Probiotic product for inhibiting salmonella in chicken body and screening method

Abstract

The invention relates to the technical field of microorganisms, in particular to a probiotic product for inhibiting salmonella in a chicken body and a screening method, the probiotic product comprises Chencity bacillus sachariensis, and the colony count of the Chencity bacillus sachariensis in the probiotic product is 107-1010 CFU / mL. Based on the screening method of the probiotics for inhibiting the salmonella in the chicken body, the screened city bacillus sachariensis is prepared into the probiotic product for inhibiting the salmonella in the chicken body, so that the salmonella infection condition of the chicken can be effectively improved, the weight of the chicken with positive salmonella infection is increased, and the salmonella in the chicken body is inhibited. The salmonella loading quantity in chicken bodies is obviously reduced, the salmonella loading quantity has the characteristics of low residual quantity and environment friendliness, and the problems of antibiotic residues, bacterial drug resistance increase and the like caused by antibiotic abuse can be avoided. By adopting the screening method of the probiotics for inhibiting the salmonella in the chicken body, provided by the invention, the dominant probiotics capable of inhibiting the salmonella can be simply, conveniently and efficiently determined.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to a probiotic product and screening method for inhibiting Salmonella in chickens BACKGROUND

[0002] Salmonella enterica serovar Enteritidis (SE) is a kind of zoonotic gram-negative bacteria, and is also one of the important foodborne pathogens. It widely exists in the living environment of various animals. As the largest reservoir of Salmonella enterica serovar Enteritidis, acute infection of Salmonella enterica serovar Enteritidis in poultry is usually accompanied by high morbidity and mortality, causing huge economic losses to poultry farming. Further, even if poultry is infected with Salmonella enterica serovar Enteritidis and does not directly cause illness, poultry farming is also plagued by Salmonella enterica serovar Enteritidis infection due to the strong survival ability and vertical transmission characteristics of Salmonella enterica serovar Enteritidis. In addition, human consumption of contaminated food, such as poultry-related products such as meat and eggs, can also lead to Salmonella enterica serovar Enteritidis infection, resulting in symptoms such as enterogastritis, and even causing death, which seriously threatens human health and safety. Therefore, controlling the infection of Salmonella enterica serovar Enteritidis in poultry is of great significance to poultry farming and human health.

[0003] In the prior art, antibiotics are often used as the main means to prevent and treat Salmonella enterica serovar Enteritidis infection in poultry. However, antibiotic treatment can lead to intestinal flora disorder, and long-term abuse can also cause a series of problems such as antibiotic residue and increased bacterial resistance. Therefore, it is necessary to seek a safer and more effective new method to replace antibiotics for improving the problem of Salmonella enterica serovar Enteritidis infection in poultry farming. SUMMARY

[0004] In view of the problems of antibiotic residue and increased bacterial resistance caused by the use of antibiotics to prevent and treat Salmonella enterica serovar Enteritidis infection in poultry in the prior art, the present application provides a probiotic product and screening method for inhibiting Salmonella enterica serovar Enteritidis in chickens.

[0005] The specific technical solutions of the present application are as follows: The present application provides a probiotic product for inhibiting Salmonella enterica serovar Enteritidis in chickens, which comprises S. enterica serovar Enteritidis, and the number of colonies of S. enterica serovar Enteritidis in the probiotic product is 10 7 ~10 10 CFU / mL.

[0006] In a possible implementation, compared with not applying the probiotic product, after the chicken positive for Salmonella enterica serovar Enteritidis is continuously applied with the probiotic product for seven days, the body weight of the chicken is increased by more than 8%, and the spleen bacterial load of the chicken is reduced by more than 50%.

[0007] The application also provides a use of the Gallibacterium salmosina in the preparation of a medicine for treating chicken Salmonella infection.

[0008] In a possible implementation, the medicine is a probiotic product. Preferably, the number of bacteria of the Gallibacterium salmosina in the probiotic product is 10 7 ~10 10 CFU / mL.

[0009] The application also provides a method for screening a probiotic for inhibiting Salmonella in a chicken body, which comprises the following steps: S1, collecting fresh feces of healthy adult Tibetan chickens that are tested negative for Salmonella, adding sterile PBS buffer, mixing and centrifuging to obtain a fecal bacteria suspension; S2, dividing the fecal bacteria suspension into two parts to obtain a first fecal bacteria suspension and a second fecal bacteria suspension, sterilizing the first fecal bacteria suspension, and culturing the first fecal bacteria suspension and the second fecal bacteria suspension with Salmonella culture solution respectively and counting the number of Salmonella to obtain the first number of Salmonella in the first fecal bacteria suspension and the second number of Salmonella in the second fecal bacteria suspension; S3, preparing the fecal bacteria suspension by using the preparation step of the fecal bacteria suspension in S1, filtering the fecal bacteria suspension through a first migration device with a first pore size into a first culture solution containing Salmonella and through a second migration device with a second pore size into a second culture solution containing Salmonella, and counting the number of Salmonella after culture to obtain the third number of Salmonella in the first culture solution and the fourth number of Salmonella in the second culture solution; the first migration device allows microorganisms and metabolites to pass through, and the second migration device only allows metabolites to pass through; S4, selecting Tibetan chickens and broiler chickens, preparing the fecal bacteria suspension of the Tibetan chickens and the fecal bacteria suspension of the broiler chickens by using the preparation step of the fecal bacteria suspension in S1, and cross-transplanting the two different fecal bacteria suspensions between the two chicken species, treating the recipient chickens for Salmonella infection after continuous administration for a preset length of time, collecting the cecal contents of the Tibetan chickens and the cecal contents of the broiler chickens for intestinal microbial analysis to obtain the first microbial abundance data in the intestinal tract of the Tibetan chickens and the second microbial abundance data in the intestinal tract of the broiler chickens; S5, determining the dominant probiotic for inhibiting Salmonella based on the size relationship between the first number of Salmonella and the second number of Salmonella, the size relationship between the third number of Salmonella and the fourth number of Salmonella, and the microbial abundance difference between the first microbial abundance data and the second microbial abundance data, wherein the dominant probiotic is the Gallibacterium salmosina provided in any of the above embodiments.

[0010] In a possible implementation, the volume of the PBS buffer in S1 is 3-5 times that of the fresh feces. In a possible implementation, the mixing method in S1 is vortex mixing. In a possible implementation, the mixing time in S1 is 2-4 min. In a possible implementation, the centrifugal force in S1 is 550-650 g. In a possible implementation, the centrifugation time in S1 is 0.5-1.5 min.

[0011] In a possible implementation, the culture time in S2 is more than 24 h. In a possible implementation, the culture time in S3 is 3.5-4.5 h.

[0012] In a possible implementation, the administration method of the fecal bacteria suspension cross transplantation in S4 comprises the following steps: taking the broiler as the recipient chicken, using a length of 7-9 cm of a disposable sterile hose to feed the broiler crop of the broiler in an empty stomach state with 0.1-0.2 mL of the fecal bacteria suspension of the Tibetan chicken; taking the Tibetan chicken as the recipient chicken, using a length of 7-9 cm of a disposable sterile hose to feed the Tibetan chicken crop of the Tibetan chicken in an empty stomach state with 0.1-0.2 mL of the fecal bacteria suspension of the broiler; and determining the length of the hose into the recipient chicken before feeding based on the crop position of each recipient chicken to ensure that the front end of the hose can reach the crop of the recipient chicken for feeding, and keeping the length of the hose into each recipient chicken the same.

[0013] In a possible implementation, the preset time length of the continuous administration in S4 is 7 days.

[0014] In a possible implementation, the processing method of the Salmonella infection in S4 is to feed the recipient chicken with Salmonella.

[0015] In a possible implementation, the collection method of the cecal contents in S4 is to respectively select the recipient chickens infected with Salmonella for 1, 3 and 7 days for slaughter sampling, and collect the cecal contents of the recipient chickens.

[0016] In a possible implementation, the Tibetan chicken and the broiler in S4 are 1-day-old.

[0017] Based on the above technical solution, the present application has at least the following beneficial effects: 1. Based on the screening method of the probiotic for inhibiting Salmonella in chickens, the Salinisphaera sp. obtained by screening is made into a probiotic product for inhibiting Salmonella in chickens, which can effectively improve the Salmonella infection of chickens, increase the body weight of chickens with Salmonella infection, significantly reduce the Salmonella load in chickens, and has the characteristics of low residual amount and environmental friendliness, which can avoid the problems of antibiotic residues and increased bacterial resistance caused by antibiotic abuse.

[0018] 2. Using the probiotic screening method for inhibiting Salmonella in chickens provided by this invention, the prepared fecal microbiota suspension is filtered through a migration device and then mixed with Salmonella culture medium for co-culture. Then, the prepared fecal microbiota suspensions of Tibetan chickens and broiler chickens are cross-transplanted. Based on the Salmonella colony count in the culture medium and the abundance of microorganisms in the Tibetan chicken intestine, the dominant probiotics that inhibit Salmonella can be identified simply and efficiently. BRIEF DESCRIPTION OF DRAWINGS

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the prior art or embodiments will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0020] Figure 1 Salmonella count was performed by co-culturing a suspension of fecal bacteria from Tibetan chickens with a Salmonella culture medium for 24 hours. Figure 2 Comparison of the inhibitory effects of fecal microbiota suspensions separated by Transwell chambers on Salmonella; Figure 3 Changes in the cecal microbiota species level in chickens after cross-transplantation of fecal microbiota suspension; Figure 4 10 7 The inhibitory effect of CFU / mL of probiotic products containing *C. marineri* on *Salmonella*. Figure 5 10 8 The inhibitory effect of CFU / mL of probiotic products containing *C. marineri* on *Salmonella*. Figure 6 10 9 The inhibitory effect of CFU / mL of probiotic products containing *C. marineri* on *Salmonella*. Figure 7 10 6 The inhibitory effect of CFU / mL of probiotic products containing *C. marineri* on *Salmonella*. Figure 8 The changes in body weight of chickens on days 1, 3, and 7 after they tested positive for Salmonella. Figure 9 The changes in liver bacterial load in chickens on days 1, 3, and 7 after Salmonella infection; Figure 10 Changes in the bacterial load in the spleen of chickens on days 1, 3, and 7 after Salmonella infection; Figure 11The changes in cecal bacterial load in chickens on days 1, 3, and 7 after Salmonella infection were observed.

[0021] Figure 12 The sequence restriction sites for sequencing the probiotics identified by the screening method of this invention.

[0022] Figure 13 The sequencing results are those of the probiotics identified by the screening method of this invention.

[0023] Figure 14 The comparison results are as follows: the probiotics identified by the screening method of the present invention are compared with Phocaeicola salanitronis strain DSM 18170. DETAILED DESCRIPTION

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] For the terms defined below, unless a different definition is given elsewhere in the claims or specification, these definitions shall apply. All numerical values, whether or not explicitly indicated, are defined herein as being modified by the term "about". The term "about" generally refers to a range of numerical values ​​that a person skilled in the art would consider equivalent to the stated values ​​to produce substantially the same properties, functions, results, etc. A range of numerical values ​​indicated by a low value and a high value is defined as encompassing all numerical values ​​included within this range and all subranges included within this range.

[0026] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising," "having," and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0027] The embodiments of the present application are described below with reference to the accompanying drawings, which are not intended to limit the disclosure as recorded in the claims.

[0028] The embodiments of the present application provide a probiotic product for inhibiting Salmonella in a chicken body, characterized in that the probiotic product comprises Phocaeicola Salanitronis, and the number of colonies of the Phocaeicola Salanitronis in the probiotic product is 10 7 ~10 10 CFU / mL.

[0029] Specifically, the Phocaeicola Salanitronis in the probiotic product is determined by the probiotic screening method for inhibiting Salmonella in a chicken body described below. The number of colonies of the Phocaeicola Salanitronis in the probiotic product can be any point value in 10 7 ~10 10 CFU / mL, for example, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, etc.

[0030] Therefore, based on the probiotic screening method for inhibiting Salmonella in a chicken body described below, the Phocaeicola Salanitronis screened is made into a probiotic product for inhibiting Salmonella in a chicken body, which can effectively improve the Salmonella infection of the chicken, increase the body weight of the chicken with Salmonella infection, and significantly reduce the Salmonella load in the chicken body, and has the characteristics of low residue and environmental friendliness, and can avoid the problems of antibiotic residue and increased bacterial resistance caused by antibiotic abuse.

[0031] In some embodiments, after the chicken with Salmonella infection is continuously administered with the probiotic product for seven days, the body weight of the chicken is increased by more than 8%, and the spleen load of the chicken is reduced by more than 50% compared with the chicken without administration of the probiotic product.

[0032] The application further provides a method for screening probiotics for inhibiting Salmonella in a chicken body, comprising the following steps: S1, collecting fresh feces of healthy adult Tibetan chickens detected as negative for Salmonella, adding sterile PBS buffer, mixing and centrifuging to obtain a fecal bacteria suspension; S2, dividing the fecal bacteria suspension into two parts to obtain a first fecal bacteria suspension and a second fecal bacteria suspension, sterilizing the first fecal bacteria suspension, and culturing the first fecal bacteria suspension and the second fecal bacteria suspension with Salmonella culture solution and counting the number of Salmonella to obtain the first number of Salmonella colonies in the first fecal bacteria suspension and the second number of Salmonella colonies in the second fecal bacteria suspension; S3, preparing the fecal bacteria suspension by the preparation step of S1, filtering the fecal bacteria suspension through a first migration device with a first pore size into a first culture solution containing Salmonella, and filtering the fecal bacteria suspension through a second migration device with a second pore size into a second culture solution containing Salmonella, counting the number of Salmonella after culture to obtain the third number of Salmonella colonies in the first culture solution and the fourth number of Salmonella colonies in the second culture solution; the first migration device allows microorganisms and metabolites to pass through, and the second migration device only allows metabolites to pass through; S4, selecting Tibetan chickens and broiler chickens, preparing the fecal bacteria suspension of the Tibetan chickens and the fecal bacteria suspension of the broiler chickens by the preparation step of S1, cross-transplanting the two different fecal bacteria suspensions between the two chicken species, infecting the recipient chickens with Salmonella after continuous administration for a preset period of time, collecting the cecal contents of the Tibetan chickens and the cecal contents of the broiler chickens for intestinal microbial analysis to obtain the first microbial abundance data in the intestinal tract of the Tibetan chickens and the second microbial abundance data in the intestinal tract of the broiler chickens; S5, determining the dominant probiotics for inhibiting Salmonella based on the size relationship between the first number of Salmonella colonies and the second number of Salmonella colonies, the size relationship between the third number of Salmonella colonies and the fourth number of Salmonella colonies, and the microbial abundance difference between the first microbial abundance data and the second microbial abundance data, and the dominant probiotics are the S. gallinarum of claim 1 or claim 2.

[0033] Specifically, S1: Salmonella detection is performed on a population of adult Tibetan chickens, and healthy adult Tibetan chickens with negative Salmonella detection in the population are selected as fecal bacteria collection samples, and a disinfected clean fecal tray is selected to receive fresh feces. The collected fresh feces is transferred to a sterile centrifuge tube and weighed, then sterile PBS buffer is added, mixed and centrifuged, and the supernatant is collected to obtain a fecal bacteria suspension. S2: The fecal bacteria suspension obtained in S1 is divided into two parts to obtain a first fecal bacteria suspension and a second fecal bacteria suspension, and the first fecal bacteria suspension is subjected to high-temperature sterilization treatment. The first fecal bacteria suspension and the second fecal bacteria suspension are respectively cultured with Salmonella culture solution for a period of time and subjected to Salmonella counting to obtain the first Salmonella colony count in the first fecal bacteria suspension and the second Salmonella colony count in the second fecal bacteria suspension. S3: The fecal bacteria suspension is prepared by the preparation step of S1, and then the fecal bacteria suspension is filtered through a first migration device with a first pore size into a first culture solution containing Salmonella, and through a second migration device with a second pore size into a second culture solution containing Salmonella. After co-culturing for a period of time, the mixed culture solution is subjected to Salmonella counting to obtain the third Salmonella colony count in the first culture solution and the fourth Salmonella colony count in the second culture solution; wherein the first migration device allows microorganisms and metabolites to pass through, and the second migration device only allows metabolites to pass through. S4: Select Tibetan chickens and broiler chickens, and respectively obtain the fecal bacteria suspension of Tibetan chickens and the fecal bacteria suspension of broiler chickens by the preparation step of S1. Then, the two different fecal bacteria suspensions are cross-transplanted between the two chicken species, i.e. the fecal bacteria suspension of Tibetan chickens is transplanted into broiler chickens, and the fecal bacteria suspension of broiler chickens is transplanted into Tibetan chickens. After continuous administration for a preset period of time, the recipient chickens are subjected to Salmonella infection treatment, and the cecal contents of Tibetan chickens and broiler chickens are collected for intestinal microbial analysis to obtain first microbial abundance data in the intestinal tract of Tibetan chickens and second microbial abundance data in the intestinal tract of broiler chickens. S5, based on the size relationship between the first Salmonella colony count and the second Salmonella colony count, the size relationship between the third Salmonella colony count and the fourth Salmonella colony count, and the microbial abundance difference between the first microbial abundance data and the second microbial abundance data, the dominant probiotic bacteria with Salmonella inhibition are comprehensively analyzed and determined, and the dominant probiotic bacteria are Phocaeicola Salanitronis.

[0034] In this way, by using the above-mentioned method for screening probiotic bacteria for inhibiting Salmonella in chickens, the prepared fecal bacteria suspension is filtered through the migration device and mixed with the Salmonella culture solution for co-culture, and the prepared fecal bacteria suspension of Tibetan chickens and the fecal bacteria suspension of broiler chickens are cross-transplanted. Based on the Salmonella colony count in the culture solution and the microbial abundance data in the intestinal tract of Tibetan chickens, the dominant probiotic bacteria with Salmonella inhibition can be determined simply and efficiently.

[0035] In order to verify that the probiotic determined by the above screening method is Phocaeicola Salanitronis, the screened probiotic is sent to Shengqiang Bioengineering (Shanghai) Co., Ltd. for sequencing using universal primers 27F and 1492R, and the sequencing sequence is as shown in Figure 12 and Figure 13 Among them, Figure 12 the sequence enzyme cutting site is shown, Figure 13 the sequencing result is shown. The sequencing result is: SEQ ID NO: 1:

[0036] The applicant then compared the above sequence to the NCBI database (https: / / blast.ncbi), and the results showed a 98.8% similarity to *Phocaeicola salanitronis* strain DSM 18170, confirming that they are the same strain. See the comparison results below. Figure 14 Phocaeicola salanitronis is commercially available from NNCC (BNCC358144).

[0037] Preferably, during the co-culture of S2, Salmonella counting is performed every 2 hours.

[0038] Preferably, a Transwell chamber is used as the migration device.

[0039] Preferably, the first pore size is 0.8 μm and the second pore size is 0.4 μm.

[0040] Preferably, the mixed culture medium is inoculated onto XLD plates for Salmonella counting.

[0041] Preferably, AA broiler chickens are selected as broiler chicken samples.

[0042] In some embodiments, the volume of PBS buffer in S1 is 3 to 5 times that of fresh feces; Specifically, the volume of PBS buffer in S1 can be any value between 3 and 5 times that of fresh feces, for example, it can be 3 times, 4 times, 5 times, etc.

[0043] Therefore, using 3 to 5 times the volume of PBS buffer can effectively dilute the collected fresh fecal samples, which is beneficial for collecting microorganisms and metabolites in the fresh fecal samples, providing a sample basis for subsequent screening steps.

[0044] Preferably, the volume of PBS buffer in S1 is 4 times that of fresh feces.

[0045] In some embodiments, the mixing method in S1 is vortex mixing; In this way, the vortex method can quickly mix fresh fecal samples with PBS buffer, which is beneficial for microorganisms and metabolites in fresh fecal samples to enter the PBS buffer, providing a better mixing effect and providing a sample basis for subsequent screening steps.

[0046] In some embodiments, the mixing time in S1 is 2 to 4 minutes; Specifically, the mixing time in S1 can be any value within 2-4 minutes, and exemplary values can be 2 minutes, 3 minutes, 4 minutes, etc.

[0047] Preferably, the mixing time in S1 is 3 minutes.

[0048] In this way, mixing the fresh fecal sample and the PBS buffer for 2-4 minutes can facilitate sufficient mixing, so that the microorganisms and metabolites in the fresh fecal sample are separated into the PBS buffer, and a better mixing effect is provided, thereby providing a sample basis for the subsequent screening step.

[0049] In some embodiments, the centrifugal force in S1 is 550-650 g. Specifically, the centrifugal force in S1 can be any value within 550-650 g, and exemplary values can be 550 g, 600 g, 650 g, etc.

[0050] In this way, centrifuging the mixed solution after mixing in S1 at a centrifugal force of 550-650 g can facilitate the microorganisms and metabolites in the fresh fecal sample to be fully separated into the PBS buffer, thereby providing a sample basis for the subsequent screening step.

[0051] Preferably, the centrifugal force in S1 is 600 g.

[0052] In some embodiments, the centrifugation time in S1 is 0.5-1.5 minutes.

[0053] Specifically, the centrifugation time in S1 can be any value within 0.5-1.5 minutes, and exemplary values can be 0.5 minutes, 1 minute, 1.5 minutes, etc.

[0054] In this way, centrifuging the mixed solution after mixing in S1 for 0.5-1.5 minutes can facilitate the mixed solution after mixing to be fully centrifuged, so that the microorganisms and metabolites in the fresh fecal sample are fully centrifuged into the PBS buffer, thereby providing a sample basis for the subsequent screening step.

[0055] Preferably, the centrifugation time in S1 is 1 minute.

[0056] In some embodiments, the culture time of S2 is 24 hours or more. In this way, mixing and co-culturing the inactivated fecal bacteria suspension and the un-inactivated fecal bacteria suspension with the culture solution containing Salmonella for 24 hours or more can facilitate the effective substances in the fecal bacteria suspension to fully contact with the Salmonella in the culture solution, so that the effective substances in the fecal bacteria suspension can fully play a role in inhibiting Salmonella, thereby providing a basis for collecting data required for subsequent analysis and screening.

[0057] In some embodiments, the culture time of S3 is 3.5-4.5 hours.

[0058] Specifically, the culture time of S3 can be any value within 3.5-4.5 hours, and exemplarily can be 3.5 hours, 4 hours, 4.5 hours, etc.

[0059] In this way, the fecal bacteria suspension filtered by the migrated device is mixed with the culture solution containing Salmonella and co-cultured for 3.5-4.5 hours, which is conducive to the full contact between the effective substances in the fecal bacteria suspension and the Salmonella in the culture solution, so that the effective substances in the fecal bacteria suspension can fully play a role in inhibiting Salmonella, and provide a basis for collecting data required for subsequent analysis and screening.

[0060] In some embodiments, the administration method of cross-transplantation of the fecal bacteria suspension of S4 includes: taking broiler chickens as the recipient chickens, using a disposable sterile hose with a length of 7-9 cm to gavage the crop of the broiler chickens in an empty stomach state with 0.1-0.2 mL of the fecal bacteria suspension of Tibetan chickens; taking Tibetan chickens as the recipient chickens, using a disposable sterile hose with a length of 7-9 cm to gavage the crop of the Tibetan chickens in an empty stomach state with 0.1-0.2 mL of the fecal bacteria suspension of broiler chickens; and determining the length of the hose entering the body of each recipient chicken based on the position of the crop of each recipient chicken before gavage, so as to ensure that the front end of the hose can reach the crop of the recipient chicken for gavage, and keep the length of the hose entering the body of each recipient chicken the same.

[0061] In this way, the crop gavage method is easy to operate, and can efficiently transplant the corresponding fecal bacteria suspension into the recipient chickens, thereby completing the cross-transplantation of the fecal bacteria suspension. Further, keeping the length of the hose entering the body of each recipient chicken the same can reduce the influence of the error of the test operation on the transplantation effect of the fecal bacteria suspension.

[0062] Preferably, the crop gavage of the recipient chickens is performed in the morning in an empty stomach state.

[0063] Preferably, 0.1 mL of the corresponding fecal bacteria suspension is gavaged into the crop of the recipient chickens.

[0064] Preferably, the length of the disposable sterile hose is 8 cm.

[0065] In some embodiments, the preset time length of the continuous administration of S4 is 7 days.

[0066] In this way, the continuous administration of the corresponding fecal bacteria suspension to the recipient chickens for 7 days is conducive to better completing the cross-transplantation of the fecal bacteria suspension between Tibetan chickens and broiler chickens, thereby providing a basis for the fecal bacteria suspension to fully play a role in inhibiting Salmonella.

[0067] In some embodiments, the method of Salmonella infection treatment of S4 is to gavage Salmonella into the recipient chickens.

[0068] Thus, the way of gavage of Salmonella is easy to operate, and the Salmonella infection of the recipient chicken can be efficiently completed, so that the recipient chicken is Salmonella positive.

[0069] In some embodiments, the cecal contents of S4 are collected by respectively selecting the recipient chickens infected with Salmonella for 1, 3 and 7 days for slaughter sampling, and collecting the cecal contents of the recipient chickens.

[0070] Thus, selecting the recipient chickens infected with Salmonella for 1, 3 and 7 days for slaughter sampling is beneficial to provide the change of the process of inhibiting Salmonella in the chicken by the fecal bacteria suspension.

[0071] In some embodiments, the Tibetan chickens and broilers of S4 are 1 day old.

[0072] The implementation effects of the technical solutions of the present application are described in detail below in combination with specific embodiments. Embodiment 1

[0073] The present embodiment provides a method for screening probiotics for inhibiting Salmonella in chickens, and the specific screening steps are as follows: 1. Preparation of fecal bacteria suspension: adult Tibetan chicken groups are detected for Salmonella, and healthy adult Tibetan chickens in the group with negative Salmonella detection are selected as fecal bacteria collection samples, and a sterilized clean plastic fecal tray is used to collect fresh feces. The collected fresh feces are transferred to a 50mL sterile centrifuge tube and weighed, then 4 times the volume of sterile PBS buffer is added, vortexed for 3min, and centrifuged at 600g to collect the supernatant to obtain the fecal bacteria suspension.

[0074] 2. High-temperature sterilization separation: the fecal bacteria suspension is divided into two equal parts to obtain the first and second fecal bacteria suspensions, and the first fecal bacteria suspension is sterilized in a pressure cooker for 20min to inactivate the microorganisms in the fecal bacteria suspension, and the second fecal bacteria suspension is not treated additionally. The two parts of fecal bacteria suspension are cultured with Salmonella: a. Take the Salmonella culture solution stored in the refrigerator at -80℃, inoculate in modified Martin, the inoculation ratio is Salmonella culture solution: modified Martin = 1:10, and place in a 37℃ constant temperature incubator, incubate at 160rpm for 16h.

[0075] b. Take the Salmonella culture solution in the constant temperature incubator, inoculate in new modified Martin, the inoculation ratio is Salmonella culture solution: modified Martin = 1:25, and place in a 37℃ constant temperature incubator, incubate at 160rpm for 4h.

[0076] c. Take out the Salmonella culture solution in the constant temperature incubator, transfer it to a centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash once with sterile PBS buffer, then centrifuge at 8000 rpm for 5 min, collect the precipitate. Dilute the precipitate to 1 x 10 10 CFU / mL with modified Martin, store it in a refrigerator at 4°C for standby.

[0077] d. Take out the Salmonella culture solution in the refrigerator at 4°C, take 3 test tubes, add 10 mL of Salmonella culture solution to each test tube, then add 10 mL of inactivated first fecal bacteria suspension and 10 mL of non-inactivated second fecal bacteria suspension to each test tube, respectively, place them in a constant temperature incubator at 37°C, every 2 h, take 50 μL of mixed solution from each of the 3 test tubes, dilute it 10 times, inoculate it on XLD plates for Salmonella counting, collect 24 h, as shown in Figure 1 .

[0078] As can be seen from Figure 1 , the number of Salmonella in the experimental group with non-inactivated second fecal bacteria suspension is reduced by more than 99%, the number of Salmonella in the experimental group with non-inactivated second fecal bacteria suspension is significantly lower than that in the experimental group with inactivated first fecal bacteria suspension and the control group with only Salmonella culture solution, and the difference between the number of Salmonella in the experimental group with inactivated first fecal bacteria suspension and the control group with only Salmonella culture solution is small, which shows that the microorganisms in the fecal bacteria suspension have a significant inhibitory effect on Salmonella.

[0079] 3. Transwell chamber separation: place the prepared fecal bacteria suspension in a Transwell chamber with a pore size of 0.8 μm and a Transwell chamber with a pore size of 0.4 μm, the microorganisms and metabolites in the fecal bacteria suspension pass through the pore size of 0.8 μm to mix with the first culture solution containing Salmonella, and only the metabolites in the fecal bacteria suspension pass through the pore size of 0.4 μm to mix with the second culture solution containing Salmonella, after 4 h of co-culture, take the mixed culture solution and inoculate it on XLD plates for Salmonella counting, get the third number of Salmonella in the first culture solution and the fourth number of Salmonella in the second culture solution, as shown in Figure 2 .

[0080] As can be seen from Figure 2 , in the presence of microorganisms and metabolites, the fecal bacteria suspension can significantly inhibit Salmonella, and the third number of Salmonella is reduced by more than 98% compared with the control group. In the presence of only metabolites, the inhibitory effect of the fecal bacteria suspension on Salmonella is not obvious, and the fourth number of Salmonella is similar to that of the control group.

[0081] 4. Fecal bacteria suspension cross transplantation: Select 1-day-old Tibetan chickens and AA broilers, and prepare the fecal bacteria suspension of Tibetan chickens and the fecal bacteria suspension of AA broilers using the above preparation steps. Then, cross transplant the two different fecal bacteria suspensions between the two chicken species. When the broiler is the recipient chicken, use a length of 8 cm of disposable sterile hose to gavage 0.1 mL of the fecal bacteria suspension of Tibetan chickens into the crop of the broiler in an empty stomach state. When the Tibetan chicken is the recipient chicken, use a length of 8 cm of disposable sterile hose to gavage 0.1 mL of the fecal bacteria suspension of broilers into the crop of the Tibetan chicken in an empty stomach state. After continuous gavage for 7 days, gavage the recipient chickens with Salmonella, and on the 1st, 3rd, and 7th days after the recipient chickens are infected with Salmonella, slaughter the recipient chickens and collect the cecal contents of the Tibetan chickens and the cecal contents of the broilers for intestinal microbial analysis to obtain the first microbial abundance data in the intestinal tract of the Tibetan chickens and the second microbial abundance data in the intestinal tract of the broilers, as shown in Figure 3 .

[0082] As can be seen from Figure 3 , after cross transplantation of the fecal bacteria suspension, at the species level, gavage of the AA broiler fecal bacteria suspension into the Tibetan chicken significantly reduced the abundance levels of Bacteroides Thetaiotaomicron, Bacteroides Salyersiae, Bacteroides Plebeius, and Bacteroides Stercoris in the intestinal tract of the Tibetan chicken, while the abundance level of Phocaeicola Salanitronis did not change significantly. Gavage of the Tibetan chicken fecal bacteria suspension into the AA broiler significantly increased the abundance levels of Bacteroides Thetaiotaomicron, Bacteroides Salyersiae, and Phocaeicola Salanitronis in the intestinal tract of the AA broiler.

[0083] 5. Comprehensive analysis: Based on the size relationship between the first Salmonella colony number and the second Salmonella colony number, and the microbial abundance difference between the first microbial abundance data and the second microbial abundance data, it is determined that Phocaeicola Salanitronis is the dominant probiotic bacteria with inhibitory effect on Salmonella. Example 2

[0084] This example verifies the inhibitory effect of the probiotic product containing 10 7 CFU / mL of Phocaeicola Salanitronis on Salmonella.

[0085] The probiotic product containing 10 7CFU / mL of Caucasus marijuana probiotic products with 10 8 CFU / mL Salmonella culture was co-cultured under anaerobic conditions at 37°C for 48 hours. The mixture was then inoculated onto XLD plates for Salmonella counting. Figure 4 As shown.

[0086] Depend on Figure 4 It can be seen that it contains 10 7 The presence of a probiotic product containing CFU / mL of Salmonella marijuana can reduce the number of Salmonella colonies by 84%. Example 3

[0087] This embodiment verifies the presence of 10 8 The inhibitory effect of CFU / mL of probiotic products containing *C. sarcodactylis marineri* on *Salmonella*.

[0088] Will contain 10 8 CFU / mL of Caucasus marijuana probiotic products with 10 8 The bacteria were co-cultured with CFU / mL Salmonella culture medium. Other settings and procedures were the same as in Example 3, and will not be repeated here. The results are as follows: Figure 5 As shown.

[0089] Depend on Figure 5 It can be seen that it contains 10 8 The presence of a probiotic product containing CFU / mL of Salmonella marijuana can reduce the number of Salmonella colonies by 82%. Example 4

[0090] This embodiment verifies the presence of 10 9 The inhibitory effect of CFU / mL of probiotic products containing *C. sarcodactylis marineri* on *Salmonella*.

[0091] Will contain 10 9 CFU / mL of Caucasus marijuana probiotic products with 10 8 The bacteria were co-cultured with CFU / mL Salmonella culture medium. Other settings and procedures were the same as in Example 3, and will not be repeated here. The results are as follows: Figure 6 As shown.

[0092] Depend on Figure 6 It can be seen that it contains 10 9 The presence of a probiotic product containing CFU / mL of Salmonella marijuana can reduce the number of Salmonella colonies by 98%.

[0093] Comparative Example 1 This embodiment verifies the presence of 10 6 The inhibitory effect of CFU / mL of probiotic products containing *C. sarcodactylis marineri* on *Salmonella*.

[0094] The probiotic product containing 10 6 CFU / mL of S. enterica was co-cultured with 10 8 CFU / mL of Salmonella culture solution, and other settings and steps were the same as those in Example 3, which are not described herein again, and the results are shown in Figure 7

[0095] As can be seen from Figure 7 , the presence of the probiotic product containing 10 6 CFU / mL of S. enterica does not significantly reduce the number of Salmonella colonies, that is, the inhibitory effect on Salmonella is weak.

[0096] As can be seen from the above Examples 3-5 and Comparative Example 1, when the number of S. enterica in the probiotic product is 10 7 CFU / mL, 10 8 CFU / mL and 10 9 CFU / mL, the probiotic product can exhibit a better inhibitory effect on Salmonella, and when the number of S. enterica is 10 6 CFU / mL, the inhibitory effect is not obvious. Example 5

[0097] This example verifies the inhibitory effect of the probiotic product of the present application on Salmonella in chickens.

[0098] 105 one-day-old AA broilers were selected and randomly divided into a control group, a probiotic product experimental group, a Salmonella experimental group and a probiotic product and Salmonella mixed experimental group, each group including 36 chickens, and all the chickens were fed with a basic diet. The chickens in the control group were not gavaged, the chickens in the probiotic product experimental group were gavaged with the probiotic product containing 10 8 CFU / mL of S. enterica for 7 consecutive days, the chickens in the Salmonella experimental group were gavaged with 0.5 mL of Salmonella culture solution with a number of 10 10 CFU / mL on the 8th day, and the chickens in the probiotic product and Salmonella mixed experimental group were gavaged with the probiotic product containing 10 8 CFU / mL of S. enterica for 7 consecutive days, and then gavaged with 0.5 mL of Salmonella culture solution with a number of 10 10 CFU / mL on the 8th day. On the 1st day, the 3rd day and the 7th day after the gavage of the Salmonella culture solution on the 8th day, 10 chickens in each group were randomly selected, weighed and then slaughtered for sampling, and the liver, spleen and cecum of the chickens were collected for microbial analysis, and the Salmonella load data of the liver, spleen and cecum of the chickens were obtained, as shown in Figures 8-11

[0099] As can be seen from Figure 8 ​​It can be seen that chickens of the same age that were only given probiotic products had a higher body weight than chickens of the same age that were not given probiotics, proving that the probiotic product of the present invention is beneficial to the weight gain of chicks. Furthermore, among the two groups of chickens that were given Salmonella by gavage on day 8, the chickens of the same age that were given probiotic products for 7 consecutive days also had a significantly higher body weight than the chickens of the same age that were only given Salmonella by gavage, with a weight gain of over 14%. Moreover, the chickens that were only given Salmonella by gavage showed a trend of weight loss in the early stages of Salmonella infection. Therefore, the probiotic product of the present invention can inhibit Salmonella and significantly increase the body weight of chickens that are positive for Salmonella infection, and also has a certain effect on increasing the body weight of chickens that are not infected with Salmonella.

[0100] Depend on Figure 9 It can be seen that, among the two groups of chickens that were administered Salmonella by gavage on the 8th day, the liver bacterial load of chickens that were administered probiotic products for 7 consecutive days was significantly lower than that of chickens that were administered Salmonella only by gavage on the same day. Specifically, compared with the liver bacterial load of chickens that were administered Salmonella only by gavage on the same day, the liver bacterial load of chickens that were administered probiotic products for 7 consecutive days decreased by more than 97% after 1 day and by more than 99% after 3 days. Furthermore, the liver of the chickens returned to Salmonella negative on the 7th day after Salmonella administration. Therefore, the probiotic product of the present invention can reduce the Salmonella bacterial load in the chicken liver and inhibit Salmonella in the chicken liver.

[0101] Depend on Figure 10 It can be seen that, among the two groups of chickens that were administered Salmonella by gavage on the 8th day, the bacterial load in the spleen of chickens that were administered probiotic products for 7 consecutive days at the same age was significantly lower than that in chickens that were administered Salmonella only by gavage at the same age. Specifically, compared with the bacterial load in the liver of chickens that were administered Salmonella only by gavage at the same age, the bacterial load in the liver of chickens that were administered probiotic products for 7 consecutive days at the same age decreased by more than 97% after 1 day, more than 37% after 3 days, and more than 99% after 7 days. Therefore, the probiotic product of the present invention can reduce the Salmonella load in the chicken spleen and inhibit Salmonella in the chicken spleen.

[0102] Depend on Figure 11 It can be seen that, among the two groups of chickens that were administered Salmonella by gavage on the 8th day, the cecal bacterial load of chickens that were administered the probiotic product for 7 consecutive days at the same age was significantly lower than that of chickens that were administered Salmonella by gavage only at the same age. Furthermore, the cecum of the chickens returned to Salmonella negative on the 3rd day after Salmonella administration. Therefore, the probiotic product of the present invention can reduce the Salmonella bacterial load in the cecum of chickens and inhibit Salmonella in the cecum of chickens.

[0103] In summary Figures 8-11 It is known that the probiotic product of the present invention can significantly increase the weight of chickens and reduce the amount of Salmonella in chickens that are positive for Salmonella infection, thereby inhibiting Salmonella.

[0104] Each of the embodiments in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments, and the same or similar parts between the embodiments can be referred to each other.

[0105] It should be noted that all the features (including the technical features recorded in different embodiments) recorded in the present application can be combined arbitrarily under reasonable circumstances, and the new technical solutions formed by the combination are all within the protection scope of the present application.

[0106] The above only describes some embodiments of the present application, and is not used to limit the present application. It should be understood by those skilled in the art that the present application can have various changes and improvements, and any modification, equivalent replacement and improvement according to the present application all fall within the protection scope of the present application.

Claims

1. A probiotic product that inhibits Salmonella in chickens, characterized in that, The probiotic product includes *C. sarcodactylis marines*, and the colony count of *C. sarcodactylis marines* in the probiotic product is 10-1. 7 ~10 10 CFU / mL.

2. The probiotic product according to claim 1, characterized in that, Compared to not using the probiotic product, after seven consecutive days of administration of the probiotic product to chickens that tested positive for Salmonella infection, the chickens' body weight increased by more than 8%, and the bacterial load in the chickens' spleens decreased by more than 50%.

3. Use of *Cryptospira marines* in the preparation of drugs for treating *Salmonella avianis* infection in chickens.

4. The use as described in claim 3, characterized in that, The drug is a probiotic product.

5. The use as described in claim 4, characterized in that, The colony count of *Cryptospira marines* in the probiotic product is 10. 7 ~10 10 CFU / mL.

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