Construction method of Pickering emulsion with stable mineralized probiotic solid particles

Abstract

The invention relates to a construction method of Pickering emulsion with stable mineralized probiotic solid particles. Metal cations (Ca < 2 + >) and urease are locally enriched through the physical property of the bacterial surface, then urea is catalyzed in situ through urease to generate carbonate ions, and the carbonate ions and Ca < 2 + > chelated on the bacterial surface are subjected to a mineralization reaction to obtain mineralized probiotics. The preparation method comprises the following steps: preparing a Pickering emulsion, modifying by using a sodium laurate solution to endow the Pickering emulsion with wettability, and constructing a water-in-oil Pickering emulsion system by using modified and mineralized probiotic particles as a stabilizer. The system synergistically resists multiple extreme stresses through physical barrier and interface regulation, so that bacteria are protected from external invasion, the storage stability of probiotics and the tolerance to severe environments (high temperature and acid) are improved, and the system is matched with a high-fat food matrix and can be used for processing and manufacturing functional foods (toast, coffee, butter, cakes and the like).

Description

Technical Field

[0001] The present invention belongs to the field of Pickering emulsions, and particularly relates to a method for constructing a W / O Pickering emulsion stabilized by mineralized probiotic solid particles. Background Art

[0002] As an active microbial preparation, when ingested in sufficient amounts, probiotics can promote microecological balance by regulating the metabolism and composition of the intestinal flora, thereby producing physiological effects of improving digestive and immune functions. In recent years, probiotics have become a research hotspot in the field of functional foods due to their regulatory effects on the host gut microbiota. Non-dairy probiotic functional foods have developed rapidly in the baking field, especially in products such as bread and cakes.

[0003] Similar to dairy fermentation functional foods, the probiotic microorganisms in these products may be naturally produced during the fermentation process or artificially added. However, a major problem faced by non-dairy probiotic functional foods is usually the thermal processing of probiotic addition after application. This obstacle has led to a trend towards low-processed foods, such as chocolate. In addition, environmental factors, such as a decrease in pH value and a decrease in sugar content, affect cell viability; this is particularly evident in functional juices and beers. The extended shelf life and adverse storage conditions of most non-dairy products pose difficulties in maintaining the survival rate of probiotics, especially exposure to higher storage temperatures will accelerate the degradation of probiotics.

[0004] Contemporary methods increasingly involve the use of stable probiotics, especially encapsulated variants. Kang et al. used porous materials such as metal-organic frameworks for encapsulation, providing unprecedented protection for biological macromolecules. (Liang K, et al. Nat Commun. 2015, DOI: 10.1038 / ncomms8240.) The Nazzaro team used an alginate-xanthan gum composite matrix to microencapsulate lactic acid bacteria through an extrusion molding process, and the survival rate retention rate of the bacteria in a simulated gastric acid environment increased by 38%, confirming that the composite wall material can effectively enhance the digestive tract delivery efficiency of probiotic preparations. (Nazzaro F, et al. Journal of Functional Foods, 2009, doi.org / 10.1016 / j.jff.2009.02.001.) Although existing embedding techniques can improve the resistance of bacteria to processing stress, there are significant limitations in the adaptability to high-fat food matrices, mainly due to the mismatch between the physical and chemical properties of traditional aqueous embedding systems and oil matrices. In view of this, the development of a new type of steady-state technology with both environmental resistance and matrix compatibility has become the key breakthrough point for expanding the development of probiotic functional foods.

[0005] The application of Pickering emulsion technology in the preparation of fat substitutes based on liquid oils provides an effective way to solve the above problems. Its uniqueness lies in that the high internal phase emulsion system constructed by protein-based particles can achieve the structural reorganization of liquid oils. This structure can not only realize the controllable structuring of liquid oils, but also provide a physical barrier protection for probiotics. Qin XS et al. demonstrated that Pickering HIPEs stabilized with whey protein isolate-epigallocatechin gallate covalent conjugates are beneficial to enhancing the storage and passage viability of Lactobacillus plantarum powder. (Qin XS, et al. Food Hydrocolloids, 2021, doi.org / 10.1016 / j.foodhyd.2021.106658.) Eslam et al. encapsulated Lactobacillus delbrueckii in the inner aqueous phase of Pickering double emulsions and compared two different surfactants, β-cyclodextrin and Tween-80, and found that the survival rate of cells encapsulated with β-cyclodextrin as an emulsifier increased significantly. (Vahabzadeh, et al. Food hydrocolloids, 2017.doi.org / 10.1016 / j.foodhyd.2016.10.035) Although the above methods use Pickering emulsions as functional carriers and improve the survival rate of probiotics to a certain extent, they do not emphasize their application in food processing, and the protection of probiotics by emulsion encapsulation is also limited. Using mineralized probiotics as stabilizers is a completely new idea, providing multiple protections for probiotics. However, there is currently no report on the application research of using probiotics to stabilize Pickering emulsions as liquid oil substitutes in the baking food system, and systematic exploration in related fields needs to be further deepened. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing Pickering emulsions stabilized by mineralized probiotic solid particles. By proposing a surface mineralization modification technology coupled with an emulsion interface engineering strategy, a W / O type Pickering emulsion system with biomineralized probiotic solid particles as interface stabilizers is constructed, overcoming the limitations of existing probiotic encapsulation and protection technologies in terms of adaptability to high-fat food matrices, while improving the survival rate of probiotics after long-term storage and in various harsh environments.

[0007] The purpose of the present invention is achieved as follows: Specifically, it includes the following steps:

[0008] Step 1: Take 2 - 10 mL of probiotic suspension in the logarithmic growth phase. After double washing with PBS buffer (pH 7.4), enrich the active bacteria by gradient centrifugation at 6000 revolutions per minute for 5 minutes under the condition of pre-cooling at 4°C. Disperse 10 - 50 mg of high-purity urease and the bacteria in 4 - 20 mL of 10 - 25 mM CaCl2 solution (containing 0.1% Tween 80 stabilizer), and perform interfacial activation treatment for 10 - 15 minutes under constant magnetic stirring at 200 revolutions per minute to achieve the directional adsorption of calcium ions on the surface of the bacteria.

[0009] Step 2: Inject an equal volume (4 - 20 mL) of 10 - 25 mM urea solution into the system of Step 1, maintain the stirring speed of 200 revolutions per minute, and carry out a bio-enzyme-controlled mineralization reaction for 30 - 60 minutes under the constant temperature condition of 25°C. The CO3 2- produced by the catalysis of urease combines with the pre-adsorbed Ca 2+ to form a CaCO3 protective layer on the surface of the bacteria. Finally, collect the mineralized probiotics by low-temperature centrifugation at 4°C and 6000 revolutions per minute for 5 minutes, and its survival rate > 90%.

[0010] Step 3: Accurately weigh 0.2 - 0.5 g of mineralized probiotics and disperse them in 100 - 350 mL of 0.5 mg / mL sodium laurate solution (containing 5 mM Tris-HCl buffer, pH 8.5), and carry out an amphiphilic molecular self-assembly reaction for 2 - 5 hours under the stirring speed of 600 revolutions per minute. Through electrostatic adsorption and hydrophobic interaction, a nanoscale sodium laurate monolayer film is formed on the surface of the mineralized layer. After purification by low-temperature centrifugation at 10000 revolutions per minute, modified mineralized probiotics with excellent oil-water interface stability are obtained.

[0011] Step 4: Ultrasonically treat 0.1 - 0.4 g of functionalized probiotics and 1 mL of refined vegetable oil (soybean oil / olive oil) for 1 minute under nitrogen protection. After injecting 1 mL of sterile deionized water, perform high-speed shear emulsification at 8000 - 10000 revolutions per minute for 1 minute (shearing gap 50 μm). After standing for 20 - 50 minutes, an oil-in-water modified mineralized probiotic Pickering emulsion is obtained.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] (1) The present invention prepares an oil-in-water modified mineralized probiotic Pickering emulsion based on the principles of biomimetic mineralization and Pickering emulsion construction. The whole process is green, mild, and biocompatible. It can simply adjust the concentration of sodium laurate to control the wettability of the modified mineralized probiotics, so as to construct a stable Pickering emulsion system;

[0014] (2) The water-in-oil modified mineralized probiotic Pickering emulsion proposed by the present invention has a significant protective effect. The outermost layer is the continuous oil phase in the water-in-oil system, the middle layer is the hydrophobic interface endued by sodium laurate, and the innermost layer is the calcium carbonate biosynthesized. They synergistically resist multiple extreme stresses through physical barrier and interface regulation to protect bacteria from external damage.

[0015] (3) After 30 minutes in a strong acid environment, the viable bacteria rate of probiotics in the water-in-oil system of the water-in-oil modified mineralized probiotic Pickering emulsion prepared by the present invention is 71.9%, while the survival rate of free probiotics is only 0.08%, which is 899 times higher. After 1 hour, there are still bacteria surviving in the water-in-oil system, while all free probiotics are dead.

[0016] (4) In the environments of 60 °C, 70 °C, 80 °C and 90 °C, 50.00%, 26.25%, 21.25% and 16.25% of the bacteria bodies of the water-in-oil modified mineralized probiotic Pickering emulsion prepared by the present invention still survive. In a high-temperature environment, as the temperature rises, only 11.67% of the bacteria bodies of free probiotics survive in the 60 °C environment, and all are dead at 70 °C, 80 °C and 90 °C.

[0017] (5) The dual barrier effects of the continuous oil phase and the calcium carbonate mineralized layer jointly induce probiotics to enter a reversible metabolic dormant state, which not only significantly improves the stability of the bacteria bodies during storage. The survival rates of probiotics in the water-in-oil emulsion system after being stored at 4 °C and 25 °C for 32 days are 35 times and 263 times higher than those of free probiotics; it also endows them with dynamic response characteristics, and the emulsion system and the mineral layer can be controllably dissociated in a specific acidic microenvironment, and then the biological activity can be restored.

[0018] Based on the excellent properties of Pickering emulsion, the present invention constructs a W / O type Pickering emulsion system with biomimetic mineralized probiotic solid particles as the interfacial stabilizer. Dense calcium carbonate and the oil phase interface jointly induce bacteria to enter a dormant state, block external environmental stresses, and improve storage stability. In a strong acid environment, the hydrophobic modified mineralized layer and the oil phase barrier jointly play a spatial barrier role, and at the same time, the calcium carbonate in the mineralized layer exhibits pH buffering performance, enabling the bacteria bodies to survive under extremely acidic conditions; during high-temperature treatment, the endothermic phase change characteristics of the mineralized shell layer and the heat insulation effect of the oil phase act synergistically, significantly reducing the heat conduction efficiency, and the survival rate of bacteria is increased. This "surface mineralization-interface engineering" coupling strategy opens up a new way for the development of heat-resistant probiotic functional foods, provides a basis for the development of related functional foods to expand the application prospects of probiotics in fat-based foods. Description of the Drawings

[0019] Figure 1Schematic diagram of the structure of a water-in-oil modified mineralized probiotic Pickering emulsion.

[0020] Figure 2 Scanning electron micrograph of mineralized probiotics.

[0021] Figure 3 Three-phase contact angle of mineralized probiotic particles modified with different concentrations of sodium laurate in an oil-water system.

[0022] Figure 4 Optical microscope photograph of a water-in-oil modified mineralized probiotic Pickering emulsion.

[0023] Figure 5 Activation of a water-in-oil modified mineralized probiotic Pickering emulsion under different acidic conditions.

[0024] Figure 6 Survival of probiotics in a water-in-oil modified mineralized probiotic Pickering emulsion in an acidic environment (pH = 2).

[0025] Figure 7 Survival of probiotics in a water-in-oil modified mineralized probiotic Pickering emulsion in a high-temperature environment.

[0026] Figure 8 Number of viable bacteria in a water-in-oil modified mineralized probiotic Pickering emulsion stored for a long time at 4 °C.

[0027] Figure 9 Number of viable bacteria in a water-in-oil modified mineralized probiotic Pickering emulsion stored for a long time at 25 °C. Detailed implementation method

[0028] A method for constructing a W / O type Pickering emulsion system using interfacial active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0029] Step 1: Take 2 - 10 mL of a probiotic suspension in the logarithmic growth phase (OD 600 = 0.6 - 0.8), perform double washing with pre-cooled PBS buffer (pH 7.4), and collect the active bacteria by centrifugation at 6000 revolutions per minute for 5 minutes at 4 °C. Disperse 10 - 50 mg / ·mL -1 urease and the bacteria together in 4 - 20 mL of 10 - 25 mM CaCl2 solution, and perform interfacial activation treatment for 10 - 15 minutes under magnetic stirring at 200 revolutions per minute to achieve uniform adsorption of Ca 2+ on the surface of the bacteria.

[0030] Step 2: Inject an equal volume (4 - 20 mL) of 10 - 25 mM urea solution into the activation system in Step 1, maintain a stirring speed of 200 revolutions per minute, and carry out a biomineralization reaction for 30 - 60 minutes under the constant temperature condition of 25°C. CO3 generated by urease catalysis 2- combines with pre-adsorbed Ca 2+ to form a CaCO3 protective layer with a controllable thickness on the surface of the bacterial cells. Finally, the mineralized probiotics are obtained by low-temperature centrifugation at 4°C and 6000 revolutions per minute for 5 minutes.

[0031] Step 3: Accurately weigh 0.2 - 0.5 g of the mineralized probiotics and disperse them in 100 - 350 mL of 0.5 mg / mL sodium laurate solution, and carry out a molecular self-assembly reaction for 2 - 5 hours at a stirring speed of 600 revolutions per minute. Through the coordination of the carboxyl group of sodium laurate with Ca 2+ in the mineralized layer, a nano-scale hydrophobic interface is constructed. After purification by low-temperature centrifugation at 10000 revolutions per minute, modified mineralized probiotics with excellent oil-water interface stability are obtained.

[0032] Step 4: Ultrasonically treat 0.1 - 0.4 g of the modified mineralized probiotics and 1 mL of refined vegetable oil for 1 minute under the protection of inert gas, and then inject 1 mL of sterile deionized water and manually shake for 30 seconds to form primary emulsion droplets.

[0033] Furthermore, use a portable high-speed disperser to shear and emulsify at 8000 revolutions per minute for 1 minute. After standing for 20 - 50 minutes, an oil-in-water modified mineralized probiotic Pickering emulsion is obtained.

[0034] Example 1

[0035] A preparation method of biomineralized probiotics with a calcium carbonate shell, comprising the following steps:

[0036] Step 1: Accurately measure 5 mL of the probiotic suspension in the logarithmic growth phase using a pipette, wash it twice with pre-cooled PBS buffer (pH 7.4), and collect the bacterial cell precipitate by centrifugation at 6000 revolutions per minute for 5 minutes at 4°C.

[0037] Step 2: Resuspend the obtained probiotic precipitate in 10 mL of sterile deionized water, add 12 mM calcium chloride and 4 mg·mL -1 urease. Magnetically stir at a speed of 200 revolutions per minute for 10 minutes to allow Ca 2+ to be fully adsorbed on the surface of the bacterial cells.

[0038] Inject 10 mL of 12 mM sterile urea solution into the reaction system in Step 2, and under the constant temperature condition of 25°C, maintain a stirring speed of 200 revolutions per minute and continuously react for 30 minutes. Construct a CaCO3 mineralized layer on the surface of the bacterial cells through urease-catalyzed reaction, and finally obtain the mineralized probiotics by low-temperature centrifugation at 4°C and 6000 revolutions per minute for 5 minutes.

[0039] The mineralized probiotics were observed under a scanning electron microscope (SEM), as specifically shown Figure 2 below.

[0040] Example 2

[0041] A preparation method of bio-mineralized probiotics with oil-water wettability includes the following steps:

[0042] Step 1: Using a pipette, accurately measure 5 mL of probiotic suspension in the logarithmic growth phase. After double washing with pre-cooled PBS buffer (pH 7.4), collect the cell precipitate by centrifuging at 6000 rpm for 5 minutes at 4 °C.

[0043] Step 2: Resuspend the obtained probiotic precipitate in 10 mL of sterile deionized water, and add 12 mM calcium chloride and 4 mg·mL -1 urease. Stir magnetically at a speed of 200 rpm for 10 minutes to allow Ca 2+ to be fully adsorbed on the cell surface.

[0044] Inject 10 mL of 12 mM sterile urea solution into the reaction system of Step 2. Under the constant temperature condition of 25 °C, maintain the stirring speed of 200 rpm and continue the reaction for 30 minutes. Construct a CaCO3 mineralized layer on the cell surface through the catalytic reaction of urease. Finally, obtain the mineralized probiotics by centrifuging at 6000 rpm at 4 °C for 5 minutes.

[0045] Step 3: Dissolve 0.08, 0.1, and 0.12 g of sodium laurate (food grade) in 200 mL of sterile deionized water respectively, and obtain uniform 0.4, 0.5, and 0.6 mg / mL surface modification solutions through vortex oscillation and ultrasonic treatment.

[0046] Step 4: Take 0.2 g of the mineralized probiotics prepared in Step 2 and add it to the surface modification solution prepared in Step 3. Stir at 25 °C and 600 rpm for 4 hours to complete the surface modification. Centrifuge at 10000 rpm at 4 °C for 5 minutes to collect the modified mineralized probiotics.

[0047] The three-phase contact angles of the modified mineralized probiotic particles at the oil-water interface were measured by the dip-coating method. As Figure 3 shown, the three-phase contact angles corresponding to the mineralized probiotics modified with 0.4, 0.5, and 0.6 mg / mL sodium laurate solutions are 81°, 90°, and 100° respectively. With the increase of the sodium silicate concentration, the three-phase contact angle of the modified mineralized probiotics also gradually increases, changing from oil-repellent and water-loving to water-repellent and oil-loving.

[0048] Example 3

[0049] A method for constructing a W / O Pickering emulsion system using interfacially active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0050] Step 1: Use a pipette to accurately measure 5 mL of probiotic suspension in the logarithmic growth phase. After double washing with pre-cooled PBS buffer (pH 7.4), centrifuge at 6000 rpm for 5 minutes at 4 °C to collect the cell precipitate.

[0051] Step 2: Resuspend the obtained probiotic precipitate in 10 mL of sterile deionized water, add 15 mM calcium chloride and 5 mg·mL -1 urease. Stir magnetically at a speed of 200 rpm for 10 minutes to allow Ca 2+ to be fully adsorbed on the cell surface.

[0052] Inject 10 mL of 15 mM sterile urea solution into the reaction system of Step 2. Under the condition of constant temperature at 25 °C, maintain the stirring speed of 200 rpm and continue the reaction for 30 minutes. Construct a CaCO3 mineralized layer on the cell surface through the catalytic reaction of urease. Finally, obtain mineralized probiotics by low-temperature centrifugation at 6000 rpm for 5 minutes at 4 °C.

[0053] Step 3: Dissolve 0.15 g of sodium laurate (food grade) in 300 mL of sterile deionized water, and obtain a uniform 0.5 mg / mL surface modification solution through vortex oscillation and ultrasonic treatment.

[0054] Step 4: Add 0.2 g of the mineralized probiotics prepared in Step 2 to the surface modification solution prepared in Step 3, and stir at 25 °C and 600 rpm for 4 hours to complete the surface modification. Centrifuge at 10000 rpm for 5 minutes at 4 °C to collect the modified mineralized probiotics.

[0055] Step 5: Add 0.2 g of the modified mineralized probiotics prepared in Step 4 to a screw-cap glass bottle containing 1 mL of edible olive oil. Under nitrogen protection, ultrasonicate for 1 minute, add 1 mL of deionized water, and shake well. (Soybean oil or corn oil can also be used instead of olive oil)

[0056] Furthermore, use a portable high-speed disperser to shear and emulsify at 10000 rpm for 1 minute. Let it stand for 30 minutes to obtain a water-in-oil modified mineralized probiotic Pickering emulsion. The obtained Pickering emulsion is as Figure 4 shown.

[0057] Example 4

[0058] A method for constructing a W / O Pickering emulsion system using interfacially active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0059] Step 1: Use a pipette to accurately measure 5 mL of probiotic suspension in the logarithmic growth phase. After double washing with pre-cooled PBS buffer (pH 7.4), collect the cell precipitate by centrifugation at 6000 rpm for 5 minutes at 4°C.

[0060] Step 2: Resuspend the obtained probiotic precipitate in 10 mL of sterile deionized water, and add 20 mM calcium chloride and 5 mg·mL -1 Urease. Stir magnetically at a speed of 200 rpm for 10 minutes to allow Ca 2+ To be fully adsorbed on the cell surface.

[0061] Inject 10 mL of 20 mM sterile urea solution into the reaction system of Step 2. Under the condition of constant temperature at 25°C, maintain the stirring speed of 200 rpm and continue the reaction for 30 minutes. Construct a CaCO3 mineralization layer on the cell surface through the urease-catalyzed reaction, and finally obtain mineralized probiotics by low-temperature centrifugation at 6000 rpm for 5 minutes at 4°C.

[0062] Step 3: Dissolve 0.1 g of sodium laurate (food grade) in 200 mL of sterile deionized water, and obtain a uniform 0.5 mg / mL surface modification solution through vortex oscillation and ultrasonic treatment.

[0063] Step 4: Take 0.25 g of the mineralized probiotics prepared in Step 2 and add it to the surface modification solution prepared in Step 3. Stir at 25°C and 600 rpm for 3 hours to complete the surface modification. Centrifuge at 10000 rpm at 4°C for 5 minutes to collect the modified mineralized probiotics.

[0064] Step 5: Take 0.3 g of the modified mineralized probiotics prepared in Step 4 and add it to a screw-cap glass bottle containing 1 mL of edible olive oil. Under nitrogen protection, ultrasonicate for 1 minute, add 1 mL of deionized water, and shake well. (Soybean oil or corn oil can also be used instead of olive oil)

[0065] Furthermore, use a portable high-speed disperser to shear and emulsify at 8000 rpm for 1 minute. Let it stand for 30 minutes to obtain a water-in-oil modified mineralized probiotic Pickering emulsion.

[0066] Evaluated the dormancy and activation conditions of the water-in-oil modified mineralized probiotic Pickering emulsion. Such as Figure 5As shown, the water-in-oil system is in a dormant state and no colonies grow. After it is treated by vortexing for 5 minutes with 10 μL, 20 μL, 30 μL, 40 μL and 50 μL of 0.01 M hydrochloric acid solution, the growth of bacteria is 5.90 log, 6.42 log, 6.82 log, 6.46 log and 3.67 log respectively, and the bacteria are reactivated. It shows that the physical barrier effect of the encapsulation system can be removed by controllably dropping acid solution (destroying the oil phase layer / demineralization treatment), the bacterial metabolic state can be reversibly regulated, and the precise recovery of the cell activity can be achieved.

[0067] Example 5

[0068] A method for constructing a W / O Pickering emulsion system using interfacial active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0069] Step 1: Accurately measure 10 mL of probiotic suspension in the logarithmic growth phase with a pipette gun. After double washing with pre-cooled PBS buffer (pH 7.4), collect the cell precipitate by centrifuging at 6000 rpm for 5 minutes at 4 °C.

[0070] Step 2: Resuspend the obtained probiotic precipitate in 10 mL of sterile deionized water, add 20 mM calcium chloride and 5 mg·mL -1 Urease. Stir magnetically at a speed of 200 rpm for 10 minutes to make Ca 2+ Fully adsorbed on the cell surface.

[0071] Inject 10 mL of 20 mM sterile urea solution into the reaction system of Step 2. Under the constant temperature condition of 25 °C, maintain the stirring speed of 200 rpm and continuously react for 60 minutes. Construct a CaCO3 mineralized layer on the cell surface through the urease-catalyzed reaction, and finally obtain mineralized probiotics by low-temperature centrifugation at 4 °C and 6000 rpm for 5 minutes.

[0072] Step 3: Dissolve 0.175 g of sodium laurate (food grade) in 350 mL of sterile deionized water, and obtain a uniform 0.5 mg / mL surface modification solution through vortex oscillation and ultrasonic treatment.

[0073] Step 4: Add 0.3 g of the mineralized probiotics prepared in Step 2 to the surface modification solution prepared in Step 3, and stir at 25 °C and 600 rpm for 4 hours to complete the surface modification. Centrifuge at 10000 rpm and 4 °C for 5 minutes to collect the modified mineralized probiotics.

[0074] Step 5: Add 0.3 g of the modified mineralized probiotics prepared in Step 4 to a screw-cap glass bottle containing 1 mL of edible olive oil, ultrasonicate for 1 minute under nitrogen protection, add 1 mL of deionized water, and shake well. (Soybean oil or corn oil can also be used to replace olive oil)

[0075] Furthermore, a hand-held high-speed disperser was used to shear and emulsify for 1 minute at 10,000 revolutions per minute. After standing for 30 minutes, the water-in-oil modified mineralized probiotic Pickering emulsion was obtained.

[0076] The tolerance of the water-in-oil modified mineralized probiotic Pickering emulsion to acidic environment was evaluated in a strongly acidic (pH = 2) environment. As Figure 6 shown, after 30 minutes in a strong acid environment, the viable bacteria rate of probiotics in the water-in-oil system was 71.9%, while the survival rate of free probiotics was only 0.08%, which was increased by 899 times. When the time increased to 1 hour, all the live cells died; while the experimental group data showed that the oil-in-water system had extremely high tolerance to acidic environment. Even when the time was extended to 1 hour and the number of live cells decreased by 1.09 log, there were still bacteria surviving.

[0077] Example 6

[0078] A method for constructing a W / O type Pickering emulsion system using interfacial active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0079] Step 1: Use a pipette to accurately measure 5 mL of probiotic suspension in the logarithmic growth phase. After double washing with pre-cooled PBS buffer (pH 7.4), centrifuge at 6000 revolutions per minute for 5 minutes at 4 °C to collect the cell precipitate.

[0080] Step 2: Resuspend the obtained probiotic precipitate in 15 mL of sterile deionized water, add 15 mM calcium chloride and 3 mg·mL -1 urease. Stir magnetically at a speed of 200 revolutions per minute for 5 minutes to allow Ca 2+ to be fully adsorbed on the cell surface.

[0081] Inject 15 mL of 15 mM sterile urea solution into the reaction system of Step 2. Under the constant temperature condition of 25 °C, maintain the stirring speed of 200 revolutions per minute and continue the reaction for 30 minutes. Construct a CaCO3 mineralized layer on the cell surface through the urease-catalyzed reaction, and finally obtain mineralized probiotics by low-temperature centrifugation at 4 °C and 6000 revolutions per minute for 5 minutes.

[0082] Step 3: Dissolve 0.125 g of sodium laurate (food grade) in 250 mL of sterile deionized water, and obtain a uniform 0.5 mg / mL surface modification solution through vortex oscillation and ultrasonic treatment.

[0083] Step 4: Add 0.2 g of the mineralized probiotics prepared in Step 2 into the surface modification solution prepared in Step 3, and stir at 25 °C and 600 rpm for 4 hours to complete the surface modification. Centrifuge at 10,000 rpm and 4 °C for 5 minutes to collect the modified mineralized probiotics.

[0084] Step 5: Take 0.25 g of the modified mineralized probiotics prepared in Step 4 and add it to a screw-capped glass bottle containing 1 mL of edible olive oil. Under nitrogen protection, ultrasonicate for 1 minute, then add 1 mL of deionized water and shake well. (Soybean oil or corn oil can also be used instead of olive oil)

[0085] Furthermore, use a hand-held high-speed disperser to shear and emulsify at 9000 rpm for 1 minute. Let it stand for 30 minutes to obtain the water-in-oil modified mineralized probiotics Pickering emulsion.

[0086] The tolerance of the water-in-oil modified mineralized probiotics Pickering emulsion to high temperature environments was evaluated in high temperature environments. As Figure 7 shown, the viable cell counts of the water-in-oil system at 60 °C, 70 °C, 80 °C and 90 °C are 6.80 log, 6.45 log, 6.40 log and 6.33 log respectively, and the viable cell rates are 50.00%, 26.25%, 21.25%, 16.25% respectively; while only 5.23 log viable cells of the free probiotics survive at 60 °C, and the viable cell rate is only 11.67%, and they all die at other temperatures. Although the survival rates of both the probiotics in the water-in-oil system and the free probiotics will decrease at high temperatures, some bacteria in the water-in-oil system still survive at 70 °C, 80 °C and 90 °C.

[0087] Example 7

[0088] A method for constructing a W / O type Pickering emulsion system using interfacial active mineralized probiotic solid particles as a stabilizer, comprising the following steps:

[0089] Step 1: Use a pipette to accurately measure 10 mL of the probiotic suspension in the logarithmic growth phase. After double washing with pre-cooled PBS buffer (pH 7.4), centrifuge at 6000 rpm for 5 minutes at 4 °C to collect the cell precipitate.

[0090] Step 2: Resuspend the obtained probiotic precipitate in 15 mL of sterile deionized water, add 12 mM calcium chloride and 3 mg·mL -1 urease. Magnetically stir at a speed of 200 rpm for 10 minutes to allow Ca 2+ to be fully adsorbed on the cell surface.

[0091] Inject 15 mL of 12 mM sterile urea solution into the reaction system of step 2, and under the constant temperature condition of 25 °C, maintain the stirring speed of 200 rpm and continuously react for 60 minutes. Construct a CaCO3 mineralization layer on the surface of the bacteria through the urease-catalyzed reaction, and finally obtain mineralized probiotics by low-temperature centrifugation at 4 °C and 6000 rpm for 5 minutes.

[0092] Step 3: Dissolve 0.1 g of sodium laurate (food grade) in 200 mL of sterile deionized water, and obtain a uniform 0.5 mg / mL surface modification solution through vortex oscillation and ultrasonic treatment.

[0093] Step 4: Add 0.3 g of the mineralized probiotics prepared in step 2 into the surface modification solution prepared in step 3, and stir at 25 °C and 600 rpm for 5 hours to complete the surface modification. Centrifuge at 10000 rpm and 4 °C for 5 minutes to collect the modified mineralized probiotics.

[0094] Step 5: Take 0.2 g of the modified mineralized probiotics prepared in step 4 and add them into a screw-cap glass bottle containing 1 mL of edible olive oil. Under nitrogen protection, ultrasonicate for 1 minute, add 1 mL of deionized water, and shake well. (Soybean oil or corn oil can also be used instead of olive oil)

[0095] Furthermore, use a portable high-speed disperser to shear and emulsify at 8000 rpm for 1 minute. Let it stand for 30 minutes to obtain a water-in-oil modified mineralized probiotic Pickering emulsion.

[0096] The effect of encapsulation of W / O Pickering emulsion combined with a biomimetic mineralization strategy on the long-term storage stability of probiotics was evaluated. As Figure 8 shown, after storage at low temperature (4 °C) for up to 32 days, the viable bacteria count in the water-in-oil system was still 8.34 log, and the viable bacteria rate was as high as 66.16%. While the viable bacteria count of free probiotics was 6.79 log, and the viable bacteria rate was only 1.9%, which was increased by about 35 times. As Figure 9 shown, even at room temperature (25 °C), the W / O Pickering emulsion system and the artificial mineral shell can still greatly improve the storage stability of bacteria. Compared with the free probiotics, only 0.23% of the viable bacteria remained after 32 days of storage, and the viable bacteria rate of the water-in-oil system was as high as 60.50%, which was increased by 263 times.

Claims

1. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles, comprising the following steps: Step 1: Preparation of a microbially induced mineralized coating. An active probiotic (1×10 8 ~5×10 8 CFU·mL -1 ) and urease (2.5~5 mg·mL -1 ) are co-dispersed in a mineralization solution containing 10~25 mM CaCl2 and 10~25 mM urea. A uniform CaCO3 protective layer is formed on the surface of the probiotic through bioenzymatic mineralization, significantly enhancing the tolerance of the bacterial cells to environmental stress; Step 2: Hydrophobic surface functionalization: 0.2-0.5g of mineralized probiotics was added to 100-350mL of 0.5mg / mL sodium laurate solution and subjected to in-situ hydrophobic modification under magnetic stirring at 600rpm for 2-5 hours. A hydrophobic interface was constructed outside the mineralized layer through fatty acid self-assembly. The precipitate was then collected by refrigerated centrifugation at 4°C and 10,000rpm for 5 minutes to obtain the hydrophobically modified mineralized probiotics. The contact angle of the hydrophobically modified probiotics was increased to 90°, significantly enhancing the anchoring ability at the oil-water interface. Step 3: Directed construction of Pickering emulsion: 0.1-0.4 g of modified probiotics were mixed with 1 mL of edible oil in a screw-cap bottle. After 1 minute of ultrasonic pretreatment, 1 mL of deionized water was added, and high-shear dynamic emulsification technology was used to form W / O colostrum. After standing for 20-50 minutes, the probiotic particles formed a dense monolayer film at the oil-water interface through the interfacial self-assembly effect, and finally a highly stable probiotic Pickering emulsion was obtained.

2. A method for constructing a stable Pickering emulsion of mineralized probiotic solid particles according to claim 1, characterized in that, In step 1, the probiotics are first mixed with calcium chloride and urease, vortexed for 5 minutes, and then urea solution is added, and magnetic stirring is performed at 200 rpm for 1 to 3 hours.

3. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that, The mineralized probiotic suspension described in step 2 is added with a series of sodium laurate solutions with a concentration gradient, sealed with plastic wrap, and fully reacted at 600 rpm with magnetic stirring for 2 to 5 hours, followed by centrifugation at 4°C and 8000 rpm for 15 minutes. After decanting the upper clear liquid, the mixture is centrifuged at 10000 rpm for 5 minutes to obtain modified mineralized probiotic particles.

4. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that, The modified mineralized probiotics after adding edible oil in step 3 are placed in a screw-capped glass bottle and uniformly dispersed by ultrasonication for 1 minute under inert gas protection. Then, 1 ml of deionized water is added to the edible oil dispersion using a pipette, and emulsified for 1 minute using a portable high-speed disperser at 6000-10000 rpm to obtain a water-in-oil modified mineralized probiotic Pickering emulsion.

5. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that, The wettability of the modified mineralized probiotics was regulated by adjusting the concentration of sodium laurate to construct a stable Pickering emulsion system.

6. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that, The outermost layer of the oil-in-water modified mineralized probiotics is the continuous oil phase in the oil-in-water system, the middle layer is the hydrophobic interface imparted by sodium laurate, and the innermost layer is biologically induced synthetic calcium carbonate. Through physical barriers and interface regulation, the bacteria are synergistically resisted from multiple extreme stresses, thereby protecting the bacteria from external invasion.

7. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, wherein, In a strong acid environment of pH = 2 for 30 minutes, the viability of probiotics in the oil-in-water system was 71.9%, while the survival rate of free probiotics was only 0.08%, an increase of 899 times. After 1 hour, bacteria were still alive in the oil-in-water system, while all the free probiotics died.

8. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that In the water-in-oil modified mineralized probiotic Pickering emulsion, at 70 °C, 80 °C, and 90 °C environments, there are still 6.45 log, 6.40 log, and 6.33 log viable bacteria, and the viable bacteria rates are 50.00%, 26.25%, 21.25%, and 16.25% respectively; while in the high-temperature environment, with the increase in temperature, the free probiotics only have 5.23 log viable bacteria at 60 °C, and the viable bacteria rate is only 11.67%, and all die at 70 °C, 80 °C, and 90 °C.

9. A method for constructing a Pickering emulsion stabilized by mineralized probiotic solid particles according to claim 1, characterized in that, After storage for up to 32 days at low temperature (4 °C), the viable bacteria rate of the water-in-oil system is as high as 66.16%, while the viable bacteria rate of the free probiotics is only 1.9%, which is about 35 times higher; even at room temperature (25 °C), compared with the free probiotics that only have 0.23% viable bacteria remaining after 32 days of storage, the viable bacteria rate of the water-in-oil system is as high as 60.50%, which is 263 times higher.

10. Application of the water-in-oil modified mineralized probiotic Pickering emulsion according to any one of claims 1 to 9 as a liquid oil substitute in the baking food industry.

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