阅读说明：本技术 基于乳液界面能诱导释放的生理毒性快速检测方法及应用 (Rapid physiological toxicity detection method based on emulsion interface energy induced release and application ) 是由 柯李晶 高观祯 余兆硕 徐阳 徐天豪 罗思浩 汪惠勤 周建武 饶平凡 于 2021-07-26 设计创作，主要内容包括：基于食用乳液界面能的诱导释放的生理毒性快速检测方法,包括以下步骤：1)取乳化剂溶于超纯水中溶解,再均匀缓慢加入食用乳液样品并采用高速剪切机剪切,剪切均质后用有机膜过滤,全过程维持温度恒定；2)按照体积配置含氧化还原酶的食用乳液样品；3)将荧光探针溶于超纯水中,并对荧光探针水溶液进行稀释,设置测定条件；4)吸取食用乳液样品于离心管内,将离心管放入样品槽中,开始测量；向样品中加入稀释后的荧光探针水溶液,得到并分析测量数据和动力学曲线。本发明的检测方法快速、高效、简单,设备要求低,结果精确,重复性高,灵敏度高,只需微量便可检测出结果；且可在体外诱导并破坏乳体系并释放出其界面能,安全便捷。(The quick physiological toxicity detection method based on the induction release of the edible emulsion interfacial energy comprises the following steps: 1) dissolving emulsifier in ultrapure water, uniformly and slowly adding an edible emulsion sample, shearing by using a high-speed shearing machine, filtering by using an organic membrane after shearing and homogenizing, and maintaining the temperature to be constant in the whole process; 2) preparing an edible emulsion sample containing oxidoreductase according to volume; 3) dissolving a fluorescent probe in ultrapure water, diluting a fluorescent probe aqueous solution, and setting a determination condition; 4) sucking an edible emulsion sample into a centrifugal tube, putting the centrifugal tube into a sample groove, and starting to measure; the diluted fluorescent probe aqueous solution was added to the sample, and measurement data and a kinetic curve were obtained and analyzed. The detection method is rapid, efficient and simple, has low equipment requirement, accurate result, high repeatability and high sensitivity, and can detect the result only by trace; and can induce and destroy the milk system in vitro and release the interface energy thereof, and is safe and convenient.)

1. The method for rapidly detecting the physiological toxicity based on the emulsion interface energy-induced release is characterized by comprising the following steps of:

1) dissolving emulsifier in ultrapure water, uniformly and slowly adding an edible emulsion sample, shearing by using a high-speed shearing machine, filtering by using an organic membrane after shearing and homogenizing, and maintaining the temperature to be constant in the whole process;

2) preparing an edible emulsion sample containing oxidoreductase according to volume;

3) dissolving a fluorescent probe in ultrapure water, diluting a fluorescent probe aqueous solution, and setting a determination condition;

4) sucking an edible emulsion sample into a centrifugal tube, putting the centrifugal tube into a sample groove, and starting to measure; the diluted fluorescent probe aqueous solution was added to the sample, and measurement data and a kinetic curve were obtained and analyzed.

2. The method for rapidly detecting physiological toxicity based on the emulsion interfacial energy induced release as claimed in claim 1, wherein the amount of the emulsifier in step 1 is 100 μ L, the amount of ultrapure water is 100mL, the amount of the edible emulsion is 500 μ L, the shearing speed is 3000-30000rpm, the time is 3-15min, and the temperature is 30-50 ℃.

3. The method for rapidly detecting physiological toxicity based on the emulsion interfacial energy induced release according to claim 1, wherein in the step 3, the fluorescent probe is a lutidine N-oxide fluorescent probe, the amount of the fluorescent probe is 0.1mg, the amount of ultrapure water is 866 μ L, and the dilution ratio is 1: 4.5;

the determination conditions are as follows: setting the preparation time of an AB-2280 illuminance sensor to be 5s, and setting the measurement time to be 120 s;

in step 4, the amount of the edible emulsion sample is 1100 muL, and the amount of the diluted fluorescent probe aqueous solution is 100 muL; the diluted aqueous solution of the fluorescent probe was added 5s after the start of the measurement.

4. The method for rapidly detecting physiological toxicity based on emulsion interfacial energy induced release according to claim 1, wherein the edible emulsion is prepared from one or more of fish oil, lard oil, olive oil, algae oil, unsaturated fatty acid and palm oil.

5. The method for rapidly detecting physiological toxicity based on emulsion interfacial energy induced release according to claim 1, wherein the edible emulsion is prepared in the form of one or more of micro-nano emulsion, liposome, micelle and vesicle.

6. The method for rapidly detecting physiological toxicity based on the release induced by interfacial energy of emulsion according to claim 1, wherein the oxidoreductase is catalase.

7. The method for rapidly detecting physiological toxicity based on the interfacial energy-induced release of emulsion according to claim 1, wherein the concentration of the reductase contained in the edible emulsion prepared in the step 2 is 0-1.0 μmol/L.

8. The method for rapidly detecting physiological toxicity based on emulsion interfacial energy induced release according to claim 1, wherein the organic membrane is 0.22-0.45 μ L organic membrane.

9. The method for rapidly detecting physiological toxicity based on emulsion interfacial energy induced release according to claim 1, wherein the emulsifier is one or more of tween, lecithin, span and glyceride.

10. Use of a method for rapidly detecting physiological toxicity based on the release induced by interfacial energy in emulsion, which is characterized in that the method for rapidly detecting physiological toxicity based on the release induced by interfacial energy in emulsion according to any one of claims 1 to 9 is used for analyzing the physiological toxicity of food emulsion.

Technical Field

The invention belongs to the field of food safety detection, and particularly relates to a method for rapidly detecting physiological toxicity based on emulsion interface energy-induced release and application thereof.

Background

Free radicals, also known as reactive oxygen species or radicals, refer to molecules, atoms or groups of atoms with unpaired valence electrons. The biological active peptide has high chemical activity, instability, short existence time, active property and extremely high reactivity, and can react with biological system components to destroy proteins, lipids and polysaccharide nucleic acid, thereby causing cell damage. The biological systems mainly encounter oxygen free radicals, and the active oxygen free radicals in the body have certain functions, such as immunity and signal transduction processes. However, excessive reactive oxygen radicals have destructive actions, resulting in damage to normal cells and tissues of the human body, thereby causing various diseases. Such as heart disease, senile dementia, Parkinson's disease and tumor. Meanwhile, the food has a certain physiological toxicity to human bodies due to the excessively high free radical content.

The edible emulsion is micro-nano emulsion which is prepared by emulsifying edible oil or fat-soluble components into O/W or W/O by a physical or chemical method, most of the micro-nano emulsion is a specific space emulsion structure formed by mechanical mixing (intensive processing such as homogenizing and shearing, and mild mechanical stirring) or ultrasonic mixing, chemical stabilization and the like, and part of energy is stored in the emulsion structure in the processing process of the micro-nano emulsion for keeping the relative stability of the structure. It has been found that when the emulsion structure is disturbed, the stored energy is released, and if the release process is carried out in the human body, it is very likely to cause inflammation of various degrees. Recently, researches show that emulsion systems such as fish oil and the like can cause inflammation of a part of organisms after being eaten, however, when tracing food safety problems, various conventional indexes such as acid value, peroxide value and the like of the prepared emulsion are normal, physiological blockage is not detected, and the operation process also meets the specifications, which brings about a plurality of questions for the safety of emulsion products. The present inventors have for the first time discovered that these safety problems are related to the free radicals that can be transformed at their interface.

In conclusion, in the face of more and more food emulsion products in life, the evaluation and detection method for physiological toxicity of the food emulsion products in the market still needs to be further improved, and a rapid evaluation method for physiological toxicity of in vitro edible emulsion is urgently needed.

Disclosure of Invention

Based on the defects and shortcomings in the prior art, the invention creatively uses catalase to induce and release the interfacial energy of the emulsion and is based on the inflammation problem caused by excessive free radicals in the food emulsion interface, a fluorescence detection technology is utilized, a rapid physiological toxicity detection method based on the induced and released interfacial energy of the emulsion is established, a detection system of the physiological toxicity of the food is supplemented, and the method has important significance for rapid detection and quality control of related products.

In order to achieve the purpose, the invention adopts the technical scheme that:

the method for rapidly detecting the physiological toxicity based on the emulsion interface energy-induced release comprises the following steps:

1) dissolving emulsifier in ultrapure water, uniformly and slowly adding an edible emulsion sample, shearing by using a high-speed shearing machine, filtering by using an organic membrane after shearing and homogenizing, and maintaining the temperature to be constant in the whole process;

2) preparing an edible emulsion sample containing oxidoreductase;

3) dissolving a fluorescent probe in ultrapure water, diluting a fluorescent probe aqueous solution, and setting a determination condition;

4) sucking an edible emulsion sample into a centrifugal tube, putting the centrifugal tube into a sample groove, and starting to measure; the diluted fluorescent probe aqueous solution was added to the sample, and measurement data and a kinetic curve were obtained and analyzed.

Preferably, the amount of the emulsifier in the step 1 is 100 μ L, the amount of ultrapure water is 100mL, the amount of the edible emulsion is 500 μ L, the shearing speed is 3000-30000rpm, the time is 3-15min, and the temperature is 30-50 ℃.

Preferably, in step 3, the fluorescent probe is a lutidine N-oxide fluorescent probe, the amount of the fluorescent probe is 0.1mg, the amount of ultrapure water is 866 μ L, and the dilution ratio is 1: 4.5;

the determination conditions are as follows: setting the preparation time of an AB-2280 illuminance sensor to be 5s, and setting the measurement time to be 120 s;

in step 4, the amount of the edible emulsion sample is 1100 muL, and the amount of the diluted fluorescent probe aqueous solution is 100 muL; the diluted aqueous solution of the fluorescent probe was added 5s after the start of the measurement.

Preferably, the edible emulsion is prepared from one or more of fish oil, lard oil, olive oil, algae oil, linoleic acid and palm oil.

Preferably, the edible emulsion is prepared in the form of one or more of nano-emulsion, liposome, micelle and vesicle.

Preferably, the oxidoreductase is a catalase. Catalase is able to induce the release of interfacial energy of the food emulsion.

Preferably, the concentration of the reductase contained in the edible emulsion prepared in the step 2 is 0-0.5 mu mol/L.

Preferably, the organic film is 0.22 to 0.45. mu.L of the organic film.

Preferably, the emulsifier is one or more of tween, lecithin, span and glyceride.

The invention also provides application of the rapid detection method for physiological toxicity based on the emulsion interface energy-induced release in analysis of the physiological toxicity of food emulsion.

The invention has the beneficial effects that:

1. the invention uses catalase to induce and release the interfacial energy of the emulsion, and establishes a quick physiological toxicity detection method based on the induced release of the interfacial energy of the emulsion by utilizing a fluorescence detection technology based on the inflammation problem caused by excessive free radicals in the food emulsion interface. The detection method is rapid, efficient and simple, and has low equipment requirement.

2. The detection method has the advantages of accurate result, high repeatability and high sensitivity, and the result can be detected only by trace amount.

3. The detection method can induce and destroy the milk system in vitro and release the interface energy of the milk system, and is safe and convenient.

Drawings

FIG. 1 shows the background ROS content of fish oil emulsions prepared with different emulsifiers and their ROS content after mixing with Catalase (CAT); (Black part: no catalase was added; gray part: catalase was added)

FIG. 2 shows the effect of different Catalase (CAT) concentrations on ROS content in fish oil emulsions (F0 is the background ROS content of fish oil emulsions, F1 is the ROS content of fish oil emulsions after hydrogen peroxide addition);

FIG. 3 effect of Catalase (CAT) addition at different concentrations on ROS content of lard emulsion (days 1, 3);

FIG. 4 effect of Catalase (CAT) addition at different concentrations on ROS content of linoleic acid emulsion;

FIG. 5 effect of different concentrations of Catalase (CAT) addition on the ROS content of DHA algal oil emulsion;

FIG. 6 is a graph showing the physiological toxicity of fish oil emulsions containing Catalase (CAT) at different concentrations (16.7. mu.g/mL for fish oil, and 0/0.24/0.48. mu. mol/L for catalase)

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

Example 1: method for rapidly detecting physiological toxicity of fish oil emulsion

Sucking 100 μ L tween 80 or lecithin, dissolving in 100ml ultrapure water, stirring, dissolving, dripping 500 μ L fish oil sample into the system, homogenizing at 18000rpm for five minutes, filtering with 0.22 μ L organic membrane, and maintaining the whole process at 40 deg.C. Respectively preparing fish oil emulsions with catalase concentration of 0/0.24/0.48 mu mol/L.

0.1mg of the lutidine N-oxide fluorescent probe was dissolved in 866. mu.L of ultrapure water, the lutidine N-oxide aqueous solution was diluted at a ratio of 1:4.5, an AB-2200 illumination sensor was turned on, the set setup time was set to 5s, and the measurement time was set to 120 s. And (3) sucking 1100 mu L of emulsion sample into a centrifuge tube, putting the centrifuge tube into a sample groove, clicking to start measurement, waiting for 5s, adding 100 mu L of diluted lutidine N-oxide fluorescent probe aqueous solution into the sample, clicking a start button, and obtaining and analyzing measurement data and a kinetic curve.

Under the conditions, the effect of different emulsifiers, different fish oil concentrations/presence/absence of catalase on the ROS content of the fish oil emulsion and the toxicity of different catalase concentrations on cells were determined, and the details are shown in the attached figure 1, the attached figure 2 and the attached figure 6.

The results show that different emulsifiers have a greater effect on the free radical content of the finished emulsion product. The important analysis in FIG. 2 shows that the free radical content of the sample without catalase is about 80000, the base number is small, and the change is not large with the increase of the fish oil concentration, while the free radical content of the fish oil emulsion added with catalase is exploded and expanded by more than 6 times, and then the analysis in FIG. 6 shows that the survival rate of the cells is reduced after the increase of the catalase concentration. Therefore, the content of free radicals and cytotoxicity are approximately related under the condition that the concentration of catalase is 0-0.48 mu mol/L, the survival rate of cells is reduced due to a large amount of burst free radicals, and therefore, the fish oil emulsion has physiological toxicity to organisms after being taken. The method has good reproducibility, and the result can be detected only by trace amount.

Example 2: method for rapidly detecting physiological toxicity of linoleic acid emulsion

Sucking 100 μ L Tween 80, dissolving in 100mL ultrapure water, stirring for dissolving, dripping 500 μ L linoleic acid sample into the system uniformly and slowly, shearing with a high-speed shearing machine at 30000rpm for 3 min, filtering the sheared homogeneous sample with 0.22 μ L organic membrane, and maintaining the whole process at 47 deg.C. Preparing linoleic acid emulsion containing catalase concentration of 0 μmol/L, 0.2 μmol/L, 0.4 μmol/L, 0.6 μmol/L, 0.8 μmol/L for use.

0.1mg of the lutidine N-oxide fluorescent probe is dissolved in 866 mu L of ultrapure water, the lutidine N-oxide fluorescent probe aqueous solution is diluted according to the proportion of 1:4.5, the measurement conditions of an AB-2200 illumination sensor control panel are set, the preparation time is set to 5s, and the measurement time is set to 120 s. And (3) sucking 1100 mu L of linoleic acid emulsion sample into a centrifuge tube, placing the centrifuge tube into a sample groove, clicking to start measurement, waiting for 5s, adding 100 mu L of diluted lutidine N-oxide fluorescent probe aqueous solution into the sample, clicking a start button, and recording the number of free radicals and the kinetic curve thereof. Partial results are shown in FIG. 4, the free radicals of the sample are exploded 2-4 times and have low base number with the increase of catalase concentration, and the sample is supposed to have no physiological toxicity to human body after being taken. The method has good reproducibility, and the result can be detected only by trace amount.

Example 3: method for rapidly detecting physiological toxicity of algae oil emulsion

Sucking 100 μ L tween 80, dissolving in 100ml ultrapure water, stirring for dissolving, dripping 500 μ L algae oil sample into the system, shearing for 15min with a high speed shearing machine at 3000rpm, filtering the sheared homogeneous sample with 0.45 μ L organic membrane, and maintaining the whole process at 32 deg.C. Preparing algae oil emulsion containing catalase concentration of 0 μmol/L, 0.2 μmol/L, 0.4 μmol/L, 0.6 μmol/L, 1.0 μmol/L for use.

0.1mg of the lutidine N-oxide fluorescent probe is dissolved in 866 mu L of ultrapure water, the lutidine N-oxide fluorescent probe aqueous solution is diluted according to the proportion of 1:4.5, the measurement conditions of an AB-2200 illumination sensor control panel are set, the preparation time is set to 5s, and the measurement time is set to 120 s. And (3) sucking 1100 mu L of algae oil emulsion into a centrifuge tube (the algae oil emulsion can be diluted when the concentration is too high), putting the centrifuge tube into a sample groove, clicking to start measurement, waiting for 5s, adding 100 mu L of diluted lutidine N-oxide fluorescent probe aqueous solution into the sample, clicking a start button, and recording the content of free radicals and the kinetic curve of the free radicals. Part of the results are shown in FIG. 4, with the increase of catalase concentration, the free radicals of the sample are exploded within 4 times and the base number is not high, and the analysis shows that the sample is basically non-physiological toxicity to human body after being taken. The method has good reproducibility, and the result can be detected only by trace amount.

Example 4: rapid detection method for physiological toxicity of lard emulsion

Sucking 100 μ L Tween 80, dissolving in 100mL ultrapure water, stirring for dissolving, dropping 500 μ L lard sample into the system uniformly and slowly, shearing for 7 min with a high speed shearing machine at 15000rpm, filtering the sheared homogeneous sample with 0.22 μ L organic membrane, and maintaining the whole process at 38 deg.C. Preparing lard emulsion with catalase concentration of 0 μmol/L, 0.1 μmol/L, 0.2 μmol/L, 0.3 μmol/L, 0.4 μmol/L, 0.5 μmol/L for use.

0.1mg of the lutidine N-oxide fluorescent probe was dissolved in 866. mu.L of ultrapure water, the lutidine N-oxide fluorescent probe aqueous solution was diluted at a ratio of 1:4.5, the measurement conditions of the AB-2200 illuminance sensor control panel were set, the preparation time was set to 5s, and the measurement time was set to 120 s. And (3) sucking 1100 mu L of lard emulsion into a centrifuge tube (the lard emulsion can be diluted when the concentration is too high), putting the centrifuge tube into a sample tank, clicking to start measurement, waiting for 5s, adding 100 mu L of diluted lutidine N-oxide fluorescent probe aqueous solution into the sample, clicking a start button, and recording the number of free radicals and the kinetic curve thereof. Partial results are shown in fig. 5, as the concentration of catalase increases, a large amount of free radicals of the sample explodes, the number of the free radicals reaches 10 at most, and the physiological toxicity of the human body after the sample is analyzed. The method has good reproducibility, and the result can be detected only by trace amount.

The above embodiment fully shows that the physiological toxicity condition of the food emulsion can be rapidly obtained through ROS detection, and the ROS content obtained based on the fluorescence detection technology is expected to be used as quality evaluation, regulation and judgment of the physiological toxicity of the food emulsion.

The foregoing has outlined rather broadly the preferred embodiments and principles of the present invention and it will be appreciated that those skilled in the art may devise variations of the present invention that are within the spirit and scope of the appended claims.