Probiotic / arachidonic acid composite microcapsule and preparation method thereof

Abstract

The application relates to a probiotic / arachidonic acid composite microcapsule and a preparation method thereof, and belongs to the technical field of microcapsule preparation, and the specific scheme is as follows: a probiotic / arachidonic acid composite microcapsule comprises wall material and core material, the core material is wrapped in the wall material, the wall material comprises sodium alginate and sodium carboxymethyl cellulose, the core material comprises arachidonic acid and probiotics, the arachidonic acid is dissolved in an oily carrier and forms a water-in-oil emulsion with a probiotic bacterial suspension, the sodium carboxymethyl cellulose wraps water-in-oil emulsion droplets, and the sodium alginate reacts with the sodium carboxymethyl cellulose to generate a gel film microcapsule wall material. The method for preparing the probiotic / arachidonic acid composite microcapsule provided by the application is simple to operate, the microfluidic chip is simple to prepare and low in cost, has a high embedding rate, the particle size of the microcapsule is controllable, and the biological activity is good.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microcapsule preparation, and particularly relates to a method for preparing probiotic / arachidonic acid composite microcapsules with controllable size by using microfluidic technology. BACKGROUND

[0002] Arachidonic acid has the effects of regulating the immune system, anti-tumor and antioxidant, etc. Probiotics can regulate the balance of intestinal flora, promote intestinal digestive absorption function and maintain intestinal microecology. The combination of arachidonic acid and probiotics has a synergistic effect, and can more efficiently exert the health care effect. However, both of them have the limitations of low bioavailability and poor stability. If the compound is coated to form a stable microcapsule form to improve the biological stability and availability, it will have important application prospects in the nutrition and health product industry.

[0003] Microcapsule technology can realize uniform encapsulation of active ingredients in the inside of microcapsules, forming a stable core-shell structure, so as to protect the active ingredients from the influence of the external environment. The advantages include protection of active ingredients, control of release speed and improvement of bioavailability, etc. Microcapsule technology can effectively solve the problems of active ingredients being easily affected by environmental factors and poor stability, improve the stability and bioavailability of the ingredients, and expand the application range. Therefore, the wide application of microcapsule technology in the fields of medicine, food, cosmetics, etc. has important significance.

[0004] The traditional method for preparing microcapsules has the limitations of high cost, complex operation, difficulty in controlling particle size and shape, etc. Microfluidic technology is a technology for manipulating fluid in micron-scale channels, which can quickly produce micron-sized monodisperse droplets. Through the design of the microfluidic system channel, probiotic W / O emulsion can be precisely coated in the droplets, and the particle size, shape and wall thickness of the microcapsules can be controlled. The microfluidic chip technology has the advantages of high throughput, low cost and high precision, etc. Therefore, the microfluidic chip technology has great potential in the preparation of microcapsules, and can promote the rapid development and application of microcapsule preparation. SUMMARY

[0005] In view of the problems of the traditional microcapsule preparation method, such as difficulty in controlling size and complex operation, the present application aims to provide a method for preparing probiotic / arachidonic acid composite microcapsules by using a microfluidic chip, which has the advantages of simple operation, high embedding rate, controllable particle size and synergistic biological activity.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A probiotic / arachidonic acid composite microcapsule, comprising a wall material and a core material, the core material being wrapped inside the wall material, the wall material comprising sodium alginate and sodium carboxymethyl cellulose, the core material comprising arachidonic acid and probiotics, the arachidonic acid being dissolved in an oily carrier and forming a water-in-oil emulsion with the probiotic bacterial suspension, the sodium carboxymethyl cellulose wrapping the water-in-oil emulsion droplets, and the sodium alginate reacting with the sodium carboxymethyl cellulose to form a gel film microcapsule wall material.

[0008] Further, the probiotics comprise Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus rhamnosus or Lactobacillus casei, and the oily carrier comprises flaxseed oil, fish oil or glycerol bisostearate.

[0009] Further, the preparation method of the probiotic bacterial suspension is as follows: inoculating the probiotics into MRS solid culture medium, culturing at 30-37°C for 22-24h, and continuously subculturing for 2-3 times to completely activate the probiotic bacterial suspension; inoculating the probiotic bacterial suspension into MRS liquid culture medium at an inoculation amount of 5-10% v / v, culturing at 30-37°C for 22-24h to collect the fermentation broth; centrifuging the fermentation broth, removing the supernatant, washing the precipitate to obtain probiotic bacterial slurry, resuspending the probiotic bacterial slurry in sterile water to obtain the probiotic bacterial suspension.

[0010] Further, the viable count of the probiotics in the fermentation broth is 1×10 8 -10×10 8 CFU / mL.

[0011] Further, the volume ratio of the arachidonic acid to the oily carrier is 20-50μL:20-50μL.

[0012] Further, the water / oil phase ratio of the water-in-oil emulsion is 1:10-2:5, and the water-in-oil emulsion further comprises 3-5% gelatin and 5-10% Tween 80 emulsifier.

[0013] A preparation method of the probiotic / arachidonic acid composite microcapsule, which utilizes a microfluidic chip to prepare the probiotic / arachidonic acid composite microcapsule, the microfluidic chip being internally provided with a main flow channel, one end of the main flow channel being provided with a core material feeding pipe, the other end being provided with a microcapsule discharging pipe, a wall material I feeding pipe and a wall material II feeding pipe being provided in the main flow channel and communicating with the main flow channel, the wall material II feeding pipe being located between the wall material I feeding pipe and the core material feeding pipe; a separation chamber being provided on the main flow channel between the wall material I feeding pipe and the microcapsule discharging pipe, the separation chamber being provided below with a wall material discharging pipe communicating with the separation chamber and above with an aeration pipe communicating with the separation chamber;

[0014] The preparation method of the probiotic / arachidonic acid composite microcapsule comprises the following steps:

[0015] The water-in-oil emulsion enters the main flow channel from the core material feeding pipe, the sodium carboxymethyl cellulose solution enters the main flow channel from the wall material II feeding pipe, the sodium carboxymethyl cellulose solution shears the water-in-oil emulsion to generate sodium carboxymethyl cellulose solution-coated water-in-oil emulsion droplets; the sodium alginate solution enters the main flow channel from the wall material I feeding pipe, the sodium alginate solution reacts with the outer sodium carboxymethyl cellulose solution to generate a gel film, and the gel film and the water-in-oil emulsion droplets jointly constitute the probiotic / arachidonic acid composite microcapsule, the excess wall material flows out from the wall material outlet pipe in the separation chamber, and the composite microcapsule flows out from the microcapsule outlet pipe.

[0016] Further, the wall material I feeding pipe and the wall material II feeding pipe are both arranged perpendicularly to the main flow channel, the outside of the core material feeding pipe is sleeved with a wall material-core material mixing pipe coaxially arranged with the core material feeding pipe, and the wall material II feeding pipe communicates with the wall material-core material mixing pipe; the outlet end of the core material feeding pipe is a constricted pointed nozzle.

[0017] Further, the microcapsule outlet pipe comprises a microcapsule outlet pipe I and a microcapsule outlet pipe II, the microcapsule outlet pipe I is coaxially arranged with the main flow channel, the microcapsule outlet pipe II is perpendicularly arranged with the main flow channel, and the connecting pipe opening of the microcapsule outlet pipe I and the main flow channel is provided with a microporous filter membrane I.

[0018] Further, the flow rate of the water-in-oil emulsion is 5-50 muL / min, the flow rate of the sodium carboxymethyl cellulose solution is 5-50 muL / min, and the flow rate of the sodium alginate solution is 50-200 muL / min; the mass percentage concentration of the sodium carboxymethyl cellulose solution is 1.0-10.0%, and the mass percentage concentration of the sodium alginate solution is 2.0-5.0%.

[0019] Compared with the prior art, the probiotic / arachidonic acid composite microcapsule has the following beneficial effects:

[0020] (1) The design of the present application can allow the core material probiotic bacterial suspension, the W / O emulsion of the oil carrier rich in arachidonic acid and the sodium carboxymethyl cellulose solution to be fed at the same time, the sodium carboxymethyl cellulose solution can wrap the W / O emulsion to generate microdroplets under the action of fluid dynamic shear, and a higher embedding rate and W / O emulsion core material utilization rate can be obtained;

[0021] (2) The outlet port of the core material feeding pipe is heated and drawn to form a pointed nozzle and present a constricted shape, which is beneficial to the rapid formation of the W / O emulsion under the action of the fluid dynamic shear flow of the sodium carboxymethyl cellulose solution, and a higher forming rate can be obtained;

[0022] (3) The present application separates the W / O emulsion gel film microcapsules from the wall material sodium alginate solution in the separation chamber by introducing nitrogen, and the separated gel film microcapsules enter the microcapsule discharge pipeline, and the two particle size ranges of the composite microcapsules are separated under the blocking action of the microporous filter membrane I, which simplifies the subsequent processing operation of the microcapsules, improves the automation degree, and realizes the effect of collecting multiple particle size microcapsules together. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of a microfluidic chip;

[0024] Figure 2 is a schematic diagram of the morphology of the composite microcapsules under a freeze scanning electron microscope;

[0025] Figure 3 is a schematic diagram of the internal structure of the composite microcapsules under a freeze scanning electron microscope;

[0026] In the figure, 1 is a main flow channel, 2 is a core material feeding pipeline, 3 is a microcapsule discharge pipeline, 4 is a wall material I feeding pipeline, 5 is a wall material II feeding pipeline, 6 is a separation chamber, 7 is a wall material discharge pipeline, 8 is a ventilation pipeline, 9 is a wall material-core material mixing pipeline, 10 is a microporous filter membrane I, 31 is a microcapsule discharge pipeline I, and 32 is a microcapsule discharge pipeline II. DETAILED DESCRIPTION

[0027] The technical solutions in the present application will be described clearly and completely below in combination with the drawings and examples. Obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the protection scope of the present application. DETAILED DESCRIPTION

[0029] The present application uses a molding method to prepare a microfluidic chip, which comprises a main flow channel 1 and a separation chamber 6. A photosensitive material is coated on the contact surface of the two microfluidic chip channels, so that the hollow microfluidic chip is prepared by sealing together through a photocuring method. The W / O emulsion of the oily carrier with arachidonic acid and the probiotic bacteria suspension is used as the microcapsule core material, and is fed at the same time with the sodium carboxymethyl cellulose solution. Under the fluid dynamic shear action of the sodium carboxymethyl cellulose solution, the composite gel microcapsules of the sodium carboxymethyl cellulose wrapped W / O emulsion are formed. The method for preparing the probiotic bacteria / arachidonic acid composite microcapsules provided by the present application is simple to operate, the microfluidic chip is simple to prepare and has low cost, has a high embedding rate, and the particle size of the microcapsules is controllable and has good biological activity.

[0030] The specific scheme is as follows:

[0031] The probiotic / arachidonic acid composite microcapsule comprises a wall material and a core material, the core material is wrapped inside the wall material, the wall material comprises sodium alginate and sodium carboxymethyl cellulose, the core material comprises arachidonic acid and probiotic bacteria, the arachidonic acid is dissolved in an oily carrier and forms a water-in-oil emulsion with the probiotic bacteria suspension, the sodium carboxymethyl cellulose wraps the water-in-oil emulsion droplets, and the sodium alginate reacts with the sodium carboxymethyl cellulose to form a gel film microcapsule wall material.

[0032] Further, the probiotic bacteria comprise Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus rhamnosus or Lactobacillus casei, and the oily carrier comprises flaxseed oil, fish oil or glycerol bisostearate.

[0033] Further, the preparation method of the probiotic bacteria suspension is as follows: inoculate the probiotic bacteria into MRS solid culture medium, cultivate at 30-37℃ for 22-24h, continuously subculture for 2-3 times to completely activate the probiotic bacteria to obtain a probiotic bacteria liquid; inoculate the probiotic bacteria liquid into MRS liquid culture medium at an inoculation amount of 5-10% v / v, cultivate at 30-37℃ for 22-24h to collect a fermentation liquid; centrifuge the fermentation liquid, remove the supernatant, wash the precipitate to obtain a probiotic bacteria slurry, add 50-100μL sterile water to resuspend the probiotic bacteria slurry to obtain the probiotic bacteria suspension, wherein the centrifugation temperature is 4-8℃, the centrifugation speed is 3000-6000rpm / min, and the centrifugation time is 10-15min. The composition of the MRS solid culture medium is: glucose 20g / L, peptone 10g / L, hydrolyzed yeast 5g / L, magnesium sulfate 0.2g / L, potassium dihydrogen phosphate 2g / L, sodium chloride 5g / L, sodium thiosulfate 0.1g / L, magnesium chloride 0.1g / L, agar 15-20g / L, and water to 1L; the composition of the MRS liquid culture medium is: glucose 20g / L, peptone 10g / L, hydrolyzed yeast 5g / L, magnesium sulfate 0.2g / L, potassium dihydrogen phosphate 2g / L, sodium chloride 5g / L, sodium thiosulfate 0.1g / L, magnesium chloride 0.1g / L, and water to 1L.

[0034] Preferably, the probiotic bacteria are Lactobacillus plantarum, genus name: Lactobacillus, species name: plantarum, scientific name: Lactobacillus plantarum; more preferably, the Lactobacillus plantarum is from tofu.

[0035] Further, the viable count of the probiotic bacteria in the fermentation liquid is 1×10 8 ~ 10×10 8 CFU / mL.

[0036] Further, the volume ratio of arachidonic acid to oily carrier is 20-50μL:20-50μL.

[0037] Furthermore, the water-in-oil emulsion has a water-to-oil ratio of 1:10 to 2:5, and further comprises 3% to 5% gelatin and 5% to 10% Tween 80 emulsifier by mass fraction. The preparation method of the water-in-oil emulsion is as follows: the components are mixed evenly according to the above proportions, and then centrifuged and ultrasonically dispersed to obtain the water-in-oil emulsion. The centrifugation temperature is 4℃ to 8℃, the centrifugation speed is 10000 to 12000 rpm / min, and the centrifugation time is 5 to 8 min. Ultrasonic dispersion is performed using a probe-type ultrasonic instrument with an ultrasonic amplitude of 40% to 45% and an ultrasonic time of 4 to 6 min. Specific Implementation Method Two

[0039] A method for preparing probiotic / arachidonic acid composite microcapsules according to a specific embodiment 1 utilizes a microfluidic chip to prepare the probiotic / arachidonic acid composite microcapsules. The microfluidic chip has a main channel 1 internally. One end of the main channel 1 has a core material inlet pipe 2, and the other end has a microcapsule outlet pipe 3. Near the core material inlet pipe 2, a wall material I inlet pipe 4 and a wall material II inlet pipe 5, connected to the main channel 1, are provided. The wall material II inlet pipe 5 is located between the wall material I inlet pipe 4 and the core material inlet pipe 2. A separation chamber 6 is provided on the main channel 1 between the wall material I inlet pipe 4 and the microcapsule outlet pipe 3. Below the separation chamber 6, a wall material outlet pipe 7, connected to the separation chamber 6, is provided, and above the separation chamber 6, a ventilation pipe 8, connected to the separation chamber 6, is provided.

[0040] The preparation method of the probiotic / arachidonic acid composite microcapsules includes the following steps:

[0041] Water-in-oil emulsion enters the main channel 1 from the core material feed pipe 2. Sodium carboxymethyl cellulose solution enters the main channel 1 from the wall material II feed pipe 5. The sodium carboxymethyl cellulose solution shears the water-in-oil emulsion, generating water-in-oil emulsion droplets encapsulated by sodium carboxymethyl cellulose solution. Sodium alginate solution enters the main channel 1 from the wall material I feed pipe 4. The sodium alginate solution reacts with the outer layer of sodium carboxymethyl cellulose solution to form a gel membrane. The gel membrane and the water-in-oil emulsion droplets together form probiotic / arachidonic acid composite microcapsules. The prepared composite microcapsules and sodium alginate solution enter the separation chamber 6 simultaneously. Nitrogen gas is introduced through the vent pipe 8. Excess sodium alginate solution flows out from the wall material discharge pipe 7, and the composite microcapsules flow out from the microcapsule discharge pipe 3.

[0042] Furthermore, both the wall material I inlet pipe 4 and the wall material II inlet pipe 5 are arranged perpendicular to the main channel 1. A wall material-core material mixing pipe 9, coaxially arranged with the core material inlet pipe 2, is sleeved outside the core material inlet pipe 2. The wall material II inlet pipe 5 is connected to the wall material-core material mixing pipe 9. The outlet end of the core material inlet pipe 2 is a constricted tip. Preferably, the core material inlet pipe 2 is a glass capillary tube, and its outlet port is heated and drawn to form a constricted tip with an inner diameter of 0.05–0.2 mm. The water-in-oil emulsion flows from the core material inlet pipe 2 into the wall material-core material mixing pipe 9, where it mixes with the sodium carboxymethyl cellulose solution introduced through the wall material II inlet pipe 5, and then flows together into the main channel 1 to mix with the sodium alginate solution.

[0043] Furthermore, the wall material and core material mixing pipe 9 is sleeved on the outer periphery of the core material feeding pipe 2. At the end away from the outlet of the wall material and core material mixing pipe 9, the inner wall of the wall material and core material mixing pipe 9 is bonded and sealed to the outer wall of the core material feeding pipe 2 using hot melt adhesive.

[0044] Furthermore, the microcapsule discharge channel 3 includes a microcapsule discharge channel I 31 and a microcapsule discharge channel II 32. The microcapsule discharge channel I 31 is coaxially arranged with the main channel 1, and the microcapsule discharge channel II 32 is perpendicular to the main channel 1. The connection points of the microcapsule discharge channels I 31 and II 32 with the main channel 1 are at the same positions. A microporous filter membrane I 10 is provided at the pipe opening of the connection point between the microcapsule discharge channel I 31 and the main channel 1. The microporous filter membrane I 10 can separate composite microcapsules of different sizes, allowing small-diameter composite microcapsules to enter the microcapsule discharge channel I 31 along the axial direction of the main channel 1, while large-diameter composite microcapsules are intercepted outside the microporous filter membrane I 10 and flow to the microcapsule discharge channel II 32. Preferably, the pore size of the microporous filter membrane I 10 is in the range of 0.1–0.4 mm; and the size of the prepared composite microcapsules is 100–800 μm.

[0045] Furthermore, the flow rate of the water-in-oil emulsion is 5–50 μL / min, the flow rate of the sodium carboxymethyl cellulose solution is 5–50 μL / min, and the flow rate of the sodium alginate solution is 50–200 μL / min.

[0046] Furthermore, the sodium carboxymethyl cellulose solution has a mass percentage concentration of 1.0% to 10.0%, and the sodium alginate solution has a mass percentage concentration of 2.0% to 5.0%.

[0047] Furthermore, the fabrication method of the microfluidic chip is as follows: a mold containing a microchannel structure is precisely prepared from single-crystal silicon material using soft lithography micro-nano fabrication technology; a polymer material is poured onto the single-crystal silicon mold and cured to prepare two microfluidic chips; a photosensitive material is coated on the contact surface of the two microfluidic chips, and ultraviolet light is used to irradiate it to cause a photochemical reaction, thereby achieving chip curing and sealing, and obtaining a hollow microfluidic chip;

[0048] Preferably, the single-crystal silicon material comprises silicon crystal (Si), silicon film, silicon-on-insulator (SOI), or silicon wafer; the polymer material comprises polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), or polycarbonate.

[0049] Furthermore, the specific steps of the soft lithography micro / nano fabrication technology are as follows: prepare a clean silicon wafer as a substrate, and prepare a transparent or semi-transparent mask containing the desired pattern; uniformly cover the silicon wafer surface with photoresist using spin coating or coating machinery; place the photoresist-coated silicon wafer in an oven to evaporate the solvent in the photoresist, causing it to harden and adhere to the silicon wafer; align the mask and the photoresist-coated silicon wafer and place them in a lithography machine, setting the exposure time to 5–15 s and the light intensity to 1–10 mW / cm². 2 The process involves exposing the pattern on the mask to the photoresist using ultraviolet light. After exposure, the silicon wafer is placed in a developing solution to dissolve or remove the portion exposed to light, thereby forming the desired pattern on the surface of the silicon wafer. The silicon wafer is then placed in a cleaning solution to remove any residual photoresist after development, resulting in a patterned photoresist template.

[0050] The protruding part of the first chip mold includes: ventilation pipe 8, upper part of separation chamber 6, upper part of main channel 1, upper part of wall material I feed pipe 4, upper part of wall material II feed pipe 5, and upper part of microcapsule discharge pipe I 31 and upper part of microcapsule discharge pipe II 32; the protruding part of the second crystal positive mold includes: lower part of separation chamber 6, wall material discharge pipe 7, lower part of main channel 1, lower part of wall material I feed pipe 4, lower part of wall material II feed pipe 5, lower part of microcapsule discharge pipe I 31 and lower part of microcapsule discharge pipe II 32.

[0051] Polymer materials and hardeners are mixed in a specific ratio to prepare a prepolymer solution. The prepolymer solution is then slowly poured onto a template using a syringe. The template containing the prepolymer solution is placed in a vacuum degassing machine for 5–10 minutes to remove bubbles. After degassing, it is placed in an oven for curing at 60–80°C for 1–5 hours. After curing, the mold is removed from the oven and allowed to cool to room temperature before gently peeling off the template to obtain two microfluidic chips. The two prepared microfluidic chips are then removed. The photosensitive material used for photochemical bonding of the two chips is photoresist. Specifically, photoresist is coated onto the surfaces of the two prepared microfluidic chips. The two microfluidic chips are then joined together and placed under an ultraviolet light source. The photosensitive material forms an adhesive under chemical reaction, firmly bonding the two chips together. The chips are then placed in a cleaning solvent to remove unreacted photosensitive material and residual adhesive. After 1–5 hours of curing, the bonded chips are inspected for quality, and microfluidic chips free of bubbles, cracks, or other defects are retained.

[0052] Example 1:

[0053] This embodiment provides a method for fabricating a microfluidic chip, the structure of which is as follows:

[0054] like Figure 1 As shown, the microfluidic chip includes: a main channel 1, a core material feed channel 2, a wall material I feed channel 4, a wall material II feed channel 5, a separation chamber 6, a ventilation channel 8, a wall material discharge channel 7, a microcapsule discharge channel I 31, and a microcapsule discharge channel II 32.

[0055] The wall material and core material mixing pipe 9 and the core material feeding pipe 2 are arranged coaxially and in the same direction as the main channel 1. The wall material I feeding pipe 4 and the wall material II feeding pipe 5 are both arranged perpendicular to the main channel 1, and the two feeding ports are 5mm apart.

[0056] The main channel 1 has a diameter of 1.5 mm; the wall material and core material mixing pipe 9 has an outer diameter of 1.0 mm and an inner diameter of 0.8 mm, and the outer wall of the wall material and core material mixing pipe 9 is fixedly and sealed to the inner wall of the main channel 1 using hot melt adhesive; the core material feeding pipe 2 has an outer diameter of 0.6 mm and an inner diameter of 0.4 mm; the discharge port of the core material feeding pipe 2 is drawn into a pointed nozzle shape by burning it with the outer flame of an alcohol torch, and the inner diameter of the pointed nozzle is 0.2 mm. The outer wall of the core material feeding pipe 2 is fixedly and sealed to the inner wall of the wall material and core material mixing pipe 9 using hot melt adhesive; the distance between the pointed port of the core material feeding pipe 2 and the discharge port of the wall material and core material mixing pipe 9 is 5 mm.

[0057] The junction of the wall material I feed pipe 4 and the main channel 1 is 3mm away from the outlet port of the wall material core material mixing pipe 9; the junction of the wall material II feed pipe 5 and the wall material core material mixing pipe 9 is 3mm away from the tip port of the core material feed pipe 2. It is necessary to use a hole punch to make horizontal holes at the corresponding positions of the wall material core material mixing pipe 9 and the main channel 1. The wall material II feed pipe 5 passes through the through hole on the side wall of the main channel 1 and connects with the wall material core material mixing pipe 9. Hot melt adhesive is used to seal the side wall of the main channel 1 and the side wall of the wall material II feed pipe 5.

[0058] The microfluidic chip is 60mm long, 20mm wide, and 10mm high. The separation chamber 6 is 40mm from the right end of the chip and 20mm from the left end. The upper part of the separation chamber 6 is connected to the ventilation pipe 8, and the lower part of the separation chamber 6 is connected to the wall material outlet pipe 7. A microporous filter membrane II with a pore size of 10μm is installed at the intersection of the separation chamber 6 and the wall material outlet pipe 7. Both the ventilation pipe 8 and the wall material outlet pipe 7 are perpendicular to the main channel 1. The microcapsule outlet pipe II 32 is perpendicular to the main channel 1, and the outlet diameter is 1mm. The microcapsule outlet pipe I 31 is coaxial with the main channel 1 and has a diameter of 1.5mm. A 400μm microporous filter membrane I is placed at the connection between the microcapsule outlet pipe I 32 and the main channel 1 to collect microcapsules with a size smaller than 400μm.

[0059] The separation chamber 6 is cylindrical, with a height of 5 mm and a diameter of 10 mm. The wall material outlet pipe 7 below it has a diameter of 2 mm. The connection between the wall material outlet pipe 7 and the separation chamber 6 is covered with a microporous filter membrane II with a pore size of 10 μm. The ventilation pipe 8 above it has a diameter of 2 mm. The microcapsule outlet pipe 3 is connected to the main channel 1 that passes through the other side of the separation chamber 6. The junction of the microcapsule outlet pipe I 31 and the microcapsule outlet pipe II 32 is located 10 mm away from the separation chamber.

[0060] Example 2:

[0061] This embodiment provides a method for preparing Lactobacillus plantarum / arachidonic acid microcapsules, specifically including the following steps:

[0062] (1) The *Lactobacillus plantarum* is derived from tofu, genus name: *Lactobacillus*, species name: *plantarum*, scientific name: *Lactobacillus plantarum*; the *Lactobacillus plantarum* was inoculated into MRS solid medium and cultured at 37℃ for 22 h, and then subcultured twice to fully activate it for use to obtain *Lactobacillus plantarum* bacterial suspension; the *Lactobacillus plantarum* bacterial suspension was transferred to MRS liquid medium at an inoculation rate of 8% v / v, and cultured at 37℃ for 22 h to collect the fermentation broth; the fermentation broth was centrifuged, the supernatant was removed, and the precipitate was washed to obtain *Lactobacillus plantarum* bacterial sludge; wherein, the viable count of *Lactobacillus plantarum* in the fermentation broth was 5 × 10⁻⁶. 8CFU / mL; centrifugation temperature: 4℃; centrifugation speed: 3500 rpm / min; centrifugation time: 15 min;

[0063] (2) The composition of the MRS solid culture medium is: 20 g / L glucose, 10 g / L peptone, 5 g / L hydrolyzed yeast, 0.2 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium chloride, 0.1 g / L sodium thiosulfate, 0.1 g / L magnesium chloride, 15-20 g / L agar, with water added to 1 L; the composition of the MRS liquid culture medium is: 20 g / L glucose, 10 g / L peptone, 5 g / L hydrolyzed yeast, 0.2 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium chloride, 0.1 g / L sodium thiosulfate, 0.1 g / L magnesium chloride, with water added to 1 L;

[0064] (3) The *Lactobacillus plantarum* bacterial sludge from step (1) was added to 80 μL of sterile water and resuspended to obtain *Lactobacillus plantarum* bacterial suspension; the linseed oil rich in arachidonic acid was obtained by adding 50 μL of arachidonic acid standard oil to 50 μL of linseed oil; the core material was a water-in-oil (W / O) primary emulsion of linseed oil rich in arachidonic acid and *Lactobacillus plantarum*; a crude mixture was prepared with a water-to-oil ratio of 1:10, a gelatin addition of 3%, and 5% Tween 80 as an emulsifier; the crude mixture was centrifuged at 4°C, 12000 rpm / min for 5 min, and then sonicated with a probe ultrasonic instrument at 40% ultrasonic amplitude for 3 min to obtain a W / O emulsion;

[0065] (4) Microcapsule production on the chip: The microcapsules were prepared on a microfluidic chip. The core material, sodium carboxymethyl cellulose solution and sodium alginate solution were respectively loaded into three 1 mL syringes and connected to the injection pump. The W / O emulsion was fed from the core material feed pipe 2 at a flow rate of 30 μL / min. The sodium carboxymethyl cellulose solution had a mass percentage concentration of 3.5% and a flow rate of 30 μL / min. The sodium alginate solution had a mass percentage concentration of 3% and a flow rate of 40 μL / min. The microcapsules were prepared in the chip and then collected in a pre-cooled sterile aqueous solution through two microcapsule discharge pipes to obtain composite microcapsules with a size range of 100-800 μm, which were stored in a refrigerator at 4°C for later use.

[0066] This invention uses cryo-electron microscopy to observe the internal morphology of microcapsules, which clearly reveals the morphology of probiotics and arachidonic acid droplets. The morphology and internal structure of the microcapsules under cryo-electron microscopy are shown below. Figure 2 and 3 As shown.

[0067] The encapsulation ability of probiotics by different sizes of Lactobacillus plantarum / arachidonic acid composite microcapsules obtained in Example 2 was evaluated.

[0068] The number of viable bacteria and the volume of the aqueous phase were measured to determine the total number of viable bacteria before encapsulation. Microcapsule samples containing *Lactobacillus plantarum* were prepared, and the total volume of the samples was recorded. The samples were kept under appropriate temperature and environmental conditions to maintain the activity of the probiotics. After centrifugation at 4°C and 4000 rpm for 5 min, the microcapsules precipitated at the bottom of the centrifuge tube, and the supernatant containing the unencapsulated aqueous phase was discarded. The microcapsules were washed with 1% v / v Tween 80 solution to remove surface impurities and unencapsulated probiotics, and then centrifuged again under the same conditions to collect the microcapsules. The washed microcapsules were resuspended in 200 μmol / L nitric oxide donor solution (SNP solution), which served as the NO donor. The resuspension solution helps maintain the activity of probiotics. Add 3-4 glass beads to the resuspension and incubate at 37°C on a shaker at 200 rpm under natural light for 30 minutes. Take 0.5 mL of the sample from the incubated suspension and serially dilute with PBS buffer. Use plate counting to determine the number of viable bacteria in the diluted sample. Based on the measured number of viable bacteria and the volume of the microcapsules, calculate the encapsulation efficiency of the probiotics using the following formula: Encapsulation efficiency = (Number of viable bacteria per unit microcapsule × Volume of microcapsule) / (Number of viable bacteria per unit aqueous phase × Volume of aqueous phase) × 100%. The results show that microcapsules with a particle size of 300 μm have the highest encapsulation efficiency and the best encapsulation effect for *Lactobacillus plantarum*.

[0069] Table 1 Encapsulation efficiency of Lactobacillus plantarum by microcapsules of different sizes

[0070] Experiment No. Particle size (μm) Embedding rate (%) 1 100 75.67±1.5 2 300 78.34±1.8 3 600 76.34±2.1 4 800 77.98±1.2

[0071] The encapsulation ability of different sizes of *Lactobacillus plantarum* / arachidonic acid composite microcapsules obtained in Example 2 for arachidonic acid was evaluated. 3.00 g of microcapsule sample was weighed, placed in a small beaker, and an appropriate amount of anhydrous ethanol, anhydrous diethyl ether, and petroleum ether (volume ratio 4:2:3) were added for ultrasonic disruption. After disruption, the sample was centrifuged at 4°C, 4000 rpm / min for 10 min, and the upper organic phase was collected and washed with distilled water. The washed sample was dried with anhydrous sodium sulfate and finally dried with hexane under nitrogen. Fatty acid extracts were obtained; the fatty acid extracts were diluted with 5 mL of n-hexane, and the absorbance was measured at a wavelength of 220 nm using a UV-Vis spectrophotometer; the method for determining the total oil content was similar to that for the surface oil content, except that the ultrasonic disruption step was not required, and the absorbance was also measured at a wavelength of 220 nm using a UV-Vis spectrophotometer; the encapsulation rate (%) was calculated using the formula: encapsulation rate = 1 - (surface oil content / total oil content) × 100%; the results showed that the microcapsules with a particle size of 300 μm had the highest encapsulation rate for arachidonic acid and the best encapsulation effect.

[0072] Table 2 Encapsulation efficiency of arachidonic acid by microcapsules of different sizes

[0073] Experiment No. Particle size (μm) Embedding rate (%) 1 100 79.57±2.3 2 300 87.66±2.7 3 600 80.52±1.6 4 800 82.73±1.9

[0074] This invention uses an in vitro simulated gastrointestinal digestion process to determine the digestibility of microcapsules. Using free *Lactobacillus plantarum* as a control group, the survival rate of *Lactobacillus plantarum* in microcapsules with a particle size of 300 μm after gastrointestinal digestion was measured. As shown in Table 3, after the simulated digestion, free *Lactobacillus plantarum* was completely inactivated, with the survival rate decreasing from an initial 8.98 log CFU / g to 0 CFU / g; the survival rate of *Lactobacillus plantarum* encapsulated in microcapsules decreased from an initial 8.32 log CFU / g to 6.54 log CFU / g. Microcapsules can delay the contact time of probiotics with digestive juices by maximally isolating them from the harsh conditions of the gastrointestinal tract by immobilizing probiotics in the core emulsion. The results show that microcapsule encapsulation can significantly alleviate the digestive degradation of probiotics in the gastrointestinal tract.

[0075] Table 3. Live cell counts of microcapsules loaded with bacteria during simulated gastrointestinal digestion.

[0076]

[0077] Example 3:

[0078] Using the microfluidic chip device in Example 1, the feed rate of the core material inlet in Example 2 was adjusted to 5 μL / min, 10 μL / min, 20 μL / min, 30 μL / min, 40 μL / min, 50 μL / min, and 60 μL / min, while keeping other conditions unchanged. The obtained microcapsule particle size range was still 100–800 μm. It was calculated that when the feed rate was 5–50 μL / min, the encapsulation rate of Lactobacillus plantarum and arachidonic acid was higher than 75%, while when the flow rate was 60 μL / min, the encapsulation rate was lower than 75%.

[0079] Example 4:

[0080] Using the microfluidic chip device in Example 1, the mass percentage concentration of the sodium alginate solution in Example 2 was adjusted to 1%, 2%, 4%, 8%, and 10%, respectively, while keeping other conditions unchanged. When the mass percentage concentration of the solution was 1%, 2%, 8%, and 10%, the calculated encapsulation rates of *Lactobacillus plantarum* and arachidonic acid were all below 70%. This is because a sodium carboxymethyl cellulose solution with a concentration below 2% cannot form a W / O emulsion encapsulating *Lactobacillus plantarum* / arachidonic acid, while an excessively high concentration of sodium carboxymethyl cellulose solution is too viscous to pass smoothly through the inlet, causing blockage. Therefore, it is impossible to encapsulate a W / O emulsion of *Lactobacillus plantarum* / arachidonic acid.

[0081] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing probiotic / arachidonic acid composite microcapsules, wherein the microcapsules comprise a wall material and a core material, the core material being encapsulated within the wall material, characterized in that: The wall material includes sodium alginate and sodium carboxymethyl cellulose, the core material includes arachidonic acid and probiotics, the arachidonic acid is dissolved in an oily carrier and forms a water-in-oil emulsion with a probiotic suspension, the sodium carboxymethyl cellulose encapsulates the water-in-oil emulsion droplets, and the sodium alginate reacts with sodium carboxymethyl cellulose to generate a gel membrane microcapsule wall material. The method is as follows: the probiotic / arachidonic acid composite microcapsules are prepared using a microfluidic chip. The microfluidic chip is provided with a main channel (1). One end of the main channel (1) is provided with a core material feeding pipe (2) and the other end is provided with a microcapsule discharge pipe (3). At one end near the core material feeding pipe (2), a wall material I feeding pipe (4) and a wall material II feeding pipe (5) are provided, which are connected to the main channel (1). The wall material II feeding pipe (5) is located between the wall material I feeding pipe (4) and the core material feeding pipe (2). A separation chamber (6) is provided on the main channel (1) between the wall material I feeding pipe (4) and the microcapsule discharge pipe (3). Below the separation chamber (6), a wall material discharge pipe (7) is provided, which is connected to the separation chamber (6). Above the separation chamber (6), a ventilation pipe (8) is provided, which is connected to the separation chamber (6). The preparation method of the probiotic / arachidonic acid composite microcapsules includes the following steps: The water-in-oil emulsion enters the main channel (1) from the core material feed pipe (2), and the sodium carboxymethyl cellulose solution enters the main channel (1) from the wall material II feed pipe (5). The sodium carboxymethyl cellulose solution shears the water-in-oil emulsion to generate water-in-oil emulsion droplets encapsulated by the sodium carboxymethyl cellulose solution. The sodium alginate solution enters the main channel (1) from the wall material I feed pipe (4). The sodium alginate solution reacts with the outer layer of sodium carboxymethyl cellulose solution to generate a gel membrane. The gel membrane and the water-in-oil emulsion droplets together form probiotic / arachidonic acid composite microcapsules. Excess wall material flows out from the wall material discharge pipe (7) in the separation chamber (6), and the composite microcapsules flow out from the microcapsule discharge pipe (3). The wall material I feed pipe (4) and wall material II feed pipe (5) are both set perpendicular to the main channel (1). The core material feed pipe (2) is fitted with a wall material core material mixing pipe (9) that is coaxially set with the core material feed pipe (2). The wall material II feed pipe (5) is connected to the wall material core material mixing pipe (9). The outlet end of the core material feed pipe (2) is a constricted pointed nozzle. The microcapsule discharge pipe (3) includes a microcapsule discharge pipe I (31) and a microcapsule discharge pipe II (32). The microcapsule discharge pipe I (31) is coaxially arranged with the main channel (1), and the microcapsule discharge pipe II (32) is perpendicular to the main channel (1). A microporous filter membrane I (10) is provided at the pipe opening where the microcapsule discharge pipe I (31) and the main channel (1) are connected.

2. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 1, characterized in that: The probiotics include Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus rhamnosus, or Lactobacillus casei, and the oily carrier includes flaxseed oil, fish oil, or glyceryl distearate.

3. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 1, characterized in that: The method for preparing the probiotic suspension is as follows: probiotics are inoculated into MRS solid medium and cultured at 30℃~37℃ for 22~24h. After continuous subculturing 2~3 times, the probiotics are fully activated to obtain the probiotic liquid. The probiotic liquid is transferred to MRS liquid medium at an inoculation rate of 5%~10% v / v and cultured at 30℃~37℃ for 22~24h to collect the fermentation broth. The fermentation broth is centrifuged, the supernatant is removed, and the precipitate is washed to obtain probiotic sludge. The probiotic sludge is added to sterile water for resuspending to obtain the probiotic suspension.

4. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 3, characterized in that: The viable count of probiotics in the fermentation broth is 1×10⁻⁶. 8 ~10×10 8 CFU / mL.

5. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 1, characterized in that: The volume ratio of arachidonic acid to the oily carrier is 20-50 μL: 20-50 μL.

6. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 1, characterized in that: The water-in-oil emulsion has a water-to-oil ratio of 1:10 to 2:5, and further includes 3% to 5% gelatin and 5% to 10% Tween 80 emulsifier by mass.

7. The method for preparing a probiotic / arachidonic acid composite microcapsule according to claim 1, characterized in that: The flow rate of the water-in-oil emulsion is 5–50 μL / min, the flow rate of the sodium carboxymethyl cellulose solution is 5–50 μL / min, and the flow rate of the sodium alginate solution is 50–200 μL / min; the mass percentage concentration of the sodium carboxymethyl cellulose solution is 1.0–10.0%, and the mass percentage concentration of the sodium alginate solution is 2.0–5.0%.

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