Method for evaluating collagen content in body wall of stichopus japonicus based on intestinal choline

Abstract

The invention discloses a method for evaluating the collagen content of the body wall of stichopus japonicus based on intestinal choline, which comprises the following steps: (1) stimulating the stichopus japonicus to spit viscera, and then separating the intestinal tract; and (2) measuring the content of choline in the intestinal tract of the stichopus japonicus, and obtaining the collagen content of the body wall of the stichopus japonicus according to the correlation model of the choline content and the collagen content of the body wall of the stichopus japonicus.

Description

Technical Field

[0001] The invention relates to a method for evaluating the collagen content of a sea cucumber body wall, and in particular to a method for evaluating the collagen content of a sea cucumber body wall based on intestinal choline. Background Art

[0002] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals. As a marine invertebrate, sea cucumbers are rich in collagen protein (40.7-63.3%) in their body walls. Therefore, sea cucumber collagen is one of the main sources of high-quality marine collagen to replace mammalian collagen. It has unique properties such as biocompatibility, immunogenicity, high biodegradability and cell growth potential, so it has a wide range of uses.

[0003] Currently, the method for determining collagen in the body wall of sea cucumbers is: obtaining a body wall sample of sea cucumbers and determining the collagen content using a hydroxyproline colorimetric method.

[0004] However, the above method has the following disadvantages:

[0005] (1) Since this method requires a large amount of sea cucumber body wall sample for determining collagen content, it is often necessary to kill the sea cucumber to obtain its body wall, resulting in the inability to retain sea cucumbers with high collagen content for breeding; even if the sea cucumbers are not killed, the risk of disease will increase after taking part of their body wall, resulting in a decrease in survival rate. Therefore, the existing technology generally adopts the method of killing the sea cucumber to obtain its body wall; therefore, the current breeding method for high collagen content traits is to take the body wall of the seed ginseng after laying eggs to determine the collagen content, so as to select eggs with high collagen content for breeding, but this method cannot continuously retain high-quality seed ginseng.

[0006] (2) This method requires the use of a large amount of hazardous chemical sulfuric acid. Summary of the invention

[0007] The present invention aims to provide a method for evaluating the collagen content of sea cucumber body wall based on intestinal choline, so as to ensure the retention and breeding of sea cucumber with high collagen content.

[0008] To achieve the above objectives, the present invention adopts the following technical solutions.

[0009] A method for evaluating the collagen content of sea cucumber body wall based on intestinal choline, comprising the following steps:

[0010] (1) Stimulate the sea cucumber to spit out the internal organs and then separate the intestines;

[0011] (2) The choline content in the intestinal tract of the sea cucumber is determined, and the collagen content of the sea cucumber body wall is obtained based on the correlation model between the choline content and the collagen content of the sea cucumber body wall.

[0012] The correlation model between the choline content and the collagen content of the sea cucumber body wall of the present invention is as follows:

[0013] Y=11.856-13.373 / X

[0014] Wherein, Y is the collagen content of the sea cucumber body wall, which refers to the mass of collagen in each gram of sea cucumber body wall (wet weight), and the unit is ug / g; X is the choline content in the sea cucumber intestine, which refers to the mass of choline in each gram of sea cucumber intestine (wet weight), and the unit is ug / g.

[0015] The present invention finds that the choline content in the intestinal tract of sea cucumbers is significantly positively correlated with the collagen content in their body wall. After a large number of experimental verifications, a correlation model between the choline content and the collagen content in the body wall of sea cucumbers is obtained, so that the collagen content in the body wall of sea cucumbers can be evaluated based on the choline content. In addition, sea cucumbers have a strong regeneration ability. After vomiting out the internal organs, they can regrow the lost parts by dividing cells and recover health in a very short time. Therefore, the present invention can evaluate the collagen content in the body wall of sea cucumbers by choline content, avoid killing sea cucumbers, and retain individuals with high nutritional quality traits, which is beneficial to scientific research and breeding.

[0016] In step (1), any method of stimulating the sea cucumber to spit out its internal organs can be used to achieve the purpose of the present invention; specifically, one of the following methods can be used to stimulate the sea cucumber to spit out its internal organs:

[0017] ① In a dry state, continuously vibrate the sea cucumber for 10 to 30 minutes;

[0018] ② The seawater environment where the sea cucumber is located undergoes a temperature or salinity change within 5 minutes, with the temperature changing by 5-10°C and the salinity changing by 5-10;

[0019] ③ Immerse the sea cucumber in a 0.8 mol / L potassium chloride solution at a temperature of 18-22°C and wait for 10-15 minutes.

[0020] In step (2), any method for determining the choline content in the intestinal tract of sea cucumbers can be used. As a specific embodiment, in step (2), the choline content in the intestinal tract of sea cucumbers is determined by enzyme colorimetry.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention finds that the choline content in the intestinal tract of sea cucumbers is significantly positively correlated with the collagen content in their body wall, determines the correlation model between the choline content and the collagen content in the body wall of sea cucumbers, and proposes for the first time a method for evaluating the collagen content in the body wall of sea cucumbers based on intestinal choline. The present invention can predict and evaluate the collagen content in the body wall of sea cucumbers by the choline content in the intestinal tract of sea cucumbers, and can obtain the collagen content in the body wall of sea cucumbers without killing the sea cucumbers, thereby screening out sea cucumbers with different collagen contents, retaining sea cucumbers with high nutritional quality traits, and solving the breeding problem of high nutritional quality sea cucumbers. The method is scientific and reasonable, and has broad development and application prospects.

[0023] (2) In the process of model construction, the correlation analysis method is used to determine the relationship between choline content and sea cucumber body wall collagen content, and finally establish an evaluation model of choline content and sea cucumber body wall collagen content according to significance; this method is scientific and has high accuracy and stability. Moreover, compared with the existing method, the present invention avoids the use of hazardous chemical sulfuric acid, ensuring operational safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the correlation model between the choline content in the intestinal tract of sea cucumber and the collagen content in the body wall of sea cucumber. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. The equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0026] In the following examples, the method for determining the choline content in the intestinal tract of sea cucumbers used in the present invention is:

[0027] 1) Preparation of choline hydroxide standard stock solution: Weigh 523.00 mg of choline bitartrate dried to constant weight at 102°C ± 2°C into a 100 mL volumetric flask and dilute to the mark with distilled water.

[0028] 2) Weigh 10 g of the mixed sea cucumber intestinal sample, add 30 mL of hydrochloric acid solution (1 mol / L) into a 100 mL conical flask, place the container containing the sample in a 70°C water bath, add a stopper to mix, hydrolyze for 3 h (shake once every 30 min), and cool; adjust the pH to 3.5-4.0 with sodium hydroxide solution, transfer to a 50 mL volumetric flask, and dilute to the mark with distilled water to obtain the sample solution to be tested.

[0029] 3) Pipette 2mL, 4mL, 6mL, 8mL, and 10mL of choline hydroxide standard stock solution into a 10mL volumetric flask, and dilute to scale with distilled water. Prepare 6 test tubes, one test tube as the reagent blank group (A), and the other five test tubes numbered from 1 to 5 for standard curve preparation, and add reagents according to Table 1; then place the test tubes in a 37℃ water bath for 15 minutes.

[0030] Table 1 Amount of reagents added when preparing the standard curve (unit: mL)

[0031]

[0032]

[0033] Note: The color developer is prepared as follows: 100-120 activity units of choline oxidase, 250-280 activity units of peroxidase, 75-100 activity units of phospholipase D, 15 mg of 4-aminoantipyrine, and 50 mg of phenol are placed in a 100 mL volumetric flask and diluted to the mark with 0.05 mol / L Tris buffer solution.

[0034] 4) Prepare two test tubes (B, C) for the sample solution to be tested, add reagents according to Table 2, cover the test tubes with sealing protective film, and mix well. Place the test tubes in a 37℃ water bath for 15 minutes.

[0035] Table 2 Amount of reagent added when measuring the sample (unit: mL)

[0036]

[0037] 5) Take the sample solution and the standard series solution out of the water bath and cool to room temperature. Measure the absorbance at a wavelength of 505nm using distilled water as a blank;

[0038] A standard curve was prepared with the concentration of the choline standard solution as the horizontal axis and the absorbance value of the standard solution minus the absorbance value of the reagent blank as the vertical axis;

[0039] Calculate the absorbance of the sample solution according to the following formula:

[0040] The absorbance of the sample solution to be tested (static absorbance) = total absorbance (absorbance of test tube C) - absorbance of reagent (absorbance of test tube A) - absorbance of sample (absorbance of test tube B)

[0041] Then, according to the absorbance of the sample solution to be tested, the choline content in the intestinal tract (wet weight) is calculated using the standard curve.

[0042] In the following examples, the steps of determining the collagen content in the body wall of sea cucumbers using the existing method are as follows:

[0043] 1) Preparation of standard stock solution (0.5 g / mL): Accurately weigh 0.0560 g of hydroxyproline standard and dissolve it in a 100 mL volumetric flask, add 1 drop of 3 mol / L sulfuric acid solution, make up to volume with water, and store at 4°C for later use.

[0044] 2) Determination of standard curve: Take 2 mL of 0.5 g / mL hydroxyproline standard stock solution and dilute it in a 100 mL volumetric flask to obtain a 10 ug / mL hydroxyproline solution. Then add 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of 10 ug / mL hydroxyproline solution to 50 mL hydrolysis tubes to make up to 4 mL. The concentrations of hydroxyproline in the 6 hydrolysis tubes are 0 ug / mL, 5 ug / mL, 10 ug / mL, 20 ug / mL, 30 ug / mL and 40 ug / mL, respectively.

[0045] Add 2.0 mL of chloramine T to each hydrolysis tube, shake well and let stand at room temperature for 20 min, then add 2.0 mL of color developer and shake well, cover with stopper, and quickly place in a 60°C water bath for 20 min. Take out the hydrolysis tube and cool it under tap water for more than 3 min. After letting stand at room temperature for 30 min, measure its absorbance at 558 nm using water as a reference, and draw a standard curve of absorbance and hydroxyproline concentration.

[0046] 3) Determination of collagen content in the body wall of sea cucumbers: Use sterile scissors to cut open the sea cucumbers, use sterile water to clean the inside of the sea cucumbers, use a scalpel to cut off the body wall at the same position on the back of the sea cucumbers, take 10g of body wall samples, freeze them with liquid nitrogen, grind the solid sample into powder with a mortar, accurately weigh 1.0g into a 50mL digestion tube, add 30mL of sulfuric acid solution (3mol / L), and then fill each hydrolysis tube with pure nitrogen near the liquid surface, blow out the air with nitrogen, and tighten the screw cap under the inflated state. Place the hydrolysis tube in a 105℃ forced air drying oven at a constant temperature for 12h. Filter the solution with filter paper into a 250mL volumetric flask while it is hot, wash the filter paper repeatedly with 15mL of sulfuric acid solution, combine the filtrate, cool to room temperature, and make up to volume with water. Pipette 30 mL of the diluted hydrolyzate into a 100 mL beaker, adjust the pH to neutral with 6 mol / L sodium hydroxide solution and 1 mol / L sodium hydroxide solution, filter into a 250 mL volumetric flask, wash the beaker and filter paper with water several times, combine the filtrate, and dilute to the mark.

[0047] Then add 2.0mL of chloramine T, shake well and let stand at room temperature for 20 minutes, then add 2.0mL of color developer, shake well and cover with a stopper, quickly place in a 60°C water bath for 20 minutes, and then cool under tap water for more than 3 minutes; after standing at room temperature for 30 minutes, the sample is measured for absorbance at 558nm, and the quantitative value is compared with the standard curve. The content of hydroxyproline in the sample is obtained by multiplying by the dilution multiple, and the content of collagen in the sample is calculated according to the following formula.

[0048] Y=X×11.1 / M

[0049] Where: Y is the content of collagen in the sample (wet weight), unit is ug / g;

[0050] X is the content of hydroxyproline in the sample (wet weight), in ug / g;

[0051] M is the mass of the sample (wet weight), in g.

[0052] Example 1 Determination of the correlation model between intestinal choline content and sea cucumber body wall collagen content.

[0053] In this example, 97 sea cucumbers from the Bohai Sea were selected; the sea cucumbers were stimulated to spit out their internal organs, the intestines were separated, and the choline content in the intestines was measured; then the collagen content in the body wall of the sea cucumbers was measured according to the above-mentioned existing method. The results are shown in Table 3.

[0054] like Figure 1 As shown, a scatter plot was drawn with choline content as the horizontal axis and collagen content as the vertical axis, and a linear fit was performed to obtain a correlation model between the choline content and the collagen content of the sea cucumber body wall as follows:

[0055] Y=11.856-13.373 / X

[0056] Wherein, Y is the collagen content of the sea cucumber body wall, which refers to the mass of collagen in each gram of sea cucumber body wall (wet weight), and the unit is ug / g; X is the choline content in the sea cucumber intestine, which refers to the mass of choline in each gram of sea cucumber intestine (wet weight), and the unit is ug / g.

[0057] The model R 2 The value was 0.386, and the P value was <0.001, which had a high prediction accuracy.

[0058] Table 3 Choline content in the intestine and collagen content in the body wall of sea cucumber

[0059]

[0060]

[0061]

[0062]

[0063] Example 2 Verification of the evaluation method of the present invention

[0064] (1) Five sea cucumbers from different breeding areas were selected and immersed in a potassium chloride solution with a concentration of 0.8 mol / L at a temperature of 18-22° C. for 10-15 min. The sea cucumber viscera vomited out during the processing were collected and the sea cucumber intestines were separated.

[0065] (2) The choline content in the intestinal tract of sea cucumbers was determined. According to the correlation model between the choline content and the collagen content in the body wall of sea cucumbers, the collagen content in the body wall of sea cucumbers was calculated. The results are shown in Table 4:

[0066] The correlation model between choline content and collagen content in the body wall of sea cucumber is:

[0067] Y=11.856-13.373 / X

[0068] Wherein, Y is the collagen content of the sea cucumber body wall, which refers to the mass of collagen in each gram of sea cucumber body wall (wet weight), and the unit is ug / g; X is the choline content in the sea cucumber intestine, which refers to the mass of choline in each gram of sea cucumber intestine (wet weight), and the unit is ug / g.

[0069] (3) The existing method was used to directly determine the collagen content of the body wall of the selected sea cucumbers. The results are shown in Table 4.

[0070] Table 4 Collagen content of sea cucumber body wall measured by the method of the present invention and the existing method

[0071]

[0072]

[0073] Through calculation, for the sea cucumbers in the above-mentioned 6 regions, the Pearson correlation between the collagen content measured by the method of the present invention and the collagen content measured by the existing method were 0.942, 0.901, 0.913, 0.990, 0.907 and 0.926, respectively, indicating that the model of the present invention has the ability to predict and evaluate the collagen content of the sea cucumber body wall with high accuracy.

Claims

1. A method for evaluating the collagen content of sea cucumber body wall based on intestinal choline, characterized in that: The following steps are involved: (1) Stimulate the sea cucumber to spit out the internal organs and then separate the intestines; (2) determining the choline content in the intestinal tract of the sea cucumber, and obtaining the collagen content of the sea cucumber body wall according to a correlation model between the choline content and the collagen content of the sea cucumber body wall; The correlation model between the choline content and the collagen content of the sea cucumber body wall is as follows: Y=11.856-13.373 / X Wherein, Y is the collagen content of the sea cucumber body wall, which refers to the mass of collagen in each gram of the sea cucumber body wall, and the unit is ug / g; X is the choline content in the sea cucumber intestine, which refers to the mass of choline in each gram of the sea cucumber intestine, and the unit is ug / g.

2. The method for evaluating the collagen content of sea cucumber body wall based on intestinal choline according to claim 1, characterized in that: Use one of the following methods to stimulate the sea cucumber to spit out its internal organs: ① In a dry state, continuously vibrate the sea cucumber for 10 to 30 minutes; ② The seawater environment where the sea cucumber is located undergoes a temperature or salinity change within 5 minutes, with the temperature changing by 5-10°C and the salinity changing by 5-10; ③ Immerse the sea cucumber in a 0.8 mol / L potassium chloride solution at a temperature of 18-22°C and wait for 10-15 minutes.

3. The method for evaluating the collagen content of sea cucumber body wall based on intestinal choline according to claim 2, characterized in that: In step (2), the choline content in the intestinal tract of the sea cucumber is determined by an enzyme colorimetric method.

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