Lactobacillus mucosa, composition and application

Abstract

The invention belongs to the field of biology, and discloses lactobacillus mucosa which is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC30899, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The lactobacillus is separated from small intestines of piglets, is one of main lactobacillus in digestive tracts of the piglets, and has certain cholate tolerance. The lactobacillus mucosa and the composition can promote the weight, the average daily gain and the weaning weight of 14-day-old suckling piglets by regulating the breast milk of the sows, promote the development of an ileum structure and the secretion of gastric mucus and gastric acid, regulate the 5-HT metabolism of intestinal tracts, and improve the immunity of the piglets. The expression of the HTR subtype in the stomach, the jejunum, the ileum and the colon is differentially regulated, and the indole level is increased by regulating tryptophan metabolism of microorganisms in the stomach, the ileum and the colon to play a role. Meanwhile, the invention further discloses application of the lactobacillus mucosa and the composition.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a mucosal lactobacillus, a composition and a use thereof. Background Art

[0002] During the lactation period, the health status of piglets, the development of intestinal function and the weaning weight reached after lactation can affect the weaning stress state and weaning mortality of piglets, and even affect the long-term growth performance of piglets. However, due to the instability of the intestinal microbiota of newborn piglets and the incomplete development of immune function, they are more susceptible to stressors such as diet and environment, as well as various pathogens, which eventually lead to intestinal dysfunction. In order to adapt to the changes in the postnatal environment and nutritional sources, the gastrointestinal structure and function of piglets begin to develop and mature rapidly after birth. At this stage, the establishment of the intestinal microbiota of newborn piglets can affect the development, maturation, metabolic homeostasis and immune defense of the gastrointestinal tract through a variety of host-microbe interactions. Therefore, the early intestinal microbiota also plays an important role in the growth and health of piglets.

[0003] In the prior art, the effects of probiotics on sows and piglets can be found in the following literature:

[0004] CN116138361A A sow feed for improving the survival rate of IUGR piglets during lactation and a preparation method thereof;

[0005] CN108079284B A medicine for improving sow production performance, its preparation method and application;

[0006] CN111032063A Combination of yeast and bacterial probiotics and methods for improving pig production;

[0007] The above scheme only superficially analyzes the changes in the production performance and immune performance of pigs caused by probiotics.

[0008] For piglets, the relevant nutrients, immune factors and other substances in sow's milk have a significant impact on piglet health.

[0009] The technical problem solved in this case is: how to regulate the growth of suckling piglets and the development of intestinal and gastric functions based on regulating sow milk. Summary of the invention

[0010] The main purpose of the present invention is to provide a mucosal lactobacillus and a composition. The mucosal lactobacillus and the composition can promote the weight, average daily weight gain and weaning weight of 14-day-old lactating piglets by regulating sow milk, promote the development of ileum structure and the secretion of gastric mucus and gastric acid, regulate intestinal 5-HT metabolism, and differentially regulate the expression of HTR subtypes in the stomach, jejunum, ileum and colon, and increase the indole level by regulating the tryptophan metabolism of gastric, ileum and colon microorganisms.

[0011] Meanwhile, the invention also discloses the use of the mucosal lactobacillus and the composition.

[0012] According to the first aspect of the present invention, the present invention provides a mucosal lactobacillus, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.30899, a deposit date of June 7, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the lactobacillus is isolated from the small intestine of piglets, is one of the main lactobacilli in the digestive tract of piglets, and has a certain bile salt tolerance.

[0013] The mucosal lactobacilli described in the examples are all mucosal lactobacilli with a deposit number of CGMCC NO.30899.

[0014] The invention also discloses the use of the mucosal lactobacillus as a sow feed additive or a piglet feed additive.

[0015] In the above-mentioned use, the sow feed additive refers to an additive added to the feed of pregnant sows; the piglet feed additive refers to an additive added to the feed of lactating piglets.

[0016] In the above-mentioned use, the mucosal lactobacillus refers to live bacteria or inactivated bacteria.

[0017] Meanwhile, the invention also discloses a composition containing the mucosal lactobacillus and tryptophan.

[0018] In the above composition, the amount of tryptophan added to the feed is 0.12 wt%.

[0019] In the above composition, the mucosal lactobacillus is administered at a rate of 1×10 per sow per day. 10 The amount of CFU is added to the feed.

[0020] It should be noted that although the present invention discloses only one point value as above, it is well known to those skilled in the art that the addition amount of the above composition fluctuates around the addition amount of ±20% and is allowed, and will not bring about a substantial performance difference;

[0021] The above limitation is made based on the inventor's preliminary experimental preparation and the limited experimental objects, but the limitation of the above point values ​​does not mean that the relevant amount of the composition is restricted.

[0022] In the above composition, the mucosal lactobacillus is also fed to piglets in the form of an oral preparation or sprayed into the living environment of piglets in the form of a spray.

[0023] In the above composition, the Lactobacillus membranaceus is a live bacterium or an inactivated bacterium.

[0024] Meanwhile, the present invention also discloses the use of any of the above-mentioned compositions as a sow feed additive or a piglet feed additive.

[0025] One of the above technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0026] The mucosal lactobacillus and the composition of the present invention can promote the weight, average daily weight gain and weaning weight of 14-day-old lactating piglets by regulating sow milk, promote the development of ileum structure and the secretion of gastric mucus and gastric acid, regulate intestinal 5-HT metabolism, and differentially regulate the expression of HTR subtypes in the stomach, jejunum, ileum and colon, and increase the indole level by regulating the tryptophan metabolism of gastric, ileum and colon microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0028] Figure 1 This is a chart showing the effects of porcine mucosal lactobacillus and tryptophan on serum antioxidant indicators in suckling piglets;

[0029] Figure 2 This is a chart showing the effects of porcine mucosal lactobacillus and tryptophan on intestinal proliferation and differentiation factors in different intestinal segments of suckling piglets;

[0030] Figure 3 This is a chart showing the effects of porcine mucosal Lactobacillus and tryptophan on the expression of key genes for 5-HT metabolism and transport in different intestinal segments of suckling piglets;

[0031] Figure 4 KEGG annotation function map of differentially expressed genes in different intestinal segments;

[0032] Figure 5 It is the KEGG signal pathway enrichment map of differentially expressed genes;

[0033] Figure 6 This is the map of common differentially expressed genes in gastric tissue regulated by age and treatment;

[0034] Figure 7 This is the common differentially expressed genes in the ileum regulated by age and treatment;

[0035] Figure 8 The figure shows the common differentially expressed genes in the colon regulated by age and treatment;

[0036] Fig. 9 Figure 2 shows differentially expressed genes specific to LM and Trp treatments, where (A) differentially expressed genes specific to LM treatment in the stomach; (B) differentially expressed genes specific to Trp treatment in the ileum;

[0037] Fig.10 This is a graph showing the effects of porcine mucosal lactobacillus and tryptophan on gastric function indices in suckling piglets;

[0038] Fig.11 It is a curve diagram of sequencing sample richness;

[0039] Fig.12 PCoA and NMDS diagrams of bacterial flora at ASV levels in different intestinal segments;

[0040] Fig.13 This is the difference in the levels of dominant bacterial phyla of microorganisms in different intestinal segments of 14-day-old suckling piglets by Lactobacillus mucosa and tryptophan;

[0041] Fig.14 This is the difference in the levels of dominant bacterial genus in the contents of different intestinal segments of 14-day-old suckling piglets by Lactobacillus mucosa and tryptophan;

[0042] Fig.15 This is a comparative analysis of multiple groups of bacteria in the contents of different intestinal segments;

[0043] Fig.16 This is the result of LEfSe analysis of microorganisms in gastric contents;

[0044] Fig.17 This is the result of LEfSe analysis of microorganisms in ileum and colon contents;

[0045] Fig.18 This is the Spearman correlation heat map between gastric HTR and 5-HT synthesis-related receptors and different bacterial genera; R values ​​are displayed in different colors in the figure. *0.01 <P≤0.05,**0.001<P≤0.01,***P≤0.001。 Fig.19 and Fig. 20 same;

[0046] Fig.19 This is the Spearman correlation heat map between ileal HTR and 5-HT synthesis-related receptors and differential bacterial genera;

[0047] Fig. 20 This is the Spearman correlation heat map between colon HTR and 5-HT synthesis-related receptors and different bacterial genera. DETAILED DESCRIPTION

[0048] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions from beginning to end. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0049] Part I. Strain Performance Verification

[0050] In the preliminary experiments, three strains of dominant lactobacilli in the intestinal tract of piglets were isolated: Lactobacillus mucosae (i.e., the strain of the present invention), L. reuteri, L. amylovorus

[0051] In this experiment, a control group and a group with 0.05% (0.5 g / L) bile salt were set up. The above inoculum was inoculated into a complete synthetic medium so that the initial bacterial concentration of fermentation was about 1.0×10 7 CFU / mL, and cultured at 37°C. Six tubes were taken out from each group at 0h, 3h, 6h, 9h, and 12h for sample collection, and the collected culture fluid was used to determine the number of viable bacteria;

[0052] The preparation method of complete synthetic medium is as follows

[0053] Complete synthetic medium (i.e., medium with defined chemical composition) was prepared according to the method of Teusink et al., and each liter of medium contained: 10 g glucose, 2.7 g sodium lactate, 0.6 g potassium chloride, 0.6 g sodium chloride, 1 g dipotassium hydrogen phosphate, 5 g potassium dihydrogen phosphate, 0.15 g calcium chloride, 0.5 g magnesium sulfate heptahydrate, 1 g sodium acetate, 0.6 g ammonium citrate, 0.5 g ascorbic acid, 0.01 g adenine, 0.01 g guanine, and 0.005 g inosine. g, orotic acid 0.005g, thymidine 0.005g, uracil 0.01g, xanthine 0.01g, 10mL heme solution (0.01%, w / v), 10mL fatty acid solution, 10mL reducing agent solution, 10mL trace element solution, 50mL sodium bicarbonate solution and 1mL resazurin solution (1%, w / v): amino acid composition reference Dai et al., where asparagine and glutamine are filtered and sterilized and added to the autoclaved culture medium. During the preparation of the culture medium, CO2 is continuously introduced into the container containing the culture medium until the culture medium is light pink to light yellow. After that, the complete synthetic culture medium is divided into Hungate anaerobic tubes into which CO2 is introduced, and each tube is divided into 9mL. After the stopper is added and the screw cap is tightened, the autoclave is sterilized at 115℃ for 15min. The culture medium after sterilization and cooling is light yellow, indicating that the environment in the anaerobic tube is in an anaerobic state.

[0054] The amino acid composition is shown in Table 1 below;

[0055] Table 1 Amino Acid Formula

[0056]

[0057] Preparation of bile salt solution

[0058] Phosphate buffer was prepared according to the method of Toouli et al.

[235] . 2.76 g of sodium dihydrogen phosphate and 3.56 g of sodium dihydrogen phosphate dihydrate were weighed and accurately diluted to 100 mL with deionized water. The pH value was adjusted to 7.4, and the solution was sterilized by filtration and stored in a refrigerator at 4°C. Bile salt solution (100×) was prepared according to Scanff et al. 0.22 g of sodium glycocholate (G7132, Sigma), 0.2 g of sodium glycochenodeoxycholate (G0759, Sigma), 0.04 g of sodium taurochenodeoxycholate (86339, Sigma), and 0.04 g of sodium taurochenodeoxycholate (T6260, Sigma). The solution was dissolved in phosphate buffer and accurately diluted to 10 mL (the final concentration of bound bile salts in the culture medium of the bile salt group was 0.05%, i.e., 0.5 g / L). The solution was sterilized by filtration and stored in a refrigerator at 4°C.

[0059] The number of viable bacteria in the fermentation broth of each strain at different time points was calculated by the plate count method or the Hungate tube rolling method, and the results are shown in Table 2. As can be seen from the table, when grown in complete synthetic medium, the growth of L. reuteri was greatly affected by the addition of bile salts, while the growth of L. mucosae and M. elsdenii was less affected by bile salts.

[0060] Table 2 Number of viable bacteria in experimental groups of different strains

[0061]

[0062] Note: Data are expressed as mean ± standard error, n = 3

[0063] Part II Feeding Experiment

[0064] Twenty-four healthy Large White × Landrace pregnant sows (255.05±3.63kg) at 109 days of gestation were selected for the experiment and divided into 6 treatment groups based on the principle of similar body weight and parity, with 4 replicates in each group and 10 piglets in each replicate.

[0065] The 6 groups were: Con group, LM group, HKLM group, Trp group, LM+Trp group and HKLM+Trp group.

[0066] LM stands for Lactobacillus mucosae, HKLM stands for killed Lactobacillus mucosae; Trp stands for tryptophan;

[0067] After sows gave birth, 0.12% Trp was added to the feed of the Trp group, LM+Trp group, and HKLM+Trp group, and the other groups were treated with nitrogen such as Ala.

[0068] In the LM group and the LM+Trp group, 1×10 10 CFU / head LM, and 1×10 10 CFU / litre LM.

[0069] HKLM group and HKLM+Trp group were given 1×10 10 CFU / head HKLM, and 1×10 10 CFU / litre HKLM. The spraying area was the sow udder and its surroundings and the piglet activity area (the floor of the farrowing bed and the fence), and the other groups were sprayed with an equal volume of 0.9% NaCl solution.

[0070] On the 7th day of lactation, creep feed was gradually added, and the total experimental period was 14 days.

[0071] Sows were fed a corn-soybean meal diet during lactation, and the suckling piglet starter feed was a mixture of Rubeile 999 (Beijing Tonglixingke Technology Co., Ltd., Beijing) and suckling pig compound feed (Beijing Lvyao Feed Technology Co., Ltd., Beijing) in proportion. The proportion of suckling pig compound feed was 37.5% and 28.6% in the second and third weeks of lactation, respectively.

[0072] Part III Study on the effect and mechanism of Lactobacillus mucosa and tryptophan in regulating the growth and intestinal function development of suckling piglets

[0073] 3.1 Test methods

[0074] 3.1.1 Sample collection

[0075] On the 14th day of the experiment, blood was collected from the anterior vena cava, and 5 mL was collected from each tube using heparin sodium anticoagulation vacuum blood collection tubes and dry vacuum blood collection tubes. The plasma and serum were separated after centrifugation at 1,500×g for 10 minutes. After the blood collection was completed, the stomach, jejunum, ileum, colon and the corresponding contents of each intestinal segment were separated and stored in liquid nitrogen. Another 2 cm of ileum was taken and transferred to 4% paraformaldehyde fixative for small intestine morphological analysis. After the start of delivery, 50 mL of colostrum was collected from the front, middle and back teats of the sows. On the 7th and 14th days, oxytocin was injected into the sows, and 50 mL of normal milk was collected from the front, middle and back teats. After mixing, the relevant indicators were measured.

[0076] 3.1.2 Growth performance and diarrhea rate

[0077] Piglets were recorded as 1 day old when they were born. The individual weights were recorded on days 1, 7, 14 and 21, and the average daily gain (ADG) of each stage was calculated using the following formula: average daily gain of each pig = (final weight of the stage - initial weight of the stage) / number of days in the experimental period.

[0078] The criteria for diarrhea were visually assessed. The anus of each piglet was checked daily to observe whether there was fecal contamination and redness and swelling, and records were kept. The fecal consistency score was 0 = formed or granular, 1 = soft feces that can be formed, 2 = thick, unformed, no separation of feces and water, 3 = liquid, unformed, and separation of feces and water. The occurrence of diarrhea was defined as a fecal consistency score of 2 or 3. The diarrhea rate (DI) was calculated based on the records. The calculation formula was: Diarrhea rate (%) = total number of diarrhea days for piglets in a single treatment group / (total number of piglets × number of days in the experimental period) × 100%.

[0079] 3.1.3 Determination of serum antioxidant indexes

[0080] Serum malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were measured using ELISA kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0081] 3.1.4 Tissue RNA extraction, reverse transcription and fluorescence quantitative PCR

[0082] (1) RNA extraction

[0083] Gastrointestinal tissue was added with liquid nitrogen and ground into powder in a mortar. About 50-100 mg of the sample was added with 1 mL of RNAiso Plus reagent to extract total RNA. The concentration and quality were detected using a Nanophotometer ultra-micro spectrophotometer (OD260 / OD280=1.8-2.0, OD230 / OD280>1.8).

[0084] (2) Two-step reverse transcription: removal of genomic DNA and reverse transcription

[0085] The extracted RNA was removed from the genomic DNA according to the instructions of the reverse transcription kit and then cDNA was synthesized. The two systems are shown in Appendix 7. The reverse transcription procedures were: 37°C, 15 min; 85°C, 5 sec; 4°C, 10 min.

[0086] (3) Quantitative PCR (qPCR)

[0087] The qPCR reaction system and procedures are shown in Appendix VII. Gene expression was quantified using the ABI 7500 real-time PCR detection system, with GAPDH as the internal reference gene. -ΔΔCt The expression level of the target gene was calculated by the method. The primer sequences are detailed in Appendix III.

[0088] 3.1.5 Determination of tryptophan, tryptophan metabolites, and free amino acids in intestinal tissue and contents

[0089] 3.1.6 Tissue sections and colon morphology examination

[0090] Paraffin embedding and tissue section preparation: (1) Tissue dehydration, transparency and wax immersion: remove the tissue from 70% ethanol and immerse it in 80% ethanol for 40 min; 95% ethanol for 1 h; anhydrous ethanol I for 30 min; anhydrous ethanol II for 30 min; xylene I for 5 min; xylene II for 5 min; wax solution I for 1 h; wax solution II for 1 h; (2) Embedding: pour the wax solution into the embedding box, place the tissue block, and install the wax holder; (3) Sectioning: use a manual slicer to cut the wax block into continuous 5 μm wax strips, then immerse the wax strips in a 43°C water bath and allow them to adhere to the surface of the slide; (4) Baking: keep warm at 37°C overnight and bake at 62°C for 1 h.

[0091] Intestinal morphology detection: (1) Dewaxing: Immerse the slices in xylene I for 10 min; xylene II for 10 min; anhydrous ethanol for 8 min; 95% ethanol for 5 min; 70% ethanol for 5 min; 50% ethanol for 5 min; distilled water for 5 min; (2) Staining: Add hematoxylin staining solution for 1 min; wash with distilled water 3 times, 2 min each time; differentiate with 1% hydrochloric acid alcohol solution for 5 sec; wash with distilled water 3 times, 5 min each time; add eosin staining solution for 1 min; wash with distilled water 3 times, 2 min each time; (3) Dehydration, transparency, and sealing: Immerse the slices in 70% ethanol for 2 sec; 95% ethanol for 5 sec; anhydrous ethanol for 5 min; xylene I for 5 min; xylene II for 5 min; add 1 drop of neutral gum on the slide, cover the slide with a cover glass, and dry overnight. Observe and take pictures under a microscope.

[0092] 3.1.7 Routine milk examination

[0093] Milk fat, milk protein, lactose, urea nitrogen and somatic cell count were determined by the Beijing Dairy Center using a 500-type milk composition and somatic cell count online analyzer (FTS+FCM) (Bentley, Bentley, USA). Milk fat, milk protein, lactose and urea nitrogen were determined by infrared spectroscopy, and somatic cell count was determined by fluorescence photoelectric counting.

[0094] 3.1.8 Determination of total amino acids in pig milk

[0095] Preparation of skim milk: Take out the milk sample at -20℃, thaw it on ice, take 1mL of whole milk in a 2mL centrifuge tube, centrifuge at 3,000g, 10min, 4℃, and aspirate the middle layer whey in a 1.5mL centrifuge tube; centrifuge again at 3,000g, 5min, 4℃, and aspirate the middle layer whey in a 1.5mL centrifuge tube.

[0096] Acid hydrolysis of skim milk: Pipette 0.5 mL skim milk into a 15 mL hydrolysis tube, add 6 mL N HCl, sonicate for 10 min, fill with N2 (1 min / tube), tighten the bottle cap after filling to prevent N2 leakage; bake at 110 ° C for 24 h (turn black), cool (observe whether the pages of all test tubes are flush), vortex low gear (3 gears) to mix; all samples in 15 mL glass tubes are transferred to 50 mL tubes, washed twice with distilled water and transferred to 50 mL tubes, and finally fixed to 50 mL with ddH2O, vortex to mix; dilute 50 times with HPLC water; filter with 0.22 μm needle filter and determine the amino acid content with amino acid analyzer.

[0097] Alkaline hydrolysis of skim milk: Pipette 50 μL skim milk into 2 mL Screw-cap tubes, add about 40 mg of hydrolyzed potato starch; add 950 μL 4.2N NaOH, 20 μL n-octanol, and sonicate for 10 min; fill with N2 (30 s / tube), and tighten the cap after filling to prevent N2 leakage; keep warm at 105°C for 20 h, cool (observe whether the pages of all test tubes are flush), mix at a low vortex gear (3rd gear) for 1 min; centrifuge at the highest speed of 21,000×g for 1 min; dilute 20 times with HPLC water; filter with a 0.22 μm needle filter and determine the amino acid content by HPLC, transfer to a 1.5 mL centrifuge tube; store at -20°C.

[0098] 3.1.9 Data Analysis

[0099] IBM SPSS Statistics software (IBM, USA) was used for data analysis. The statistical method for diarrhea rate data in this trial was the chi-square test. ANOVA model was used for one-way or two-way analysis of variance, and Duncan multiple comparisons were used. P < 0.05 indicated a significant difference.

[0100] 3.2 Results

[0101] 3.2.1 Effects of porcine mucosal lactobacillus and tryptophan on growth performance of suckling piglets

[0102] As shown in Table 3, LM and LM+Trp treatments increased the body weight (P<0.05) and ADG (P<0.05) of suckling piglets in the first two weeks, and the weaning weight of suckling piglets in the LM and LM+Trp groups was significantly higher than that in the Con group (P<0.05). In the two-way ANOVA, LM could alleviate the diarrhea rate of suckling piglets (P<0.05).

[0103] Table 3 Effects of LM and Trp on growth performance of suckling piglets

[0104]

[0105]

[0106] Note: ac, data in the same row with different letters on the shoulders indicate significant differences (P<0.05).

[0107] 3.2.2 Effects of porcine mucosal lactobacillus and tryptophan on routine milk routine of sows at 7 and 14 days

[0108] As shown in Table 4, the combined treatment of LM and Trp increased the milk fat content of sows on the 14th day (P<0.05), and decreased the milk somatic cell count of sows on the 7th day (P<0.05).

[0109] Table 4 Effects of porcine mucosal lactobacillus and tryptophan on routine milk of sows on days 7 and 14

[0110]

[0111]

[0112] Note: SCC Somatic cell count; UN Urea nitrogen.

[0113] 4.2.3 Effects of porcine Lactobacillus mucosa and tryptophan on amino acid levels in sow milk on day 14

[0114] As shown in Tables 5 and 6, LM and Trp treatments alone increased the level of free serine in sow milk, while LM treatment increased the level of free histidine in sow milk (P<0.05). LM and Trp treatments had no significant effect on the level of total amino acids in sow milk (P<0.05). The total level of free amino acids in sow colostrum was significantly lower than that in normal milk (P<0.05), while the total amino acids in colostrum were significantly higher than those in normal milk (P<0.05).

[0115] Table 5 Effects of porcine mucosal lactobacillus and tryptophan on free amino acids in sow milk

[0116]

[0117] Note: EAA Essential amino acid; NAA Nonessential amino acid.

[0118] Table 6 Effects of porcine mucosal Lactobacillus and tryptophan on total amino acid concentration in sow milk

[0119]

[0120] Note: EAA Essential amino acid; NAA Nonessential amino acid.

[0121] 3.2.4 Effects of porcine mucosal lactobacillus and tryptophan on ileum morphology in suckling piglets

[0122] As shown in Table 7, LM and Trp alone or in combination increased the villus height (P<0.05) and V / C (P<0.05) of pre-suckling piglets, but had no significant effect on the crypt depth (P>0.05).

[0123] Table 7 Effects of porcine mucosal lactobacillus and tryptophan on ileum morphology of suckling piglets

[0124]

[0125] Note: Data are expressed as mean, n=6, a，b The differences in shoulder letters indicate significant differences (P<0.05).

[0126] 3.2.5 Effects of porcine mucosal lactobacillus and tryptophan on serum antioxidant indices in suckling piglets

[0127] like Figure 1 It can be seen that LM and Trp alone or in combination can significantly reduce the serum MDA level of suckling piglets (P<0.05) and significantly increase the SOD level (P<0.05).

[0128] 3.2.6 Effects of porcine mucosal lactobacillus and tryptophan on intestinal proliferation and differentiation factors in different intestinal segments of suckling piglets

[0129] like Figure 2 It can be seen that LM as a main effect can significantly upregulate the expression of LGR5 in the stomach and colon (P<0.05). For TGFβ isoforms, LM can upregulate the expression of TGFβ2 (P<0.05) and TGFβ3 (P<0.05) in the stomach, and TGFβ1 (P<0.05) in the ileum.

[0130] 3.2.7 Effects of porcine mucosal Lactobacillus and tryptophan on key enzymes of 5-HT metabolism, vector expression, and HTR expression in different intestinal segments of suckling piglets

[0131] like Figure 3 It can be seen that LM and Trp alone and in combination can significantly down-regulate the expression of 5-HT synthase TPH1 in the ileum (P<0.05). LM as a main effect can significantly up-regulate the expression of 5-HT reuptake transporter SERT (P<0.05) and 5-HT degradation enzyme MAOA (P<0.05) in the stomach. Trp as a main effect can significantly up-regulate the expression of SERT in the three intestinal segments of jejunum, ileum and colon (P<0.05).

[0132] As shown in Table 8, LM as a main effect significantly promoted the expression of HTR2A, HTR2B and HTR4 in the stomach (P<0.05). Trp as a main effect can downregulate the expression of HTR2B in the jejunum (P<0.05) and upregulate the expression of HTR4 in the jejunum (P<0.05). LM and Trp alone or in combination can upregulate the expression of HTR4 in the ileum and colon (P<0.05), and the addition of LM and Trp can inhibit the expression of HTR7 in the colon (P<0.05).

[0133] Table 8 Effects of porcine mucosal lactobacillus and tryptophan on the expression of 5-HT receptor subtypes in different intestinal segments of piglets

[0134]

[0135]

[0136] Note: Data are expressed as mean, n=6, a-c The differences in shoulder letters indicate significant differences (P<0.05).

[0137] 3.2.8 Effects of porcine mucosal Lactobacillus and tryptophan on tryptophan metabolites and amino acid concentrations in different parts of lactating piglets

[0138] The results of the detection of tryptophan metabolites in plasma, gastrointestinal tissues and intestinal cavity showed that (Table 9) compared with other groups, the 5-hydroxyindoleacetic acid and 5-HT in the gastric tissue of piglets in the LM+Trp treatment group were higher than those in other groups (P<0.05), and LM alone could also increase the level of 5-HT in gastric tissue (P<0.05). LM and Trp alone or in combination reduced the level of 3-indolepropionic acid in ileal contents (P<0.05), while LM alone reduced the level of 3-methylindole in this part (P<0.05). The concentrations of indole and 3-methylindole in the colon contents of piglets in the LM+Trp group were significantly higher than those in other groups (P<0.05).

[0139] Table 9 Effects of porcine mucosal lactobacillus and tryptophan on the concentrations of tryptophan metabolites in different parts of piglets

[0140]

[0141]

[0142] Table 2-12

[0143]

[0144] Note: Data are expressed as mean, n=6, a，b The differences in shoulder letters indicate significant differences (P<0.05).

[0145] As shown in Table 10, compared with other groups, LM treatment can significantly increase the concentration of plasma methionine (P<0.05). Trp treatment can increase the concentration of gastric tryptophan (P<0.05), and LM can increase the concentration of gastric and colonic valine (P<0.05). In colonic tissue, the concentrations of phenylalanine, isoleucine and leucine were increased by LM treatment (P<0.05), while lysine was significantly decreased by Trp treatment (P<0.05).

[0146] Table 10 Effects of porcine mucosal lactobacillus and tryptophan on free amino acid concentrations in piglet plasma

[0147]

[0148]

[0149] Note: Data are expressed as mean, n=6, different letters in a and b indicate significant difference (v<005).

[0150] 3.3 Results Analysis

[0151] The test results show that:

[0152] 1) Supplementation of LM and Trp can promote the weight, average daily weight gain and weaning weight of 14-day-old lactating piglets. LM, as a main effect, has an alleviating effect on the diarrhea rate during the lactation period. In addition, the combined use of LM and Trp can increase the milk protein and milk fat content in sow milk and has a potential regulatory effect on mammary inflammation, which is manifested in a lower somatic cell count.

[0153] 2) LM and Trp added alone or in combination can promote the development of ileum structure and the secretion of gastric mucus and gastric acid, promote the expression of LGR5, a gene for proliferation of gastric and colonic intestinal stem cells, and differentially regulate the expression of TGFβ family subtypes in different intestinal segments;

[0154] 3) LM and Trp treatment modulate intestinal 5-HT metabolism and differentially regulate the expression of HTR isoforms in the stomach, jejunum, ileum, and colon;

[0155] 4) The combined use of LM and Trp can increase indole levels by regulating tryptophan metabolism in the stomach, ileum, and colon.

[0156] 5) This experiment supplemented the dominant lactobacillus LM and Trp in the intestine of piglets to explore the effects of the two alone and in combination on the growth performance and intestinal development of offspring piglets. Similar to the above results, supplementation with LM and Trp promoted the body weight, average daily weight gain and weaning weight of 14-day-old suckling piglets, and LM as the main effect alleviated the diarrhea rate during the lactation period. In addition, Trp and LM alone and in combination increased the villus height and villus height crypt depth ratio of suckling piglets. These two apparent indicators indicate that Trp and LM treatment may promote the overall health of piglets by promoting the absorption of nutrients.

[0157] 6)Trp, LM and LM+Trp treatments can all downregulate the MDA level in the serum of piglets and upregulate the SOD level. Therefore, the addition of Trp to sows during lactation, oral and spraying treatment of mother and child LM, and the combination of the above methods can enhance the antioxidant capacity of the offspring.

[0158] 7) The prior art reported that immunohistochemical co-localization found that TGF-β3 in human gastric tissue can be localized in parietal cells, chief cells and mucous cells, and this result is consistent with the upregulation of ATP4A, a marker of gastric acid-secreting parietal cells in gastric tissue in this experiment; the research results of the present invention prove that there are spatial differences in the distribution of TGFβ in different parts of the gastrointestinal tract;

[0159] In summary, supplementation with LM and Trp can improve the growth performance of lactating piglets and promote the development of gastric function and ileal structure. In addition, LM and Trp have spatial differences in the regulation of development-related genes and 5-HT metabolic homeostasis in different intestinal segments, among which HTR4 is a key regulatory target of LM. LM can promote the expression of Trp indole metabolic pathway and upregulate the levels of indole and its derivatives in the ileum and colon. The conventional nutrients in sow milk are affected by the composition of the diet, and Trp as a main effect can promote the increase of milk fat and milk protein concentration in normal milk.

[0160] Part IV: Regulation of Lactobacillus mucosa and tryptophan on the gastrointestinal microflora and tissue gene expression in piglets

[0161] 4.1 Test methods

[0162] 4.1.1 RNA extraction, library construction and DNBSEQ-T7 sequencing

[0163] RNA extraction: Total RNA was extracted from gastric, jejunal, ileal and colonic tissue samples, with 6 replicates per group. The concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was determined by Agilent2100. The total RNA amount required for single library construction was ≥1μg, the concentration was ≥35ng / μL, OD260 / 280 ≥1.8, and OD260 / 230 ≥1.0.

[0164] Transcriptome library construction and DNBSEQ-T7 sequencing: After the RNA sample was qualified, 1 μg of total RNA was used for library construction. The sequencing of this study was entrusted to Shanghai Meiji Biomedical Technology Co., Ltd. The specific method is to use magnetic beads with Oligo (dT) to perform AT base pairing with ployA, separate mRNA from total RNA, add fragmentation buffer, and randomly break mRNA into small fragments of about 300 bp; under the action of reverse transcriptase, use random primers to reversely synthesize one-strand cDNA with mRNA as a template, and then perform two-strand synthesis to form a stable double-stranded structure; the double-stranded cDNA structure is a sticky end, and End Repair Mix is ​​added to make it a blunt end, and then an A base is added to the 3' end to facilitate the addition of the adapter sequence later; the product after connecting the adapter is purified and fragmented, and the sorted product is amplified by PCR, and the final library is purified. Each library was quantified using Qubit 4.0 (Invitrogen, Carlsbad, CA, USA), and the insert size was confirmed using Agilent 2100. Finally, the DNA sequencing was performed on the DNBSEQ-T7 platform.

[0165] 4.1.2 Bioinformatics analysis

[0166] Seqprep software was used to filter and quality control the raw data of double-end sequencing to obtain high-quality sequencing data for subsequent analysis. The specific parameters were as follows: 1) remove the adapter sequence in the sequence; 2) use Sickle software to remove bases with a 5' end quality <20 and a 3' end quality <3; 3) remove ambiguous base sequences; 4) remove the adapter and sequences with a length of less than 30 bp after quality trimming.

[0167] The high-quality sequences were aligned to the Sus_scrofa genome for positioning analysis using Hisat2 software (Version 2.1.0). The saturation and coverage of reads aligned to the genome were analyzed using RSeQC software (Version 2.3.6). RSEM software was used to calculate the FPKM value (Fragments per kilobases per million reads) of each gene in the sample, and this value was used as the expression of the gene / transcript in the sample and the differentially expressed genes (DEGs) between the two groups were calculated. The screening criteria for DEGs were corrected P value < 0.05 and the fold change (FC) of the differentially expressed genes ≥ 2.

[0168] KOBAS software (Version 2.1.1) was used to perform KEGG functional annotation analysis on DEGs, and R script was used to perform KEGG metabolic pathway enrichment analysis on DEGs. When the corrected P value was ≤ 0.05, the KEGG function or KEGG metabolic pathway was considered to be significantly enriched.

[0169] 4.1.3 qPCR verification of differentially expressed genes in the transcriptome

[0170] In order to verify the reliability of transcriptome sequencing to identify differentially expressed genes, qPCR was performed on gastric tissues of different treatment groups.

[0171] 4.1.4 Extraction of total DNA from different intestinal segments

[0172] The stomach, ileum and colon chymes of 14-day-old suckling piglets slaughtered in the feeding experiment were selected and mixed evenly. 180-220 mg of chyme was used to extract total genomic DNA from chyme samples using QIAamp Fast DNA Stool Mini Kit (Qiagen, Tübingen, Germany), and the concentration and quality were detected using Nanophotometer ultra-micro spectrophotometer.

[0173] 4.1.5 Bacterial 16S rRNA gene sequencing analysis

[0174] Universal primers were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. The primer sequences for the target fragment were as follows: forward primer 338F: 5'-ACTCCTACGGGAGGCAGCAG-3', reverse primer 806R: 5'-GGACTACHVGGGTWTCTAAT-3'. Each sample was repeated three times, and the PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were excised and recovered using the AxyPrep DNA Gel Recovery Kit (AXYGEN), eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Referring to the preliminary quantitative results of electrophoresis, the PCR products were quantified by QuantiFluor TM -ST blue fluorescence quantitative system (Promega) was used for detection and quantification, and then the samples were mixed in corresponding proportions according to the sequencing requirements of each sample. Then, 2×250bp double-end sequencing was performed on the Illumina MiSeq PE300 sequencing platform (Illumina, San Diego, USA) after library construction.

[0175] After the PE reads obtained by Illumina sequencing are split into samples, the paired-end reads are first quality controlled and filtered according to the sequencing quality, and then spliced ​​according to the overlap relationship between the paired-end reads to obtain the optimized data after quality control splicing. Then, the sequence denoising method (DADA2 / Deblur, etc.) is used to process the optimized data to obtain ASV (Amplicon Sequence Variant) representing the sequence and abundance information.

[0176] Mothur (Version 1.30) software was used for Ace, Sobs, Chao and Shannon alpha diversity analysis, and R language (Version 3.3.1) was used for principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) to explore the differences in microbial community structure between samples and the contribution of different classifications to samples. R language (Version 3.3.1) was used for LEfSe (Linear discriminant analysis effect size) analysis to analyze the significant differences between groups.

[0177] 4.1.6 Spearman correlation heat map of host 5-HT homeostasis-related transcriptome and differential bacterial genera

[0178] The Spearman correlation coefficient was used to calculate the correlation between the top 50 most abundant differential bacterial genera in each intestinal segment and the expression levels of 5-HT homeostasis-related genes. Spearman coefficient |r|>0.8, P<0.05 indicated a significant correlation.

[0179] 4.1.7 Data Analysis

[0180] The PCR validation results were analyzed using IBM SPSS Statistics software (IBM, USA). ANOVA model was used for one-way or two-way analysis of variance, and Duncan multiple comparisons were used. P < 0.05 indicated significant differences. Transcriptome and 16S rRNA gene sequencing analysis was completed by Majorbio (https: / / cloud.majorbio.com / ), an online analysis website of Meiji Bioinformatics Cloud.

[0181] 4.2 Results

[0182] 4.2.1 Sample transcriptome sequencing data quality assessment

[0183] After the samples were sequenced, the raw data was obtained. After filtering, each group obtained no less than 4.00×107 Reads, the base amount of each sample was greater than 6G, the Q20 was no less than 96.96%, and the Q30 was no less than 91.05%. The results show that the sequencing quality of the samples is high and suitable for further analysis. The transcriptome sequencing data of each sample is shown in Appendix VIII.

[0184] 4.2.2 Comparison and analysis results of sample sequencing data and reference genome data

[0185] The comparison results showed that the proportion of the total number of reads that could be aligned to the reference sequence for all samples was between 93.9% and 95.8%, the proportion of reads aligned to multiple positions in the reference sequence was less than 3.83%, and the proportion of reads aligned to a unique position in the reference sequence was greater than 90.91%.

[0186] 4.2.3 Screening of differentially expressed genes

[0187] As shown in Table 11, in the stomach, compared with the Con group, a total of 171 differential genes were found in the Trp group, including 79 up-regulated genes and 92 down-regulated genes; a total of 236 differential genes were found in the LM group, including 167 up-regulated genes and 69 down-regulated genes. In the ileum, compared with the Con group, a total of 2459 differential genes were found in the Trp group, including 1470 up-regulated genes and 989 down-regulated genes; a total of 1380 differential genes were found in the LM group, including 1030 up-regulated genes and 350 down-regulated genes. In the colon, compared with the Con group, a total of 593 differential genes were found in the Trp group, including 175 up-regulated genes and 418 down-regulated genes; a total of 524 differential genes were found in the LM group, including 156 up-regulated genes and 368 down-regulated genes.

[0188] Table 11 Statistics of the number of up- and down-regulated genes in different intestinal segments

[0189]

[0190]

[0191] Note: D14, day 14; CT, Con; TP, Trp; LM, Limosilactobacillus mucosae; TL, LM+Trp; ST, Stomach; IL, Ileum;

[0192] 4.2.4 Biological function analysis of differentially expressed genes among different treatment groups

[0193] Since the annotation information of the pig reference genome is still incomplete, the biological function analysis of differentially expressed genes can only be performed within the limited annotation information ( Figure 4 ). Through KEGG pathway analysis, the differentially expressed genes in gastric tissue were enriched in four categories: biological systems, cellular processes, environmental information processing, and metabolism; the differentially expressed genes in ileum were enriched in four categories: biological systems, cellular processes, environmental information processing, and genetic information processing; and the differentially expressed genes in colon were enriched in five categories: biological systems, cellular processes, environmental information processing, genetic information processing, and metabolism.

[0194] 4.2.5 KEGG functional enrichment analysis of intestinal transcriptome

[0195] like Figure 5As shown in the figure, the Kyoto encyclopedia of genes and genomes (KEGG) database analyzed the functional pathways involved in the differentially expressed genes. The results showed that compared with the gastric tissue of the Con group, the significantly differentially expressed genes upregulated by LM treatment were enriched in two biological system pathways, including pancreatic juice secretion and renin secretion. Compared with the colon tissue of the Con group, the significantly differentially expressed genes downregulated by Trp were enriched in two environmental information processing pathways and one disease-related pathway, including the TNF signaling pathway, the JAK-STAT signaling pathway, and the inflammatory bowel disease signaling pathway. Compared with the ileum tissue of the Con group, the significantly differentially expressed genes upregulated by Trp were enriched in 5 biological system pathways, 2 metabolism-related pathways, 3 environmental information processing pathways, and 1 cellular process pathway, including intestinal immune network and IgA production, hematopoietic cell lineage, B cell receptor signaling pathway, Fc gamma R-mediated phagocytosis, phospholipase D signaling pathway, phagosome, calcium signaling pathway, P13K-Akt signaling pathway, mucus-type O-glucan biosynthesis, retinol metabolism, and synaptic vesicle cycle. Compared with the ileum tissue of the Con group, the significantly differentially expressed genes downregulated by Trp were enriched in 3 metabolism-related pathways and 2 cellular process pathways, including lysosome, sphingolipid metabolism, other sugar degradation, glycosaminoglycan degradation, and ferroptosis. Comprehensive analysis found that the number of pathways significantly altered by LM or Trp in the ileum was significantly higher than that in other intestinal segments.

[0196] 4.2.6 Common differentially regulated genes by age and treatment

[0197] Combined with the transcriptome data of 2-day-old piglets, the up- and down-regulation of common differentially expressed genes in each intestinal segment caused by different treatments at 14 days of age were analyzed ( Figure 6 ). Compared with the gene expression in the stomach of 2-day-old newborn piglets, RBP4 and CLCA1 were significantly downregulated, while DUOX2 and LDHB were significantly upregulated in the stomach of 14-day-old suckling piglets. In the piglets of different treatment groups at 14 days old, CLCA1 expression was significantly upregulated in the LM group, while RBP4 expression was significantly upregulated in the LM+Trp group, while DUOX2 and LDHB were significantly downregulated.

[0198] In the ileum, ADIPOQ was significantly upregulated in 14-day-old suckling piglets compared with the gene expression in 2-day-old newborn piglets, and ADIPOQ was also significantly upregulated in LM piglets compared with the 14-day-old Con group ( Figure 7 ).

[0199] Compared with the colon gene expression of 2-day-old newborn piglets, NOD2, CCL8, CCR1 and CCL4 were significantly upregulated in 14-day-old suckling piglets. Compared with the 14-day-old Con group, the above four genes in the Trp group were significantly downregulated ( Figure 8 ).

[0200] As Fig. 9 shown, compared with the gene expression in the stomach of piglets in the control group, LM treatment significantly up-regulated the expression of SLC28A3 in the stomach, while Trp had no significant effect on the expression of SLC28A3. Compared with the gene expression in the ileum of piglets in the control group, Trp treatment significantly up-regulated the expression of BGN in the ileum, while LM had no significant effect on the expression of BGN.

[0201] 4.2.7 qPCR verification of differentially expressed genes in the stomach function by transcriptome sequencing

[0202] As Fig.10 shown, treatment with LM and Trp alone or in combination could significantly increase the expression of GAST and ATPase H+ / K+ transporting subunit alpha (ATP4A) in the stomach (P<0.05). Trp tended to inhibit the expression of PCSK1 (0.05<P<0.1), while LM tended to promote the expression of PCSK1 (0.05<P<0.1).

[0203] 4.2.8 Analysis of sample sequencing volume and dilution curve

[0204] Before analyzing the intestinal microbial diversity, we analyzed the sequencing volume and depth of the samples measured in this experiment to determine whether the sequencing results of this experiment were reasonable. It can be seen from the rank abundance curves of each sample in each intestinal segment that the sequencing sample volume in this experiment was sufficient to perform data analysis and predict species richness ( Fig.11 ).

[0205] 4.2.9 Effects of Lactobacillus mucosae and tryptophan on the changes of α-diversity and β-diversity of the flora in the contents of different intestinal segments

[0206] As shown in Table 12, the results of the Alpha diversity analysis of the flora in the stomach, ileum and colon contents showed that LM treatment could up-regulate the Alpha diversity of the flora in the stomach contents of piglets, manifested as an increase in the Ace index and Chao index at the genus level. There were no significant changes in the Alpha diversity of the ileum and colon flora. By comparing the microbial structure similarities of individual samples in different treatment groups in different intestinal segments through PCoA images, it was found that compared with the Con group, the samples in the LM and Trp treatment groups alone or in combination were more concentrated, indicating that the microbial community structures between samples were more similar, and the distances between samples in different treatment groups were not far, indicating that the microbial community structures between samples were highly similar. Further Anosim similarity analysis showed that the stress values were <0.2 and the P values were <0.05 among the treatment groups in different intestinal segments, indicating that the treatment groups with LM and Trp alone or in combination had more similar species compositions ( Fig.12 ).

[0207] Table 12 Comparison of diversity and richness of microbiota in different intestinal segments

[0208]

[0209]

[0210] Note: Data are expressed as mean, n = 6. ac, data in the same row with different letters in the shoulder indicate significant difference (P < 005), the same as in the following table.

[0211] 4.2.10 Effects of porcine mucosal lactobacilli and tryptophan on the flora structure of different intestinal segments

[0212] According to the analysis of species composition, at the phylum level, the gastric and ileal contents were dominated by Firmicutes, Proteobacteria and Bacteroidetes, while the colonic contents were dominated by Bacteroidetes, Firmicutes and Fusobacterium. At the genus level, the most abundant species in the gastric contents were Lactobacillus, Bacillus and Actinobacillus; the most abundant species in the ileal contents were Romboutsia, Actinobacillus and Lactobacillus; the most abundant species in the colonic contents were Prevotella, norank_f_norank_o_Clostridia_vadinBB60_group and Alloprevotella. For different treatments, Trp could upregulate the abundance of Lactobacillus in gastric contents, and LM could upregulate the abundance of Roseburia in ileum ( Fig.13 , Fig.14 ).

[0213] 4.2.11 Effects of porcine mucosal lactobacilli and tryptophan on different bacterial genera in different intestinal segments

[0214] In gastric contents, the LM and LM+Trp groups had higher proportions of Lactobacillus agilis and Muribaculaceae. The LM group had higher proportions of Lachnospiraceae XPB1014, Blautia, Coprococcus, Eubacterium brachy, Eubacterium hallii, and Muribaculaceae. In ileal contents, the Trp and LM+Trp groups had higher proportions of Veillonella, and the LM group had higher proportions of Turicibacter. In colonic contents, LM and Trp treatments upregulated Bacillus, and the LM+Trp group had higher proportions of Prevotella ( Fig.15 ).

[0215] 4.2.12 Effects of porcine mucosal lactobacilli and tryptophan on differences in microbial communities in different intestinal segments

[0216] The marker strains in different treatment groups were screened by LEfSe multi-level species analysis, and then linear discriminant analysis (LDA) was used to discriminate the differences between different treatment groups ( Fig.16 , Fig.17). The results showed that 44 bacterial genera were detected in the gastric contents, which could be used as potential microbial markers to distinguish the differences in gastric contents among the Trp group, the LM group, and the LM+Trp group. Among them, 2 genera were relatively abundant in the Con group, 1 genera was relatively abundant in the Trp group, 21 genera were relatively abundant in the LM group, and 20 genera were relatively abundant in the LM+Trp group. The enrichment ratio of g_Foumierella in the Trp group was significantly higher than that in other groups, while the enrichment ratios of f_Fibrobacteraceae, f_Ruminococcaceae, and g_Lachnospiraceae_XPB1014 in the LM group were significantly higher than those in other groups. Prevotellaceae_UCG-003, o_Burkholderiales, and Bacilli.o_RF39 were significantly enriched in the LM+Trp group. Comparison of ileal content microorganisms in different treatment groups revealed that o_Campylobacterales, f_Clostridiaceae, and f_Veillonellaceae were significantly enriched in the Con group, c_Negativicutes and g_Peptoniphilus were significantly enriched in the Trp group, c_Clostridia, g_Turicibacter, and o_Erysipelotrichales were more abundant in the LM group, and c_Gammaproteobacteria were more abundant in the LM+Trp group. Comparison of colonic content microorganisms in different treatment groups revealed that 16 genera were detected as potential microbial markers for distinguishing differences in colonic content between the Trp group, LM group, and LM+Trp group, of which 3 genera were relatively abundant in the Con group, 5 genera were relatively abundant in the Trp group, 5 genera were relatively abundant in the LM group, and 3 genera were relatively abundant in the LM+Trp group. The enrichment ratio of o_Izemoplasmatales in the Con group was significantly higher than that in other groups, the enrichment ratio of g_Anaerovibrio in the Trp group was significantly higher than that in other groups, the enrichment ratio of o_Campylobacterales, f_Peptostreptococcaceae, and p_Campilobacterota in the LM group was significantly higher than that in other groups, and the enrichment ratio of g_Odoribacter in the LM+Trp group was significantly higher than that in other groups.

[0217] 4.2.13 Spearman correlation heat map of 5-HT homeostasis-related genes and differential bacterial genera in different intestinal segments

[0218] The heat map results show that ( Fig.18 , Fig.19 , Fig. 20) Among the differential bacterial genera in the top 50 of the contents of different intestinal segments, Lactobacillus was significantly negatively correlated with HTR3A in the ileum (0.001 < P ≤ 0.01), while it was positively correlated with TPH1 and HTR2A in the colon (0.01 < P ≤ 0.05) and negatively correlated with HTR7 (0.01 < P ≤ 0.05). For the differential bacterial genera common to different intestinal segments, such as Actinobacillus, it was significantly positively correlated with the expression of HTR1A in the stomach (0.001 < P ≤ 0.01), while it was negatively correlated with HTR3A and HTR7 in the colon (0.01 < P ≤ 0.05). Similarly, Fusobacterium was significantly positively correlated with the expression of HTR4 in the stomach (0.001 < P ≤ 0.01), while it was negatively correlated with HTR4 in the colon (0.01 < P ≤ 0.05).

[0219] 4.3 Results and Discussion

[0220] 1) In this experiment, the intake of LM and Trp could change the intestinal flora structure and diversity of nursing piglets. Transcriptome sequencing data showed that the regulation of the intestine by LM and Trp was spatially specific, which was related to the specific functions of intestinal segments.

[0221] 2) In this experiment, Trp treatment down-regulated the TNF signaling pathway and JAK-STAT signaling pathway in the colon, and at the same time had an inhibitory effect on the inflammatory bowel disease pathway. This suggests that the occurrence of inflammation in the hindgut segment may be closely related to the regulation of colonic immune function by Trp. The number of pathways significantly changed by diet regulation in the ileum was significantly higher than that in other intestinal segments. Trp can promote the development of immune network pathways, such as the intestinal IgA production immune network and B cell receptor signaling pathway, and down-regulate the glycosaminoglycan degradation and ferroptosis pathways.

[0222] 3) LM and Trp have a promoting effect on the main functions of the stomach;

[0223] The main function of the stomach is to secrete gastric acid to digest food and resist foodborne microorganisms.

[0224] Oxygen secretory glands are functional glands of the stomach, in which endocrine cells and parietal cells are two types of cells that secrete mucus and gastric acid, respectively, and their specific genes are GAST and ATP4A, respectively. CLCA1 can be highly expressed in pig stomach parietal cells. In addition, CLCA1 is one of the main components of gastrointestinal mucus and is regarded as a mucin marker. Serum retinol binding protein 4 (RBP4) is a retinol carrier in the circulatory system, and the expression of RBP4 in the stomach is considered to be one of the important derivatives of ghrelin synthesis. With the increase of age, the expression of RBP4 in the stomach of piglets decreased, while RBP4 expression was upregulated under the combined treatment of LM and Trp. The possible reason is that in order to adapt to the law of piglet development, newborn piglets have a high demand for colostrum intake, and after gradually consuming solid feed, the demand for food is suppressed under the changes in the internal and external environment. The above transcriptome results are consistent with the qPCR verification results. The expression of PCSK1, a marker of appetite-related enzyme ghrelin, was upregulated in piglets in the LM+Trp group. LM can upregulate the abundance of endocrine cells and parietal cells and promote normal gastric function.

[0225] Trp supplementation alone suppressed the appetite of suckling piglets, which is consistent with previous studies. RBP4 is closely related to insulin sensitivity in the body, which may be because ghrelin cell-derived RBP4 in gastric tissue may not affect systemic insulin resistance or thyroid hormone transport.

[0226] DOX2 and LDHB increased with age and were downregulated in the LM+Trp group, indicating that the combined treatment of LM and Trp was beneficial to the positive regulation of gastric genes, thereby reducing the risk of gastric diseases.

[0227] Compared with newborn piglets, ADIPOQ expression was significantly upregulated in suckling piglets, and the LM+Trp group further upregulated the expression of this gene, indicating that the combination of LM and Trp may have a potential effect on fat synthesis and metabolism.

[0228] 4) In this experiment, compared with other gastrointestinal segments, the pathways in the colon that underwent more differential changes during dietary regulation were immune-related pathways. CCL4, CCL8, CCR1, and NOD2 are all immune-related proinflammatory factors, and Trp intake inhibited the trend of increasing inflammatory factors with age. Comprehensive consideration of the differential gene expression caused by age differences is of great reference significance for exploring the changes brought about by LM and Trp to piglets.

[0229] 5) Comparison of the common effects of LM or Trp alone and in combination on the transcriptional level of the digestive tract showed that LM may have a regulatory effect on nucleotide absorption and metabolism in the stomach; the probiotic effects of LM and Trp on gastrointestinal microorganisms can be determined by the intestinal microbial marker flora after ingestion. On the one hand, LM can promote the enrichment of flora related to the digestion and decomposition of polysaccharides, carbohydrates, lipids and other substances in the stomach, while in the ileum and colon, strains with the main functions of fatty acid metabolism and immune regulation are enriched. On the other hand, Trp can promote the enrichment of flora related to lactose metabolism and lipid decomposition in the stomach, and promote the balance of microbial communities.

Claims

1. A strain of Lactobacillus mucosa, characterized in that The mucosal lactobacillus is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number: CGMCC NO.30899, the deposit date: June 7, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. Use of the mucosal lactobacillus as claimed in claim 1 as a sow feed additive or a piglet feed additive.

3. The use according to claim 2, characterized in that: The sow feed additive refers to an additive added to the feed of pregnant sows; the piglet feed additive refers to an additive added to the feed of lactating piglets.

4. The use according to claim 2, characterized in that: The mucosal lactobacillus refers to live bacteria or inactivated bacteria.

5. A composition, characterized in that Contains the mucosal lactobacillus according to claim 1 and tryptophan.

6. The composition according to claim 5, characterized in that The amount of tryptophan added to the feed is 0.12wt%.

7. The composition according to claim 5, characterized in that The mucosal lactobacillus was fed at a rate of 1×10 10 The amount of CFU is added to the feed.

8. The composition according to claim 7, characterized in that The mucosal lactobacillus is also fed to piglets in the form of an oral preparation or sprayed into the living environment of piglets in the form of a spray.

9. The composition according to claim 5, characterized in that The membrane lactobacillus is a live bacterium or an inactivated bacterium.

10. Use of the composition according to any one of claims 5 to 9 as a sow feed additive or a piglet feed additive.

Images