Probiotic microcapsules based on droplet microfluidics technology and preparation method and application thereof

Abstract

The present application belongs to the technical field of probiotic preparation, and relates to a probiotic microcapsule based on droplet microfluidic technology and a preparation method and application thereof.The raw material of the microcapsule wall material comprises separated whey protein and polyglutamic acid derivative, and the raw material of the microcapsule core material comprises probiotic bacteria.Whey protein is crosslinked with polyglutamic acid derivative under the action of transglutaminase, and the prepared probiotic microcapsule has uniform particle size, can provide stronger moisture resistance and better stress resistance.The double-crosslinked probiotic microcapsule prepared by the present application has higher freeze-drying survival rate, room temperature storage property and gastrointestinal fluid tolerance, and has very wide application prospect and potential social and economic value.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, and relates to a probiotic microcapsule based on droplet microfluidics technology, its preparation method and application. Background Technology

[0002] In the development of traditional livestock farming, the widespread use of antibiotics has gradually exposed its drawbacks, such as disrupting the balance of gut microbiota, causing decreased immunity in livestock, and drug residues in the food chain. Most seriously, the overuse of antibiotics can lead to the spread of drug-resistant bacteria and the transmission of drug-resistant genes. Therefore, the development and promotion of antibiotic alternatives for livestock farming is driven by market demand.

[0003] Clostridium butyricum is a beneficial intestinal microorganism that plays a significant role in the animal gut, regulating intestinal flora, improving intestinal health and function, and promoting digestion and absorption. Its metabolic products mainly include butyric acid, vitamins, proteases, and antimicrobial polypeptides. Clostridium butyricum produces fatty acids such as butyric acid in the intestine, effectively lowering intestinal pH, promoting the growth of beneficial bacteria such as lactobacilli and bifidobacteria, and inhibiting the growth of pathogenic bacteria such as Salmonella typhi, Salmonella cholerae, and Staphylococcus aureus. The vitamins in its metabolic products can activate immune cells and enhance animal immunity. Various enzymes can function in the intestine, promoting absorption and digestion. Furthermore, Clostridium butyricum is a Gram-positive anaerobic endophytic spore-forming bacillus with a certain degree of resistance to environmental changes, giving it the potential to be a good probiotic. It can be widely used in clinical medicine, agricultural production, aquaculture, and animal husbandry, making it an ideal biological agent with broad development prospects. The quantity and activity of probiotic live bacteria products such as Clostridium butyricum will decrease after processing, storage and transportation to the gastrointestinal tract in harsh acid and alkaline environments.

[0004] Microencapsulation technology, as an effective means of protecting probiotics from adverse external environments, has received increasing attention and is widely used. However, common probiotic microencapsulation technologies, such as emulsification, complex coagulation, or spray drying, generally suffer from poor uniformity and uncontrollable size. Therefore, this paper aims to develop a method for preparing polyglutamic acid and its derivatives microcapsules with adjustable size and good uniformity based on droplet microfluidic technology. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the wall materials for encapsulating probiotic microcapsules in the prior art are mostly common traditional materials, and the resulting microcapsules do not have sufficient resistance to adverse conditions such as gastric acid and bile salts. This invention provides a method for preparing probiotic microcapsules based on polyglutamic acid derivatives. Starting from polyglutamic acid, through modification, drying and other steps, a microcapsule wall material PGA-GMA was developed and successfully applied to the encapsulation of probiotic microcapsules in order to obtain a microcapsule that is resistant to gastric acid and has high stability.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned probiotic microcapsules.

[0007] Furthermore, the technical problem to be solved by the present invention is to provide the application of the above-mentioned probiotic microcapsules.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] This invention discloses a probiotic microcapsule based on droplet microfluidics technology. The microcapsule wall material of the probiotic microcapsule includes isolated whey protein and polyglutamic acid derivatives, and the microcapsule core material includes probiotics.

[0010] In some embodiments, the polyglutamic acid derivative is prepared according to the following method:

[0011] Polyglutamic acid and glycidyl methacrylate were dissolved in deionized water to obtain an aqueous solution of polyglutamic acid. The pH of the aqueous solution of polyglutamic acid was adjusted to 3.5-4.0 with sulfuric acid solution. Then, the reaction was heated. After the reaction was completed, the reaction solution was dialyzed and freeze-dried to obtain PGA-GMA.

[0012] Preferably, the concentration of sulfuric acid in the sulfuric acid aqueous solution is 3 mol / L.

[0013] In some embodiments, the polyglutamic acid is any one of γ-polyglutamic acid, sodium γ-polyglutamic acid, and potassium γ-polyglutamic acid, preferably sodium γ-polyglutamic acid; the number average molecular weight of the polyglutamic acid is 500-2000 kDa; in the aqueous solution of the polyglutamic acid, the mass concentration of polyglutamic acid is 10%-20%, and the mass concentration of glycidyl methacrylate is 5%-10%; the heating reaction is carried out at a temperature of 60-80°C for 8-10 hours.

[0014] In some embodiments, preferably, the polyglutamic acid aqueous solution has a polyglutamic acid mass concentration of 10% and a glycidyl methacrylate mass concentration of 5%; the heating reaction is carried out at a temperature of 80°C for 10 hours.

[0015] The polyglutamic acid (γ-PGA) is an anionic polypeptide polymer synthesized by microorganisms using L-glutamic acid and D-glutamic acid monomers through γ-amide bonds.

[0016] In some embodiments, the probiotic is Clostridium butyricum; the Clostridium butyricum is pretreated by the following method:

[0017] Clostridium butyricum was inoculated into RCM solid medium and anaerobically cultured at 30℃~37℃ for 20~24h to obtain single colonies. These single colonies were then inoculated into RCM liquid medium using an inoculation loop and anaerobically cultured at 30℃~37℃ for 20~24h to obtain Clostridium butyricum seed culture. The seed culture was then transferred to RCM liquid medium at an inoculation rate of 5%~10% v / v and anaerobically cultured at 30℃~37℃ for 20~24h to obtain fermentation broth. The fermentation broth was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain Clostridium butyricum sludge. The viable count of Clostridium butyricum in the fermentation broth was 1×10⁻⁶. 8 ~10×10 8 CFU / mL; the centrifugation is carried out at a temperature of 4℃~8℃, a speed of 4000~8000rpm, and a time of 5~15min.

[0018] The *Clostridium butyricum* mentioned above is a genus of *Clostridium*, species name *butyricum*, scientific name *Clostridium butyricum*, and has the accession number CCTCC AB 2017089.

[0019] The RCM solid culture medium consists of the following components: 10.0g peptone, 10.0g beef powder, 3.0g yeast powder, 5.0g glucose, 1.0g soluble starch, 5.0g sodium chloride, 3.0g sodium acetate, 0.5g L-cysteine ​​hydrochloride, and 20g agar.

[0020] The RCM liquid culture medium consists of the following components: 10.0 g / L peptone, 10.0 g / L beef meal, 3.0 g / L yeast extract, 5.0 g / L glucose, 1.0 g / L soluble starch, 5.0 g / L sodium chloride, 3.0 g / L sodium acetate, and 0.5 g / L L-cysteine ​​hydrochloride. The solvent is deionized water or ultrapure water.

[0021] Furthermore, this invention discloses a method for preparing the above-mentioned probiotic microcapsules, comprising the following steps:

[0022] (1) Dissolve whey protein and transglutaminase in deionized water, mix well, and obtain the first mixture;

[0023] (2) Dissolve the degradable crosslinking agent in an aqueous solution of dimethyl sulfoxide to obtain a second mixture;

[0024] (3) Dissolve the polyglutamic acid derivative and the photoinitiator in deionized water to obtain a third mixture;

[0025] (4) Disperse the probiotics in sterile water and resuspend them to obtain a probiotic suspension; mix the first mixture obtained in step (1) with the second mixture obtained in step (2) to obtain a fourth mixture; then mix the fourth mixture with the third mixture obtained in step (3), and add the probiotic suspension to the mixture. After mixing, obtain an aqueous phase.

[0026] (5) The silicone oil and emulsifier are shaken and mixed to obtain a continuous phase;

[0027] (6) The aqueous phase obtained in step (4) and the continuous phase obtained in step (5) are simultaneously pumped into the T-shaped chip of droplet microfluidics for shearing to obtain microdroplets; the microdroplets are photocured to obtain probiotic microcapsules.

[0028] In some embodiments, in step (1), the transglutaminase activity is 45-55 U / g; the protein content in the isolated whey protein is greater than 80%; and in the first mixture, the mass concentration of the isolated whey protein is 2%-5% and the mass concentration of the transglutaminase is 3%-5%.

[0029] In some embodiments, preferably, in step (1), the transglutaminase activity is 50 U / g; in the first mixture, the mass concentration of the separated whey protein is 4%, and the mass concentration of the transglutaminase is 4%.

[0030] In step (1), the transglutaminase is defined as having an enzyme activity of one enzyme unit (U) as the amount of enzyme required to produce 1 μmol of L-glutamic acid-γ-monohydroxyxamic acid per minute at 37°C.

[0031] In some embodiments, in step (2), the degradable crosslinking agent is N,N′-(2-amino-1,4-phenylene)acrylamide; the volume ratio of dimethyl sulfoxide to water in the dimethyl sulfoxide aqueous solution is 4% to 6%:1; and the mass concentration of the degradable crosslinking agent in the second mixture is 0.1% to 0.5%.

[0032] In some embodiments, preferably, in step (2), the volume ratio of dimethyl sulfoxide to water in the dimethyl sulfoxide aqueous solution is 5%:1; and the mass concentration of the decomposable crosslinking agent in the second mixture is 0.1% to 0.2%.

[0033] The N,N′-(2-amino-1,4-phenylene)diacrylamide is abbreviated as APD.

[0034] In some embodiments, in step (3), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate or trimethylbenzoyl-diphenylphosphine oxide; in the third mixture, the mass concentration of the polyglutamic acid derivative is 5% to 10%, and the mass concentration of the photoinitiator is 0.5% to 2%.

[0035] In some embodiments, preferably, in step (3), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; in the third mixture, the mass concentration of the polyglutamic acid derivative is 10%, and the mass concentration of the photoinitiator is 1%.

[0036] Wherein, the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is abbreviated as LAP; and the trimethylbenzoyl-diphenylphosphine oxide is abbreviated as TPO.

[0037] In some embodiments, in step (4), the volume ratio of the first mixture to the second mixture is 0.5–1.5:1; the volume ratio of the third mixture to the fourth mixture is 0.5–2:1; and the viable count of probiotics in the aqueous phase is 6 × 10⁻⁶. 8 ~10×10 8 CFU / mL.

[0038] In some embodiments, preferably, in step (4), the volume ratio of the first mixture to the second mixture is 1:1; and the volume ratio of the third mixture to the fourth mixture is 1:1.

[0039] In step (4), the probiotics are dispersed in sterile water and resuspended to obtain a probiotic suspension. The amount of sterile water used is just enough to resuspend the probiotics.

[0040] In some embodiments, in step (5), the silicone oil is DOWSIL RSN-0749; the emulsifier is Span 80; and the volume ratio of the emulsifier to the silicone oil is 0.5% to 2.0%:1, preferably 1.0%:1.

[0041] In some embodiments, in step (6), the flow rate of the aqueous phase pumped into the T-shaped chip of the droplet microfluidic system is 2-6 μL / min; the flow rate of the continuous phase pumped into the T-shaped chip of the droplet microfluidic system is 5-40 μL / min; the shearing is performed at a temperature of 25-30°C; the photocuring is performed using ultraviolet light as the light source, with a wavelength of 320-400 nm, a curing temperature of 30°C-37°C, and a curing time of 3-5 min; and the size of the probiotic microcapsules is 90-610 μm.

[0042] In step (6), the diameter of the channel in the T-shaped chip is 100μm to 500μm, and the material of the T-shaped chip is polydimethylsilane (PDMS).

[0043] In step (6), during the photocuring process, the light source is positioned 10cm to 15cm directly above the microdroplets to ensure sufficient illumination.

[0044] The application of the above-mentioned probiotic microcapsules in the preparation of drugs or feeds for improving the intestinal flora of livestock and poultry is also within the scope of protection of this invention.

[0045] Beneficial effects:

[0046] Compared with existing probiotic microcapsule technology, this invention has the following advantages:

[0047] (1) The microcapsule wall material of *Clostridium butyricum* described in this invention is mainly composed of isolated whey protein and polyglutamic acid. Polyglutamic acid contains a large number of carboxyl groups, while whey protein, under the action of transglutaminase, exposes more lysine and glutamine residues. Under acidic conditions, the two polymers carry opposite charges, generating electrostatic attraction, thereby increasing the gel viscosity and strength, and improving the physical properties of the microcapsules. In addition, whey protein has good film-forming and emulsifying properties. Therefore, *Clostridium butyricum* microcapsules prepared using polyglutamic acid derivatives and whey protein provide better low-temperature tolerance and improve the survival rate of probiotics.

[0048] (2) Polyglutamic acid (γ-PGA) is an anionic polypeptide polymer synthesized by microorganisms using L-glutamic acid and D-glutamic acid monomers through γ-amide bonds. It is a protective capsule secreted by some microorganisms. Recent studies have found that in addition to its good hydrophilicity, high swelling ratio, good biocompatibility, and biodegradability, polyglutamic acid hydrogels can also effectively shield light radiation and alleviate photoaging. Therefore, using polyglutamic acid as the main material for loading Clostridium butyricum can ensure that the prepared product is safe and environmentally friendly.

[0049] (3) While conventional emulsification or spray drying methods for preparing monolayer microcapsules offer simple processing, they also result in uneven microcapsules, questionable encapsulation effects, and room for improvement in oxygen barrier properties and the protective effect exhibited during drying. Droplet microfluidics, as an emerging microcapsule preparation technology, uses chip fabrication and selection to adjust the flow rates of the oil and water phases to prepare uniform microcapsules of different particle sizes. The prepared microcapsules achieve higher encapsulation rates. Simultaneously, the whey protein introduced into the aqueous phase undergoes cross-linking of polyglutamic acid derivatives under the action of transglutaminase, resulting in Clostridium butyricum microcapsules with essentially uniform particle size, providing stronger moisture barrier properties and better stress resistance. Attached Figure Description

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0051] Figure 1 This is a schematic diagram of the droplet microfluidics of the present invention.

[0052] Figure 2 This is a flow path diagram of the droplet microfluidic flow of the present invention.

[0053] Figure 3 Optical morphology image of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 2.

[0054] Figure 4 The particle size distribution of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 2 is shown in the figure.

[0055] Figure 5 Optical morphology image of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 3.

[0056] Figure 6 The particle size distribution of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 3 is shown in the figure.

[0057] Figure 7 Optical morphology image of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 4.

[0058] Figure 8 The particle size distribution of the double-crosslinked Clostridium butyricum microcapsules prepared in Example 4 is shown in the figure.

[0059] Figure 9 The survival rate of Clostridium butyricum in freeze-dried formulations of Clostridium butyricum microcapsules of different sizes during freeze-drying.

[0060] Figure 10 To assess the gastric tolerance of Clostridium butyricum microcapsules.

[0061] Figure 11 To assess the tolerance of Clostridium butyricum microcapsules to intestinal fluid.

[0062] Figure 12 The isothermal hygroscopic curve of Clostridium butyricum microcapsules.

[0063] Figure 13 This refers to the composition of the gut microbiota at the phylum level in broilers.

[0064] Figure 14 This represents the genus-level gut microbiota composition of broiler chickens.

[0065] Figure 15 These are immunological parameters in broiler serum. Detailed Implementation

[0066] To make the technical problems solved, technical solutions, and beneficial effects of this invention clearer, the following examples, in conjunction with embodiments, illustrate preferred embodiments of this invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent technologies discovered by the inventors that can be used to implement this invention, and therefore can be considered preferred solutions for implementing this invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this invention.

[0067] The RCM solid culture medium used in this embodiment of the invention consists of: 10.0g peptone, 10.0g beef powder, 3.0g yeast powder, 5.0g glucose, 1.0g soluble starch, 5.0g sodium chloride, 3.0g sodium acetate, 0.5g L-cysteine ​​hydrochloride, and 20g agar.

[0068] The RCM liquid culture medium used in this embodiment of the invention consists of: 10.0 g / L peptone, 10.0 g / L beef meal, 3.0 g / L yeast extract, 5.0 g / L glucose, 1.0 g / L soluble starch, 5.0 g / L sodium chloride, 3.0 g / L sodium acetate, and 0.5 g / L L-cysteine ​​hydrochloride. The solvent is deionized water or ultrapure water.

[0069] The simulated gastric juice used in this invention is prepared by taking 16.4 mL of dilute hydrochloric acid (equivalent to 3.84 mL of hydrochloric acid), adding about 800 mL of sterile water and 10 g of pepsin, shaking well, and then diluting with sterile water to 1000 mL to obtain simulated digestive gastric juice with pH = 2.0 to 3.0. The mixture is then filtered through a 0.22 μm sterile filter membrane for sterilization.

[0070] Simulated intestinal fluid: Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of sterile water, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; dissolve 10g of pancreatic enzyme in an appropriate amount of sterile water, mix the two solutions, and dilute with sterile water to 1000mL to obtain simulated digestive intestinal fluid, which is then filtered through a 0.22μm sterile filter membrane for sterilization.

[0071] The butyric acid bacterium used in this embodiment of the invention is a genus name: Clostridium, species name: butyricum, scientific name: Clostridium butyricum, accession number: CCTCC AB 2017089, purchased from Nanjing Ansute Biotechnology Co., Ltd.

[0072] The transglutaminase used in this embodiment of the invention has an enzyme activity of 50 U / g. The enzyme activity of transglutaminase is defined as the amount of enzyme required to produce 1 μmol of L-glutamate-γ-monohydroxyxamic acid per minute at 37°C as one enzyme unit (U).

[0073] The isolated whey protein used in this embodiment of the invention has a protein content greater than 80% by mass.

[0074] The T-shaped chips used in this invention have channel diameters of 100μm, 300μm, and 500μm. The T-shaped chips are made of polydimethylsilane (PDMS), and all chips can be purchased from the Cambridge University Nanjing Science and Technology Innovation Center.

[0075] In the photocuring process of this embodiment of the invention, the light source is set 10cm to 15cm directly above the microdroplets to ensure sufficient light conditions.

[0076] The phosphate-buffered saline (PBS) used in this invention has a concentration of 2 mol / L and a pH of 6.8.

[0077] Example 1

[0078] This embodiment provides a method for preparing polyglutamic acid derivative monolayer microcapsules, the method of preparing monolayer microcapsules includes the following steps;

[0079] Preparation of polyglutamic acid derivatives:

[0080] Sodium γ-polyglutamate (number average molecular weight of 500 kDa) and glycidyl methacrylate were dissolved in deionized water to obtain an aqueous solution of polyglutamate. The concentration of sodium γ-polyglutamate in the aqueous solution was 10 wt%, and the concentration of glycidyl methacrylate was 5 wt%. The pH of the aqueous solution of polyglutamate was adjusted to 3.5-4.0 with 3 mol / L sulfuric acid solution. The reaction was then heated in a water bath at 80 °C for 10 h. After the reaction was completed, the reaction solution was dialyzed for 48 h and then lyophilized to obtain the PGA-GMA powder sample.

[0081] Clostridium butyricum pretreatment:

[0082] Clostridium butyricum preserved in glycerol tubes was inoculated into sterilized RCM solid medium and anaerobically cultured at 37°C for 24 h to obtain single colonies. These single colonies were then inoculated into RCM liquid medium using an inoculation loop and anaerobically cultured at 32°C for 24 h to obtain a Clostridium butyricum seed culture. The seed culture was then transferred to sterile, fresh RCM liquid medium at a 10% v / v inoculation rate and anaerobically cultured at 32°C for 24 h to obtain a fermentation broth (approximately 2–3 mL). The viable cell count in the fermentation broth was approximately 9.0 × 10⁻⁶. 8 CFU / mL ~10×10 8CFU / mL; After centrifuging the fermentation broth at 6000 rpm for 10 min at 4℃, the supernatant was removed, and the precipitate was washed twice with sterile physiological saline to obtain Clostridium butyricum sludge for later use.

[0083] Preparation of monolayer Clostridium butyricum microcapsules:

[0084] (1) Dissolve the weighed degradable crosslinking agent APD in a dimethyl sulfoxide aqueous solution (the volume ratio of dimethyl sulfoxide to water is 5%:1) to obtain a crosslinking agent solution; wherein, the concentration of the degradable crosslinking agent APD in the crosslinking agent solution is 0.1wt%.

[0085] (2) Dissolve the weighed PGA-GMA and photoinitiator LAP in deionized water to obtain a polyglutamic acid derivative solution containing LAP; wherein, in the polyglutamic acid derivative solution containing LAP, the concentration of PGA-GMA is 10wt% and the concentration of photoinitiator LAP is 1wt%.

[0086] (3) The *Clostridium butyricum* sludge obtained above was dispersed in 50 μL of sterile water and resuspended to obtain a *Clostridium butyricum* suspension; the cross-linking agent solution obtained in step (1) and the polyglutamic acid derivative solution containing LAP obtained in step (2) were mixed in equal volumes, and then the *Clostridium butyricum* suspension was added. After mixing, a droplet microfluidic aqueous phase was obtained and stored for later use. The viable count of *Clostridium butyricum* in the aqueous phase was 8.5 × 10⁻⁶. 8 CFU / mL.

[0087] (4) Mix DOWSIL RSN-0749 and Span 80 by shaking to obtain a continuous phase of droplet microfluidics, and store it for later use; wherein the volume ratio of Span 80 to DOWSIL RSN-0749 is 1.0%:1.

[0088] (5) The aqueous phase obtained in step (3) and the continuous phase obtained in step (4) are simultaneously pumped into the T-shaped chip of droplet microfluidics and sheared at 25°C. The shearing force provided by the continuous phase is used to shear the aqueous phase to form microdroplets. Then the microdroplets are collected in centrifuge tubes for photocuring to obtain monolayer Clostridium butyricum microcapsules.

[0089] In this study, when the diameter of the droplet microfluidic T-chip was 100 μm, the flow rate of the aqueous phase pumped into the droplet microfluidic T-chip was 4 μL / min, and the flow rate of the continuous phase pumped into the droplet microfluidic T-chip was 12 μL / min. The microdroplets were collected in centrifuge tubes and photocured using ultraviolet light with a wavelength of 320–400 nm at 30 °C for 3 min to obtain 100 μm monolayer Clostridium butyricum microcapsules.

[0090] In this study, when the diameter of the droplet microfluidic T-chip was 300 μm, the flow rate of the aqueous phase pumped into the droplet microfluidic T-chip was 5 μL / min, and the flow rate of the continuous phase pumped into the droplet microfluidic T-chip was 20 μL / min. The microdroplets were collected in centrifuge tubes and photocured using ultraviolet light with a wavelength of 320–400 nm at 30°C for 3 min to obtain 400 μm monolayer Clostridium butyricum microcapsules.

[0091] In this study, when the diameter of the droplet microfluidic T-chip was 500 μm, the flow rate of the aqueous phase pumped into the droplet microfluidic T-chip was 6 μL / min, and the flow rate of the continuous phase pumped into the droplet microfluidic T-chip was 30 μL / min. The microdroplets were collected in centrifuge tubes and photocured using ultraviolet light with a wavelength of 320–400 nm at 30°C for 3 min to obtain 600 μm monolayer Clostridium butyricum microcapsules.

[0092] Figure 1 This is a schematic diagram of microfluidic operation, illustrating how a continuous phase pumped in from the top and bottom shears the aqueous phase pumped in from the left to form microdroplets. Figure 2 To prepare a microdroplet flow path diagram for a laboratory microfluidic device.

[0093] Example 2

[0094] This embodiment provides a method for preparing Clostridium butyricum microcapsules that are doubly cross-linked with polyglutamic acid derivatives and whey protein isolate.

[0095] Preparation of polyglutamic acid derivatives:

[0096] Sodium γ-polyglutamate (number average molecular weight of 500 kDa) and glycidyl methacrylate were dissolved in deionized water to obtain an aqueous solution of polyglutamate. The concentration of sodium γ-polyglutamate in the aqueous solution was 10 wt%, and the concentration of glycidyl methacrylate was 5 wt%. The pH of the aqueous solution of polyglutamate was adjusted to 3.5-4.0 with 3 mol / L sulfuric acid solution. The reaction was then heated in a water bath at 80 °C for 10 h. After the reaction was completed, the reaction solution was dialyzed for 48 h and then lyophilized to obtain the PGA-GMA powder sample.

[0097] Clostridium butyricum pretreatment:

[0098] Clostridium butyricum preserved in glycerol tubes was inoculated into sterilized RCM medium and anaerobically cultured at 37°C for 24 h to obtain single colonies. These single colonies were then inoculated into RCM liquid medium using an inoculation loop and anaerobically cultured at 32°C for 24 h to obtain a Clostridium butyricum seed culture. The seed culture was then transferred to sterile, fresh RCM liquid medium at a 10% v / v inoculation rate and anaerobically cultured at 32°C for 24 h to obtain a fermentation broth (approximately 2–3 mL). The viable cell count in the fermentation broth was approximately 9.0 × 10⁻⁶.8 CFU / mL ~10×10 8 CFU / mL; After centrifuging the fermentation broth at 6000 rpm for 10 min at 4℃, the supernatant was removed, and the precipitate was washed twice with sterile physiological saline to obtain Clostridium butyricum sludge for later use.

[0099] Preparation of double-layer cross-linked Clostridium butyricum microcapsules:

[0100] (1) Weigh whey protein isolate (protein content > 80%) and commercial grade transglutaminase (enzyme activity 50 U / g) and dissolve them in deionized water, stir and mix at room temperature to obtain the first mixture, and store it for later use; wherein, the concentration of whey protein isolate in the first mixture is 4 wt% and the concentration of transglutaminase is 4 wt%.

[0101] (2) Dissolve the weighed degradable crosslinking agent APD in a dimethyl sulfoxide aqueous solution (the volume ratio of dimethyl sulfoxide to water is 5%:1) to obtain a second mixture; wherein the concentration of the degradable crosslinking agent APD in the second mixture is 0.2wt%.

[0102] (3) The weighed polyglutamic acid derivative (PGA-GMA prepared in Example 1) and photoinitiator LAP were dissolved in deionized water to obtain a third mixture; wherein the concentration of polyglutamic acid derivative in the third mixture was 10 wt% and the concentration of photoinitiator LAP was 1 wt%.

[0103] (4) Disperse the above-obtained Clostridium butyricum sludge in 50 μL of sterile water and resuspend it to obtain a Clostridium butyricum suspension; mix the first mixture obtained in step (1) with the second mixture obtained in step (2) in equal volumes to obtain a fourth mixture; then mix the fourth mixture with the third mixture obtained in step (3) in equal volumes, and add the Clostridium butyricum suspension to the mixture, mix well to obtain an aqueous phase, and store it for later use; wherein, the viable count of Clostridium butyricum in the aqueous phase is 8.5 × 10⁻⁶. 8 CFU / mL.

[0104] (5) Mix DOWSIL RSN-0749 and Span 80 by shaking to obtain a continuous phase of droplet microfluidics, and store it for later use; wherein the volume ratio of Span 80 to DOWSIL RSN-0749 is 1.0%:1.

[0105] (6) The aqueous phase obtained in step (4) and the continuous phase obtained in step (5) are simultaneously pumped into a T-shaped chip (channel diameter of 100 μm) of droplet microfluidics. Shearing is performed at 25 °C, with the continuous phase providing shearing force to shear the aqueous phase to obtain microdroplets. The flow rate of the aqueous phase pumped into the T-shaped chip of droplet microfluidics is 4 μL / min, and the flow rate of the continuous phase pumped into the T-shaped chip of droplet microfluidics is 12 μL / min. The microdroplets are collected in centrifuge tubes for photocuring. The ultraviolet light wavelength is 320–400 nm, and curing is performed at 30 °C for 5 min to obtain double-crosslinked (97.7 ± 6.0) μm Clostridium butyricum microcapsules.

[0106] Figure 3 This is an optical morphology image of Clostridium butyricum microcapsules under a microscope. Figure 4 This is a size distribution diagram of Clostridium butyricum microcapsules. Figure 3 It can be seen that the size of the Clostridium butyricum dual microcapsules prepared under these conditions is uniform. Figure 4 The size of the prepared double-layer Clostridium butyricum microcapsules is (97.7±6.0) μm.

[0107] Example 3

[0108] The preparation method of this embodiment is the same as that of embodiment 2. The difference is that in step (6) of the preparation of the double-layer cross-linked Clostridium butyricum microcapsules, the diameter of the droplet microfluidic T-shaped chip is 300 μm, the flow rate of the aqueous phase pumped into the droplet microfluidic T-shaped chip is 5 μL / min, and the flow rate of the continuous phase pumped into the droplet microfluidic T-shaped chip is 20 μL / min. The continuous phase with different flow rates provides shear forces of different sizes, so that the aqueous phase is sheared into microdroplets of different sizes. After collecting the microdroplets, they are solidified to obtain double-cross-linked Clostridium butyricum microcapsules with a size of (396.5±12.1) μm. Figure 5 This is an image of the optical morphology of the microcapsules under a microscope. Figure 6 This is a size distribution diagram of the microcapsules. Figure 5 It can be seen that the size of the Clostridium butyricum dual microcapsules prepared under these conditions is uniform. Figure 6 It can be seen that the size of the prepared double-layer Clostridium butyricum microcapsules is (396.5±12.1)μm.

[0109] Example 4

[0110] The preparation method of this embodiment is the same as that of embodiment 2. The difference is that in step (6) of the preparation of the double-layer cross-linked Clostridium butyricum microcapsules, the diameter of the droplet microfluidic T-shaped chip is 500 μm, the flow rate of the aqueous phase pumped into the droplet microfluidic T-shaped chip is 6 μL / min, and the flow rate of the continuous phase pumped into the droplet microfluidic T-shaped chip is 30 μL / min. The continuous phase with different flow rates provides shear forces of different sizes, so that the aqueous phase is sheared into microdroplets of different sizes. After collecting the microdroplets, they are solidified to obtain double-cross-linked Clostridium butyricum microcapsules with a size of (596.8±12.1) μm. Figure 7 This is an image of the optical morphology of the microcapsules under a microscope. Figure 8 This is a size distribution diagram of the microcapsules. Figure 7 It can be seen that the size of the Clostridium butyricum dual microcapsules prepared under these conditions is uniform. Figure 8 The size of the prepared double-layer Clostridium butyricum microcapsules is (596.8±12.1) μm.

[0111] Example 5: Clostridium butyricum loading rate test

[0112] The butyric acid loading capacity of Clostridium butyricum microcapsules of different sizes prepared in Examples 1-4 was evaluated.

[0113] (1) The number of viable bacteria per unit aqueous phase and the volume of aqueous phase before shearing are used as the total number of viable bacteria before embedding.

[0114] (2) After centrifuging the solidified reaction solution containing microcapsules at 4000 rpm for 5 min, the precipitate (the precipitate is the solidified microcapsules, and the supernatant is the oil phase) was collected. The precipitate was washed with 1% v / v Tween 80 solution and then resuspended in 10 mL of 200 μmol / L nitric oxide donor solution (SNP solution is NO donor solution) to obtain a suspension. 3-4 glass beads were added to the suspension to ensure that the microcapsules were fully in contact with the nitric oxide donor solution during shaking and stirring. The suspension was then incubated at 37°C and 200 rpm under natural light for 30 min. Subsequently, 0.5 mL of the sample solution was serially diluted with PBS buffer and the viable count was determined. The butyric acid citrate loading capacity of the microcapsules prepared in Examples 1-4 was calculated. The butyric acid citrate loading capacity of the butyric acid citrate microcapsules is shown in Table 1.

[0115] Calculate the survival rate using the following formula:

[0116] Survival rate = (Number of viable bacteria per unit microcapsule * Volume of microcapsule) / (Number of viable bacteria per unit aqueous phase * Volume of aqueous phase) × 100%

[0117] Table 1 Loading capacity of microcapsules of different sizes for Clostridium butyricum

[0118]

[0119] Table 1 shows that two types of microcapsules with different particle sizes were prepared by varying the flow rate ratios of the aqueous phase and the continuous phase. The monolayer microcapsules cross-linked with PGA-GMA alone (100 μm, 400 μm, and 600 μm) achieved loading rates of Clostridium butyricum of 64.3%, 68.6%, and 67.3%, respectively. In contrast, the bilayer microcapsules cross-linked with whey protein achieved loading rates of 72.8%, 74.5%, and 72.9% for Clostridium butyricum, all higher than those of the monolayer cross-linked microcapsules. Furthermore, as shown in Table 1, the 400 μm microcapsules exhibited the best loading capacity, with the bilayer cross-linked microcapsules showing a 5.9% higher loading rate than the monolayer cross-linked microcapsules.

[0120] Example 6: Evaluation of the performance of microcapsule freeze-drying

[0121] The freeze-drying properties of Clostridium butyricum microcapsules of different sizes prepared in Examples 1-4 were evaluated, and the bacterial survival ability during the process was also evaluated.

[0122] In the early stages of the experiment, 10 wt.% trehalose aqueous solution, 10 wt.% maltodextrin aqueous solution, and 10 wt.% skim milk aqueous solution were used as freeze-drying protectants for Clostridium butyricum microcapsules (prepared in Examples 1-4). Preliminary screening revealed that skim milk may be the carrier with the best protective effect for Clostridium butyricum.

[0123] Control group: Clostridium butyricum suspension. Clostridium butyricum preserved in glycerol tubes was inoculated into sterilized RCM solid medium and anaerobically cultured at 37°C for 24 h to obtain single colonies. These single colonies were then inoculated into RCM liquid medium using an inoculation loop and anaerobically cultured at 32°C for 24 h to obtain Clostridium butyricum seed culture. The seed culture was then transferred to sterile fresh RCM liquid medium at a 10% v / v inoculation rate and anaerobically cultured at 32°C for 24 h to obtain fermentation broth (approximately 2–3 mL). The viable cell count in the fermentation broth was approximately 9.0 × 10⁻⁶. 8 CFU / mL ~10×10 8 CFU / mL; After centrifuging the fermentation broth at 6000 rpm for 10 min at 4℃, the supernatant was discarded, and the precipitate was washed twice with sterile physiological saline to obtain Clostridium butyricum sludge. The bacterial cells were resuspended in an equal volume of 10 wt% skim milk aqueous solution to the Clostridium butyricum sludge to obtain a suspension. The suspension was pre-cooled at -20℃ and then dried at -40℃ and 0.01 Mba for 24 h to obtain free Clostridium butyricum. The total viable count of Clostridium butyricum before drying was confirmed before adding skim milk. After freeze-drying, the number of viable cells per unit mass and the total mass of the freeze-dried bacterial powder were measured. The survival rate of Clostridium butyricum in the control group was calculated using the following formula:

[0124] Survival rate = (Number of viable bacteria per unit mass × Total mass of lyophilized bacterial powder) / (Total number of viable bacteria before drying) × 100%

[0125] Microcapsule group: A 10 wt% skim milk aqueous solution was used as the protective carrier for the lyophilization of *Clostridium butyricum* microcapsules (prepared in Examples 1-4). The solidified microcapsules were resuspended and dispersed by adding an equal volume of 10 wt% skim milk aqueous solution to the fermentation broth, and then pre-cooled at -20°C. Subsequently, they were dried at -40°C and 0.01 Mba for 24 h to obtain the lyophilized *Clostridium butyricum* microcapsule formulation. The total viable count of *Clostridium butyricum* before drying was confirmed before adding skim milk (refer to Example 5). After lyophilization, 0.5 g of the lyophilized *Clostridium butyricum* microcapsule formulation was weighed and resuspended in 10 mL of 200 μmol / L SNP solution as a nitric oxide donor solution to obtain a suspension. Add 3-4 glass beads to the suspension, incubate the suspension at 37℃ and 200 rpm in a shaker under natural light for 30 min, then take 0.5 mL of the sample solution and perform serial dilution with PBS buffer to determine the viable bacterial count. Calculate the ability of microcapsules of different particle sizes to protect Clostridium butyricum during lyophilization. The experimental results are shown below. Figure 9 .

[0126] Calculate the survival rate using the following formula:

[0127] Survival rate = (Number of viable bacteria per unit mass of microcapsule * Mass of lyophilized sample) / (Total number of viable bacteria before drying) × 100%

[0128] like Figure 9 As shown, the freeze-drying survival rate of Clostridium butyricum in the free bacteria (control group, CK) was 36.9%. The freeze-drying survival rates of Clostridium butyricum in the 100μm, 400μm, and 600μm double cross-linked Clostridium butyricum microcapsule freeze-dried formulations were 81.7%, 88.2%, and 83.5%, respectively, while the freeze-drying survival rates of Clostridium butyricum in the single-layer Clostridium butyricum microcapsule freeze-dried formulations were 70.1%, 72.8%, and 69.0%, respectively. The freeze-drying survival rates of Clostridium butyricum in the 100μm, 400μm, and 600μm double cross-linked Clostridium butyricum microcapsule freeze-dried formulations were 11.6%, 15.4%, and 14.5% higher than those in the single-layer Clostridium butyricum microcapsule freeze-dried formulations, respectively.

[0129] Example 7: Evaluation of the gastric juice tolerance performance of microcapsules

[0130] 0.5 g of *Clostridium butyricum* microcapsules (free *Clostridium butyricum* lyophilized preparation and microcapsule lyophilized preparation prepared in Example 6) were taken and the viable count was determined. Then, the microcapsules were added to 49.5 mL of simulated gastric fluid and incubated at 37°C under anaerobic conditions for 3 h. The precipitate was then collected by centrifugation at 4000 r / min for 5 min, washed with sterile water, and resuspended in 10 mL of 200 μmol / L SNP aqueous solution as a nitric oxide donor solution to obtain a suspension. 3–4 glass beads were added to the suspension, and the suspension was incubated at 37°C and 200 rpm in a shaker under natural light for 30 min. 0.5 mL of the sample solution was serially diluted with PBS buffer and the viable count was determined. The tolerance of *Clostridium butyricum* cells to different sized microcapsules under gastric and intestinal fluid conditions was calculated. The experimental results are as follows: Figure 10 , Figure 11 .

[0131] like Figure 10 As shown, compared to the free group (control group, CK), which had a survival rate of only 43.5% after 3 hours of simulated gastric juice treatment, the survival rates of *Clostridium butyricum* in the lyophilized formulations of double-crosslinked *Clostridium butyricum* microcapsules (100 μm, 400 μm, and 600 μm) were 93.0%, 95.6%, and 89.6%, respectively. In contrast, the survival rates of *Clostridium butyricum* in the lyophilized formulations of single-layer *Clostridium butyricum* microcapsules were 77.2%, 79.0%, and 75.3%. The survival rates of *Clostridium butyricum* in the double-crosslinked microcapsules were increased by 15.8%, 16.6%, and 14.3% compared to those in the single-layer formulations. These results indicate that the double-crosslinked *Clostridium butyricum* microcapsules with added whey protein exhibit better gastric juice tolerance.

[0132] like Figure 11 As shown, compared to the free group (control group, CK), where only 60.0% of *Clostridium butyricum* survived after 3 hours of treatment with simulated intestinal fluid, the survival rates of *Clostridium butyricum* in lyophilized formulations of 100μm, 400μm, and 600μm double-crosslinked *Clostridium butyricum* microcapsules were 94.1%, 94.6%, and 92.0%, respectively. In contrast, the survival rates of *Clostridium butyricum* in lyophilized formulations of monolayer *Clostridium butyricum* microcapsules were 80.7%, 84.9%, and 78.0%. The survival rates of *Clostridium butyricum* in lyophilized formulations of 100μm, 400μm, and 600μm double-crosslinked *Clostridium butyricum* microcapsules were 13.4%, 9.7%, and 14.0% higher than those in monolayer formulations. These results indicate that double-crosslinked *Clostridium butyricum* microcapsules with added whey protein exhibit better intestinal fluid tolerance.

[0133] Overall, the double-crosslinked Clostridium butyricum microcapsules exhibited good in vitro simulated digestion performance.

[0134] Example 8: Evaluation of the hygroscopic properties of microcapsules

[0135] The moisture isotherm of the freeze-dried microcapsules was measured using a dynamic vapor adsorption system at 25℃ ± 0.2℃. Approximately 5 mg of sample (the freeze-dried microcapsule formulation prepared in Example 6, with a size of 400 μm) was weighed onto a sample tray and placed in a humidity-controlled chamber for measurement. The relative humidity (RH) was increased from 0% to 95% RH, and the results were automatically measured and recorded at 10% intervals. The results are as follows: Figure 12 As shown.

[0136] from Figure 12 The dynamic water vapor adsorption experiment showed that when the relative humidity rose to 95%, the mass of the single-layer Clostridium butyricum microcapsule freeze-dried preparation reached 100.9%, while the mass of the double-crosslinked Clostridium butyricum microcapsule freeze-dried preparation changed by 84.7%, a relative decrease of 16.2%. The double-crosslinked Clostridium butyricum microcapsules have lower hygroscopic properties, meaning they have stronger moisture-blocking properties.

[0137] Example 9: Storage stability of Clostridium butyricum powder

[0138] Using a 10 wt% skim milk aqueous solution as the Clostridium butyricum carrier for lyophilization, two sets of microcapsules (single-layer and double-layer Clostridium butyricum microcapsules, prepared in Example 6) with a particle size of 400 μm were lyophilized. The resulting Clostridium butyricum powder was stored in a desiccator at 25°C. Samples were taken out periodically, and the number of viable bacteria in the samples was counted on plates. The storage performance was evaluated by calculating the survival rate.

[0139] Survival rate = Current number of surviving bacteria / Initial number of viable bacteria in the dried sample

[0140] As shown in Table 2, under room temperature storage conditions, the microcapsule groups all provided higher survival rates compared to the free bacterial group (the control group sample prepared in Example 6), which had a survival rate of only 1.3% for Clostridium butyricum after 90 days of storage. Specifically, the monolayer microcapsules showed a survival rate of only 29% for Clostridium butyricum after 3 months, while the double-crosslinked Clostridium butyricum microcapsules achieved a survival rate of 41%, representing a 12% improvement in survival rate compared to the monolayer microcapsule group.

[0141] Table 2 Storage performance of bacterial powder at 25℃

[0142]

[0143] Example 10: Clostridium butyricum microcapsule group improves the intestinal flora of broilers

[0144] This embodiment provides the application of the prepared double-crosslinked Clostridium butyricum microcapsules in broiler farming and the analysis of the intestinal flora of the cecum of broilers.

[0145] (1) Broiler chicken farming and grouping

[0146] One hundred and eighty one-day-old broilers were randomly divided into three large treatment groups. Each large treatment group was further divided into three small treatment groups, with 20 chickens in each small treatment group. The experiments were the same for all three small treatment groups and were parallel experiments. The three large treatment groups were the blank control group, the bacterial powder group, and the double cross-linked Clostridium butyricum microcapsule group.

[0147] Among them, the blank control group was fed with a full-course basal diet; the bacterial powder group was fed with a full-course basal diet plus 10 6 CFU / day Clostridium butyricum powder; Microencapsulated group: 10 CFU / day of basal diet throughout the entire feeding period. 6 Double-crosslinked Clostridium butyricum microcapsules (CFU / day)

[0148] (2) Feeding and Management

[0149] The experiment was conducted in the animal facility, where all broilers were raised in cages with free access to feed and water. Deworming and disinfection were carried out in accordance with the standard chicken farm feeding regulations.

[0150] (3) Intestinal flora detection

[0151] Cecal chyme was collected at 21 days of age and stored in a sterile PE tube at -80°C. The cecal contents were scraped onto a sterile glass slide and transferred to a 2mL sterile cryovial, which was then stored in liquid nitrogen for later use.

[0152] Genomic DNA was extracted from the contents of the cecum and subjected to 16SV4 amplicon sequencing. The DNA extraction and sequencing flowchart is as follows: Sample preparation → DNA extraction and detection → PCR amplification → Product purification → Library preparation and library inspection → sequencing on a lonS5™ XL. The raw sequencing data was spliced, filtered, and the effective data were obtained. Based on the effective data, OTU (Operational Taxonomic Unit) clustering and species classification analysis were performed. According to the OTU clustering results, on the one hand, species annotation was performed on the sequence of each OTU to obtain the corresponding species information and species-based abundance distribution. At the same time, OTU abundance, alpha diversity calculation, and petal diagram analysis were performed to obtain information on species richness and evenness within the sample, as well as information on shared and unique OTUs among different samples or groups. On the other hand, multiple sequence alignment of OTUs and construction of phylogenetic trees were performed, and the differences in community structure among different samples or groups were explored through dimensionality reduction analysis and sample clustering tree display. To further explore the differences in community structure among the grouped samples, statistical analysis methods were used to test the significance of differences in species composition and community structure among the grouped samples.

[0153] (4) Detection of serum immune levels in broilers

[0154] At 21 days of age, two broiler chickens were randomly selected from each of the three parallel sub-treatment groups in each large treatment group, for a total of six chickens in each large treatment group. The chickens were weighed after fasting, and 10 mL of blood was collected from the anterior vena cava. The blood was allowed to stand for 1 hour, then centrifuged at 3500 r / min for 5 minutes to collect the serum, which was then stored at -20℃ for later testing.

[0155] The results are as follows Figure 13 As shown, Group C was the control group, Group T (bacterial powder group) was the commercially available Clostridium butyricum group, and Group H was the double-crosslinked Clostridium butyricum microcapsule group. The experimental results showed that the average composition of Firmicutes in the control group, bacterial powder group, and double-crosslinked Clostridium butyricum microcapsule group were 41.81%, 48.57%, and 56.97%, respectively; the average composition of Bacteroidetes was 54.34%, 44.49%, and 39.29%, respectively; and the average composition of Cyanobacteria was 0.32%, 0.44%, and 1.08%, respectively. It can be seen that after adding Clostridium butyricum to the diet, the Firmicutes phylum in the cecum of broilers increased, while the Bacteroidetes phylum decreased. This may be one of the reasons for the improved growth performance of broilers. At the same time, the upward trend of Cyanobacteria is consistent with the changing trend of intestinal microorganisms when probiotic preparations are applied to animal husbandry. Compared to commercially available Clostridium butyricum powder, the double-layer Clostridium butyricum microcapsule group showed an 8.40% increase in Firmicutes and a 5.20% decrease in Bacteroidetes. This means that the double-layer Clostridium butyricum microcapsule group resulted in a higher proportion of Firmicutes and a lower proportion of Bacteroidetes. Firmicutes and Bacteroidetes are two phyla in the gut that are related to animal digestion and nutrient metabolism. A higher Firmicutes / Bacteroidetes ratio leads to more efficient absorption of food calories in broilers, and a higher proportion of Firmicutes also results in a healthier gut environment for broilers.

[0156] Figure 14The diagram shows the intestinal microbial composition under genus-level conditions. The average percentages of Bacteroides in the blank group (C1-C6), the powder group (T1-T6), and the double-crosslinked Clostridium butyricum microcapsule group (H1-H6) were 30.32%, 27.56%, and 21.37%, respectively; the average percentages of Oscillatoria were 3.71%, 4.50%, and 5.57%, respectively; the average percentages of Lactobacillus acidophilus were 3.46%, 5.84%, and 8.29%, respectively; and the average percentages of Faecalibacterium were 1.69%, 3.42%, and 4.35%, respectively. After feeding Clostridium butyricum powder or Clostridium butyricum microcapsules, the percentages of Bacteroides decreased, while the percentages of Oscillatoria, Lactobacillus acidophilus, and Faecalibacterium increased. This indicates that Clostridium butyricum, as a probiotic, effectively improves the intestinal environment of broilers by reducing the proportion of harmful bacteria and increasing the proportion of beneficial bacteria. Oscillatoria, Lactobacillus acidophilus, and Faecalibacterium are typical probiotics. Compared with the Clostridium butyricum powder group, the proportions of Oscillatoria, Lactobacillus acidophilus, and Faecalibacterium increased by 1.07%, 2.45%, and 0.95% respectively in the double-layer Clostridium butyricum microcapsule group. The increase of these three genera in the double-layer Clostridium butyricum microcapsule group indicates that it improved the proportion of beneficial bacteria in the intestine. Bacteroides is a pathogenic bacterium in the human intestine. Compared with the Clostridium butyricum powder group, the proportion of Bacteroides in the double-layer Clostridium butyricum microcapsule group decreased by 6.19%, indicating that it reduced the proportion of harmful bacteria in the intestine. This shows that the double-layer Clostridium butyricum microcapsule group can better improve intestinal health than the Clostridium butyricum powder group.

[0157] Figure 15 The statistical chart shows the serum immune levels (C represents the control group, T represents the bacterial powder group, and H represents the double-layer Clostridium butyricum microcapsule group). The results indicate that the serum interleukin levels in the double-layer Clostridium butyricum microcapsule group were significantly higher than those in the control group, with increases of 66.27%, 58.79%, and 46.97% in IL-1β, IL-6, and TNF-α, respectively. Furthermore, the serum interleukin levels in the double-layer microcapsule group were significantly higher than those in the commercially available Clostridium butyricum powder group, with increases of 26.36%, 34.50% in IL-1β, IL-6, and TNF-α, respectively. The antibody levels in the serum of the double-layer Clostridium butyricum microcapsule group were significantly higher than those in the control group. Compared with the control group, the serum IgA, IgM, and IgG levels in the double-layer Clostridium butyricum microcapsule group increased by 68.73%, 39.64%, and 52.44%, respectively. The antibody levels in the serum of the double-layer Clostridium butyricum microcapsule group were also significantly higher than those in the commercially available Clostridium butyricum powder group. Compared with the commercially available Clostridium butyricum powder group, the serum IgA, IgM, and IgG levels in the double-layer Clostridium butyricum microcapsule group increased by 29.32%, 22.03%, and 20.78%, respectively. The experimental results indicate that the double-layer Clostridium butyricum microcapsule group can significantly improve the immunological indicators in broiler serum, and its effect is far superior to that of the commercially available Clostridium butyricum powder group.

[0158] This invention provides a probiotic microcapsule based on droplet microfluidics technology, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A probiotic microcapsule based on droplet microfluidic technology, characterized in that, The probiotic microcapsules mentioned above have microcapsule wall material made of isolated whey protein and polyglutamic acid derivatives, and microcapsule core material made of probiotics. The polyglutamic acid derivative was prepared according to the following method: Polyglutamic acid and glycidyl methacrylate were dissolved in deionized water to obtain an aqueous solution of polyglutamic acid. The pH of the aqueous solution of polyglutamic acid was adjusted to 3.5-4.0 with sulfuric acid solution. Then, the reaction was heated. After the reaction was completed, the reaction solution was dialyzed and lyophilized to obtain PGA-GMA. The probiotic mentioned is Clostridium butyricum; The method for preparing the probiotic microcapsules includes the following steps: (1) Dissolve whey protein and transglutaminase in deionized water, mix well, and obtain the first mixture; (2) Dissolve the pyrolytic crosslinking agent in an aqueous solution of dimethyl sulfoxide to obtain a second mixture; (3) Dissolve the polyglutamic acid derivative and the photoinitiator in deionized water to obtain a third mixture; (4) Disperse the probiotics in sterile water and resuspend them to obtain a probiotic suspension; mix the first mixture obtained in step (1) with the second mixture obtained in step (2) to obtain a fourth mixture; then mix the fourth mixture with the third mixture obtained in step (3), and add the probiotic suspension to the mixture system. After mixing, obtain an aqueous phase. (5) The silicone oil and emulsifier are shaken and mixed to obtain a continuous phase; (6) The aqueous phase obtained in step (4) and the continuous phase obtained in step (5) are simultaneously pumped into the T-shaped chip of droplet microfluidics for shearing to obtain microdroplets; the microdroplets are photocured to obtain probiotic microcapsules. The polyglutamic acid mentioned is γ-polyglutamic acid.

2. The probiotic microcapsule according to claim 1, characterized in that, The polyglutamic acid has a number-average molecular weight of 500-2000 kDa; the heating reaction is carried out at a temperature of 60-80°C for 8-10 hours.

3. The probiotic microcapsule according to claim 1, characterized in that, The *Clostridium butyricum* was pretreated using the following method: Clostridium butyricum was inoculated into RCM solid medium and anaerobically cultured at 30℃~37℃ for 20~24 h to obtain single colonies. These single colonies were then inoculated into RCM liquid medium using an inoculation loop and anaerobically cultured at 30℃~37℃ for 20~24 h to obtain Clostridium butyricum seed culture. The seed culture was then transferred to RCM liquid medium at an inoculation rate of 5%~10% v / v and anaerobically cultured at 30℃~37℃ for 20~24 h to obtain fermentation broth. The fermentation broth was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain Clostridium butyricum sludge. The viable count of Clostridium butyricum in the fermentation broth was 1×10⁻⁶. 8 ~10×10 8 CFU / mL; the centrifugation is carried out at a temperature of 4℃~8℃, a speed of 4000~8000 rpm, and a time of 5~15 min.

4. The probiotic microcapsule according to claim 1, characterized in that, In step (1), the transglutaminase has an enzyme activity of 45-55 U / g; the isolated whey protein has a protein content of more than 80%.

5. The probiotic microcapsule according to claim 1, characterized in that, In step (2), the decomposable crosslinking agent is N,N'-(2-amino-1,4-phenylene)diacrylamide.

6. The probiotic microcapsule according to claim 1, characterized in that, In step (3), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate or trimethylbenzoyl-diphenylphosphine oxide.

7. The probiotic microcapsule according to claim 1, characterized in that, In step (4), the volume ratio of the first mixture to the second mixture is 0.5~1.5:1; the volume ratio of the third mixture to the fourth mixture is 0.5~2:1; and the viable count of probiotics in the aqueous phase is 6×10⁻⁶. 8 ~10×10 8 CFU / mL.

8. The probiotic microcapsule according to claim 1, characterized in that, In step (5), the silicone oil is DOWSILRSN-0749; the emulsifier is Span 80.

9. The probiotic microcapsule according to claim 1, characterized in that, In step (6), the flow rate of the aqueous phase pumped into the T-shaped chip of the droplet microfluidic system is 2~6 μL / min; the flow rate of the continuous phase pumped into the T-shaped chip of the droplet microfluidic system is 5~40 μL / min; the shearing is performed at a temperature of 25~30℃; the photocuring is performed using ultraviolet light as the light source, with a wavelength of 320~400 nm, a curing temperature of 30℃~37℃, and a curing time of 3~5 min; and the size of the probiotic microcapsules is 90~610 μm.

10. The use of the probiotic microcapsules according to any one of claims 1 to 9 in the preparation of drugs or feeds for improving the intestinal flora of livestock and poultry.

Images