Method and kit for evaluating the quality of akkermansia muciniphila products

Abstract

The present application relates to mucinophilic akkermansia product quality grading evaluation method and kit. The method detects the content of P9 protein, Amuc_1100 protein and Amuc_1409 protein, calculates the comprehensive index of functional protein EPI=0.4×CP9+0.35×CAmuc1100+0.25×CAmuc1409, and determines the product quality grade: S level (EPI is greater than or equal to 80), A level (60 is less than EPI and is less than or equal to 80), B level (40 is less than EPI and is less than or equal to 60), C level (EPI is less than 40). EPI is greater than or equal to 40 and a single protein is not less than 3 ng / mL is qualified product. The present application establishes a scientific and quantitative quality grading evaluation system for AKK product.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method and reagent kit for quality grading and evaluation of Akkermansia myxophilus products. Background Technology

[0002] With the rapid development of the market for metabiotic products related to AKK (Akkermansia muciniphila), the industry's demand for product quality evaluation is becoming increasingly urgent. Currently, most AKK products on the market use traditional "bacterial count indicators" (CFU / g) or viable cell counts to indicate product quality. However, numerous studies have shown that the core efficacy of AKK bacteria, such as metabolic regulation, immune enhancement, and intestinal barrier repair, does not solely depend on the quantity of the bacteria themselves, but is jointly determined by the expression levels of key functional proteins secreted by the bacteria—including the metabolic regulation core protein P9, the outer membrane functional protein Amuc_1100, and the intestinal barrier repair protein Amuc_1409. Therefore, traditional "bacterial count evaluation" cannot scientifically reflect the true efficacy of the product.

[0003] While some companies in the industry have begun to explore the detection of single functional proteins (for example, Suncon has launched a detection kit for P9 protein, and brands such as GT&G have proposed using "functional protein detection" as a supplementary evaluation dimension), current technology still lacks a quality grading system that can integrate the three core functional proteins—P9, Amuc_1100, and Amuc_1409—into a unified scoring standard. This results in inconsistent quality of AKK bacteria products on the market, a lack of authoritative industry grading standards, and difficulty for downstream brands and consumers to effectively distinguish the true quality of products.

[0004] To address the aforementioned issues, this invention provides a method and kit for quality grading and evaluation of AKK bacteria products based on the Efficacy Protein Index (EPI). This invention objectively classifies products into four grades—S, A, B, and C—by quantifying the relative expression levels of three core proteins, thereby providing the AKK bacteria industry with a unified, scientific, and quantitative standard for quality evaluation and grading. Summary of the Invention

[0005] ① Problem to be solved: Lack of a unified quality grading and evaluation standard for AKK bacteria products.

[0006] ② Technical solution: Establish a four-level quality grading system based on the Efficacy Protein Index (EPI).

[0007] ③ Beneficial effects: Establishing a scientific and quantitative quality grading and evaluation system for AKK bacteria products. Detailed Implementation

[0008] Example 1: Sample Detection

[0009] (1) Sample pretreatment: Take 100 mg of the AKK bacteria sample to be tested, add 1 mL of lysis buffer (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% SDS, and 1× protease inhibitor mixture), vortex to mix, incubate on ice for 30 minutes, sonicate (power 200W, work for 2 seconds and stop for 2 seconds, for a total of 3 minutes), centrifuge at 12,000g at 4℃ for 20 minutes, and take the supernatant as the test solution.

[0010] (2) ELISA detection: Take 50 μL of the test solution and add it to the double antibody sandwich ELISA detection plates of P9 protein, Amuc_1100 protein and Amuc_1409 protein respectively. Perform incubation, washing, color development and termination of reaction according to the operating procedures of each detection system, and read the OD450 value on the microplate reader.

[0011] (3) Concentration calculation: Plot a standard curve with the logarithm of the concentration of the three protein standards as the x-axis and the OD450 value as the y-axis. Fit the four-parameter Logistic equation and calculate the measured concentration (ng / mL) of the three proteins in each sample.

[0012] Example 2: Result Calculation and Classification

[0013] (1) Data correction: Divide the measured concentrations of the three proteins obtained in Example 1 by the corresponding reference concentrations of the protein standards to obtain the relative content of each protein.

[0014] (2) Calculation of the comprehensive index: The efficacy protein comprehensive index (EPI) is calculated according to the following formula:

[0015] EPI = 0.4 × (CP9 / SP9) × 100 + 0.35 × (CAmuc1100 / SAmuc1100) ×100 + 0.25 × (CAmuc1409 / SAmuc1409) × 100

[0016] CP9, CAmuc1100, and CAmuc1409 represent the measured concentrations (ng / mL) of the three proteins; SP9, SAmuc1100, and SAmuc1409 represent the reference concentrations (ng / mL) of the corresponding protein standards. Weighting coefficients (0.4, 0.35, and 0.25) were determined based on the functional importance of each protein.

[0017] (3) Grading: Based on the calculated EPI value, the quality grade of the product is determined by referring to the grading standard of Example 3.

[0018] Example 3: Result Judgment and Quality Grading Standards

[0019] Based on the EPI value, the product quality grading standards are as follows:

[0020]

[0021] Criteria for determining pass / fail:

[0022] Qualified products must simultaneously meet the following requirements: EPI ≥ 40, and the detection values ​​of all three proteins must not be lower than 3 ng / mL.

[0023] Non-conforming products: Products meeting any of the following conditions are considered non-conforming:

[0024] (1) EPI < 40;

[0025] (2) 40 ≤ EPI < 60, but the detection value of any protein is less than 3 ng / mL;

[0026] (3) The detection value of any one of the three proteins is below the detection limit (0.1 ng / mL).

[0027] Seriously unqualified: The detection values ​​of two or more proteins are all below the detection limit (0.1 ng / mL).

Claims

1. A method for quality grading and evaluation of Akkermansia muciniphila products, characterized in that, Includes the following steps: (a) Detect the content of P9 protein, Amuc_1100 protein and Amuc_1409 protein in the sample to be tested; (b) Calculate the Efficacy Protein Index (EPI) based on the detection results of the three proteins: EPI = 0.4×CP9 + 0.35×CAmuc1100 + 0.25×CAmuc1409; (c) Compare the EPI with a preset grading standard to determine the product quality grade.

2. The quality grading and evaluation method according to claim 1, characterized in that, The grading standard divides products into four grades: S (superior), A (qualified), B (basically qualified), and C (unqualified).

3. The quality grading and evaluation method according to claim 2, characterized in that, S-Class: EPI ≥ 80; Grade A: 60 ≤ EPI < 80; Grade B: 40 ≤ EPI < 60; Grade C: EPI < 40.

4. The quality grading and evaluation method according to claim 2, characterized in that, Products with an EPI ≥ 60 and a P9 protein, Amuc_1100 protein, and Amuc_1409 protein detection value of not less than 3 ng / mL are considered qualified products.

5. The quality grading and evaluation method according to claim 4, characterized in that, Products meeting any of the following conditions are considered non-conforming: (a) EPI < 40; (b) 40 ≤ EPI < 60 and the detection value of any protein is less than 3 ng / mL; (c) The detection value of any one of the three proteins is less than 0.1 ng / mL.

6. A kit for grading and evaluating the quality of AKK bacteria products for implementing the method according to any one of claims 1 to 5, characterized in that, It includes test reagents for detecting P9 protein, Amuc_1100 protein, and Amuc_1409 protein, as well as instructions that describe the product quality grading and evaluation standards.

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