Method for evaluating polysaccharide content in body wall of stichopus japonicus based on octopamine in intestinal tract

Abstract

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

Description

Technical Field

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

[0002] Sea cucumber polysaccharide is a sulfated polysaccharide in the body wall of sea cucumbers. It has multiple biological activities and pharmacological effects. It not only has strong free radical scavenging and reducing abilities, but also has multiple medicinal potentials such as anti-thrombotic, anti-inflammatory, anti-cancer and hypoglycemic. As a high-value biologically active factor unique to sea cucumbers, sea cucumber polysaccharide is an important manifestation of the medicinal value of sea cucumbers.

[0003] At present, the method for determining the polysaccharide content of the sea cucumber body wall is to obtain a sea cucumber body wall sample and directly determine the polysaccharide content using the phenol-sulfuric acid method. The above method has the following main disadvantages:

[0004] (1) Since the method for determining the polysaccharide content requires a large amount of sea cucumber body wall sample (more than 10 g), it is often necessary to kill the sea cucumber to obtain its body wall, and the sea cucumber with high polysaccharide content cannot be retained for breeding; even if the sea cucumber is not killed, the risk of disease in the sea cucumber will increase after taking part of its body wall, resulting in a decrease in survival rate. Therefore, the prior art generally adopts the method of killing the sea cucumber to obtain its body wall;

[0005] Therefore, the current breeding method for high polysaccharide content traits is to measure the polysaccharide content of the body wall of the ginseng after it lays eggs, so as to select eggs with high polysaccharide content for breeding. However, this method cannot continuously retain high-quality ginseng.

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

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

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

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

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

[0011] (2) The content of octopamine in the intestine of sea cucumber is determined, and the polysaccharide content of the sea cucumber body wall is obtained according to the correlation model between the octopamine content and the polysaccharide content of the sea cucumber body wall.

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

[0013] Y=0.039+0.001X-0.000004X2,

[0014] Wherein, Y is the polysaccharide content of the sea cucumber body wall, which refers to the percentage of polysaccharide in the dry weight of the sea cucumber body wall, in %; X is the octopamine content in the sea cucumber intestine, which refers to the mass of octopamine per gram of sea cucumber intestine (wet weight), in ug / g.

[0015] Octopamine is a marine bioactive substance present in the intestinal tract of sea cucumbers. It is usually present in high levels in invertebrates and plays a vital role in regulating multiple metabolic pathways. The present invention finds that the octopamine content in the intestinal tract of sea cucumbers is significantly positively correlated with the polysaccharide content in its body wall. After a large number of experimental verifications, a correlation model between the octopamine content and the polysaccharide content in the body wall of sea cucumbers is obtained, so that the polysaccharide content in the body wall of sea cucumbers can be evaluated based on the octopamine content.

[0016] 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 polysaccharide content of the sea cucumber body wall by the octopamine content, avoid killing the sea cucumber to determine its body wall polysaccharide content, and can retain individuals with high nutritional quality traits, which is beneficial to scientific research and breeding.

[0017] 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:

[0018] ① In a dry state, continuously vibrate the sea cucumber for 10 to 30 minutes;

[0019] ② The seawater environment where the sea cucumber is located undergoes a temperature or salinity change within 5 minutes, with the temperature change range being 5 to 10°C and the salinity change range being 5 to 10;

[0020] 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.

[0021] In step (2), the content of octopamine in the intestinal tract of sea cucumber is determined by high performance liquid chromatography.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention finds that the octopamine content in the intestine of sea cucumbers is significantly positively correlated with the polysaccharide content in their body wall, determines the correlation model between the octopamine content and the polysaccharide content in the body wall of sea cucumbers, and proposes for the first time a method for evaluating the polysaccharide content in the body wall of sea cucumbers based on intestinal octopamine. The present invention can evaluate the polysaccharide content in the body wall of sea cucumbers by the octopamine content, and can obtain the polysaccharide content in the body wall of sea cucumbers without killing the sea cucumbers, thereby screening out sea cucumbers with different polysaccharide 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.

[0024] (2) In the process of model construction, the correlation analysis method is used to determine the relationship between the octopamine content and the polysaccharide content of the sea cucumber body wall, and finally an evaluation model of the octopamine content and the polysaccharide content of the sea cucumber body wall is established according to the significance; the method is scientific and has high accuracy and stability. Moreover, compared with the existing method, the present invention avoids the use of hazardous chemicals sulfuric acid, ensuring the safety of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the correlation model between the octopamine content in the intestine of sea cucumber and the polysaccharide content in the body wall of sea cucumber. DETAILED DESCRIPTION

[0026] 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.

[0027] In the following examples, the method for determining the octopamine content in the intestinal tract of sea cucumber is:

[0028] 1) Preparation of octopamine standard stock solution: Take a 25 mL brown volumetric flask, add 0.25 g of octopamine standard, dissolve it with ultrapure water and make up to volume to prepare an octopamine standard stock solution with a concentration of 10 mg / mL.

[0029] 2) Chromatographic conditions: AQ-C18 liquid chromatography column (4.6×250 mm, 5 μm); mobile phase: acetonitrile: 0.02 mol / L sodium dihydrogen phosphate = 4:6, pH = 7.5; column temperature: 30°C; flow rate: 0.8 mL / min; injection volume: 10 μL; UV detection wavelength: 284 nm.

[0030] 3) Accurately pipette an appropriate amount of the octopamine standard stock solution, use the mobile phase to prepare a standard series of solutions with octopamine concentrations of 0.1, 0.5, 1.0, 1.5, 2.0 and 2.5 mg / mL, and store in the dark. Perform analysis by high performance liquid chromatography to draw a standard curve of octopamine concentration.

[0031] 4) Determination of octopamine content in the intestinal tract of sea cucumber: accurately weigh 1.0g of sea cucumber intestinal homogenate, place it in a brown centrifuge tube, add 25mL 5% trichloroacetic acid, shake and extract at 30°C, centrifuge at 5000r / min for 10min, and collect the supernatant; accurately transfer 5mL of supernatant to a 15mL brown centrifuge tube, adjust the solution pH to 12, add 5mL of a mixed solution of n-butanol / chloroform (1:1) in equal proportions, shake and extract, continue to centrifuge at 5000r / min for 10min, and collect the supernatant. The obtained extract is concentrated, added with 0.1mol / L hydrochloric acid for shaking, and passed through a 0.45μm microporous filter for testing. According to the above chromatographic conditions, the test is carried out, and each sample is analyzed 3 times, and the octopamine content in the intestinal tract (wet weight) is calculated according to the octopamine concentration standard curve.

[0032] In the following examples, the steps of determining the polysaccharide content of the sea cucumber body wall using the existing method are:

[0033] (1) 0, 0.02, 0.04, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.2 mL of standard glucose solution (0.2 mg / mL) were respectively placed in test tubes and made up to 0.2 mL with deionized water; 0.1 mL of 6% phenol solution was added to each test tube, and then 0.5 mL of concentrated sulfuric acid was quickly added perpendicular to the liquid surface. After standing for 10 min, the absorbance value was measured at 490 nm; a standard curve was drawn with the mass of glucose as the horizontal axis and the absorbance value as the vertical axis.

[0034] (2) Use sterile scissors to cut open the sea cucumber, use sterile water to clean the inside of the sea cucumber, use a scalpel to cut the body wall at the same position on the back of the sea cucumber, and take 20g of body wall sample; place the collected body wall sample in an oven (80°C) to dry thoroughly, then grind it with a mortar, accurately weigh 2mg and dissolve it in 2mL of deionized water, shake it for 5min, and after it is fully dissolved, add 1mL of 6% phenol solution to each tube of test solution, and then quickly add 5mL of concentrated sulfuric acid perpendicular to the liquid surface. After standing for 10min, measure the absorbance value at 490nm; according to the standard curve, obtain the polysaccharide content of the sea cucumber body wall (dry weight).

[0035] Example 1 Determination of the correlation model between octopamine content and sea cucumber body wall polysaccharide content.

[0036] In this example, 118 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 octopamine content in the intestines was measured; then the polysaccharide content in the body wall of the sea cucumbers was measured according to the above-mentioned existing method. The results are shown in Table 1.

[0037] like Figure 1 As shown, a scatter plot was drawn with the octopamine content as the horizontal axis and the polysaccharide content as the vertical axis, and a linear fit was performed to obtain a correlation model between the octopamine content (wet weight) and the polysaccharide content of the sea cucumber body wall as follows:

[0038] Y=0.039+0.001X-0.000004X2

[0039] Wherein, Y is the polysaccharide content of the sea cucumber body wall, which refers to the percentage of polysaccharide in the dry weight of the sea cucumber body wall, in %; X is the octopamine content in the sea cucumber intestine, which refers to the mass of octopamine per gram of sea cucumber intestine (wet weight), in ug / g.

[0040] The model R 2 The value was 0.366, and the P value was <0.001, which had a high prediction accuracy.

[0041] Table 1 Octopamine content in the intestine and polysaccharide content in the body wall of sea cucumber

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] Example 2 Verification of the evaluation method of the present invention

[0048] (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.

[0049] (2) The content of octopamine in the intestinal tract of sea cucumbers was determined. According to the correlation model between the octopamine content and the polysaccharide content of the sea cucumber body wall, the polysaccharide content of the selected sea cucumber body wall was calculated. The results are shown in Table 2:

[0050] The correlation model between octopamine content and sea cucumber body wall polysaccharide content is:

[0051] Y=0.039+0.001X-0.000004X2

[0052] Wherein, Y is the polysaccharide content of the sea cucumber body wall, which refers to the percentage of polysaccharide in the dry weight of the sea cucumber body wall, in %; X is the octopamine content in the sea cucumber intestine, which refers to the mass of octopamine per gram of sea cucumber intestine (wet weight), in ug / g.

[0053] (3) The existing method was used to directly determine the polysaccharide content of the body wall of the selected sea cucumbers. The results are shown in Table 2.

[0054] Table 2 The content of polysaccharide in the body wall of sea cucumber determined by the method of the present invention and the existing method

[0055]

[0056]

[0057] Through calculation, for the sea cucumbers in the above-mentioned 6 regions, the Pearson correlation between the polysaccharide content measured by the method of the present invention and the polysaccharide content measured by the existing method were 0.995, 0.970, 0.914, 0.944, 0.918 and 0.900, respectively, indicating that the model of the present invention has the ability to predict and evaluate the polysaccharide content of the sea cucumber body wall with high accuracy.

Claims

1. A method for evaluating the polysaccharide content of sea cucumber body wall based on intestinal octopamine, 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 content of octopamine in the intestinal tract of the sea cucumber, and obtaining the polysaccharide content of the sea cucumber body wall according to a correlation model between the octopamine content and the polysaccharide content of the sea cucumber body wall; The correlation model between the octopamine content and the sea cucumber body wall polysaccharide content is as follows: Y=0.039+0.001X-0.000004X2, Wherein, Y is the polysaccharide content of the sea cucumber body wall, which refers to the percentage of polysaccharide in the dry weight of the sea cucumber body wall, in %; X is the octopamine content in the sea cucumber intestine, which refers to the mass of octopamine per gram of sea cucumber intestine, in ug / g.

2. The method for evaluating the content of polysaccharides in the body wall of sea cucumbers based on intestinal octopamine 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 temperature or salinity changes within 5 minutes, with the temperature changing by 5 to 10°C and the salinity changing by 5 to 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 content of polysaccharides in the body wall of sea cucumbers based on intestinal octopamine according to claim 2, characterized in that: The content of octopamine in the intestine of sea cucumber was determined by high performance liquid chromatography.

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