O / W type milk-like fat globules, preparation method based on microfluidics and application of embedded probiotics

Abstract

The invention discloses an O / W type milk-like fat globule, a preparation method based on microfluidics and application of embedded probiotics. According to the O / W type milk-like fat globules, OPO structural ester serves as a core material, a lactoferrin-lecithin-HMO compound serves as a wall material, and the wall material is obtained by dissolving lactoferrin, lecithin and HMO in ultrapure water according to the mass ratio of 1: (0.01-0.5): 1 and adjusting the pH of a system to be 5-7. Tests show that the freeze-drying survival rates of the lactobacillus rhamnosus and the bifidobacterium embedded in the O / W type milk-like balls are 98.6 + / -3.5% and 96.1 + / -5.8% respectively, and the survival rates of the lactobacillus rhamnosus and the bifidobacterium after gastrointestinal digestion are 96.6 + / -2.7% and 92.9 + / -6.9% respectively.

Description

Technical Field

[0001] This invention relates to milk fat globule mimicry, and particularly to an O / W type milk fat globule mimicry microcapsule and its microfluidic-based preparation method and application in encapsulating probiotics, belonging to the field of probiotic encapsulation technology. Background Technology

[0002] As a live microbial preparation, the efficacy of probiotics depends on the survival ability of the live bacteria. However, in practical applications, they face multiple challenges: temperature fluctuations and mechanical stress during processing may damage bacterial activity; changes in water activity and oxygen permeation during storage can affect the stability of the bacterial community; and oral delivery requires overcoming digestive tract barriers such as gastric acid erosion and bile dissolution. These factors collectively limit the practical application value of probiotic products.

[0003] Existing technologies for mimicking milk fat globule probiotic microcapsules have significant limitations. These limitations primarily lie in their structural design, which is confined to a single-layer biomimetic model of the milk fat globule membrane, resulting in limited protective effects. Furthermore, the preparation process faces significant technical bottlenecks. Freeze-drying methods can easily damage the bacterial structure due to ice crystal formation, while the high-temperature process of spray drying severely depletes bacterial activity. Fluidized bed drying causes bacterial inactivation due to mechanical stress. Extraction-fusion methods not only suffer from low process precision but also pose safety risks due to chemical residues. For example, Chinese invention patent CN114532541B discloses a milk fat globule membrane-mimicking probiotic microcapsule prepared using a milk fat globule membrane composite wall material via spray, freeze-drying, or fluidized bed drying techniques. However, the probiotic survival rate is consistently below 30%, confirming the insufficient protective effect of a single-layer biomimetic model. Chinese invention patent CN113892649B uses organic solvents to extract milk fat globule membrane proteins and constructs microcapsules through enzymatic cross-linking. This process also only achieves a single-layer biomimetic structure and suffers from difficulty in controlling process precision and the risk of chemical residues, severely restricting its application prospects in the food industry.

[0004] Existing probiotic microcapsules based on droplet microfluidics technology are mainly W / O (water-in-oil) structures. These microcapsules have the following limitations: First, probiotics encapsulated in an aqueous phase are still affected by changes in internal water activity and external oxygen permeation, threatening the long-term stability of the bacterial community. Second, the three-dimensional network structure of the W / O type is insufficient to effectively block the permeation of digestive enzymes and bile salts during gastrointestinal digestion, leading to a significant decrease in bacterial activity. Furthermore, droplets prepared by droplet microfluidics require solidification to form microcapsules for application, and some research on solidification often involves chemical cross-linking agents or organic solvents, introducing food safety risks. For example, Chinese invention patent application CN118452469A prepared a three-dimensional network W / O type emulsion gel using arachidonic acid as the oil phase and a mixture of sodium carboxymethyl cellulose and sodium alginate containing probiotics as the aqueous phase; however, after gastrointestinal digestion, the bacterial activity decreased by two orders of magnitude. Chinese invention patent application CN116407568A mixes whey protein, transglutaminase, a cleavable crosslinking agent, a photoinitiator, polyglutamic acid derivatives, and dimethyl sulfoxide with a probiotic suspension. The mixture is then sheared in a two-phase microchannel with silicone oil and an emulsifier to form droplets, which are then cured by ultraviolet light to obtain W / O type microcapsules. However, this process does not further remove organic reagents, which poses a food safety risk. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an O / W type biomimetic milk fat globules that are similar to natural milk fat globules by using a dual structure design of "triglyceride core-milk fat globule membrane" and an aqueous phase formed by lactoferrin-lecithin-HMO complex as the outer membrane; and to provide a method for preparing O / W type biomimetic milk fat globules using microfluidic technology.

[0006] Another objective of this invention is to incorporate probiotics into the oil-phase core formed in the core material, providing an O / W-type imitation milk fat globule for the encapsulation of probiotics, thereby improving the survival stability of probiotics under lyophilization and oral delivery conditions such as gastric acid and bile salts.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An O / W type milk fat globul, the core material of which includes an OPO structure ester, and the wall material is a lactoferrin-lecithin-HMO complex. The wall material is obtained by dissolving lactoferrin, lecithin and HMO in ultrapure water at a mass ratio of 1:(0.01-0.5):1 and adjusting the pH of the system to 5-7.

[0009] To further achieve the purpose of this invention, preferably, in the preparation of the wall material, the concentration of lactoferrin is 5-20 mg / mL, the concentration of lecithin is 0.1-10 mg / mL, and the concentration of HMO is 5-20 mg / mL.

[0010] Preferably, the HMO is one or more of 2'-fucosylated lactose (2'-FL), lactose-N-neotetrasaccharide (LNnT), 6'-sialylated lactose (6'-SL), and 3'-sialylated lactose (3'-SL);

[0011] The microfluidic preparation method for the O / W type mimicry milk fat globules includes the following steps:

[0012] 1) Dissolve lactoferrin, lecithin, and HMO in sterile ultrapure water, adjust the pH to 5-7, and obtain the aqueous phase; melt the OPO structured ester to obtain the oil phase;

[0013] 2) In a microfluidic platform at 37-40℃, the aqueous phase and oil phase are introduced into the aqueous phase channel and oil phase channel of the microfluidic platform, respectively. The oil phase channel is a horizontal pipe, and there are two aqueous phase channels. The outlet points are located on the front and rear sides or the top and bottom sides of the oil phase channel outlet point, respectively. The flow rate ratio of the oil phase and the aqueous phase is adjusted to 1:1-1:5. The aqueous phase shears the outer periphery of the oil phase from both sides, so that the oil phase is dispersed in the aqueous phase to form O / W type imitation emulsion fat globules with uniform particle size.

[0014] 3) O / W type imitation emulsion fat globule droplets are solidified by temperature difference-induced phase transition to obtain O / W type imitation emulsion fat globules.

[0015] Preferably, the diameter of the oil phase channel is 50-400 μm, and the diameter of the water phase channel is 60-480 μm.

[0016] Preferably, the aqueous phase channel has undergone hydrophilic treatment before use.

[0017] Preferably, the hydrophilic treatment involves immersing the aqueous channel in a 1-1.5% PVA solution for 40-60 minutes, extracting to remove excess solution, and then drying in an oven at 80-85°C for 24-48 hours.

[0018] Preferably, the temperature difference-induced phase change curing involves transferring O / W-type imitation emulsion fat globules formed in a microfluidic platform at 37-40℃ to an environment at 0-4℃ and allowing them to stand and solidify for 12-24 hours, then filtering to remove excess aqueous phase to obtain solid O / W-type imitation emulsion fat globules; the OPO structure ester melting is achieved by heating and melting in a water bath at 37-40℃.

[0019] Application of the O / W type milk fat globules in encapsulating probiotics: The core material also includes probiotics; the OPO structure ester is heated and melted, and then lyophilized probiotic powder is added and dispersed to obtain the core material.

[0020] Preferably, the probiotics are one or more of the genera *Lactobacillus*, *Bifidobacterium*, *Escherichia coli*, *Streptococcus*, and *Bacillus*; specifically, *Lactobacillus rhamnosus* GG (LGG) and *Bifidobacterium animalis* subsp. *lactis* BB-12.

[0021] The heating and melting process is carried out in a water bath at 37-40℃;

[0022] The dispersion is described as vortex dispersion;

[0023] The mass-to-volume ratio of the probiotic freeze-dried powder to the OPO structured ester is 20-30:1, with the mass and volume units being milligrams and milliliters, respectively.

[0024] Compared with the prior art, the present invention has the following advantages and superior effects:

[0025] 1) This invention, for the first time, constructs O / W-type mimicry milk fat globules with an oil phase composed of OPO-structured esters (1,3-dioleoyl-2-palmitoylglycerol triglyceride) as the core and an aqueous phase formed by a lactoferrin-lecithin-HMO complex as the outer membrane, giving the resulting O / W-type mimicry milk fat globules an O / W structure similar to natural milk fat globules. The oil phase core, formed by incorporating probiotics into the core material, provides a stable hydrophobic environment, effectively mitigating the impact of changes in water activity and oxygen permeation on the probiotics. The outer mimicry milk fat globule membrane possesses excellent barrier properties, improving the survival stability of the oil phase and probiotics under lyophilization and oral delivery conditions involving gastric acid and bile salts. Tests showed that the lyophilized survival rates of *Lactobacillus rhamnosus* and *Bifidobacterium* embedded in the O / W-type mimicry milk fat globules were 98.6±3.5% and 96.1±5.8%, respectively, and their survival rates after gastrointestinal digestion were 96.6±2.7% and 92.9±6.9%, respectively.

[0026] 2) This invention uses droplet microfluidics to prepare O / W type milk fat globule droplets. The preparation process is gentle and can be achieved by controlling the flow parameters of the aqueous and oil phases. The parameters are well controllable, avoiding the damage to probiotics caused by high temperature or mechanical stress in traditional processing. This technical approach helps maintain the physiological state of the oil phase during the preparation process and promotes the consistency of microcapsule structure and function.

[0027] 3) This invention uses a temperature difference-induced phase change curing method to solidify droplets into microspheres. The entire process does not require chemical crosslinking agents or organic solvents, thus avoiding residual risks. At the same time, the raw materials for the preparation of the outer layer of this invention, such as lactoferrin and HMO, are all derived from breast milk, which have good biocompatibility and food-grade safety, making the resulting O / W type milk fat globules have higher biocompatibility and safety in food-grade applications.

[0028] 4) The O / W type milk fat globule-like probiotics of this invention only require the addition of probiotics to the oil phase. The OPO structure ester is used as the core of the oil phase, and the lactoferrin-lecithin-HMO complex is used as the outer membrane of the aqueous phase, thereby achieving dual protection for the probiotics: the oil phase core provides a hydrophobic environment, which can effectively reduce the impact of water and oxygen on the probiotics; the outer milk fat globule membrane can enhance the probiotics' tolerance to the freeze-drying process and the gastrointestinal acid and bile salt environment.

[0029] 5) This invention is the first to utilize droplet microfluidics technology to construct O / W type probiotic microcapsules. The preparation process is mild and highly controllable, avoiding problems such as high temperature, ice crystal damage and crosslinking agent residue that exist in traditional spray drying, freeze drying or chemical crosslinking and curing, thereby better maintaining the activity of probiotics.

[0030] 6) The O / W type milk fat globules containing probiotics of the present invention can be used in probiotic delivery formulations and can be applied to the preparation of food supplements, nutritional interventions, intestinal microecological regulation or medical nutrition support products. Attached Figure Description

[0031] Figure 1 This is a schematic diagram and a real-world pathway diagram of the preparation of O / W type droplets using a microfluidic platform according to the present invention.

[0032] Figure 2 Photographs of O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) using lactoferrin-lecithin-2'-FL as the wall material in Example 1.

[0033] Figure 3 Photographs of O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) using lactoferrin-lecithin-6'-FL as the wall material in Example 2.

[0034] Figure 4 Photographs of O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) using lactoferrin-lecithin-LNnT as the wall material in Example 3.

[0035] Figure 5 This is a confocal laser-guided microscopy (CLSM) image of an O / W type mimicry milk fat globule droplet from Example 1.

[0036] Figure 6 This is a scanning electron microscope (SEM) image of the O / W type milk fat globule-like solid microspheres from Example 6.

[0037] Figure 7 The diagram (A) and optical microscope image (B) show the formation process of O / W type mimic milk fat globule droplets encapsulating probiotics in Example 3.

[0038] Figure 8This is a SEM image of the O / W type milk fat-mimicking solid microspheres encapsulated with probiotics from Example 3. Detailed Implementation

[0039] To better understand the present invention, it will be further described below with reference to the accompanying drawings and specific embodiments. However, the implementation of the present invention is not limited thereto. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Milk fat globules (MFGs) are a key component of breast milk. Their structure consists of a core of triglycerides (OPO and OPL structural esters) and an outer layer of a complex biomembrane—the milk fat globule membrane (MFGM)—composed of glycoproteins and phospholipids. The present invention provides a mimicry of milk fat globules, specifically an oil-in-water (O / W) type mimicry. The core material comprises OPO structural esters, and the wall material is a lactoferrin-lecithin-HMO complex. The wall material is obtained by dissolving lactoferrin, lecithin, and HMO in ultrapure water at a mass ratio of 1:(0.01-0.5):1, and adjusting the pH of the system to 5-7. The oil-phase core formed by adding probiotics to the core material provides a stable hydrophobic environment, effectively mitigating the impact of changes in water activity and oxygen permeation on the probiotics. The outer mimicry of milk fat globule membrane possesses excellent barrier properties, improving the survival stability of probiotics under lyophilization and oral delivery conditions involving gastric acid and bile salts.

[0041] The O / W type mimicry milk fat globule preparation method of the present invention is a microfluidic preparation method, comprising the following steps:

[0042] 1) Dissolve lactoferrin, lecithin, and HMO in sterile ultrapure water, adjust the pH to 5-7, and obtain the aqueous phase; melt the OPO structured ester to obtain the oil phase;

[0043] 2) In a microfluidic platform at 37-40℃, the aqueous phase and oil phase are introduced into the aqueous phase channel and oil phase channel of the microfluidic platform, respectively. The oil phase channel is a horizontal pipe, and there are two aqueous phase channels. The outlet points are located on the front and rear sides or the top and bottom sides of the oil phase channel outlet point, respectively. The flow rate ratio of the oil phase and the aqueous phase is adjusted to 1:1-1:5. The aqueous phase shears the outer periphery of the oil phase from both sides, so that the oil phase is dispersed in the aqueous phase to form O / W type imitation emulsion fat globules with uniform particle size.

[0044] 3) O / W type imitation emulsion fat globule droplets are solidified by temperature difference-induced phase transition to obtain O / W type imitation emulsion fat globules.

[0045] This preparation method utilizes a microfluidic platform device and microfluidic chip technology. Microfluidics integrates the basic operational units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a single micrometer-scale chip, automating the entire analytical process. For the microfluidic platform device, please refer to [reference needed]. Figure 1 In the figure, A on the left is a schematic diagram of O / W type droplet formation, and B on the right is a real-world pathway diagram. The oil phase channel is a horizontal pipe, and there are two water phase channels. The outlet points are located on the front and back sides or the top and bottom sides of the oil phase channel outlet point, respectively. The water phase (blue) pumped in from the front and back or top and bottom sides shears the outer periphery of the oil phase (red) pumped in from the left side, so that the oil phase (blue) droplets are evenly dispersed in the water phase (red) and covered with blue rings. A bilayer structure of milk fat globules was successfully constructed, with OPO structure ester as the oil phase core and milk fat globule membrane composed of lactoferrin-lecithin-HMO complex as the outer layer.

[0046] The raw materials for the O / W type milk fat globule-like protein of this invention have good biocompatibility and food-grade safety. For example, lactoferrin is an iron-binding glycoprotein in the transferrin family, found in mammalian milk and secretions, with the highest content in colostrum. It inhibits the growth of pathogenic microorganisms by chelating iron ions and directly destroys the cell walls of Gram-negative bacteria, exhibiting broad-spectrum resistance to bacteria, fungi, rotavirus, and adenovirus. HMOs, also known as human milk oligosaccharides, are the third largest solid component in breast milk. HMOs have functions such as regulating intestinal flora, maintaining intestinal barrier integrity, promoting immune system development, promoting brain development, and fighting infection. Lecithin, also known as egg yolk lecithin, is an oily substance found in animal and plant tissues and egg yolks. Its components include phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids, and it is hailed as the "third nutrient" alongside protein and vitamins. OPO structured lipid, chemically named 1,3-dioleoyl-2-palmitoylglycerol triglyceride, is a structured fat that mimics the molecular structure of breast milk lipids through enzymatic lipid exchange technology. Its palmitic acid content at position 2 is over 40%, closely resembling the OPO structure of breast milk. It is primarily used in infant formula to replace ordinary vegetable oils, and has the effects of reducing calcium soap formation, promoting calcium absorption, and reducing the incidence of constipation. Therefore, the O / W-type mimicry breast milk fat globules prepared in this invention exhibit higher biocompatibility and safety for food-grade applications.

[0047] Example 1: Preparation of O / W type mimicking milk fat globule droplets

[0048] 1) Preparation of aqueous phase: Lactoferrin, lecithin and 2'-FL were dissolved in 100 mL of sterile ultrapure water at a mass ratio of 1:0.05:1, with 0.5 g of protein. The mixture was magnetically stirred until completely dissolved. The pH of the system was adjusted to 5.0 using 1 M HCl and 1 M NaOH solution to obtain the aqueous phase for later use.

[0049] 2) Preparation of oil phase: 10 mL of OPO structured ester was heated in a 37°C water bath until completely melted to obtain the oil phase for later use.

[0050] 3) Preparation of O / W-type mimicry emulsion fat globule droplets: The processing temperature of the microfluidic platform was controlled at a constant 38℃. The aqueous phase from step 1) and the oil phase from step 2) were introduced into the oil phase channel and aqueous phase channel of the microfluidic platform, respectively. The diameter ratio of the oil phase channel to the aqueous phase channel was 1:1.2, with the oil phase channel diameter being 200 μm and the aqueous phase channel diameter being 240 μm. The flow rate ratio of the oil phase to the aqueous phase was controlled at 1:3 by the microfluidic chip, so that the oil phase was dispersed in the aqueous phase to form O / W-type mimicry emulsion fat globule droplets. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, the aqueous phase channel was immersed in a 1% (w / v, g / L) polyvinyl alcohol (PVA) solution for 50 min, and after removing excess solution, it was dried in an oven at 85℃ for 24 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the aqueous phase channel. Figure 1 This diagram illustrates the formation of O / W droplets within a microfluidic platform and provides a real-world pathway diagram. Within the microfluidic platform, as shown... Figure 1 As shown in the figure, A on the left is a schematic diagram of O / W type droplet formation, and B on the right is a real-world pathway diagram. The oil phase channel is a horizontal pipe, and there are two water phase channels. The outlet points are located on the front and back sides or the top and bottom sides of the oil phase channel outlet point, respectively. The water phase (blue) pumped in from the front and back or top and bottom sides shears the outer periphery of the oil phase (red) pumped in from the left, so that the oil phase (blue) droplets are evenly dispersed in the water phase (red) and covered with blue rings. A bilayer structure of milk fat globules was successfully constructed, with OPO structure ester as the oil phase core and milk fat globule membrane composed of lactoferrin-lecithin-HMO complex as the outer layer.

[0051] 4) Curing of O / W-type mimicry emulsion fat globules: The O / W-type mimicry emulsion fat globules obtained in step 3) are cured by temperature difference-induced phase change method. Specifically, the droplets formed in the 38℃ microfluidic platform are transferred to the 0-4℃ environment and left to stand for 12 hours to allow the OPO core to undergo phase change and solidify. Then, the excess aqueous phase is removed by filtration to obtain solid microspheres, completing the transformation from liquid phase to solid phase, and obtaining O / W-type mimicry emulsion fat globules solid microspheres. Figure 2 In this embodiment, O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) with lactoferrin-lecithin-2'-FL as the wall material are shown.

[0052] Morphology of O / W-type milk-like fat globule droplets: The morphology of the O / W-type milk-like fat globule droplets obtained in step 3) was observed using a confocal laser microscope (CLSM). First, Nile red (0.2 mg / mL, labeled as oil), FITC (1 mg / mL, labeled as lactoferrin and 2'-FL) were mixed in isopropanol, and Rhodamine-DHPE (1 mg / mL, labeled as lecithin) was dissolved in chloroform. 10 μL of the fluorescent dye was mixed with 1 mL of fresh droplet for staining. Then, 30 μL of the stained droplet was added to a confocal culture dish. Subsequently, Ar / K and He / Ne dual-channel lasers were excited at 488 nm and 633 nm, respectively, to obtain CLSM images. Finally, the images were smoothed and labeled with scale bars using ZEN software. Figure 5 The image shows a CLSM diagram of the O / W-type mimicry milk fat globule droplets in this embodiment. Green fluorescence represents the oil phase of the OPO-structured ester, red fluorescence represents lactoferrin and 2'-FL in the aqueous phase, and blue fluorescence represents lecithin in the aqueous phase. As can be seen from the image, the oil phase (green) droplets are uniformly dispersed in the aqueous phase (red) and coated with a blue ring. This result visually confirms that the present invention successfully constructed a mimicry milk fat globule with a bilayer structure: an OPO-structured ester as the oil phase core, and an outer layer coated with a milk fat globule membrane composed of a lactoferrin-lecithin-HMO complex.

[0053] Morphology of O / W-type milk fat globule-like solid microspheres: The microstructure of O / W-type milk fat globule-like solid microspheres was observed using a scanning electron microscope (SEM). A clean copper stage was prepared and conductive adhesive was applied. A suitable amount of solid powder sample was taken with a clean key and attached to the conductive stage. Excess powder on the surface was blown away with a syringe, and then gold sputtering was performed for 60 seconds. Finally, the sample was observed using SEM. Figure 6 This is a SEM image of the O / W type imitation milk fat solid microspheres in this embodiment. As can be seen from the figure, the surface morphology of the cured microspheres is regular, with no obvious structural collapse or cracking, and the particle size distribution is uniform. This demonstrates the feasibility of the temperature difference-induced phase change curing method used in this invention. The regular spherical structure and controllable particle size distribution are more conducive to product development and application.

[0054] In summary, the results clearly demonstrate that this invention successfully constructed an O / W-type microbial fat globule dual-protection structure based on droplet microfluidic technology. The structure consists of an OPO-structured ester as the oil phase core material and a milk fat globule membrane composed of lactoferrin, lecithin, and HMO as the aqueous phase wall material. The microspheres retain their structural integrity after curing.

[0055] Example 2: Preparation of O / W type mimicking milk fat globule droplets

[0056] A method for preparing O / W-type mimicry milk fat globules based on droplet microfluidic technology includes the following steps:

[0057] 1) Aqueous phase preparation: Lactoferrin, lecithin and 6'-SL were dissolved in 100mL of sterile ultrapure water at a mass ratio of 1:0.3:1, with 1.5g of protein. The mixture was magnetically stirred until completely dissolved. The pH of the system was adjusted to 7.0 using 1M HCl and 1M NaOH to obtain the aqueous phase for later use.

[0058] 2) Preparation of oil phase: The OPO structure ester was heated in a 40°C water bath until completely melted to obtain the oil phase, which was then set aside.

[0059] 3) Preparation of O / W-type mimicry emulsion fat globules: The processing temperature of the microfluidic platform was controlled at a constant 40℃. The aqueous phase from step 1) and the oil phase from step 2) were introduced into the oil phase channel and aqueous phase channel of the microfluidic platform, respectively. The diameter ratio of the oil phase channel to the aqueous phase channel was 1:1.2, with the oil phase channel having a diameter of 50 μm and the aqueous phase channel having a diameter of 60 μm. The flow rate ratio of the oil phase to the aqueous phase was controlled at 1:1 using a microfluidic chip, allowing the oil phase to disperse in the aqueous phase to form O / W-type mimicry emulsion fat globules. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, the aqueous phase channel was immersed in a 1.5% (w / v, g / L) polyvinyl alcohol (PVA) solution for 40 min, and after removing excess solution, it was dried in an oven at 80℃ for 48 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the aqueous phase channel. The operation of the microfluidic platform was as described in Example 1.

[0060] 4) Curing of O / W-type mimicry emulsion fat globules: The O / W-type mimicry emulsion fat globules obtained in step 3) are cured by temperature difference-induced phase change method. Specifically, the droplets formed in the 38℃ microfluidic platform are transferred to the 0-4℃ environment and left to stand for 16 hours to allow the OPO core to undergo phase change and solidify. Then, the excess aqueous phase is removed by filtration to obtain solid microspheres, completing the transformation from liquid phase to solid phase, and obtaining O / W-type mimicry emulsion fat globules solid microspheres. Figure 3 The images show O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) with lactoferrin-lecithin-6'-FL as the wall material in this embodiment.

[0061] Example 3: Preparation of O / W type mimicking milk fat globule droplets

[0062] A method for preparing O / W-type mimicry milk fat globules based on droplet microfluidic technology includes the following steps:

[0063] 1) Aqueous phase preparation: Lactoferrin, lecithin and LNnT were dissolved in 100mL of sterile ultrapure water at a mass ratio of 1:0.5:1, with 2g of protein. The mixture was magnetically stirred until completely dissolved. The pH of the system was adjusted to 6.0 using 1M HCl and 1M NaOH to obtain the aqueous phase for later use.

[0064] 2) Oil phase preparation: The OPO structured ester was heated in a 37°C water bath until completely melted to obtain the oil phase, which was then set aside.

[0065] 3) Preparation of O / W-type mimicry emulsion fat globules: The processing temperature of the microfluidic platform was controlled at a constant 38℃. The aqueous phase from step 1) and the oil phase from step 2) were introduced into the oil phase channel and aqueous phase channel of the microfluidic platform, respectively. The diameter ratio of the oil phase channel to the aqueous phase channel was 1:2, with the oil phase channel having a diameter of 100 μm and the aqueous phase channel having a diameter of 200 μm. The flow rate ratio of the oil phase to the aqueous phase was controlled at 1:5 using a microfluidic chip, allowing the oil phase to disperse in the aqueous phase to form O / W-type mimicry emulsion fat globules. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, the aqueous phase channel was immersed in a 1.5% (w / v, g / L) polyvinyl alcohol (PVA) solution for 40 min, and after removing excess solution, it was dried in an oven at 80℃ for 48 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the channel. The operation of the microfluidic platform was as described in Example 1.

[0066] 4) Curing of O / W-type mimicry emulsion fat globules: The O / W-type mimicry emulsion fat globules obtained in step 3) are cured by temperature difference-induced phase change method. Specifically, the droplets formed in the 37℃ microfluidic platform are transferred to the 0-4℃ environment and left to stand for 24 hours to allow the OPO core to undergo phase change and solidify. Then, the excess aqueous phase is removed by filtration to obtain solid microspheres, completing the transformation from liquid phase to solid phase, and obtaining O / W-type mimicry emulsion fat globules solid microspheres. Figure 4 The images show O / W type imitation milk fat globule droplets (A) and solidified microspheres (B) with lactoferrin-lecithin-LNnT as the wall material in this embodiment.

[0067] As seen in Examples 1-3, O / W-type imitation milk fat spheres were successfully prepared based on droplet microfluidic technology, with OPO structured ester as the oil phase core material and milk fat globule membrane composed of lactoferrin-lecithin-HMO as the aqueous phase wall material. The phase transition from droplets to solid microspheres was achieved by temperature difference induced phase change curing method, and the entire process flow was fully established.

[0068] Example 4:

[0069] A method for preparing O / W-type milk fat globules encapsulated with probiotics based on droplet microfluidics technology includes the following steps:

[0070] 1) Aqueous phase preparation: Lactoferrin, lecithin and 2'-FL were dissolved in 100 mL of sterile ultrapure water at a mass ratio of 1:0.25:1, with 1 g of protein. The mixture was magnetically stirred until completely dissolved. The pH of the system was adjusted to 7.0 using 1 M HCl or 1 M NaOH to obtain the aqueous phase for later use.

[0071] 2) Oil phase preparation: The OPO structured ester was heated and melted in a 40°C water bath, and Lactobacillus rhamnosus GG (LGG) was added. The mass-volume ratio of the probiotic freeze-dried powder to the OPO structured ester was 20:1, with mass and volume units of milligrams and milliliters, respectively. The mixture was then vortexed to form a probiotic-oil phase mixture.

[0072] 3) Formation of O / W-type mimicry milk fat globule droplets encapsulating probiotics: In a microfluidic platform maintained at a constant temperature of 38°C, the aqueous phase from step 1) and the oil phase from step 2) were introduced into a microfluidic platform with a diameter ratio of 1:1.2, forming oil phase channels and aqueous phase channels, respectively. The oil phase channel had a diameter of 400 μm, and the aqueous phase channel had a diameter of 480 μm. The flow rate ratio of the oil and aqueous phases was controlled at 1:3 using a microfluidic chip, allowing the oil phase to disperse in the aqueous phase to form O / W-type mimicry milk fat globule droplets. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, the aqueous phase channel was immersed in a 1.25% (g / L) polyvinyl alcohol (PVA) solution for 60 min, then extracted to remove excess solution, and dried in an oven at 80°C for 20 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the aqueous phase channel. In the microfluidic platform, such as... Figure 1 As shown, the oil phase channel is a horizontal pipe, and there are two water phase channels with outlets located on the front and back sides of the oil phase channel outlet. The water phase (blue) pumped in from the front and back sides shears the outer periphery of the oil phase (red) pumped in from the left side, so that the oil phase (blue) droplets are evenly dispersed in the water phase (red) and covered with blue rings. A bilayer structure of milk fat globules was successfully constructed, with OPO structure ester as the oil phase core and milk fat globule membrane composed of lactoferrin-lecithin-HMO complex as the outer layer.

[0073] 4) Solidification of O / W-type mimicry milk fat globules encapsulating probiotics: The O / W-type mimicry milk fat globules droplets encapsulating probiotics obtained in step 3) were solidified by temperature difference induced phase change method. Specifically, the droplets formed in the 38℃ microfluidic platform were transferred to the 0-4℃ environment and allowed to stand for 12 hours to solidify. Then, the excess aqueous phase was removed by filtration to obtain solid microspheres of O / W-type mimicry milk fat globules encapsulating probiotics, thus completing the transformation from liquid phase to solid phase.

[0074] 5) Initial viable count of O / W type imitation milk fat globules containing probiotics: Take an appropriate amount of the O / W type imitation milk fat globules sample containing probiotics after solidification in step 4) and place it in 1 mL of physiological saline. Vortex dissolve and break the cell wall, and then perform serial dilution. The initial viable count is determined according to the plate count method.

[0075] 6) Morphology of O / W-type mimicry milk fat globules containing probiotics: The morphology of the O / W-type mimicry milk fat globules solid microspheres containing probiotics (from step 4) was observed using a scanning electron microscope (SEM). A clean copper stage was prepared and conductive adhesive was applied. A small amount of powder sample was applied to the conductive stage using a clean key, and excess powder was blown off with a syringe. Then, gold sputtering was performed for 60 seconds. Finally, the sample was observed using SEM.

[0076] Figure 7 Optical microscope image showing the formation process of O / W-type mimicry milk fat globule droplets encapsulating probiotics. Figure 7 On the left side, A shows probiotics in the oil phase being sheared into droplets by the aqueous phase of the lactoferrin-lecithin-HMO complex, and... Figure 7 On the right, B clearly shows that probiotics are present inside the droplet, with a droplet size of 480.38±9.70μm. This result indicates that the O / W type milk fat globules constructed in this invention can successfully encapsulate probiotics.

[0077] Figure 8 SEM images of O / W-type milk fat-mimicking solid microspheres encapsulating probiotics are shown. The solidified microspheres maintained a regular morphology, with no obvious structural collapse or breakage, and exhibited a uniform particle size distribution. Furthermore, the initial viable count of the O / W-type milk fat-mimicking microspheres encapsulating probiotics after solidification was 3.5 x 10⁻⁶. 8 cfu / mL. This indicates that the present invention ensures a high survival rate of probiotics.

[0078] In summary, the O / W-type milk fat spheres constructed based on droplet microfluidic technology in this invention can successfully encapsulate probiotics and maintain a high survival rate after solidification.

[0079] Example 5:

[0080] A method for preparing O / W-type milk fat globules encapsulated with probiotics based on droplet microfluidics technology includes the following steps:

[0081] 1) Preparation of aqueous phase: Dissolve 1g of lactoferrin, 40mg of lecithin and 1g of 2'-FL in 100mL of sterile ultrapure water, stir magnetically until completely dissolved, and adjust the pH to 6.0 to obtain the aqueous phase.

[0082] 2) Oil phase preparation: The OPO structured ester was melted by heating in a 37°C water bath, and Lactobacillus rhamnosus (LGG) lyophilized powder was added. The mass-volume ratio of Lactobacillus rhamnosus lyophilized powder to OPO structured ester was 30:1, with mass and volume units of milligrams and milliliters, respectively. The mixture was then vortexed to form a probiotic-oil phase mixture.

[0083] 3) Formation of O / W-type mimicry milk fat globule droplets encapsulating probiotics: In a microfluidic platform maintained at a constant temperature of 40℃, the aqueous phase from step 1) and the probiotic-oil phase from step 2) were introduced into a microfluidic platform with a diameter ratio of 1:1.3, consisting of an oil phase channel and an aqueous phase channel. The oil phase channel had a diameter of 200 μm, and the aqueous phase channel had a diameter of 260 μm. The flow rate ratio of the oil and aqueous phases was controlled at 1:3 using a microfluidic chip, allowing the oil phase to disperse in the aqueous phase to form O / W-type mimicry milk fat globule droplets. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, the aqueous phase channel was immersed in a 1.5% (g / L) polyvinyl alcohol (PVA) solution for 50 min, then extracted to remove excess solution, and dried in an oven at 85℃ for 24 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the channel. In the microfluidic platform, such as... Figure 1 As shown, the oil phase channel is a horizontal pipe, and there are two water phase channels with outlets located on the front and back sides of the oil phase channel outlet. The water phase (blue) pumped in from the front and back sides shears the outer periphery of the oil phase (red) pumped in from the left side, so that the oil phase (blue) droplets are evenly dispersed in the water phase (red) and covered with blue rings. A bilayer structure of milk fat globules was successfully constructed, with OPO structure ester as the oil phase core and milk fat globule membrane composed of lactoferrin-lecithin-HMO complex as the outer layer.

[0084] 4) Solidification of O / W-type mimicry milk fat globules encapsulating probiotics: The O / W-type mimicry milk fat globules droplets encapsulating probiotics obtained in step 3) were solidified by temperature difference induced phase change method. Specifically, the droplets formed in the 40℃ microfluidic platform were transferred to the 0-4℃ environment and allowed to stand for solidification for 24 hours. Then, the excess aqueous phase was removed by filtration to obtain solid microspheres of O / W-type mimicry milk fat globules encapsulating probiotics, thus completing the transformation from liquid phase to solid phase.

[0085] Example 6: O / W type milk fat globule freeze-dried probiotic-encapsulated bacteria and their survival rate in gastrointestinal digestion

[0086] A method for preparing O / W-type milk fat globules encapsulating probiotics based on droplet microfluidic technology includes the following steps:

[0087] 1) Aqueous phase preparation: Dissolve 1g lactoferrin, 50mg lecithin and 1g 2'-FL in 100mL sterile ultrapure water, stir magnetically until homogeneous, and adjust the pH of the system to 6.0.

[0088] 2) Oil phase preparation: The OPO structured ester was melted by heating in a water bath at 40°C. Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis subsp. lactis BB-12 bacterial powders were added separately. The mass-to-volume ratio of the two probiotic freeze-dried powders to the OPO structured ester was 30:1, with the volume units being milligrams and milliliters, respectively. The mass ratio of Lactobacillus rhamnosus freeze-dried powder to Bifidobacterium animalis subsp. lactis freeze-dried powder was 1:1. The mixture was vortexed to form a probiotic-oil phase mixture.

[0089] 3) Formation of O / W-type mimicry milk fat globule droplets encapsulating probiotics: In a microfluidic platform maintained at a constant temperature of 38℃, the aqueous phase from step 1) and the probiotic-oil phase from step 2) were introduced into the platform, which consisted of an oil phase channel and an aqueous phase channel with a diameter ratio of 1:1.2, respectively. The oil phase channel had a diameter of 300 μm, and the aqueous phase channel had a diameter of 360 μm. The flow rate ratio of the oil and aqueous phases was controlled at 1:5 using a microfluidic chip, causing the oil phase to disperse in the aqueous phase and form O / W-type mimicry milk fat globule droplets, thus encapsulating the probiotics. The aqueous phase channel underwent hydrophilic treatment before use. Specifically, it was immersed in a 1% (g / L) polyvinyl alcohol (PVA) solution for 60 min, then extracted to remove excess solution, and dried in an oven at 80℃ for 20 h to achieve hydrophilicity, forming a stable hydrophilic layer on the inner wall of the aqueous phase channel. In the microfluidic platform, such as... Figure 1 As shown, the oil phase channel is a horizontal pipe, and there are two water phase channels with outlets located on the front and back sides of the oil phase channel outlet. The water phase (blue) pumped in from the front and back sides shears the outer periphery of the oil phase (red) pumped in from the left side, so that the oil phase (blue) droplets are evenly dispersed in the water phase (red) and covered with blue rings. A bilayer structure of milk fat globules was successfully constructed, with OPO structure ester as the oil phase core and milk fat globule membrane composed of lactoferrin-lecithin-HMO complex as the outer layer.

[0090] 4) Solidification of O / W-type mimicry milk fat globules encapsulating probiotics: The O / W-type mimicry milk fat globules droplets encapsulating probiotics obtained in step 3) were solidified by temperature difference induced phase change method. Specifically, the droplets formed in the 38℃ microfluidic platform were transferred to the 0-4℃ environment and allowed to stand for solidification for 20 hours. Then, the excess aqueous phase was removed by filtration to obtain solid microspheres of O / W-type mimicry milk fat globules encapsulating probiotics, thus completing the transformation from liquid phase to solid phase.

[0091] 5) Freeze-drying of O / W-type mimic milk fat globules encapsulating probiotics: The solid O / W-type mimic milk fat globules obtained in step 4) were freeze-dried at -60℃ for 16 hours to obtain freeze-dried O / W-type mimic milk fat globules encapsulating probiotics.

[0092] 6) Survival rate of O / W type mimic milk fat globules encapsulated with probiotics after freeze-drying: Take an appropriate amount of the freeze-dried O / W type mimic milk fat globules encapsulated with probiotics from step 5) and place it in 1 mL of physiological saline. Vortex dissolve and break the cell wall, and then perform serial dilution. Measure the number of viable probiotics according to the plate count method. At the same time, use unencapsulated probiotics as a control test.

[0093] 7) In vitro gastrointestinal simulation digestion: The O / W type microbial fat globules containing probiotics obtained in step 4) were used for in vitro gastrointestinal simulation testing. Simulated gastric juice preparation: An appropriate amount of O / W type microbial fat globules containing probiotics were mixed with simulated gastric juice (SGF) containing 3.2 mg / mL pepsin and 2 mg / mL NaCl to simulate gastric digestion. The mixture was then adjusted to pH 2.0 and continuously shaken at medium speed at 37°C for 2 hours. Simulated intestinal juice preparation: The pH of the above-mentioned gastric chyme was rapidly adjusted to 7.0 to stop pepsin activity. Subsequently, the gastric chyme was mixed with simulated intestinal juice (SIF) containing 8.8 mg / mL NaCl, 5 mg / mL bile salts, 3 mg / mL pancreatic enzymes, and 6.8 mg / mL potassium dihydrogen phosphate. The mixture was then adjusted to pH 7.0 and continuously shaken at medium speed at 37°C for 2 hours to simulate small intestinal digestion. At the endpoint, 1 mL of the digestion solution was taken out, serially diluted, and the number of viable probiotics was determined by plate count method. At the same time, unencapsulated probiotics were used as a control test.

[0094] As shown in Table 1, after freeze-drying, the survival rates of unencapsulated *Lactobacillus rhamnosus* and *Bifidobacterium rhamnosus* were only 17.8±1.2% and 12.4±3.6%, respectively, while the survival rates of *Lactobacillus rhamnosus* and *Bifidobacterium rhamnosus* encapsulated in O / W-type milk fat globules were 98.6±3.5% and 96.1±5.8%, respectively. Furthermore, after gastrointestinal digestion, the survival rates of unencapsulated *Lactobacillus rhamnosus* and *Bifidobacterium rhamnosus* were essentially 0%, while the survival rates of *Lactobacillus rhamnosus* and *Bifidobacterium rhamnosus* encapsulated in O / W-type milk fat globules were 96.6±2.7% and 92.9±6.9%, respectively.

[0095] The results showed that the constructed novel milk fat globule-like structure, with OPO-structured esters as the oil phase core and a milk fat globule membrane composed of lactoferrin-lecithin-HMO as the aqueous phase wall material, forms a dual protective structure. This significantly enhances the resistance of probiotics to freeze-drying damage and the gastrointestinal environment, providing key technical support for the development of probiotic delivery formulations that maintain high activity during processing, storage, and gastrointestinal delivery. The probiotic-encapsulated O / W type milk fat globule-like structure prepared by this invention can be used as a probiotic delivery formulation for the preparation of products related to food supplementation, nutritional intervention, intestinal microecological regulation, or medical nutritional support. In application, the probiotic-encapsulated O / W type milk fat globule-like structure can be directly introduced into the corresponding formulation system as a probiotic source. After mixing, drying, or filling, the corresponding probiotic delivery formulation or its product can be obtained.

[0096] Table 1

[0097]

[0098] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An O / W type mimicking milk fat globules, characterized in that, The core material includes an OPO structure ester, and the wall material is a lactoferrin-lecithin-HMO complex. The wall material is obtained by dissolving lactoferrin, lecithin and HMO in ultrapure water at a mass ratio of 1:(0.01-0.5):1 and adjusting the pH of the system to 5-7.

2. The O / W type mimicking milk fat globules according to claim 1, characterized in that, In the preparation of the wall material, the concentration of lactoferrin is 5-20 mg / mL, the concentration of lecithin is 0.1-10 mg / mL, and the concentration of HMO is 5-20 mg / mL.

3. The O / W type mimicking milk fat globules according to claim 1, characterized in that, The HMO is one or more of 2'-fucosylated lactose, lactose-N-neotetrasaccharide, 6'-sialylated lactose, and 3'-sialylated lactose.

4. The microfluidic preparation method of O / W type mimicry milk fat globules according to any one of claims 1-3, characterized in that... Includes the following steps: 1) Dissolve lactoferrin, lecithin, and HMO in sterile ultrapure water, adjust the pH to 5-7, and obtain the aqueous phase; melt the OPO structured ester to obtain the oil phase; 2) In a microfluidic platform at 37-40℃, the aqueous phase and oil phase are introduced into the aqueous phase channel and oil phase channel of the microfluidic platform, respectively. The oil phase channel is a horizontal pipe, and there are two aqueous phase channels. The outlet points are located on the front and rear sides or the top and bottom sides of the oil phase channel outlet point, respectively. The flow rate ratio of the oil phase and the aqueous phase is adjusted to 1:1-1:

5. The aqueous phase shears the outer periphery of the oil phase from both sides, so that the oil phase is dispersed in the aqueous phase to form O / W type imitation emulsion fat globules with uniform particle size. 3) O / W type imitation emulsion fat globules droplets are solidified by temperature difference-induced phase transition to obtain O / W type imitation emulsion fat globules.

5. The microfluidic preparation method for O / W-type mimicry milk fat globules according to claim 4, characterized in that, The diameter of the oil phase channel is 50-400 μm, and the diameter of the water phase channel is 60-480 μm.

6. The microfluidic preparation method of O / W type mimicry milk fat globules according to claim 4 or 5, characterized in that, The aqueous phase channel underwent hydrophilic treatment before use.

7. The microfluidic preparation method for O / W-type mimicry milk fat globules according to claim 6, characterized in that, The hydrophilic treatment involves immersing the aqueous channel in a 1-1.5% PVA solution for 40-60 minutes, extracting to remove excess solution, and then drying in an oven at 80-85°C for 24-48 hours.

8. The microfluidic preparation method of O / W type mimicry milk fat globules according to claim 4, characterized in that, The temperature difference induced phase change curing involves transferring O / W-type imitation emulsion fat globules formed in a microfluidic platform at 37-40℃ to an environment at 0-4℃ and allowing them to stand and solidify for 12-24 hours. Excess aqueous phase is then removed by filtration to obtain solid O / W-type imitation emulsion fat globules. The OPO structure ester melting is achieved by heating and melting in a water bath at 37-40℃. The diameter of the O / W type imitation emulsion fat globules droplets is 50-500 μm, and the diameter of the solidified O / W type imitation emulsion fat globules is 30-300 μm.

9. The application of the O / W type mimicry milk fat globules according to any one of claims 1-3 in the encapsulation of probiotics, characterized in that, The core material also includes probiotics; the OPO structure ester is heated and melted, and then probiotic freeze-dried powder is added and dispersed to obtain the core material.

10. The application of the O / W type mimicry milk fat globules according to claim 9 in the encapsulation of probiotics, characterized in that, The probiotics mentioned are one or more of the genera Lactobacillus, Bifidobacterium, Escherichia coli, Streptococcus, and Bacillus; The heating and melting process is carried out in a water bath at 37-40℃; The dispersion is described as vortex dispersion; The mass-to-volume ratio of the probiotic freeze-dried powder to the OPO structured ester is 20-30:1, with the mass and volume units being milligrams and milliliters, respectively.

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