A probiotic-loaded oil-in-water double gel and its preparation method and application

Abstract

The present invention provides an oil-in-water (O / W) double gel loaded with probiotics, as well as its preparation method and application, and relates to the field of probiotic products. The raw materials of the probiotic-loaded O / W double gel include an aqueous phase and an oil phase. The aqueous phase includes linseed gum and a solvent; the oil phase includes lecithin, beeswax, oil, and probiotic slurry. Through the coordinated action of linseed gum as an aqueous phase gelling agent and lecithin and beeswax as oil phase gelling agents, a stable O / W double gel system can be formed. The probiotics are encapsulated in the inner oil phase, achieving a preliminary encapsulation effect. The outer aqueous phase further prevents the migration of the probiotics, resulting in a high encapsulation rate, improved thermal stability, storage stability, and stability during digestion of the probiotics, and thus increased survival rate of the probiotics. Furthermore, the outer phase of the O / W double gel system is an aqueous phase, which significantly reduces the greasy feel compared to the O / W double gel, and the product is more refreshing.

Description

Technical Field

[0001] The present invention relates to the field of probiotic products, and in particular to an oil-in-water type oil-water double gel loaded with probiotics, and a preparation method and application thereof. Background Art

[0002] Probiotics are living microorganisms that, when consumed in sufficient quantities, can have a positive impact on the human body. They form a symbiotic relationship with the human intestinal flora, regulating it to facilitate digestion and absorption, and maintaining a dynamic balance in the microbial ecosystem. Probiotics can enhance immunity by boosting both specific and nonspecific immunity, regulating the immune system and improving the host's ability to resist disease.

[0003] As people’s lifestyles and eating habits change, more and more people are beginning to pay attention to healthy eating, and the probiotics industry is also booming. It is generally believed that probiotic products should contain at least 10 6 ~10 7 A high number of viable bacteria (CFU / mL or CFU / g) is required to produce beneficial health effects. However, probiotics are very sensitive to the external environment and are often inactivated during storage, processing, and gastrointestinal digestion. Therefore, probiotics must be encapsulated in a protective system to enhance their activity and extend their shelf life.

[0004] A gel is a semisolid system composed of a solid and a liquid. The gelling agent in the system forms a network structure that prevents the flow of the liquid phase, thereby improving the stability of the system. Traditional hydrogels contain a polar solvent as the continuous phase, typically water, and are generally used to deliver hydrophilic substances. Oleogels contain a non-polar liquid (organic solvent, mineral oil, or vegetable oil) as their continuous phase and are generally used to deliver lipophilic substances. However, their oiliness and viscosity limit their application in production. An oil-water bigel is a dual-continuous phase system formed by mixing a hydrogel and an oil-phase gel. This bigel combines the advantages of both emulsions and gels, enabling the simultaneous delivery of both lipophilic and hydrophilic active substances. Unlike conventional emulsions, this bigel does not contain surfactants, making it more environmentally friendly. Furthermore, since both the oil and water phases are gels, they can better lock active substances into the network structure, improving system stability and exhibiting better physicochemical properties. This bigel is gaining increasing attention, development, and application in various industries, including food, pharmaceuticals, cosmetics, and biomaterials.

[0005] Currently, there are few reports on methods for loading probiotics using a dual-gel system. Patent document CN117044949A discloses a method for preparing probiotic-encapsulated particles. Specifically, it involves constructing a water-in-oil dual-gel emulsion comprising a hydrogel phase and an oil-gel phase. The oil-gel phase includes oil, an oil-soluble emulsifier, and an auxiliary oil-gelling agent, while the hydrogel phase includes probiotics, a gel solution, and an acidity regulator and / or a cationic salt. While this method discloses the use of a dual-gel system for loading probiotics, it still requires the subsequent preparation of multiple emulsions and embedded particles, resulting in a complex process. Furthermore, the dual-gel system produced is a water-in-oil dual-gel. When used alone, the external oil phase can make the product feel more greasy, limiting its application.

[0006] Therefore, how to prepare a probiotic-loaded double gel with a refreshing texture and a stable system has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] Therefore, the object of the present invention is to provide a probiotic-loaded oil-in-water double gel with a refreshing texture and a stable system, thereby improving the storage properties of the probiotics in the oil-in-water double gel and their resistance to adverse conditions.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides an oil-in-water double gel loaded with probiotics, the raw materials of which include an aqueous phase and an oil phase.

[0010] The aqueous phase comprises: flaxseed gum and a solvent;

[0011] The oil phase comprises: lecithin, beeswax, oil and probiotic mud.

[0012] Furthermore, the mass concentration of flaxseed gum in the aqueous phase is 0.1% to 10%, preferably 10%.

[0013] Furthermore, the mass concentration of lecithin in the oil phase is 0.1% to 10%, the mass concentration of beeswax is 0.1% to 10%, and the mass concentration of probiotic sludge is 0.1% to 3%. Preferably, the mass concentration of lecithin in the oil phase is 1%, the mass concentration of beeswax is 3%, and the mass concentration of probiotic sludge is 0.5%.

[0014] Furthermore, the mass ratio of the water phase to the oil phase is 5-9:1-5, preferably 9:1, 7:3 or 5:5.

[0015] Furthermore, the probiotic slurry is obtained by activating the probiotics, centrifuging the culture medium after culture, and discarding the supernatant. Preferably, the centrifugation condition is: centrifugation at 3500 rpm for 10 minutes.

[0016] Furthermore, in the oil-in-water double gel loaded with probiotics, the loading concentration of probiotics is at least 1×10 6 CFU / g.

[0017] Furthermore, the solvent in the aqueous phase is water.

[0018] Furthermore, the oil includes at least one of soybean oil, corn oil, peanut oil, and rapeseed oil.

[0019] Furthermore, the probiotic species in the probiotic suspension include at least one of yeast, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, and Bifidobacterium.

[0020] In a second aspect, the present invention provides a method for preparing the probiotic-loaded oil-in-water dual-gel, comprising the following steps:

[0021] (1) mixing flaxseed gum and a solvent, heating and dissolving the mixture to obtain an aqueous solution;

[0022] (2) mixing lecithin, beeswax and oil, heating and dissolving, adding probiotic mud, and mixing uniformly to obtain an oil phase solution;

[0023] (3) The aqueous phase solution and the oil phase solution are mixed uniformly in proportion, sheared at high speed, and cooled to room temperature to obtain the oil-in-water type oil-water double gel loaded with probiotics.

[0024] Furthermore, in step (1), the heating and dissolving conditions are: heating at a temperature of 80 to 85° C. for 2 to 4 hours, preferably heating at a temperature of 85° C. for 2 hours.

[0025] Furthermore, in step (2), the heating and dissolving conditions are: heating at a temperature of 60 to 65° C. for 15 to 30 minutes, preferably heating at a temperature of 65° C. for 20 minutes.

[0026] Furthermore, in step (3), the temperatures of the aqueous phase solution and the oil phase solution during mixing are both 35-40°C, preferably 37°C; the high-speed shearing conditions are: shearing at a speed of 2000-15000 rpm for 1-5 min, preferably shearing at a speed of 4000-11000 rpm for 1-3 min, and more preferably shearing at a speed of 10000 rpm for 3 min.

[0027] In a third aspect, the present invention provides the use of the probiotic-loaded oil-in-water bigel or the probiotic-loaded oil-in-water bigel obtained by the preparation method in the preparation of food, health products, feed, medicine, and cosmetics.

[0028] The technical solution of the present invention has the following advantages:

[0029] The water-in-oil type oil-water double gel loaded with probiotics provided by the present invention comprises an aqueous phase and an oil phase, wherein the aqueous phase comprises linseed gum and a solvent; and the oil phase comprises lecithin, beeswax, oil and a probiotic suspension. During the invention process, the inventors discovered that by the cooperation of the aqueous phase gelling agent linseed gum and the oil phase gelling agents lecithin and beeswax, a stable water-in-oil type double gel system can be formed, wherein the probiotics are coated in the inner oil phase, achieving a preliminary encapsulation effect, and the outer aqueous phase further prevents the migration of the probiotics, resulting in a high embedding rate, thereby improving the thermal stability, storage stability and stability of the probiotics during digestion, thereby improving the survival rate of the probiotics. In addition, the outer phase of the water-in-oil type double gel system is an aqueous phase, which greatly reduces the greasy feeling compared to the water-in-oil type double gel, and the product is more refreshing.

[0030] The lecithin and beeswax in the oil phase have a synergistic effect on the stability of the oil-in-water dual-gel system. Using either alone does not form a stable dual-gel at all. Adjusting the ratio of the water phase to the oil phase also fails to form a stable water-in-oil dual-gel. This further demonstrates the crucial role of flaxseed gum, lecithin, and beeswax in the stability of the oil-in-water dual-gel system.

[0031] The probiotic-loaded oil-in-water bigel provided by the present invention can be inverted to form a gel and maintain a stable state; has good rheological behavior and also shows obvious shear-thinning behavior; has a high encapsulation rate; has strong thermal stability, and the oil-in-water structure can effectively inhibit heat transfer, reduce the loss of probiotics during water evaporation, and thus improve their survival rate; has high stability when stored at room temperature, reduces the rate of probiotic migration to the water phase, and thus increases its storage time; the system has stronger stability and mechanical strength, and the compact structure of the bicontinuous phase during the digestion process makes the system more stable, delays the erosion of the system by lipase and bile salts, and thus improves the survival rate of the probiotics.

[0032] The oil-in-water double gel loaded with probiotics provided by the present invention has a simple preparation process, is easy to operate, and has controllable costs, and can be widely used in the fields of food, health products, feed, medicine, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is a comparison diagram of the appearance of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0035] Figure 2 This is a frequency scanning analysis chart of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0036] Figure 3 Graphs showing the apparent viscosities of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0037] Figure 4 are differential scanning calorimetry curves of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0038] Figure 5 This is a comparison chart of the embedding efficiency of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0039] Figure 6 3 is a comparison chart of the thermal stability of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 and free probiotics during pasteurization;

[0040] Figure 7 This is a comparison chart of the storage stability of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 at room temperature;

[0041] Figure 8 This is a CLSM image of the product obtained in Example 3 of the present invention, wherein green represents probiotics and blue represents flaxseed gum;

[0042] Figure 9 is a comparison chart of changes in free fatty acid release rates of the products obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0043] Figure 10 3 is a graph comparing the stability of the products and free probiotics obtained in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 in simulated gastrointestinal digestion. DETAILED DESCRIPTION

[0044] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0045] The probiotic bacteria used in the embodiments of the present invention are specifically selected from Lactobacillus plantarum LP90, which is deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection under the deposit number CGMCC No. 10453. Information on this strain is disclosed in patent document CN104928208A. A commercially available bacterial liquid product of this strain is used in this embodiment.

[0046] The probiotic suspension used in the embodiments of the present invention is prepared by the following method:

[0047] Lactobacillus plantarum LP90 was inoculated into MRS broth culture medium, cultured in a 37°C constant temperature incubator for 18 hours, and then passaged for secondary activation. After secondary activation, the culture was continued in a 37°C constant temperature incubator for 18 hours. The cultured MRS broth culture medium was centrifuged at 3500 rpm for 10 minutes, and the supernatant was discarded to obtain Lactobacillus plantarum slurry.

[0048] If specific experimental steps or conditions are not specified in the examples, the procedures or conditions of conventional experimental steps described in the literature in the field can be followed. All raw materials and instruments used are commercially available, including but not limited to those used in the examples of this application.

[0049] Example 1

[0050] This embodiment provides an oil-in-water double gel loaded with probiotics, and the specific preparation steps are as follows:

[0051] (1) Preparation of aqueous phase: Add 1 g of flaxseed gum powder to 99 g of distilled water and heat in an 85°C water bath for 2 h to completely dissolve the powder to obtain an aqueous phase solution.

[0052] (2) Preparation of oil phase: 1 g of soybean lecithin and 3 g of beeswax were added to 96 g of soybean oil and heated in a 65°C water bath for 20 min to completely dissolve the mixture. When the temperature of the mixture dropped to 37°C, 0.5 g of probiotic slurry was added and mixed uniformly to obtain an oil phase solution.

[0053] (3) Preparation of double gel: The temperature of the aqueous phase solution and the oil phase solution were lowered to 37°C, the aqueous phase solution and the oil phase solution were mixed in a mass ratio of 9:1, stirred evenly, sheared at 10,000 rpm for 3 minutes in a high-speed shearing machine, and cooled to room temperature to obtain a double gel product.

[0054] Example 2

[0055] This embodiment provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are similar to those in Example 1, with the only difference being that in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 7:3.

[0056] Example 3

[0057] This embodiment provides an oil-in-water type oil-water dual gel loaded with probiotics. The specific preparation steps are similar to those in Example 1, with the only difference being that in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0058] Comparative Example 1

[0059] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are referred to Example 1, except that: in step (2), 1g of soybean lecithin is added to 99g of soybean oil, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0060] Comparative Example 2

[0061] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are referred to Example 1, except that: in step (2), 3g of beeswax is added to 97g of soybean oil, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0062] Comparative Example 3

[0063] This comparative example provides a probiotic-loaded water-in-oil double gel. The specific preparation steps refer to Example 1, with the only difference being that in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 3:7.

[0064] Comparative Example 4

[0065] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are referred to Example 1, except that: in step (2), 1g of soybean lecithin and 3g of rice bran wax are added to 96g of soybean oil, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0066] Comparative Example 5

[0067] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are referred to Example 1, except that: in step (2), 1g of soybean lecithin and 3g of candelilla wax are added to 96g of soybean oil, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0068] Comparative Example 6

[0069] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are similar to those in Example 1, except that: in step (1), 1 g of pectin is added to 99 g of distilled water, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0070] Comparative Example 7

[0071] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are similar to those in Example 1, except that: in step (1), 1 g of gum arabic is added to 99 g of distilled water, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0072] Comparative Example 8

[0073] This comparative example provides an oil-in-water type oil-water double gel loaded with probiotics. The specific preparation steps are similar to those in Example 1, except that: in step (1), 1 g of xanthan gum is added to 99 g of distilled water, and in step (3), the aqueous phase solution and the oil phase solution are mixed in a mass ratio of 5:5.

[0074] Experimental Example 1

[0075] This experimental example aims to study the inversion gelling properties of the products of Examples 1 to 3 and Comparative Examples 1 to 3, and to observe the stability of the products prepared in Comparative Examples 4 to 8.

[0076] 7 g of the freshly prepared finished product was placed in a 10 mL container and the lid was tightened. Comparative Examples 4 to 8 were left to stand and their product states were observed. Examples 1 to 3 and Comparative Examples 1 to 3 were placed upside down and their gelling properties were observed.

[0077] Depend on Figure 1 It can be seen that Comparative Examples 1 to 2 cannot be inverted to form a gel, indicating that a single soybean lecithin or beeswax cannot form a stable oil-water double gel. However, in Examples 1 to 3 and Comparative Example 3, adding both to the oil phase can make the oil-water double gel inverted to form a gel, indicating that in the double gel system provided by the present invention, soybean lecithin and beeswax have a synergistic effect on the stability of the system.

[0078] In Comparative Examples 4-5, the beeswax added to the oil phase was replaced with rice bran wax and candelilla wax, respectively. The resulting oil-water bigels were observed to be significantly yellowed, with oil droplets precipitating on the surface, and failed to form a stable bigel system. In Comparative Examples 6-8, the flaxseed gum added to the aqueous phase was replaced with pectin, gum arabic, and xanthan gum, respectively. The results showed that the oil-water bigel prepared in Comparative Example 6 exhibited significant stratification after 2 hours of storage. The stratification in the oil-water bigel prepared in Comparative Example 7 was even more pronounced, with the oil and water phases completely separated. The oil-water bigel prepared in Comparative Example 8 also exhibited significant stratification after 3 hours of storage. This demonstrates that the oil-water bigels prepared using flaxseed gum as the aqueous phase gelling agent and soybean lecithin and beeswax as the oil phase gelling agents exhibited good stability and demonstrated a strong synergistic effect. Substituting other types of gelling agents failed to form a stable bigel system.

[0079] Experimental Example 2

[0080] This experimental example is intended to study the rheological properties of the products of Examples 1 to 3 and Comparative Examples 1 to 3.

[0081] The measurements were performed on a MARS 60 rheometer using a geometric parallel plate (P20, 20 mm). A certain amount of sample was placed on the lower measuring plate, the gap was set to 1 mm, and the test temperature was 25°C. First, the linear viscoelastic region (LVR) of the sample was tested in the strain range between 0.01% and 20% (constant frequency 1 Hz), and the stress was determined to be 1%. Then, a frequency sweep was performed in the range of 0.1-10 Hz under a stress condition of 1%, and the storage modulus (G′) and loss modulus (G”) of the oil-water dual gel were recorded. The apparent viscosity of different oil-water dual gels was measured at a constant frequency of 1 Hz in the shear rate range of 0.1 to 1001 / s.

[0082] Depend on Figure 2 It can be seen that G' of the comparative example and the embodiment is greater than G", indicating that they have similar gel behavior. Compared with comparative examples 1 to 2, embodiments 1 to 3 and comparative example 4 have higher G' and G" values, indicating that their dual gel structures have better rheological behavior.

[0083] Depend on Figure 3 As can be seen, Examples 1-3 and Comparative Example 3 exhibit significant shear-thinning behavior, with lower apparent concentrations of the oil-water bigels associated with faster shear rates. Comparative Examples 1-2 exhibit weaker resistance to shear forces. The combination of soy lecithin and beeswax in the oil phase of Examples 1-3 and Comparative Example 3 results in stronger physical properties and increased stability for the oil-water bigels, demonstrating a synergistic effect between soy lecithin and beeswax.

[0084] Experimental Example 3

[0085] This experimental example is intended to study the thermodynamic properties of the products of Examples 1 to 3 and Comparative Examples 1 to 3.

[0086] Measurements were performed using a differential scanning calorimeter. 3-5 mg of the oil-water dual gel was sealed in a crucible, with an empty crucible used as a control. The thermodynamic curves were measured at a heating rate of 10°C / min over a temperature range of 30-120°C under an inert nitrogen atmosphere.

[0087] Depend on Figure 4 It can be seen that Figure 2 and Figure 3 The results are similar to those of the literature. Single soybean lecithin or beeswax has a lower phase transition temperature, while the phase transition temperature of the oil-water double gel after the two are compounded is significantly increased, which proves that soybean lecithin and beeswax play a synergistic role in the oil gel and improve its thermal stability. Moreover, as the proportion of the oil phase increases, the phase transition temperature gradually increases, which also shows that its structure has strong heat resistance.

[0088] Experimental Example 4

[0089] This experimental example aims to study the encapsulation efficiency of probiotics by the products of Examples 1 to 3 and Comparative Examples 1 to 3.

[0090] The encapsulation efficiency of Lactobacillus plantarum was obtained by measuring the number of probiotics before and after encapsulation, and the encapsulation efficiency was calculated using the following formula:

[0091]

[0092] Depend on Figure 5 As can be seen, the encapsulation efficiency of probiotics in oil-water gels prepared using soy lecithin or beeswax as the oil-phase gelling agent was lower than that of the combined oil-water gels. The encapsulation efficiency gradually increased with increasing the oil-phase ratio in the oil-water gel. This increase in the oil-phase ratio increased the viscosity of the system, which effectively reduced damage to the probiotics during homogenization. However, when the oil-phase ratio increased to 70%, a phase transition occurred, exposing the probiotics in the oil phase to the outer layer. This resulted in greater mechanical damage, freeing more probiotics and significantly reducing the encapsulation efficiency.

[0093] Experimental Example 5

[0094] This experimental example aims to study the thermal stability of the products of Examples 1 to 3 and Comparative Examples 1 to 3 and free probiotics during pasteurization.

[0095] Free probiotics and the products of Examples 1 to 3 and Comparative Examples 1 to 3 were heated in a water bath at 72° C. for 15 seconds under simulated pasteurization conditions. The samples before and after heating were subjected to gradient dilution and Lactobacillus plantarum counts.

[0096] Depend on Figure 6 It can be seen that the thermal stability of the oil-water double gel prepared by using soy lecithin or beeswax alone as the oil phase gelling agent is lower than that of the oil-water double gel prepared after compounding. As the proportion of the oil phase in the oil-water double gel increases, the thermal stability gradually increases. The tight coverage of the oil droplets and the network structure of the hydrogel effectively delay the diffusion of heat. Studies have shown that thermal damage to probiotics is mainly caused by their binding with water molecules. Therefore, the oil-in-water structure can effectively inhibit heat transfer and reduce the loss of probiotics during water evaporation, thereby improving their survival rate. The product obtained in Comparative Example 3 is an oil-in-water double gel. The probiotics in the outer layer are easily affected by heat treatment and inactivated, so the number of live bacteria is significantly reduced compared with Examples 1 to 3.

[0097] Experimental Example 6

[0098] This experimental example is intended to study the storage stability of the products of Examples 1 to 3 and Comparative Examples 1 to 3.

[0099] The products of Examples 1 to 3 and Comparative Examples 1 to 3 were stored at room temperature, and the number of surviving probiotics was measured every week.

[0100] Depend on Figure 7 As can be seen, during the 6-week storage experiment, the colony counts of Comparative Examples 1-2 decreased by 2.18 and 1.99 log CFU / g, respectively, while the colony counts of Examples 1-3 decreased by 1.66, 1.55, and 1.43 log CFU / g, respectively. This indicates that Examples 1-3 better protect the probiotics encapsulated in the dual-gel system than Comparative Examples 1-2. The colony count of Comparative Example 3 decreased by 1.76 log CFU / g during storage. This is because the oil-in-water dual-gel, an oil-in-water dual-gel, provides less protection for the probiotics in the outer layer. As the proportion of the oil phase in the dual-gel increases, the storage stability of Examples 1-3 gradually increases. This is because as the oil phase ratio increases, the system becomes more viscous, thus the encapsulated probiotics are better fixed in the oil-in-water dual gel structure, reducing their migration rate into the water phase and thus increasing their storage time. However, in Comparative Examples 1 and 2, the oil phase contains only soy lecithin or beeswax, which is unable to effectively stabilize the system, resulting in a significant decrease in the colony count over time. The colony count in Comparative Example 3 dropped sharply during storage because when the oil phase ratio increased to 70%, the structure underwent a phase transition, resulting in a water-in-oil dual gel. The outer phase is the oil phase, exposing the probiotics to air and failing to effectively protect them.

[0101] Experimental Example 7

[0102] This experimental example aims to study the microstructure of the product of Example 3.

[0103] The CLSM image of the product of Example 3 was taken using a laser confocal microscope.

[0104] Depend on Figure 8 It can be seen that the structure of the product of Example 3 is an oil-in-water double gel, in which the probiotics are encapsulated in the oil droplets and wrapped by the outer layer of hydrogel, which can effectively improve the survival rate of the probiotics in harsh environments.

[0105] Experimental Example 8

[0106] This experimental example aims to study the release of free fatty acids from the products of Examples 1 to 3 and Comparative Examples 1 to 3 in simulated intestinal digestion.

[0107] The simulated intestinal fluid (pH=7.0) consisted of 2.0 mg / mL pancreatic enzyme, 3.2 mg / mL pancreatic lipase, and 12.0 mg / mL bile salts.

[0108] During the simulated intestinal digestion process, 0.1 M NaOH was added to maintain the pH of the mixture at 7.00 ± 0.02, and the volume of NaOH consumed within 2 h was recorded. The free fatty acid (FFA) release rate was calculated using the following formula:

[0109]

[0110] Among them, V NaOH is the volume of NaOH consumed (L), m NaOH is the molar concentration of NaOH (M), M liqid is the molecular weight of soybean oil (g / mol), w liqid is the initial mass of soybean oil (g).

[0111] Depend on Figure 9 As can be seen, the free fatty acid release rate of the oil-water bigel prepared with soy lecithin or beeswax alone as the oil phase gelling agent increases rapidly in the first 30 minutes of intestinal digestion. This may be because soy lecithin or beeswax alone cannot effectively stabilize the oil-water system, resulting in rapid digestion and poor structural properties, which increases the contact area between the oil phase and lipase. The high free fatty acid release rate of Comparative Example 3 may be due to the fact that the external phase is an oil phase. Unlike Examples 1-3, this is a water-in-oil bigel, which ensures that the oil phase is always in contact with lipase, thereby increasing the free fatty acid release rate.

[0112] Experimental Example 9

[0113] This experimental example aims to study the stability of the products of Examples 1 to 3 and Comparative Examples 1 to 3 during gastrointestinal digestion.

[0114] The simulated gastric fluid (pH=2.0) consisted of 3.2 mg / mL pepsin and 1.0 M hydrochloric acid; the simulated intestinal fluid (pH=7.0) consisted of 2.0 mg / mL pancreatic enzyme, 3.2 mg / mL pancreatic lipase and 12.0 mg / mL bile salts.

[0115] The sample was placed in simulated gastric fluid and incubated at 100 rpm in a 37°C air bath shaker for 2 hours. Samples were taken every half hour to measure the survival rate of the probiotics. After each sampling and gastric digestion, the mixture was placed in ice water and the pH was adjusted to 7.0 to terminate the reaction. The gastric digestion fluid that terminated the reaction was added to the simulated intestinal fluid and continued to incubate at 100 rpm in a 37°C air bath shaker for 2 hours. Samples were taken every half hour to measure the survival rate of the probiotics. After sampling, they were placed in an ice water bath to prevent further reaction.

[0116] Depend on Figure 10It can be seen that in simulated gastric juice digestion, the viable bacterial counts of Comparative Examples 1 to 2 decreased from 7.99±0.03 and 7.95±0.03 log CFU / g to 6.24±0.05 and 6.11±0.13 log CFU / g, respectively. The viable bacterial counts of Examples 1 to 3 and Comparative Example 3 decreased from 8.10±0.01, 8.21±0.01, 8.31±0.02, and 7.97±0.04 log CFU / g to 7.57±0.05, 7.84±0.04, 7.95±0.02, and 6.19±0.10 log CFU / g, respectively, and the free probiotics decreased from 8.00±0.02 log CFU / g to 4.69±0.04 log CFU / g. During the simulated intestinal digestion process, the viable bacterial counts of Comparative Examples 1 to 2 decreased from 6.24±0.05 and 6.11±0.13 log CFU / g to 5.57±0.08 and 5.32±0.23 log CFU / g, respectively. The viable bacterial counts of Examples 1 to 3 and Comparative Example 3 decreased from 7.57±0.05, 7.84±0.04, 7.95±0.02, and 6.19±0.10 log CFU / g to 6.80±0.08, 7.13±0.02, 7.33±0.04, and 4.99±0.11 log CFU / g, respectively, and the free probiotic counts decreased from 4.69±0.04 log CFU / g to 0.62±0.15 log CFU / g.

[0117] Comparative Examples 1 to 3 have a relatively poor protective effect on probiotics during gastrointestinal digestion, while Examples 1 to 3 have a significantly increased probiotic survival rate. The samples of Comparative Examples 1 to 2 gradually disintegrated in gastric juice, while the sample structure of Comparative Example 3 did not disintegrate. However, due to the phase transition, the oil phase was directly exposed to the gastric juice, and the hydrogel network was unable to provide a good protective effect, resulting in a decrease in survival rate. During the simulated intestinal digestion process, the decrease in probiotic content was mainly related to the decomposition and destruction of the oil gel structure by lipase and the erosion of bile acid. Since the oil-water double gel of Comparative Examples 1 to 2 had already disintegrated in gastric juice, it lost its protective effect on probiotics. However, the oil-water double gel of Examples 1 to 3 had a synergistic effect of its oil phase gelling agent, and the system had stronger stability and mechanical strength, ensuring the integrity of the system during the digestion process. Similar to the digestion situation in gastric juice, the probiotic content in Comparative Example 3 decreased the most during intestinal digestion, while in Examples 1 to 3, the decrease in probiotic content gradually decreased as the oil phase ratio increased. This indicates that the compact structure of the bicontinuous phase in Examples 1 to 3 makes the system more stable, delays the erosion of the system by lipase and bile salts, and thus improves the survival rate of probiotics.

[0118] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An oil-in-water double gel loaded with probiotics, characterized in that: Its raw materials include water phase and oil phase, and the mass ratio of the water phase to the oil phase is 5-9:1-5. The aqueous phase comprises: flaxseed gum and a solvent, wherein the mass concentration of the flaxseed gum in the aqueous phase is 0.1% to 1%, and the solvent in the aqueous phase is water; The oil phase comprises: lecithin, beeswax, oil and probiotic mud, wherein the mass concentration of lecithin in the oil phase is 0.1% to 1%, the mass concentration of beeswax is 3% to 10%, the mass concentration of probiotic mud is 0.1% to 3%, and the lecithin is soybean lecithin; The preparation method of the oil-in-water type oil-water dual gel loaded with probiotics comprises the following steps: (1) mixing flaxseed gum and a solvent, heating and dissolving the mixture to obtain an aqueous solution; (2) mixing lecithin, beeswax and oil, heating and dissolving, adding probiotic mud, and mixing uniformly to obtain an oil phase solution; (3) The aqueous phase solution and the oil phase solution are mixed uniformly in proportion, sheared at high speed, and cooled to room temperature to obtain the oil-in-water type oil-water double gel loaded with probiotics.

2. The probiotic-loaded oil-in-water double gel according to claim 1, characterized in that: The mass concentration of flaxseed gum in the aqueous phase is 1%; The mass concentration of lecithin in the oil phase is 1%, the mass concentration of beeswax is 3%, and the mass concentration of probiotic slurry is 0.5%; The mass ratio of the water phase to the oil phase is 9:1, 7:3 or 5:

5.

3. The probiotic-loaded oil-in-water double gel according to claim 1, characterized in that: The probiotic slurry is obtained by activating the probiotics, centrifuging the culture medium after culture, and discarding the supernatant.

4. The probiotic-loaded oil-in-water double gel according to claim 3, characterized in that: The centrifugation conditions were: centrifugation at 3500 rpm for 10 min.

5. The probiotic-loaded oil-in-water double gel according to claim 1, characterized in that: The oil comprises at least one of soybean oil, corn oil, peanut oil, and rapeseed oil; The probiotics in the probiotic slurry include at least one of yeast, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, and Bifidobacterium; In the oil-in-water double gel loaded with probiotics, the loading concentration of probiotics is at least 1×10 6 CFU / g.

6. The method for preparing the oil-in-water type oil-water dual gel loaded with probiotics according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) mixing flaxseed gum and a solvent, heating and dissolving the mixture to obtain an aqueous solution; (2) mixing lecithin, beeswax and oil, heating and dissolving, adding probiotic mud, and mixing uniformly to obtain an oil phase solution; (3) The aqueous phase solution and the oil phase solution are mixed uniformly in proportion, sheared at high speed, and cooled to room temperature to obtain the oil-in-water type oil-water double gel loaded with probiotics.

7. The method for preparing the oil-in-water double gel loaded with probiotics according to claim 6, characterized in that: In step (1), the heating and dissolving conditions are: heating at a temperature of 80 to 85° C. for 2 to 4 hours; In step (2), the heating and dissolving conditions are: heating at 60-65° C. for 15-30 min; In step (3), the temperatures of the aqueous solution and the oily solution during mixing are both 35-40° C.; the high-speed shearing conditions are: shearing at a rotation speed of 2000-13000 rpm for 1-3 min.

8. The method for preparing the oil-in-water double gel loaded with probiotics according to claim 7, characterized in that: In step (1), the heating and dissolving conditions are: heating at 85° C. for 2 h; In step (2), the heating and dissolving conditions are: heating at 65°C for 20 minutes; In step (3), the temperatures of the aqueous phase solution and the oil phase solution during mixing are both 37° C.; the high-speed shearing condition is: shearing at a speed of 10,000 rpm for 3 minutes.

9. Use of the probiotic-loaded oil-in-water bigel according to any one of claims 1 to 5 or the probiotic-loaded oil-in-water bigel obtained by the preparation method according to any one of claims 6 to 8 in the preparation of foods, health products, feeds, medicines, and cosmetics.

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