High-throughput reverse screening method of functional probiotics for preventing salmonella typhimurium

Abstract

The invention discloses a high-throughput reverse screening method for functional probiotics for preventing salmonella typhimurium, belongs to the technical field of microorganisms, and is a high-throughput reverse screening method based on a plate agglutination test, and the functional probiotics capable of secreting salmonella typhimurium immune analogues can be rapidly and efficiently screened out. The screening efficiency is greatly improved, the screening cost is greatly reduced, and the screened functional probiotics can prevent salmonella typhimurium infection, are safe and non-toxic, have no side reaction, have long immune duration and have important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products, infection of salmonella typhimurium in clinical breeding is effectively prevented, the death rate of ducks is remarkably reduced, and economic benefits are improved.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and in particular relates to a high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium. Background Art

[0002] Salmonella Typhimurium is a Gram-negative bacillus belonging to the genus Salmonella in the family Enterobacteriaceae. It is widely present in nature and is an intestinal pathogen that can cause zoonotic diseases. The mortality rate of infected ducklings can reach 30-80%, with the main manifestations being mental depression, diarrhea (yellow-green loose feces), difficulty breathing, neurological symptoms, and death, posing a huge threat to the healthy development of the duck industry.

[0003] The antigenic composition of Salmonella typhimurium is very complex. Its outer membrane protein, O antigen, H antigen, polysaccharide, etc. have good immunogenicity and can all be used as candidate immunogens for its drug development. Probiotics secrete a variety of extracellular polysaccharides, lipopeptides, polypeptides, proteins, etc. during the fermentation process, and have multiple immunological functions. Screening the immune analogs of Salmonella typhimurium from its fermentation broth can provide important value for the development of drugs for this disease. Currently, the anti-pathogen (toxin) functional probiotics obtained by screening mostly contain direct antiviral and antibacterial substances in their metabolites, which have a short protection period for animals and require multiple doses to achieve sustained and effective effects. Ordinary screening methods require sample collection first, sample processing and then screening, and the steps are cumbersome and time-consuming. Moreover, the current screening method can only screen one or several bacteria at a time, and the detection efficiency is low. 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 for preventing Salmonella typhimurium, so as to solve the technical problems of low efficiency, complicated steps and low success rate of Salmonella typhimurium screening in the prior art. Salmonella typhimurium immune analogues can be screened quickly and efficiently from a large number of probiotics. The drugs prepared using the immune analogues can prevent the occurrence of Salmonella typhimurium disease in ducks for a long time.

[0005] To achieve the above purpose, the technical solution adopted in this application is: to provide a high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium, which specifically includes the following steps: (I) Activation and cultivation of candidate probiotics: inoculating the candidate probiotics into a liquid culture medium for activation to obtain a candidate probiotic culture solution; centrifuging the candidate probiotic culture solution and collecting the supernatant to obtain a candidate probiotic test solution; (II) High-throughput screening of functional probiotics: The candidate probiotic test solution was diluted two-fold, and the candidate probiotic test solution, Salmonella typhimurium standard antigen, and physiological saline at different dilution multiples were respectively aspirated and dropped onto a clean glass plate; then Salmonella typhimurium positive serum was added to mix the Salmonella typhimurium positive serum with the liquid on the glass plate evenly, spread it on the liquid surface, and gently shake the glass plate to observe the results; the dilution multiple of the gradient dilution was 2 1 -2 10 The specific dilution factor is adjusted according to the experimental results and is not limited to 2 1 -2 10 between.

[0006] (III) Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the physiological 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.

[0007] In one embodiment, The candidate probiotic is Lactobacillus acidophilus.

[0008] In one embodiment, The liquid culture medium is MRS liquid culture medium.

[0009] In one embodiment, The activation conditions in step (i) are: constant temperature incubation at 37°C for 18 h.

[0010] In one embodiment, In step (i), the centrifugal speed is 8000 r / min and the centrifugal time is 10 min.

[0011] In one embodiment, Step (ii) The diluent used for diluting the candidate probiotic test solution is sterile saline.

[0012] In one embodiment, The aspirated volume of candidate probiotic test solution, Salmonella typhimurium standard antigen, normal saline and Salmonella typhimurium positive serum was 30 μl.

[0013] In one embodiment, Step (ii) The diameter of the liquid surface is 1-2 cm.

[0014] In one embodiment, Step (ii) After coating the liquid surface, observe the results after 2 minutes.

[0015] In one embodiment, Screening methods also include duckling immune challenge tests.

[0016] Beneficial technical effects: The present application provides a high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium. It is a high-throughput reverse screening method based on 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 screened functional probiotics can prevent Salmonella typhimurium infection, are safe and non-toxic, have no side effects, and have a long immune duration, and have important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products, which can effectively prevent Salmonella typhimurium infection in clinical breeding, significantly reduce duck mortality, and improve economic benefits. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear, 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.

[0018] The candidate probiotics used in this embodiment are Lactobacillus acidophilus, and the strains are all from the strain resource library of Weifang Huazhuo Biotechnology Co., Ltd. In this experiment, 200 candidate probiotics (numbered S1-S200) were randomly selected; the standard positive serum of Salmonella typhimurium and the standard antigen of Salmonella typhimurium are from the standard serum and antigen library of Weifang Huazhuo Biotechnology Co., Ltd.

[0019] Example 1 The high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium specifically comprises the following steps: 1. Activation and cultivation of candidate probiotics: S1. Prepare MRS liquid culture 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 hydrogen 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 them in deionized water, add 1 ml of Tween 80, make up to 1 L, sterilize at 121 ℃ for 15 min, cool to room temperature and set aside; S2. The candidate probiotics S1-S200 frozen in a -80 ℃ refrigerator were inoculated into MRS liquid culture medium respectively, and cultured at 37 ℃ for 18 h for activation to obtain candidate probiotic culture solution; the candidate probiotic culture solution was centrifuged at 8000 r / min for 10 min, and the fermentation supernatant was collected to obtain candidate probiotic test solution S1-S200; (II) High-throughput screening of functional probiotics: dilute the candidate probiotic test solution with sterile saline solution by two times, and take 30 μl of the candidate probiotic test solution, Salmonella typhimurium standard antigen, and saline solution at different dilution multiples, and drop them on a clean glass plate; then drop the Salmonella typhimurium positive serum, and use the bent surface of the gun tip to evenly mix the Salmonella typhimurium positive serum with the liquid already coated on the glass plate, and spread it to a liquid surface with a diameter of 1-2 cm. Gently shake the glass plate continuously, and observe the results after 2 minutes; (III) Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the physiological saline does not agglutinate, the screening method is established; the probiotics corresponding to the candidate probiotic test solution that forms agglutination with the positive serum of Salmonella typhimurium are the screened functional probiotics; the dilution multiple of the candidate probiotic test solution and the standard positive serum is the agglutination titer, and the higher the titer, the stronger the immune response ability of the two. According to the above-mentioned plate agglutination test screening method, a total of 16 candidate probiotics were screened, of which 3 strains had an agglutination titer of 2 5 or above, which are S23, S77 and S125 respectively. The results are shown in Table 1.

[0020] Table 1 Screening results of candidate functional probiotics

[0021] Example 2 Verification of the Functionality of Functional Probiotics in Preventing Salmonella Typhimurium In this example, three functional probiotic strains S23, S77 and S125 screened in Example 1 were selected to study the effects of samples prepared from these three strains on preventing Salmonella typhimurium infection; at the same time, one strain of Lactobacillus acidophilus S37 in Example 1 that had no agglutination titer with the standard positive serum of Salmonella typhimurium was selected as a control bacterium; the verification steps were as follows: (I) Sample preparation: The screened probiotics S23, S77, S125 and S37 were inoculated into MRS liquid culture medium at a rate of 5% and cultured at 37°C. When the bacterial count reached 10 8.0 When the CFU / mL was above, the fermentation was stopped, and the mixture was centrifuged at 8000 r / min for 10 min. The fermentation supernatant was collected to prepare the fermentation supernatants of S23, S77, S125 and S37, respectively. The supernatants were sterile filtered with a 0.22 μm filter membrane. The sterile fermentation supernatants of S23, S77 and S125 were functional probiotic samples, recorded as S23 samples, S77 samples and S125 samples, and the sterile fermentation supernatant of S37 was the control bacteria sample, recorded as S37 sample. (II) Sample inspection: Each sample shall be subjected to a plate agglutination test with a standard positive serum of Salmonella typhimurium; among them, the plate agglutination titer of S23, S77 and S125 samples with the standard positive serum of Salmonella typhimurium shall not be less than 25, and the control bacteria S37 sample shall have no plate agglutination titer; (III) Duckling immune challenge test S1. Experimental grouping: 100 Cherry Valley ducklings were randomly divided into 5 groups, 20 ducklings in each group, of which S23, S77 and S125 groups were all functional probiotic sample groups, S37 group was the control bacteria sample group, and the blank control group was the non-challenge control group; each group was immunized and challenged with viruses; after the challenge, the ducklings were observed for 7 days, and the clinical changes such as spirit, drinking water, feeding, and death of each group were recorded, and the number of deaths was used as the standard to calculate the protection rate; S2. Design of immune challenge test: S23 group: S23 sample of step (I) was used; S77 group: S77 sample; S125 group: S125 sample; S37 group: S37 sample; ducklings were 5 and 6 days old, and the dosage was 0.5 ml / bird. The challenge strain was Salmonella typhimurium HZ strain, and the challenge age was 12 days. The challenge method was intramuscular injection, and the challenge dose was 1.0×10 9 CFU / mouse; The blank control group was raised normally without any manipulation; S3. Test results: The ducklings in the blank control group were stable throughout the whole process, and no abnormalities were found. The ducklings in the S23, S77 and S125 groups basically recovered on the second day after the challenge, eating normally and in good spirits. However, the ducklings in the S37 group had obvious clinical reactions after the challenge, with depression and unwillingness to move. Some ducks had not recovered until the seventh day after the challenge. The mortality after the challenge is shown in Table 2. The protection rates of the functional probiotics S23, S77 and S125 groups against Salmonella typhimurium were 65%, 90% and 75%, respectively, while the protection rate of the control bacteria S37 group was only 20%.

[0022] Table 2 Statistics of deaths in each group

[0023] The above results fully prove that the functional probiotics S23, S77 and S125 screened in the present application all have significant efficacy in preventing Salmonella Typhimurium, among which probiotic S77 has the best effect, with a protection rate of 90%; at the same time, it is shown that the functional probiotic screening method for preventing Salmonella Typhimurium based on the plate agglutination test in the present application is efficient, simple to operate and has a high success rate.

[0024] Example 3 Functional probiotics prevent the immune persistence of Salmonella typhimurium The functional probiotic S77 sample prepared in Example 2 was mixed and emulsified with a white oil adjuvant at a ratio of 1:2 to prepare a functional probiotic vaccine as a bacterial liquid group; a control bacteria S37 vaccine was prepared according to the above method as a control bacteria group; and an immunity duration test was conducted, and the test was as follows: (I) Experimental grouping: 140 Cherry Valley ducklings were randomly divided into 7 groups, 20 ducks in each group. The challenge strains were all Salmonella typhimurium HZ strains, and the challenge method was intramuscular injection. The challenge dose was 1.0×10 9 CFU / each, the specific groups are shown in Table 3; the ducklings in each group were observed for 7 days after the infection, and the clinical changes such as spirit, drinking water, feeding, and death of each group were recorded, and the number of deaths was used as the standard to calculate the protection rate.

[0025] Table 3 Design of duckling immune challenge experiment

[0026] (II) Test results: No abnormality was observed in the ducklings in the bacterial liquid group after immunization. They ate normally and were in good spirits after challenge at 21, 28 and 35 days of age. The ducks in the control bacterial group showed obvious clinical reactions, with listlessness and lying on the ground without moving. The mortality after challenge is shown in Table 4. The protection rates of the functional probiotic S77 samples against Salmonella typhimurium after challenge at 21, 28 and 35 days of age were 85%, 90% and 90%, respectively, while the protection rates of the control bacteria S37 samples were 15%, 25% and 15%, respectively.

[0027] Table 4 Statistics of deaths in each group

[0028] The substances in the probiotic fermentation broth that directly kill pathogens are generally antimicrobial peptides, etc., which are completely metabolized in the body within 2-3 days. The functional probiotic S77 screened in the present application is used to prevent Salmonella typhimurium, and the immune duration is at least 28 days, indicating that the immune protection effect is the effect of the immune analogs in the functional probiotic fermentation broth, rather than directly killing pathogenic substances; the results of this embodiment fully prove that the functional probiotic screening method for preventing Salmonella typhimurium based on the plate agglutination test of the present invention is not only highly efficient, simple to operate, and has no side effects in immune application, but also has a long immune duration, and is of great value in clinical application development.

[0029] Example 4 Application test of functional probiotics as oral products to prevent Salmonella typhimurium infection (I) Experimental design: A meat duck farm in Shandong Province has 4 meat duck breeding sheds with a total of 30,000 Cherry Valley ducks, which are divided into experimental and control groups according to the sheds, with 7,500 ducks in each shed; S1, experimental group (shed 1 and shed 3): the functional probiotic sample S77 prepared in Example 2 was applied to the drinking water of Cherry Valley ducks at 5, 6, 15, and 16 days of age, 0.5 ml / bird; S2, control group (shed 2 and shed 4): when Cherry Valley ducks were 5, 6, 15, and 16 days old, the control bacteria sample S37 prepared in Example 2 was applied to the drinking water, 0.5 ml / bird; the rest was in accordance with the normal drug prevention procedure; (II) Test results: Experimental group: The ducks were slaughtered normally at 35 days of age, with no obvious disease. The mortality rate of the ducks during the entire breeding period was 4.8%; Control group: At 30 days old, the ducks began to show signs of lethargy, neck shrinkage, and reduced appetite. Two days later, the number of deaths increased, with a mortality rate of about 8.3%. Autopsy of the dead ducks showed that the livers were brittle and swollen, and the spleens were swollen with necrotic spots on the surface. Laboratory tests showed that the pathogen was Salmonella typhimurium. Conventional antibiotic treatment alleviated the mortality rate, with about 50 deaths per day. The ducks were slaughtered at 35 days old, and the mortality rate of the entire breeding period was 10.5%; The above test data show that the functional probiotic S77 screened by the present invention is prepared into a product and applied to drinking water at 5, 6, 15 and 16 days of age in clinical practice, which can effectively reduce the probability of ducks contracting Salmonella typhimurium in clinical breeding, reduce the mortality rate of meat ducks, and improve economic benefits; at the same time, it is explained that the functional probiotic screening method for preventing Salmonella typhimurium based on the plate agglutination test of the present application is efficient, simple to operate and has a high success rate, and the screened functional probiotic strains have outstanding clinical application effects.

[0030] The present application provides a high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium. The method is a high-throughput reverse screening method based on a 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 screened functional probiotics can prevent Salmonella typhimurium infection, are safe and non-toxic, have no side effects, and have a long immune duration, and have important clinical application value; the functional probiotics can be prepared into functional probiotic vaccines or oral products, which can effectively prevent Salmonella typhimurium infection in clinical breeding, significantly reduce duck mortality, and improve economic benefits.

[0031] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0032] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium, characterized in that: The specific steps include: (i) Activating and culturing candidate probiotics: inoculating candidate probiotics into liquid culture medium for activation to obtain candidate probiotic culture solution; centrifuging the candidate probiotic culture solution and collecting the supernatant to obtain candidate probiotic test solution; (II) High-throughput screening of functional probiotics: the candidate probiotic test solution is diluted two-fold, and the candidate probiotic test solution, Salmonella typhimurium standard antigen, and physiological saline of different dilution multiples are respectively aspirated and dropped on a glass plate; then Salmonella typhimurium positive serum is added dropwise to evenly mix the Salmonella typhimurium positive serum with the liquid on the glass plate, and the serum is spread on the liquid surface to observe the results; (III) Screening of functional probiotics: When the standard antigen of Salmonella typhimurium agglutinates and the physiological saline does not agglutinate, the probiotics corresponding to the candidate probiotic test solution that agglutinates with the positive serum of Salmonella typhimurium are screened out, which are the screened functional probiotics.

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

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

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

5. The high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that: The centrifugal speed in step (a) is 8000 r / min and the centrifugal time is 10 min.

6. The high-throughput reverse screening method for 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 (ii) is sterile saline.

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

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

9. The high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that: Step (ii) After coating the liquid surface, observe the results after 2 minutes.

10. The high-throughput reverse screening method for functional probiotics for preventing Salmonella typhimurium according to claim 1, characterized in that: Screening methods also include duckling immune challenge tests.

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