High-throughput reverse screening method for functional probiotics against Salmonella typhimurium

Abstract

The present application discloses a high-throughput reverse screening method for functional probiotics against Salmonella typhimurium, which belongs to the field of microbial technology. This method is a high-throughput reverse screening method based on the plate agglutination test, which can quickly and efficiently screen out functional probiotics that can secrete Salmonella typhimurium immune analogs, greatly improving the screening efficiency and reducing the screening cost. The functional probiotics obtained by screening can prevent Salmonella typhimurium infection, are safe and non-toxic, have no side reactions, and have a long immune persistence period, and have important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products to effectively prevent Salmonella typhimurium infection in clinical breeding, significantly reduce the duck mortality rate, and improve economic benefits.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and particularly relates to a high-throughput reverse screening method for functional probiotics against Salmonella typhimurium. Background Art

[0002] Salmonella Typhimurium is a Gram-negative bacillus belonging to the genus Salmonella of the Enterobacteriaceae family. It is widely present in nature and is an intestinal pathogen that can cause zoonotic diseases. After ducklings are infected, the mortality rate can reach 30 - 80%, mainly manifested as listlessness, diarrhea (yellowish-green loose stools), dyspnea, neurological symptoms, and death, causing great harm to the healthy development of the duck farming industry.

[0003] The antigen composition of Salmonella Typhimurium is very complex. Its outer membrane proteins, O antigens, H antigens, polysaccharides, etc. have good immunogenicity and can all be used as candidate immunogens for its drug development; probiotics secrete various extracellular polysaccharides, lipopeptides, polypeptides, proteins, etc. during fermentation and have various immunological functions. Screening for immune analogs of Salmonella Typhimurium from its fermentation broth can provide important value for the drug development of this disease; currently, most of the functional probiotics against pathogenic bacteria (toxins) obtained by screening contain direct antiviral and antibacterial substances in their metabolites, and the protection period for animals is short, and multiple doses are required to achieve a continuous and effective effect; the ordinary screening method needs to first collect samples, process the samples and then screen, with cumbersome and time-consuming steps; and the current screening method can only screen one or several bacteria at a time, with low detection efficiency. Summary of the Invention

[0004] The purpose of the implementation of this application is to provide a high-throughput reverse screening method for functional probiotics against Salmonella typhimurium, so as to solve the technical problems of low screening efficiency, cumbersome steps, and low success rate of Salmonella typhimurium in the prior art, and can quickly and efficiently screen immune analogs of Salmonella typhimurium from a large number of probiotics. The drug prepared using this immune analog can prevent the occurrence of duck Salmonella typhimurium disease for a long time.

[0005] To achieve the above purpose, the technical solution adopted by this application is: to provide a high-throughput reverse screening method for functional probiotics against Salmonella typhimurium, which specifically includes the following steps:

[0006] (1) Activation and culture of candidate probiotics: Inoculate candidate probiotics into a liquid medium for activation to obtain a candidate probiotic culture solution; centrifuge the candidate probiotic culture solution and collect the supernatant to obtain a candidate probiotic test solution;

[0007] (2) High-throughput screening of functional probiotics: Dilute the candidate probiotic test solution by two-fold serial dilution. Pipette the candidate probiotic test solution at different dilution multiples, the standard antigen of Salmonella typhimurium, and physiological saline onto a clean glass plate; then add the positive serum of Salmonella typhimurium and mix the positive serum of Salmonella typhimurium with the liquid on the glass plate evenly to form a liquid surface, and gently shake the glass plate continuously to observe the results; the dilution multiples of the serial dilution are between 2 1 -2 10 Specifically, the dilution multiples are adjusted according to the experimental results and are not limited to between 2 1 -2 10 .

[0008] (3) Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the physiological saline does not agglutinate, screen out the probiotics corresponding to the candidate probiotic test solution that forms agglutination with the positive serum of Salmonella typhimurium, which are the screened functional probiotics.

[0009] In one embodiment,

[0010] the candidate probiotic is Lactobacillus acidophilus.

[0011] In one embodiment,

[0012] the liquid medium is MRS liquid medium.

[0013] In one embodiment,

[0014] the activation condition in step (1) is: constant temperature cultivation at 37 °C for 18 h.

[0015] In one embodiment,

[0016] the centrifugation speed in step (1) is 8000 r / min and the centrifugation time is 10 min.

[0017] In one embodiment,

[0018] the diluent used for diluting the candidate probiotic test solution in step (2) is sterilized physiological saline.

[0019] In one embodiment,

[0020] the pipetting volumes of the candidate probiotic test solution, the standard antigen of Salmonella typhimurium, physiological saline, and the positive serum of Salmonella typhimurium are all 30 μl.

[0021] In one embodiment,

[0022] the diameter of the liquid surface in step (2) is 1 - 2 cm.

[0023] In one embodiment,

[0024] After step (2) of coating to form a liquid surface, observe the result after 2 minutes.

[0025] In one of the embodiments,

[0026] The screening method further includes a duckling immunization challenge test.

[0027] Beneficial technical effects: The present application provides a high-throughput reverse screening method for functional probiotics against Salmonella typhimurium, which is a high-throughput reverse screening method based on the plate agglutination test. It can quickly and efficiently screen out functional probiotics that can secrete Salmonella typhimurium immune analogs, greatly improving the screening efficiency and reducing the screening cost. The screened functional probiotics can prevent Salmonella typhimurium infection, are safe, non-toxic, have no side reactions, and have a long immune persistence period, with important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products to effectively prevent Salmonella typhimurium infection in clinical breeding, significantly reduce the duck mortality rate, and improve economic benefits. Specific embodiments

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer and more understandable, the present application is further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0029] The probiotic candidate bacteria used in this embodiment are Lactobacillus acidophilus, and the strains are all from the strain resource library of Weifang Huazhuo Biotechnology Co., Ltd. 200 candidate probiotics (numbered S1-S200) were randomly selected for this experiment; the standard positive serum of Salmonella typhimurium and the standard antigen of Salmonella typhimurium were from the standard serum and antigen library of Weifang Huazhuo Biotechnology Co., Ltd.

[0030] Example 1

[0031] A high-throughput reverse screening method for functional probiotics against Salmonella typhimurium specifically includes the following steps:

[0032] (1) Activation and culture of candidate probiotics:

[0033] S1. Prepare MRS liquid medium: Weigh 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium citrate, 2.6 g of K2PO4•3H2O, 0.1 g of MgSO4•7H2O, 0.05 g of MnSO4 and 0.5 g of cysteine hydrochloride, dissolve with deionized water, then add 1 ml of Tween 80, make up the volume to 1 L, sterilize at 121 °C for 15 minutes, and wait to cool to room temperature for standby;

[0034] S2. Respectively inoculate the candidate probiotics S1 - S200 stored in an -80 °C refrigerator into MRS liquid medium, and culture them at a constant temperature of 37 °C for 18 h for activation to obtain candidate probiotic culture solutions; centrifuge the candidate probiotic culture solutions at 8000 r / min for 10 min, collect the fermentation supernatant, and obtain candidate probiotic test solutions S1 - S200;

[0035] (II). High-throughput screening of functional probiotics: Dilute the candidate probiotic test solutions with sterilized saline by two-fold serial dilution. Respectively pipette 30 μl of candidate probiotic test solutions with different dilution multiples, standard antigen of Salmonella typhimurium, and saline, and drop them onto a clean glass plate; then drop the positive serum of Salmonella typhimurium, and use the bent surface of the pipette tip to evenly mix the positive serum of Salmonella typhimurium with the liquid already coated on the glass plate, and spread it into a liquid surface with a diameter of 1 - 2 cm. Gently shake the glass plate continuously, and the results can be observed after 2 min;

[0036] (III). Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the saline does not agglutinate, the screening method is established; the probiotic corresponding to the candidate probiotic test solution that forms agglutination with the positive serum of Salmonella typhimurium is the screened functional probiotic; among them, the dilution multiples of the candidate probiotic test solution and the standard positive serum are the agglutination titers. The higher the titer, the stronger the immune reaction ability between the two. According to the above plate agglutination test screening method, a total of 16 candidate probiotics were screened, and the agglutination titers of 3 of them reached 2 5 or above, which are S23, S77, and S125 respectively, and the results are shown in Table 1.

[0037] Table 1 Screening results of candidate functional probiotics

[0038]

[0039] Example 2 Verification of the function of functional probiotics in preventing Salmonella typhimurium

[0040] In this example, 3 functional probiotics S23, S77, and S125 screened in Example 1 were selected to study the effect of the samples prepared from these 3 strains on preventing Salmonella typhimurium challenge; at the same time, 1 strain of Lactobacillus acidophilus S37 with no agglutination titer with the standard positive serum of Salmonella typhimurium in Example 1 was selected as the control strain; the verification steps are as follows:

[0041] (I). Sample preparation: Inoculate the screened probiotics S23, S77, S125, and S37 into MRS liquid medium at an inoculation amount of 5%, and culture them at 37 °C. When the bacterial count reaches 10 8.0When it reaches CFU / mL or above, stop fermentation, centrifuge at 8000 r / min for 10 min, collect the fermentation supernatant, and obtain the fermentation supernatants of S23, S77, S125, and S37 respectively. Filter them aseptically through a 0.22 μm filter membrane. Among them, the aseptic fermentation supernatants of S23, S77, and S125 are functional probiotic samples, denoted as S23 sample, S77 sample, and S125 sample, and the aseptic fermentation supernatant of S37 is a control bacteria sample, denoted as S37 sample;

[0042] (II) Sample inspection: Perform a plate agglutination test on each sample with the standard positive serum of Salmonella typhimurium. Among them, the plate agglutination titers of S23 sample, S77 sample, and S125 sample with the standard positive serum of Salmonella typhimurium should all be not less than 25, and the control bacteria S37 sample should have no plate agglutination titer;

[0043] (III) Duckling immunization and challenge test

[0044] S1. Test grouping: 100 Cherry Valley ducklings are randomly divided into 5 groups, with 20 ducklings in each group. Among them, the S23 group, S77 group, and S125 group are all functional probiotic sample groups, the S37 group is a control bacteria sample group, and the blank control group is a non-challenged control. Immunize and challenge each group. Observe for 7 days after challenge, record the clinical changes such as the spirit, drinking water, feeding, and death of each group of ducklings, and calculate the protection rate based on the number of deaths;

[0045] S2. Immunization and challenge test design: For the S23 group, use the S23 sample in step (I). For the S77 group, use the S77 sample. For the S125 group, use the S125 sample. For the S37 group, use the S37 sample. The duckling age is 5 and 6 days old. The administration method is oral, and the dosage is 0.5 ml per duckling. The challenge strain is Salmonella typhimurium strain HZ. The challenge age is 12 days old. The challenge method is intramuscular injection, and the challenge dosage is 1.0×10 9 CFU per duckling; The blank control group is raised normally without any operations;

[0046] S3. Test results: The ducklings in the blank control group were stable throughout the process and showed no abnormalities. The ducklings in the S23 group, S77 group, and S125 group basically recovered on the 2nd day after challenge, ate normally, and had good mental states. However, the ducklings in the S37 group had obvious clinical reactions after challenge, were listless and reluctant to move, and some ducks still did not recover until the 7th day after challenge. The death situation after challenge is shown in Table 2. The protection rates of the functional probiotics S23 group, S77 group, and S125 group against Salmonella typhimurium were 65%, 90%, and 75% respectively, while the protection rate of the control bacteria S37 group was only 20%.

[0047] Table 2 Statistical table of death situations in each group

[0048]

[0049] The above results fully prove that the functional probiotics S23, S77, and S125 screened in this application all have a significant effect in preventing Salmonella typhimurium. Among them, the probiotic S77 has the best effect, with a protection rate of 90%; at the same time, it shows that the screening method of functional probiotics for preventing Salmonella typhimurium based on the plate agglutination test in this application has the characteristics of high efficiency, simple operation, and high success rate.

[0050] Example 3 Immune persistence period of functional probiotics in preventing Salmonella typhimurium

[0051] Mix the functional probiotic S77 sample prepared in Example 2 with white oil adjuvant in a ratio of 1:2 and emulsify it to obtain a functional probiotic vaccine, which is used as the bacterial liquid group; prepare the vaccine of the control bacterium S37 according to the above method as the control bacterium group; conduct an immune persistence period test as follows:

[0052] (I). Test grouping: 140 Cherry Valley ducklings were randomly divided into 7 groups, with 20 ducks in each group. The challenge strain was Salmonella typhimurium strain HZ, and the challenge method was intramuscular injection, and the challenge dose was 1.0×10 9 CFU / duck. The specific grouping is shown in Table 3; observe for 7 days after challenge, record the clinical changes of the ducklings in each group, such as spirit, drinking water, feeding, and death, and calculate the protection rate based on the number of deaths.

[0053] Table 3 Design of duckling immunization and challenge test

[0054]

[0055] (II). Test results: No abnormalities were observed in the ducklings in the bacterial liquid group after immunization. They ate normally and had good mental states after challenge at 21, 28, and 35 days of age; the clinical reactions of the ducklings in the control bacterium group were obvious, with listless spirits and lying on the ground without moving; the death situations after challenge are shown in Table 4. The protection rates of the functional probiotic S77 sample against Salmonella typhimurium were 85%, 90%, and 90% respectively after challenge at 21, 28, and 35 days of age, while the protection rates of the control bacterium S37 sample were 15%, 25%, and 15% respectively.

[0056] Table 4 Statistical table of death situations in each group

[0057]

[0058] The substances that directly kill pathogenic bacteria in the probiotic fermentation broth are generally antibacterial peptides, etc., which are all metabolized within 2-3 days in vivo. The functional probiotic S77 screened in this application has an immune persistence period of at least 28 days when applied to prevent Salmonella typhimurium, indicating that this immune protection effect is exerted by the immune analogues in the functional probiotic fermentation broth, rather than the substances that directly kill pathogenic bacteria. The results of this example fully prove that the method for screening functional probiotics for preventing Salmonella typhimurium based on the plate agglutination test of the present invention not only has the characteristics of high efficiency, simple operation, and no side effects in immune application, but also has a long immune persistence period and has important value in clinical application development.

[0059] Example 4 Application test of functional probiotics as an oral product to prevent Salmonella typhimurium infection

[0060] (I). Experimental design: A meat duck farm in Shandong, with 4 meat duck breeding sheds, a total of 30,000 Cherry Valley ducks. Divided into experimental group and control group according to the shed, with 7,500 ducks in each shed;

[0061] S1. Experimental group (Shed No. 1 and Shed No. 3): On the 5th, 6th, 15th, and 16th days of age of Cherry Valley ducks, drink the functional probiotic sample S77 prepared in Example 2, 0.5 ml per duck;

[0062] S2. Control group (Shed No. 2 and Shed No. 4): On the 5th, 6th, 15th, and 16th days of age of Cherry Valley ducks, drink the control bacteria sample S37 prepared in Example 2, 0.5 ml per duck; Other follow the normal drug prevention procedures;

[0063] (II). Experimental results:

[0064] Experimental group: Reached normal slaughter age at 35 days old, no obvious disease occurred, and the mortality rate of the duck flock during the entire breeding period was 4.8%;

[0065] Control group: At 30 days old, the duck flock began to show listlessness, neck retraction, and reduced food intake. The number of dead and culled ducks increased after 2 days, and the mortality rate was about 8.3%. The livers of the dead ducks were brittle and enlarged during autopsy, and the spleens were enlarged with necrotic spots on the surface. After laboratory testing, the pathogen was Salmonella typhimurium. After treatment with conventional antibiotics, the death rate was alleviated, and about 50 ducks died every day. By the time of slaughter at 35 days old, the mortality rate of the duck flock during the entire breeding period was 10.5%;

[0066] The above test data indicate that the functional probiotic S77 screened by the present invention is prepared into a product and applied to drinking water at the 5th, 6th, 15th, and 16th days of age clinically, which can effectively reduce the probability of ducks getting Salmonella typhimurium disease in clinical breeding, reduce the mortality rate of meat ducks, and improve economic benefits; at the same time, it shows that the screening method of functional probiotics for preventing Salmonella typhimurium based on the plate agglutination test in this application has the characteristics of high efficiency, simple operation, and high success rate, and the clinical application effect of the screened functional probiotic strains is prominent.

[0067] This application provides a high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium, which is a high-throughput reverse screening method based on the plate agglutination test. It can quickly and efficiently screen out functional probiotics that can secrete Salmonella typhimurium immune analogs, greatly improving the screening efficiency and reducing the screening cost. The screened functional probiotics can prevent Salmonella typhimurium infection, are safe, non-toxic, and have no side reactions, and have a long immune persistence period, with important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products to effectively prevent Salmonella typhimurium infection in clinical breeding, significantly reduce the duck mortality rate, and improve economic benefits.

[0068] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, "a plurality" means two or more, unless otherwise specifically defined.

[0069] The above are only the preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims

1. A high-throughput reverse screening method for functional probiotics against Salmonella typhimurium, characterized in that, Specifically, it includes the following steps: (1) Activation and culture of candidate probiotics: The candidate probiotics are inoculated into a liquid medium for activation to obtain a candidate probiotic culture solution; the candidate probiotic culture solution is centrifuged, and the supernatant is collected to obtain a candidate probiotic test solution; (2) High-throughput screening of functional probiotics: The candidate probiotic test solution is serially diluted two-fold with sterilized saline. 30 μl of the candidate probiotic test solution at different dilution multiples, the standard antigen of Salmonella typhimurium, and saline are respectively pipetted and dropped onto a clean glass plate; then the positive serum of Salmonella typhimurium is dropped, and the bent surface of the pipette tip is used to evenly mix the positive serum of Salmonella typhimurium with the liquid already coated on the glass plate, and a liquid surface with a diameter of 1 - 2 cm is spread. The glass plate is gently shaken continuously, and the result can be observed after 2 minutes; (3) Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the saline does not agglutinate, the probiotics corresponding to the candidate probiotic test solution that forms agglutination with the positive serum of Salmonella typhimurium are screened out, which are the screened functional probiotics.

2. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that, The candidate probiotics are Lactobacillus acidophilus.

3. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that The liquid medium is MRS liquid medium.

4. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, wherein The conditions for activation in step (1) are: constant temperature culture at 37 °C for 18 h.

5. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, wherein The rotation speed of centrifugation in step (1) is 8000 r / min, and the centrifugation time is 10 min.

6. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that The diluent used for diluting the candidate probiotic test solution in step (2) is sterilized saline.

7. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that The pipetting volume of the candidate probiotic test solution, the standard antigen of Salmonella typhimurium, saline, and the positive serum of Salmonella typhimurium is all 30 μl.

8. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that The diameter of the liquid surface in step (2) is 1 - 2 cm.

9. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that After spreading the liquid surface in step (2), the result is observed after 2 minutes.

10. The high-throughput reverse screening method of functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that, The screening method also includes a duckling immunization challenge test.

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