obtained an extractant.

2. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The power of the ultrasonic dispersion is 350-400W, and the ultrasonic time is 20-30min.

3. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The mass fraction of the sodium alginate solution is 10-15%.

4. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The mass fraction of the ammonium acetate solution is 3-5%.

5. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The temperature of the SO3 stirring reaction is 55-65°C, the stirring time is 10-20min, and the stirring speed is 1000-1500r / min.

6. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The specific operation steps of the extraction are: The filtrate is passed through an extractor with a filler content of 500 mg. The column of the extractor can accommodate 8 to 12 mL of solution. 5 to 8 mL of 5 to 10% ammoniated methanol is used to activate and elute the extractor, and the eluent is collected.

7. The method for extracting and purifying nitrosamines in food according to claim 1, characterized in that: The steps of the gel purification process are: The product of step three is sent to a gel permeation chromatography column for elution and purification, wherein the gel permeation chromatography column is 30 cm×2.5 cm and the mobile phase is methanol-water solution.

8. The method for extracting and purifying nitrosamines in food according to claim 7, characterized in that: The mass fraction of the methanol aqueous solution is 95-97%.

9. The method for extracting and purifying nitrosamines in food according to claim 8, characterized in that: The flow rate of the mobile phase is 4.5-5.0 mL / min.

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