阅读说明：本技术 一种检测发酵酱油制品中毛发水的方法 (Method for detecting hair water in fermented soy product ) 是由 李丰 毓志超 文永贤 于 2020-09-28 设计创作，主要内容包括：本发明公开了一种检测发酵酱油制品中毛发水的方法,包括以下：步骤a,前处理；步骤b,第一样液测定；步骤c,第二样液测定；步骤d,第三样液测定；步骤e,定性判定。本发明的检测方法以氯丙醇作为毛发水的指示物,将氯丙醇通过反应转化为乙酰丙酮作为检测目标物,来判定酱油样品是否含有毛发水。检测方法操作简便,现象稳定、快速、明显,专属性强,灵敏度高,不需要大型的分析仪器,对实验环境要求低,极大的降低了假阳性的情况产生,适用于对发酵酱油制品中是否含有毛发水进行大批量快速检测。(The invention discloses a method for detecting hair water in a fermented soy product, which comprises the following steps: step a, pretreatment; step b, measuring the first sample solution; step c, measuring the second sample liquid; d, measuring the third sample liquid; and e, qualitatively judging. The detection method provided by the invention uses chloropropanol as an indicator of hair water, and converts chloropropanol into acetylacetone through reaction as a detection target object to judge whether a soy sauce sample contains hair water. The detection method is simple and convenient to operate, stable, rapid and obvious in phenomenon, strong in specificity, high in sensitivity, free of large-scale analytical instruments, low in requirement on experimental environment, capable of greatly reducing the generation of false positive conditions, and suitable for rapidly detecting whether the fermented soy sauce product contains the hair water in a large scale.)

1. A method for detecting hair water in fermented soy products is characterized by comprising the following steps:

step a, pretreatment: weighing a certain amount of soy sauce sample in a container, adding sodium chloride, activated carbon, scrap iron and water, uniformly mixing, adding anhydrous ether, mixing, centrifuging, and dividing the upper solution into three equal parts, namely a first sample solution, a second sample solution and a third sample solution;

step b, first sample solution determination: adding a tetrahydrofuran solution of methyl magnesium chloride into the first sample solution, oscillating and mixing, adding a first catalyst and anhydrous ether, carrying out magnetic stirring, filtering to obtain a first solution to be detected, dropwise adding a silver nitrate solution into the first solution to be detected, and observing whether a precipitate is generated in the first solution to be detected or not when each drop of the silver nitrate solution is added, wherein the first catalyst is a cuprous halide compound;

step c, measuring the second sample solution: adding a tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether into the second sample solution, oscillating, uniformly mixing, standing to obtain a second solution to be detected, adding a silver nitrate solution into the second solution to be detected, and observing whether a precipitate is generated in the second solution to be detected or not when one drop of the silver nitrate solution is added;

step d, measuring a third sample solution:

d1, drying the third sample liquid with nitrogen, adding a sodium hydroxide solution, oscillating, uniformly mixing, and standing to obtain a third liquid to be detected;

d2, adding an acetic acid solution into the third solution to be detected, and adjusting the pH value of the third solution to be detected to 7.0 +/-0.1;

d3, adding a second catalyst, heating and reacting under the condition of uninterrupted oxygen introduction and under the condition of a certain temperature, filtering, and removing insoluble substances to obtain a third filtrate, wherein the second catalyst is a metal simple substance;

d4, evaporating the third filtrate to dryness, adding a tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether, and performing first redissolution to obtain a first redissolution;

d5, drying the first redissolution by blowing, adding an acetic acid-sodium acetate buffer solution for second redissolution after drying, and adding a sodium hydroxide solution to adjust the pH value to 7.0 +/-0.1 to obtain a second redissolution;

d6, adding the second catalyst into the second redissolution, heating and reacting under the condition of uninterrupted introducing oxygen and under the condition of a certain temperature, filtering, and removing insoluble substances to obtain a fourth filtrate;

d7, adding formaldehyde solution and acetic acid-sodium acetate buffer solution into the fourth filtrate, and observing whether the fourth filtrate is discolored;

step e, qualitative judgment:

if the following three conditions are satisfied simultaneously: and if white precipitates are generated in the first solution to be tested, no precipitates are generated in the second solution to be tested, and the fourth filtrate is changed from colorless to yellow, the soy sauce sample is judged to contain hair water.

2. The method of claim 1, comprising the steps of:

step a, pretreatment: weighing 1.0-3.0ml of soy sauce sample into a centrifuge tube, adding 2-4g of sodium chloride, 2.0-2.5g of activated carbon, 0.2-0.5g of scrap iron and 5-10ml of water, uniformly mixing, adding 5ml of anhydrous ether, mixing, centrifuging for 5-6 minutes at the rotating speed of 3500 plus 4000 rpm, and dividing 4.5ml of upper solution into three equal parts which are respectively a first sample solution, a second sample solution and a third sample solution;

step b, first sample solution determination: adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride into the first sample solution, oscillating and uniformly mixing, adding 0.2-0.4g of first catalyst, supplementing and adding anhydrous ether to enable the volume of the solution to be 20ml, stirring for 15-20 minutes at room temperature at 500 rpm, filtering to obtain a first solution to be detected, dropwise adding a silver nitrate solution into the first solution to be detected, and observing whether a precipitate is generated in the first solution to be detected or not every time one drop of the silver nitrate solution is added, wherein the first catalyst is a cuprous halide compound;

step c, measuring the second sample solution: adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether into the second sample solution to make the volume of the solution be 20ml, uniformly mixing by oscillation, standing for 10-15 minutes to obtain a second solution to be detected, adding a silver nitrate solution into the second solution to be detected, and observing whether a precipitate is generated in the second solution to be detected or not every time one drop of the silver nitrate solution is added;

step d, measuring a third sample solution:

d1, drying the third sample liquid with nitrogen, adding 2-4ml of sodium hydroxide solution, oscillating, mixing uniformly, and standing to obtain a third liquid to be detected;

d2, adding 1mol/L acetic acid solution into the third solution to be detected, and adjusting the pH value of the third solution to be detected to 7.0 +/-0.1;

d3, adding a second catalyst, heating and reacting for 25-35 minutes at 45-65 ℃ under the condition of continuously introducing oxygen, filtering, and removing insoluble substances to obtain a third filtrate, wherein the second catalyst is a metal simple substance;

d4, evaporating the third filtrate to dryness, adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether, and carrying out first redissolution to obtain a first redissolution;

d5, drying the first redissolution by using nitrogen, adding 4-6ml of acetic acid-sodium acetate buffer solution for second redissolution after drying, and adding sodium hydroxide solution to adjust the pH value to 7.0 +/-0.1 to obtain a second redissolution;

d6, adding the second catalyst into the second redissolution, heating to react for 25-35 minutes at 45-65 ℃ under the condition of continuously introducing oxygen, filtering, and removing insoluble substances to obtain a fourth filtrate;

d7, adding 0.1-0.3ml of formaldehyde solution and 4-6ml of acetic acid-sodium acetate buffer solution into the fourth filtrate, and observing whether the fourth filtrate is discolored or not;

step e, qualitative judgment:

if the following three conditions are satisfied simultaneously: and if white precipitates are generated in the first solution to be tested, no precipitates are generated in the second solution to be tested, and the fourth filtrate is changed from colorless to yellow, the soy sauce sample is judged to contain hair water.

3. The method of claim 1, wherein the first catalyst is any one of cuprous chloride, cuprous bromide, or cuprous iodide.

4. The method of claim 1, wherein the second catalyst is any one of copper, zinc or iron.

5. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the mass fraction of formaldehyde in the formaldehyde solution is in the range of 37% to 40%.

6. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the concentration of the sodium hydroxide solution is 1.0 mol/L.

7. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the flow rate of oxygen in step d is 7-8 m/s.

8. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the pH of the acetic acid-sodium acetate buffer solution is 6.0.

9. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the concentration of methyl magnesium chloride in the tetrahydrofuran solution of methyl magnesium chloride is 3 mol/L.

10. The method for detecting hair water in fermented soy sauce products as claimed in any of claims 1 to 4, wherein the concentration of the silver nitrate solution is 0.05 mol/L.

Technical Field

The invention relates to the field of soy sauce detection, in particular to a method for detecting hair water in a fermented soy sauce product.

Background

In recent years, hair hydrolysate is added into products such as soy sauce by partial lawless persons instead of grains such as soybeans for fermentation, so that the content of false amino acid substances in the products is increased, and the false soy sauce is artificially synthesized. The soy sauce processed by the hair has low production cost, has price competitiveness in the market and deceives consumers. Hair water, namely human or animal hair, is rich in protein, and is subjected to acid hydrolysis to produce amino acid solution, and the amino acid solution is illegally added into food to serve as a nutrient. In 2009, hair water was specifically listed as inedible substances in the list of inedible substances that may be illegally added to foods (third batch), published by the ministry of health, and the key product was soy sauce seasoning.

Hair and the like are non-food materials, and hair itself contains many harmful substances such as lead, arsenic and the like, and cannot be used as a material for processing food and seasonings at all. Industrial hydrochloric acid used in reactions such as hair hydrolysis and other reagents also contain excessive toxic substances such as arsenic, lead, mercury and the like. In addition, toxic and harmful substances such as carcinogenic trichloropropanol and the like can be formed in the process of hydrolyzing the vegetable protein by hydrochloric acid, so that the 'hair water' after cystine is extracted contains a large amount of toxic and harmful substances. If consumers eat the 'hair water soy sauce', arsenic poisoning and other acute and chronic poisoning can be caused, and the health can be hurt.

The method for detecting the content of chloropropanol and fatty acid ester thereof in hair water by taking chloropropanol as a main index is measured in GB 5009.191-2016 national food safety standard, wherein the first method adopts the operation principle that an isotope dilution technology is adopted and D5-3-chloro-1, 2-propanediol (D5-3-MCPD) is taken as an internal standard. Adding an internal standard into the sample, performing solid-phase support liquid-liquid extraction by using kieselguhr as an adsorbent, leaching by using n-hexane-anhydrous ether solution (9+1) to remove non-polar lipid components, and eluting 3-chloro-1, 2-propanediol (3-MCPD) by using anhydrous ether. Deriving with heptafluorobutyrylimidazole, measuring by using a gas chromatography-mass spectrometer, and quantifying by using an internal standard method. However, the method has complex pretreatment, needs a large expensive instrument such as a gas chromatography-mass spectrometer, has high operation difficulty, high cost and higher requirement on the level of a laboratory, is only limited to routine detection in a laboratory, and is not beneficial to quickly detecting the target and the requirement of obtaining a result.

Therefore, a rapid detection method which is simple and rapid to operate, does not need a large-scale analysis instrument, consumes less time, has strong specificity, high sensitivity, high accuracy of detection results, low analysis cost and low requirement on experimental environment and is suitable for detecting whether the fermented soy sauce product contains the hair water or not needs to be developed.

Disclosure of Invention

The invention aims to provide a method for detecting hair water in a fermented soy sauce product. According to one aspect of the present invention, there is provided a method of detecting hair water in a fermented soy sauce product, comprising the following:

step a, pretreatment: weighing a certain amount of soy sauce sample in a container, adding sodium chloride, activated carbon, scrap iron and water, uniformly mixing, adding anhydrous ether, mixing, centrifuging, and dividing the upper solution into three equal parts, namely a first sample solution, a second sample solution and a third sample solution;

step b, first sample solution determination: adding a tetrahydrofuran solution of methyl magnesium chloride into the first sample solution, oscillating and uniformly mixing, adding a first catalyst and anhydrous ether, magnetically stirring, filtering to obtain a first solution to be detected, dropwise adding a silver nitrate solution into the first solution to be detected, and observing whether a precipitate is generated in the first solution to be detected or not when each drop of the silver nitrate solution is added, wherein the first catalyst is a cuprous halide compound;

step c, measuring the second sample solution: adding a tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether into the second sample solution, oscillating, uniformly mixing, standing to obtain a second solution to be detected, adding a silver nitrate solution into the second solution to be detected, and observing whether a precipitate is generated in the second solution to be detected or not when one drop of the silver nitrate solution is added;

step d, measuring a third sample solution:

d1, drying the third sample liquid with nitrogen, adding a sodium hydroxide solution, oscillating, uniformly mixing, and standing to obtain a third liquid to be detected;

d2, adding an acetic acid solution into the third solution to be detected, and adjusting the pH value of the third solution to be detected to 7.0 +/-0.1;

d3, adding a second catalyst, heating and reacting under the condition of uninterrupted oxygen introduction and under the condition of a certain temperature, filtering, and removing insoluble substances to obtain a third filtrate, wherein the second catalyst is a metal simple substance;

d4, evaporating the third filtrate to dryness, adding a tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether, and performing first redissolution to obtain a first redissolution;

d5, drying the first redissolution by blowing, adding an acetic acid-sodium acetate buffer solution for second redissolution after drying, and adding a sodium hydroxide solution to adjust the pH value to 7.0 +/-0.1 to obtain a second redissolution;

d6, adding the second catalyst into the second redissolution, heating and reacting under the condition of uninterrupted introducing oxygen and under the condition of a certain temperature, filtering, and removing insoluble substances to obtain a fourth filtrate;

d7, adding formaldehyde solution and acetic acid-sodium acetate buffer solution into the fourth filtrate, and observing whether the fourth filtrate is discolored;

step e, qualitative judgment:

if the following three conditions are satisfied simultaneously: and if white precipitates are generated in the first solution to be tested, no precipitates are generated in the second solution to be tested, and the fourth filtrate is changed from colorless to yellow, the soy sauce sample is judged to contain hair water.

The invention has the beneficial effects that: the detection method takes chloropropanol as an indicator of the hair water, and converts chloropropanol into acetylacetone through reaction as a detection target object to judge whether the sample contains the hair water. Iron chips and active carbon are used for removing color of the soy sauce sample, sodium chloride is used for improving the precipitation degree of chloropropanol, and anhydrous ether is used for extracting chloropropanol in the sample into an upper layer solution for determination. And during the measurement of the first sample liquid, cuprous halide is used as a first catalyst to catalyze the reaction of the chloropropanol and the methyl magnesium chloride, and after the reaction, chloride ions in the chloropropanol are released to react with the silver nitrate solution to generate a precipitate, which indicates that the first sample liquid contains chlorine element. When the second sample liquid is measured, the first catalyst is not added, the chloropropanol and the methyl magnesium chloride do not react, and neither the chloropropanol nor the methyl magnesium chloride has a precipitation reaction with the silver nitrate, so that the fact that the precipitate generated in the first sample liquid is generated by the precipitation reaction of chloride ions released after the chloropropanol reaction and the silver nitrate. And when the third sample liquid is measured, converting chloropropanol into acetylacetone through reaction, judging that the fourth filtrate contains acetylacetone through formaldehyde-acetylacetone color reaction, and indicating that the third sample liquid contains a propanol group, so that the sample is judged to contain chloropropanol, and further the sample is judged to contain hair water.

The detection method provided by the invention is simple and convenient to operate, stable, rapid and obvious in phenomenon, strong in specificity and high in sensitivity, does not need a large-scale analytical instrument, has low requirements on experimental environment, greatly reduces the generation of false positive conditions, and is suitable for rapidly detecting whether the fermented soy sauce product contains the hair water or not in a large batch.

In some embodiments, a method of the invention for detecting hair water in a fermented soy product comprises the following:

step a, pretreatment: weighing 1.0-3.0ml of soy sauce sample into a centrifuge tube, adding 2-4g of sodium chloride, 2.0-2.5g of activated carbon, 0.2-0.5g of scrap iron and 5-10ml of water, mixing, adding 5ml of anhydrous ether, mixing, centrifuging for 5-6 minutes at the rotating speed of 3500 plus 4000 rpm, and dividing 4.5ml of upper solution into three equal parts which are respectively a first sample solution, a second sample solution and a third sample solution;

step b, first sample solution determination: adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride into the first sample solution, oscillating and uniformly mixing, adding 0.2-0.4g of first catalyst, supplementing and adding anhydrous ether to enable the volume of the solution to be 20ml, stirring for 15-20 minutes at room temperature at 500 rpm, filtering to obtain a first solution to be detected, dropwise adding a silver nitrate solution into the first solution to be detected, and observing whether a precipitate is generated in the first solution to be detected or not every time one drop of the silver nitrate solution is added, wherein the first catalyst is a cuprous halide compound;

step c, measuring the second sample solution: adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether into the second sample solution to make the volume of the solution be 20ml, uniformly mixing by oscillation, standing for 10-15 minutes to obtain a second solution to be detected, adding a silver nitrate solution into the second solution to be detected, and observing whether a precipitate is generated in the second solution to be detected or not every time one drop of the silver nitrate solution is added;

step d, measuring a third sample solution:

d1, drying the third sample liquid with nitrogen, adding 2-4ml of sodium hydroxide solution, oscillating, mixing uniformly, and standing to obtain a third liquid to be detected;

d2, adding 1mol/L acetic acid solution into the third solution to be detected, and adjusting the pH value of the third solution to be detected to 7.0 +/-0.1;

d3, adding a second catalyst, heating and reacting for 25-35 minutes at 45-65 ℃ under the condition of continuously introducing oxygen, filtering, and removing insoluble substances to obtain a third filtrate, wherein the second catalyst is a metal simple substance;

d4, evaporating the third filtrate to dryness, adding 0.05-0.10ml of tetrahydrofuran solution of methyl magnesium chloride and anhydrous ether, and carrying out first redissolution to obtain a first redissolution;

d5, drying the first redissolution by using nitrogen, adding 4-6ml of acetic acid-sodium acetate buffer solution for second redissolution after drying, and adding sodium hydroxide solution to adjust the pH value to 7.0 +/-0.1 to obtain a second redissolution;

d6, adding the second catalyst into the second redissolution, heating to react for 25-35 minutes at 45-65 ℃ under the condition of continuously introducing oxygen, filtering, and removing insoluble substances to obtain a fourth filtrate;

d7, adding 0.1-0.3ml of formaldehyde solution and 4-6ml of acetic acid-sodium acetate buffer solution into the fourth filtrate, and observing whether the fourth filtrate is discolored or not;

step e, qualitative judgment:

if the following three conditions are satisfied simultaneously: and if white precipitates are generated in the first solution to be tested, no precipitates are generated in the second solution to be tested, and the fourth filtrate is changed from colorless to yellow, the soy sauce sample is judged to contain hair water.

In some embodiments, the first catalyst is any one of cuprous chloride, cuprous bromide, or cuprous iodide. Cuprous halide is used as a first catalyst, and methyl magnesium chloride can form an organic copper complex with the cuprous halide, so that the following reaction is promoted to be carried out in the positive reaction direction, the production of magnesium chloride is promoted, chlorine in chloropropanol is converted into chloride ions to be released into a first solution to be detected, the subsequent precipitation reaction of the chloride ions and a silver nitrate solution is facilitated, and the judgment is carried out.

In some embodiments, the second catalyst is any one of copper, zinc, or iron. Any metal simple substance of copper, zinc or iron is used as a second catalyst to perform catalytic oxidation on propylene glycol, and a hydroxyl group is converted into an aldehyde group to generate malondialdehyde, so that subsequent reaction is facilitated.

In some embodiments, the mass fraction of formaldehyde in the formaldehyde solution is 37% to 40%. The formaldehyde solution with the mass fraction has obvious color development effect and is easy to obtain when formaldehyde-acetylacetone color development reaction is carried out.

In some embodiments, the concentration of the sodium hydroxide solution is 1.0 mol/L. The 1.0mol/L sodium hydroxide solution reacts with chloropropanol to generate propylene glycol, which is convenient for subsequent reaction to generate acetylacetone. The 1.0mol/L sodium hydroxide solution can also be used for adjusting the pH value of the second complex solution to be in a neutral pH environment.

In some embodiments, the flow rate of oxygen in step d is from 7 to 8 m/s. Oxygen is introduced to facilitate the reaction of catalytic oxidation of alcohol to aldehyde.

In some embodiments, the pH of the acetic acid-sodium acetate buffer solution is 6.0. The acetic acid-sodium acetate buffer solution provides an acidic environment for the formaldehyde-acetylacetone color reaction.

In some embodiments, the concentration of methyl magnesium chloride in the tetrahydrofuran solution of methyl magnesium chloride is 3 mol/L. Sufficient methyl magnesium chloride is provided to react with the chloropropanol.

In some embodiments, the concentration of the silver nitrate solution is 0.05 mol/L. And performing precipitation reaction on the silver nitrate solution and chloride ions in the first solution to be detected to prove that the first sample solution contains chlorine element.

Detailed Description

The present invention will be described in further detail with reference to examples.