Method for preparing functional biological flora by coupling target biological flora and host original flora and application of functional biological flora
By targeting the coupling of biological bacteria with the host's original flora, combined with low-temperature vacuum freeze-drying technology and synergistic excipients, the problem of colonization of exogenous strains in the host's intestines was solved, achieving stable improvement of intestinal microecology and degradation of harmful substances.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-04-03
AI Technical Summary
Existing probiotic products are mostly composed of one or a few exogenous strains, which are difficult to colonize effectively in the host's gut, resulting in unstable effects, poor targeting, and insufficient persistence. Moreover, existing technologies neglect the importance of the host's original microbiota.
By coupling exogenous targeted microorganisms with the host's original microbial community, a freeze-dried powder form of functional microbial community is prepared using a low-temperature vacuum freeze-drying process. The exogenous strains and the host's original microbial community work together, supplemented by modified montmorillonite and chitosan oligosaccharide as synergistic excipients, to form a stable microbial community.
It enabled the rapid adaptation and colonization of exogenous strains in the host gut, improved the stability of the gut microbiota, significantly reduced heavy metal and antibiotic residues in eggs, and inhibited the proliferation of Salmonella, with better effects than single-strain products.
Abstract
Description
Technical Field
[0001] The invention relates to the fields of microbial application and livestock and poultry breeding technology, specifically a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbiota and its application. Background Technology
[0002] With the development of intensive egg-laying hen farming, problems such as heavy metal pollution in feed and overuse of veterinary antibiotics have become increasingly prominent, leading to excessive levels of heavy metals and antibiotic residues in eggs, as well as an increased risk of contamination by pathogenic bacteria such as Salmonella, which seriously threaten food safety and public health.
[0003] Currently, probiotic preparations are often added to feed to improve the gut health of laying hens, thereby indirectly reducing harmful residues. However, most existing probiotic products consist of one or a few exogenous probiotics. After entering the host, these strains face the challenge of the host-specific environment and are difficult to colonize and function effectively in the complex gut microbiota, resulting in unstable effects, poor targeting, and insufficient persistence.
[0004] Furthermore, existing technologies often overlook the importance of the host's own original microbiota. The host's original microbiota is a micro-ecological system that has been formed through long-term co-evolution and is most adapted to the host's internal environment. Coupled with exogenous target strains, it is expected to solve the bottleneck problem of the difficulty in colonizing exogenous strains.
[0005] Therefore, there is an urgent need in this field for a targeted solution that can achieve efficient integration of exogenous functional microbiota with the host's internal environment, thereby stably and efficiently reducing egg residues and inhibiting pathogens. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a method for formulating functional microbial communities by coupling targeted microorganisms with the host's original microbiota, and its application. This method solves the problem that most existing probiotic products consist of single or several exogenous probiotics. After entering the host, these strains face challenges from the host-specific environment, making it difficult for them to effectively colonize and exert their effects in the complex intestinal flora. This results in unstable effects, poor targeting, and insufficient persistence. Existing technologies often neglect the importance of the host's own original microbiota, which is a microecological system that has been formed through long-term co-evolution and is most adapted to the host's internal environment. Coupling it with exogenous targeted strains is expected to solve the bottleneck problem of the difficulty in colonizing exogenous strains.
[0007] To achieve the above objectives, the invention provides the following technical solution: a method for preparing functional microbial communities through a coupling mechanism between targeted microbes and the host's original microbial community, comprising the following steps: (1) Provide a functional microbial composition, wherein the functional microbial composition consists of exogenous targeted microbial communities, host original microbial communities and synergistic excipients; (2) Each strain in the exogenous targeted microbial community was subjected to high-density fermentation culture and the bacterial cells of each strain were collected; (3) The bacterial cells of each strain collected in step (2) are mixed evenly with the original host flora and the synergistic excipients to form a mixture; (4) The mixture is processed by low-temperature vacuum freeze-drying process to prepare a functional biological microbial community in freeze-dried powder form. The conditions of low-temperature vacuum freeze-drying process are: freeze-drying temperature between -45℃ and -35℃, vacuum degree below 10Pa, and drying time between 24 and 48 hours.
[0008] Preferably, the exogenous targeted microbial community is composed of Bacillus licheniformis, Pseudomonas NJ-62 strain, Saccharomyces boulardii, Lactobacillus plantarum, and Bacillus subtilis, and the synergistic excipients include modified montmorillonite and chitosan oligosaccharide; based on the total mass of the functional microbial community, the modified montmorillonite accounts for 15% of the total mass, and the chitosan oligosaccharide accounts for 5% of the total mass.
[0009] Preferably, based on the total mass of the prepared functional microbial community, the content of each active ingredient is as follows: The content of Bacillus licheniformis is not less than 5 × 10⁻⁶. 9 CFU / g; The content of the Pseudomonas NJ-62 strain is not less than 3×10. 9 CFU / g; The content of *Saccharomyces boulardii* is not less than 2 × 10⁻⁶ 10 CFU / g; The content of *Lactobacillus plantarum* is not less than 1×10⁻⁶. 10 CFU / g; The content of Bacillus subtilis is not less than 8 × 10⁻⁶. 9 CFU / g; The content of the original host flora is not less than 2×10 10 CFU / g.
[0010] Preferably, the original host microbiota is derived from the intestinal contents or feces of healthy laying hens and obtained after isolation, culture and enrichment.
[0011] A functional microbial community formulated by coupling targeted microorganisms with the host's original microbial community.
[0012] The application of a functional microbial community formulated by coupling targeted microbes with the host's original microbial community, the purpose of which is to reduce heavy metal residues in eggs, degrade antibiotic residues in eggs, and / or inhibit Salmonella in the digestive tract of laying hens.
[0013] Preferably, the application method is feed addition, specifically: the functional microbial community is mixed into the feed at a ratio of 0.2% to 0.5% of the total weight of the laying hen feed.
[0014] Preferably, the application method is water replenishment, specifically including the following steps: The functional microbial community, brown sugar and water are mixed in a mass ratio of 1:(1-3):(80-120) and fermented at 25℃-37℃ for 18-36 hours to make a fermentation liquid; The fermentation liquid is diluted with water at a mass ratio of 1:(300-700) and then given to laying hens for drinking.
[0015] Preferably, the service life of the functional microbial community is: Routine preventative use: Use for 5 to 10 consecutive days each month; High-residue treatment use: Use continuously for 14 to 28 days. After the residue detection index in the eggs meets the standard, switch to the routine preventive use plan.
[0016] Beneficial effects The invention provides a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbiota, and its application. The invention has the following advantages: This invention is the first to systematically couple exogenous targeted functional microbiota with the host (laying hen) original microbiota. The host original microbiota, as the "vanguard" and "ecological cornerstone", can quickly adapt to the intestinal environment, creating a favorable micro-ecological environment for the colonization and function of exogenous functional microbiota. This solves the technical problem of exogenous strains "not adapting to the environment" and achieves a synergistic effect of "1+1>2". The selected exogenous strains have clearly defined functions and strong synergistic effects, jointly constructing a healthy gut microbiota and reducing harmful residues in eggs from the source; By employing a specific low-temperature vacuum freeze-drying process and synergistic excipients (modified montmorillonite as a carrier and adsorbent, and chitosan oligosaccharide as a prebiotic), the activity of the microbial community is protected to the greatest extent, thereby improving the stability of the product during storage and use. Detailed Implementation
[0017] The present invention will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0018] Example 1: Preparation of functional microbial communities The freeze-dried powder strains of Bacillus licheniformis, Pseudomonas NJ-62, Saccharomyces boulardii, Lactobacillus plantarum and Bacillus subtilis were inoculated into the corresponding slant culture medium for activation. Bacillus licheniformis, Pseudomonas aeruginosa, Lactobacillus plantarum, and Bacillus subtilis: Incubate at 37°C for 24-48 hours using LB slant agar. Yeast (Saccharomyces boulardii): Incubate at 30°C for 48-72 hours using YPD slant medium; The activated bacterial strains were inoculated into the corresponding liquid seed culture media and cultured on a shaker (bacteria: 37℃, 180 rpm, 24 hours; yeast: 30℃, 150 rpm, 36 hours) to prepare primary seed liquid.
[0019] Preparation of host progeny microbiota Cecal contents were aseptically collected from multiple healthy Hy-Line Brown hens at their peak egg production and which had not been treated with antibiotics. The contents were immediately placed in an anaerobic sampling bag and filled with nitrogen. The collected samples were mixed thoroughly with pre-reduced PBS buffer at a ratio of 1:10 (w / v) in an anaerobic workstation (85% N2, 10% H2, 5% CO2). Uses 4 layers of sterile gauze for filtration to remove large particles; Centrifuge the filtrate (1000 g, 10 min, 4 °C) and collect the mixture of supernatant and precipitate containing bacteria.
[0020] The collected bacterial mixture was resuspended in anaerobic PBS containing 10% glycerol, aliquoted, and stored in an ultra-low temperature freezer at -80°C for later use. This is the original host bacterial culture stock solution.
[0021] High-density fermentation Each primary seed culture was transferred at a 5% inoculation rate into fermenters containing the corresponding fermentation medium for high-density fermentation. Specific fermentation parameters are shown in the table below: Table 1: High-density fermentation process parameters for each strain strain name Fermentation medium Temperature (°C) pH control Ventilation rate (vvm) Stirring speed (rpm) Fermentation time (h) <![CDATA[End point OD 600 / Viable cell count]]> Bacillus licheniformis Corn starch, soybean meal, etc. 37 7.0-7.2 1.0-1.5 300-400 24-30 <![CDATA[OD 600 ≥ 60]]> Pseudomonas NJ-62 peptone, yeast extract, etc. 30 7.2-7.4 1.2-1.8 350-450 36-40 <![CDATA[≥5.0×10 10 CFU / mL]]> Saccharomyces boulardii yeast extract, glucose, etc. 30 nature 0.8-1.2 200-300 48-60 <![CDATA[≥2.5×10 10 CFU / mL]]> Lactobacillus plantarum MRS medium 37 <![CDATA[6.2 - 6.5 (regulated by NaCO3)]]> Nitrogen protection 100 (mix only) 24-36 <![CDATA[≥1.2×10 10 CFU / mL]]> Bacillus subtilis Bacillus licheniformis 37 7.0-7.2 1.0-1.5 300-400 24-30 <![CDATA[OD 600 ≥ 65]]> After fermentation, the bacterial cells were collected using a continuous flow centrifuge (bacteria: 8000 rpm, yeast: 6000 rpm) and washed twice with sterile physiological saline to obtain a high-concentration bacterial sludge.
[0022] Weigh each component precisely according to the final product formula shown in the table below.
[0023] Table 2: Final Product Formulation of Functional Microbial Community Components effect Content requirements Feeding calculation (based on the preparation of 1 kg of product) Bacillus licheniformis mud Functional bacteria <![CDATA[≥5×10 9 CFU / g]]> <![CDATA[Providing a bacterial sludge volume of ≥ 5.0×10 12 CFU viable bacteria]]> Pseudomonas NJ-62 mycelium mud Functional bacteria <![CDATA[≥3×10 9 CFU / g]]> <![CDATA[Provide a bacterial sludge volume of ≥ 3.0×10 12 CFU viable bacteria]]> Saccharomyces boulardii sludge Functional bacteria <![CDATA[≥2×10 10 CFU / g]]> <![CDATA[Provide a bacterial sludge volume of ≥ 2.0×10 13 CFU viable bacteria]]> Lactobacillus plantarum mud Functional bacteria <![CDATA[≥1×10 10 CFU / g]]> <![CDATA[Providing a sludge volume of viable bacteria ≥ 1.0×10 13 CFU]]> Bacillus subtilis sludge Functional bacteria <![CDATA[≥8×10 9 CFU / g]]> <![CDATA[Provide a bacterial sludge volume of ≥ 8.0×10 12 CFU viable bacteria]]> Host original microbial culture stock solution Core co-bacteria <![CDATA[≥2×10 10 CFU / g]]> <![CDATA[Provide a bacterial liquid volume of ≥ 2.0×10 13 CFU viable bacteria]]> Modified montmorillonite Carrier / Adsorbent 15% (w / w) 150g Chitosan oligosaccharide prebiotics 5% (w / w) 50g maltodextrin Lyophilization protectant / filler Supplement to 100% Supplement to 1kg The aforementioned bacterial sludge, host original bacterial culture stock solution, and auxiliary materials such as modified montmorillonite, chitosan oligosaccharide, and maltodextrin are added together into a three-dimensional motion mixer. Mix for 60 minutes at a temperature below 15℃ to ensure the materials are fully homogeneous.
[0024] Low-temperature vacuum freeze drying Spread the evenly mixed wet material evenly in the freeze-drying tray, controlling the thickness to within 1 cm, and then freeze-dry: Pre-freezing stage: The material is rapidly cooled to -40°C and maintained at this temperature for 2 hours to completely freeze the material.
[0025] Main drying stage: Start the vacuum system to stabilize the vacuum level inside the chamber at 5-8 Pa. Then slowly raise the temperature of the partition from -40℃ to -20℃. This stage lasts for about 20 hours to sublimate and remove most of the free water.
[0026] Analysis and drying stage: Gradually increase the temperature of the partition to 30°C and maintain this temperature for 8-10 hours, while keeping the vacuum degree <10Pa, in order to remove bound water.
[0027] Discharge: After freeze-drying, nitrogen gas is introduced to ventilate the air, and the material is immediately transferred to an ultra-low humidity environment (relative humidity <10%). After being pulverized by a pulverizer and passed through an 80-mesh sieve, a freeze-dried powder formulation with good flowability is obtained. Finally, it is packaged in aluminum foil bags under nitrogen protection.
[0028] Example 2: Verification of the application effect of functional microbial communities Experimental Materials and Methods Product of this invention: freeze-dried functional microbial community powder prepared according to the method of Example 1.
[0029] Commercially available comparison product: A certain brand of compound probiotics (mainly containing Bacillus subtilis and Enterococcus faecalis), with a live bacteria count labeled as 2.0 × 10⁻⁶. 10 CFU / g.
[0030] Four hundred and fifty healthy Hy-Line Brown chickens aged 220 days with similar egg production rates were selected and randomly divided into three treatment groups, with five replicates in each group and thirty chickens in each replicate. The groupings are as follows: Control group: fed with a basal diet.
[0031] Commercially available product group: Add 0.3% of the commercially available control product to the basal diet.
[0032] Product group of this invention: 0.3% of the product of this invention is added to the basal diet.
[0033] Feeding and Management The trial lasted for 8 weeks. All laying hens were housed in stacked cages with free access to feed and water. The immunization program was carried out according to standard procedures. The basic diet formula was formulated with reference to the NRC (1994) nutritional requirements for laying hens.
[0034] Detection indicators and methods Egg heavy metal residue (lead): Sample preparation: At the end of week 4 and week 8 of the experiment, 10 eggs were randomly collected from each replicate. After removing the eggshells, the egg liquid was homogenized, and 2.0 g of sample was accurately weighed into a digestion vessel. 5 mL of nitric acid was added, and the sample was digested using a microwave digester.
[0035] Detection method: After the digestion solution was diluted with water, the lead content was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900) in accordance with GB5009.268-2016.
[0036] Antibiotic residues in eggs (oxytetracycline): Modeling: Before the start of the formal experiment, all groups were fed a diet supplemented with 20 mg / kg oxytetracycline for 2 weeks to establish a residual model.
[0037] Sample processing and detection: Egg samples were collected on days 0, 7, and 14 of the experiment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used for detection. The chromatographic column was a C18 column; the mobile phase consisted of acetonitrile and 0.1% formic acid aqueous solution.
[0038] Count of Salmonella in the cecum: Sample collection: At the end of the experiment, two chickens were randomly selected from each replicate, and cecal contents were aseptically collected.
[0039] Detection method: After serial dilution of the sample with sterile physiological saline, spread it on XLD agar plates and incubate at 37°C for 24-48 hours to count typical colonies.
[0040] Test results Table 3: Results of lead residue detection in eggs (μg / kg, ±SD) Group Week 0 Week 4 Week 8 control group 125.4 ± 10.2ᵃ 118.7 ± 9.5ᵃ 110.3 ± 8.8ᵃ Commercially available product group 126.1 ± 11.5ᵃ 85.6 ± 7.2ᵇ 65.4 ± 6.1ᵇ Product Group of this Invention 124.8 ± 9.8ᵃ 52.3 ± 5.4ᶜ 28.1 ± 3.2ᶜ Note: Different letters in the superscript of data in the same column indicate significant differences (p<0.05).
[0041] Results Analysis: As shown in Table 3, compared with the control group, both additive groups significantly reduced lead residue in eggs (p<0.05). At week 8, the lead residue in the product group of this invention was significantly lower than that in the commercially available product group (p<0.01), and was far below the national standard limit of 100 μg / kg, indicating that the product of this invention has excellent effect in reducing heavy metal lead pollution.
[0042] Effect on degradation of oxytetracycline residues in eggs Table 4: Changes in oxytetracycline residues in eggs after discontinuation of medicated feed (μg / kg, ±SD) Group Day 0 Day 7 Day 14 control group 155.5 ± 12.8ᵃ 95.8 ± 8.9ᵃ 52.3 ± 5.7ᵃ Commercially available product group 157.2 ± 13.5ᵃ 68.4 ± 6.5ᵇ 25.1 ± 3.1ᵇ Product Group of this Invention 156.0 ± 11.9ᵃ 35.6 ± 4.1ᶜ Not detected Results Analysis: As shown in Table 4, after discontinuing the use of medicated feed, the degradation rate of oxytetracycline in the eggs of the product group of this invention was significantly faster than that of the other two groups. By day 7, its residual amount was significantly lower than that of the commercially available product group (p<0.05). By day 14, the oxytetracycline content in the samples of the product group of this invention was lower than the detection limit of the method (1.0 μg / kg), while it was still detected in the other two groups. This indicates that the multiple functional strains contained in the product of this invention have a high efficiency in degrading oxytetracycline.
[0043] Inhibitory effect against Salmonella cecum Table 5: Salmonella count in cecal contents of laying hens at the end of the experiment (log) 10 CFU / g, ±SD) Group Salmonella count Positive rate control group 3.52 ± 0.41ᵃ 80% Commercially available product group 2.88 ± 0.35ᵇ 60% Product Group of this Invention 1.95 ± 0.28ᶜ 20% Results Analysis: As shown in Table 5, compared with the control group, the product of this invention can significantly reduce the number of Salmonella in the cecum (p<0.01) and reduce the positive detection rate of Salmonella from 80% to 20%. This effect is significantly better than that of commercially available products, which proves that the bacterial flora of this invention can effectively inhibit the proliferation of pathogenic bacteria such as Salmonella in the intestine through multiple mechanisms such as competitive exclusion and acid production, and improve intestinal health.
[0044] The above-described specific embodiments detail the preparation process, application method, and verification results of the present invention. Through the standardized process of Example 1, functional biological flora products with high viable bacterial counts can be stably prepared. The animal test data of Example 2 fully demonstrate that the product of the present invention can effectively reduce the residues of heavy metal lead and antibiotic oxytetracycline in eggs, and can significantly inhibit Salmonella in the digestive tract of laying hens. The overall effect is significantly better than similar products on the market, providing an effective technical solution for achieving safe egg production.
[0045] It should be noted that in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.
Claims
1. A method for preparing functional microbial communities by coupling targeted microorganisms with the host's original microbial community, characterized in that, Includes the following steps: (1) Provide a functional microbial composition, wherein the functional microbial composition consists of exogenous targeted microbial communities, host original microbial communities and synergistic excipients; (2) Each strain in the exogenous targeted microbial community was subjected to high-density fermentation culture and the bacterial cells of each strain were collected; (3) The bacterial cells of each strain collected in step (2) are mixed evenly with the original host flora and the synergistic excipients to form a mixture; (4) The mixture is processed by low-temperature vacuum freeze-drying process to prepare a functional biological microbial community in freeze-dried powder form. The conditions of low-temperature vacuum freeze-drying process are: freeze-drying temperature between -45℃ and -35℃, vacuum degree below 10Pa, and drying time between 24 and 48 hours.
2. The method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbiota according to claim 1, characterized in that, The exogenous targeted microbial community is composed of Bacillus licheniformis, Pseudomonas NJ-62 strain, Saccharomyces boulardii, Lactobacillus plantarum and Bacillus subtilis, and the synergistic excipients include modified montmorillonite and chitosan oligosaccharide; based on the total mass of the functional microbial community, the modified montmorillonite accounts for 15% of the total mass and the chitosan oligosaccharide accounts for 5% of the total mass.
3. The method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbiota according to claim 1, characterized in that, Based on the total mass of the prepared functional microbial community, the content of each active ingredient is as follows: The content of Bacillus licheniformis is not less than 5 × 10⁻⁶. 9 CFU / g; The content of the Pseudomonas NJ-62 strain is not less than 3×10. 9 CFU / g; The content of *Saccharomyces boulardii* is not less than 2 × 10⁻⁶. 10 CFU / g; The content of *Lactobacillus plantarum* is not less than 1×10⁻⁶. 10 CFU / g; The content of Bacillus subtilis is not less than 8 × 10⁻⁶. 9 CFU / g; The content of the original host flora is not less than 2×10⁻⁶. 10 CFU / g.
4. The method for preparing functional microbial communities according to claim 1, characterized in that, The original host microbiota is derived from the intestinal contents or feces of healthy laying hens and is obtained through isolation, culture, and enrichment.
5. A functional microbial community formulated according to any one of claims 1-4 by coupling the targeted microbial community with the original host microbial community.
6. The application of a functional microbial community formulated according to the coupling method of targeted microorganisms and the original host microbiota as described in claim 5, characterized in that, The purpose of this application is to reduce heavy metal residues in eggs, degrade antibiotic residues in eggs, and / or inhibit Salmonella in the digestive tract of laying hens.
7. The application of the functional microbial community formulated according to the coupling method of targeted microorganisms and the original host microbiota as described in claim 6, characterized in that, The application method is feed addition, specifically: the functional biological flora is mixed into the feed at a ratio of 0.2% to 0.5% of the total weight of the laying hen feed.
8. The application of the functional microbial community formulated according to the coupling method of targeted microorganisms and the original host microbiota as described in claim 7, characterized in that, The application method is to replenish fluids through drinking water, specifically including the following steps: The functional microbial community, brown sugar and water are mixed in a mass ratio of 1:(1-3):(80-120) and fermented at 25℃-37℃ for 18-36 hours to make a fermentation liquid; The fermentation liquid is diluted with water at a mass ratio of 1:(300-700) and then given to laying hens for drinking.
9. The application of the functional microbial community formulated according to the coupling method of targeted microorganisms and the original host microbiota as described in claim 1, characterized in that, The usage cycle of the aforementioned functional microbial community is: Routine preventative use: Use for 5 to 10 consecutive days each month; High-residue treatment use: Use continuously for 14 to 28 days. After the residue detection index in the eggs meets the standard, switch to the routine preventive use plan.