Method for rapidly identifying lactic acid bacteria with alpha-amylase inhibitory activity

Abstract

The invention relates to a high-throughput screening method based on starch-iodine chromogenic reaction, which is used for rapidly identifying lactic acid bacteria strains with alpha-amylase inhibitory activity, and belongs to the technical field of microbial engineering and biological medicine. The method comprises the following steps: preparing an MRS solid culture medium containing soluble starch, preparing a flat plate, performing punching treatment, mixing an alpha-amylase solution and a to-be-detected bacterial liquid in an isovolumetric manner, adding the mixture into holes of the flat plate, performing constant-temperature culture at 37 DEG C for 24-48 hours by taking a non-inoculated alpha-amylase solution as a negative control, and detecting the content of the to-be-detected bacterial liquid in the to-be-detected bacterial liquid. After the culture is finished, uniformly dropwise adding an iodine-potassium iodide solution on the surface of the culture medium for chromogenic reaction, and judging the alpha-amylase inhibitory activity of the strain by observing the formation condition and the diameter of a transparent circle around a bacterial colony. The method has the advantages of simplicity and convenience in operation, rapidness in detection, intuitive result, safety, environment friendliness and the like, is suitable for screening the alpha-amylase inhibitory activity of various lactic acid bacteria and other microorganisms, and provides an efficient and reliable technical scheme for developing a probiotic preparation with a hypoglycemic function; the important application value is realized in the aspects of prevention and treatment of related diseases such as diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the field of microbial engineering and biomedical technology, and specifically relates to a method for rapid screening of lactic acid bacteria with α-amylase inhibitory activity based on a novel identification platform. More specifically, the present invention provides a high-throughput screening system integrating microbial culture and enzyme activity detection, which is used to quickly and efficiently screen functional lactic acid bacteria strains with α-amylase inhibitory activity. The method innovatively combines microbial culture, enzyme activity detection and visual identification, providing reliable technical support for the development of new blood sugar-lowering probiotic preparations. Background Art

[0002] Starch, as the main energy source in human diet, participates in energy metabolism through enzymatic reactions in the body. Among them, α-amylase (EC 3.2.1.1), as the key enzyme for starch digestion, specifically hydrolyzes the α-1,4-glycosidic bonds within starch molecules, breaking down starch into small molecules such as oligosaccharides. While this biochemical process provides the body with the energy it needs, it also leads to a significant increase in blood sugar levels after meals. Long-term high blood sugar levels may induce diabetes and its related complications.

[0003] According to the latest statistics from the World Health Organization (WHO), diabetes has become a major global public health problem, and its complication spectrum covers more than 100 clinical diseases, including retinopathy, diabetic nephropathy, peripheral neuropathy and diabetic foot. It is worth noting that among the diabetic population, type 2 diabetes mellitus (T2DM) accounts for as much as 90%, and its main pathological characteristics are insulin resistance and pancreatic β-cell dysfunction. At present, the clinical treatment strategies for T2DM mainly focus on regulating insulin secretion and delaying carbohydrate absorption. Among them, α-amylase inhibitors have attracted much attention because they can effectively control postprandial blood sugar.

[0004] Although the existing clinically used α-amylase inhibitor drugs (such as acarbose, miglitol and voglibose, etc.) have been approved by regulators, they are often accompanied by gastrointestinal adverse reactions during use, including symptoms such as nausea, abdominal pain and bloating. In terms of α-amylase inhibitory activity detection, the existing technology mainly relies on in vitro detection methods such as 3,5-dinitrosalicylic acid (DNS) method and glucose oxidase (GOD) method. However, these traditional methods have obvious limitations: first, the experimental process is cumbersome and requires multiple steps such as strain culture, centrifugation, enzyme reaction, etc.; second, the detection cycle is long and the reagent consumption is large, resulting in high screening costs; finally, the complexity of the experimental operation also limits its application in large-scale screening. The present invention provides a new α-amylase inhibitory activity detection method based on the starch-iodine colorimetric principle. Compared with the existing technology, this method has the following significant advantages: (1) The experimental system is safe and environmentally friendly, and the reagents used are non-toxic and harmless; (2) The operation process is simple and efficient, and the strain culture and target bacteria identification are organically combined; (3) The test results are intuitive and reliable, and the differences in α-amylase inhibitory activity of different strains can be compared simultaneously; (4) The experimental cost and detection time are significantly reduced, and the screening efficiency is improved. This innovative method provides a new technical solution for the screening of α-amylase inhibitors and has important application value. Summary of the invention

[0005] The purpose of the present invention is to provide an efficient and convenient method for screening lactic acid bacteria with α-amylase inhibitory activity. The method has the characteristics of simple operation, rapid detection, safe and readily available reagents, etc., can realize large-scale strain screening, and can be widely used in the field of screening microbial resources with α-amylase inhibitory activity.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0007] A rapid screening method for lactic acid bacteria with α-amylase inhibition activity, characterized by comprising the following steps:

[0008] (1) Preparation of culture medium: MRS culture medium was used as the base medium, 1-2% (w / v) soluble starch and 2.0% (w / v) agar powder were added, and the medium was sterilized by high pressure at 121°C for 15 minutes and kept warm in a water bath at 55°C for later use;

[0009] (2) Plate preparation: Pour the incubation culture medium into a sterile culture dish. After solidification, use a sterile hole puncher to evenly punch holes on the plate.

[0010] (3) Sample treatment: Mix 2 mg / mL α-amylase solution with the bacterial culture to be tested at a ratio of 1:1 (v / v), take an appropriate amount of the mixture and add it to the plate wells. Use a mixture of α-amylase solution without bacterial culture and MRS liquid culture medium as a control, and culture at 37°C for 24-48 hours.

[0011] (4) Color development reaction: Evenly apply iodine solution (0.08 mol I2 dissolved in 3.2 mol / L KI solution) to the surface of the plate after incubation and let stand at room temperature for color development reaction;

[0012] (5) Result determination: Observe the color development area and measure the diameter of the transparent zone, and evaluate the α-amylase inhibitory activity of the strain by comparing it with the control group. Among them, the strain with no transparent zone or a transparent zone diameter significantly smaller than that of the control group is a positive strain with α-amylase inhibitory activity, and the transparent zone diameter is negatively correlated with the inhibitory activity.

[0013] The present invention has the following significant advantages and innovations:

[0014] Innovativeness of the method: For the first time, the starch-iodine color development principle was combined with lactic acid bacteria screening to establish a new α-amylase inhibition activity lactic acid bacteria identification platform;

[0015] Ease of operation: By optimizing the culture medium formula and color development conditions, complex enzyme activity detection is simplified to an intuitive color development reaction;

[0016] High detection efficiency: Multiple samples can be tested in parallel in a single experiment, which significantly improves the screening efficiency;

[0017] Reliability of results: Based on the quantitative relationship between starch and iodine color development, it can accurately reflect the α-amylase inhibitory activity of the strain;

[0018] Wide application: Applicable to the screening of α-amylase inhibitory activity of various lactic acid bacteria and other microorganisms;

[0019] Safety and environmental protection: All reagents used are conventional experimental reagents, non-toxic and harmless, and comply with laboratory safety regulations.

[0020] The present invention provides a rapid and reliable technical solution for screening lactic acid bacteria with α-amylase inhibition activity, and has important application value in the field of functional probiotic development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Results of direct identification of lactic acid bacteria with α-amylase inhibitory activity on solid culture medium (L3, L4, and L7 have no transparent circles, indicating that α-amylase activity is inhibited and the strain has a strong α-amylase inhibitory effect. The transparent circles of L1, L2, and L6 are smaller than those of the control group, indicating that these strains have a certain α-amylase inhibitory effect) DETAILED DESCRIPTION

[0022] The following is a detailed description with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0023] Example 1: Isolation of lactic acid bacteria with α-amylase inhibitory activity from kimchi

[0024] Sample pretreatment: Take 5 g kimchi sample, add 50 mL YPD liquid medium, and culture at 37 °C with shaking for 48 h;

[0025] Serial dilution plating: dilute the pre-culture with sterile water to 10 -6 ~10 -8 , 200 μL was spread on MRS solid medium and cultured at 37°C for 48 h;

[0026] Purification culture: colonies with different morphologies were picked and streaked on plates to purify to single colonies, and 7 candidate strains (L1-L7) were obtained.

[0027] Medium preparation: prepare agar plates containing 1.6% starch and punch holes (pore diameter 6 mm);

[0028] Strain inoculation: Take the candidate strain culture solution and mix it with α-amylase solution in equal volumes, take 200 μL and inject it into the well, and use MRS+α-amylase solution as negative control; culture at 37℃ for 24 hours;

[0029] Interpretation of the initial screening results: Add iodine standard solution to develop color. The diameter of the transparent circle around the colony is inversely proportional to the degree of starch hydrolysis. Figure 1 As shown, L3, L4, and L7 have no transparent circles, indicating that the α-amylase activity is inhibited and the strain has a strong α-amylase inhibitory effect. The transparent circles of L1, L2, and L6 are smaller than those of the control group, indicating that these strains have a certain α-amylase inhibitory effect.

Claims

1. A method for rapid identification of lactic acid bacteria with α-amylase inhibitory activity, characterized in that: The steps include: (1) Prepare a solid culture medium using MRS and starch as raw materials and keep it warm at 55°C for later use. (2) Pour the insulated solid culture medium into a sterile culture dish to prepare a solid plate, and then punch holes after it solidifies. (3) Add α-amylase solution and the bacterial solution to be identified into the wells and incubate at 37°C for 24-48 hours. (5) Add iodine-potassium iodide aqueous solution to the plate after incubation in step (3) to carry out a color reaction. (6) Observe the color development and the size of the transparent circle. Lactobacillus with no transparent circle or a smaller transparent circle than the control group is capable of inhibiting amylase activity.

2. The method for rapid identification of lactic acid bacteria with α-amylase inhibitory activity according to claim 1, characterized in that: The amount of starch added in step (1) is 1-2 g / 100 ml of culture medium.

3. The method for rapid identification of lactic acid bacteria with α-amylase inhibitory activity according to claim 1, characterized in that: The culture medium in step (1) is MRS culture medium, which is used to identify lactic acid bacteria.

4. The method for rapid identification of lactic acid bacteria having α-amylase inhibitory activity according to claim 1, characterized in that: Step (3) also includes setting a negative control well of pure α-amylase solution without inoculation of any bacteria + MRS liquid culture medium to correct the experimental results.

Images