阅读说明：本技术 奶粉中脂肪酸检测方法 (Method for detecting fatty acid in milk powder ) 是由 蔡德玲 黄晶 蔡勤仁 彭碧宁 曾川 吕梦莎 林加燕 赵乃漫 于 2020-12-14 设计创作，主要内容包括：本发明提供了奶粉中脂肪酸检测方法,包括步骤如下：步骤1：准确称取奶粉,并加入甲苯及含5-15％酰基化试剂的醇溶液；步骤2：振荡混合,并在80±1℃下反应2h；步骤3：加入碳酸盐溶液,并离心,采集上清液；步骤4：取上清液进行色谱分析,确定上清液中脂肪酸酯含量,计算奶粉中脂肪酸检测方法。现有奶粉中脂肪酸检测过程繁琐,且要求严格,导致效率低下。本发明通过对脂肪酸进行酯化,并色谱分析,获得溶液中脂肪酸含量,从而可以快速有效的采集脂肪酸总量,进而检测奶粉中脂肪酸的含量。(The invention provides a method for detecting fatty acid in milk powder, which comprises the following steps: step 1: accurately weighing milk powder, and adding toluene and an alcoholic solution containing 5-15% of an acylation agent; step 2: shaking and mixing, and reacting for 2h at 80 +/-1 ℃; and step 3: adding carbonate solution, centrifuging and collecting supernatant; and 4, step 4: and (4) taking the supernatant for chromatographic analysis, determining the content of fatty acid ester in the supernatant, and calculating the fatty acid detection method in the milk powder. The fatty acid detection process in the existing milk powder is complicated and strict in requirements, so that the efficiency is low. According to the invention, the fatty acid content in the solution is obtained by esterifying the fatty acid and carrying out chromatographic analysis, so that the total fatty acid amount can be rapidly and effectively collected, and the content of the fatty acid in the milk powder can be further detected.)

1. The method for detecting fatty acid in milk powder is characterized by comprising the following steps:

step 1: accurately weighing milk powder, and adding toluene and an alcoholic solution containing 5-15% of an acylation agent;

step 2: shaking and mixing, and reacting for 2h at 80 +/-1 ℃;

and step 3: adding carbonate solution, centrifuging and collecting supernatant;

and 4, step 4: and (4) taking the supernatant for chromatographic analysis, determining the content of fatty acid ester in the supernatant, and calculating the fatty acid detection method in the milk powder.

2. The method for detecting fatty acid in milk powder according to claim 1, wherein the chromatographic analysis conditions are as follows:

sample inlet temperature: 250 ℃; detector temperature: 270 ℃; column temperature program: starting to maintain the temperature of 130 ℃ for 1min, increasing the temperature to 170 ℃ at 6.5 ℃/min, increasing the temperature to 215 ℃ at 2.75 ℃/min, and maintaining the temperature for 10 min; carrier gas: high purity N₂。

3. The method for detecting fatty acid in milk powder according to claim 2, wherein the chromatographic analysis conditions further comprise a sample injection mode: split-flow sample injection is carried out according to the split-flow ratio of 25: 1; constant voltage mode: 29.885 psi; tail blowing: 60 mL/min; sample introduction amount: 1.0. mu.L.

4. The method for detecting fatty acids in milk powder according to claim 3, wherein the chromatographic conditions further comprise a chromatographic column: BPX 70.

5. The method for detecting fatty acids in milk powder according to claim 4, wherein the size of the chromatographic column is 60m x 0.25mm x 0.25 μm.

6. The method for detecting fatty acid in milk powder according to claim 1, wherein the step 3 specifically comprises: cooling the solution to room temperature, transferring into a centrifuge tube, washing the reaction equipment of the solution with carbonate solution for 2-4 times, mixing uniformly, centrifuging at high speed, and collecting the supernatant.

7. The method for detecting fatty acid in milk powder according to claim 1, wherein the ratio of the mass of the milk powder to the volume of toluene is 0.1g/mL, and the volume ratio of toluene to the alcoholic solution of the acylating reagent is 5: 5-10.

8. The method for detecting fatty acids in milk powder according to claim 1, wherein the alcohol solution of the acylating agent is a methanol solution containing 10% of alkyl acyl halide.

9. The method for detecting fatty acid in milk powder according to claim 8, wherein the volume ratio of the carbonate solution to the toluene is 5-10: 5.

10. the method for detecting fatty acid in milk powder according to claim 1, wherein the steps 1 and 2 are performed in parallel reaction tubes.

Technical Field

The invention relates to a method for detecting fatty acid in food, in particular to a method for detecting fatty acid in milk powder.

Background

The milk powder is used as a substitute of milk and is an important food in the growth process of infants, so whether the nutrition components in the milk powder are balanced or not is an important index for measuring the quality of the milk powder, fat is used as an important nutrient substance of a human body and mainly exists in the form of fatty acid in the milk powder, the nutrition components are essential for infants, and in order to determine the quality of the milk powder, the fatty acid in the milk powder of each brand, variety and production batch needs to be tracked and detected for a long time, so the detection process of the fatty acid in the milk powder needs to be carried out for many times, and the detection process of the fatty acid in the existing milk powder is complicated and strict in requirement, so that the.

Disclosure of Invention

The invention provides a method for detecting fatty acid in milk powder, which is used for rapidly detecting the content of the fatty acid in the milk powder.

The invention provides a method for detecting fatty acid in milk powder, which comprises the following steps:

step 1: accurately weighing milk powder, and adding toluene and an alcoholic solution containing 5-15% of an acylation agent;

step 2: shaking and mixing, and reacting for 2h at 80 +/-1 ℃;

and step 3: adding carbonate solution, centrifuging and collecting supernatant;

and 4, step 4: and (4) taking the supernatant for chromatographic analysis, determining the content of fatty acid ester in the supernatant, and calculating the fatty acid detection method in the milk powder.

Further, the chromatographic conditions are as follows:

sample inlet temperature: 250 ℃; detector temperature: 270 ℃; column temperature program: initial temperature 130 ℃ hold 1min, rising to 170 ℃ at 6.5 ℃/min, rising to 215 ℃ at 2.75 ℃/min, and keeping for 10 min; carrier gas: high purity N₂。

Further, the chromatographic analysis conditions further comprise a sample injection mode: split-flow sample injection is carried out according to the split-flow ratio of 25: 1; constant voltage mode: 29.885 psi; tail blowing: 60 mL/min; sample introduction amount: 1.0. mu.L.

Still further, the chromatographic conditions further comprise column: BPX 70.

Still further, the column size is 60m × 0.25mm × 0.25 μm.

Further, the step 3 specifically includes: cooling the solution to room temperature, transferring into a centrifuge tube, washing the reaction equipment of the solution with carbonate solution for 2-4 times, mixing uniformly, centrifuging at high speed, and collecting the supernatant.

Further, the ratio of the mass of the milk powder to the volume of the toluene is 0.1g/mL, and the volume ratio of the toluene to the alcoholic solution of the acylation reagent is 5: 5-10.

Further, the alcoholic solution of the acylation reagent is a methanol solution containing 10% of alkyl acyl halide.

Further, the volume ratio of the carbonate solution to the toluene is 5-10: 5.

further, the step 1 and the step 2 are both carried out in parallel reaction tubes.

Compared with the prior art, the method has the advantages that the fatty acid content in the solution is obtained through esterification and chromatographic analysis of the fatty acid, so that the total fatty acid amount can be rapidly and effectively collected, and the content of the fatty acid in the milk powder can be further detected.

Drawings

FIG. 1 is a chromatogram of fatty acids according to an example of the present invention;

FIG. 2 is a graph showing the standard ARA recovery rate in example 1 of the present invention;

FIG. 3 is a graph of EPA recovery standard for example 1.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

The parallel reactors used in examples 1-4 of the present invention were parallel synthesis reactors MJN12-25 ML.

Example 1

A sample of 0.500g of milk powder (to the nearest 0.1mg) was weighed out accurately into a parallel reactor and 5.0ml of toluene was added. Adding 6.0mL of 10% acetyl chlorocresol solution into a sample, opening cooling water of a parallel reactor, not filling inert gases such as nitrogen, oscillating, mixing, placing on a parallel evaporator for reaction at 80 +/-1 ℃ for 2 hours, cooling, taking out, transferring a sample solution after reaction into a 50mL centrifuge tube, respectively washing a glass tube with 3.0mL of saturated sodium carbonate solution for three times, combining the sodium carbonate solution, uniformly mixing, centrifuging at a high speed, and taking a supernatant for determination.

Example 2

A sample of 0.500g of milk powder (to the nearest 0.1mg) was weighed out accurately into a parallel reactor and 5.0ml of toluene was added. Adding 6.0mL of 10% acetyl chloroformic alcohol solution into a sample, opening cooling water of a parallel reactor, flushing nitrogen, oscillating, mixing, placing on a parallel evaporator at 80 +/-1 ℃ for reacting for 2h, cooling, taking out, transferring the reacted sample solution into a 50mL centrifuge tube, respectively washing a glass tube with 3.0mL of saturated sodium carbonate solution for three times, combining the sodium carbonate solutions, mixing uniformly, centrifuging at a high speed, and taking the supernatant for determination.

Example 3

A sample of 0.500g of milk powder (to the nearest 0.1mg) was weighed out accurately into a parallel reactor and 5.0ml of toluene was added. Adding 6.0mL of 15% acetyl chlorocresol solution into a sample, opening cooling water of a parallel reactor without nitrogen flushing, oscillating, mixing, placing on a parallel evaporator for reaction at 80 +/-1 ℃ for 2 hours, cooling, taking out, transferring a sample liquid after reaction into a 50mL centrifuge tube, respectively washing a glass tube with 3.0mL of saturated sodium carbonate solution for three times, combining the sodium carbonate solution, mixing uniformly, centrifuging at a high speed, and taking a supernatant for determination.

Example 4

A sample of 0.500g of milk powder (to the nearest 0.1mg) was weighed out accurately into a parallel reactor and 5.0ml of toluene was added. Adding 6.0mL of 5% acetyl chlorocresol solution into a sample, opening cooling water of a parallel reactor without nitrogen flushing, oscillating, mixing, placing on a parallel evaporator for reaction at 80 +/-1 ℃ for 2 hours, cooling, taking out, transferring a sample liquid after reaction into a 50mL centrifuge tube, respectively washing a glass tube with 3.0mL of saturated sodium carbonate solution for three times, combining the sodium carbonate solution, mixing uniformly, centrifuging at a high speed, and taking a supernatant for determination.

Comparative example 1

A sample of 0.5g of milk powder (to an accuracy of 0.1mg) was accurately weighed into a 15mL dry screw glass tube and 5.0mL of toluene was added. To the sample was added 6.0mL of 10% solution of acetochlorocresol and the screw cap was tightened. Shaking and mixing, placing in 80 + -1 deg.C water bath for 2h, taking out every 20min, shaking for 1 time, taking out after water bath, cooling to room temperature, taking out, cooling to room temperature, transferring the reacted sample liquid to 50mL centrifuge tube, washing glass tube with 3.0mL sodium carbonate solution for three times, mixing sodium carbonate solution, mixing, centrifuging at high speed, and taking supernatant for determination.

Comparative example 2

A sample of 0.5g of milk powder (to an accuracy of 0.1mg) was accurately weighed into a 15mL dry screw glass tube and 5.0mL of toluene was added. 6.0mL of 10% solution of acetylchloride in water was added to the sample, and after introducing nitrogen gas, the screw cap was tightened. Shaking and mixing, placing in 80 + -1 deg.C water bath for 2h, taking out every 20min, shaking for 1 time, taking out after water bath, cooling to room temperature, taking out, cooling to room temperature, transferring the reacted sample liquid to 50mL centrifuge tube, washing glass tube with 3.0mL sodium carbonate solution for three times, mixing sodium carbonate solution, mixing, centrifuging at high speed, and taking supernatant for determination.

The chromatographic conditions of examples 1 to 4 of the present invention and comparative examples 1 to 2 were as follows:

a chromatographic column: BPX70(60 m.times.0.25 mm. times.0.25 μm); sample inlet temperature: 250 ℃; detector temperature: 270 ℃; column temperature program: starting to maintain the temperature of 130 ℃ for 1min, rising to 170 ℃ at 6.5 ℃/min, rising to 215 ℃ at 2.75 ℃/min, and maintaining for 10 min; carrier gas: high purity N₂(the purity is more than or equal to 99.999%); and (3) sample introduction mode: split-flow sample injection (split-flow ratio 25: 1); constant voltage mode: 29.885 psi; tail blowing: 60 mL/min; sample introduction amount: 1.0. mu.L. Under these conditions 37 lipidsThe chromatogram of the fatty acid mixture is shown in FIG. 1, and only 37 fatty acids can be completely separated within 25 minutes.

To test the recovery efficiency of example 1 of the present invention, the recovery rate of example 1 of the present invention was tested by adding ARA standards, and the standard curve of the ARA recovery rate is shown in fig. 2, and the recovery rate is shown in the following table.

The recovery rate of the ARA of the first milk powder (Xibao 3 paragraph) is 99.00-118.28% under the condition that the addition standard amount is 60-120 mg/L, and the coefficient of variation is 0.016-0.081; the recovery rate of the ARA of the second milk powder (formula goat milk) is 94.25-106.12% under the condition that the addition standard amount is 80-150 mg/L, the coefficient of variation is 0.026-0.063, and the stability is good.

To test the recovery efficiency of example 1 of the present invention, the recovery rate of example 1 of the present invention was tested by adding EPA standard, and the EPA recovery standard curve is shown in fig. 3, which is shown in the following table.

As shown in fig. 3, the EPA standard solution concentrations were 15, 30, 50, 100, 250, and 500mg/L, respectively, and the EPA standard curve was y-0.4269 x +0.9455, R-0.9982, which showed good linear relationship. The recovery rate of EPA of the first milk powder (Xibao 3 section) is 91.11-120.27% under the condition that the addition amount is 15-60 mg/L, and the variation coefficient is 0.042-0.102; the recovery rate of EPA of the second milk powder (formula goat milk) is 101.45-121.33% under the condition that the addition standard amount is 15-60 mg/L, and the variation coefficient is 0.031-0.069.

The fatty acid contents of the Xibao 1-stage milk powder measured in the examples 1 and 2 are compared as shown in the table below.

The two methods of example 1 and example 2 were performed in the same experimental environment, using the same reagents, with 3 replicates of each sample. In the fatty acid detection of Xibao 1-stage milk powder, the errors of AA, DHA and alpha-linoleic acid measured by the two methods are 7.08%, 9.65%, 4.79% and 2.03%, respectively; the coefficient of variation of the whole measured without nitrogen charging is between 0.0128 and 0.0572, and the coefficient of variation of the whole measured during nitrogen charging is between 0.0524 and 0.0749.

The fatty acid contents of Xibao 3-stage milk powder measured in examples 1 and 2 are compared as shown in the table below.

In the fatty acid detection of Xibao 3-stage milk powder, the errors of AA, DHA and alpha-linoleic acid measured by the two methods of the example 1 and the example 2 are 3.56%, 4.47%, 4.20% and 3.40%, respectively; the coefficient of variation of the whole measured without nitrogen charging is between 0.0293 and 0.0434, and the coefficient of variation of the whole measured during nitrogen charging is between 0.0318 and 0.0448; in addition, the measured data is more stable when the nitrogen is not filled in the embodiment 1 of the invention, and the difference is smaller; and the error of the two methods is less than 10 percent, which meets the requirement of detection on the error.

And by adopting the comparative experiment conditions, fatty acid tests are respectively carried out on the Xibao 1-section milk powder and the Xibao 3-section milk powder according to the experiment conditions of the comparative example 1 and the comparative example 2, and the results show that DHA errors in the Xibao 1-section milk powder and the Xibao 3-section milk powder of the comparative example 1 and the comparative example 2 are respectively over 10 percent and can maximally reach 12.31 percent (the testing result of the Xibao 1-section milk powder), and ARA errors in the Xibao 1-section milk powder and the Xibao 3-section milk powder are respectively over 9 percent and can maximally reach 10.47 percent (the testing result of the Xibao 1-section milk powder).

Therefore, compared with the comparative example, the embodiment of the invention uses the parallel reactor to replace the common water bath, avoids shaking every 20 minutes, and liberates manpower; and the method has good recovery rate and precision without filling inert gases such as nitrogen, reduces operation steps and improves detection efficiency while not influencing detection precision.

Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the modifications and equivalents of the specific embodiments of the present invention can be made by those skilled in the art after reading the present specification, but these modifications and variations do not depart from the scope of the claims of the present application.