A saliva-combined lactobacillus 8-2 strain and its application

Abstract

The present invention provides a salivarius lactobacillus 8-2 strain and an application thereof, wherein the salivarius lactobacillus 8-2 strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC NO.30612. The salivarius lactobacillus described in the present invention has good acid resistance, is non-hemolytic, has strong antibacterial ability, can reduce the damage caused by salmonella infection, and can effectively promote the absorption function of intestinal mucosa, improve the production performance of laying hens, has the potential to be developed into a high-quality probiotic preparation for laying hens with both "antibiotic replacement" function and the function of promoting the repair of intestinal mucosal damage, and can be used in the preparation of laying hen antibiotic substitutes, including the application of the fermentation supernatant of the strain in the preparation of laying hen antibiotic substitutes.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a salivarius-associated lactobacillus 8-2 strain and an application thereof. Background Art

[0002] Poultry eggs are an important source of high-quality animal protein. my country is a major consumer of poultry eggs, producing and consuming about 40% of the world's poultry eggs. However, many laying hen companies in my country still face problems such as backward feeding and management methods, unsatisfactory egg-laying performance of laying hens, and unreasonable use of antibiotics. Probiotics have been valued in the livestock and poultry farming industry due to their safety, effectiveness, and low cost.

[0003] Several studies have shown that lactic acid bacteria exhibit excellent antibacterial activity in poultry farming. Ligilactobacillus salivarius LI01 has been shown to protect the intestinal mucosal barrier, reduce the levels of inflammatory factors and bacterial transfer in serum, and increase the abundance of intestinal flora (Yao et al., 2021). The combination of Lactobacillus salivarius (L. salivarius) and Lactobacillus reuteri (L. reuteri) can reduce the colonization of Campylobacter jejuni and Salmonella enteritidis (S. enteritidis) in broilers (Ghareeb et al., 2012). Therefore, lactic acid bacteria preparations can be used as a potential high-efficiency alternative to antibiotics in the laying hen industry.

[0004] In addition to its strong antibacterial activity, Lactobacillus also has a variety of regulatory functions on intestinal mucosal function. Many studies have shown that improving the intestinal mucosal morphology of laying hens is closely related to the improvement of egg production performance and egg quality. For example, feeding Lactobacillus reuteri and Lactobacillus plantarum can reduce the intestinal mucosal permeability of chickens (Meyer et al., 2020); Lactobacillus reuteri D8 improves intestinal mucosal damage caused by inflammatory enteritis and increases the number of EdU-positive cells and Lgr5-positive cells (Hou et al., 2018), promoting intestinal stem cell regeneration. Therefore, Lactobacillus preparations can be used as a feed additive to regulate intestinal mucosal function in laying hen farming.

[0005] At present, there are many types of probiotics used in poultry production and breeding, but there is no consensus on probiotic products that can simultaneously play an antibiotic replacement function, promote intestinal mucosal damage repair, and enhance intestinal mucosal absorption. Discovering more new microecological preparations that can replace traditional veterinary drugs has broad prospects and will provide strong support for green laying hen breeding. Summary of the invention

[0006] The purpose of the present invention is to provide a salivary Lactobacillus 8-2 strain, which is classified and named as: salivary Lactobacillus (Ligilactobacillus salivarius), which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on May 13, 2024, with a deposit number of CGMCC NO.30612, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The 16S rDNA full sequence of the salivary Lactobacillus (Ligilactobacillus salivarius) 8-2 strain provided by the present invention is shown in SEQ ID No.1.

[0007] 1. Screening and physicochemical properties analysis of saliva-associated Lactobacillus 8-2 strain

[0008] (1) Colony morphology: It grows well on MRS solid medium, with round, raised, milky white colonies with neat edges, plump, moist and opaque.

[0009] (2) Bacterial morphology: Gram-positive bacteria, short rod-shaped, blunt-ended, non-spore-forming;

[0010] (3) Growth characteristics: Facultative anaerobic, a single colony was cultured in MRS liquid medium for 12 h to enter the logarithmic growth phase and 20 h to enter the stable phase;

[0011] (4) Non-hemolytic;

[0012] (5) The strain was identified as Ligilactobacillus salivarius by 16S rDNA sequencing and named as Lactobacillus salivarius strain 8-2.

[0013] Another object of the present invention is to provide the use of the saliva combined Lactobacillus 8-2 strain in the preparation of an antibiotic substitute for laying hens. It is mainly used to inhibit Salmonella, Escherichia coli and Staphylococcus aureus. The saliva combined Lactobacillus 8-2 strain or its fermentation supernatant can promote the repair of damaged intestinal mucosa of laying hens and enhance the absorption function of the intestinal mucosa of laying hens.

[0014] The saliva-associated Lactobacillus 8-2 strain and its fermentation supernatant have antibacterial ability against common pathogenic bacteria of laying hens. The saliva-associated Lactobacillus 8-2 strain and its fermentation supernatant have good inhibitory effect on Escherichia coli, Salmonella typhimurium, Salmonella enteritidis and Staphylococcus aureus, and the antibacterial effect is stronger than that of the commercial strain - Lactobacillus salivarius (CGMCC1.1881).

[0015] The saliva combined with Lactobacillus 8-2 strain and its fermentation supernatant have a promoting effect on the repair process of damaged intestinal mucosa of laying hens. Feeding saliva combined with Lactobacillus 8-2 strain can significantly reduce intestinal damage caused by Salmonella typhimurium infection, reduce the level of pro-inflammatory factors in serum, increase the level of anti-inflammatory factors, restore intestinal barrier function, and improve intestinal flora.

[0016] The saliva combined with Lactobacillus 8-2 strain and its fermentation supernatant have an enhancing effect on the intestinal mucosal absorption function of laying hens. Feeding saliva combined with Lactobacillus 8-2 strain can increase the intestinal mucosal absorption area of ​​laying hens, increase the expression of intestinal mucosal amino acid transporters, increase serum free amino acid levels, and improve laying hens' production performance and egg quality.

[0017] The Lactobacillus salivarius described in the present invention has good acid resistance, is non-hemolytic, has strong antibacterial ability, can reduce the damage caused by Salmonella infection, and can effectively promote the absorption function of the intestinal mucosa, improve the production performance of laying hens, and has the potential to be developed into a high-quality probiotic preparation for laying hens that has both "antibiotic replacement" function and the function of promoting the repair of intestinal mucosal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of salivary Lactobacillus 8-2 strain.

[0019] Figure 2 This is the Gram staining result of saliva combined with Lactobacillus 8-2 strain.

[0020] Figure 3 The results of 16s rDNA electrophoresis of saliva combined with Lactobacillus 8-2 strain.

[0021] Figure 4 The results of the hemolysis experiment of saliva combined with Lactobacillus 8-2 strain.

[0022] Figure 5 The results of the antibacterial experiment of saliva combined with Lactobacillus 8-2 strain.

[0023] Figure 6 Pre-feeding chicks with saliva combined with Lactobacillus 8-2 strain can reduce intestinal damage when Salmonella is infected.

[0024] Figure 7 Pre-feeding with saliva combined with Lactobacillus 8-2 strain can protect the intestinal mucosal barrier function of chicks when infected with Salmonella.

[0025] Figure 8 Pre-feeding with saliva combined with Lactobacillus 8-2 strain can protect the intestinal mucosal structure of chicks when infected with Salmonella.

[0026] Fig. 9 Pre-feeding with saliva combined with Lactobacillus 8-2 strain can reduce intestinal epithelial cell apoptosis in chicks infected with Salmonella.

[0027] Fig.10 Pre-feeding with saliva combined with Lactobacillus 8-2 strain can protect local mitochondria in the intestinal crypts of chicks when infected with Salmonella.

[0028] Fig.11 Analysis of intestinal flora in chicks.

[0029] Fig.12 The effect of saliva combined with Lactobacillus 8-2 strain on the abundance of three amino acid transporter genes in the intestine of laying hens.

[0030] Fig.13 To investigate the effect of saliva combined with Lactobacillus 8-2 strain on serum free amino acid levels in laying hens.

[0031] Fig.14 The effect of saliva combined with Lactobacillus 8-2 strain on the intestinal villi morphology of laying hens.

[0032] Fig.15 The effect of saliva combined with Lactobacillus 8-2 strain on the absorption area of ​​intestinal villi of laying hens.

[0033] Fig.16 The effect of salivary combined Lactobacillus 8-2 strain on laying hen egg quality. DETAILED DESCRIPTION

[0034] The present invention is further described in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0035] Example 1: Isolation and identification of strains

[0036] 1. Isolation of Lactobacillus 8-2 from Saliva

[0037] (1) Sample source

[0038] The strain of the invention is separated from the intestinal tract of healthy Zhenning native chickens.

[0039] (2) Isolation and purification of strains

[0040] Take 0.1g of fresh feces and add 1mL of sterile saline, mix well, dilute 10 times, and draw 100μL of 10 -4 The dilution was evenly spread on the MRS solid agar plate, and after culturing at 37℃ for 48h, a single colony with good growth was picked and cultured in MRS liquid medium for 24h, and then streaked with the bacterial liquid for culture. After repeating the purification culture for 3 times, a relatively pure single colony was obtained. A single colony was picked and cultured in MRS liquid medium for expansion, and 20% glycerol was added and frozen at -80℃.

[0041] The fermentation medium of the present invention is: 1000mL double distilled water, 10g peptone, 10g beef powder, 5g yeast powder, 20g glucose, 0.1g magnesium sulfate, 5g sodium acetate, 2g ammonium citrate, 2g dipotassium hydrogen phosphate, 0.05g manganese sulfate, 1mL Tween 80. The solid medium needs to be added with 2% agar powder. The pH is adjusted to 5.5-6.5, and sterilized at 121°C for 30min.

[0042] 2. Identification of Saliva-associated Lactobacillus 8-2

[0043] (1) Colony characteristics

[0044] It grows well on MRS solid medium, with round, raised, milky white colonies with neat edges, plump, moist and opaque. Figure 1 .

[0045] (2) Bacterial morphology

[0046] The purified strains were subjected to Gram staining. Staining method: (1) Smear fixation, use a sterile inoculation loop to take a loop of double distilled water and apply it to the center of the slide, then pick a single colony and evenly apply it to the double distilled water area. After the liquid on the slide dries, you can start staining; (2) Primary staining, add ammonium oxalate crystal violet to stain for 1 minute, wash with water; (3) Mordant staining, add iodine solution to stain for 1 minute, wash with water; (4) Decolorization, add 95% ethanol, shake and decolorize for 45 seconds, then wash with water; (5) Restaining, add fuchsin staining solution for 1 minute, wash with water. After staining, observe under a microscope and screen out blue-purple Gram-positive bacteria.

[0047] Saliva-associated Lactobacillus 8-2 strain was Gram-positive, did not produce spores, and had a short rod-shaped body with blunt ends. Figure 2 .

[0048] (3) 16S rDNA sequencing

[0049] The target strain genomic DNA was extracted as a PCR amplification template, and the bacterial universal primers 27F and 1492R were used to perform 16SrDNA PCR experiments. After the PCR reaction was completed, the PCR product was subjected to agarose gel electrophoresis. The length of the amplified fragment was about 1500 bp. Figure 3. The PCR product was sequenced by Meiji Biotechnology Co., Ltd., and its 16S rDNA full sequence is shown in SEQID No. 1. BLAST sequence alignment was performed on the NCBI website and identified as Ligilactobacillus salivarius. The strain was named Ligilactobacillus salivarius 8-2, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 13, 2024, with a deposit number of CGMCC NO.30612, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0050] Example 2: Acid resistance and safety of saliva-lactobacillus

[0051] (1) Acid resistance test

[0052] Adjust the pH of the MRS liquid culture medium to 3, filter and sterilize with a 0.22μm filter membrane, and store at 4℃ for later use. Pick a single colony of saliva-associated Lactobacillus 8-2 and expand it in MRS liquid culture medium at 37℃ for 12h. Take 0.5mL of the bacterial solution and transfer it to 4.5mL of MRS liquid culture medium with a pH of 3.0. Continue to culture at 37℃ for 3h. Use the dilution plate coating method to determine the number of viable bacteria in the 0h and 3h samples, and use the MRS measurement value without adjusting the pH as a control to calculate the survival rate. The calculation formula is: Survival rate = N t / N0×100%. Where N0 represents the number of viable bacteria at 0h (CFU / mL), N t The results showed that the survival rate of Lactobacillus salivarius 8-2 strain in MRS medium with a pH of 3.0 was 92.3%, indicating that the strain can survive in the gastric acid environment.

[0053] (2) Hemolysis test

[0054] Use an inoculation loop to pick up the bacterial liquid and inoculate it on an MRS agar plate containing 5% (w / v) sheep blood. Place it in a 37°C incubator and incubate it for 48 hours. Observe the hemolysis around the colonies. Hemolysis is divided into three types: β-hemolysis (transparent band), α-hemolysis (green band), and γ-hemolysis (no hemolysis). The results are as follows Figure 4 As shown, the salivary Lactobacillus 8-2 strain did not show α-hemolysis and β-hemolysis, and was highly safe for laying hens.

[0055] Example 3: Antibacterial ability of saliva combined with Lactobacillus 8-2 strain

[0056] Pour 20 μL of sterilized LB solid medium into each culture dish. After the solid medium cools and solidifies, remove the sterile Oxford cup that was placed in advance. Activate the four frozen pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Salmonella enteritidis) on the LB solid medium for 2 to 3 times, pick a single colony and place it in LB liquid medium, and culture it at 37°C for 10 hours. Collect the bacteria by centrifugation and resuspend them in sterile PBS to a concentration of 10 8 CFU / mL. Take 200 μL and evenly apply it on LB solid medium with a sterile cotton swab. Inoculate the activated saliva combined with Lactobacillus 8-2 strain into MRS medium and expand the culture at 37℃ for 13h. Adjust the concentration to 10 8 CFU / mL, the fermentation supernatant was added to the cup wells (200μL / well), and the MRS liquid culture medium without bacteria was used as a blank control. The diameter of the inhibition zone was measured after culturing at 37℃ for 24h. Each sample was repeated 3 times. The commercial strain - Lactobacillus salivarius (CGMCC1.1881) was used as a control. The results are shown in Figure 5 As shown in Table 1, Lactobacillus saliva 8-2 can inhibit Escherichia coli, Salmonella typhimurium, Salmonella enteritidis and Staphylococcus aureus, and its antibacterial effect is stronger than that of the commercial strain (Lactobacillus salivarius CGMCC1.1881), indicating that Lactobacillus saliva 8-2 can inhibit common intestinal pathogens in laying hens and has the potential to replace antibiotics.

[0057] Table 1 Antibacterial effect of saliva combined with Lactobacillus 8-2 strain

[0058]

[0059] Example 4: In vivo anti-infection ability of saliva combined with Lactobacillus 8-2 strain

[0060] (1) Experimental animals and experimental design

[0061] The experimental animals were Hy-Line brown chicks. After hatching, they were first adaptively fed for 3 days and then randomly divided into 7 groups, namely CON (blank control), CS (Salmonella infection), MS (MRS + Salmonella infection), AS (antibiotics + Salmonella infection), LS (8-2 supernatant + Salmonella infection), LP (8-2 live bacteria + Salmonella infection), with 15 chicks in each group. During the experiment, the MS, AS, LS and LP groups were fed with MRS liquid culture medium, antibiotics (enrofloxacin), 8-2 supernatant and 8-2 live bacteria, respectively. On the 15th day of the experiment, each chicken was fed with 1×10 9 The CFU Salmonella typhimurium liquid was administered orally to explore the anti-Salmonella infection effect of 8-2 live bacteria and its fermentation supernatant.

[0062] (2) Basic diet and feeding management

[0063] The temperature of the rearing environment was maintained at 30°C, the light system was 16L:8D, the chicks were free to eat and drink, the feeding amount was adjusted according to the age of the chicks, and the feces were cleaned every day to ensure a good living environment. Among them, the CON group and the CS group were fed with a basic diet; the MS group was fed with a basic diet + MRS liquid medium (1mL / chicken); the AS group was fed with a basic diet + enrofloxacin (0.5μg / chicken); the LS group was fed with a basic diet + 8-2 supernatant (1mL / chicken, composed of 1×10 10 cfu / mL concentration of bacterial solution was centrifuged and the supernatant was taken); the LP group was fed with a basic diet + 8-2 live bacteria (1×10 10 cfu / piece). Other routine management was followed. At the end of the experiment, the chicks were necropsied, pathological scores were performed according to the severity of intestinal lesions, jejunal tissues were collected for subsequent morphological observation and protein and RNA extraction, and feces were collected for 16S rDNA sequencing.

[0064] (3) Pathological scoring

[0065] During autopsy, intestinal pathology scores were performed according to Table 2. The statistical results are shown in Table 3. Antibiotics, 8-2 fermentation supernatant and 8-2 live bacteria can significantly reduce intestinal damage caused by Salmonella infection, among which the fermentation supernatant has the best effect.

[0066] Table 2 Scoring criteria for intestinal pathology

[0067] score Lesions 0 No obvious lesions 0.5 Serous layer and mesentery are severely congested 1 The intestinal wall is thin and brittle, with a few bleeding spots 2 There is a small amount of gas and local necrosis 3 There are a lot of gas-filled and necrotic plaques in the intestine

[0068] Table 3 Intestinal pathology scoring results

[0069] CS MS AS LS LP Pathological scoring <![CDATA[1.70±0.19 a ]]> <![CDATA[1.73±0.20 a ]]> <![CDATA[0.80±0.13 b ]]> <![CDATA[0.47±0.11 b ]]> <![CDATA[0.63±0.10 b ]]>

[0070] Note: Mean values ​​with different superscript letters indicate significant differences (p < 0.05). CON (blank control), CS (Salmonella infection), MS (MRS + Salmonella infection), AS (antibiotics + Salmonella infection), LS (8-2 supernatant + Salmonella infection), LP (8-2 live bacteria + Salmonella infection) group.

[0071] (4) Changes in the abundance of genes related to inflammatory factors in intestinal tissue

[0072] Real-time fluorescence quantitative PCR (qPCR) was used to analyze the changes in the abundance of intestinal inflammatory factors (TNF-α, IL-1β, IL-4, IL-10). Intestinal tissue RNA was extracted and reverse transcribed into cDNA. The cDNA of each sample was diluted 5 times and the qPCR reaction system was prepared according to Table 4. The statistical results are shown in Table 4. Figure 6As shown in the data, after infection with Salmonella, the levels of intestinal proinflammatory factors (TNF-α, IL-1β) increased significantly, and the levels of anti-inflammatory factors (IL-4, IL-10) decreased significantly. Both the pre-feeding 8-2 fermentation supernatant and live bacteria precipitate could reduce the levels of proinflammatory factors (TNF-α, IL-1β) and increase the levels of anti-inflammatory factors (IL-4, IL-10), among which the fermentation supernatant had better effect.

[0073] The mucus layer of the intestinal mucosa is an important line of defense for the intestine against external pathogens. MUC2 is a secretory mucin produced by intestinal epithelial goblet cells. The results of the analysis of the abundance of MUC2 mRNA in intestinal tissue ( Figure 7 ) showed that after infection with Salmonella, the abundance of MUC2 decreased significantly, while pre-feeding with 8-2 ​​fermentation supernatant could significantly increase the abundance of MUC2, indicating that 8-2 fermentation supernatant can effectively enhance the intestinal mucosal barrier function.

[0074] Table 4qRCR reaction system

[0075] Element Sample volume 2×SYBR qPCR Master Mix 7.5μL Forward Primer (10μM) 0.3μL Reverse Primer (10μM) 0.3μL cDNA 1.5μL ddH2O Up to 15 μL

[0076] After the system was mixed and centrifuged, it was placed in the CFX96 Touch real-time PCR system for qPCR reaction. The reaction procedure is as follows:

[0077] Stage 1: (1×)

[0078] Step 1.1: 95℃, 30s

[0079] Stage 2: (40×)

[0080] Step 2.1: 95℃, 10s

[0081] Step 2.2: 60℃, 30s

[0082] Figure 6 Middle: CON (blank control) group, CS (Salmonella infection) group, MS (MRS + Salmonella infection) group, AS (antibiotics + Salmonella infection) group, LS (8-2 supernatant + Salmonella infection) group, LP (8-2 live bacteria + Salmonella infection) group.

[0083] Figure 7 Middle: CON (blank control) group, CS (Salmonella infection) group, MS (MRS + Salmonella infection) group, AS (antibiotics + Salmonella infection) group, LS (8-2 supernatant + Salmonella infection) group, LP (8-2 live bacteria + Salmonella infection) group.

[0084] (5) Intestinal mucosal morphology analysis

[0085] Fresh jejunal tissue was fixed in 4% paraformaldehyde. After fixation for 48 hours, it was dehydrated with gradient alcohol, permeabilized with xylene, and then embedded in paraffin. After cooling, it can be sliced. After slicing, the slices were placed at 60℃ for 2 hours, and then dewaxed, hydrated and HE stained. The process is: xylene (Ⅰ) 5 minutes; xylene (Ⅱ) 5 minutes; anhydrous ethanol (Ⅰ) 2 minutes; anhydrous ethanol (Ⅱ) 2 minutes; 90% ethanol 2 minutes; 70% ethanol 2 minutes; hematoxylin staining 5 minutes; tap water rinse; eosin staining 5 seconds; tap water rinse. Finally, dehydration, transparency and sealing were carried out: 95% ethanol 1 minute; anhydrous ethanol (Ⅰ) 2 minutes; anhydrous ethanol (Ⅱ) 2 minutes; xylene (Ⅰ) 3 minutes; xylene (Ⅱ) 3 minutes; use an appropriate amount of neutral resin to seal the slice. The HE staining results are as follows: Figure 8 As shown, compared with the CON group, the intestinal villi in the MS group were severely damaged and broken; the degree of intestinal villi damage in the LS group was lighter than that in the MS group, and the villi were basically intact, indicating that 8-2 fermentation supernatant can reduce the damage of Salmonella infection to the intestinal mucosal structure of laying hens.

[0086] Figure 8 Middle: CON (blank control) group; MS (MRS + Salmonella infection) group; LS (8-2 fermentation supernatant + Salmonella infection) group.

[0087] (6) Analysis of apoptosis of intestinal mucosal epithelial cells

[0088] The procedure was performed according to the instructions of the TUNEL cell apoptosis detection kit, and the process was as follows: dewaxing and hydrating paraffin sections; permeabilization (proteinase K); washing samples 2 to 3 times; equilibration at room temperature for 30 minutes; labeling with labeling solution at 37°C for 60 minutes; washing; DAPI counterstaining for 5 minutes; washing samples 2 to 3 times. Fig. 9 As shown, cell apoptosis mainly occurs at the top of the villi. After Salmonella infection, intestinal epithelial cell apoptosis is more serious. Pre-feeding with saliva combined with Lactobacillus 8-2 fermentation supernatant can significantly reduce the degree of intestinal epithelial cell apoptosis.

[0089] Fig. 9 Middle: TUNEL positive cells are apoptotic cells, emitting green fluorescence; cell nuclei emit blue fluorescence. CON (blank control) group; MS (MRS + Salmonella infection) group; LS (8-2 fermentation supernatant + Salmonella infection) group.

[0090] (7) Ultrastructural analysis of local mitochondria in intestinal crypts

[0091] 1) Take out the sample immersed in glutaraldehyde and rinse it with 1 mL 0.1 M PBS three times, 10 min each time;

[0092] 2) Add 50-100 μL (covering the sample) of 1% osmium acid and fix for 1 hour;

[0093] 3) Rinse with 1 mL of double distilled water three times, 10 min each time;

[0094] 4) Fix / stain once with 100 μL 2% uranyl acetate for 30 min;

[0095] 5) Add 1 mL of 50%, 70%, and 90% ethanol, once for 15 min each time, 100% ethanol, once for 20 min, and 100% acetone, twice for 20 min each time;

[0096] 6) Add 300-500 μL of embedding agent + acetone mixture (1:1) and place in a drying oven for 2 h;

[0097] 7) Add 300-500 μL of embedding agent + acetone mixture (3:1) to the tissue sample and place it in a drying oven overnight, then embed the sample the next day.

[0098] 8) Embed, slice, stain and load onto the machine after the sample solidifies.

[0099] Transmission electron microscopy results Fig.10 As shown, the local mitochondrial damage in the intestinal crypts of the Salmonella infection group was severe, manifested by the breakage and disappearance of mitochondrial cristae, mitochondrial autophagy, and internal vacuolation; while the mitochondrial cristae of the group pre-fed with 8-2 ​​fermentation supernatant were only slightly broken, and the autophagy phenomenon was milder than that of the Salmonella infection group.

[0100] Fig.10 Middle: MS (MRS + Salmonella infection) group; LS (8-2 fermentation supernatant + Salmonella infection) group.

[0101] (9) Intestinal flora analysis

[0102] like Fig.11 As shown, the species diversity and richness of the intestinal flora in the Salmonella (S8) infection group after pre-feeding with 8-2 ​​fermentation supernatant were higher than those in the Salmonella infection (SM) group, indicating that 8-2 fermentation supernatant can improve the intestinal flora.

[0103] Fig.11 Middle: S8 refers to LS group (8-2 fermentation supernatant + Salmonella infection group), SM refers to MS group (MRS + Salmonella infection group).

[0104] In conclusion, pre-feeding saliva combined with fermentation supernatant of Lactobacillus 8-2 strain can effectively reduce the damage of intestinal mucosa of laying hens caused by Salmonella typhimurium infection.

[0105] Example 5: The promoting effect of saliva combined with Lactobacillus 8-2 on intestinal mucosal absorption function

[0106] (1) Experimental animals and experimental design

[0107] The experimental animals were 450-day-old Hy-Line brown laying hens. 108 of them were randomly divided into 4 groups (27 birds / group): (1) CON group: fed with a basal diet; (2) MRS group: fed with a basal diet containing MRS liquid medium; (3) 8-2 supernatant group: fed with a basal diet containing saliva combined with Lactobacillus 8-2 strain (1×10 10 CFU / bird / day) fermentation supernatant; (4) 8-2 precipitation group: fed with saliva combined with Lactobacillus 8-2 strain (1×10 10 CFU / bird / day) of live bacteria as the basic diet. Feed continuously for 14 days.

[0108] (2) Analysis of intestinal mucosal absorption function

[0109] During the feeding period, egg production and egg weight were recorded daily. After feeding, blood was collected from the wing vein of each chicken to collect serum for amino acid level testing; duodenum tissue was taken for RNA extraction and morphological observation; and eggs were taken to test egg quality.

[0110] (1) Expression levels of amino acid transporters in the duodenum

[0111] The method is the same as in Example 4 (4), and the results are as follows Fig.12 As shown, feeding saliva combined with fermentation supernatant of Lactobacillus 8-2 strain can significantly increase the gene abundance of three amino acid transporters in intestinal tissue, suggesting that it can promote the amino acid absorption capacity of intestinal mucosa.

[0112] (2) Serum amino acid levels

[0113] Serum samples were taken, protein was precipitated with 5% trichloroacetic acid, centrifuged at 7000-10000rpm / min in a high-speed refrigerated centrifuge for 15min, the supernatant was taken and stored, and filtered with a 0.45μm+0.22μm filter membrane before being analyzed on the machine. The samples were detected by Hitachi L8900 fully automatic amino acid analyzer, chromatographic column: Na+ type cation exchange column (4.6mm ID×60mm, 3μm particles); ion exchange resin 2622, detector was UV-visible detector; color developer was ninhydrin / sodium acetate buffer; buffer system was citric acid buffer B1 (pH3.2), B2 (pH3.0), B3 (pH 4.0), B4 (pH4.9); buffer flow rate was 0.4mL / min; column temperature was 55℃, reaction room temperature was 135℃. The free amino acid content in the sample solution was determined by external standard method, where the detection wavelength of proline was 440nm and the detection wavelength of other amino acids was 570nm.

[0114] Serum total amino acid levels Fig.13As shown in Table 5, feeding 8-2 supernatant and live bacterial precipitate can significantly increase the level of free amino acids in serum. The levels of various amino acids are shown in Table 5. After feeding saliva combined with Lactobacillus 8-2 strain, the levels of free amino acids in serum were all increased. Among them, 8-2 fermentation supernatant significantly increased the levels of acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu), neutral amino acids such as alanine (Ala), isoleucine (Ile), tyrosine (Tyr) and basic amino acids such as histidine (His); 8-2 live bacterial precipitate significantly increased the levels of neutral amino acids such as valine (Val), leucine (Leu) and basic amino acids such as lysine (Lys). It is worth noting that among these significantly upregulated amino acids, 7 are essential amino acids for the human body: isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), and valine (Val).

[0115] Table 5 Serum amino acid levels

[0116]

[0117]

[0118] (3) Analysis of intestinal villus morphology

[0119] The staining method is the same as in Example 4.5. The HE staining results are as follows Fig.14 As shown in the figure, compared with the control group (CON), the surface of the intestinal villi of 8-2 fermentation supernatant and 8-2 live bacteria precipitation had more wrinkles, and the increase in wrinkles indicated that the intestinal mucosa had a larger absorption area. Fig.15 ) showed that feeding 8-2 fermentation supernatant significantly increased the villus circumference, suggesting that the intestinal mucosal absorption area was larger. The above results confirmed that saliva combined with Lactobacillus 8-2 strain can increase the intestinal mucosal absorption area.

[0120] (4) Egg production

[0121] The egg production was recorded daily. The average daily egg production rate is shown in Table 6. The total egg production during the test period is shown in Table 7. Feeding 8-2 fermentation supernatant and live bacteria precipitate can increase the daily egg production rate and egg production of chickens.

[0122] Table 6 Daily average egg production rate (%)

[0123] CON MRS 8-2 Supernatant 8-2 Live bacteria Egg production rate 86.78 88.89 90.12 91.36

[0124] Table 7 Total egg production (eggs / 27 birds / 14 days)

[0125] CON MRS 8-2 Supernatant 8-2 Precipitation Egg production 319 320 327 331

[0126] (5) Egg quality

[0127] Egg quality generally refers to the appearance (size, shape, gloss) and the quality of the contents (viscosity and color of the egg white, etc.). Among them, Haversian units and albumen height are important indicators for egg quality assessment internationally, used to measure egg white quality and egg freshness.

[0128] Egg quality test results Fig.16 As shown, after feeding 8-2 fermentation supernatant and live bacteria precipitate, the Haversian unit, albumen height, eggshell thickness and eggshell strength of eggs were improved to varying degrees, indicating that the 8-2 strain can improve egg quality.

Claims

1. A salivary Lactobacillus 8-2 strain, characterized in that: The strain is named as: Lactobacillus salivarius ( Ligilactobacillus salivarius ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 13, 2024, with the deposit number: CGMCC NO.30612.

2. The use of the salivary Lactobacillus 8-2 strain according to claim 1 in the preparation of an antibiotic substitute for laying hens, characterized in that: The invention comprises the application of the fermentation supernatant of the strain in preparing an antibiotic substitute for laying hens; the antibiotic substitute inhibits the activity of salmonella, escherichia coli and staphylococcus aureus.

Images