Application of chicken-derived saliva combined lactobacillus in laying hens in later laying period

Abstract

The invention discloses application of chicken-derived saliva combined lactobacillus in laying hens in the later egg laying period. The chicken-derived saliva combined lactobacillus is used for preparing products for improving the production performance of the laying hens in the later egg laying period, improving the oxidation resistance of the laying hens in the later egg laying period and / or improving the egg quality of the laying hens in the later egg laying period. The chicken source combined lactobacillus salivarius is RS-11-1, is classified and named as the combined lactobacillus salivarius, is registered and preserved in China General Microbiological Culture Collection Center (CGMCC) on November 12, 2024, and has the preservation number of CGMCC No.32589. The chicken source combined lactobacillus salivarius has the advantages that the chicken source combined lactobacillus salivarius can be used for preparing the chicken source combined lactobacillus salivarius, and the chicken source combined lactobacillus salivarius can be used for preparing the chicken source combined lactobacillus salivarius; the chicken-derived saliva combined with lactobacillus can improve the intestinal health of laying hens in the late egg laying period, enhance the oxidation resistance of intestinal tracts, and improve the egg laying performance and egg quality of the laying hens in the late egg laying period.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. More specifically, the present invention relates to the application of chicken-derived Ligilactobacillus salivarius in laying hens in the late laying period. Background Art

[0002] High-yielding hens usually experience problems such as ovarian aging, decreased egg production, and poor egg quality after 400 days of age. During the aging process of laying hens, the estrogen effect and the liver antioxidant function gradually decline, and the ability of the liver to secrete yolk precursor substances decreases, resulting in a decline in laying performance. Therefore, improving the antioxidant capacity of laying hens can alleviate oxidative damage to the liver or ovaries of laying hens, promote the synthesis of yolk precursor substances and the expression of related genes, thereby improving the production performance and egg quality of laying hens. From the perspective of antioxidant, exploring how to improve the production performance and egg quality of laying hens in the late laying period is of great significance for extending the laying cycle of laying hens, realizing the "extended breeding" of laying hens, and improving the economic benefits of laying hen farming. Summary of the Invention

[0003] An object of the present invention is to solve at least the above problems and / or deficiencies and provide at least the advantages described hereinafter.

[0004] To achieve these objects and other advantages of the present invention, there is provided an application of chicken-derived Ligilactobacillus salivarius in laying hens in the late laying period, wherein the chicken-derived Ligilactobacillus salivarius is used for preparing a product for improving the production performance of laying hens in the late laying period;

[0005] The chicken-derived Ligilactobacillus salivarius is RS-11-1, classified as Ligilactobacillus salivarius, and has been registered and preserved in the China General Microbiological Culture Collection Center on November 12, 2024, with the preservation number CGMCC No. 32589.

[0006] An application of chicken-derived Ligilactobacillus salivarius in laying hens in the late laying period, wherein the chicken-derived Ligilactobacillus salivarius is used for preparing a product for improving the egg quality of laying hens in the late laying period.

[0007] An application of chicken-derived Ligilactobacillus salivarius in laying hens in the late laying period, wherein the chicken-derived Ligilactobacillus salivarius is used for preparing a product for improving the antioxidant capacity of laying hens in the late laying period.

[0008] Preferably, the product is a drug or an animal feed additive.

[0009] Preferably, the product contains chicken-derived Ligilactobacillus salivarius, the fermentation supernatant of chicken-derived Ligilactobacillus salivarius, the bacterial liquid of chicken-derived Ligilactobacillus salivarius, and / or the cell-free extract of chicken-derived Ligilactobacillus salivarius.

[0010] Preferably, improving the production performance of laying hens in the late laying period includes: increasing the egg production rate, increasing the average egg weight, and / or reducing the feed-to-egg ratio.

[0011] Preferably, improving the egg quality of laying hens in the late laying period includes: increasing the eggshell strength, increasing the yolk color, increasing the Haugh unit, and / or increasing the albumen height.

[0012] Preferably, improving the antioxidant capacity of laying hens in the late laying period includes: reducing the content of malondialdehyde in the serum, increasing the content of total superoxide dismutase in the serum, increasing the content of glutathione peroxidase in the serum, and / or increasing the total antioxidant capacity in the serum.

[0013] Preferably, the 16S rDNA sequence of the Lactobacillus salivarius - associated strain from chicken is as shown in SEQ ID NO.1.

[0014] Preferably, the addition amount of the animal feed additive is such that the viable count of the Lactobacillus salivarius - associated strain from chicken in the feed is 0.5×10 7 ~2×10 7 CFU / g.

[0015] A probiotic agent contains the Lactobacillus salivarius - associated strain from chicken as described above.

[0016] Preferably, in the probiotic agent, the viable count of the Lactobacillus salivarius - associated strain from chicken is 1×10 10 CFU / g.

[0017] Preferably, the preparation method of the probiotic agent is as follows: The Lactobacillus salivarius - associated strain from chicken is spread and inoculated on MRS solid medium, cultured at a constant temperature of 37°C for 24 h, and then sub - cultured for three generations. The strain is further fermented at 37°C and pH = 5.0 - 5.5 for 24 h to prepare a bacterial liquid. The viable count of the Lactobacillus salivarius - associated strain from chicken in the bacterial liquid is adjusted to 1×10 10 CFU / g to obtain the probiotic agent.

[0018] A post - biotic contains the inactivated cells, fermentation metabolites, and / or inactivated fermentation broth of the Lactobacillus salivarius - associated strain from chicken as described above.

[0019] Preferably, the preparation method of the post - biotic includes the following steps:

[0020] Step 1: Add bletilla striata polysaccharide and shikimic acid to sterile water and stir evenly to obtain a mixed solution; wherein, the mass - to - volume ratio of bletilla striata polysaccharide, shikimic acid, and sterile water is 1 g:0.3 - 0.8 g:50 - 150 mL;

[0021] Step 2: Inoculate the activated Lactobacillus salivarius combined with chicken source RS-11-1 into MRS liquid medium at 1-5% (v / v). After culturing at a constant temperature of 37°C for 24 h, add the mixed solution obtained in Step 1 and continue culturing for 12 h. Inactivate the fermentation broth at 80-100°C for 10-30 min to obtain postbiotics; wherein, the mass-volume ratio of bletilla striata polysaccharide to MRS liquid medium is 0.5-1.5 g:1 L.

[0022] Preferably, the product contains the probiotic agent and / or postbiotics as described above.

[0023] The present invention has at least the following beneficial effects: The present invention provides an application of Lactobacillus salivarius combined with chicken source in laying hens in the late laying period. Lactobacillus salivarius combined with chicken source RS-11-1 has good probiotic characteristics, has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus and Salmonella, has a certain ability to scavenge DPPH free radicals, superoxide anion free radicals and hydroxyl free radicals, and has a certain reducing ability. Adding it as a feed additive to the feed of laying hens in the late laying period can improve the intestinal health of laying hens in the late laying period, enhance the antioxidant capacity of the intestine, and improve the laying performance and egg quality of laying hens in the late laying period.

[0024] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the bacteriostatic circle effect diagram of Lactobacillus salivarius combined with chicken source RS-11-1 of the present invention, wherein: A Escherichia coli; B Salmonella; C Staphylococcus aureus;

[0026] Figure 2 It is the growth state diagram of Lactobacillus salivarius combined with chicken source RS-11-1 of the present invention;

[0027] Figure 3 It is the Gram staining diagram of Lactobacillus salivarius combined with chicken source RS-11-1 of the present invention;

[0028] Figure 4 It is the genetic evolutionary tree of Lactobacillus salivarius combined with chicken source RS-11-1 of the present invention;

[0029] Figure 5 It is the growth curve of Lactobacillus salivarius combined with chicken source RS-11-1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The following further detailed description of the present invention is provided in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the text of the specification.

[0031] It should be understood that terms such as "having", "comprising", and "including" as used herein do not preclude the presence or addition of one or more other elements or combinations thereof.

[0032] In the following examples, the specific component ratio of the MRS solid medium used is: peptone 10 g, beef extract 10 g, yeast powder 5 g, K 2 HPO 4 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1 mL, MgSO 4 ·7H 2 O 0.58 g, MnSO 4 ·4H 2 O 0.25 g, agar 15 g, distilled water 1000 mL.

[0033] The specific component ratio of the MRS liquid medium is: peptone 10 g, beef extract 10 g, yeast powder 5 g, K 2 HPO 4 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1 mL, MgSO 4 ·7H 2 O 0.58 g, MnSO 4 ·4H 2 O 0.25 g, distilled water 1000 mL.

[0034] The specific component ratio of the MRS solid medium containing calcium carbonate is: peptone 10 g, beef extract 10 g, yeast powder 5 g, K 2 HPO 4 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1 mL, MgSO 4 ·7H 2 O 0.58 g, MnSO 4 ·4H 2 O 0.25 g, CaCO 3 7.5 g, agar 15 g, distilled water 1000 mL.

[0035] Example 1

[0036] A chicken-derived Ligilactobacillus salivarius RS-11-1, classified and named as Ligilactobacillus salivarius, was registered and preserved in the China General Microbiological Culture Collection Center on November 12, 2024, with the preservation number CGMCC No. 32589.

[0037] Isolation and identification of chicken-derived Ligilactobacillus salivarius:

[0038] (1) Screening and isolation of chicken-derived lactic acid bacteria

[0039] Using the fresh chicken manure of local chickens in Mianyang, Sichuan as a sample, 0.1 g of chicken manure was weighed aseptically and serially diluted with PBS solution. The diluted solution was evenly spread on MRS solid medium containing calcium carbonate and incubated at 37 °C for 24 h. Single colonies with calcium dissolution zones were picked and streaked onto MRS solid medium and incubated at 37 °C for 24 h. This purification was repeated three times until pure strains were obtained. A total of 114 Gram-positive lactic acid bacteria strains were initially screened and isolated, numbered from RS-1-1 to RS-21-8.

[0040] (2) Screening of the antagonistic properties of chicken-derived lactic acid bacteria against pathogenic bacteria

[0041] Inhibitory test against pathogenic bacteria: 1 mL of Staphylococcus aureus, Escherichia coli, and Salmonella with a bacterial concentration of 1×10 9 cfu / mL was respectively dropped onto the prepared sterile LB agar medium. The bacterial solution was evenly spread over the surface of the medium with a disposable spreading rod; after standing for 1 min, the excess bacterial solution was aspirated with a pipette, and then holes were punched with a sterile punch with a diameter of 7 mm and the plate was sealed under a flame; after solidification, 100 μL of each lactic acid bacteria suspension with a concentration of 1×10 9 cfu / mL was added to the holes and incubated at 37 °C for 24 h. Each probiotic was done in 3 parallels. The diameter (mm) of the inhibition zone was measured with a vernier caliper, and the size of the inhibition zone diameter was used to represent the antibacterial activity of the probiotic.

[0042] Through the inhibition of Escherichia coli, Staphylococcus aureus, and Salmonella, 2 lactic acid bacteria strains with certain antibacterial effects were initially screened, namely RS-11-1 and RS-21-1. Among them, RS-11-1 had the best antibacterial effect and could be used to prepare products that inhibit Escherichia coli, Staphylococcus aureus, and Salmonella. The antibacterial results are shown in Table 1, Figure 1 . The dominant strains obtained through the antibacterial test were preserved for the next rescreening of tolerance to the digestive tract environment.

[0043] Table 1

[0044]

[0045] (3) Screening of acid and bile salt tolerance of chicken-derived lactic acid bacteria

[0046] To further rescreen the candidate strains for tolerance to the digestive tract environment, an acid and bile salt tolerance test was conducted.

[0047] Bile salt tolerance test: The overnight cultured bacterial solution was inoculated into MRS liquid medium with different bile salt levels (0.15% and 0.3%) at 5% (v / v), and placed at 37°C for 2 h. At 0 and 2 h, 100 μL of the bacterial solution was taken and serially diluted, and the diluted solution was spread on MRS solid medium and cultured at 37°C for 24 h, and the colonies were counted; the survival rate was calculated, and each sample had 3 replicates.

[0048] Survival rate (%) = number of colonies at "2 h" / number of colonies at "0 h" × 100%

[0049] Acid tolerance test: The overnight cultured bacterial solution was inoculated into MRS liquid medium with pH 2 and 3 at 5% (v / v), and placed at 37°C for 2 h. At 0 and 2 h, 100 μL of the bacterial solution was taken and serially diluted (10 -3 、10 -5 、10 -7 ), the diluted solution was spread on MRS plates, and cultured at 37°C for 24 h, and the colonies were counted; the survival rate was calculated, and each sample had 3 replicates.

[0050] Survival rate (%) = number of colonies at "2 h" / number of colonies at "0 h" × 100%

[0051] The test results are shown in Table 2. In the environments of pH = 2 and pH = 3, the survival rates of RS-11-1 and RS-21-1 were both relatively high; in summary, it shows that both RS-11-1 and RS-21-1 have good acid tolerance. In the environment of 0.15% bile salt, both strains had a certain tolerance; while in the environment of 0.3% bile salt, the survival rate of RS-11-1 was higher. Considering the results of acid and bile salt tolerance, RS-11-1 has good acid and bile salt tolerance characteristics.

[0052] Table 2

[0053]

[0054]

[0055] (4) Identification of chicken-derived lactic acid bacterium RS-11-1

[0056] After isolating a single colony of pure lactic acid bacterium, the colony was identified. According to the colonies of the lactic acid bacterium of the present application obtained after isolation and purification, the cells were processed, and the lactic acid bacterium obtained after isolation and purification was identified by its cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence.

[0057] Morphological identification

[0058] Figure 2 is a schematic diagram of the growth state of chicken-derived Lactobacillus RS-11-1, and its colony morphological characteristics are shown in Table 3. The Gram staining observation is as Figure 3, it can be found that the screened chicken-derived lactic acid bacterium RS-11-1 is rod-shaped.

[0059] Table 3

[0060] Edge regularity Smoothness Texture Color Transparency Surface protrusion Colony morphology Size RS-11-1 Neat Yes Moist White Opaque Yes Round 1.2 - 1.5 mm

[0061] Physiological and biochemical characteristics identification

[0062] The physiological and biochemical characteristics of this isolated strain are shown in Table 4. Refer to the "Manual for Systematic Identification of Common Bacteria" and the "Bergey's Manual of Determinative Bacteriology" for comparative analysis, and it is preliminarily determined that RS-11-1 is a bacterium belonging to the genus Lactobacillus delbrueckii subsp. lactis.

[0063] Table 4

[0064] Item Result Item Result Catalase - Sorbitol - VP reaction - Mannitol + Methyl red reaction + Glucose + Starch hydrolysis - Raffinose + Cellobiose - Sucrose + Maltose + Gelatin -

[0065] Note: + indicates positive; - indicates negative

[0066] Molecular biology identification

[0067] The universal primers 27F and 1492R for bacterial identification of 16S rDNA were used for PCR amplification. The primer sequences are shown in Table 5, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0068] Table 5

[0069]

[0070]

[0071] The PCR amplification template was the activated fresh bacterial solution, and the system was 50 μL. The reaction system is shown in Table 6;

[0072] Table 6

[0073]

[0074] The PCR amplification program was as follows: 95 °C for 5 min; 95 °C for 45 s; 55 °C for 45 s; 72 °C for 1 min; repeat 35 cycles; 72 °C for 8 min. The PCR amplification products were identified by 1% agarose gel electrophoresis and then sequenced by Shanghai Sangon Biotech Co., Ltd., and BLAST alignment analysis was performed in GenBank.

[0075] The 16S rDNA sequence of RS-11-1 is shown in SEQ ID NO.1. BLAST alignment was performed in GenBank, and a phylogenetic tree was drawn, as Figure 4As shown, RS-11-1 and the reference strain Ligilactobacillus salivarius (JCM 1231) are on the same branch and have the closest genetic relationship. Therefore, RS-11-1 can be determined to be Ligilactobacillus salivarius.

[0076] (5) Biological characteristic analysis of chicken-derived Ligilactobacillus salivarius RS-11-1

[0077] After activating the strain for one generation, inoculate it into 100 mL of MRS liquid medium at an inoculation amount of 2% (v / v), and culture it with shaking at 37 °C. Take 5 mL of the bacterial liquid for testing every 4 h, and measure the bacterial liquid concentration (OD value) at a wavelength of 600 nm using an ultraviolet spectrophotometer for a total of 24 h. Through the determination of the growth curve of chicken-derived Ligilactobacillus salivarius RS-11-1, as Figure 5 shown, it can be found that RS-11-1 enters the logarithmic growth phase after 4 h and the stationary phase after 12 h.

[0078] (6) In vitro antioxidant properties of chicken-derived Ligilactobacillus salivarius RS-11-1

[0079] 1) Reagents

[0080] Absolute ethanol, potassium ferricyanide, PBS buffer solution, acetic acid, ferric chloride, ascorbic acid, hydrogen peroxide, ferrous sulfate, 3,5-dinitrosalicylic acid (DNS), pyrogallol (pyrogallic acid), tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution (Tris), phosphate buffer solution, o-phenanthroline solution, etc. All of the above reagents are of analytical grade.

[0081] 2) Extraction of sample bacterial liquid

[0082] Inoculate the activated strain into MRS liquid medium at 2% (v / v), and after culturing at 37 °C for 8 h, collect the bacterial liquid for testing.

[0083] 3) Determination of DPPH free radical scavenging ability

[0084] Take 1 mL of the sample and place it in a test tube, add 2 mL of DPPH absolute ethanol solution (concentration 0.2 mmol / L), mix well, react in the dark at room temperature for 30 min, centrifuge at 4 °C, 8000 g for 10 min, take the supernatant, measure its absorbance at 517 nm, zero with deionized water, do 3 replicates for each group, and calculate the average value.

[0085] DPPH scavenging rate (%) = [1 - (A sample - A blank) / A control] × 100%

[0086] (Blank group: Equal volume of anhydrous ethanol is used instead of DPPH; Control group: Equal volume of distilled water is used instead of the sample solution, and the blank is zeroed with an equal volume mixture of distilled water and anhydrous ethanol.)

[0087] Preparation of liquid: 0.2 mmol / L DPPH anhydrous ethanol solution: Weigh 50 mg of DPPH, add 634 mL of anhydrous ethanol, and shake well.

[0088] Determination of hydroxyl radical (·OH) scavenging activity (Fenton method)

[0089] The Fenton method utilizes the reaction between H 2 O 2 and Fe 2+ to generate Fenton reaction, producing highly reactive ·OH. Salicylic acid can capture ·OH and generate a colored substance (dihydroxybenzoic acid), which has the maximum absorption at 510 nm. However, if a certain amount of scavenging substance is added, it will compete with the reaction of salicylic acid, resulting in a decrease in the production of the colored substance (dihydroxybenzoic acid) and causing a certain change in the absorbance of the solution. The activity of the antioxidant can be measured based on the change in absorbance.

[0090] H 2 O 2 +Fe 2+ →·OH + OH - +Fe 3+

[0091] Preparation of liquid: ① FeSO 4 solution: Weigh 0.05 g of ferrous sulfate heptahydrate accurately and place it in a 100 mL volumetric flask. Add distilled water to the scale and shake well to obtain a 1.8 mmol / L ferrous sulfate solution;

[0092] ② Salicylic acid - ethanol solution: Weigh 0.0249 g of salicylic acid accurately and place it in a 100 mL volumetric flask. Add anhydrous ethanol to the scale. After shaking and stabilizing, a 1.8 mmol / L salicylic acid - ethanol solution is obtained;

[0093] ③ 0.3% H 2 O 2 solution: Accurately measure 1.0 mL of 30% hydrogen peroxide and place it in a 100 mL volumetric flask. Add distilled water to the scale and mix well to obtain a 0.3% H 2 O 2 solution.

[0094] The specific steps are as follows: Add 2 mL of 1.8 mmol / L FeSO 4 solution, 1 mL of the sample, and 0.3% H 2 O2 Take 0.1 mL of the solution, shake well, then add 1.5 mL of 1.8 mmol / L salicylic acid - ethanol solution, shake well, let it stand at 30 °C for 30 min, and then measure the absorbance value Ai of the sample group at 510 nm; replace the H 2 O 2 in the solution with an equal amount of distilled water, carry out the reaction, use it as the sample reference group and measure its absorbance value Aj; replace the sample in the sample group with an equal amount of distilled water, carry out the reaction, and use it as the blank control group to measure the absorbance value Ao. Calculate the hydroxyl radical scavenging rate according to the following formula:

[0095] Hydroxyl radical scavenging rate (%) = [Ao - (Ai - Aj) / Ao] × 100%

[0096] Measurement of superoxide anion radical (O 2 - ·) scavenging activity (pyrogallol method)

[0097] Pyrogallol is extremely prone to autoxidation in an alkaline environment, producing colored intermediate products and superoxide anion O 2 - ·, and O 2 - · also has a catalytic effect on the autoxidation reaction. The amount of O 2 - · generated is judged according to the amount of colored substances produced. When an antioxidant active substance is added, it can weaken the autoxidation of pyrogallol, so as to judge the antioxidant activity size by the decreasing trend of colored substances.

[0098] Preparation of the liquid: ① Pyrogallol: Weigh 0.315 g of pyrogallol precisely, place it in a 100 mL volumetric flask, then add 10 mL of 0.1 mol / L hydrochloric acid, and finally add distilled water to the scale, shake well, and obtain 25 mmol / L pyrogallol; pyrogallol should be prepared and used immediately, and the reaction is carried out at 20 °C;

[0099] ② Tris - HCl: Weigh 3.0285 g of Tris reagent precisely and place it in a 500 mL volumetric flask, then add 114.5 mL of 0.1 mol / L hydrochloric acid, and finally add distilled water to the scale, shake well, and obtain 0.05 mol / L Tris - HCl buffer solution.

[0100] The specific steps are as follows: Take out a test tube, add 7.5 mL of 0.05 mol / L Tris-HCl buffer solution, place it in a water bath at 25 °C and preheat for 20 min. Then, add 1 mL of sample solution and 0.5 mL of 25 mmol / L pyrogallol solution respectively. After mixing, place it in a water bath at 25 °C and react for 5 min. Add 1 mL of concentrated HCl to terminate the reaction. As the sample group, measure the absorbance Ai of the solution at a wavelength of 320 nm with a UV-visible spectrophotometer; for the sample reference group, use the same volume of distilled water to replace the pyrogallol solution and measure the absorbance Aj of the solution; for the blank control group, use the same volume of distilled water to replace the sample solution and measure the absorbance Ao of the solution. Calculate the scavenging rate of O 2 - · according to the following formula:

[0101] Scavenging rate of superoxide anion radical (%) = [Ao - (Ai - Aj)] / Ao × 100%

[0102] Determination of reducing power

[0103] Preparation of solutions: ① 0.2 mol / L phosphate buffer solution with pH 6.6: First, prepare stock solution A of 0.2 M Na 2 HPO 4 (17.19 g of Na 2 HPO 4 ·12H 2 O + 240 mL of H 2 O) and stock solution B of 0.2 M NaH 2 PO 4 (11.23 g of NaH 2 PO 4 ·2H 2 O + 360 mL of H 2 O); Take 40 mL of stock solution A and 60 mL of stock solution B, and mix them to obtain 0.2 mol / L phosphate buffer solution with pH 6.6;

[0104] ② 1% potassium ferricyanide: 1 g of potassium ferricyanide + 100 mL of H 2 O;

[0105] ③ 10% trichloroacetic acid: 1 mL of trichloroacetic acid + 9 mL of H 2 O;

[0106] ④ 0.1% ferric chloride: 0.1 g of ferric chloride + 100 mL of H 2 O.

[0107] Take 0.5 mL of the sample and place it in a test tube. Add 0.5 mL of a phosphate buffer solution with a concentration of 0.2 mol / L and a pH of 6.6. Then add 0.5 mL of 1% potassium ferricyanide. After water bath at 50 °C for 20 min, quickly cool it in an ice bath. Then add 0.5 mL of 10% trichloroacetic acid, centrifuge at 4000 r / min for 10 min. Take 1 mL of the supernatant, add 1 mL of distilled water and 1 mL of 0.1% ferric chloride, mix well, let it stand at room temperature for 10 min, and measure its absorbance at a wavelength of 700 nm. Each sample has 3 replicates, and calculate the average value. The greater the absorbance, the stronger the reducing ability of the sample to be measured.

[0108] The in vitro antioxidant results are shown in Table 7. RS-11-1 has certain DPPH radical, superoxide anion radical, hydroxyl radical scavenging abilities and certain reducing ability, and its antioxidant ability is stronger than that of 1% vitamin C, and it can be used to prepare antioxidant products.

[0109] Table 7

[0110]

[0111] Example 2

[0112] Application of Lactobacillus salivarius combined with Lactobacillus in laying hens in the late laying period:

[0113] Preparation method of probiotic agent of Lactobacillus salivarius combined with Lactobacillus RS-11-1: Spread and inoculate 100 μL of RS-11-1 on MRS solid medium, incubate at 37 °C for 24 h, continue to subculture for three generations, and then inoculate the strain into a 20 L automatic fermenter (37 °C, pH = 5.0 - 5.5, 24 h) to prepare the bacterial liquid. Use the plate counting method to calculate the viable count of the produced bacterial liquid, and adjust the viable count of Lactobacillus RS-11-1 in the bacterial liquid to 1×10 10 CFU / g to obtain the probiotic agent; the dosage of MRS solid medium is to spread 3 mm thick MRS solid medium in a 10 mm culture plate.

[0114] Animal experiment treatment: Select 180 healthy Roman laying hens at 60 weeks of age with similar laying rates (about 85%), and randomly divide them into 3 groups, namely the control group (fed with the basal diet), the positive control group (adding a certain manufacturer's lactic acid bacteria (Yihong Biology, lactic acid bacteria) to the basal diet to adjust the viable count in the feed to 1×10 7 CFU / g), and the RS-11-1 group (adding RS-11-1 probiotic agent to the basal diet to adjust the viable count in the feed to 1×10 7(CFU / g), with 6 replicates in each group and 10 chickens in each replicate. The preliminary trial period was 7 days, and the formal trial period was 56 days. The test chickens were all raised and managed according to the conventional procedures of the chicken farm. During the trial, immunization, disinfection and other procedures were carried out according to the normal procedures of the chicken farm. The feed consumption and egg production of laying hens in each group were recorded every day, the egg weight of each group was weighed, and the feed-to-egg ratio of each group was calculated.

[0115] (1) Effects of Lactobacillus salivarius combined with chicken origin on production performance of laying hens

[0116] As can be seen from Table 8, compared with the control group, adding RS-11-1 to the diet of laying hens could significantly increase the egg production rate and average egg weight during the whole period, reduce the feed-to-egg ratio (P<0.05), and was better than adding lactic acid bacteria from a certain manufacturer. This indicates that Lactobacillus salivarius combined with chicken origin RS-11-1 can significantly improve the production performance of laying hens in the late laying period, and Lactobacillus salivarius combined with chicken origin RS-11-1 of the present invention can be used to prepare products for improving the production performance of laying hens in the late laying period.

[0117] Table 8

[0118]

[0119]

[0120] Note: Data are expressed as mean ± standard error of the mean (SEM). Data in the same row with no superscript or the same superscript letter indicate no significant difference (P>0.05), and different superscript letters indicate significant difference (P<0.05). The same applies to the following tables.

[0121] (2) Effects of Lactobacillus salivarius combined with chicken origin on egg quality of laying hens

[0122] As can be seen from Table 9, compared with the control group, adding Lactobacillus salivarius combined with chicken origin RS-11-1 to the diet of laying hens could significantly improve the yolk color and Haugh unit at the 8th week, and increase the egg weight and eggshell strength during the whole period (P<0.05). This indicates that adding Lactobacillus salivarius combined with chicken origin RS-11-1 to the diet can significantly improve the egg quality of laying hens in the late laying period, and Lactobacillus salivarius combined with chicken origin RS-11-1 of the present invention can be used to prepare products for improving the egg quality of laying hens in the late laying period.

[0123] Table 9

[0124] Item Control group Positive control group RS-11-1 group 4 weeks Egg shape index 1.32±0.01 1.32±0.01 1.32±0.01 <![CDATA[Eggshell strength, kg / cm 2 > <![CDATA[3.45±0.01 c > <![CDATA[3.53±0.01 b > <![CDATA[3.62±0.02 a > Egg weight, g <![CDATA[62.91±0.53 b > <![CDATA[62.94±0.75 a > <![CDATA[63.54±0.94 a > Albumen height, mm 8.02±0.09 7.77±0.06 7.8±0.09 Yolk color 6.55±0.12 7.5±0.12 7.71±0.12 Haugh unit 86.34±1.08 87.39±0.81 88.72±1.13 8 weeks Egg shape index 1.31±0.01 1.31±0.01 1.32±0.01 <![CDATA[Eggshell strength, kg / cm 2 > 3.68±0.13 3.74±0.11 3.81±0.11 Egg weight, g <![CDATA[60.03±0.53 b > <![CDATA[61.12±0.84 b > <![CDATA[65.68±0.98 a > Albumen height, mm <![CDATA[7.13±0.12 b > <![CDATA[7.32±0.11 b > <![CDATA[7.8±0.08 a <!-- 9 -->]]> Yolk color <![CDATA[4.73±0.1 c > <![CDATA[5.19±0.1 b > <![CDATA[5.74±0.09 a > Haugh unit <![CDATA[83.35±1.07 b > <![CDATA[86.45±0.99 ab > <![CDATA[88.01±0.99 a >

[0125] (3) Effects of Lactobacillus salivarius combined with chicken origin on antioxidant capacity of laying hens

[0126] As can be seen from Table 10, compared with the control group, adding chicken-derived saliva combined with Lactobacillus RS-11-1 to the laying hen diet can significantly reduce the content of MDA in the serum (P<0.05), while the content of T-SOD increases significantly (P<0.05). This indicates that adding chicken-derived saliva combined with Lactobacillus RS-11-1 to the feed can improve the antioxidant capacity of laying hens in the late laying period. The chicken-derived saliva combined with Lactobacillus RS-11-1 of the present invention can be used to prepare products for improving the antioxidant capacity of laying hens in the late laying period.

[0127] Table 10

[0128]

[0129]

[0130] Example 3

[0131] A preparation method of chicken-derived saliva combined with Lactobacillus postbiotics, comprising the following steps:

[0132] Step 1: Add 1 g of Bletilla striata polysaccharide and 0.5 g of shikimic acid to 100 mL of sterile water, and stir evenly to obtain a mixed solution;

[0133] Step 2: Inoculate the activated chicken-derived saliva combined with Lactobacillus RS-11-1 into 1 L of MRS liquid medium at 2% (v / v). After culturing at a constant temperature of 37 °C for 24 h, add the mixed solution obtained in Step 1, and continue to culture for 12 h. Inactivate the fermentation broth at 90 °C for 20 min to obtain the inactivated fermentation broth of chicken-derived saliva combined with Lactobacillus, that is, postbiotics.

[0134] In this example, during the fermentation and culture of RS-11-1, adding Bletilla striata polysaccharide and shikimic acid is beneficial to the growth and metabolism of RS-11-1 on the one hand, and improves the activity of the fermentation broth. On the other hand, using shikimic acid and Bletilla striata polysaccharide with antibacterial and antioxidant effects as ingredients, the prepared postbiotics can further enhance the antioxidant capacity, egg quality and egg production rate of laying hens.

[0135] Example 4

[0136] A preparation method of chicken-derived saliva combined with Lactobacillus postbiotics, comprising the following steps:

[0137] Step 1: Add 1 g of Bletilla striata polysaccharide to 100 mL of sterile water, and stir evenly to obtain a mixed solution;

[0138] Step 2: Inoculate the activated Lactobacillus salivarius combined with chicken origin RS-11-1 into 1 L of MRS liquid medium at 2% (v / v). After culturing at a constant temperature of 37 °C for 24 h, add the mixed solution obtained in Step 1 and continue culturing for 12 h. Inactivate the fermentation broth at 90 °C for 20 min to obtain the inactivated fermentation broth of Lactobacillus salivarius combined with chicken origin, namely postbiotics.

[0139] Example 5

[0140] A preparation method of postbiotics of Lactobacillus salivarius combined with chicken origin, comprising: inoculating the activated Lactobacillus salivarius combined with chicken origin RS-11-1 into 1 L of MRS liquid medium at 2% (v / v), culturing at a constant temperature of 37 °C for 36 h, and inactivating the fermentation broth at 90 °C for 20 min to obtain the inactivated fermentation broth of Lactobacillus salivarius combined with chicken origin, namely postbiotics.

[0141] According to the method in Example 1, measure the in vitro antioxidant activity of the postbiotics of Lactobacillus salivarius combined with chicken origin prepared in Examples 3-5. As shown in Table 11, the postbiotics of Lactobacillus salivarius combined with chicken origin prepared by the present invention have excellent antioxidant activity, and Example 3 is the best, which can be used to prepare antioxidant products.

[0142] Table 11

[0143] Antioxidant index Example 3 Example 4 Example 5 DPPH free radical scavenging rate (%) 95.68 93.55 92.74 Superoxide anion free radical scavenging rate (%) 94.07 93.19 91.63 Hydroxyl free radical scavenging rate (%) 98.72 98.56 98.41 Reducing power (OD value) 0.065 0.060 0.057

[0144] According to the method in Example 2, use the postbiotics prepared in Examples 3-5 for animal experiments. The postbiotics group is fed a basal diet supplemented with 1 wt‰ postbiotics. As shown in Table 12, it can be seen that adding the postbiotics prepared by the present invention to the feed for laying hens in the late laying period can further improve the laying rate, Haugh unit of eggs and antioxidant capacity of laying hens. The postbiotics of the present invention can be applied to laying hens in the late laying period to prepare products that improve the production performance of laying hens in the late laying period, improve the antioxidant capacity of laying hens in the late laying period and / or improve the egg quality of laying hens in the late laying period.

[0145] Table 12

[0146]

[0147] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. An application of chicken saliva combined with lactobacillus in laying hens in the late egg-laying period, characterized in that: The chicken saliva combined with lactobacillus is used to prepare a product that improves the production performance of laying hens in the late egg-laying period; The chicken-derived saliva-associated Lactobacillus is RS-11-1, classified and named as saliva-associated Lactobacillus Ligilactobacillus salivarius, and was registered and preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 12, 2024, with the preservation number CGMCC No.32589.

2. The use of chicken saliva combined with lactobacillus in late egg-laying hens as claimed in claim 1, characterized in that: The chicken-derived saliva combined with lactobacillus is used to prepare a product for improving the egg quality of laying hens in the late egg-laying period.

3. The use of chicken saliva combined with lactobacillus in laying hens in the late egg-laying period as claimed in claim 1, characterized in that: The chicken-derived saliva combined with lactobacillus is used to prepare a product for improving the antioxidant capacity of laying hens in the late egg-laying period.

4. The use of chicken saliva combined with lactobacillus in late laying hens as claimed in any one of claims 1 to 3, characterized in that: The product is a medicine or an animal feed additive.

5. The use of chicken saliva combined with lactobacillus in late laying hens as claimed in any one of claims 1 to 3, characterized in that: The product contains chicken saliva combined with lactobacillus, chicken saliva combined with lactobacillus fermentation supernatant and / or chicken saliva combined with lactobacillus bacterial liquid.

6. The use of chicken saliva combined with lactobacillus in late egg-laying hens as claimed in claim 1, characterized in that: The method of improving the production performance of laying hens in the late egg-laying period includes: improving the egg-laying rate, increasing the average egg weight and / or reducing the feed-to-egg ratio.

7. The use of chicken saliva combined with lactobacillus in late laying hens as claimed in claim 2, characterized in that: The method for improving the egg quality of laying hens in the late laying period includes: improving eggshell strength, improving yolk color, improving Haugh unit and / or improving albumen height.

8. The use of chicken saliva combined with lactobacillus in laying hens in the late egg-laying period as claimed in claim 3, characterized in that: The method for improving the antioxidant capacity of laying hens in the late egg-laying period includes: reducing the malondialdehyde content in serum, increasing the total superoxide dismutase content in serum, increasing the glutathione peroxidase content in serum and / or improving the total antioxidant capacity in serum.

9. The use of chicken saliva combined with lactobacillus in late laying hens as claimed in claim 1, characterized in that: The 16S rDNA sequence of the chicken saliva-associated lactobacillus is shown in SEQ ID NO.

1.

10. A postbiotic, characterized in that: The postbiotics contain the inactivated bacteria, fermentation metabolites and / or inactivated fermentation liquid of the chicken saliva-combined lactobacillus according to claim 1.

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