Composite microecological preparation, fermentation liquor and application of composite microecological preparation in laying hen production

Abstract

The invention discloses a compound microecological preparation, fermentation liquor and application of the compound microecological preparation in laying hen production. The compound micro-ecological preparation is prepared from clostridium butyricum NIP1021, bacillus cereus 2022B1, bacillus licheniformis 2022B2 and enterococcus faecium 2022B6. The fermentation liquor of the compound micro-ecological preparation can promote the growth performance levels of the Haillan grey laying hens, such as the final weight and the like; the development of thymus and bursa of Fabricius immune organs of laying hens can be promoted; the content of serum immune globulin IgG and serum-related immune cell factors IL-1beta, IL-6 and TNF-alpha of laying hens is remarkably increased, so that the serum nonspecific immune level of animal bodies is improved; according to the method, the intestinal villus area of the laying hens is increased, the nutrient absorption capacity of the laying hens is improved, cecum flora diversity and species abundance are increased, dominant flora categories are thick-wall flora and bacteroides to regulate intestinal flora balance, and the method has positive significance in maintaining intestinal health.

Description

Technical Field

[0001] The present invention belongs to the technical field of composite microbial preparations, and more specifically, relates to a composite microecological preparation, a fermentation liquid and an application thereof in laying hen production. Background Art

[0002] China's egg production accounts for about 40% of the world's total egg production, making it the world's largest egg producer and consumer. At the same time, eggs are an important source of protein for humans. In recent years, the advocacy of green and nutritious poultry products has received great attention, and the awareness of food safety has also been further strengthened.

[0003] At present, laying hens are usually raised in cages and fed, which are prone to diseases. It is not easy for farmers to prevent and control diseases in laying hens. When a certain disease occurs in the flock and medication is required, some commonly used drugs such as aminophylline, chlortetracycline, sulfonamides and other drugs will inhibit or damage the immune system of laying hens. In particular, the drug resistance and drug residues caused by the abuse of antibiotics will bring great harm to human health. In recent years, my country has also announced and issued relevant policies and measures on "banning antibiotics" in livestock and poultry production and application. Therefore, the research and development of natural and harmless livestock and poultry feed products that replace antibiotics is a current research hotspot. It is in the context of the national "ban on antibiotics" that finding efficient and safe antibiotic substitutes is the biggest opportunity and challenge facing the livestock and poultry industry.

[0004] Compound microbial preparations, also known as "compound microecological preparations", are microbial preparations that are cultivated in appropriate proportions to give full play to the advantages of the group and achieve the best application effect. Compound microecological preparations are most widely reported in the research and application of poultry broilers and laying hens. Compared with pure live bacteria containing only a single bacterium, compound microecological preparations present a more complex composition structure, more comprehensive efficacy and higher homeostasis. Compound microecological preparations are more adaptable to the complexity of agricultural and pastoral environments. For example, Chinese patent CN114921372A discloses a probiotic preparation, preparation method and application for improving egg quality, and Zhang Wei et al. disclose the effects of a composite microecological preparation on laying performance, egg quality, serum biochemical indicators and intestinal morphology of laying hens (Zhang Wei, Zuo Fangrui, Qiu Quan, et al. Effects of composite microecological preparations on laying performance, egg quality, serum biochemical indicators and intestinal morphology of laying hens [J]. Feed Research, 2022, 45(02): 40-45.), however, the sources of these strains are unknown, and the strains will have a harmful effect on poultry due to individual differences, and the growth performance of laying hens needs to be further improved. Therefore, providing a composite microecological preparation from a chicken source that can promote the growth performance of laying hens, improve the immunity of laying hens, improve the balance of intestinal flora of laying hens, promote intestinal health of laying hens, and reduce the use of antibiotics is of great significance to the healthy breeding of laying hens. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a composite microecological preparation.

[0006] The second object of the present invention is to provide the use of the composite microecological preparation in preparing brown sugar fermentation broth.

[0007] The third object of the present invention is to provide a method for preparing the fermentation broth of the composite microecological preparation.

[0008] The fourth object of the present invention is to provide a fermentation broth of the composite microecological preparation prepared by the preparation method.

[0009] The fifth object of the present invention is to provide an application of the fermentation broth of the composite microecological preparation.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] A composite microecological preparation, composed of Clostridium butyricum NIP1021, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6, wherein the bacterial amount ratio of Clostridium butyricum NIP1021, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6 is (0.5-2): (0.5-2): (0.5-2); the Bacillus cereus 2022B1 was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64738; the Bacillus licheniformis is 2022B2, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64739; the Enterococcus faecalis is 2022B6, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64740.

[0012] The present invention separates and purifies several purified strains from the intestinal flora and feces of healthy broilers. After subsequent Gram staining microscopy, morphological observation, and biochemical identification of the strains, strains that roughly meet the characteristics of probiotics are selected. The bacterial 16S rDNA gene sequencing comparison and the construction of the evolutionary tree obtain strains with a similarity of 99% or more, determine the species, and finally screen out three chicken-derived probiotics: Bacillus cereus 2022B1, Bacillus licheniformis 2022B2, and Enterococcus faecium 2022B6; and these three chicken-derived probiotics are combined with Clostridium butyricum NIP1021 strains known in the prior art to form a composite microecological preparation. Each strain has excellent probiotic properties, good drug resistance, acid resistance, bile salt resistance, agglutination in the intestine (self-agglutination and other agglutination) and inhibition of the growth of common intestinal pathogens; it can improve the growth performance of laying hens and enhance immunity. At the same time, the composite microbial preparation can be propagated in a low pH environment to avoid contamination by miscellaneous bacteria and reduce fermentation costs.

[0013] Furthermore, the bacterial amount ratio of Clostridium butyricum NIP1021, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6 is 1:1:1:1.

[0014] Furthermore, the viable count of the composite microecological preparation is 10 8 ~10 9 cfu / mL.

[0015] The present invention also provides application of the composite microecological preparation in preparing brown sugar fermentation broth.

[0016] The present invention also provides a method for preparing a fermentation liquid of a composite microecological preparation, which is to first use molasses as a raw material, ferment the seed liquid of each strain in the composite microecological preparation alone (first fermentation) to obtain the fermentation seed liquid of each strain, and then use brown sugar as a raw material, compound the seed liquid of the first fermentation according to the bacterial amount ratio, and then ferment it for the second time to prepare the fermentation liquid. The pH is 4.8-4.9. Furthermore, the pH of the first fermentation culture is 4.4-4.6, and the inoculated bacterial amount is 10 8 CFU / mL.

[0017] Furthermore, the composition ratio of the brown sugar fermentation liquid in the second fermentation culture is: the first fermentation seed liquid (mL): brown sugar (g): water at about 30°C (28-32°C) (mL) = 1:5:200, and the fermentation bacteria amount is 10 8 ~10 9 CFU / mL, fermentation time is 24 to 48h.

[0018] The present invention also provides a fermentation broth of a composite probiotic preparation prepared by any of the above methods. The fermentation broth of the composite probiotic preparation of the present invention can promote the growth performance level of Hy-Line gray laying hens, such as the final weight; can promote the development of the immune organs of the thymus and bursa of Fabricius of laying hens; significantly increase the serum immunoglobulin IgG and serum-related immune cytokines IL-1β, IL-6, and TNF-α content of laying hens to improve the serum nonspecific immunity level of the animal body; increase the villus area of ​​the small intestine of laying hens to improve the ability to absorb nutrients and increase the diversity and species abundance of the cecal flora, and regulate the balance of intestinal flora with the dominant flora being Firmicutes and Bacteroidetes.

[0019] Therefore, the present invention also provides the use of the fermentation broth of the above-mentioned composite microecological preparation in the preparation of products that promote the growth performance of laying hens, improve the balance of intestinal flora of laying hens, promote the intestinal health of laying hens, and enhance the immunity of laying hens.

[0020] Furthermore, the application dosage of the fermentation liquid of the composite microecological preparation is 1 mL / kg body.

[0021] Furthermore, the product is feed.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention combines the screened chicken-derived probiotics Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6 with the existing Clostridium butyricum NIP1021 to prepare a composite microecological preparation. The chicken-derived probiotics are derived from the intestinal flora and feces of healthy broilers, and can be directly used in the composite formula of poultry probiotics, thereby avoiding the harmful effects of individual differences in the strains on poultry, and having the ability to reproduce in a low pH environment; each strain has excellent probiotic properties, good drug resistance, acid resistance, bile salt resistance, agglutination effect in the intestine (self-agglutination and other agglutination) and inhibition of the growth of common intestinal pathogens. The composite microecological preparation prepared by fermenting brown sugar with the composite microbial agent fermentation liquid can promote the growth performance level of Hy-Line gray laying hens, such as the final weight; can promote the development of the immune organs of the thymus and bursa of Fabricius of laying hens; significantly increase the content of serum immunoglobulin IgG and serum-related immune cytokines IL-1β, IL-6, and TNF-α of laying hens to improve the serum nonspecific immunity level of the animal body; increase the villus area of ​​the small intestine of laying hens to improve the ability to absorb nutrients and increase the diversity and species abundance of cecal flora, and regulate the balance of intestinal flora with the dominant flora being Firmicutes and Bacteroidetes; that is, the brown sugar fermentation liquid of the composite microecological preparation has the functions of promoting the growth performance of laying hens, improving the balance of intestinal flora of laying hens, promoting the intestinal health of laying hens, improving the immunity of laying hens, and reducing the use of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology of each bacterial species in the composite microecological preparation. (a) Bacillus cereus, (b) Bacillus licheniformis, (c) Enterococcus faecium, (d) Clostridium butyricum.

[0025] Figure 2 The morphological diagrams of the bacteria in the composite microecological preparation are as follows: (a) Bacillus cereus, (b) Bacillus licheniformis, (c) Enterococcus faecium, and (d) Clostridium butyricum.

[0026] Figure 3 Phylogenetic tree constructed for the 16S rDNA of the chicken-derived probiotic Bacillus cereus.

[0027] Figure 4 Phylogenetic tree constructed for the 16S rDNA of the chicken-derived probiotic Bacillus licheniformis.

[0028] Figure 5 Phylogenetic tree constructed for the 16S rDNA of the chicken-derived probiotic Enterococcus faecium.

[0029] Figure 6 This is the growth curve of each strain in the composite microecological preparation.

[0030] Figure 7This is a comparison chart of the antibacterial ability of various strains of the composite microecological preparation against pathogens.

[0031] Figure 8 This is a comparison chart of the acid resistance of various strains in the composite microecological preparation.

[0032] Fig. 9 This is a comparison chart of the bile salt resistance of various strains in the composite microecological preparation.

[0033] Fig.10 This is the pH change curve of brown sugar fermentation of the composite microecological preparation.

[0034] Fig.11 This is a graph evaluating the growth performance of laying hens fed with compound proecological preparations for 21 days and 42 days.

[0035] Fig.12 This is a graph evaluating the immune organ index of laying hens.

[0036] Fig.13 To establish the standard curves of serum immunoglobulins and related immune cytokines: (a) IgM standard curve, (b) IgG standard curve, (c) IL-1β standard curve, (d) IL-6 standard curve, (e) TNF-α standard curve, (f) IFN-α standard curve.

[0037] Fig.14 Changes in serum immunoglobulin levels in laying hens fed with compound probiotic preparations for 21 days and 42 days. (a) Changes in serum immunoglobulin levels at 21 days, (b) Changes in serum immunoglobulin levels at 42 days.

[0038] Fig.15 Changes in serum immune cytokine levels of laying hens fed with compound probiotics for 21 days and 42 days. (a) Changes in serum immune cytokine levels at 21 days, (b) Changes in serum immune cytokine levels at 42 days.

[0039] Fig.16 This is a schematic diagram of the intestinal villi of the duodenum of laying hens.

[0040] Fig.17 This is a schematic diagram of the jejunal villi of laying hens.

[0041] Fig.18 This is a schematic diagram of the ileum villi of laying hens.

[0042] Fig.19 is the relative abundance of phylum level of cecal flora in laying hens.

[0043] Fig. 20 is the relative abundance of cecal flora at the genus level in laying hens. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] The Clostridium butyricum NIP1021 strain has a deposit number of FERM BP-5795 and was deposited on January 23, 1995 at the Institute of Biotechnology, Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry, Room 3, 1 Higashi 1-jo Street, Tsukuba City, Ibaraki Prefecture, Japan. The patent protection period has now expired and it is deposited in the Sericulture Comprehensive Utilization Laboratory, College of Animal Science, South China Agricultural University.

[0047] Example 1 Isolation and identification of chicken-derived probiotics

[0048] Chicken probiotics are derived from the intestinal flora and feces of healthy broilers. Slaughter healthy broilers and collect feces, process the intestinal contents and immerse them completely in salt water and ensure that the contents and feces are fully mixed with the salt water. Treat it in a static water bath at 80°C for 20 minutes, 3000-4000r / min, 10 minutes, then take 1 ml of the supernatant and put it into a sterile test tube. Then dilute it according to the concentration gradient by 10 -1 -10 -6 , prepare suspensions of different concentrations, and then take 100 μL of samples from each dilution and spread them evenly on LB agar plates. After constant temperature culture at 37°C for 24-48 hours, use a disposable sterile inoculation loop to pick a single colony with a diameter of 2 to 3 mm, and then continuously dilute, streak, and spread the picked colony to purify it so that several fields of view under the microscope are single bacteria, and obtain several purified strains.

[0049] (1) After subsequent Gram staining, morphological observation, and biochemical identification, strains that roughly meet the characteristics of probiotics were selected. Finally, three types of chicken-derived probiotics were selected, Bacillus cereus, Bacillus licheniformis, and Enterococcus faecium. They were numbered 2022B1, 2022B2, and 2022B6, respectively. The colony morphology is shown in Figure 1 (a, b, c), Gram staining microscopy Figure 2 (a, b, c), the results are summarized in Tables 1 and 2 below.

[0050] Table 1 Gram staining microscopy and morphological observation of chicken-derived probiotics

[0051]

[0052] from Figure 1 (a, b, c), Figure 2 (a, b, c) and Table 1 show that all three strains are Gram-positive bacteria. In terms of morphological observation: 2022B1 is white, round, waxy, rod-shaped, and has spores; 2022B2 is white, irregularly round, rough, sticky, straight rod-shaped, and has spores; 2022B6 is white, round, dry, shiny, spherical or chain-shaped.

[0053] Table 2 Biochemical reaction identification results of chicken-derived probiotics

[0054]

[0055]

[0056] As can be seen from Table 2, referring to the Bergey's Manual of Bacterial Identification and the Manual of Common Bacterial Identification, Bacillus 2022B1, 2022B2; Streptococcus 2022B6 that decomposes sugars and produces acid.

[0057] (2) Bacterial 16S rDNA gene sequencing: The genomic DNA of each strain was extracted according to the method for extracting genomic DNA of Gram-positive bacteria. PCR amplification used the 16S rDNA bacterial strain identification PCR kit of Shanghai Biotech. In order to ensure that more comprehensive 16S rDNA sequence information was obtained, three sequencing primers were provided at the same time. The primer sequences are shown in Table 3. The PCR reaction system is shown in Table 4.

[0058] Table 3 16S rDNA bacterial species identification PCR sequencing primer sequences

[0059]

[0060] Note: Forward sequencing primer Seq F, middle sequencing primer Seq I, and reverse sequencing primer Seq R.

[0061] Table 4 PCR amplification reaction system

[0062]

[0063] Note: PCR primer F / R1 is used to amplify the first 500 bp of bacterial 16S rDNA, and F / R2 is used to amplify the full sequence DNA fragment, which is about 1500 bp in size.

[0064] The PCR products of each strain were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequences were compared with NCBI database by BLAST, and the strains and sequences with similarity greater than 99% were obtained, as shown in Table 5. The phylogenetic tree was constructed by the neighbor method using MEGA6.0 software, as shown in Table 5. Figure 3 , Figure 4 , Figure 5. The species of probiotics from chicken can be determined.

[0065] Table 5 BLAST comparison of chicken probiotic sequence results

[0066]

[0067] Example 2 Strain Performance Test

[0068] The strains screened in Example 1 were subjected to growth curve determination, drug sensitivity test, antibacterial test and in vitro evaluation of acid and bile resistance with Clostridium butyricum NIP1021 preserved in the laboratory. First, the Clostridium butyricum preserved by the glycerol preservation method was revived and activated, and the purity and characteristics of the revived strains should be confirmed. The propagation method of each strain is: pick fresh colonies or bacterial mosses, inoculate into a 50mL sterilized conical flask under aseptic operation, generally select 25mL of TSB liquid culture medium or LB liquid culture medium, 37℃180-200rpm, and culture for 18h-24h to obtain the bacterial stock solution.

[0069] Clostridium butyricum Figure 1 (d) and Figure 2 (d) It is a Gram-positive bacterium that forms white or creamy shiny, large or small round colonies on LB agar plates, which are slightly protruding and appear as straight or curved rods or short chains under the microscope.

[0070] (1) Growth curve determination

[0071] To ensure that the OD600 absorbance value of the bacterial growth curve is between 0.10 and 0.65, the dilution multiple of the bacterial stock solution can be obtained after preliminary experiments. The operation method of the bacterial growth curve determination is as follows: First, in this experiment, each bacterial stock solution is diluted 1000 times with uninoculated LB liquid culture medium, and 200 μL of each bacterial dilution is taken in any well of a 96-well plate, and two duplicate wells are made. Each bacterial dilution is added in sequence to avoid confusion and should be marked; at least three wells of the 96-well plate are left with two uninoculated culture media as blank control solutions. Control the addition time well to avoid affecting the results. The fully automatic microbial growth curve determination instrument on the plate after the sample is added determines the changes in the OD600 value of the strain at different times of growth. The instrument determination setting starts from time 0 and determines the OD600 absorbance value of the bacterial solution every 2 hours for 24 hours. The OD600 value measured by each bacterial solution is repeated three times and the average value is taken; the OD value measured after dilution must be multiplied by the dilution multiple to obtain the actual OD value. The growth curves of each strain were plotted using OD600 values ​​at different times. Figure 6 .

[0072] from Figure 6It can be obtained that the logarithmic phases of Bacillus cereus 2022B1 and Clostridium butyricum are 4h-20h and 2h-18h respectively, and the bacterial culture time should be harvested within 20h and 18h ​​respectively; the logarithmic phase of Bacillus licheniformis 2022B2 is 2h-22h, and the bacteria can be harvested within 22h of culture; the logarithmic phase of Enterococcus faecalis 2022B6 is 0h-20h, and the bacterial culture time is controlled within 20h of culture.

[0073] (2) Drug sensitivity test

[0074] First, a fresh single colony was selected and cultured in LB broth medium for 16-18 h to prepare a 0.5 McFarland turbidimeter (1-1.5×10 8 CFU / mL). 100 μL of the suspension was applied to LB solid culture medium and the surface was completely dry. Use sterile tweezers to select each antibiotic susceptibility sheet and stick it on the surface of the plate. The susceptibility sheet and the culture medium should be tightly attached. Each antibiotic should be repeated twice. Invert the plate and incubate at 37°C for 24 hours. Use a vernier caliper to measure the diameter of the inhibition ring, observe and record. The results were determined by measuring the inhibition zone in mm. The inhibition zone is usually over 20 mm in size for extremely sensitive; 15-20 mm for highly sensitive; 10-14 mm for moderately sensitive; and insensitive or no inhibition zone if the diameter is less than 10 mm. The inhibition zone diameter is over 15 mm for sensitive. The results are shown in Table 6.

[0075] Table 6 Drug sensitivity results

[0076]

[0077]

[0078] From Table 6, it can be seen that Bacillus cereus 2022B1 is insensitive, i.e. resistant, to penicillin, ampicillin, ceftriaxone and lincomycin, but is sensitive to the other antibiotics; Bacillus licheniformis 2022B2 is resistant to penicillin, erythromycin and lincomycin, but is sensitive to the other antibiotics; Enterococcus faecalis 2022B6 is resistant to ceftazidime and lincomycin, but is sensitive to the other antibiotics; Clostridium butyricum is resistant to penicillin, ceftazidime, piperacillin and polymyxin, but is sensitive to the other antibiotics.

[0079] (3) Antibacterial test

[0080] This experiment uses a bacterial agglutination test. The pathogenic bacteria Salmonella (Salmonellatyphimurium SED-1) and Staphylococcus aureus (Staphylococcus aureus SA) preserved in the laboratory were revived and activated. After pure culture, fresh single colonies were selected and shaken in LB liquid culture medium for 16-18 hours to prepare a bacterial suspension of the pathogen; for the strains and pathogens, the bacterial solution was mixed with PBS buffer in a certain proportion so that the OD value of the mixed solution was about 0.6-0.8 at a wavelength of 600nm, and the absorbance value of each bacterial solution was recorded as Ax, and the pathogen was recorded as Ay. An equal volume (2mL) of each bacterial suspension and a pathogenic bacteria suspension were mixed and incubated at room temperature for 5h. A control group containing 4mL of a single bacterial suspension was cultured under the same growth conditions, and the OD value was measured at 600nm. The absorbance value of each bacterial suspension and the mixed solution of pathogens was A 样 , OD600 is used to measure the absorbance of the upper layer of the bacterial mixture. Calculation formula: Inhibition rate (%) = [(Ax + Ay) / 2-A 样 ] / (Ax+Ay)×100%, the results are shown in Table 7 and Figure 7 .

[0081] Table 7 Antibacterial rate

[0082]

[0083] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different lowercase letters indicate that the difference is significant (P<0.05).

[0084] From Table 7 and Figure 7 It can be seen that Clostridium butyricum had the best agglutination ability against SED-1 and SA, and its SED-1 inhibition rate of 38.62±2.07% was significantly higher than that of 2022B1 (25.58±2.14%) (P<0.05), and the inhibition rate of Clostridium butyricum against SA was 54.29±4.22%, which was significantly higher than that of other strains (P<0.05).

[0085] The agglutination ability of 2022B2 on SED-1 and SA was higher than that of 2022B1 and 2022B6. ​​Its SED-1 inhibition rate of 34.00±3.88% was significantly higher than that of 2022B1 (25.58±2.14%) (P<0.05) and that of 2022B6 (32.82±3.73%) (P>0.05). The inhibition rate of 2022B2 on SA was 44.89±1.02%, which was significantly higher than that of 2022B1 (39.96±1.41%) (P<0.05).

[0086] (4) Acid resistance test

[0087] The bacterial count reached 10 8CFU / mL of bacterial solution was added to MRS liquid culture medium at a 1% (v / v) inoculation rate. The pH value of the experimental group culture medium was adjusted to 1.5, 2.5, 3.5, and 4.5 with artificial gastric juice, and the blank group culture medium was not adjusted. After culturing at 37°C for 4 hours, each bacterial solution was diluted 10 -1 -10 -6 , and then 100 μL of the bacterial dilution was spread on MRS solid medium and cultured at 37°C for 48 hours. Three appropriate dilution plates were selected to calculate the colony count and strain survival rate, and each sample was repeated 3 times.

[0088] Strain survival rate (%) = number of viable bacteria in different pH medium / number of viable bacteria in conventional medium × 100%

[0089] Table 8 Survival rate of each bacteria under different acidic conditions

[0090]

[0091] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different lowercase letters indicate that the difference is significant (P<0.05).

[0092] From Table 8 and Figure 8 It can be obtained that in the pH 1.5 environment, the 18±1.18% survival rate of 2022B6 was significantly higher than that of 2022B1 (P<0.05). In the pH 2.5 environment, the 37±0.62% survival rate of 2022B6 and the 38±1.02% survival rate of 2022B2 were significantly higher than those of the other strains (P<0.05). In the pH 3.5 environment, the 52±2.30% survival rate of 2022B2 and the 53±4.01% survival rate of 2022B6 were significantly higher than those of the other strains (P<0.05). In the pH 4.5 environment, the 73±1.03% survival rate of 2022B6 was significantly higher than that of the other strains (P<0.05). The best performance in the acid resistance test was 2022B2 and 2022B6, followed by butyric acid bacteria and 2022B1.

[0093] (5) Bile salt tolerance test. MRS liquid medium was added with artificial intestinal fluid to prepare medium containing 0.03%, 0.1%, 0.3%, 0.5%, and 1% bile salt concentrations. 8 CFU / mL of bacterial solution was added to MRS liquid medium at a 1% (v / v) inoculation rate. The medium was added with artificial intestinal fluid to prepare 5 experimental groups with different bile salt concentrations. The control group was not treated. After static culture at 37°C for 24 hours, each bacterial solution was diluted 10 with PBS buffer. -1 -10 -6, and then take 100 μL of the bacterial dilution solution and spread it on MRS solid medium, and culture it at 37℃ for 48 hours. Select the appropriate three dilution plates to calculate the colony count and strain survival rate, and repeat each sample 3 times. Calculate the survival rate.

[0094] Survival rate (%) = number of viable bacteria in the bile salt group / number of viable bacteria in the control group × 100%

[0095] Table 9 Survival rate of each bacteria under different bile salt conditions

[0096]

[0097] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different lowercase letters indicate that the difference is significant (P<0.05).

[0098] From Table 9 and Fig. 9 It can be obtained that under the five bile salt conditions of 1%, 0.5%, 0.3%, 0.1% and 0.03%, the survival rate of 2022B2 was significantly higher than that of the other strains (P < 0.05), which were 60 ± 1.37%, 77 ± 1.06%, 87 ± 1.17% and 98 ± 3.78% respectively. Under the bile salt conditions of 1%, 0.5%, 0.3% and 0.03%, the survival rate of 2022B6 was significantly higher than that of Clostridium butyricum and 2022B1 (P < 0.05), which were 45 ± 1.17%, 59 ± 0.41%, 67 ± 2.19% and 88 ± 1.97% respectively. 2022B2 had the best bile salt tolerance, followed by 2022B6, Clostridium butyricum and 2022B1. Example 3 Preparation of fermentation broth of composite microecological preparation and its effect on laying hen production performance

[0099] The screening strains were preserved by glycerol method. The prepared fresh bacterial suspension and sterilized glycerol were divided into cryopreservation tubes at a ratio of 7:3 and marked. After being fully mixed, they were stored in a -80°C refrigerator.

[0100] 1. Preparation of compound microecological preparation fermentation broth

[0101] First, take fresh colonies or bacterial mosses of each strain and inoculate them into TSB culture medium, shake and culture them at 37℃, 180-200rpm for 18-24h, perform Gram staining and microscopic examination to ensure that there is no contamination by foreign bacteria, and then prepare the seed solution of LB broth culture medium and store it at -4℃ for later use. Then, for the first fermentation culture, prepare 500mL molasses culture medium in a 1L micro-fermenter, adjust the pH to about 4.5, sterilize at 115℃ for 15min, and wait until the temperature drops to 37℃. Inoculate 10 8 CFU / mL seed solution, ferment for 24 hours, pay attention to the pH change and adjust it appropriately. The first fermentation is for each bacteria to adapt to the acidic fermentation environment independently. The amount of each bacteria can reach 10 in 24 hours of the first fermentation. 5CFU / mL or so.

[0102] The second fermentation is brown sugar fermentation. The bacterial combinations are mixed and fermented. The amount of each bacterial combination before fermentation is 10% of that achieved in the first fermentation. 5 The ratio of the bacterial amount of each fermentation liquid is 1:1:1:1. The ratio of the first fermentation seed liquid (mL): brown sugar (g): water at about 30°C (mL) = 1:5:200. The second fermentation condition of brown sugar is to seal the fermentation container, and then stand at room temperature for 24 hours before the fermentation is completed. The fermentation bacterial amount is 10 8 -10 9 CFU / mL.

[0103] The fermented seed liquid of the composite probiotics was inoculated into the correspondingly configured brown sugar fermented liquid and fermented for 48 hours. Starting from time 0, the pH value change was measured with a pH meter at intervals of 2 hours, and the change curve was drawn.

[0104] from Fig.10 The pH value of the composite microecological preparation increased from 4.7 at 0h to 5.4 at 14h, dropped to below 5.0 at 24h, and remained within 4.8 at 48h, indicating that the composite microecological preparation can ensure normal fermentation under a low pH environment during brown sugar fermentation.

[0105] 2. Test on the effect of compound microecological preparation fermentation liquid on laying hen production performance

[0106] (1) Animal husbandry. 90 healthy 7-day-old Hy-line grey laying hens were randomly divided into three groups: B, Y, and K. Each group had 3 replicates, with 10 hens in each replicate. In the experimental group B, the strain combination was the above-mentioned Clostridium butyricum, Bacillus cereus, Bacillus licheniformis, and Enterococcus faecium composite microecological preparation. The positive control group in group Y was a product provided by a certain company, which was Bacillus subtilis and Enterococcus faecium (1:1, 10 each). 5 CFU / g) composite microecological preparation, which was also added to drinking water after secondary brown sugar fermentation. Group K was a blank negative control group. The volume ratio (v / v) of the fermented liquid of each group of bacteria to drinking water was 1%. The weight of each group of laying hens was weighed and recorded before the experiment. The feeding cycle was 42 days. During the whole feeding process, each group of laying hens had free access to food (basic feed) and free drinking water. The feeding method was flat breeding in cages. The entire feeding cycle was 42 days, and the feed consumption of each group of laying hens was recorded throughout the whole process. The initial weight of each group of laying hens on an empty stomach was weighed and recorded before the experiment, recorded as the starting weight. At the same time, the fasting weight of each group of laying hens after feeding for 21 days and after feeding for 42 days was weighed and recorded as the final weight data, referred to as the final weight. Based on this data, the average daily weight gain (ADG), average daily feed intake (ADFI), feed-to-weight ratio (F / G) and mortality rate of laying hens in each growth period were evaluated. The calculation formula is as follows:

[0107] ADG = (final weight - initial weight) / number of days of feeding

[0108] ADFI = total feed intake / number of days of feeding

[0109] F / G = average daily feed intake / average daily weight gain

[0110] Before the start of this study, the empty stomach weight of each group of laying hens was weighed and recorded as the initial weight, which was 61.46±1.25g. According to the records, the final weight, average daily weight gain (ADG), average daily feed intake (ADFI) and feed-to-weight ratio (F / G) of laying hens fed with compound probiotic preparations in two stages (1-21d and 1-42d) were calculated as shown in Table 10 and Fig.11 The mortality rate is shown in Table 11.

[0111] Table 10 Evaluation of growth performance of laying hens fed with compound probiotics for 21 days and 42 days

[0112]

[0113]

[0114] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05). "21B" refers to group B on day 21, and "42B" refers to group B on day 42.

[0115] From Table 10 and Fig.11 It can be seen that the final weight at 21 days: Group B was higher than the positive control group Y and the negative blank control group K (P>0.05); ADG: Group B was higher than the control group Y and K (P>0.05); ADFI: Group B was higher than the control group Y and K (P>0.05); F / G: Group B was higher than the control group Y and K (P>0.05). The final weight at 42 days: Group B was higher than the positive control group Y (P>0.05), and significantly higher than the negative blank control group K (P<0.05); ADG: Group B was higher than the control group Y and K (P>0.05); ADFI: Group B was higher than the control group Y and K (P>0.05); F / G: Group B was higher than the control group Y and K (P>0.05).

[0116] The results showed that after 42 days of feeding with the compound microecological preparation, the growth performance of laying hens in group B was significantly better than that in the control group K (P < 0.05). The growth performance ADG, ADFI, F / G and final weight of laying hens fed with the compound microbial preparation were higher than those of the control groups Y and K, and their final weight was significantly higher than that of the control group K at 42 days of feeding with the compound microbial preparation (P < 0.05).

[0117] Table 11 Mortality rate of chickens fed with compound probiotic preparations

[0118]

[0119] As can be seen from Table 11, the mortality rate of chickens has no direct relationship with feeding compound microecological preparations.

[0120] (2) After feeding the composite microbial agent for 28 days, 5 laying hens were randomly selected from each group and repeated, and their fasting body weights were recorded. Three laying hens were randomly selected from each repeated group and killed, and their thymus, spleen, and bursa of Fabricius were removed of fat and connective tissue and weighed and recorded. The organ indexes of the thymus, spleen, and bursa of Fabricius were calculated.

[0121] Immune organ index = immune organ weight (g) / animal body weight (kg)

[0122] Table 12 Immune organ index of laying hens fed with compound probiotics for 28 days

[0123]

[0124]

[0125] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05).

[0126] From Table 12 and Fig.12 The immune organ development of laying hens fed with compound probiotics for 28 days can be obtained. Thymus index: The experimental group B was 8.95±0.49, significantly higher than the blank group K (P<0.05). Spleen index: The experimental group B was 7.07±0.12, higher than the control groups Y and K, but the differences between the groups did not reach a significant level (P>0.05). Bursa index: The group B was 6.68±0.07, significantly higher than the blank group K (P<0.05).

[0127] The immune organ development index of experimental group B was higher than that of blank group K, among which the thymus index and bursa index were significantly higher than those of blank group K (P < 0.05), indicating that the composite microecological preparation has a certain promoting effect on the development of immune organs of laying hens.

[0128] (3) Three laying hens were randomly selected from each replicate after being fed with the compound probiotic preparation for 21 days and 42 days, and 5-8 mL of blood was collected from the wing vein. The blood collection tube was tilted at 45 degrees and allowed to stand at room temperature. The blood was centrifuged at 3000 r / min for 20 minutes at low temperature. The serum was aliquoted and stored at -80°C for later use.

[0129] Competitive enzyme-linked immunosorbent assay kit was used to measure the levels of immunoglobulins IgM and IgG in laying hen serum; the levels of related immune cytokines in serum, such as IL-1β and IL-6 in chicken interleukin (Interleukin), TNF-α in chicken tumor necrosis factor (Tumor Necrosis Factor), and IFN-α in chicken interferon (Interferon).

[0130] All kits were equilibrated at room temperature for 30 minutes before use. Washing solution, standard, biotin antigen, and avidin-HRP dilution were prepared before the test. The original solution, 10-fold dilution, and 100-fold dilution were also prepared for sample processing. The regression equation of the standard curve was calculated based on the concentration and OD value, and the calculation was performed using the dedicated calculation software ELISAcale. The fitting model can be a logistic curve (four parameters). See the standard curve establishment for details. Fig.13 For specific operations, please refer to the ELISA kit instructions of Nanjing Jiancheng Company.

[0131] Table 13 Serum immunoglobulin expression

[0132]

[0133]

[0134] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05). "21B" refers to group B on day 21, and "42B" refers to group B on day 42.

[0135] From Table 13 and Fig.14 It can be seen that at 21 days, the IgM of each group had no significant difference (P>0.05), and the content of the experimental group B was higher than that of the control group Y and K, 7.14±2.01 mg / mL in group B. IgG: The content of the experimental group B was higher than that of the control group Y (P>0.05), and significantly higher than that of the blank group K (P<0.05), 44.71±6.63 mg / mL in group B.

[0136] IgM at 42 days: There was no significant difference among the groups (P>0.05), and the content of group B in the experimental group was higher than that of control groups Y and K, with group B at 6.70±0.52 mg / mL. IgG: The content of group B in the experimental group was higher than that of control groups Y and K (P>0.05), with group B at 27.97±6.34 mg / mL. The content of serum immunoglobulin IgM and IgG in laying hens at 42 days decreased compared with that at 21 days.

[0137] Table 14 Expression of serum-related immune cytokines

[0138]

[0139] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05). "21B" refers to group B on day 21, and "42B" refers to group B on day 42.

[0140] From Table 14 and Fig.15 It can be seen that at 21 days, the content of IL-1β in the experimental group B was higher than that in the control group Y (P>0.05), and significantly higher than that in the blank group K (P<0.05), and the content of group B was 63.88±7.82ng / L. IL-6: The content of the experimental group B was lower than that in the control group Y (P>0.05), and significantly higher than that in the blank group K (P<0.05), and the content of group B was 70.28±9.02ng / L. TNF-α: There was no significant difference among the groups (P>0.05), and the content of the experimental group B was higher than that in the control group K, and the content of group B was 98.85±14.98ng / L. IFN-α: There was no significant difference among the groups (P>0.05), and the content of group B was lower than that of the control groups Y and K (P>0.05), and the content of group B was 53.48±3.74ng / L.

[0141] IL-1β at 42 days: There was no significant difference among the groups (P>0.05), and the content of the experimental group B was higher than that of the blank group K, 46.79±6.99ng / L in group B. IL-6: The content of the experimental group B was higher than that of the blank group K (P>0.05), and there was no significant difference compared with the positive group Y (P>0.05), 30.58±5.57ng / L in group B. TNF-α: The experimental group B was significantly higher than the blank group K (P<0.05), and higher than the positive group Y (P>0.05), 61.92±5.10ng / L in group B. IFN-α: The content of the experimental group B was higher than that of the blank group K (P>0.05), and lower than that of the Y group (P<0.05), 86.82±3.35ng / L in group B. The levels of serum-related immune cytokines IL-1β, IL-6, and TNF-α in laying hens at 42 days old decreased compared with those at 21 days old, while the level of IFN-α increased.

[0142] The serum immunoglobulin IgG content of laying hens fed with the compound microbial agent for 21 days was significantly higher than that of the blank group K (P < 0.05), and the serum related immune cytokines IL-1β and IL-6 were higher than those of the blank group K (P < 0.05). The serum related immune cytokines TNF-α of laying hens fed with the compound microbial agent for 42 days were significantly higher than those of the blank control group K (P < 0.05). Overall, it was shown that feeding compound microecological preparations can enhance the nonspecific humoral immune expression of serum by increasing the content of serum immunoglobulins and related immune cytokines.

[0143] (4) Three laying hens fed for 28 days were randomly selected from each group, and 3-5 cm of intestinal segments such as the duodenum, jejunum, and ileum near the cecum were quickly collected. After each intestinal segment was washed, it was placed in a test tube containing 4% tissue formaldehyde fixative for fixation. The tissues fixed for no less than 24 hours were sliced ​​into paraffin sections according to tissue detection requirements, and finally the small intestinal villus height and crypt depth of the samples were measured using Image J analysis software, and the ratio of small intestinal villus height to crypt depth was calculated. The average value of three fields of view was taken for each slice.

[0144] Villus height (V) = vertical distance from the villus tip to the crypt opening

[0145] Small intestinal crypt depth (C) = distance from crypt base to crypt opening

[0146] The ratio of the two (V / C) = villus height / crypt depth

[0147] Table 15 Changes in duodenal villi and intestinal crypts

[0148]

[0149] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05).

[0150] From Table 15 and Fig.16 It can be seen that the intestinal villus height (VH) of the duodenum fed for 28 days: the VH of each group was not significantly different (P>0.05), and the experimental group B was higher than the control group Y and K, and B was 1374.22±129.45μm. Crypt depth (CD): the CD of each group was not significantly different (P>0.05), and the CD of the experimental group B was shallower than that of the control group Y and K, and B was 185.11±46.42μm. The ratio of the two (V / C): the V / C of each group was not significantly different (P>0.05), and the experimental group B was higher than the control group Y and K, and B was 7.43±2.02.

[0151] Table 16 Changes in jejunal villi and crypts

[0152]

[0153] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05).

[0154] From Table 16 and Fig.17It can be seen that the VH of the jejunum: the VH of each group was not significantly different (P>0.05), the experimental group B was lower than the blank group K, B was 1012.67±166.15μm. Crypt depth (CD): the CD of each group was not significantly different (P>0.05), the CD of the experimental group B was shallower than that of the control group K, B was 158.50±23.89μm. The ratio of the two (V / C): the V / C of each group was not significantly different (P>0.05), the experimental group B was lower than the control group K, B was 6.41±1.59.

[0155] Table 17 Changes in ileal villi and crypts

[0156]

[0157] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05).

[0158] From Table 17 and Fig.18 It can be seen that the VH of ileum: the VH of each group was not significantly different (P>0.05), and the VH of the experimental group B was increased compared with the control group Y and K, and B was 702.61±162.85μm. CD: the CD of each group was not significantly different (P>0.05), and the experimental group B was shallower than the control group Y and K, and B was 154.09±28.75μm. V / C: the V / C of each group was not significantly different (P>0.05), and the V / C of the experimental group B was increased compared with the control group Y and K, and B was 4.50±0.88.

[0159] After feeding the compound probiotic preparation for 28 days, there was no significant difference in VH, CD and V / C between the experimental group B and the blank group K (P>0.05). However, compared with the blank group K, the experimental group B may have a certain degree of damage to the jejunum, with reduced VH, reduced V / C, and reduced intestinal villus area in this intestinal segment. However, the VH and V / C of the duodenum and ileum of the small intestine increased. The increase in the villus area of ​​the small intestine increases the ability of laying hens to absorb nutrients.

[0160] (5) Aseptic collection: 3 cecal samples of laying hens were randomly selected from each group after feeding for 28 days. The samples were numbered and stored in a -80℃ refrigerator. The samples were stored in dry ice and sent to Wuhan Baiyihui Biotechnology Co., Ltd. for analysis of changes in intestinal flora using bacterial 16SrRNA high-throughput sequencing technology. Total DNA extraction from cecal microbial tissue was performed by PCR amplification of the bacterial 16s rDNA V3-V4 region: the primers used in the first round of PCR amplification have been integrated with the 16S rDNA V3-V4 region specific sequence of the Miseq sequencing platform. At the same time, the genomic DNA was accurately measured using the Qubit3.0 DNA detection kit, and the appropriate amount of DNA was added to the PCR reaction. For specific promoter information, see Table 18; and the reaction system required for PCR is listed in Table 19; finally, the first round of PCR amplification was completed according to the PCR conditions shown in Table 20.

[0161] Table 18 16S rDNA V3-V4 universal primers

[0162]

[0163] Table 19 16s rDNA V3-V4 region PCR amplification reaction system 1

[0164]

[0165] Table 20 PCR system - reaction conditions

[0166]

[0167]

[0168] Note: The reaction lasts for 5 cycles from denaturation to extension, and 25 cycles from the next denaturation to final extension.

[0169] After the first round of amplification, the second round of amplification was carried out, and Illumina bridge PCR compatible primers were introduced. The PCR system was carried out according to Table 21; the configured PCR system 2 was used for PCR amplification according to the reaction conditions in Table 22.

[0170] Table 21 PCR system 2

[0171]

[0172] Table 22 PCR system II reaction conditions

[0173]

[0174] Note: The reaction was performed for 35 cycles from denaturation to extension.

[0175] The PCR products were identified by 2% agarose gel electrophoresis, recovered and purified, and the recovered PCR products were mixed and quantified using the Quant-iT PicoGreen dsDNA Assay Kit. The Miseq PE300 platform was used for sequencing to construct the Miseq library. The PEreads obtained by Miseq sequencing were quality controlled, filtered and spliced ​​using FLASH software, and OTUs (Operational Taxonomic Units) cluster analysis and species taxonomy analysis were performed based on the valid data obtained.

[0176] The Alpha diversity of the microbial community in the cecum of each group after feeding the compound preparation for 28 days was analyzed by using 16S rRNA high-throughput measurement technology. It was found that the greater the Shannon diversity index of the sample and the smaller the Simpson index, the higher the diversity of the cecal microorganisms can be inferred; at the same time, the Chao index and ACE index increase with the increase in the richness of biological species, and when the sample coverage index value reaches close to 1, it can be shown that the established library has a high accuracy in revealing the composition of cecal microorganisms. See Table 23 Alpha Diversity Index of Cecal Microbiome.

[0177] Table 23 Alpha diversity index of cecal microorganisms

[0178]

[0179] Note: The same lowercase letters in each column indicate that the difference is not significant (P>0.05), and different letters indicate that the difference is significant (P<0.05).

[0180] As can be seen from Table 23, the diversity of cecal samples of laying hens fed for 28 days is represented by Shannon index and Simpson index. Shannon index: experimental group B is higher than control group Y and K (P>0.05); Simpson index: experimental group is lower than control group Y and K (P>0.05); indicating that the diversity of cecal microbial samples in the experimental group is higher than that in the control group. The species abundance analysis of cecal microbial samples is represented by Chao and ACE indexes. Chao index: experimental group B is higher than control group Y and K (P>0.05); ACE: experimental group B is higher than control group Y and K (P>0.05); the abundance of cecal microbial species in the experimental group is higher than that in control group Y and K. From the perspective of describing the authenticity of cecal samples, that is, the coverage of the sample library, the Coverage index of the experimental group and the control group is >0.99, close to 1, indicating that the three groups of samples in this experiment can reflect the authenticity of the microbial flora.

[0181] It shows that feeding laying hens with compound probiotic preparations can increase the diversity and species abundance of the cecal intestinal flora. Compound probiotic preparations can regulate changes in the intestinal flora of animals and reduce the occurrence of "dysbacteriosis".

[0182] Table 24 Relative abundance of caecal flora at the phylum level (%)

[0183]

[0184]

[0185] From Table 24 and Fig.19 It can be seen that after feeding the composite probiotics for 28 days, the main dominant bacteria at the cecal flora level of each group of laying hens were Firmicutes and Bacteroidetes, accounting for 95.76%-98.41% of the total flora, and the top ten cecal flora levels were Firmicutes A, Bacteroidetes, Firmicutes D, Desulfobacterota_I, Campylobacterota, Cyanobacteria, Actinobacteria, Proteobacteria, Firmicutes B, and Verrucomicrobiota. Most of the harmful intestinal bacteria belong to the Desulfobacteria phylum. The bacterial flora of experimental group B, Firmicutes and Bacteroidetes, accounted for 98.41% of the total flora, which was higher than 95.76% of the blank control group K and 98.31% of the positive group Y. This shows that feeding compound probiotic preparations in this study can regulate the relative abundance of Firmicutes and Bacteroidetes, improve the balance of intestinal flora to maintain the intestinal health of laying hens, and the effect of experimental group B in regulating intestinal flora is better than that of group Y.

[0186] Table 25 Relative abundance of cecal flora at genus level (%)

[0187]

[0188] From Table 25 and Fig. 20It can be seen that the main dominant bacteria at the genus level of the cecal flora of laying hens in each group after feeding for 28 days were Faecalibacterium, Enterococcus, Phocaeicola, Mediterraneibacter, Faecousia and Bacteroides, accounting for 57.81%-65.30% of the total flora, and the top ten genus levels of the cecal flora were Faecalibacterium, Enterococcus, Phocaeicola, Mediterraneibacter, Faecousia, Bacteroides, Lawsonibacter, Ruthenibacterium, Rikenella and Borkfalkia.

[0189] The main dominant genera in the cecal intestinal flora were Faecalibacterium, Enterococcus, Phocaeicola, Faecousia and Bacteroides of the Oscillospiraceae family. These five genera accounted for 62.17% of the total flora in the experimental group B, which was higher than 57.81% in the blank control group K. This showed that the compound microecological preparation could regulate the relative abundance of bacterial genera in the flora and improve the balance of intestinal flora to maintain the intestinal health of laying hens.

[0190] In summary, 1. The present invention screened chicken-derived probiotics Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6, and combined them with Clostridium butyricum NIP1021 to form a composite bacterial agent, and the fermentation liquid with a pH value of 4.8-4.9 was prepared by brown sugar fermentation. 2. The production application effect of the composite bacterial agent fermentation liquid fed to healthy 7-day-old Hy-line gray laying hens at 1% (v / v) in drinking water was evaluated as follows:

[0191] (1) The growth performance ADG, ADFI, F / G and final weight of the laying hens fed with the compound microbial agent in the experimental group B were higher than those of the control groups Y and K. The final weight of the laying hens fed with the compound microbial agent for 42 days was significantly higher than that of the control group K (P < 0.05), indicating that the compound microbial agent can promote animal growth performance.

[0192] (2) The immune organ development index of experimental group B was higher than that of blank group K, among which the thymus index and bursa index were significantly higher than those of blank group K (P < 0.05), indicating that the composite microecological preparation has a certain promoting effect on the development of the immune organs of laying hens.

[0193] (3) The serum immunoglobulin IgG content of laying hens fed with the compound microbial agent for 21 days was significantly higher than that of the blank group K (P < 0.05), and the serum related immune cytokines IL-1β and IL-6 were higher than those of the blank group K (P < 0.05). The serum related immune cytokine TNF-α of laying hens fed with the compound microbial agent for 42 days was significantly higher than that of the blank control group K (P < 0.05). This shows that feeding compound microecological preparations can enhance the nonspecific humoral immune expression of serum by increasing the content of serum immunoglobulins and related immune cytokines.

[0194] (4) Feeding compound bacterial agents can increase the villi area of ​​the small intestine of laying hens, thereby improving the ability of laying hens to absorb nutrients and increasing the diversity and species abundance of the cecal flora. The dominant flora phyla are Firmicutes and Bacteroidetes, which regulate the balance of intestinal flora and have a positive significance for maintaining intestinal health.

[0195] The results showed that the brown sugar fermented liquid of the composite microecological preparation can promote the growth performance of laying hens, improve the balance of intestinal flora of laying hens, promote intestinal health of laying hens, enhance the immunity of laying hens, and thus improve the production performance of laying hens. The composite microecological preparation is of great significance to the healthy breeding of laying hens.

Claims

1. A composite microecological preparation, characterized in that: The invention is composed of Clostridium butyricum NIP1021, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6, wherein the bacterial amount ratio of Clostridium butyricum NIP102, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6 is (0.5-2): (0.5-2): (0.5-2); the Bacillus cereus 2022B1 was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64738; the Bacillus licheniformis is 2022B2, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64739; the Enterococcus faecalis is 2022B6, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 11, 2024, and the culture collection number is GDMCC NO: 64740.

2. The composite microecological preparation according to claim 1, characterized in that: The bacterial quantity ratio of Clostridium butyricum NIP1021, Bacillus cereus 2022B1, Bacillus licheniformis 2022B2 and Enterococcus faecium 2022B6 is 1:1:1:

1.

3. The composite microecological preparation according to claim 1 or 2, characterized in that: The viable bacteria count of the composite microecological preparation is 10 8 ~10 9 cfu / mL.

4. Use of the composite microecological preparation according to claim 1 or 2 in preparing brown sugar fermentation broth.

5. A method for preparing a fermentation broth of a composite microecological preparation, characterized in that: First, molasses is used as a raw material, and the seed liquid of each bacterial species in the composite microecological preparation according to claim 1 or 2 is fermented separately to obtain fermented seed liquid; then brown sugar is used as a raw material, and the seed liquids fermented for the first time are compounded according to the bacterial amount ratio and then fermented for the second time to prepare the fermentation liquid.

6. The preparation method according to claim 5, characterized in that: The pH of the first fermentation culture is 4.4-4.6, and the inoculated bacterial amount is 10 8 CFU / mL.

7. The preparation method according to claim 5, characterized in that: The composition ratio of the brown sugar fermentation liquid in the second fermentation culture is the first fermentation seed liquid: brown sugar: water = 1: 5: 200, and the fermentation bacteria amount is 10 8 ~10 9 CFU / mL, fermentation time is 24 to 48h.

8. The fermentation broth of the composite microecological preparation prepared by the method according to any one of claims 5 to 7.

9. Use of the fermentation broth of the composite probiotic preparation according to claim 8 in the preparation of products for promoting the growth performance of laying hens, improving the balance of intestinal flora of laying hens, promoting intestinal health of laying hens, and improving the immunity of laying hens.

10. The use according to claim 9, characterized in that: The application dosage of the fermentation liquid of the composite microecological preparation is 1 mL / kg body.

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