Lactobacillus salivarius lsf12 and use thereof

Abstract

The present application relates to the technical field of microorganism and fermentation, and particularly relates to a saliva combined lactobacillus (Lactobacillus salivarius, LSF12) and application thereof. Ligilactobacillus salivarius ​ The saliva combined lactobacillus LSF12 provided by the present application has a preservation number of CGMCC NO.33008, can effectively inhibit the growth and reproduction of escherichia coli, and has good tolerance to high temperature, high cholesteric salt and low pH environment. The present application also studies a fermentation method of the saliva combined lactobacillus LSF12, and provides a fermentation method suitable for rapid production of the saliva combined lactobacillus LSF12. The obtained fermentation product can be used for development and production of downstream products. Further research shows that the fermentation product can improve the egg production performance and egg quality of old laying hens.

Description

Technical Field

[0001] This invention relates to the field of microbiology and fermentation technology, and more particularly to a type of Lactobacillus salivarius (…). Ligilactobacillus salivarius LSF12 and its applications. Background Technology

[0002] Avian colibacillosis refers to a focal or systemic infection caused by specific serotypes of pathogenic Escherichia coli, such as O1, O2, O35, and O78. Some common symptoms include: Escherichia coli septicemia, omphalitis, yolk sac peritonitis, upper respiratory tract infection, enteritis, panophthalmitis, fibrinous airsacculitis, perihepatitis and pericarditis, swollen head syndrome, cellulitis, arthritis, synovitis, encephalitis, Escherichia coli granuloma, yolk sac peritonitis, and salpingitis. Early infection of chicks with Escherichia coli often results in high infection and mortality rates; broilers infected with Escherichia coli experience slow growth, diarrhea, gradual dehydration, emaciation, and death; laying hens infected with Escherichia coli often exhibit sporadic deaths, decreased egg production, and reduced egg quality. Avian colibacillosis occurs globally, and chickens of different ages and breeds can be infected year-round. Air, water, feed, and related equipment can all serve as transmission vectors for the disease. It is roughly estimated that avian colibacillosis causes hundreds of millions of US dollars in economic losses to the global poultry industry each year.

[0003] For a long time, antibiotics have been the primary means of preventing and treating avian colibacillosis. However, prolonged and high-dose use of antibiotics can easily lead to drug resistance in E. coli, weaken the immune system, and increase the risk of mixed infections such as mycoplasma and Salmonella, making treatment more difficult. Furthermore, drug residues in livestock and poultry products are prone to exceeding national standards. With the ban on antibiotics, finding natural antibiotic alternatives that are green, efficient, and cost-effective has become a pressing issue for the livestock and poultry farming industry. Probiotics, as microorganisms that improve and regulate the intestinal microbiota to produce a series of beneficial effects on the body, have gradually become a substitute for antibiotics in veterinary drugs and feed additives. Currently, there is an urgent need to develop a probiotic preparation with good inhibitory effects on E. coli, which can play a role in treating avian colibacillosis.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a strain of Lactobacillus salivae LSF12 and its application. This strain exhibits strong resistance and can effectively inhibit Escherichia coli under various environments.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides *Lactobacillus salivarius* LSF12, with accession number CGMCC NO.33008. *Lactobacillus salivarius* LSF12 can effectively inhibit the growth and reproduction of *Escherichia coli* and exhibits good tolerance to high temperature, high bile salt, and low pH environments.

[0007] In a second aspect, the present invention provides a *Lactobacillus salivarius* preparation containing at least one of the following: *Lactobacillus salivarius* LSF12 cells, fermentation broth, fermentation supernatant, fermentation broth precipitate, and lyophilized powder.

[0008] Preferably, the saliva-infused lactobacillus preparation is an Escherichia coli inhibitor.

[0009] Thirdly, the present invention provides the application of the aforementioned *Lactobacillus salivarius* LSF12 or the *Lactobacillus salivarius* preparation in the inhibition of *Escherichia coli* for non-disease diagnostic purposes. Based on the stress-resistant properties of the aforementioned *Lactobacillus salivarius* LSF12, it exhibits significant advantages in the inhibition of *Escherichia coli* under specific environmental conditions.

[0010] Fourthly, the present invention provides an optimized fermentation process for Lactobacillus salivarius LSF12 and provides a fermentation method for Lactobacillus salivarius LSF12, including the step of inoculating Lactobacillus salivarius LSF12 into a fermentation medium for fermentation culture.

[0011] Preferably, the fermentation medium comprises: molasses 22.5±5 g / L, beef extract 15.0±5 g / L, yeast powder 2.5±0.5 g / L, peptone 15.0±5 g / L, triammonium citrate 2±0.5 g / L, anhydrous sodium acetate 5±1 g / L, dipotassium hydrogen phosphate 2±0.5 g / L, magnesium sulfate dodecahydrate 0.58±0.1 g / L, manganese sulfate tetrahydrate 0.25±0.05 g / L, and Tween-80 1±0.2 mL / L.

[0012] Preferably, the inoculum size of Lactobacillus saliva-associated (LSF12) is 5% ± 1%.

[0013] Preferably, the fermentation temperature of Lactobacillus saliva-associated (LSF12) is 37±2℃ and the fermentation time is 20h.

[0014] Preferably, the initial pH value of Lactobacillus saliva-associated salivarius LSF12 fermentation is 6.0±0.5.

[0015] Fifthly, the present invention provides a fermentation product obtained by the fermentation method described above. This fermentation product can be used in the development and production of downstream products.

[0016] Sixthly, this invention provides the application of the aforementioned *Lactobacillus salivarius* LSF12 and the fermentation product in improving the intestinal flora or intestinal health of aged laying hens. The aged laying hens can be 70 weeks or older. This invention has found through research that feeding aged laying hens with chicken feed containing the aforementioned *Lactobacillus salivarius* LSF12 or the fermentation product as an additive can improve the egg production performance and egg quality of aged laying hens.

[0017] Preferably, the amount of *Lactobacillus saliva-associated* LSF12 added per 1 kg of chicken feed is 1 × 10⁻⁶. 8 -1×10 10 CFU.

[0018] Beneficial effects: This invention provides *Lactobacillus salivarius* LSF12, with accession number CGMCC NO. 33008. The *Lactobacillus salivarius* LSF12 provided by this invention can effectively inhibit the growth and reproduction of *Escherichia coli* and exhibits good tolerance to high temperature, high bile salt, and low pH environments. This invention also studies the fermentation method of the *Lactobacillus salivarius* LSF12 and provides a fermentation method suitable for the rapid production of *Lactobacillus salivarius* LSF12. The obtained fermentation product can be used for the development and production of downstream products and has broad application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0020] Figure 1 The colony morphology of Lactobacillus salivarius LSF12 strain.

[0021] Figure 2 The antibacterial effect of Lactobacillus salivans strain LSF12 on Escherichia coli was investigated.

[0022] Figure 3 The results are from the acid resistance test of Lactobacillus salivae LSF12 strain.

[0023] Figure 4 The results of the bile salt tolerance test for Lactobacillus salivae LSF12 strain are shown.

[0024] Figure 5 The results are from the temperature resistance test of Lactobacillus salivae LSF12 strain.

[0025] Figure 6This is a phylogenetic tree diagram of Lactobacillus salivae LSF12 strain.

[0026] Figure 7 The growth curve of Lactobacillus salivae LSF12 strain is shown.

[0027] Figure 8 Selection of carbon source for fermentation of Lactobacillus saliva-associated strain LSF12.

[0028] Figure 9 Optimize the carbon source concentration for fermentation of Lactobacillus saliva-associated strain LSF12.

[0029] Figure 10 Selection of nitrogen source for fermentation of Lactobacillus saliva-associated strain LSF12.

[0030] Figure 11 Optimize the inoculum size for fermentation of Lactobacillus saliva-associated LSF12 strain.

[0031] Figure 12 Optimize the initial pH for fermentation of Lactobacillus saliva-associated strain LSF12.

[0032] Figure 13 Optimize the fermentation temperature for Lactobacillus saliva-associated strain LSF12. Detailed Implementation

[0033] This invention involves selecting poultry fecal samples, diluting and spreading the samples; isolating, purifying, and preserving the cultured bacterial strains; initially screening the isolated and preserved strains to obtain avian lactic acid bacteria that inhibit Escherichia coli; then further screening the lactic acid bacteria to identify their acid, bile salt, temperature, and artificial gastrointestinal fluid tolerance; identifying the species of the screened lactic acid bacteria; and optimizing the fermentation process of the identified lactic acid bacteria, including culture medium optimization (carbon and nitrogen source screening) and fermentation condition optimization (inoculum size, initial pH, and temperature). Ultimately, this provides a new probiotic product and its fermentation method for treating avian E. coli infection.

[0034] Specifically, this invention provides an avian-derived *Lactobacillus salivarius* strain, LSF12, which can inhibit *Escherichia coli*. It was isolated and screened from poultry fecal samples and effectively inhibits the growth and reproduction of *E. coli*, exhibiting good tolerance to high temperature, high bile salt, and low pH environments. The strain LSF12 was identified as *Lactobacillus salivarius* (…). Ligilactobacillus salivarius This strain was deposited on December 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as *Lactobacillus salivarius*. Ligilactobacillus salivariusThe collection number is CGMCC NO. 33008.

[0035] The avian-derived Lactobacillus saliva-associated LSF12 of the present invention was obtained by isolation and screening using the following method.

[0036] (1) Select donor chickens and collect the contents of the duodenum, jejunum, ileum and cecum.

[0037] (2) Weigh 0.5g of intestinal contents and add 4.5mL of sterile saline to prepare a suspension.

[0038] (3) The suspension is serially diluted, and then 10 -3 10 -4 10 -5 10 -6 10 -7 The suspensions were spread onto MRS solid medium containing 1.5% CaCO3 and anaerobically cultured at 37°C for 24 h.

[0039] (4) After the colonies grow, select a single colony with a clear lysis zone and culture it in MRS broth. Then, perform streak purification on MRS solid medium and repeat three times.

[0040] (5) Cultivate the strain obtained in step (4), centrifuge at 12000r for 10min, and filter the supernatant with a 0.22μm sterile filter.

[0041] (6) The Oxford cup method was used to detect the inhibitory effect of the supernatant on Escherichia coli, and lactic acid bacteria with large inhibition zone diameter were selected.

[0042] (7) Identify the acid resistance, bile salt resistance and temperature resistance of the lactic acid bacteria screened in step (6), and identify their species.

[0043] (8) Optimize the fermentation process of the lactic acid bacteria screened in step (7), including the selection and concentration ratio of carbon and nitrogen sources in the fermentation medium and fermentation conditions, including fermentation temperature, initial pH and inoculum size.

[0044] Ultimately, this invention screened and obtained a strain of avian-derived Lactobacillus saliva-associated LSF12, which can effectively inhibit the growth and reproduction of Escherichia coli. Furthermore, its fermentation process was optimized, enabling it to be cultured and fermented using suitable and inexpensive carbon and nitrogen sources. The resulting probiotic product can be put into production and application.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0048] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0049] Example 1 This embodiment describes the isolation and purification process of Lactobacillus saliva-associated strain LSF12, as well as the research process and results of its biological characteristics.

[0050] 1. Experimental steps.

[0051] 1.1 Isolation and purification of strains.

[0052] First, the necessary tools for separation, such as scissors and tweezers, were autoclaved and prepared for use. The contents of the duodenum, jejunum, ileum, and cecum of healthy adult Hy-Line Brown chickens were collected, and aseptic techniques were employed throughout the experiment. 0.5g (±0.001g) of sample was taken, and the contents of each intestinal segment were purified with physiological saline for 10 minutes. -2 times, 10-3 times, 10 -4 times, 10 -5 times, 10 -6 The culture was diluted 100 μL at different dilutions and added to the surface of prepared MRS agar medium containing 1.5% CaCO3, and then spread evenly using a disposable spreader. The culture was incubated at 37°C for 24 h. Colonies were purified and subcultured using disposable inoculation loops based on their size, morphology, and surface smoothness. After multiple streak purification cultures using the triple-strike method, single colonies were picked and placed in MRS liquid medium for enrichment.

[0053] Take 0.7 ml of well-enriched MRS liquid culture medium, add 0.7 ml of 50% glycerol saline, repeatedly pipette, freeze at -20℃ for one day, then transfer to -80℃ for freezing to preserve the bacterial strain.

[0054] 1.2 Antibacterial test.

[0055] Preparation of pathogen indicator bacteria suspension: Pathogenic *E. coli* was inoculated into sterilized ordinary broth medium and incubated at 37°C for 24 hours, then stored at 4°C for later use. Preparation of centrifugal supernatant from isolated strains: The isolated strains were inoculated into sterilized MRS liquid medium and anaerobically cultured at 37°C for 24 hours. The cultured suspension was then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected. Oxford cup inhibition test: 100 µL of *E. coli* was spread evenly on an EMB agar plate. After the pathogen suspension had completely penetrated the plate, Oxford cups were placed horizontally on top. 200 µL of centrifugal supernatant was added to each Oxford cup. The plates were then stored horizontally at 4°C for 12–16 hours, then incubated at 37°C for 24 hours. The diameter of the inhibition zone was observed and measured. The experiment was performed in triplicate.

[0056] 1.3 Acid resistance test.

[0057] The pH of the MRS liquid culture medium was adjusted to 3.5, 4.0, 4.5, 5.0, and 5.5 respectively using 1 mol / L HCl. The medium was then autoclaved at 115°C for 30 min. The activated lactic acid bacteria were inoculated into the MRS liquid culture medium at different pH values ​​at a 2% (v / v) inoculation rate and cultured at 37°C for 24 h. Samples were then taken to determine the OD of the bacterial culture. 600 .

[0058] 1.4 Bile salt tolerance test.

[0059] Porcine bile salts were added to MRS liquid medium at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / v), respectively. The medium was thoroughly shaken to ensure complete dissolution, and then autoclaved at 115°C for 30 min. The activated lactic acid bacteria were then inoculated into the MRS liquid medium at different bile salt concentrations at an inoculum rate of 2% (v / v) and incubated at 37°C for 24 h. Samples were then taken to determine the OD of the bacterial culture. 600 .

[0060] 1.5 Temperature resistance test.

[0061] The activated lactic acid bacteria were inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 30°C, 37°C, 42°C, 45°C, and 50°C for 24 h. Then, samples were taken to measure the OD of the bacterial culture. 600 .

[0062] 1.6 Simulated gastric and intestinal fluid tolerance test.

[0063] Preparation of artificial gastric fluid: Add 9.5%–10.5% hydrochloric acid (v / v) to a sterile aqueous solution, adjust the pH to 3.0, then add 2.5 g / L pepsin, dissolve thoroughly, and filter through a 0.22 μm sterile membrane for sterilization. Preparation of artificial intestinal fluid: Dissolve 3.4 g potassium dihydrogen phosphate in 250 mL of sterile water, adjust the pH to 6.8 with 0.4% sodium hydroxide solution, add 1 g trypsin per 100 mL of liquid, mix thoroughly, and filter through a 0.22 μm sterile membrane for sterilization.

[0064] 500 μL of activated lactic acid bacteria fermentation broth was mixed with 4.5 mL of artificial gastric fluid in a 5 mL shaker tube and incubated at 37°C for 4 h. Viable bacteria were then counted. Subsequently, 500 μL of the cultured lactic acid bacteria was inoculated into 4.5 mL of artificial intestinal fluid and incubated at 37°C for 4 h. Viable bacteria were counted again. The experiment was performed in triplicate. The survival rate was calculated using the following formula: Survival rate (%) = (Number of viable bacteria in gastric or intestinal fluid after 4 h / Number of viable bacteria after 0 h) × 100%.

[0065] 1.7 16S rRNA sequence analysis.

[0066] Based on the 16S rDNA gene sequences of various lactic acid bacteria in GenBank, and by comparing the 16S rDNA gene sequences of lactic acid bacteria using DNAMAN software, a pair of primers was obtained (Table 1), which were then synthesized by Sangon Biotech (Shanghai) Co., Ltd. [1]

[0067] Extraction of genomic DNA from the strain: DNA from the isolated strain was extracted using a DNA extraction kit, strictly following the steps outlined in the kit's instructions.

[0068] PCR amplification: Using the extracted bacterial DNA as a template, PCR amplification was performed using the designed 16S rDNA primers. The PCR reaction system is shown in Table 2, and the PCR reaction program and its cycling parameters are shown in Table 3.

[0069]

[0070]

[0071] PCR gel electrophoresis verification: Add 10 mL of TAE and 0.9 g of agarose powder to 80 mL of double-distilled water. After the mixture is fully dissolved, transfer it to an Erlenmeyer flask and microwave for 3 min. Once the agarose powder and TAE are completely combined, add 10 μl of GVII and pour into a mold. After the liquid cools and solidifies, add 3 μl each of PCR product, Marker 2000, and empty control in sequence. Then start the electrophoresis apparatus and set the program to 120V / 20 min.

[0072] 16S rRNA sequencing: The PCR amplification products were sent to Biomarker Biotechnology Co., Ltd. for next-generation sequencing of the strain's 16S rRNA. The sequenced full-length 16S rRNA of the strain was entered into the GenBank database for BLAST homology alignment with known standard strain sequences.

[0073] 1.8 Determination of the growth curve of lactic acid bacteria.

[0074] After the strain was activated and expanded, it was inoculated into a 100ml Erlenmeyer flask containing 25ml of MRS liquid medium and incubated for 24h. Samples were taken every 4h to measure the absorbance value. The growth curve was plotted with the incubation time as the x-axis and the corresponding absorbance value as the y-axis. Each group was replicated three times.

[0075] 1.9 Optimization of carbon source types.

[0076] Based on MRS, only the carbon source was changed, while other components of the MRS liquid medium remained unchanged. The original carbon source (glucose) in the MRS medium was replaced with 2% (w / v) maltose, sucrose, glucose, molasses, and fructooligosaccharides, respectively, with a medium without any added carbon source serving as a control group. A 2% inoculum was inoculated into 100 ml Erlenmeyer flasks containing 25 ml of liquid medium. The initial pH of the medium was adjusted to 6.0, and the mixture was incubated at 37°C for 20 hours. The bacterial density and viable cell count of the fermentation broth were then measured to screen for the optimal carbon source.

[0077] 1.10 Carbon source concentration optimization.

[0078] The optimal carbon source concentrations were set to 7.5 g / L, 12.5 g / L, 17.5 g / L, 22.5 g / L, and 27.5 g / L, respectively. Other components of the MRS liquid culture medium remained unchanged. The medium was inoculated at a rate of 2% into 25 ml of culture medium. The initial pH of the culture medium was adjusted to 6.0. The medium was then incubated at 37°C for 20 h. The bacterial density and viable cell count were then measured to determine the optimal amount of carbon source to be added.

[0079] 1.11 Optimization of nitrogen source types.

[0080] Based on the optimal carbon source and concentration optimization, other components of the MRS medium remained unchanged. The main nitrogen sources in the MRS medium—peptone, beef extract, and yeast extract—were replaced with 1.5% (w / v) of beef extract, yeast extract, NH4Cl, urea, and soybean meal, respectively. MRS medium without any added nitrogen source served as a control. A 2% inoculum was inoculated into 25 ml of medium, with an initial pH of 6.0. Fermentation was carried out statically at 37°C for 20 h. The bacterial density and viable cell count of the fermentation broth were then measured to screen for the optimal nitrogen source.

[0081] 1.12 Nitrogen source optimization orthogonal experiment.

[0082]

[0083] 1.13 Selection of vaccination amount.

[0084] According to the obtained optimized culture medium, the initial pH of the culture medium was adjusted to 6.0, and the inoculation amount was selected as 1%, 2%, 3%, 4% and 5% respectively. The culture medium was inoculated and fermented in a constant temperature incubator at 37℃ for 20 hours. The bacterial density and viable number of the fermentation broth were measured to obtain the optimal inoculation amount.

[0085] 1.14 Temperature selection.

[0086] The initial pH of the culture medium was adjusted to 6.0. The culture medium was inoculated into the optimized fermentation medium at the optimized inoculation amount. The fermentation temperatures were set at 28℃, 31℃, 34℃, 37℃, and 40℃, respectively. After static fermentation for 20 hours, the bacterial density and viable cell count of the fermentation broth were measured to determine the optimal temperature.

[0087] 1.15 Selection of initial pH.

[0088] The initial pH of the culture medium was adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0. The culture medium was inoculated at the optimized inoculum size and at the optimized temperature. After 20 hours of static fermentation, the bacterial density and viable cell count of the fermentation broth were measured to determine the optimal initial pH.

[0089] 2. Experimental results.

[0090] 2.1 Isolation of strains and their antibacterial effects.

[0091] The strain was isolated from chicken manure. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 The inhibitory effect of the strain on Escherichia coli.

[0092] 2.2 Acid resistance test.

[0093] The strain exhibits good tolerance to low pH environments. Figure 3 The values ​​represent the OD600 values ​​of the strain under different pH conditions.

[0094] 2.3 Bile salt tolerance test.

[0095] The strain can survive in bile salt concentrations of 0.3%, 0.4%, and 0.5%, and has good tolerance to high concentrations of bile salts. Figure 4 The values ​​are OD600 values ​​of the strain at different bile salt concentrations.

[0096] 2.4 Temperature resistance test.

[0097] The cell density of the strain decreased with increasing culture temperature, and it showed a certain degree of tolerance to 45℃ and 50℃. Figure 5 The values ​​are OD600 values ​​of the strain at different temperatures.

[0098] 2.5 Simulated gastric and intestinal fluid tolerance test.

[0099] The strain exhibited good tolerance to both artificial gastric and intestinal fluids, with a high survival rate after 4 hours. Table 5 shows the survival rates of the strain in artificial gastric and intestinal fluids.

[0100]

[0101] 2.6 Results of 16S rRNA sequencing experiments.

[0102] The strain was identified as *Lactobacillus salivarius* by 16S rRNA sequencing and BLAST comparison. Figure 6 This is a phylogenetic tree diagram.

[0103] The measured sequence is as follows:

[0104] 2.7 Growth curve of Lactobacillus saliva-associated with saliva.

[0105] Lactobacillus salivarius LSF12 is in the lag phase from 0 to 2 hours, during which the bacterial growth is slow. After 2 hours, it enters the logarithmic phase, during which the number of viable bacteria increases rapidly. It enters the stationary phase from 12 to 20 hours and enters the death phase after 20 hours. Figure 7 The growth curve for [the product / product].

[0106] 2.8 Selection of carbon source for fermentation.

[0107] The fermentation effects of different carbon sources on Lactobacillus saliva-associated LSF12 were in the order of sucrose > glucose > molasses. Sucrose had the best fermentation effect, but considering its cost and availability, molasses was ultimately chosen as the most suitable carbon source for LSF12. Figure 8 The viable count of Lactobacillus saliva-associated LSF12 under various fermentation carbon sources.

[0108] 2.9 Selection of fermentation carbon source concentration.

[0109] The highest number of viable bacteria in the fermentation broth of Lactobacillus salivarius LSF12 was observed at a molasses concentration of 22.5 g / L. Figure 9 The viable count of LSF12 of Lactobacillus salivae at various carbon source concentrations.

[0110] 2.10 Selection of nitrogen source for fermentation.

[0111] The fermentation effects of different nitrogen sources for Lactobacillus saliva-associated LSF12 were as follows: yeast extract > beef extract > peptone. Since mixed nitrogen sources provide richer nutrition than single nitrogen sources, yeast extract, beef extract and peptone were selected as mixed nitrogen sources. Figure 10 The viable count of Lactobacillus saliva-associated LSF12 under various fermentation nitrogen sources.

[0112] 2.11 Optimization of nitrogen source ratio and concentration.

[0113] The results of the orthogonal experiment (Table 6) show that under these conditions, the highest viable cell count (3.15 × 10⁻⁶) was achieved with beef extract (15.0 g / L), yeast powder (2.5 g / L), and peptone (15.0 g / L). 9 CFU / mL.

[0114]

[0115] 2.12 Optimization of fermentation inoculum size.

[0116] The optimal inoculum size for Lactobacillus saliva-associated (LSF12) is 5%. Figure 11 The viable count of LSF12 saliva-associated lactobacillus at different inoculation amounts.

[0117] 2.13 Optimization of initial fermentation pH.

[0118] The optimal initial pH for *Lactobacillus salivarius* LSF12 is 6.0. Figure 12 The viable count of LSF12 *Lactobacillus salivarius* at different initial pH values.

[0119] 2.14 Fermentation temperature optimization.

[0120] The optimal fermentation temperature for Lactobacillus salivarius LSF12 is 37℃. Figure 13 The viable count of Lactobacillus salivarius LSF12 at different fermentation temperatures.

[0121] Therefore, the optimal culture conditions for *Lactobacillus salivarius* LSF12 are as follows: molasses 22.5 g / L, beef extract 15.0 g / L, yeast extract 2.5 g / L, peptone 15.0 g / L, triammonium citrate 2 g, anhydrous sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate dodecahydrate 0.58 g, manganese sulfate tetrahydrate 0.25 g, Tween-80 1 mL / L, initial pH 6.0, inoculum size 5%, and culture temperature 37℃.

[0122] 3. Study on the improvement of egg production performance and egg quality in aged laying hens by combining saliva with Lactobacillus LSF12 3.1 Experimental Design and Feeding Program The experiment employed a completely randomized design, selecting Hy-Line Brown laying hens aged 67 weeks or older of the same breed, with similar weights, good health, and consistent origin. These hens were randomly divided into four treatment groups, with six replicates per treatment group (one replicate per cage). Each replicate contained six hens, totaling approximately 144 laying hens. The experiment lasted eight weeks. The treatments included: 1) a control group; 2) group T1 (1×10⁻⁶ chickens were added to each 1 kg of feed). 8 3) T2 group (add 1×10 to every 1 kg of feed) 9 4) T3 group (add 1×10 CFU bacterial solution per 1 kg of feed) 10 (CFU bacterial solution). Throughout the experiment, the room temperature was maintained at 25°C, with 16 hours of light and 8 hours of darkness per day. The laying hens had free access to food and water for a total of 8 weeks. Eggs were collected daily, and egg quality was determined at the end of the fourth and eighth weeks.

[0123] 3.2 Data Analysis Data are presented as mean ± standard error (SEM). SPSS 20.0 (Chicago, USA) was used for analysis. One-way ANOVA was performed, followed by Duncan's multiple comparison test. Significance levels are indicated by asterisks, with * indicating 0.01 < 0.01. pValue < 0.05, ** indicates 0.001 < p Value < 0.01, *** indicates p A value < 0.001, or significance is indicated by a letter. If both groups have the same letter, the difference between them is not significant; if the two groups have different letters, the difference between them is significant.

[0124] 3.3 Experimental Results 1) Effects of Lactobacillus saliva-associated with LSF12 on the laying performance of hens in the late laying period Adding Lactobacillus salivarius LSF12 to feed can improve the rate of qualified eggs and average daily feed intake in laying hens during the late laying period. P <0.05%, reducing the egg breakage rate ( P <0.05).

[0125]

[0126] 2) Effects of Lactobacillus saliva-associated with LSF12 on egg quality during late laying period Adding LSF12 to the saliva of Lactobacillus saliva during weeks 0-4 and 0-8 both reduced egg yolk color. P <0.05), adding a low dose of saliva combined with Lactobacillus LSF12 from 0-8 weeks can increase the yolk ratio of eggs. P <0.05).

[0127]

[0128]

[0129] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Lactobacillus salivarius ( Ligilactobacillus salivarius LSF12, characterized in that, Its accession number is CGMCC NO. 33008.

2. A saliva-based lactobacillus preparation, characterized in that, The product contains at least one of the following: Lactobacillus salivarius LSF12 cells, fermentation broth, fermentation broth precipitate, and lyophilized powder as described in claim 1.

3. The use of the Lactobacillus salivae-associated salivae LSF12 of claim 1 or the Lactobacillus salivae-associated salivae preparation of claim 2 in the inhibition of Escherichia coli for non-disease treatment or diagnostic purposes.

4. A fermentation method for Lactobacillus saliva-associated with LSF12, characterized in that, The Lactobacillus salivans LSF12 of claim 1 was inoculated into a fermentation medium and fermented.

5. The fermentation method according to claim 4, characterized in that, The fermentation medium comprises the following components: molasses 22.5±5 g / L, beef extract 15.0±5 g / L, yeast powder 2.5±0.5 g / L, peptone 15.0±5 g / L, triammonium citrate 2±0.5 g / L, anhydrous sodium acetate 5±1 g / L, dipotassium hydrogen phosphate 2±0.5 g / L, magnesium sulfate dodecahydrate 0.58±0.1 g / L, manganese sulfate tetrahydrate 0.25±0.05 g / L, and Tween-80 1±0.2 mL / L.

6. The fermentation method according to claim 4 or 5, characterized in that, The inoculation amount of Lactobacillus saliva-associated saliva LSF12 was 5% ± 1%.

7. The fermentation method according to claim 6, characterized in that, The fermentation temperature of Lactobacillus saliva-associated LSF12 was 37±2℃, and the fermentation time was 20h.

8. The fermentation method according to claim 6, characterized in that, The initial pH value for fermentation of Lactobacillus saliva-associated LSF12 was 6.0 ± 0.

5.

9. The application of Lactobacillus salivae LSF12 as described in claim 1 in improving the egg production performance and egg quality of older laying hens.

10. The application according to claim 9, characterized in that, The dosage of *Lactobacillus saliva-associated* LSF12 added per 1 kg of chicken feed is 1 × 10⁻⁶. 8 -1×10 10 CFU.

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