阅读说明：本技术 一种仿生评估甲壳类水产品致敏性的方法 (bionic method for evaluating allergenicity of crustacean aquatic products ) 是由 刘光明 刘萌 韩天娇 刘庆梅 刘思寒 刘红 于 2019-09-11 设计创作，主要内容包括：本发明涉及一种仿生评估甲壳类水产品致敏性的方法,该方法包括：(1)仿生消化：利用体外胃肠液连续消化法处理甲壳类水产品可食用部分得到连续消化产物；(2)仿生转运：将连续消化产物加入含有小肠上皮粘膜Caco-2细胞的转运小室进行肠道细胞吸收转运；(3)体外评估：采用LAD2人肥大细胞脱颗粒模型,评估甲壳类水产品经仿生消化吸收后的致敏性；本发明采用体外胃肠液连续消化结合肠道上皮细胞吸收转运的方法,仿生处理甲壳类水产品；通过检测处理后样品诱导免疫细胞产生过敏介质的情况,以评估甲壳类水产品的致敏性；该方法简单易操作,通用性强。(The invention relates to a bionic method for evaluating the sensitization of crustacean aquatic products, which comprises the following steps: (1) bionic digestion: treating edible part of shellfish aquatic product by in vitro gastrointestinal fluid continuous digestion method to obtain continuous digestion product; (2) bionic transfer: adding the continuous digestion product into a transport chamber containing small intestinal epithelial mucosa Caco-2 cells for intestinal cell absorption and transport; (3) in vitro evaluation: evaluating the allergenicity of the crustacean aquatic products after bionic digestion and absorption by adopting an LAD2 human mast cell degranulation model; the method adopts a method of combining in-vitro gastrointestinal fluid continuous digestion with intestinal epithelial cell absorption and transportation to perform biomimetic treatment on the crustacean aquatic products; detecting the condition that the treated sample induces immune cells to generate allergic media so as to evaluate the allergenicity of the crustacean aquatic products; the method is simple and easy to operate, and has strong universality.)

1. A bionic method for evaluating the sensitization of crustacean aquatic products is characterized by comprising the following steps:

(1) Bionic digestion: treating the edible part of the crustacean aquatic product by an in vitro gastrointestinal fluid continuous digestion method to obtain a continuous digestion product;

(2) Bionic transfer: adding the continuous digestion product into a transfer chamber containing small intestinal epithelial mucosa Caco-2 cells to carry out intestinal cell absorption and transfer to obtain a transfer solution;

(3) In vitro evaluation: the sensitization of the transport fluid was evaluated using the LAD2 human mast cell degranulation assay.

2. The method for biomimetically assessing sensitization of crustacean aquatic products according to claim 1, wherein in step (1), said in vitro gastrointestinal fluid continuous digestion method comprises:

Simulating digestion in the stomach for 0.5-1.5 h;

Simulating digestion in the intestines for 2-6 h.

3. the method for biomimetic evaluation of the sensitization of crustacean aquatic products according to claim 2, wherein said simulated gastric digestion is: edible parts of crustacean aquatic products are processed according to the following steps of: adding the protein into a pepsin liquid with the pH of 1.0-2.0 according to the proportion of 1: 25-1: 100(w/w), placing the mixture in a shaking table at 37 ℃ for digestion for 0.5-1.5 h, adjusting the pH to 7.2-7.4, stopping digestion in the stomach, and obtaining a stomach digestion product; the simulated intestinal digestion is: trypsin and chymotrypsin mixtures were mixed according to protease: adding the protein into the gastric digestion product according to the ratio of 1: 100-1: 300(w/w), placing the gastric digestion product into a shaker at 37 ℃ for digestion, taking out the gastric digestion product after 2-6 h, and inactivating the enzyme in boiling water bath for 5min to obtain the continuous digestion product.

4. The method for biomimetically assessing the sensitization of crustacean aquatic products according to claim 1, wherein said step (2) comprises: inoculating human small intestine epithelial mucosa cell Caco-2 into 24-pore plate transport chamber, and pre-culturing to transmembrane resistance of more than 500 Ω/cm²constructing an intestinal epithelial monolayer cell transport model; and adding the continuous digestion product from the top side inner chamber, adding a cell-free buffer solution to the substrate side, incubating in a cell culture box at 37 ℃ for 2-6 h, and collecting a substrate side transfer solution to obtain the transfer solution.

5. the method according to claim 1, wherein in step (3), the transfer solution is added to LAD2 cells sensitized with the serum of the crustacean allergic patient, incubated, centrifuged, the supernatant is collected and the cells are lysed, and the cell degranulation is detected to evaluate the sensitization of the transfer solution.

6. The biomimetic assessment method for the sensitization of crustacean aquatic products according to claim 5, wherein in the step (3), the incubation time is 0.5-2.0 h.

Technical Field

the invention relates to the technical field of food detection, in particular to a bionic evaluation method for sensitization of class A aquatic products.

Background

due to the rich protein content and delicious taste, the crustacean aquatic products always occupy a seat on the dining table of human beings. The food and agriculture organization of the united nations showed that the consumption of the crustacean per capita reaches 1.7 kg/year in an investigation report of 2009. The consumption and the processing amount of the crustacean aquatic products in China are steadily increased year by year, and the total amount of the aquatic products in 2017 reaches 2196.3 ten thousand tons. However, the abundant protein stores contain a considerable variety of allergenic proteins in addition to the high-quality proteins that supply body energy.

The sensitization of a novel protein is routinely evaluated internationally using pepsin resistance, serum binding capacity in allergic patients and models of cellular or mouse allergy and is used as a standard for the identification of food allergens. In real life, however, the body takes in whole food rather than a single protein; the whole food comprises other components such as carbohydrate, protein and lipid, and complex interaction occurs between the components in the thermal processing process of the food, such as degradation and polymerization of protein. After processed food enters an organism and is digested by a series of enzymes of the gastrointestinal tract, large immunogenic peptide and complete protein can be absorbed through the lumen of the small intestine to trigger immune cells of mucosa, so that the whole anaphylactic reaction generating mechanism is started, and the organism has multiple system symptoms such as vomiting, diarrhea, urticaria and even shock.

therefore, the complex internal environment of the body and the participation of immune cells cause the internationally recognized research mode of the single purified protein to have great limitation, and the digestive absorption condition of the gastrointestinal tract of the body cannot be comprehensively reproduced; the influence of the digested food on the immune cells of the organism cannot be measured, and the correct measurement of the sensitization condition of the complex crustacean aquatic product matrix after multi-component ingestion can also have certain influence.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a bionic method for evaluating the sensitization of crustacean aquatic products, which evaluates the sensitization of crustacean aquatic products by detecting digestion, absorption and transportation after food is taken into an organism and stimulating immune cells to generate an allergic medium. The method is simple and easy to operate and has strong universality.

therefore, in one aspect of the invention, the invention provides a method for bionically evaluating the sensitization of crustacean aquatic products, which comprises the following steps:

(1) Bionic digestion: treating the edible part of the crustacean aquatic product by an in vitro gastrointestinal fluid continuous digestion method to obtain a continuous digestion product;

(2) Bionic transfer: adding the continuous digestion product into a transfer chamber containing small intestinal epithelial mucosa Caco-2 cells to carry out intestinal cell absorption and transfer to obtain a transfer solution;

(3) In vitro evaluation: the sensitization of the transport fluid was evaluated using the LAD2 human mast cell degranulation assay.

According to the method for bionically evaluating the allergenicity of the crustacean aquatic products, an intestinal epithelial cell model and an immune cell model are innovatively combined outside a gastrointestinal tract digestion model recognized at home and abroad, and the whole process of allergenicity of an organism due to the fact that a digestive tract barrier and a cell barrier are reproduced after allergic components in food enter the organism is further caused; compared with a conventional single protein or component digestion and sensitization detection model, the method can accurately measure the sensitization condition of the crustacean aquatic product matrix after multi-component ingestion; the method is also suitable for the sensibility assessment of the whole food after being ingested by the body.

In addition, the method for biomimetically evaluating the sensitization of the crustacean aquatic product provided by the embodiment of the invention can also have the following additional technical characteristics:

According to an embodiment of the present invention, in step (1), the in vitro gastrointestinal fluid continuous digestion method comprises:

Simulating digestion in the stomach for 0.5-1.5 h;

Simulating digestion in the intestines for 2-6 h.

According to an embodiment of the present invention, the simulated gastric digestion is: edible parts of crustacean aquatic products are processed according to the following steps of: adding the protein into a pepsin liquid with the pH of 1.0-2.0 according to the proportion of 1: 25-1: 100(w/w), placing the mixture in a shaking table at 37 ℃ for digestion for 0.5-1.5 h, adjusting the pH to 7.2-7.4, stopping digestion in the stomach, and obtaining a stomach digestion product; the simulated intestinal digestion is: trypsin and chymotrypsin mixtures were mixed according to protease: adding the protein into the gastric digestion product according to the ratio of 1: 100-1: 300(w/w), placing the gastric digestion product into a shaker at 37 ℃ for digestion, taking out the gastric digestion product after 2-6 h, and inactivating the enzyme in boiling water bath for 5min to obtain the continuous digestion product.

According to an embodiment of the present invention, the step (2) includes: inoculating human small intestine epithelial mucosa cell Caco-2 into 24-pore plate transport chamber, and pre-culturing to transmembrane resistance of more than 500 Ω/cm²constructing an intestinal epithelial monolayer cell transport model; and adding the continuous digestion product from the top side inner chamber, adding a cell-free buffer solution to the substrate side, incubating in a cell culture box at 37 ℃ for 2-6 h, and collecting a substrate side transfer solution to obtain the transfer solution.

According to an embodiment of the present invention, in step (3), the transport solution is added to LAD2 cells that have been sensitized with serum from a shell-allergy-free patient, incubated, centrifuged, the supernatant collected and the cells lysed, and the cells are tested for degranulation to assess the sensitization of the transport solution.

According to the embodiment of the invention, in the step (3), the incubation time is 0.5-2.0 h.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a graph showing the sensitization of Penaeus vannamei evaluated by the LAD2 cell model of example 1;

FIG. 2 is a LAD2 cell model of example 2 to evaluate the sensitization of Scylla paramamosain.

Detailed Description

the technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

According to the embodiment of the invention, the invention provides a bionic evaluation method for the allergenicity of crustacean aquatic products, which comprises the following steps:

(1) Bionic digestion: the edible part of the crustacean aquatic product is treated by an in vitro gastrointestinal fluid continuous digestion method to obtain a continuous digestion product. The shellfish aquatic product can be directly eaten fresh or cooked; the edible part of the edible part comprises but is not limited to a treatment mode of removing heads, shells and the like. Edible part of crustacean aquatic products is treated with protease: adding the protein into a pepsin liquid with the pH of 1.0-2.0 according to the proportion of 1: 25-1: 100(w/w), placing the mixture in a shaking table at 37 ℃ for digestion for 0.5-1.5 h, adjusting the pH to 7.2-7.4, stopping digestion in the stomach, and obtaining a stomach digestion product; then, the trypsin and chymotrypsin mixture was mixed according to protease: adding the protein into the gastric digestion product according to the ratio of 1: 100-1: 300(w/w), placing the gastric digestion product into a shaker at 37 ℃ for digestion, taking out the gastric digestion product after 2-6 h, and inactivating the enzyme in boiling water bath for 5min to obtain the continuous digestion product.

(2) Bionic transfer: will be describedThe continuous digestion product is added into a transfer chamber containing Caco-2 cells of small intestinal epithelial mucosa (the cells are purchased from ATCC) to carry out intestinal cell absorption and transfer, and transfer liquid is obtained. Inoculating human small intestine epithelial mucosa cell Caco-2 into 24-pore plate transport chamber, and pre-culturing to transmembrane resistance of more than 500 Ω/cm²Constructing a compact intestinal epithelial monolayer cell transport model; and adding a gastrointestinal fluid continuous digestion product from the inner chamber at the top side, adding a cell-free buffer solution at the substrate side, incubating for 2-6 h in a cell culture box at 37 ℃ to complete absorption and transportation, and collecting a substrate side transportation fluid to obtain the transportation fluid.

3) In vitro evaluation: and evaluating the sensitization of the crustacean aquatic products after biomimetic digestion, transportation and absorption by adopting an LAD2 degranulation test of human mast cells. Mast cells are important immune cells in the human body and play an important role in the development of allergic reactions. The method comprises the steps of taking human mast cells LAD2 (purchased from ATCC) as immune cell representatives, establishing a degranulation model, adding basolateral external liquid subjected to gastrointestinal fluid continuous digestion and intestinal epithelial cell transfer into LAD2 cells sensitized by serum of a shell allergy patient, incubating and stimulating for 0.5-2.0 hours in a cell culture box at 37 ℃, and detecting the condition that the intestinal epithelial absorption and transfer liquid stimulates LAD2 cells to generate an allergic medium.

therefore, crustacean aquatic products are subjected to biomimetic treatment by a method of combining in-vitro gastrointestinal fluid continuous digestion with intestinal epithelial cell absorption and transportation; and then, the allergenicity of the crustacean aquatic products is evaluated by detecting the condition that the treated sample induces immune cells to generate allergic mediums, so that the allergenicity of the crustacean aquatic products after multi-component ingestion in matrixes can be accurately measured. The invention completely simulates a series of physiological and biochemical reactions experienced by the human body after the human body ingests the thermally processed crustacean aquatic products, comprehensively considers the interaction among the components in the complex food and the evaluation of the sensitization under the special microenvironment in the body, provides scientific basis for the sensitization detection of the substrate of the whole food, and also provides theoretical basis for the production and processing of the low-sensitization food. The method is simple and easy to operate and has strong universality.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.