Probiotic-loaded water-in-oil type emulsion gel and preparation method thereof

Abstract

The invention provides water-in-oil type emulsion gel loaded with probiotics and a preparation method of the water-in-oil type emulsion gel, and belongs to the technical field of probiotic products, the water-in-oil type emulsion gel loaded with the probiotics comprises a water phase and an oil phase, and the internal water phase is composed of gel and probiotic suspension; and the external oil phase consists of edible oil, a lipophilic emulsifier and at least one oil gelator. According to the water-in-oil emulsion gel, the stability of entrapped probiotics can be remarkably improved, meanwhile, the activity of the entrapped probiotics in the processing, storage and digestion processes is improved, particularly, the normal-temperature storage time is prolonged by 3 months, and a scientific basis is provided for normal-temperature food development.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic products, and in particular relates to a water-in-oil emulsion gel loaded with probiotics and a preparation method thereof. Background Art

[0002] Probiotics are a class of beneficial live microorganisms that colonize the human intestinal tract and reproductive system and produce health benefits. They help maintain the balance of intestinal microorganisms, enhance immunity, improve digestive health, and may have a positive impact on cardiovascular health and weight management. In addition, probiotics can enhance the body's immunity and resist the invasion of pathogens by forming an intestinal barrier. By consuming foods or supplements rich in probiotics, the number of probiotics in the body can be increased, the intestinal flora can be adjusted, and thus overall health can be promoted.

[0003] One of the main difficulties faced in the development of the probiotics industry is the reduction of the activity of probiotics during processing. This problem is mainly because probiotics are extremely sensitive to the temperature, humidity, oxygen and other conditions of the processing environment. For example, high temperature can cause protein denaturation and reduce the activity of probiotics; while too low a temperature may cause damage to the cell structure. In order to improve the activity of probiotics, encapsulation technology is widely used. This technology encapsulates probiotics through physical and chemical means to isolate them from the outside world and reduce the impact of adverse environments on the bacteria. Common probiotic delivery systems include microcapsules, hydrogels, emulsions, etc., which have been proven to effectively improve the survival rate of probiotics in vitro and in vivo. Although there have been breakthroughs in probiotic encapsulation technology, there is still a lack of development of room temperature probiotic foods. Because probiotics are extremely sensitive to temperature, at room temperature, the number of live probiotics will drop sharply with the extension of storage time, seriously affecting the stability and probiotic effect of the product. Therefore, how to maintain the high activity and stability of probiotics at room temperature has become a key technical problem restricting the development of this industry. Currently, existing research is mostly focused on the screening and functional evaluation of probiotic strains and the maintenance of activity in low-temperature environments. There is insufficient research on the processing technology of probiotic foods at room temperature and methods to maintain the activity of probiotics.

[0004] Oil-in-water emulsions have shown great potential as probiotic delivery carriers. They can effectively encapsulate and control the release of hydrophilic bioactive substances and expand the application range of probiotics in the food industry. However, the thermodynamic instability of oil-in-water emulsions is one of the main challenges they face. This instability is usually manifested as the emulsion is prone to stratification during storage, flocculation and agglomeration, etc., which leads to emulsion instability and affects the activity and delivery efficiency of probiotics. Therefore, improving the stability of oil-in-water emulsions to protect probiotics from adverse environmental influences is a key issue that needs to be addressed in current research and applications. Summary of the invention

[0005] The present invention provides a method for preparing a water-in-oil emulsion gel loaded with probiotics. The stability of the water-in-oil emulsion gel is improved by two-phase gelation of an oil phase and an aqueous phase. The dense network structure in the aqueous phase and the stacked crystal structure in the oil phase can improve the resistance of the encapsulated probiotics to extremely adverse conditions. The probiotics can be stably stored for 3 months at room temperature, thereby improving the development potential of the probiotics as room temperature food.

[0006] The present invention provides a probiotic-loaded water-in-oil emulsion gel, comprising an aqueous phase and an oil phase, wherein the internal aqueous phase consists of a gelling agent and a probiotic suspension; and the external oil phase consists of an edible oil, a lipophilic emulsifier and at least one oil gel factor.

[0007] Furthermore, the mass ratio of the oil phase to the water phase is (5-9):(1-5); Preferably, the mass ratio of the oil phase to the water phase is (6-8):(2-4).

[0008] Further, in the aqueous phase, the gelling agent includes a protein or polysaccharide capable of forming a gel; Preferably, the gelling agent comprises at least one of gellan gum, arabinoxylan, sodium alginate, carrageenan, whey protein isolate, soy protein isolate, and pea protein isolate; More preferably, the mass concentration of the gelling agent in the aqueous phase is 0.1% to 20%.

[0009] Further, in the aqueous phase, the probiotic suspension is formed by mixing the probiotics with physiological saline; Preferably, the mass of the probiotic suspension is 0.1% to 5% of the mass of the aqueous phase; More preferably, the probiotics include at least one of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium.

[0010] Further, in the oil phase, the oil gel factor includes at least one of β-sitosterol, γ-oryzanol, beeswax, candelilla wax, monoglyceride, and glyceryl monostearate; Preferably, the oleogels include at least one of β-sitosterol or γ-oryzanol; More preferably, in the oil phase, the mass concentration of the oil gel factor is 0.1% to 20%.

[0011] Furthermore, in the oil phase, the lipophilic emulsifier includes at least one of polyglycerol ricinoleate, lecithin, and Span; preferably, the mass concentration of the lipophilic emulsifier is 1% to 10%; In the oil phase, the edible oil includes at least one of soybean oil, corn oil, peanut oil and rapeseed oil.

[0012] The present invention also provides a method for preparing any of the above-mentioned probiotic-loaded water-in-oil emulsion gels, comprising the following steps: S1, adding the gelling agent to the aqueous phase, first mixing, refrigerating for hydration reaction, performing gelation treatment, and then adding the probiotic suspension, second mixing, to obtain the aqueous phase; S2, adding the oil gel factor and the lipophilic emulsifier to the edible oil, heating and mixing, to obtain an oil phase; S3, mixing the above water phase and the above oil phase, homogenizing, and obtaining a water-in-oil emulsion. Furthermore, in step S1, the hydration reaction is carried out under refrigeration at 4° C. for 8 to 15 hours; In step S1, the gelation treatment method includes at least one of heat treatment, enzyme induction, ion induction, and pH induction; Preferably, the gelation treatment method is heat treatment; More preferably, the heat treatment is heating at 80-90° C. for 20-40 min and then cooling.

[0013] Furthermore, in step S2, the heating and mixing is specifically heating and stirring in a constant temperature water bath at 80-85° C. for 15-30 minutes.

[0014] Furthermore, in step S3, the high-speed shearing condition is 2000-15000 rpm for 1-5 min.

[0015] The present invention has the following advantages: The invention provides a method for preparing a biphasic gelled oil-in-water emulsion gel loaded with probiotics. The oil phase is improved in structure by adding an oil gelling factor to the oil phase, thereby improving the stability of the emulsion system. The biphasic gelled oil-in-water emulsion gel of the oil phase and the water phase can significantly improve the stability of the encapsulated probiotics and improve the activity of the loaded probiotics during processing, storage and digestion, especially prolonging the storage time at room temperature to 3 months, thereby providing a scientific basis for the development of room temperature food. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 These are the appearance observation images of the emulsions obtained in Comparative Example 1 and Examples 1 to 9 in Test Example 1 of the present invention when placed normally (A) and inverted (B).

[0018] Figure 2 It is the 6-hour stability index image of Comparative Example 1 and Examples 1-5 (A), and Examples 3, 6-9 (B) in Test Example 1 of the present invention.

[0019] Figure 3 These are the apparent viscosity, frequency sweep analysis, and temperature frequency sweep analysis images of Comparative Example 1 and Examples 1 to 5 (A, B, C), and Examples 3, 6 to 9 (D, E, F) in Test Example 1 of the present invention.

[0020] Figure 4 These are Fourier transform infrared spectroscopic images of Comparative Example 2 and Examples 3, 6 to 9 in Experimental Example 1 of the present invention.

[0021] Figure 5 These are the x-ray diffraction images of Comparative Example 2 and Examples 3, 6 to 9 in Experimental Example 1 of the present invention.

[0022] Figure 6 These are polarizing microscope images of Comparative Example 2 and Examples 3, 6 to 9 in Experimental Example 1 of the present invention.

[0023] Figure 7 These are cryo-scanning electron microscope images of Comparative Example 1 and Example 3 in Experimental Example 1 of the present invention.

[0024] Figure 8 The probiotic survival numbers of Comparative Example 1 and Examples 1-5 (A, B), and Examples 3, 6-9 (C, D) in Test Example 1 of the present invention stored at 4° C. and room temperature for 3 months, respectively.

[0025] Fig. 9 It is the number of probiotics surviving after simulated pasteurization treatment of Comparative Example 1 and Examples 1-5 (A), Examples 3, 6-9 (B) in Test Example 1 of the present invention.

[0026] Fig.10 It is the number of probiotics surviving after freeze-thaw treatment of Comparative Example 1 and Examples 1-5 (A), Examples 3, 6-9 (B) in Test Example 1 of the present invention.

[0027] Fig.11 It is the number of probiotics surviving in the gastrointestinal simulated digestion process of Comparative Example 1 and Examples 1-5 (A), Examples 3, 6-9 (B) in Test Example 1 of the present invention.

[0028] Fig.12 It is the free fatty acid release rate during the gastrointestinal simulated digestion of Comparative Example 1 and Examples 1-5 (A), Comparative Example 2, and Examples 3, 6-9 (B) in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0030] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

[0031] The embodiment of the present invention provides a probiotic-loaded water-in-oil emulsion gel, comprising an aqueous phase and an oil phase, wherein the internal aqueous phase is composed of a gelling agent and a probiotic suspension; and the external oil phase is composed of an edible oil, a lipophilic emulsifier, and at least one oil gel factor.

[0032] In an embodiment of the present invention, the two-phase gelled water-in-oil emulsion gel shows a significant effect in the protection and delivery of probiotics. In this system, by combining the three-dimensional network structure of the hydrogel and the stacking of oil crystals in the oil phase, double protection is provided for probiotics. The hydrogel network structure is formed by physical or chemical cross-linking of proteins, polysaccharides, etc., which can protect probiotics from the stimulation of adverse external environments. At the same time, nutrients are provided for probiotics during processing and storage. Adding an oil gel factor in the oil phase allows the stacking of oil crystals to enhance the structural stability of the emulsion and improve the mechanical properties of the emulsion. This two-phase gelled water-in-oil emulsion gel can play a key role in extending the storage period and improving the gastrointestinal activity of probiotics, thereby improving its survival rate in normal temperature probiotic food processing and the human digestive tract.

[0033] Furthermore, in the aqueous phase, the gelling agent includes a protein or polysaccharide that can form a gel. Preferably, the gelling agent includes at least one of gellan gum, arabinoxylan, sodium alginate, carrageenan, whey protein isolate, soy protein isolate, and pea protein isolate. Furthermore, in the water phase, the mass concentration of the gelling agent is 0.1% to 20%.

[0034] Furthermore, in the aqueous phase, the probiotics include at least one of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium.

[0035] Further, in the aqueous phase, the probiotic suspension is a mixture of probiotics and physiological saline. The physiological saline is a NaCl solution with a mass concentration of 0.2% to 1.0%. Preferably, the physiological saline is a NaCl solution with a mass concentration of 0.8% to 0.9%. Furthermore, in the water phase, the mass of the probiotic suspension is 0.1% to 5% of the mass of the water phase. Preferably, the mass of the probiotic suspension is 1% of the mass of the water phase.

[0036] Further, in the oil phase, the oil gel factor includes at least one of β-sitosterol, γ-oryzanol, beeswax, candelilla wax, monoglyceride, and glyceryl monostearate. In the embodiment of the present invention, the addition of the oil gel factor can form a dense crystal network structure in the continuous phase, which plays an important role in improving the stability of the emulsion and enhancing the protection of the probiotics, improving the resistance of the encapsulated probiotics to extremely adverse conditions, and enabling them to be stably stored for 3 months at room temperature.

[0037] Preferably, the oleogel factor comprises at least one of β-sitosterol or γ-oryzanol.

[0038] Further, in the oil phase, the mass concentration of the oil gel factor is 0.1% to 20%. Preferably, the mass concentration of the oil gel factor is 2% to 10%. In the embodiment of the present invention, adding an appropriate concentration of the oil gel factor to the oil phase is beneficial to the stability of the emulsion system. However, too high a concentration is prone to instability. This is mainly because the oil gel factor can form a stronger crystal structure in the oil phase, and such larger crystals will squeeze and puncture the droplets, causing the emulsion to become unstable.

[0039] Furthermore, in the oil phase, the lipophilic emulsifier includes at least one of polyglycerol ricinoleate, lecithin, and span. Furthermore, in the oil phase, the mass concentration of the lipophilic emulsifier is 1% to 10%, preferably 2% to 8%. Furthermore, in the oil phase, the edible oil includes at least one of soybean oil, corn oil, peanut oil and rapeseed oil.

[0040] Further, the mass ratio of the oil phase to the water phase is (5-9): (1-5). Preferably, the mass ratio of the oil phase to the water phase is (6-8): (2-4). In the embodiment of the present invention, as the proportion of the oil phase gradually increases, the emulsion becomes more stable. When the concentration of the oil phase is too high, the emulsion is prone to instability. This is mainly because the oil phase is too much so that it is not completely adsorbed around the droplets, causing the instability of the system.

[0041] The present invention also provides a method for preparing any of the above-mentioned water-in-oil emulsion gels loaded with probiotics, comprising the following steps: S1, adding the gelling agent to the aqueous phase, first mixing, refrigerating for hydration reaction, performing gelation treatment, and then adding the probiotic suspension, second mixing, to obtain the aqueous phase; S2, adding the oil gel factor and the lipophilic emulsifier to the edible oil, heating and mixing, to obtain an oil phase; S3, mixing the above water phase and the above oil phase, homogenizing, and obtaining a water-in-oil emulsion. Furthermore, in step S1, the hydration reaction is carried out under refrigeration for 8 to 15 hours at 4°C. Specifically, the hydration reaction can be carried out in a refrigerator at 4°C overnight.

[0042] Furthermore, in step S1, the first mixing and the second mixing are mixed by magnetic stirring. Furthermore, in step S1, during the first mixing, the magnetic stirring rate is 300-700 rpm, and the stirring time is 2-4 h. Preferably, stirring is performed at 500 rpm for 3 h. Furthermore, in step S1, during the second mixing, the magnetic stirring rate is 300-700 rpm, and the stirring time is 10-40 min. Preferably, stirring is performed at 500 rpm for 30 min.

[0043] Furthermore, in step S1, the gelation treatment method includes at least one of heat treatment, enzyme induction, ion induction, and pH induction. Preferably, the gelation treatment method is heat treatment. More preferably, the heat treatment is specifically heating at 80-90°C for 20-40 min and then cooling.

[0044] Furthermore, in step S1, the minimum concentration of probiotics after loading is 1×10 6 CFU / g.

[0045] Furthermore, in step S2, the heating and mixing is specifically heating and stirring in a constant temperature water bath at 80-85°C for 15-30 minutes to fully dissolve the raw oil gel factor and lipophilic emulsifier in the edible oil.

[0046] Furthermore, in step S3, the mass ratio of the oil phase to the water phase is (5-9):(1-5). Preferably, the mass ratio of the oil phase to the water phase is (6-8):(2-4).

[0047] Furthermore, in step S3, the high-speed shearing condition is 2000-15000 rpm for 1-5 min.

[0048] Furthermore, in step S3, a high-speed shearing machine is used for homogenization. On the other hand, the embodiments of the present invention also provide the use of any of the above-mentioned probiotic-loaded water-in-oil emulsion gels in the fields of food, feed, biology, pharmaceutical encapsulation, and cosmetics.

[0049] The present invention will be described in detail below with reference to embodiments and drawings.

[0050] Example 1 A method for preparing a water-in-oil emulsion loaded with probiotics comprises the following steps: (1) Preparation of probiotic suspension: Will Lactobacillus plantarum LP90 Culture in MRS broth medium in advance and place in a 37℃ incubator for 18 h. Lactobacillus plantarum LP90The culture medium was removed by multiple centrifugation (3500 rpm, 10 min) and washing (0.85% saline, i.e., NaCl solution, w / w). Add 0.85wt.% physiological saline and mix well to obtain a probiotic suspension.

[0051] (2) Preparation of aqueous phase: 16 g of whey protein isolate was added to 84 g of distilled water and magnetically stirred at 500 rpm for 3 h to obtain a 16 wt.% whey protein isolate solution.

[0052] 0.4 g of high acyl gellan gum was added to 99.6 g of distilled water, and the mixture was stirred magnetically at 500 rpm for 3 h to obtain a 0.4 wt.% high acyl gellan gum solution.

[0053] Both solutions were placed at 4°C overnight for hydration. The two solutions were mixed in a 1:1 ratio and magnetically stirred at 500 rpm for 1 h to mix them evenly. The mixed solution was placed in a water bath, stirred at 85°C for 30 min, and cooled.

[0054] When cooled to room temperature, a probiotic suspension with a mass concentration of 1% was added, and magnetic stirring was performed at 500 rpm for 30 min to obtain an aqueous phase.

[0055] (3) Preparation of oil phase: Add 6 g of polyglycerol ricinoleate, 2.4 g of β-sitosterol and 3.6 g of γ-oryzanol to 92 g of soybean oil, so that the final concentrations are 6 wt.%, 0.8 wt.% and 1.2 wt.%, respectively. Heat in a water bath at 85 °C for 30 min to mix well, and obtain an oil phase; (4) Preparation of emulsion: The oil phase and the water phase were mixed in a mass ratio of 6:4 and sheared for 3 min at 6000 rpm using a high-speed shearing machine (IKA T25 digitalUltraturrax, GmbH, Staufen, Germany) to prepare an oil-in-water emulsion. 6 CFU / g.

[0056] Example 2 The same as Example 1, except that the concentration of β-sitosterol in the oil phase is 1.6 wt.%, and the concentration of γ-oryzanol is 2.4 wt.%.

[0057] Example 3 The same as Example 1, except that the concentration of β-sitosterol in the oil phase is 2.4wt.%, and the concentration of γ-oryzanol is 3.6wt.%.

[0058] Example 4 Same as Example 1, except that the concentration of β-sitosterol in the oil phase is 3.2 wt.%, and the concentration of γ-oryzanol is 4.8 wt.%.

[0059] Example 5 The same as Example 1, except that the concentration of β-sitosterol in the oil phase is 4 wt.%, and the concentration of γ-oryzanol is 6 wt.%.

[0060] Example 6 The same as Example 3, except that, in the emulsion preparation process, the mass ratio of the oil phase to the water phase used is 9:1. Example 7 The same as Example 3, except that, in the emulsion preparation process, the mass ratio of the oil phase to the water phase used is 8:2. Example 8 The same as Example 3, except that, during the emulsion preparation process, the mass ratio of the oil phase to the water phase used was 7:3.

[0061] Example 9 The same as Example 3, except that, during the emulsion preparation process, the mass ratio of the oil phase to the water phase used was 5:5.

[0062] Comparative Example 1 The same as Example 1, except that the oil phase contains only 94 g soybean oil and 6 g lipophilic emulsifier polyglycerol ricinoleate. Comparative Example 2 An oil gel comprises the following steps: The same operation process as step 3 in Example 3.

[0063] Test Example 1 Emulsion performance test 1. Storage stability (appearance) Experimental method: The prepared comparative example 1 and embodiments 1 to 9 were placed into observation bottles and photographed.

[0064] Experimental results: The results show that when the sterol concentration is low (Comparative Example 1, Examples 1 and 2), the emulsion is liquid, and when the sterol concentration is 6% or more, an emulsion gel structure is formed and can be inverted. When the oil-water ratio of the emulsion is changed (Examples 3, 6-9), the structure of the emulsion is not affected, and a stable inverted emulsion gel can be formed.

[0065] 2. Storage stability (TSI) Experimental method: The physical stability of W / O emulsions was measured by Turbiscan Lab Expert (Formulaction, France). 20 mL of sample was placed in a test bottle and scanned every 30 minutes for a total of 6 hours. The stability index (TSI) was obtained using Towersoft 2.0.0.9 software.

[0066] Experimental results: Figure 2 (A) It can be seen that as the concentration of sterols increases, the TSI value of the emulsion decreases from 0.81 to 0.53, and the TSI value of Example 5 is 0.60. A lower TSI value represents a higher stability. The experimental results show that adding sterols to the oil phase is beneficial to the stability of the emulsion system, and as the concentration of sterols increases, the TSI value of the emulsion becomes lower and the emulsion becomes more stable. When the sterol concentration increases to 10%, the TSI value increases, which may be because the higher sterol concentration forms a stronger crystal structure in the oil phase, and this larger crystal will squeeze and pierce the droplets, causing the emulsion to become unstable.

[0067] Depend on Figure 2 (B) It can be seen that as the oil phase ratio continues to increase, the TSI value of the emulsion decreases from 0.63 to 0.38. When the oil phase mass fraction reaches 90% (Example 6), the TSI value is 0.76. The experimental results show that as the oil phase ratio gradually increases, the emulsion becomes more stable. When the oil phase concentration increases to 90%, the emulsion becomes unstable. This is mainly due to the excessive amount of oil phase, which is not completely adsorbed around the droplets, causing the instability of the system.

[0068] 3. Rheological properties Experimental methods: The apparent viscosity, gel properties, and temperature-dependent properties of different W / O emulsions were measured using a MARS 60 rheometer (Thermo Scientific, Karlsruhe, Germany). The test temperature was set at 25°C, and geometric parallel plates (20 mm diameter, 1.0 mm gap) were used for the measurements. First, strain-stress sweep tests were performed to determine the linear viscoelastic region (LVR) of the W / O emulsions in the strain range of 0.1–20%. The apparent viscosity of the different samples was measured at shear rates ranging from 0.1 s-1 to 100 s-1 at a constant frequency of 1.0 Hz. Frequency sweep tests (0.1–10 Hz) were performed at a constant strain of 0.5% to measure the elastic modulus (G') and viscous modulus (G''). Temperature frequency sweep tests were performed in the range of 25°C to 80°C to measure the changes in the G' and G'' values ​​of the samples at different temperatures.

[0069] Experimental results: Figure 3(A) It can be found that the viscosity of all samples decreases with the increase of shear rate, showing obvious shear thinning behavior. And the viscosity gradually increases with the increase of sterol concentration in the oil phase. Figure 3 (B) It can be found that the G' of all samples is greater than G'', showing obvious gel behavior. With the increase of sterol concentration, the G' and G'' values ​​gradually increase, which indicates that the gel strength of the emulsion is gradually increasing. Figure 3 (C) It can be found that with the increase of temperature, G' and G'' in Comparative Example 1 and Examples 1 and 2 where no emulsion gel is formed do not show obvious changes, while G' and G'' in Examples 3, 4 and 5 show obvious decreases. This is mainly due to the melting of sterols in the oil phase during the heating process, which leads to the weakening of the gel strength. In addition, with the increase of sterol concentration, the intersection position of G' and G'' also gradually increases, which are 52°C, 57°C and 62°C, respectively. This shows that the increase of sterol concentration helps to increase the phase transition temperature of the emulsion, thereby improving the thermal stability of the emulsion.

[0070] Depend on Figure 3 (D) It can be found that all samples are non-Newtonian fluids, showing obvious shear thinning behavior. Among them, Example 6 has the lowest viscosity and shows instability. As the oil phase ratio increases, the emulsions of the other samples gradually become viscous, proving that increasing the oil phase ratio can improve the viscoelasticity of the emulsion. Figure 3 (E) It can be found that G' of all samples is greater than G'', showing obvious gel behavior. Example 6 shows obvious frequency dependence, proving that the strength of the gel formed is weak. As the proportion of oil phase increases, the G' and G'' values ​​of the remaining samples gradually increase, indicating that a stronger gel structure is formed. Figure 3 (F) It can be found that with the increase of temperature, G' and G'' in all samples decreased significantly, and the intersection of G' and G'' was 52°C. It is worth noting that when the heating temperature is greater than 52°C, only G' of Example 3 and Example 9 is greater than G'', and G' of the other samples is less than G'', which is mainly related to the whey protein isolate in the internal water phase. Since the gel formed by the thermal denaturation of whey protein isolate is a thermally irreversible gel, it is less affected by heat during the heating process. Therefore, during long-term high-temperature heating, a higher water phase ratio will have stronger stability.

[0071] 4. Fourier transform infrared spectroscopy Experimental method: The crystal structures of Comparative Example 2, Examples 3, and 6 to 9 were analyzed by Fourier transform infrared spectrometer (Nicolet IS50, Thermo Fisher Scientific, Madison, USA). Spectra were collected in total reflection mode at 400-4000 cm-1 The scan was repeated 32 times within the scanning range with a resolution of 4 cm -1 OMNIC software was used to process and analyze the spectral data. Figure 4 .

[0072] Experimental results: It was found that at 3690-3000 cm -1 Broad peaks were observed at 2953, 2853, and 1463 cm, which are mainly related to the stretching vibration of OH in the hydrogel. As the amount of oil gel increases, the OH bond stretching vibration intensity tends to decrease, indicating that the hydrogen bonds in the sample decrease. -1 The signals at 1743, 1160 cm-1 are mainly related to the stretching vibrations of CH or C=H bonds. These peaks mainly correspond to the saturated and unsaturated fatty acyl chains in vegetable oils. -1 The absorption peak observed at 1639-1640 cm -1 The signal at 1639 cm-1 is related to the stretching vibration of C=O in gellan gum. As the proportion of oil phase increases, the signal at 1639 cm-1 increases. -1 The blue shift to 1640 cm -1 This is mainly related to the decrease in the concentration of the water phase in the system, which leads to the weakening of the hydrogen bonding force and the C=O force. In general, the change in the oil-water ratio in the emulsion does not generate new chemical bonds, but only changes the strength of the absorption peak. As the proportion of the oil phase increases, the strength of the oil phase group increases, and the strength of the water phase group weakens.

[0073] 5. Crystal structure Experimental method: Use X-ray diffractometer (XRD-6100, Shimadzu, Tokyo, Japan) to measure the crystal structure of different samples. Take an appropriate amount of sample and spread it on the center groove of the test plate. Set the scanning speed to 1° / min and the scanning step to 0.02°. Use Jade software to analyze the diffraction pattern. The results are shown in Figure 5 .

[0074] Experimental results: Both Comparative Example 2 and Examples 3, 6-9 show similar broad diffuse peaks at 4.58Å, which is a typical β-crystal form, indicating that sterols form a stable crystal structure in the oil phase to enhance the stability of the emulsion, and the formation of the emulsion does not change the interplanar spacing of the β-sitosterol-γ-oryzanol crystal structure. As the oil phase ratio increases, the peak area of ​​XRD in the emulsion gradually increases, proving that the emulsion with a high oil phase ratio forms a stronger crystal structure.

[0075] 6. Microstructure 6.1 Polarized light microscope Experimental method: The microscopic morphology of the emulsions of Comparative Example 2, Examples 3, and 6 to 9 was observed by polarizing microscope (DM4000, Leica, Weztlar, German) at a magnification of 50×. Figure 6 .

[0076] Experimental results: It can be seen from the figure that more needle-shaped crystal structures are formed in the oil phase, and the droplets are fixed on the crystal surface or between the crystal networks. As the proportion of the oil phase increases, the size and number of crystals gradually increase, and a strong fat crystal network is formed in the oil phase. The crystal network structure in the oil phase tightly binds the droplets in the inner water phase, preventing them from migrating and agglomerating, thereby improving the stability of the emulsion.

[0077] 6.2 Cryo-SEM Experimental method: After the emulsion samples were pre-treated by cryofixation and gold spraying, they were placed under a cryo-scanning electron microscope by a cryo-transfer system for observation. The cryo-scanning electron microscope (Sigema300, Zeiss, Oberkochen, Germany) was used to take cryo-electron microscope images of the emulsions prepared in Comparative Example 1 and Example 3 at magnifications of 1000× and 20000×, respectively. The results are shown in Figure 7 .

[0078] Experimental results: Dense network structures are distributed in the droplets of Comparative Example 1 and Example 3, and the dense double network structure in the inner water phase tightly wraps the probiotics. The continuous phase of Comparative Example 1 is a smooth structure, while the continuous phase in Example 3 is a rough structure, and small crystal structures can be found stacked in the continuous phase. It is proved that sterols form a dense crystal network structure in the continuous phase, which plays an important role in improving the stability of the emulsion and enhancing the protection of the probiotics.

[0079] 7. Storage stability of water-in-oil emulsions containing probiotics Experimental method: Fresh samples and free probiotics were stored at 4°C and room temperature for 3 months, and the number of colonies in the emulsion was measured every 14 days using the plate count method. Figure 8 .

[0080] Experimental results: Figure 8 (A) It can be found that after storage at 4°C for three months, the comparative example 1 decreased by 2.00 log CFU / g, and the examples 1 to 5 decreased by 1.63, 1.58, 0.88, 0.85, and 0.89 log CFU / g, respectively; Figure 8(B) It can be found that after three months of storage at room temperature, the comparative example 1 decreased by 5.38 log CFU / g, and the examples 1 to 5 decreased by 4.03, 2.97, 1.65, 1.41, and 1.41 log CFU / g, respectively. With the increase of sterol concentration, especially when the sterol concentration is ≥ 6%, the survival rate of probiotics during storage decreases the least. It is proved that the addition of sterol improves the stability of the emulsion system, reduces the stimulation of the adverse external environment to the probiotics, and plays a positive role in extending the storage period, especially room temperature storage.

[0081] Depend on Figure 8 (C) It can be found that after storage at 4°C for three months, the free probiotics were completely inactivated, and Examples 3, 6 to 9 decreased by 1.21, 2.64, 0.85, 1.06, and 1.31 log CFU / g, respectively; Figure 8 (D) It can be found that the free probiotics are completely inactivated after storage for 2 months. After storage at room temperature for three months, Examples 3, 6 to 9 respectively decreased by 2.02, 3.42, 1.35, 2.03, and 2.52 log CFU / g. The activity of probiotics delivered by emulsion is greater than that of free probiotics, proving that the emulsion system encapsulation has a positive effect on improving the activity of probiotics. For emulsions, the storage stability of probiotics is mainly consistent with the emulsion stability results. With the increase of the proportion of oil phase in the emulsion, the emulsion crystal structure becomes more compact, which improves the stability of the emulsion. When the mass fraction of the oil phase in the emulsion reaches 90% (Example 6), the crystal structure in the system is too large to be adsorbed on the oil-water interface, so that it appears unstable, resulting in a decrease in the storage activity of probiotics.

[0082] 8. Temperature stability (pasteurization, freeze-thaw stability) 8.1 Simulated pasteurization stability Experimental method: W / O emulsion and free probiotic solution were heated under two pasteurization conditions: 65℃-30min and 72℃-15s. The heated samples were then placed in ice water to quickly terminate the reaction. Free Lactobacillus plantarum was used as a control. The simulated pasteurization stability of the samples was evaluated by measuring the number of surviving probiotics in the emulsion. The results are shown in Fig. 9 .

[0083] Experimental results: Fig. 9(A) It can be found that after heating at 72°C for 15 s, Comparative Example 1 decreased by 1.18 log CFU / g, and Examples 1 to 5 decreased by 1.08, 0.99, 0.84, 0.51, and 0.39 log CFU / g, respectively; after heating at 65°C for 30 min, Comparative Example 1 decreased by 4.01 log CFU / g, and Examples 1 to 5 decreased by 3.56, 3.55, 3.33, 2.93, and 2.65 log CFU / g, respectively. The experimental results show that short-term high-temperature heating may be more suitable for the processing of probiotic products. With the increase of sterol concentration, the heat resistance of the emulsion is better and the survival rate of probiotics is higher. This is mainly because the increase of sterol concentration increases the viscosity and phase transition temperature of the emulsion, reduces the collision of droplets, and the higher sterol concentration increases the crystal network structure in the continuous phase, increases the thickness of the interfacial film, and prevents the emulsion from agglomerating.

[0084] Depend on Fig. 9 (B) It can be found that after heating at 72°C for 15 s, the free probiotics decreased by 3.33 log CFU / g, and Examples 3, 6-9 decreased by 0.84, 1.11, 0.65, 0.69, and 0.99 log CFU / g, respectively; after heating at 65°C for 30 min, the free probiotics were completely inactivated, and Examples 3, 6-9 decreased by 3.33, 3.59, 2.91, 3.19, and 3.42 log CFU / g, respectively. Compared with free probiotics, the emulsion-encapsulated probiotics have higher activity after simulated pasteurization, proving that the emulsion system plays an important role in improving the heat resistance of probiotics. With the increase of the proportion of the oil phase (except 90%), the survival rate of probiotics also gradually increased. This is mainly because the dense crystal network structure in the oil phase can effectively slow down or isolate the effect of heating on the probiotics, and the dense network structure in the inner water phase also improves the thermal stability of the probiotics.

[0085] 8.2 Freeze-thaw stability Experimental method: The freshly prepared emulsion gel and free probiotic solution were placed in a -20°C refrigerator for 22 h to achieve freezing, and then the samples were taken out and placed in a 37°C water bath for 2 h to complete thawing, completing a freeze-thaw cycle. The total number of freeze-thaw cycles was 3 times, and the number of surviving probiotics was measured after each freeze-thaw cycle. The freeze-thaw stability of the sample was evaluated by observing the number of surviving probiotics in the emulsion. Results are shown in Fig.10 .

[0086] Experimental results: Fig.10(A) It can be found that after the freeze-thaw cycle is completed for 3 times, Comparative Example 1 has decreased by 2.07 logCFU / g, and Examples 1 to 5 have decreased by 1.68, 1.25, 0.42, 0.40, and 0.66 log CFU / g, respectively. The experimental results show that the addition of sterols in the oil phase can improve the freeze-thaw stability of plant lactobacillus to a certain extent, and has more viable counts after three freeze-thaw cycles as the concentration of sterols increases. This is mainly due to the dense crystal network in the oil phase and the dense gel network in the water phase that hinder the aggregation of droplets, inhibiting ice crystals from piercing the interfacial film.

[0087] Depend on Fig.10 (B) It can be found that after the freeze-thaw cycle is completed 3 times, the free probiotics decrease by 3.91 log CFU / g, and Examples 3, 6-9 decrease by 0.42, 1.21, 0.37, 0.39, and 0.72 log CFU / g, respectively. The experimental results show that the emulsion system plays an important role in protecting the freeze-thaw stability of probiotics. With the increase of the oil phase ratio (except 90%), the freeze-thaw stability of the emulsion becomes better, which is mainly because the reduction of the water content in the system does not generate too many ice crystals to pierce the droplets during the freeze-thaw process. In addition, a larger oil phase ratio also has more crystal network structures, which helps to improve the stability of the emulsion and protect the activity of the probiotics. When the oil phase ratio is 90% (Example 6), due to the excessive oil phase ratio in the system, the huge crystal structure may backlog the droplets, causing the instability of the system and thus reducing the survival rate of the probiotics.

[0088] 9. Simulated gastrointestinal digestion experiment Experimental method: Simulated gastric fluid is composed of 3.2 mg / mL pepsin and 1.0M hydrochloric acid; simulated intestinal fluid (pH=7.0) is composed of 2.0 mg / mL pancreatic enzyme, 3.2 mg / mL pancreatic lipase and 12.0 mg / mL bile salt. The emulsion / free probiotic solution is mixed with simulated gastric fluid to form gastric digestion fluid, and the pH of the mixed solution is adjusted to 2.0, and incubated in a 37°C water bath shaker at 100 rpm for 2 h. Samples are taken every half an hour, and the pH of the solution is adjusted to 7 after sampling to terminate the reaction; after gastric digestion, the pH of the gastric digestion fluid mixture is adjusted to 7, and simulated intestinal fluid is added. The mixed solution is kept at a pH of 7 and incubated in a 37°C water bath shaker at 100 rpm for 2 h. Samples are taken every half an hour, and the solution is placed in an ice water bath after sampling to terminate the reaction. The number of probiotic colonies was measured by the plate count method. Results are shown in Fig.11 .

[0089] Experimental results: Fig.11(A) It can be found that after the simulated gastric digestion, the comparative example 1 decreased by 0.65 logCFU / g, and the examples 1 to 5 decreased by 0.52, 0.99, 0.32, 0.22, and 0.16 logCFU / g, respectively; after the simulated intestinal digestion, the comparative example 1 decreased by 0.92 logCFU / g, and the examples 1 to 5 decreased by 0.63, 0.62, 0.53, 0.43, and 0.28 logCFU / g, respectively. The experimental results show that the biphasic gelled W / O emulsion can improve the survival rate of probiotics during the simulated gastrointestinal digestion. This is mainly because the simultaneous gelation of the aqueous phase and the oil phase can effectively inhibit the penetration of gastric acid, pepsin, bile salts, etc., and prevent inactivation caused by contact with the probiotics. As the sterol concentration increases, the gastrointestinal stability of probiotics increases, which indicates that the oil phase gel plays a more dominant role in improving the gastrointestinal activity of probiotics. The increase in the strength of the oil phase gel can reduce the rate of fat decomposition, while the gel network structure in the inner water phase reduces the contact between probiotics and bile salts, thereby improving the survival rate of probiotics during gastrointestinal simulated digestion. Depend on Fig.11 (B) It can be found that after simulated gastric digestion, the free probiotics decreased by 4.15 log CFU / g, and Examples 3, 6-9 decreased by 0.32, 0.56, 0.23, 0.23, and 0.35 log CFU / g, respectively; after simulated intestinal digestion, the free probiotics were completely inactivated, and Examples 3, 6-9 decreased by 0.53, 0.97, 0.33, 0.49, and 0.79 log CFU / g, respectively. The experimental results show that the probiotics encapsulated in the emulsion can effectively improve their survival rate during gastrointestinal simulated digestion compared with free probiotics. The gastrointestinal digestion activity of probiotics is similar to the stability results of the emulsion. With the increase of the oil phase ratio (except 90%), the emulsion stability gradually increases, and the digestion survival rate of probiotics is also higher. This is mainly because the increase in the oil phase ratio makes the crystal network structure in the oil phase more dense, which plays a vital role in delaying gastric acid erosion and lipase decomposition. At the same time, since Example 6 mainly relies on the stacking of large crystals to stabilize the emulsion structure, the structure is more likely to become loose when faced with the erosion of lipase and gastric acid, thereby reducing the gastrointestinal digestive activity.

[0090] 10. Determination of free fatty acids (FFA) Experimental method: The specific steps of simulating gastrointestinal digestion are the same as those of Experiment 9. When the digestion reaches the intestinal digestion process, 0.1M NaOH solution is added to the digestion mixture every half an hour to maintain the pH of the mixture at 7.0, and the amount of NaOH solution added is recorded. The FFA release rate is calculated by the following formula:

[0091] Among them, VNaOH represents the volume of NaOH solution added (L), m NaOH represents the molecular weight of NaOH (M), M liquid represents the molar mass of soybean oil (g / mol), w liquid represents the mass of soybean oil (g). Fig.12 .

[0092] Experimental results: Fig.12 It can be found in (A) that the free fatty acid release rate of Comparative Example 1 is 87.27%, and the free fatty acid release rates of Examples 1 to 5 are 61.99%, 54.41%, 30.80%, 22.73%, and 14.96%, respectively. As the sterol concentration increases, the FFA release rate gradually decreases, which is mainly due to the fact that a higher sterol concentration will form a denser crystal network structure, which helps to reduce the decomposition rate of lipase, thereby reducing the release rate of free fatty acids.

[0093] Depend on Fig.12 It can be found in (B) that the free fatty acid release rate of Comparative Example 2 is 48.25%, and the free fatty acid release rates of Examples 3, 6 to 9 are 30.80%, 44.95%, 26.73%, 27.66%, and 40.25%, respectively. As the proportion of oleogel increases (except for the 90% sample), the FFA release rate gradually decreases, which is mainly due to the fact that a higher oleogel fraction will enhance the viscoelasticity of the sample, hinder the penetration of lipase, and thus reduce the degree of fat decomposition.

[0094] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A water-in-oil emulsion gel loaded with probiotics, comprising an aqueous phase and an oil phase, characterized in that: The internal water phase is composed of a gelling agent and a probiotic suspension; the external oil phase is composed of edible oil, a lipophilic emulsifier and at least one oil gelling factor.

2. The water-in-oil emulsion gel according to claim 1, characterized in that: The mass ratio of the oil phase to the water phase is (5-9):(1-5); Preferably, the mass ratio of the oil phase to the water phase is (6-8):(2-4).

3. The water-in-oil emulsion gel according to claim 1, characterized in that In the aqueous phase, the gelling agent includes a gel-forming protein or polysaccharide; Preferably, the gelling agent comprises at least one of gellan gum, arabinoxylan, sodium alginate, carrageenan, whey protein isolate, soy protein isolate, and pea protein isolate; More preferably, the mass concentration of the gelling agent in the aqueous phase is 0.1% to 20%.

4. The water-in-oil emulsion gel according to claim 1, characterized in that: In the aqueous phase, the probiotic suspension is a mixture of probiotics and saline; Preferably, the mass of the probiotic suspension is 0.1% to 5% of the mass of the aqueous phase; More preferably, the probiotics include at least one of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium.

5. The water-in-oil emulsion gel according to claim 1, characterized in that: In the oil phase, the oil gel factor includes at least one of β-sitosterol, γ-oryzanol, beeswax, candelilla wax, monoglyceride, and glyceryl monostearate; Preferably, the oleogels include at least one of β-sitosterol or γ-oryzanol; More preferably, in the oil phase, the mass concentration of the oil gel factor is 0.1% to 20%.

6. The water-in-oil emulsion gel according to claim 1, characterized in that: In the oil phase, the lipophilic emulsifier includes at least one of polyglycerol ricinoleate, lecithin, and Span; preferably, the mass concentration of the lipophilic emulsifier is 1% to 10%; In the oil phase, the edible oil includes at least one of soybean oil, corn oil, peanut oil and rapeseed oil.

7. The method for preparing the water-in-oil emulsion gel loaded with probiotics according to any one of claims 1 to 6, comprising the following steps: S1, adding the gelling agent to the water phase, first mixing, refrigerating for hydration reaction, performing gelation treatment, and then adding the probiotic suspension, second mixing, to obtain the water phase; S2, adding the oil gel factor and the lipophilic emulsifier to the edible oil, heating and mixing, to obtain an oil phase; S3, mixing the above water phase and the above oil phase, homogenizing, and obtaining a water-in-oil emulsion.

8. The preparation method according to claim 7, characterized in that: In step S1, the hydration reaction is carried out under refrigeration, specifically at 4° C. for 8 to 15 hours; In step S1, the gelation treatment method includes at least one of heat treatment, enzyme induction, ion induction, and pH induction; Preferably, the gelation treatment method is heat treatment; More preferably, the heat treatment is heating at 80-90° C. for 20-40 min and then cooling.

9. The preparation method according to claim 7, characterized in that: In step S2, the heating and mixing is specifically heating and stirring in a constant temperature water bath at 80-85° C. for 15-30 min.

10. The preparation method according to claim 7, characterized in that: In step S3, the high-speed shearing condition is 2000-15000 rpm for 1-5 min.

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