CN118028155B

CN118028155B - Novel probiotics, fermented coconut water metaplasia and application thereof in poultry feed additive

Info

Publication number: CN118028155B
Application number: CN202410122547.2A
Authority: CN
Inventors: 刘四新; 康佳木; 蔡坤; 张家超; 李从发; 郑磊杰; 庞情; 袁亚倩
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2024-01-30
Filing date: 2024-01-30
Publication date: 2024-09-06
Anticipated expiration: 2044-01-30
Also published as: CN118028155A

Abstract

The invention provides a novel probiotic, fermented coconut water metazoan and application thereof in a feed additive for poultry, wherein the probiotic lactobacillus is Lactiplantibacillus plantarum MS c, and the preservation number is GDMCC No:64184 the probiotic yeast is Saccharomyces CEREVISIAE Y301, accession number GDMCC No:64183 and then lactobacillus MS2c and/or saccharomycete Y301 are used as strains to ferment coconut water to prepare the feed additive which is beneficial to self-healing recovery of enteritis of poultry, especially Wenchang chickens. The invention better utilizes the resource characteristics and component characteristics of the coconut water which is a byproduct of the coconut processing industry, realizes low-cost high-density fermentation culture of excellent probiotics, reduces waste and protects the environment according to local conditions, and achieves the purpose of green culture.

Description

Novel probiotics, fermented coconut water metaplasia and application thereof in poultry feed additive

Technical Field

The invention belongs to the technical field of biological medicines, and relates to novel probiotics, fermented coconut water metazoan and application thereof in a feed additive for poultry.

Background

Wenchang chickens are indigenous characteristic chicken species in Hainan, are also excellent varieties in yellow feather chickens, and are required to be fattened due to unique 'white cutting chicken' making and eating modes, so that the grown-up chickens are required to be square and round in shape, tender in meat quality and long in fattening, the time for marketing is longer, generally more than 100 days, the risk of chicken infection of enteritis is increased compared with other chickens, and the environment-friendly breeding mode which is favorable for intestinal health and immunity is more urgent for using microbial preparations such as probiotics.

Coconut (Cocos nucifera l.) is one of the important commercial crops in the south of the sea, and because of its natural green, unique and delicious nature, coconut related products are continuously in hot sale and in short supply. The coconut processing products in the past mainly comprise coconut meat (solid endosperm of coconut), and the products comprise coconut juice, coconut slurry, coconut oil, coconut sugar, coconut chips and the like, and the coconut water is a byproduct of coconut shell breaking and meat taking, and belongs to the liquid endosperm part. In Hainan, enterprises for breaking shells of coconuts to obtain meat and supply the coconuts to downstream coconut meat processing are very common, and mainly used are raw coconuts imported from southeast Asia, and almost all of 20 hundred million coconuts consumed by annual processing adopt a traditional manual shell breaking mode, so that coconut water is in a state of scattered and inconvenient collection and inconvenient centralized storage when released from individual coconuts, and therefore, the coconut water after coconut meat is obtained in the coconut processing industry is often a byproduct which is not fresh enough, loses freshness and can not be drunk any more, and can only be processed and utilized industrially. Over ten years ago, this coconut water was used for the fermentative production of coconut (nata de coco, a bacterial cellulose) and the byproduct coconut water produced during this process was still to be utilized for high value processing due to the fact that the traditional technology in the real industry was dominant.

Disclosure of Invention

The invention aims to provide novel probiotics, fermented coconut water metazoan and application thereof in poultry feed additives, and based on high-value utilization of less fresh and less fresh coconut water, probiotics with excellent probiotic characteristics and suitable for intestinal health of poultry are screened based on a replacement strategy of poultry cultivation, and the probiotics are combined with local coconut resources and fermented to prepare the high-activity coconut water metazoan, so that the high-biomass coconut water fermented feed additives are prepared, and a novel approach is explored for green efficient cultivation of poultry, particularly Wenchang chickens.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The invention provides a novel probiotic lactic acid bacteria, which is named Lactiplantibacillus plantarum MS c and has the preservation number of GDMCC No:64184.

The invention also provides a novel probiotic microzyme, named Saccharomyces CEREVISIAE Y301, accession number GDMCC No:64183.

The invention also provides a prebiotic of the coconut water fermented by probiotics, which comprises a lactic acid bacteria fermentation coconut water metazoan taking lactic acid bacteria MS2c single bacteria as a fermentation strain, a yeast fermentation coconut water metazoan taking yeast Y301 single bacteria as a fermentation strain, and a compound bacterial strain fermentation coconut water metazoan taking lactic acid bacteria MS2c and yeast Y301 together as fermentation strains; the lactobacillus MS2c has a preservation number of GDMCC No:64184; the preservation number of the saccharomycete Y301 is GDMCC No:64183.

The invention also provides a preparation method of the prebiotics after the coconut water is fermented by probiotics, which comprises the following steps: simultaneously inoculating lactobacillus MS2c and saccharomycete Y301 according to the following lactobacillus: the yeast is in a ratio of 1:2, 4 percent of total inoculum size is removed from the compound strain to ferment coconut water, the fermentation temperature is 33-37 ℃, and the fermentation time is 24-30 hours; the lactobacillus MS2c has a preservation number of GDMCC No:64184; the preservation number of the saccharomycete Y301 is GDMCC No:64183.

The invention also provides a feed additive for poultry, which comprises the probiotic fermented coconut water metaplasia.

The invention also provides application of the prebiotics obtained by fermenting coconut water with the probiotics in the growth of poultry and the promotion of enteritis self-healing recovery.

Further, the lactobacillus fermented coconut water metagen can relieve the increase of IL-6, IL-10, TNF-alpha, IFN-gamma, D-lactic acid, uric acid, GPT and GOT contents caused by enteritis in serum, the lactobacillus fermented coconut water metagen can relieve the increase of TNF-alpha, IFN-gamma and D-lactic acid contents caused by enteritis in serum, and the compound strain fermented coconut water metagen can relieve the increase of IL-6, IL-10, IFN-gamma and D-lactic acid contents caused by enteritis in serum.

Further, the yeast fermented coconut water metaplasia and the lactic acid bacteria fermented coconut water metaplasia can improve intestinal villus structure and intestinal wall thickness and recover damaged intestinal mucosa.

Further, the yeast fermented coconut water metazoan is capable of modulating amino acid metabolism, reducing the abundance of potential pathogenic bacteria including alanine, aspartic acid, and glutamic acid, increasing the abundance of potential beneficial bacteria including achalamin, deoxynucleotide, lauroyl glycine, guanidinopropionic acid, and methyltryptophan, increasing the abundance of anti-inflammatory and anti-inflammatory compounds including ALISTIPES, the potential beneficial bacteria including chaetoceraceae Lachnospiraceae, and improving the growth rate of birds; the lactic acid bacteria fermented coconut water metazoan can regulate glycerophospholipid metabolic pathway, remodel intestinal flora structure, reduce abundance of potential pathogenic bacteria, increase abundance of potential beneficial bacteria, inhibit production of potential carcinogen epsilon-caprolactam and cause production of specific intestinal metabolites, wherein the potential pathogenic bacteria comprise ALISTIPES genus, and the potential beneficial bacteria comprise lactobacillus plantarum, krigine Teng Senshi family CHRISTENSENELLACEAE, LACHNOSPIRACEAE and bifidobacterium ragon; the compound strain fermented coconut water metazoan can enhance the growth performance of poultry, relieve intestinal wall damage and increase the abundance of specific intestinal metabolites, thereby relieving enteritis symptoms; the specific intestinal metabolites include glyceride CDP-DG, gentamicin C and polymyxin B2.

In the invention, the poultry is preferably Wenchang chicken.

The invention has the beneficial effects that:

The present invention discusses the possibility of using coconut water as a probiotic medium to produce a metaproduct. In addition, the invention also researches the effectiveness of the probiotic products in promoting recovery of the acute enteritis of Wenchang chickens. This study not only demonstrates the impact and mechanism of fermentation of probiotics in coconut water on the self-recovery of chickens suffering from acute enteritis, but also provides a valuable route to utilizing non-fresh, ineligible coconut water.

The invention firstly screens lactobacillus from the intestinal canal of the indigenous Wenchang chicken, screens saccharomycetes from natural fermentation coconut water in a processing plant, obtains a plurality of high-activity strains with excellent properties through a large number of probiotics and antibacterial activity tests, screens a strain of saccharomyces cerevisiae Y301 and a strain of lactobacillus plantarum MS2c which can grow rapidly in the coconut water and have excellent probiotics, respectively cultures lactobacillus and saccharomycetes by-product coconut water as culture matrixes, simultaneously respectively cultures the strains at high density through fermentation condition optimization, carries out fermentation culture of compound strains, obtains a metaplasia product of the metaplasia products of the high-activity thalli and the metabolites of the metaplasia products, namely the high-activity fermentation coconut water metaplasia products, and is used as a feed additive to irrigate the Wenchang chicken suffering from acute enteritis, and has obvious promotion effects on growth performance of the Wenchang chicken, biochemical and immune indexes and self-healing recovery of enteritis. The invention better utilizes the resource characteristics and component characteristics of the byproduct coconut water, realizes the high-density fermentation culture of excellent probiotics, prepares the feed additive which is favorable for the enteritis self-healing recovery of Wenchang chickens, realizes the local material-taking protection and resource utilization, and achieves the purpose of green cultivation. The invention is simple, convenient, efficient, ingenious and scientific, and is a new idea for carefully screening, developing and innovatively applying according to local conditions.

Drawings

FIG. 1 shows the survival rate of the strain of example 1 of the present invention in a pH2.5 and 0.3% bile salt environment for 3 hours, and the different cases indicate differences in tolerance to pH2.5 and 0.3% bile salt concentration (P < 0.05) between the different strains.

FIG. 2 shows the surface hydrophobicity and self-polymerizing ability of the strains of example 1 of the present invention, different cases indicating differences in group between different strains (P < 0.05).

FIG. 3 shows the antibacterial activity of the lactic acid bacteria against Salmonella bacteria of example 1 of the present invention, the letters being quite different representing a significant difference (p < 0.05).

FIG. 4 shows the bacteriostatic activity of the lactic acid bacteria against E.coli in example 1 of the present invention, the letters being quite different representing a significant difference (p < 0.05).

FIG. 5 shows that the size of the inhibition zone of the lactic acid bacteria against Staphylococcus aureus in example 1 of the present invention, the letters are completely different, representing the significant difference (p < 0.05).

FIG. 6 shows the acid-resistant survival rate of rescreened lactic acid bacteria of example 1 of the present invention, capital letters are the differences between groups, lowercase letters are the differences within groups, and complete differences in letters represent significant differences (p < 0.05).

FIG. 7 shows the bile salt-resistant viability of rescreened lactic acid bacteria of example 1 of the invention, capital letters being the difference between groups and lowercase letters being the difference within groups, the letters being completely different representing significant differences (p < 0.05).

FIG. 8 is a graph showing the effect of different amounts of each component added on the number of living MS2c bacteria in example 1 of the present invention, and the difference is remarkable (p < 0.05) as the letters are completely different.

FIG. 9 is a microscopic image of several yeasts of different morphologies and a single colony morphology according to example 2 of the present invention.

FIG. 10 shows the results of comparing the viable count of each candidate yeast in coconut water in example 2 of the present invention.

FIG. 11 shows acid resistant survival of the re-screened yeasts in example 2 of the present invention.

FIG. 12 shows the bile salt-tolerant survival of the rescreened yeasts of example 2 of the present invention.

FIG. 13 is a graph showing the relationship between the inoculation ratio and the viable count of the composite fermentation in example 3 of the present invention.

FIG. 14 is a graph showing the relationship between the inoculation sequence and the viable count of the composite fermentation in example 3 of the present invention.

FIG. 15 is a graph showing the relationship between the inoculum size and the viable count of the composite fermentation in example 3 of the invention.

FIG. 16 shows the number of viable bacteria of each of the single strain fermentation and the composite strain fermentation in example 3 of the present invention.

FIG. 17 is a schematic diagram showing the experimental design of the intervention of the prebiotics of the coconut water fermented by the probiotics on the acute enteritis chickens in the test example of the invention.

FIG. 18 shows the effect of probiotic fermented coconut water metazoan on the growth performance of acutely enteritis chickens in test cases of the invention, (a) average daily feed intake; (b) average daily body weight; (c) an average DAI disease activity index.

FIG. 19 shows the effect of fermented coconut water metazoan on serum immunobiochemical markers of acute enteritis chickens in a test example of the invention, (a) serum immunometric content profile; (b) serum biochemical index content map.

FIG. 20 is a graph showing the effect of prebiotic intervention on repair of acute enteritis chicken intestinal wall injury after fermentation of coconut water by probiotics in a test example of the invention; (left) morphological effect comparison of different magnifications of intestinal mucosa tissue; comparison of intestinal wall thickness (right).

FIG. 21 shows shannon index (a) and beta diversity (b) of chicken intestinal microbiome alpha diversity in test cases of the present invention.

FIG. 22 is a block diagram of the microbiome of the cecum content of chickens in a test case of the present invention.

FIG. 23 illustrates comparative analysis of the effect of post-fermentation coconut water metazoan intervention on the microbiota of the cecal content of each group of chickens, (C) CK1, CK2 and CK3; (D) CK1, CK2, and M2; (E) CK1, CK2 and Y3; (F) CK1, CK2, and MY5.

FIG. 24 is a principal component analysis plot between six groups and a partial least squares-discriminant analysis plot for comparison between each group and model groups, (a) a principal component analysis plot between six groups; (b) CK1 and CK2; (c) CK2 and CK3; (d) CK2 and M2; (e) CK2 and Y3; (f) CK2 and MY5.

FIG. 25 shows a topology of KEGG metabolic pathways, (left) M2 group, (right) Y3 group.

FIG. 26 shows a network analysis of the correlation between differential flora and differential metabolites in test cases of the invention, (a) shows a correlation analysis with the intervention of single plant MS2c fermented coconut water (group M2); (b) Related analysis under the intervention of single strain Y301 fermented coconut water (group Y3) is shown. (based on p <0.05 and correlation coefficient r > 0.4).

FIG. 27 illustrates the mechanisms of MS2c fermented coconut water intervention and Y301 fermented coconut water intervention to promote recovery from chicken colitis, (a) outlines the mechanism of correlation between differential flora, differential metabolites and biochemical immune indicators of MS2c intervention; (b) The mechanism of correlation between the differential flora, differential metabolites and biochemical immune index of Y301 intervention is outlined.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

The research thought of the invention mainly comprises the following aspects:

Firstly, based on acid and bile salt resistance, bacteriostasis activity and the like, probiotic lactobacillus and saccharomycetes are widely screened, and then further re-screening is carried out on the condition that the rapid propagation in coconut water reaches high biomass, so as to obtain probiotic strains capable of growing vigorously in the coconut water.

And secondly, researching the process conditions of single-strain fermentation and composite-strain fermentation of the probiotics based on the coconut water matrix, and optimizing the composite fermentation process of the probiotics in the coconut water to obtain a high-activity fermented coconut water metaplasia product with the advantages of both the probiotics microzyme and the probiotic lactic acid bacteria.

Thirdly, taking the feed additive as a feed additive for poultry, and feeding the Wenchang chicken enteritis model by three fermented coconut water metazoans, and researching the effect of the fermented coconut water metazoans on self-healing recovery and the mechanism of the fermented coconut water metazoans on the intestinal flora structure and composition, the intestinal metabolite difference, the physiological biochemistry and immune indexes and the correlation relationship between the intestinal flora structure and composition and the physiological biochemistry and immune indexes.

Example 1 screening and identification of Lactobacillus plantarum MS2c

The strain has been deposited at the collection of microorganism strains (GDMCC) of Guangdong province at 12/24 of 2023 under the deposit number GDMCCNo:64184, deposit address: building 5, road 100, university, building 59, university of Guangzhou City martyr, university of Guangdong, institute of microorganisms, taxonomic designation: lactobacillus plantarum (Lactiplantibacillusplantarum).

Experimental material

1.1 Sieving, culturing and fermenting Experimental Material

And (5) taking intestinal contents for lactobacillus separation after bloodletting and slaughtering of the local domestic adult Wenchang chickens.

Coconut water: the mature coconut in the coconut processing factory is immediately filtered by two filter cloths of more than 400 meshes and more than 1000 meshes after being broken and taken, and is quickly placed at a low temperature of 3-4 ℃ for refrigeration. For preparing culture medium.

Other experimental materials such as tryptone, agar powder, etc. are all conventional experimental materials unless otherwise specified.

1.2 Preparation of Medium and Sterilization

MRS medium: 54g of MRS dry powder is added into distilled water 1L, stirred and heated until the MRS dry powder is completely dissolved, and the pH is natural and about 6.4. Is used as a seed culture medium for lactobacillus screening, preservation and fermentation.

LB medium: 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride are added with 1L of distilled water, stirred and heated until the mixture is completely dissolved, and the pH is natural. The strain is used as a strain seed culture medium for bacteriostasis experiment indication.

Coconut water basal medium: coconut water, and a refractive sugar degree of about 5.0-5.4 degrees Bx, and adding a glucose solution to adjust the sugar degree to 6.0 degrees Bx, and using the mixture as a fermentation basic culture medium.

1.2 To 1.5 percent of agar powder is added when the solid culture medium is prepared. All media were sterilized at 121℃for 15min.

1.3 Major instrumentation

And (3) a microscope: orinbas Inc., japan; high-speed refrigerated centrifuge: thermo company, germany; pure water meter: taiwan Ai Ke of china; colony analyzer (model MF 4) was from shanghai science and technology limited, china, and the enzyme-labeled instrument (model Synergy LX) was from united states BIOTEK, and the hand-held meter (model PAL-1) was from japan aiton.

Test methods and results

2.1 Screening and identification of lactic acid bacteria in the intestinal tract of Wenchang chickens in Hainan province

All experiments were performed in triplicate and the data are shown as mean ± SD. Carrying out data statistics analysis by adopting SPSS22 software; drawing by using Origin2019 software; significance was analyzed using one-way analysis of variance (ANOVA). The different letters on the bar graph represent significant differences (P < 0.05). 2.1.1 preliminary separation and purification of lactic acid bacteria

The sample Wenchang chicken is brought back to a laboratory, the blood is taken by pricking, the duodenum, the cecum and the ileum and the content are collected in a 50mL centrifuge tube, 25g of the cecum and the jejunum content are respectively diluted by 10 times with sterile physiological saline (0.85% w/v, pH 7.2-7.4) in an ultra clean bench, and homogenized for 20 minutes. The colony with large transparent ring of dissolved calcium is selected for streak purification by coating and separating on MRS plate containing 2% (w/v) CaCO ₃ by dilution coating method, culturing at 37 ℃ for 48 hours, selecting colony with quick growth and large transparent ring for gram staining, selecting positive strain, and then carrying out thixotropic enzyme test. All G ⁺ and thixotropic strains are found to be straight, thick, rod-shaped or short-rod-shaped, and the two ends of the strain are straight or round, and are mostly arranged singly, in pairs or in short chain shape, so that the strain accords with the general characteristics of lactobacillus, and 11 strains are initially selected for subsequent screening through primary screening. 2.1.2 acid and bile salt resistance testing of Strain

Acid and bile tolerance is one of the important criteria for screening probiotic strains. The activated 11 primary screened test strains were centrifuged at 4500r/min for 10min, respectively, and the cells were washed twice with sterile Phosphate Buffered Saline (PBS), resuspended, and incubated at 37℃for 3h in MRS medium at pH 2.5 and MRS medium containing 0.3% (w/v) bile salts, respectively. Plate counts were performed at 0h and 3h, respectively, with reference to national standard GB 4789.2-2016, and survival rates were calculated to evaluate the acid and bile salt resistance of each strain.

As shown in FIG. 1, strains K111, MS2c, MZ2, MS2 and MS111 show good acid tolerance, the survival rate is close to or more than 100%, wherein the acid tolerance of the strain K111 is highest, and the survival rate is close to 120%; the bile salt tolerance of the strains MS2c, MZ2, S311, K111 and MS111, MY1 is significantly higher than that of other strains (P < 0.05), wherein the strain MS2c has the highest bile salt tolerance and the survival rate is 70.41%.2.1.3 cell surface hydrophobicity test of lactic acid bacteria

The surface hydrophobicity of bacteria can be indirectly evaluated for its ability to adhere to intestinal epithelial cells, and is generally high and generally has a high ability to adhere to intestinal mucosal cells, and is generally tested for hydrophobicity with the affinity of lactic acid bacteria for hydrocarbons. This example was measured using the cell adhesion hydrocarbon method with a slight modification. The test strain activation culture solution is centrifuged at 4500r/min for 10min, washed by PBS buffer solution for 2 times and resuspended in 3mL PBS, and the initial absorbance is controlled to OD ₆₀₀ within the range of 0.6-0.8. 1mL of dimethylbenzene and n-dodecane are added into the bacterial suspension, the bacterial suspension is vortexed for 1-2min, is taken out after standing and warm bath for 15min at 37 ℃, is vortexed for 1-2min again, is subjected to standing for 1h, and the OD ₆₀₀ of an upper organic phase is measured to calculate the surface hydrophobicity of the bacterial strain.

To comprehensively examine the performances of all strains and realize no screening, 11 strains are tested, and as shown in fig. 2, the surface hydrophobicity of the strain MS2c, which is shown by the paraxylene and n-dodecane solvent, is obviously higher than that of other strains (P < 0.05), which are respectively 43.82 percent and 54.12 percent; the surface hydrophobicity of the other strains is lower than 40%, wherein the surface hydrophobicity of the strain K111 and the surface hydrophobicity of the strain MS111 in two solvents are similar and are about 40% and about 20% respectively, so that the hydrophobicity of the strain MS2c is highest, and the strain K111, the strain MS111 and the strain S311 possibly have better adhesion to intestinal tracts and are hydrophobic times; other strains were very hydrophobic.

2.1.4 Automatic polymerization ability test of lactic acid bacteria

The ability of bacteria to colonise the gut and form their biofilms is inseparable, and the high self-polymerizability of the strains helps to form biofilms to protect their own activity. The self-polymerization capability of the lactobacillus has strain specificity, and different strains have larger difference. With a slight modification in reference (RAHMAN M,KIM W S,KUMURA H,et al.In vitro effects of bovine lactoferrin on autoaggregation ability and surface hydrophobicity of bifidobacteria[J].Anaerobe,2008,14(2):73-77.), the strain culture was centrifuged at 4500r/min for 10min, washed with PBS, resuspended, and after vortexing, the initial turbidity OD ₆₀₀ was measured, and after standing at 37℃for 24h, the supernatant turbidity OD ₆₀₀ was measured, and the self-polymerization percentage was calculated.

As shown in fig. 2, a difference was found in the autopolymerization ability of all strains, with the autopolymerization ability of strain MS2c still being highest, 88.52%, significantly higher than that of the other strains (P < 0.05); the strains K111, MS111, MZ2, MZ6 and S311 all have an autopolymerization force exceeding 60%, and also have good autopolymerization ability.

2.1.5 Antibacterial Activity test of lactic acid bacteria

The bacteriostasis ability of probiotics is particularly important, and as a probiotic strain for enteritis curing application, the probiotic strain must have good ability of inhibiting the growth of pathogenic bacteria, and the bacteriostasis test indicator used in the embodiment is common pathogenic bacteria, such as escherichia coli ECXT, salmonella SXS, staphylococcus aureus JH, listeria monocytogenes and pseudomonas aeruginosa, and the indicator is cultivated for 24 hours at the inoculation amount of 2% (v/v) in an LB culture medium at 37 ℃ for later use, and is slightly modified according to the agar diffusion method described by Muhammad et al. Adding the strain to be tested into MRS culture medium according to the inoculation amount of 2% (v/v), culturing at 37 ℃ for 24 hours to obtain a first-generation culture, activating again to obtain a second-generation culture, adding 0.5mL of indicator bacteria into a sterile empty culture dish, pouring LB agar culture medium cooled to about 50 ℃ after sterilization into the culture dish, quickly and fully mixing uniformly, cooling and solidifying, preparing holes by using a sterile puncher (6 mm), adding 70 mu L of activated 2-generation culture of the strain to be tested into each hole, culturing at 37 ℃ for 24 hours, and measuring the diameter (mm) of a transparent bacteriostasis ring.

For no screening, the 11 strains were all subjected to bacteriostasis capability test (see table 1), and the diameter of the bacteriostasis zone was found to be between 10.52 and 18.65mm, and significant differences exist. Wherein, the bacteria inhibition zones of the strains MS2c, K111 and MS111 on salmonella, staphylococcus aureus and escherichia coli are all obviously superior to other strains (P < 0.05); the bacteria inhibition zone of the strain MS2c on the listeria monocytogenes is obviously larger than that of other strains (P < 0.05); the bacteriostasis circle of the strains MS2c and K111 on the pseudomonas aeruginosa is obviously larger than that of other strains (P < 0.05); the strains MS2c, K111, MS111 and S311 have larger bacteriostasis circles for five indicator bacteria in a combined way, which indicates that the bacteria have wider bacteriostasis spectrums and better bacteriostasis capacity.

TABLE 1 antibacterial conditions of fermentation supernatants of Strain on five indicator bacteria

Note that: different lowercase letters indicate significant differences between the same column of data (P < 0.05), and the same letter indicates insignificant differences.

2.1.6 16S rDNA sequencing

Through the probiotic characteristic test, the 4 strains of lactic acid bacteria MS2c, K111, MS111 and S311 are found to have excellent performance, and then the molecular biological species identification of the 16S rDNA is carried out. Strain identification was done by Qingdao peng Xiang biotechnology Co., ltd, using 16S universal primers for amplification and sequencing analysis. Wherein the molecular sequence of the 16S rDNA gene of the strain MS2c is 1391bp, and the sequence of the molecular sequence is shown as SEQ ID NO. 1.

The results of the BLAST on-line analysis were compared with the gene sequences in NCBI database, and are shown in Table 2, wherein the homology of the strains MS2c, K111 and MS111 with Lactobacillus plantarum (Lactobacillus plantarum, which is designated Lactiplantibacillus plantarum recently) is highest, the homology of the strain S311 with enterococcus faecalis (Enterococcus faecalis) is highest, the homology and coverage percentage of all strains with the reference bacteria in the gene library are over 99%, and the E value is 0 (see Table 2).

Table 24 16S rDNA homology comparison analysis of strains

2.2 Comparison and rescreening of Excellent probiotics based on Strong bacteriostatic Activity

Candidate lactic acid bacteria: lactobacillus plantarum (Lactiplantibacillus plantarum) MS2c, MS111 and K111 obtained by the preliminary screening; screening and identifying antagonistic lactobacillus in natural fermented coconut water and antibacterial property [ J ]. Food research and development. 2021,42 (23) DOI: 10.12161/j.issn.1005-6521.2021.23.025.); lactobacillus acidophilus (Lactobacillus acidophilus) LA71, a commercial strain of probiotic bacteria, for control; W417-W421 are lactobacillus which is screened from coconut water after natural fermentation (more than 48 hours of standing fermentation in an open container at the temperature of 25-35 ℃) in coconut processing factories and is preliminarily confirmed, but the lactobacillus is not identified by species.

The SPSS21.0 software is adopted to carry out statistical analysis on experimental data, the average value of the experimental data is represented by +/-standard error, the F test is adopted to carry out difference significance analysis on the experimental result, P is smaller than 0.05 and represents the difference is significant, and P is smaller than 0.01 and represents the difference is extremely significant.

2.2.1 Strain and its activation and seed liquid preparation

Test strain: MS2c, MS111, K111, A32, W417 to W421, LA71

Indicator strain: coli ECXT, salmonella SXS, staphylococcus aureus JH

Taking frozen strain at-80 deg.C, and respectively culturing with MRS (lactic acid bacteria) or LB (indicator bacteria) liquid culture medium at 37 deg.C for two generations to make absorbance OD ₆₀₀ reach about 0.8, and using as seed solution.

2.2.2 Antibacterial prescreening and tolerating rescreening

The bacteria with strong antibacterial activity are selected by performing antibacterial activity test on the 10 lactobacillus with different sources, and then acid and bile salt resistance are compared to re-screen out excellent probiotics with high probiotic characteristics for developing a chicken enteritis cure test.

(1) Antibacterial screening

And (3) carrying out in-vitro antibacterial activity test on 10 strains of lactic acid bacteria, and measuring the diameter of a bacteriostasis ring by adopting a perforation method.

Bacteriostasis test by punching method: adding 100 mu L (total viable count is about 10 ⁵～10⁶ cfu/mL) of indicator bacteria liquid into a flat plate, pouring and cooling to about 50 ℃ and 20mL of LB solid medium, rapidly and fully mixing, punching by a puncher after solidification, adding 80 mu L of test bacteria fermentation liquor into the hole, standing and culturing for 16-18h, observing a bacteriostasis result, and measuring the diameter of a bacteriostasis ring.

Strains with higher antibacterial activity are screened through antibacterial action on three pathogenic indicator bacteria, and as shown in figures 3-5, three lactic acid bacteria MS2c, A32 and MS111 have good antibacterial activity compared with a control strain LA 71. Wherein MS2c is significantly stronger than a32, also significantly stronger than LA71 strain (p < 0.05), and the three strains have a stronger inhibitory effect on gram-positive staphylococcus aureus (p < 0.05). MS111 and MS2c are Lactobacillus plantarum and A32 is Lactobacillus fermentum, so these three strains were selected for fermentation in coconut water for further screening.

(2) Acid-resistant and bile salt-resistant rescreening

Acid resistance experiment: the pH of the MRS culture solution was adjusted to 2.0, 3.0 and 4.0 with 1mol/L HCl solution, respectively. Three lactic acid bacteria to be compared are respectively inoculated into MRS liquid culture media with different pH values, and are cultured for 16 hours at 37 ℃, and OD ₆₀₀ values are measured to calculate the survival rate.

Bile salt resistance experiment: three lactic acid bacteria to be compared are respectively inoculated into MRS liquid culture media with different bile salt concentrations (0, 0.3%, 0.6% and 0.9%), and are cultured for 16 hours at 37 ℃, and OD ₆₀₀ is measured to calculate the survival rate.

The experimental results (as shown in fig. 6 and 7) show that the acid resistance of lactobacillus plantarum MS2c and MS111 is significantly higher than that of lactobacillus fermentum a32 (p < 0.05) under different pH environments. In terms of cholate resistance, the survival rate of MS2c is extremely higher than A32 and also is obviously higher than that of MS111 (p < 0.05) at three concentrations, three strains are comprehensively evaluated through antibacterial activity and acid-resistant cholate-resistant characteristics, and MS2c is selected as a preferred strain for subsequent further experiments. The fermentation culture condition of the strain in coconut water is optimized.

2.2.3 Optimization of fermentation conditions of high bacteriostatic Activity probiotics in coconut Water

And (3) selecting the experimental condition with the highest viable count by carrying out optimization experiments on different culture medium components.

Strain density determination: the strain was cultured at MRS 37℃for 18 hours. 200. Mu.L to 96-well plates were pipetted under sterile conditions and OD ₆₀₀ was measured with a microplate reader, 3 replicates per group.

Viable cell count determination: the assay was performed by gradient dilution-plate coating, 3 replicates per group.

The optimization experiment process is as follows:

Setting a certain range for each nutrient element affecting the growth and reproduction of lactobacillus, and optimizing conditions based on coconut water basic culture medium, wherein the nutrient elements are as follows: the sugar degree adding range is set to be 1, 2.5 and 4 degrees Bx; the peptone addition range is set to be 5, 10 and 15g/L; the adding range of the yeast extract powder is 1, 3 and 5g/L, the adding range of sodium acetate is 1, 3 and 5g/L, and the adding range of the citric acid triammonium is 0, 0.5, 1 and 1.5g/L. The viable count and turbidity of the bacterial liquid were measured after culturing at 37℃for 18 hours at an inoculum size of 2%.

As is clear from FIG. 8, when the sugar degree was 4℃Brix, the addition amount of yeast extract was 5g/L, the addition amount of peptone was 5g/L, the addition amount of sodium acetate was 5g/L, and the addition amount of triammonium citrate was 0.5g/L, the viable count was 1.8X10 ⁸ CFU/mL, and the absorbance was about 1.25, which were all at or near the highest value. In terms of cost saving, the fermentation conditions were taken as those of the strain MS2c in coconut water medium.

In the embodiment, firstly, high-activity probiotics are screened from the intestinal tracts of Wenchang chickens, then 10 lactic acid bacteria including a laboratory pre-screened strain and a commercial control strain are subjected to in vitro antibacterial activity comparison to obtain lactobacillus plantarum MS111 and MS2c from the intestinal tracts of chickens and lactobacillus fermentum A32 from fermented foods, three lactic acid bacteria with higher antibacterial activity are screened through acid and bile salt tolerance re-screening, one lactobacillus plantarum MS2c is selected as a preferred strain, and the culture medium component of the strain MS2c is optimized, so that the viable count reaches more than 10 ⁸ CFU/mL, the activity of the fermented coconut water metazoan is greatly improved, and a basis is provided for the subsequent application experiment on enteritis chickens. The research is to comprehensively utilize the byproduct coconut water, so that the coconut water becomes a culture medium suitable for lactobacillus plantarum fermentation, and a new thought and a new way are provided for grading treatment and efficient utilization of the coconut water.

Example 2 screening and identification of probiotic Yeast Y301

The strain has been deposited at the collection of microorganism strains (GDMCC) of Guangdong province at 12/24 of 2023 under the deposit number GDMCCNo:64183, deposit address: building 5, road 100, university, building 59, university of Guangzhou City martyr, university of Guangdong, institute of microorganisms, taxonomic designation: saccharomyces cerevisiae (Saccharomyces cerevisiae).

Separating and purifying strain

Naturally fermenting (at room temperature and 25-35 ℃ for more than 48h in an open container) coconut water in a coconut processing factory, taking a naturally fermented coconut water sample, fully and uniformly mixing, and then sucking 2mL as a bacteria screening sample to screen saccharomycetes with probiotic characteristics.

The natural fermentation coconut water sample is added into YPD culture medium to carry out enrichment culture at 30 ℃, and then is subjected to coating separation in coconut water solid culture medium by serial dilution method to carry out culture at 30 ℃. And (3) screening colony morphology of the single colonies, picking the single colonies to be larger, carrying out streak separation on the suspected saccharomycetes colonies again, picking out the single colonies to carry out microscopic examination, and carrying out YPD inclined plane preservation on the colonies judged to be saccharomycetes by budding propagation.

Second, microscopic examination of the strain

And selecting single colonies from the plate after streak purification, and observing the single colonies by a crystal violet single-staining method in a microscopic way to finally obtain 40 saccharomycete isolates which are all round, oval or sausage-shaped in form and all bud and reproduce. Subsequently, a coconut water liquid culture was carried out, and a culture which was capable of rapidly clouding coconut water but which had no floating film on the liquid surface was selected, and 7 strains were obtained, of which the morphology of strains Y301, S1171 and 134 was representative, and the colony and fungus morphology thereof were as shown in FIG. 9.

Screening of yeasts based on growth Properties in coconut Water

Screening fast growing strains according to the turbidity change speed of the bacterial liquid during the culture in coconut water, and selecting a plurality of strains for viable count: yeast was inoculated into 30mL of sterilized coconut water medium at an inoculum size of 2%, and fermented at 30℃for 15 hours, and the viable count was measured by plate viable count method, as shown in FIG. 10.

Statistical analysis showed that the viable count of strains Y301 and S1171 was significantly higher than the other strains (p < 0.05), and that strain 134 was also selected as an alternative strain.

Fourth, acidity and bile salt tolerance screening

Acid resistance culture experiment: liquid YPD medium was prepared, and pH was adjusted to 2.0, 3.0 and 4.0 with 1mol/L HCl, respectively, and a set of pH was set to be natural and non-adjusted. The yeasts obtained by the preliminary screening were inoculated at 2%, cultured at 30℃for 15 hours, and OD ₆₀₀ values of each strain were measured.

Bile salt resistance experiment: the primary screening yeasts are inoculated into YPD liquid culture media with the concentration of bile salts of 0, 0.3%, 0.6% and 0.9%, respectively, and are cultured for 15 hours at 30 ℃, and OD ₆₀₀ is measured. The survival rate of each strain was calculated as follows:

survival = Na/Nb x 100%, na is OD ₆₀₀ value after application of treatment, nb is OD ₆₀₀ without treatment (pH natural and without bile salts).

From the results of fig. 11, it was found that strain Y301 exhibited the highest acid resistance: the survival rate of Y301 was significantly higher than 134 and S1171 (p < 0.05) in different pH environments. At pH4, the survival rate of Y301 was highest, the difference was insignificant compared to the survival rate at pH3, and at pH2 the survival rate was halved but still around 40%, indicating that strain Y301 had a higher acid resistance. But the acid resistance of strains 134 and S1171 is significantly lower than Y301.

In the bile salt tolerance experiment, Y301 survival at 0.3% bile salt concentration is significantly higher than S1171 and 134 (p < 0.05) as reflected in figure 12. However, at both 0.6% and 0.9% bile salt concentration, the viability of the three strains was significantly reduced (p < 0.05). This suggests that the yeast is sensitive to bile salts, but the Y301 bile salt tolerance is highest among the three strains, so that the strain Y301 is to be selected as the target test strain for the subsequent chicken enteritis self-healing recovery test. In order to determine the species relationship, strain identification is performed first.

Fifth, species identification of Strain Y301

And (3) carrying out ITS sequencing identification on the strain Y301, wherein the ITS gene sequence 673bp is shown as SEQ ID NO. 2. The NCBI alignment was submitted, and the results are shown in Table 3, with the combination of morphological features, to comprehensively determine that strain Y301 was Saccharomyces cerevisiae (Saccharomyces cerevisiae).

TABLE 3 ITS gene sequence alignment of Strain Y301

Example 3 preparation of lactic acid bacteria and Yeast composite probiotic fermented coconut Water metaplasia and Process optimization

When the conditions are optimized below, the composite probiotics of lactic acid bacteria and saccharomycetes are fermented at the temperature of about 35 ℃. The fermentation temperature can be between 33 and 37 ℃ without affecting the fermentation effect.

Determination of the inoculation ratio of the fermentation of the Complex Strain

Referring to fig. 13, it can be found that substantially similar variation trend is exhibited by setting different inoculation ratios of lactobacillus and yeast, wherein when the ratio is lactobacillus: when the saccharomycetes are 1:2, the number of viable bacteria in the fermentation water of the compound strain reaches the highest, and the compound strain can be maintained for a longer time in a stable period and can be obviously higher than other ratios when being cultured in a compound way, and the total number of viable bacteria is 4.05X10 ⁸ CFU/mL. Thus lactic acid bacteria were selected: yeast is 1:2, and is used as the inoculation ratio of the metazoan after the preparation of the compound strain fermented coconut water.

Determining the fermentation and inoculation sequence of the composite strain

From the inoculation sequence and viable bacteria count chart (FIG. 14), the viable bacteria count in the simultaneous inoculation mode was significantly higher than that of the other groups at the time points 18h, 24h, 30h and 36h, and the viable bacteria count was 2.62X10 ⁸ CFU/mL. Therefore, the mode of simultaneous inoculation is selected, and the subsequent popularization and application are also facilitated.

Third, determination of total inoculation amount of composite strain fermentation

The effect of inoculum size on viable count is shown in FIG. 15, wherein 4% and 5% inoculum sizes are significantly higher than the other two groups, 4% of the groups are in 12-20 h, the viable count is extremely fast increased and reaches a level (p > 0.05) equivalent to 5% inoculum size, and the larger inoculum size can maintain good strain advantage of the target strain, and is beneficial to fast propagation in coconut water to inhibit infectious microbe, so 4% inoculum size is selected, and the viable count is 4. 10 ⁸ CFU/mL. Determining the number of viable bacteria and fermentation time of single-strain fermentation and composite-strain fermentation

Based on the above screening experiments, fermentation conditions for different strains were set as follows: MS2c is inoculated into a sterilized coconut water culture medium according to the inoculation amount of 2 percent, and fermentation culture is carried out at 37 ℃; y301 was also inoculated in a sterilized coconut water medium at an inoculum size of 2%, and cultured by fermentation at 30 ℃; the composite strain is mixed in a ratio of 1:2, inoculated into sterilized coconut water culture medium according to a total inoculum size of 4 percent, and fermented and cultured at 35 ℃.

As can be seen by comparing trend graphs of single bacteria and composite strain fermentation (see FIG. 16), after 18 hours of single bacteria fermentation, the viable count reaches the highest value of 2.00×10 ⁸ CFU/mL, while the viable count of composite strain fermentation is significantly higher than that of single bacteria fermentation (p < 0.05) after 18 hours, and still presents a slow rising trend after 24 hours and reaches the highest value of 2.86×10 ⁸ CFU/mL after 30 hours, and can maintain a higher viable count level within 36 hours, which indicates that the composite culture of the two is favorable for significantly prolonging the stable period of the total viable count. Therefore, the optimal biomass time for single-strain fermentation of the saccharomycetes and the lactobacillus is determined to be 18h and 24h, and the optimal biomass time for single-strain fermentation of the compound strain is determined to be 24-30 h.

Therefore, through the experimental results, and based on the consideration of cost and efficiency, the optimal fermentation conditions for the composite fermentation are selected as follows: two bacteria are inoculated simultaneously, and lactic acid bacteria are used for: the yeast is in a ratio of 1:2, 4% of total inoculum size is used for fermenting coconut water by the compound strain, and the compound strain is prepared into a compound strain fermented coconut water metaplasia after being cultured for 24-30 hours at 35 ℃ and is prepared into a feed additive for further application experiments.

Test example production and self-healing promotion of post-natal element of coconut water fermented by probiotics on Wenchang chickens with DSS induced enteritis

In the present invention, animal experiments and protocols were approved by the university of Hainan ethical Committee and followed the guidelines of "university of Hainan laboratory animal protection and utilization guidelines (approval number HNUAUCC-2023-00210)".

Data analysis was performed using GraphPad (san diego, california, usa GraphPad Software) and R software. Data are presented as mean ± Standard Deviation (SD), significance is marked using the difference letters: at p <0.05, the differences between the letters were considered significant. In the partial large graph, the differences are represented significantly as: i.e. p <0.05; the difference is very significant expressed as: i.e. p <0.01; the most significant difference is expressed as: i.e. p <0.001.

Experimental methods and designs

Modeling experiment of chicken enteritis model: in order to fully study the effect of two probiotics on the self-healing recovery of the acute enteritis of poultry, we induced the acute enteritis in chickens. First, a modeling experiment was performed on 14-day-old chickens to evaluate the effect of two different concentrations (1.5% and 2.5% w/v) and two intake methods (gavage and free drinking) of Dextran Sodium Sulfate (DSS) on the chicken intestinal tract. The results showed that 14 day old chickens, who were free to drink 1.5% DSS solution, exhibited diarrhea symptoms, some also had bloody stool. At the same time, higher concentrations of 2.5% DSS solution caused more severe symptoms, a higher blood to stool ratio, and even death of individual chickens. Therefore, 1.5% DSS (w/v, 40,000kda, macklin) solution was selected for free drinking and modeling experiments were performed for 7 days to induce acute enteritis in the chickens tested.

The experimental process of constructing acute enteritis model chicken and fermenting coconut water metaplasia by probiotics comprises the following steps: 60 Wenchang chicken cocks with a day age of 2 weeks and a weight range of 90-110 g are purchased from Wenchang in Hainan province. Randomly dividing into six groups (n=10), and raising in a laboratory at 28-30 ℃ in chicken cages, wherein each group is fed with the recommended common feed of the breeding plant every day, and only the additives in drinking water are different. Adding 1.5% DSS solution, performing enteritis molding for 1 week, stopping using DSS after molding success, and entering into prebiotic intervention feeding experimental stage after fermenting coconut water by probiotics for 2 weeks. Each group is free to feed the same daily ration and purified water every day, but each group is fed with 2mL of corresponding solution every morning, and the specific conditions are as follows: blank control (CK 1) and model control (CK 2): purified water is provided; unfermented coconut water control group (CK 3): feeding 2mL of unfermented coconut water; feeding 2mL S.cerevisiae Y301 fermented coconut water into yeast group (Y3 group); the bacterial group (M2 group) is irrigated with 2mL L.plantarum MS2c fermented coconut water; the mix group (MY 5) was fed with L.plantarium and S.cerevisiae mix fermented coconut water. The experiment was ended after 2 weeks of intervention feeding, i.e. 21 days of total test time, weighed after 12 hours of fasting, and blood was collected from the winged vein while euthanasia was performed by neck bleeding. The sections of the intestinal tract and cecum stool specimens were opened and subjected to biochemical and immunological index measurement, intestinal microbiology diversity and cecum metabonomics analysis, and the simplified procedure is shown in fig. 17.

Second, growth performance, physiological and biochemical and immune index measurement

Physiological indicators are measured to assess the health differences in chickens, including daily ration intake, daily gain, and Disease Activity Index (DAI). Fecal occult blood levels were determined using a test kit. Blood samples of ten chickens from each group were collected before euthanasia, and serum was isolated after 30 minutes of natural clotting. Serum levels of cytokines IL-10, IL-6, TNF- α and IFN- γ were determined using ELISA kit (Solabo, beijing, china). Meanwhile, serum biochemical indexes including glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), uric Acid (UA) and D-lactic acid (D-LA) were measured using a detection kit (Solabo, beijing, china).

2.1 Effect of prebiotic intervention of coconut Water fermentation by probiotics on growth performance recovery of acute enteritis model chickens

As shown in fig. 18, after 7 days of molding with DSS, all other groups, except the control group CK1, showed lower daily intake, preliminarily demonstrating that molding was effective. After the start of the intervention experiment, the rate of recovery of the ration intake was faster for MY5 and Y3 groups than for CK2, CK3 and M2 groups by day 12. Until the end of the full course experiment on day 21, the recovery of ration intake was higher for the MY5 and Y3 groups than for the M2, CK2 and CK3 groups. The average weight of the chickens and the feed intake tended to be similar. Demonstrating that the intervention of saccharomycete Y301 in fermented coconut water metazoans (Y3 group and MY5 group) is more beneficial to the weight recovery of the chickens with acute enteritis.

However, feed intake and weight gain tended to return well for the 3 coconut water metazoan intervention groups (Y3, M2, MY 5) compared to model group CK2, with the 3 groups returning faster than CK2 (fig. 18a and b). In addition, disease Activity Index (DAI), in particular scores associated with intestinal disease, are improved. And the observation of the conditions of stool color, humidity and the like in the test process shows that the prebiotic intervention after the fermentation of coconut water by probiotics effectively improves the abnormal stool, and the recovery speed of the conditions of stool such as loose stool, bloody stool, coccidian stool and the like is obviously faster than that of the CK2 group (figure 18 c).

2.2 Effect of prebiotic intervention of coconut Water fermentation by probiotics on the serum Biochemical and immune index of acute enteritis model chickens

The content of various cytokines and biochemical indexes in the serum of each group of 10 experimental chickens was determined by analysis (data average). The results (fig. 19) show that post-prebiotic intervention of probiotic fermented coconut water resulted in reduced levels of both serum inflammatory factors and anti-inflammatory factors. For the IL-10 index, the M2 and MY5 groups were significantly lower than the CK2 group (p < 0.05), with no significant difference (p > 0.05) from the control group CK 1. For the IL-6 index, MY5 group was significantly lower than CK2 group (p < 0.05), and M2 and Y3 groups were also reduced. As for the TNF-. Alpha.index, the level of Y3 group was significantly lower than that of CK2 group (p < 0.05). On the IFN-gamma index, the levels of M2, Y3 and MY5 groups were significantly lower than for the CK2 group (p < 0.05). It was found that the level of immune cytokines in serum correlated closely with the inflammatory response in chickens and was substantially consistent with the growth performance status (fig. 19 a).

The level of serum D-lactic acid (D-LA) can be used as a characteristic index of chicken intestinal mucosa injury and permeability increase. Experimental results showed that the CK2 group D-lactate levels were significantly higher than the probiotic intervention group Y3 and MY5 groups (p < 0.05). Indicating that the intervention of coconut water fermented by saccharomycete Y301 helps recovery of intestinal damage and reduces the level of D-lactic acid in serum. Uric Acid (UA) levels in serum can reflect kidney injury and load conditions. The serum uric acid difference between the model group and the control group is not obvious, which indicates that DSS modeling has little influence on the kidney and the function thereof. GOT and GPT enzymes are used as markers for assessing liver damage in chickens. Each probiotic fermented coconut water (M2, Y3, MY 5) significantly reduced GOT levels (p < 0.05) compared to control CK2 and inhibited GPT elevation in experimental chickens (fig. 19 b).

From comprehensive analysis, the influence rule trend of D-LA and IL-10 and IFN-gamma in the experiment is highly correlated, and the success of the modeling experiment is indirectly indicated. From the perspective of data trend, the probiotics intervention group effectively promotes the immune and biochemical indexes to develop towards good or obvious change direction, and the experimental group promotes the strengthening of intestinal immune biochemical indexes and barrier functions, thereby promoting the self-healing recovery process of acute enteritis inflammation induced by DSS.

Third, pathological index measurement

After the blood sampling experiment, the cecum 1cm section at the end of the intestine was retrieved. This section was used to evaluate cecal wall thickness and structural integrity, as observed by HE staining. The intestinal tissue of the chicken was treated according to the method of O.Srinal for staining and the cecal tissue was fixed with 4% (w/v) formaldehyde for 24 hours. Followed by dehydration, paraffin embedding, and cutting into 3 μm sections. Colon samples were stained for Hematoxylin and Eosin (HE) as described previously. Histopathological conditions were assessed using an optical microscope (Nikon, japan) and the extent of tissue damage was analyzed using Image-Pro Plus 6.0 analysis software and the results were reported in millimeters.

Histological examination of the ileum by HE staining (fig. 20, left) revealed that DSS-induced acute enteritis group (CK 2 group) resulted in severe ileal epithelial cell loss, villus structural destruction and massive villus fragmentation shedding. However, after two weeks of intervention of the prebiotics after fermentation of coconut water by probiotics, the intestinal mucosal structure of the experimental group (M2, Y3, MY 5) developed towards improvement. According to the results of fig. 20 (right), the approach between MY5 group and control group was close, without significant difference in intestinal wall thickness (p > 0.05). This shows that the MS2c and Y301 composite strain fermented coconut water metazoan has a remarkable recovery promoting effect on the damaged intestinal wall of the acute enteritis chicken. The intestinal wall thickness of the Y3 and M2 groups, although not yet fully recovered, was significantly lower than that of the control group (CK 1), but significantly higher than that of the model group (CK 2). Indicating that intervention of single strain fermented coconut water metazoans also helps to restore impaired intestinal wall structure.

Extraction and analysis of microbial diversity of the content of the cecum of chickens

After euthanasia, six chickens were randomly selected from each group and the cecal content was rapidly extracted. The cecum content samples were mixed and stored in dry ice and sent to the Shanghai Meiji biomedical technology Co., ltd for cecum microorganism diversity analysis. Analysis high throughput sequencing involving DNA extraction, PCR amplification of the V3-V4 variable region of the 16S rDNA gene, library construction and 16S functional predictive analysis.

The upstream primer used was 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer was 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). The 16S rDNA gene V3-V4 variable region of the sample was amplified by PCR, and the PCR reaction system was as follows: 5X TRANSSTART FASTPFU buffer 4. Mu.L, 2.5mM dNTPs 2. Mu.L, upstream primer (5. Mu.M) 0.8. Mu.L, downstream primer (5. Mu.M) 0.8. Mu.L, TRANSSTART FASTPFU DNA POLYMERASE 0.4. Mu.L, template DNA 10ng was adjusted to 20. Mu.L. Three replicates were prepared for each sample.

4.1 Effect of prebiotic intervention of coconut Water fermentation by probiotics on intestinal microbiota diversity and flora composition of acute enteritis model chickens

The present invention determines the microbial diversity of the experimental chicken cecal content by high throughput sequencing and analyzes the alpha diversity and beta diversity (fig. 21a and b), finding that there is a very significant difference in alpha diversity between CK1 and CK2 groups (p < 0.01). This shows that DSS modeling (CK 2 group) significantly reduced the microbiota diversity of the cecal contents of chickens, which again indirectly demonstrated the success of modeling experiments, with confidence in all experimental data and analytical results. While the microbial community α diversity of all experimental groups had a good trend (fig. 21 a), demonstrating that the intervention of the prebiotics after fermentation of coconut water by probiotics could play a role in intestinal microbial diversity recovery and protection. No significant differences were shown between the six groups of samples in terms of β diversity, indicating that the differences in microbial community structure at the genus level between these test groups were not large by 2 week time intervention. Some differences were still observed between the intervention group and model group CK2, and were further analyzed below.

After analyzing the relative abundance of each group of microorganism composition (FIG. 22), it was found that Bacteroides, faecalis, ruminococcus, bifidobacterium, majoranferae, odormitomyces, paralobacter, shigella and the like exert an important effect in the intestinal microorganism structure. Shigella coli is a common pathogenic bacterium, and results in bacillary dysentery, the content of M2 and MY5 groups (accounting for 0.3% and 0.16% respectively) is lower than the abundance in CK1 (2.7%) and Y3 (3.6%), which indicates that the relative content of parent pathogenic bacteria which exist in a certain amount (CK 1 group accounting for 2.7%) can be reduced when MS2c intervenes, and the potential improvement of the functions of intestinal flora structures is predicted.

In the group analysis chart (fig. 23C), it can be seen that bacteroides multinomial (Bacteroides dorei) is more abundant in the three control CK1, CK2 and CK3 groups and relatively lower in the CK2 group, which is a beneficial bacterium that can effectively break down carbohydrates into glucose and other sugars, helping digestion and absorption of nutrients from the feed. The second important gut is the gram negative another genus (ALISTIPES), which is reported to be positively associated with certain intestinal diseases, with a high abundance in the CK2 group. On the other hand, the abundance of lactococcus taiwanensis (Lactococcus taiwanensis), kriging Teng Senshi family R7 group (CHRISTENSENELLACEAE R group) was significantly higher in the control CK1 group and CK3 (p < 0.01), and lower in CK 2. Lactococcus taiwanensis is a potential probiotic commonly used for fermentation of dairy products; the bacteria kries Teng Senshi are reported to be inversely related to intestinal diseases. These data indicate that, after DSS treatment, CK2 group is used as model group of acute enteritis, and intestinal bacteria feelings of the masses condition corresponds to the detection result of clinical manifestation, immunology and histological index in the culture. Meanwhile, the intestinal tracts of Wenchang chickens are sensitive to DSS-induced intestinal flora imbalance, so that the DSS induction is a simple and effective enteritis model modeling method of the Wenchang chickens.

For the analysis of the M2 group, the heat map (fig. 23D) showed that the abundance of the cladosporium (ALISTIPES) highly correlated with intestinal disease was higher in the model group, whereas the abundance in the M2 group was lower, close to the control group, indicating that intervention by lactobacillus plantarum MS2c could effectively reduce the abundance of the potentially harmful bacteria cladosporium in the cecum of chickens. In addition, the comparison analysis of CK1 and CK2 groups shows that the abundance of the FSC020 group (Lachnospiraceae FSC020 group) and the Lactobacillus plantarum (L.plantarum) in the M2 group is higher than that of the model group, and even the abundance of the Lactobacillus plantarum is higher than that of the CK1 group, so that the MS2c intervention can cause the chicken intestinal tract to better colonize, thereby effectively increasing the abundance of beneficial Lactobacillus plantarum in the intestinal tract, repairing unbalanced intestinal flora and promoting the self-healing recovery of enteritis. The high abundance results of lactobacillus plantarum also highly correspond to the screening results of the multiple probiotics in the embodiment 1, and prove that the invention has high reliability and strong innovation in research content.

For group Y3, the heat map (fig. 23E) shows that the relative abundance of the other clade in the saccharomyces cerevisiae Y301-interfered group is lower than that of the model group, and is close to that of the control group. Whereas the group R7 of the family Krilsieidae Teng Senshi and the group NK3A20 of the family Maspiriaceae (Lachnospiraceae NK A20 group) were less abundant in model group CK2, but significantly more abundant in group Y3, which was closer to control group CK1. The intervention of the saccharomycete Y301 is also beneficial to the balance and recovery of intestinal flora in enteritis chickens.

For MY5 group, fig. 23F shows: the abundance of the krigineus Teng Senshi mycological R7 group in the model group CK2 is significantly lower than that of the control group CK1, and the group MY5 shows higher relative abundance and is closer to the control group, which indicates that the intestinal microbiomes of the CK1 group and the MY5 group are more stable. The abundance of the group NK3A20 in the trichomonadaceae is lower in the model group CK2, but the abundance of the group MY5 is obviously higher and is closer to the group CK 1. The bacillaceae is a genus that is known to produce short chain fatty acids and is inversely related to acute enteritis, with higher abundance in CK1 and MY5 groups than in CK 2. This suggests that the intestinal microbiome of the MY5 group is more similar to a healthy intestinal microbiome. Lactiplantibacillus plantarum also tended to be more abundant in the MY5 group than CK2 and CK1, suggesting that complex strain fermentation of coconut water post-primordial intervention is beneficial for colonization and stabilization of beneficial bacteria in the chicken gut microbiome.

Metabonomics determination and analysis of the content of the cecum of chickens

5.1 Effect of prebiotic intervention of coconut Water fermentation by probiotics on the metabolites of the content of the cecum of acute enteritis model chickens

As shown in FIG. 24, principal Component Analysis (PCA) was performed on the integrated data for each group of metabolites, and the results showed that the metabolites of the different groups aggregated together, indicating a high similarity between models. However, by performing partial least squares-discriminant analysis (PLS-DA) on each experimental group, it was found that the experimental groups M2 and MY5 were separated from the model group CK2 at a higher degree, as in the control group CK1, and showed a significant difference (p < 0.05), which indicates that the intervention of coconut water fermented with lactic acid bacteria MS2c was able to significantly recover the metabolites in the intestinal tract to the near control group. On the other hand, the degree of separation between CK3 group, Y3 group and model group CK2 was relatively low, indicating limited effect on intestinal metabolites after intervention of unfermented coconut water group (CK 3) and yeast fermented water alone (Y3). This also suggests that the intervention of strain MS2c in fermenting coconut water metazoans may be a major factor in the differentiation of intestinal metabolites, suggesting that probiotic MS2c has intrinsically better regulatory efficacy of the metabolites in improving intestinal flora balance and repair.

5.2 Path analysis of intestinal metabolism in model enteritis chickens by prebiotic intervention in coconut Water fermentation by probiotics

In the topology, the horizontal axis represents importance, and the vertical axis represents p-value. Metabolic pathways with p-values less than 0.05 were selected for analysis, with the most important of the key pathways identified as affecting the relevant metabolite concentration or activity. In the case of the lactobacillus MS2c intervention (group M2), the key metabolic pathway with the most significant difference was glycerophospholipid metabolism (fig. 25, left), suggesting that possibly the intestinal wall mucosal cells of the enteritis model chickens were stimulated by the intestinal beneficial metabolites to be repaired faster and better. In the case of the intervention of the yeast Y301 (group Y3), the main pathways affecting the differences in metabolites include alanine, aspartic acid and glutamic acid metabolic pathways (fig. 25, right), in which alanine is an important precursor substance of bile acids, aspartic acid is mainly digested and metabolized in the liver to produce urea and supply energy to the body, and glutamic acid can form α -ketoglutarate with pyruvic acid, participating in the regulation of tricarboxylic acid cycle, having an effect on both energy metabolism and substance metabolism in chicken digestion.

5.3 Correlation analysis between differential flora and differential metabolites

Correlation analysis of differential metabolites and differential flora (fig. 26 a) showed a significant negative correlation between MS2c intervention (M2 group) and ALISTIPES genus. There are several reports showing ALISTIPES to exhibit pathogenic characteristics in enteritis and bowel cancer. The invention discovers that MS2c fermented coconut water metaplasia can reduce the abundance of ALISTIPES in cecum, and further supports the repair or inhibition effect of the metaplasia on intestinal inflammation. In addition, a negative correlation was observed between MS2c fermented coconut water metazoan and epsilon-caprolactam, which was classified by the WHO as one of the 4 classes of carcinogens. This suggests that the strain MS2c of the present invention fermented coconut water metazoan may have an inhibitory effect on the production of certain potentially carcinogenic compounds in the chicken intestinal tract, thereby advantageously promoting the recovery of intestinal health.

In the analysis of differential metabolites and differential bacteria of intestinal contents, several substances were found to be positively correlated with MS2c fermented coconut water metazoans, which are CHRISTENSENELLACEAE, DESULFUROCOCCUS, gentamicin gentamicin) C, CDP-DG (a-25:0/a-21:0), lachnospiraceae, polymyxin (polymyxin) B2, berberine (berberine) and Botrytis halophila (halophilic arcobacter), and the like. Of these, CHRISTENSENELLACEAE, known for its negative correlation with inflammation, is a bacterial family with potential probiotic properties, and Lachnospiraceae promotes intestinal health by metabolizing short chain fatty acids.

In the correlation analysis of group Y3 (FIG. 26 b), 5-methoxytryptophan was positively correlated with ALISTIPES. This may be due to its role in the signalling pathway, leading to an increase in its level in the gut in severe inflammation.

Sixth, promote the probiotic intervention mechanism that acute enteritis model chicken resumes

Through the comprehensive analysis of the series of related indexes and intestinal flora and metabolite data, two potential mechanisms for promoting acute enteritis recovery are revealed. After passing MS2c fermented coconut water metazoans through 2 weeks of drinking water for dry prognosis, the following key changes were observed:

first, the daily ration intake, weight gain and DAI values of enteritis chickens are all trended well after intervention. Meanwhile, the abundance of beneficial bacteria in the intestinal tract, such as lactobacillus plantarum, CHRISTENSENELLACEAE, LACHNOSPIRACEAE, bifidobacterium ragum and the like, is increased, and the intestinal microecological balance is maintained.

Secondly, the MS2c intervention reduces the level of D-lactic acid and GOT enzyme in the serum of enteritis chickens, and reduces IL-10 and IL-6 indexes and TNF-alpha and IFN-gamma indexes of the enteritis chickens. These indicators are all associated with DSS-induced intestinal inflammation, and a decrease in the level of the indicator indicates that the intervention is effective and helps to reduce the inflammatory response.

Again, MS2c intervention also reduced the abundance of the potentially pathogenic ALISTIPES genera associated with colon cancer in the gut. More importantly, the intervention increased the levels of the beneficial metabolites gentamicin C, polymyxin B2 in the gut. These metabolites play an important role in promoting inflammatory recovery and maintaining intestinal health (fig. 27 a).

In addition, MS2c intervention affects the glycerophospholipid metabolic pathway, resulting in increased CDP-DG (a-25:0/a-21:0) levels, potentially stimulating rapid repair of intestinal mucosal cells, reducing intestinal inflammation.

An increase in abundance of Lachnospiraceae families was indirectly observed due to intervention of the metazoan of Y301 fermented coconut water. These bacteria can produce probiotic metabolites such as short chain fatty acids, inhibiting the growth of ALISTIPES genus associated with inflammation. This change resulted in increased abundance of anti-inflammatory and inflammation-reducing compounds such as melatonin, deoxyinosine, lauroyl glycine, guanidinopropionic acid, and methyltryptophan (fig. 27 b).

In addition, intervention of Y301 reduces the levels of pro-inflammatory metabolites such as tetradecanoic acid, thereby reducing the levels of intestinal inflammatory factors including TNF- α, IFN- γ, and D-lactic acid. This series of changes affects the alanine, aspartate and glutamate metabolic pathways, increasing the levels of methyltryptophan and melatonin, thereby enhancing the antioxidant capacity of the gut and maintaining gut health.

Thus, the findings of the present application suggest that the intervention feeding of probiotic fermented coconut water may help to promote slow recovery of chickens from acute enteritis. Relevant data results of improving physical, biochemical and immunological barriers of intestinal tracts are observed, thereby helping recovery of Wenchang chickens in DSS induced acute enteritis.

In conclusion, the study of the invention shows that the metazoan formed by probiotics and fermented coconut water has positive effects on the recovery of acute enteritis of Wenchang chickens: saccharomyces CEREVISIAE Y301 regulates amino acid metabolism, results in higher levels of melatonin, promotes cell proliferation and development, intervenes to improve chicken growth rate, lactiplantibacillus plantarum MS c intervenes to regulate glycerophospholipid metabolic pathways, remodels intestinal flora structure of acute enteritis chicken, reduces the abundance of potential pathogenic bacteria of ALISTIPES genus, and increases the abundance of potentially beneficial species such as L.plantarum, CHRISTENSENELLACEAE, and the like. But also results in the production of beneficial intestinal metabolites such as gentamicin C and polymyxin B2, which are recognized for their therapeutic effect on acute enteritis. The combined intervention of S.cerevisiae Y301 and L.plantarum MS2C not only enhances the growth performance, but also reduces the damage of intestinal walls, and increases the abundance of intestinal metabolites such as gentamicin C, polymyxin B2 and the like, thereby reducing enteritis symptoms. In addition, this combined intervention also reduced serum immune marker levels, including IL-10, IL-6, TNF- α, IFN- γ and D-lactate, thereby alleviating intestinal epithelial cell damage and promoting recovery from acute enteritis. The research provides important insights for probiotics and the action mechanism of the coconut water fermented by the probiotics in recovery of acute enteritis, and provides a new view for sustainable green cultivation practice of Wenchang chickens.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.

Claims

1. A novel probiotic lactic acid bacterium, named lactobacillus plantarum (Lactiplantibacillus plantarum) MS2c, deposit No. GDMCC No:64184.

2. A novel probiotic yeast, named Saccharomyces cerevisiae (Saccharomyces cerevisiae) Y301, accession number GDMCC No:64183.

3. The prebiotics of the coconut water fermented by the probiotics are selected from lactobacillus fermented coconut water prebiotics which take lactobacillus MS2c single bacteria as fermentation strains, or saccharomycetes fermented coconut water prebiotics which take saccharomycetes Y301 single bacteria as fermentation strains, or compound bacterial strains fermented coconut water prebiotics which take lactobacillus MS2c and saccharomycetes Y301 together as fermentation strains; the lactobacillus MS2c has a preservation number of GDMCC No:64184; the preservation number of the saccharomycete Y301 is GDMCC No:64183.

4. A method for preparing a prebiotic after fermentation of coconut water by probiotics, comprising: simultaneously inoculating lactobacillus MS2c and saccharomycete Y301 according to the following lactobacillus: the yeast is in a ratio of 1:2, 4% of total inoculum size is used for fermenting fresh coconut water by the composite strain, the fermentation temperature is 33-37 ℃, and the fermentation time is 24-30 hours; the lactobacillus MS2c has a preservation number of GDMCC No:64184; the preservation number of the saccharomycete Y301 is GDMCC No:64183.

5. A poultry feed additive comprising the probiotic fermented coconut water metazoan of claim 3.

6. Use of the probiotic fermented coconut water metazoan of claim 3 in the manufacture of a medicament for use in the growth and promotion of self-healing recovery of enteritis in birds, such as Wenchang chickens.