阅读说明：本技术 一种抑制病原菌的潜在益生菌株的快速筛选方法 (Rapid screening method of potential probiotic strains for inhibiting pathogenic bacteria ) 是由 李锋 单明旭 谢英杰 杨春旭 单安山 于 2019-10-22 设计创作，主要内容包括：本发明公开了一种对致病性细菌具有抑菌活性的菌株筛选方法,如下：将冻存致病性指示菌活化、富集培养后制成指示菌液；取待筛选供试菌株,活化培养,收集上清液,制成待筛选供试菌上清液；取过滤后的待筛选供试菌上清液与指示菌液等量混合为待测样品组,加入到细胞培养板中,以指示菌液与灭菌PBS溶液的混合物作为阳性对照组,液体培养基为阴性对照组；利用酶标仪对混合待测样品的吸光度值进行测定,以灭菌PBS溶液与指示菌液混合作为对照,抑菌活性即抑菌率为：<Image he="86" wi="700" file="DDA0002242505390000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>根据抑菌率的大小判断抑菌活性的强弱,抑菌率越大表明抑菌活性越强。本发明具有筛选样本量大,检测速度及敏感性提高,耗费时间减少的优点。(The invention discloses a screening method of a strain with bacteriostatic activity on pathogenic bacteria, which comprises the following steps: activating and enriching the cryopreserved pathogenicity indicating bacteria to prepare indicating bacteria liquid; taking a test strain to be screened, carrying out activation culture, collecting supernatant, and preparing the supernatant into the test strain to be screened; taking filtered supernatant of the test bacteria to be screened and an indication bacterial solution, mixing the supernatant and the indication bacterial solution in equal amount to form a sample group to be tested, adding the sample group to a cell culture plate, taking a mixture of the indication bacterial solution and a sterilized PBS solution as a positive control group, and taking a liquid culture medium as a negative control group; the absorbance value of the mixed sample to be detected is measured by using an enzyme-labeling instrument, the sterilized PBS solution and the indicating bacteria liquid are mixed as a control, and the bacteriostatic activity, namely the bacteriostatic rate, is as follows: judging the strength of the bacteriostatic activity according to the bacteriostatic rate, wherein the greater the bacteriostatic rate is, the bacteriostatic activity isThe stronger the activity. The invention has the advantages of large screening sample amount, improved detection speed and sensitivity and reduced time consumption.)

1. A method for screening strains with bacteriostatic activity on pathogenic bacteria is characterized by comprising the following steps:

(1) activating and culturing the cryopreserved pathogenicity indicating bacteria by using a solid culture medium, and then performing enrichment culture by using a liquid culture medium with the same formula as the solid culture medium to prepare an indicating bacteria liquid;

(2) taking a test strain to be screened, carrying out activated culture by using a liquid culture medium suitable for the growth of the test strain to be screened, centrifuging, collecting supernatant, filtering and sterilizing to prepare the supernatant of the test strain to be screened;

(3) the screening method comprises the following steps: taking filtered supernatant of the test bacteria to be screened and an indication bacterial solution which are equivalently mixed to form a sample group to be tested, adding the sample group to a cell culture plate, taking a mixture of the indication bacterial solution and a sterilized PBS solution as a positive control group, and taking the liquid culture medium in the step 1 as a negative control group;

(4) and (3) calculating the bacteriostatic activity: the absorbance value of the mixed sample to be detected is measured by using an enzyme-labeling instrument, the sterilized PBS solution and the indicating bacteria liquid are mixed as a control, and the bacteriostatic activity, namely the bacteriostatic rate, is as follows:

and judging the strength of the antibacterial activity according to the size of the antibacterial rate, wherein the higher the antibacterial rate is, the stronger the antibacterial activity is.

2. The method for screening a strain having bacteriostatic activity against pathogenic bacteria according to claim 1, wherein the following bacteria can be screened as indicator bacteria: including Escherichia coli, Salmonella, Staphylococcus aureus, and Pseudomonas aeruginosa.

3. The method for screening a strain having bacteriostatic activity against pathogenic bacteria according to claim 1, wherein: the solid culture is brain heart infusion agar.

Technical Field

The invention belongs to the technical field of probiotic bioassay, and particularly relates to a rapid screening method of potential probiotic strains for inhibiting pathogenic bacteria.

Background

In recent years, diseases of piglets caused by infection with pathogenic microorganisms have been increasing worldwide, and such damage may be repeated during growth and development. The research shows that: after piglets are infected with pathogenic bacteria such as escherichia coli and salmonella, a series of hazards are usually accompanied, including: serious intestinal villus atrophy, increased bacterial displacement from intestinal cavities to lymph nodes, increased depth of intestinal crypts, slow growth and development speed, reduced cholesterol transport capacity, death of piglets and the like, and have caused serious economic loss for livestock breeding production. During the production process, the piglet bacterial infection is usually treated by antibiotics, but the use of the antibiotics not only causes bacterial resistance, but also has adverse effects on the environment, animals and even human beings. Therefore, there is a need to find alternative products that can replace antibiotics for the treatment of bacterial infections in piglets. The conventional method for screening probiotic strains with bacteriostatic activity mostly adopts an oxford cup method, namely a bacteriostatic circle method, and is the most common method for determining the bacteriostatic activity. The principle is as follows: placing an Oxford cup on an indicator bacterium flat plate with a certain concentration, adding a bacterial metabolite into the Oxford cup, culturing for a certain time under a proper culture condition, adding sterilized water as a control, and selecting a strain with high-efficiency bacteriostatic activity by comparing the size of a bacteriostatic zone and combining the concentration of the indicator bacterium. Although this method is suitable for bacteria with fast growth characteristics, it also has some drawbacks, including: the position of the Oxford cup is easy to be dislocated, the adding amount of the supernatant is limited to about 100-200 microliter, the transfer operability of the culture dish is poor, the supernatant needs 2 hours or even longer for diffusion, the single operable sample size is small, the test takes time and the like. The existence of the problems not only increases the pollution probability and prolongs the screening time, but also increases the complexity of the probiotic screening work. Therefore, the oxford cup method is not suitable for large-scale rapid screening and operation of probiotics with bacteriostatic activity.

Disclosure of Invention

In view of the problems, the invention aims to provide a method for rapidly screening potential probiotic strains for inhibiting pathogenic bacteria, which can replace the traditional oxford cup method.

The purpose of the invention is realized by the following technical scheme: a method for screening a strain with bacteriostatic activity on pathogenic bacteria comprises the following steps:

(1) activating and culturing the cryopreserved pathogenicity indicating bacteria by using a solid culture medium, and then performing enrichment culture by using a liquid culture medium with the same formula as the solid culture medium to prepare an indicating bacteria liquid;

(2) taking a test strain to be screened, carrying out activated culture by using a liquid culture medium suitable for the growth of the test strain to be screened, centrifuging, collecting supernatant, filtering and sterilizing to prepare the supernatant of the test strain to be screened;

(3) the screening method comprises the following steps: taking filtered supernatant of the test bacteria to be screened and an indication bacterial solution which are equivalently mixed to form a sample group to be tested, adding the sample group to a cell culture plate, taking a mixture of the indication bacterial solution and a sterilized PBS solution as a positive control group, and taking the liquid culture medium in the step 1 as a negative control group;

(4) and (3) calculating the bacteriostatic activity: the absorbance value of the mixed sample to be detected is measured by using an enzyme-labeling instrument, the sterilized PBS solution and the indicating bacteria liquid are mixed as a control, and the bacteriostatic activity, namely the bacteriostatic rate, is as follows:

and judging the strength of the antibacterial activity according to the size of the antibacterial rate, wherein the higher the antibacterial rate is, the stronger the antibacterial activity is.

The invention can be used for screening the following bacteria as indicator bacteria: including Escherichia coli, Salmonella, Staphylococcus aureus, and Pseudomonas aeruginosa.

The solid culture is brain-heart infusion agar.

The invention has the advantages that: compared with the conventional Oxford cup bacteriostasis ring method, the contact area and sensitivity of the Escherichia coli to the probiotic metabolite (supernatant) are obviously improved, the bacteriostasis activity efficiency is truly reflected, the accuracy is improved, and the determination error is reduced; the operability is stronger, the misoperation rate is reduced, the test time is reduced, and the sample amount can be greatly increased.

Detailed Description

The invention is further illustrated by the following examples: