Double emulsion gel beads embedding beta-carotene and probiotic bacteria and methods of making the same

Abstract

The application relates to the embedding technical field, and particularly discloses a double emulsion gel bead embedding beta-carotene and probiotics and a preparation method thereof. The double emulsion gel bead comprises a composite layer, an oil phase and an inner water phase from outside to inside. The composite layer comprises an outer water phase and calcium alginate which are fused with each other. The outer water phase comprises a product after carrageenan and sodium caseinate are subjected to a Maillard reaction. The oil phase comprises beta-carotene, vegetable oil and a food emulsifier. The inner water phase comprises probiotics. In the gel bead, the beta-carotene and the probiotics are simultaneously embedded in the double emulsion gel bead, the calcium alginate hydrogel shell starts to decompose when entering the intestinal tract, then the double-layer emulsion is slowly decomposed after being subjected to chemical digestion of pancreatic juice, bile and small intestinal juice and mechanical digestion of the small intestine in the small intestine, and the beta-carotene and the probiotics are gradually released, the beta-carotene is converted into vitamin A in the intestinal tract and is absorbed, and the probiotics are delivered to the colon to play a probiotic effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of embedding technology, more particularly, to a double emulsion gel bead embedding β-carotene and probiotics and a preparation method thereof. BACKGROUND

[0002] Probiotics have beneficial effects such as treating diarrhea and improving intestinal flora, and are widely used in food. Probiotic products need to reach a concentration of at least 10 6 CFU / mL to exert probiotic effects. However, probiotics have poor environmental resistance, and probiotics are affected by external environments such as pH, oxygen content, temperature, and gastric acid and bile salts in the human digestive tract, resulting in a substantial reduction in the number of viable bacteria and a loss of probiotic effects. Therefore, it is of great significance to establish a stable probiotic protection system.

[0003] β-carotene in carotenoids is a fat-soluble natural active ingredient that can be converted into vitamin A in small intestine and liver cells. Its nutritional value and disease prevention effects are increasingly attracting attention. β-carotene can be oxidized by oxygen and its nutritional value can be reduced by gastric acid. β-carotene has a highly hydrophobic skeleton and is difficult to disperse in the body fluid environment of the small intestine, which reduces its absorption rate.

[0004] β-carotene and probiotics complement each other in taste and are easily combined to produce a better mouthfeel. This combination can also provide a higher level of nutritional value to the human body, for example, probiotics can help the human body better absorb β-carotene.

[0005] In order to ensure the survival rate of probiotics and their colonization and release in the human intestinal tract, embedding technology is one of the most effective methods. Embedding technology uses physical and chemical means to encapsulate the bacterial body, isolates the bacterial body from the outside world, and reduces the impact of adverse environments and other related factors on the bacterial body. It is an effective method for maintaining the activity and stability of the bacterial body and exerting probiotic effects.

[0006] Traditional probiotic embedding technologies mainly include extrusion, freeze-drying, and spray drying. These technologies are very mature and widely used, but there are some significant defects in their use that cannot meet the requirements of bacterial embedding. For example, the extrusion method produces microcapsules with large particle sizes and poor uniformity, and the formation of microbeads is slow and the yield is low, making it difficult to use for large-scale production. Vacuum freeze-drying method inevitably causes mechanical damage to cell membranes, cell walls, solutes, and other factors during the freeze-drying process, and also affects cell metabolism regulation, resulting in cell death and reduced survival rate. The spray drying method exposes the bacterial body to a high-temperature environment for a short time during the preparation process, which easily damages the cell wall and cell membrane, and has a negative impact on the structural integrity of live bacterial cells, subsequent processing and processing, and probiotic effects.

[0007] At present, there are some products using gelatin and β-carotene as raw materials to improve the absorption rate of β-carotene by means of processes such as dissolution, rotary evaporation, mixing and shearing, high-pressure homogenization, and spray drying, but there is still a lack of a scheme that can embed probiotics and β-carotene at the same time. SUMMARY

[0008] In view of the lack of edible products and preparation methods that can embed probiotics and β-carotene at the same time, the present application proposes a double emulsion gel bead embedding β-carotene and probiotics and a preparation method thereof.

[0009] In the first aspect, the present application proposes a double emulsion gel bead embedding β-carotene and probiotics, and adopts the following technical scheme.

[0010] A double emulsion gel bead embedding β-carotene and probiotics comprises a composite layer, an oil phase, and an inner water phase from outside to inside. The composite layer comprises an outer water phase and calcium alginate that are fused with each other; the outer water phase comprises a product after carrageenan and sodium caseinate undergo a Maillard reaction. The oil phase comprises β-carotene, vegetable oil, and a food emulsifier. The inner water phase comprises probiotics.

[0011] By adopting the above technical scheme, β-carotene and probiotics are embedded in the double emulsion gel bead at the same time. The calcium alginate hydrogel shell starts to decompose as soon as it enters the intestine, then the double emulsion (the composite layer wraps the oil phase, and the oil phase wraps the inner water phase to form a double emulsion) is slowly decomposed in the small intestine after being subjected to chemical digestion by pancreatic juice, bile, and small intestinal juice and mechanical digestion by the small intestine, while β-carotene and probiotics are gradually released. The vegetable oil and the food emulsifier promote the dissolution of β-carotene in the intestinal juice. β-carotene will be converted into vitamin A in the intestine and be absorbed. Probiotics will be delivered to the colon to exert a probiotic effect. The probiotics can be Lactobacillus, Bifidobacterium, and some gram-positive bacteria, etc.

[0012] As an improvement of the double emulsion gel bead embedding β-carotene and probiotics, in the outer water phase, the mass ratio of the sodium caseinate and the carrageenan put in is (0.4-2.5):1.

[0013] By adopting the above technical scheme, the grafting degree of sodium caseinate and carrageenan is high, the emulsification effect is good, and it is easy to disperse, which is conducive to the formation of gel beads with small and uniform particle sizes.

[0014] As a further improvement of the double emulsion gel bead embedding β-carotene and probiotics, in the outer water phase, the mass ratio of the sodium caseinate and the carrageenan put in is (1.8-2.2):1.

[0015] By adopting the technical scheme, the grafting degree of sodium caseinate and carrageenan is high, the emulsification effect is good, it is beneficial to be combined with calcium alginate to be wrapped outside the oil phase, there is a difference between the external water phase and the oil phase in the solubility of beta-carotene, and the external water phase can promote beta-carotene to be protected by being transferred into the oil phase.

[0016] Optionally, the vegetable oil is soybean oil, and the food emulsifier is polyglycerol ricinoleate; the mass of the polyglycerol ricinoleate relative to the volume of the soybean oil is 2-4 g / 100 mL. Beta-carotene is easily dissolved in soybean oil, and polyglycerol ricinoleate promotes the emulsification between soybean oil and intestinal juice, so that beta-carotene is easily dispersed in intestinal juice and is converted and absorbed.

[0017] As an improvement of the double emulsion gel bead embedding beta-carotene and probiotics, the internal water phase includes bacteria liquid, and the bacteria liquid includes the probiotics. The internal water phase further includes a probiotic protective agent; the probiotic protective agent includes skimmed milk powder, inulin, and glycerol; the mass of the skimmed milk powder is 5-10% of the mass of the bacteria liquid; the mass of the inulin is 5-10% of the mass of the bacteria liquid; and the mass of the glycerol is 1-3% of the mass of the bacteria liquid.

[0018] By adopting the technical scheme, the skimmed milk powder can form a protective film outside the bacteria, which blocks the harsh external environment; the inulin can promote the growth and reproduction of intestinal beneficial bacteria in the body; and the glycerol can reduce the damage of active substances in the bacteria under freeze-drying conditions in the preparation and preservation process of the gel bead.

[0019] In a second aspect, the application further provides a preparation method of a double emulsion gel bead embedding beta-carotene and probiotics, and the following technical scheme is adopted.

[0020] The preparation method of the double emulsion gel bead embedding beta-carotene and probiotics includes:

[0021] Preparation of a primary emulsion: mixing a probiotic bacteria liquid and a probiotic protective agent to obtain an internal water phase; dispersing the internal water phase in an oil phase to obtain the primary emulsion in which the oil phase wraps the internal water phase;

[0022] Preparation of an external water phase: mixing a product after Maillard reaction of carrageenan and sodium caseinate and water to obtain the external water phase;

[0023] Preparation of a secondary emulsion: dispersing the primary emulsion in the external water phase, and adding beta-carotene to obtain the secondary emulsion;

[0024] Preparation of the gel beads: the sodium alginate and the secondary emulsion are mixed to obtain a mixed emulsion, and the mixed emulsion is added into a calcium ion solution by dropwise addition or microfluidic technology to obtain the gel beads.

[0025] By adopting the technical scheme, the inner water phase is dispersed in the oil phase to obtain a primary emulsion; the primary emulsion is dispersed in the outer water phase to obtain a secondary emulsion; and the mixed emulsion is added into a calcium ion solution by dropwise addition or microfluidic technology, so that the gel speed is fast, the gel beads can be quickly obtained, the gel beads are small and have uniform particle sizes. In the gel beads, the inner water phase is a concentrated bacterial solution, the oil phase wraps the inner water phase to enable the probiotic bacteria to survive in a micro-water environment, the outer water phase can make the emulsion more stable, and the beta-carotene is wrapped in the oil phase to realize the co-embedding of the probiotic bacteria and the fat-soluble natural active ingredient beta-carotene. In the process of preparing the secondary emulsion, the beta-carotene is transferred from the outer water phase to the oil phase. The present scheme does not adopt a mode of directly adding the beta-carotene into the oil phase, because if the beta-carotene is directly added into the primary emulsion and dissolved in the oil phase, the oil phase is exposed to the air, so that the beta-carotene is easily oxidized by contacting the air. Therefore, the outer water phase is wrapped around the oil phase to protect the beta-carotene, and then the beta-carotene is added into the outer water phase to be introduced into the oil phase.

[0026] The process of preparing the double-emulsion gel beads does not need freezing or opening a vacuum, and the probiotic bacteria are basically not damaged. The processes of preparing the primary emulsion, preparing the secondary emulsion and preparing the gel beads are directly related to the probiotic bacteria, and are all carried out at normal temperature and pressure, for example, at an air pressure of 0.9-1.1 atm and a temperature of 5-45 ℃, and more preferably at a temperature of 15-38 ℃. The above processes do not need heating, and the survival rate of the probiotic bacteria is high. The process of preparing the outer water phase can be heated, but the process of dispersing the primary emulsion in the outer water phase can be carried out after the outer water phase is cooled to normal temperature. Therefore, the probiotic bacteria do not directly contact high-temperature substances, are basically not damaged, have an intact structure and a high survival rate.

[0027] When the outer water phase is prepared, the content of hydroxyl groups in the protein molecules (sodium caseinate) increases after the sodium caseinate and carrageenan are covalently grafted, and the content of amino groups decreases and the molecular weight increases during the glycosylation reaction. The protein-polymer covalent complex prepared by the Maillard reaction has higher solubility and emulsification than the raw materials.

[0028] It should be noted that the order of preparing the primary emulsion and preparing the outer water phase can be arbitrary, such as simultaneous preparation.

[0029] As an improvement of the preparation method of the double emulsion gel beads embedding beta-carotene and probiotics, the dispersing of the inner water phase in the oil phase to obtain the primary emulsion wrapped by the oil phase from the inner water phase specifically comprises: mixing the inner water phase and the oil phase at a volume ratio of 2:(7~9) and homogenizing at a speed of 10000~20000 rpm for 5~7 min to obtain the primary emulsion.

[0030] By adopting the above technical scheme, the inner water phase can be fully fine, and one or more uniform fine liquid particles with a particle size of about 1~5 μm are dispersed in the oil phase and wrapped by the oil phase to form the primary emulsion.

[0031] As an improvement of the preparation method of the double emulsion gel beads embedding beta-carotene and probiotics, the preparation of the outer water phase specifically comprises: dissolving sodium caseinate and carrageenan in water at a mass ratio of (2 ± 0.2):1, then freeze-drying, grinding into powder, and placing the powder in a saturated potassium bromide solution, controlling the solution temperature to be 50~70 ℃, and reacting for 16~32 h, then drying and cooling to obtain dry powder, and mixing the dry powder with water to obtain the outer water phase.

[0032] By adopting the above technical scheme, the grafting degree of carrageenan and sodium caseinate is high, the outer water phase has high viscosity and is uniform and stable, and the emulsification effect is good.

[0033] As an improvement of the preparation method of the double emulsion gel beads embedding beta-carotene and probiotics, in the preparation of the secondary emulsion, the volume ratio of the added primary emulsion and the outer water phase is 1:(1.4~1.6).

[0034] By adopting the above technical scheme, the volume of the outer water phase is about half more than that of the primary emulsion, forming a complex of the outer water phase wrapping the primary emulsion. If the outer water phase is too much, more materials are consumed, and it is also not conducive to the release of the oil phase and the inner water phase in the intestinal tract. If the outer water phase is too little, it is easy to cause incomplete wrapping, affecting the protection effect.

[0035] As an improvement of the preparation method of the double emulsion gel beads embedding beta-carotene and probiotics, the preparation of the secondary emulsion specifically comprises: adding the primary emulsion to the outer water phase, adding beta-carotene, and the addition ratio of the beta-carotene to the primary emulsion is (4~6) mg / mL, and then homogenizing to obtain the secondary emulsion.

[0036] By adopting the above technical scheme, a complex of the outer water phase wrapping the primary emulsion is formed, the beta-carotene is transferred from the outer water phase into the oil phase outside the primary emulsion, and is protected and conducive to the conversion and absorption in the intestinal tract.

[0037] As an improvement of the preparation method of the double emulsion gel beads embedding beta-carotene and probiotics, the preparation of the gel beads specifically comprises: uniformly mixing a 1.5-2.5 wt% sodium alginate aqueous solution and the secondary emulsion at a volume ratio of 1: (0.9-1.1) to obtain a mixed emulsion, and adding the mixed emulsion into a 4-6 wt% calcium chloride solution by dropwise addition or a microfluidic process to obtain the gel beads.

[0038] By adopting the technical solution, during the dropwise addition or the microfluidic process, sodium alginate and calcium chloride react to generate calcium alginate which is insoluble in water, and the size of the droplets formed by the dropwise addition or the microfluidic process is uniform, and since the size of the droplets basically determines the particle size of the gel beads, the gel beads with uniform particle size are formed,

[0039] In summary, the double emulsion gel beads embedding beta-carotene and probiotics and the preparation method thereof have the following beneficial effects:

[0040] The inner water phase is a concentrated bacterial solution, the oil phase wraps the inner water phase to enable the probiotics to survive in a micro-water environment, the outer water phase can make the emulsion more stable, and the beta-carotene is wrapped in the oil phase to achieve the co-embedding of the probiotics and the fat-soluble natural active ingredient. The beta-carotene is easier to store after being embedded in the emulsion.

[0041] After taking the gel beads, the alginate hydrogel shell begins to decompose as soon as it enters the intestine; then, the double emulsion is slowly decomposed after being subjected to chemical digestion by pancreatic juice, bile and small intestinal juice and mechanical digestion by the small intestine, and the beta-carotene and the probiotics are gradually released, the beta-carotene is converted into vitamin A in the intestine, and the probiotics are delivered to the colon.

[0042] The double emulsion gel system can embed the probiotics and the fat-soluble natural active ingredient together in the double emulsion gel system to achieve a slow-release effect in the gastrointestinal tract, and has a good market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 A particle size and PDI comparison chart of the gel beads prepared in Examples 1-3 for Test Example 1.

[0044] Figure 2 A grafting degree comparison chart of the reaction product of carrageenan and sodium caseinate prepared in Examples 1-3 for Test Example 2.

[0045] Figure 3 An electrophoresis comparison chart of sodium caseinate, a physical mixture of unreacted carrageenan and sodium caseinate, and a reaction product of carrageenan and sodium caseinate for Test Example 3.

[0046] Figure 4W1 / O / W2 double emulsion particle size distribution chart for Test Example 4.

[0047] Figure 5 Optical microscope image of W1 / O / W2 double emulsion for Test Example 5.

[0048] Figure 6 Laser confocal microscope image of W1 / O / W2 double emulsion for Test Example 6.

[0049] Figure 7 Swelling rate of gel beads at different pH for Test Example 7.

[0050] Figure 8 SEM image of gel beads for Test Example 8.

[0051] Figure 9 Release rate chart of β-carotene in in-vitro gastrointestinal simulation experiment for Test Example 9.

[0052] Figure 10 Release amount chart of probiotic bacteria in in-vitro gastrointestinal simulation experiment for Test Example 9. DETAILED DESCRIPTION

[0053] Some embodiments of the double emulsion gel beads embedding β-carotene and probiotic bacteria and the preparation method thereof are described in detail as follows. Example 1

[0054] Preparation of primary emulsion: First, the inner water phase (W1) is prepared by mixing the lactic acid bacteria liquid with the lactic acid bacteria protective agent. The lactic acid bacteria protective agent includes skimmed milk powder, inulin and glycerol, the proportion of skimmed milk powder is 7.5wt% relative to the pure lactic acid bacteria liquid, the proportion of inulin is 7.5wt% relative to the pure lactic acid bacteria liquid, and the proportion of glycerol is 2wt% relative to the pure lactic acid bacteria liquid. In this embodiment, 20 g (about 20 mL) of lactic acid bacteria liquid, 1.5 g of skimmed milk powder, 1.5 g of inulin and 0.4 g of glycerol are put in. The oil phase (O) is prepared by dissolving 3 g of PGPR (polyglyceryl ricinoleate) in 100 mL of soybean oil, and the addition ratio of the mass of PGPR to the volume of soybean oil is 3 g / 100 mL. Then, the W1 phase (20 g) and the O phase (80 g) mixed in a volume ratio of 2:8 are homogenized using an Ultra-Turrax homogenizer at a speed of 15,000 rpm at 25 ℃ for 6 min to obtain the primary emulsion (W1 / O).

[0055] Preparation of the external water phase: 10 g of sodium caseinate and carrageenan with a mass ratio of 1 / 2 were completely dissolved in 500 mL of distilled water, and then the mixture was freeze-dried for 48 h. Subsequently, the sample was ground into powder and sieved (80 mesh). Then, the Maillard reaction was carried out using a dry heating method. 10 g of the powder was placed in a reaction container containing 500 mL of saturated potassium bromide solution, which can maintain the relative humidity of the environment at about 79%, and the pH of the solution is about 7.4. The temperature of the mixture was controlled at 60°C, and the reaction was terminated after 24 h of cooling. The sodium caseinate and carrageenan graft product was obtained, and the dry powder was obtained after drying, which was the external emulsifier. The dry powder obtained by the Maillard reaction of carrageenan and sodium caseinate was dispersed in distilled water at a proportion of 30wt% to form the external water phase (W2).

[0056] Preparation of the secondary emulsion: 20 mL of the primary emulsion (W1 / O) was added to 30 mL of the external water phase (W2), and 100 mg of β-carotene was added. The solution was mixed uniformly and homogenized using a homogenizer at a speed of 11,000 rpm at 25°C for 3 min to obtain a secondary emulsion (W1 / O / W2 double emulsion). The obtained W1 / O / W2 double emulsion was stored at a temperature of 25°C.

[0057] Preparation of the gel beads: sodium alginate was dissolved in water to obtain a sodium alginate solution with a concentration of 2wt%. The sodium alginate solution was mixed with the secondary emulsion prepared above at a volume ratio of 1:1 to obtain a mixed emulsion. The mixed emulsion was added dropwise to a 5wt% calcium chloride solution to form gel beads dispersed in water. Example 2

[0058] This example uses a technical solution basically the same as that of Example 1, the only difference being that in the preparation of the external water phase step: the mass ratio of sodium caseinate to carrageenan is 1 / 1, and the total amount is 10 g.

[0059] Example 2 ultimately also prepared a kind of gel beads. Example 3

[0060] This example uses a technical solution basically the same as that of Example 1, the only difference being that in the preparation of the external water phase step: the mass ratio of sodium caseinate to carrageenan is 2 / 1, and the total amount is 10 g.

[0061] Example 3 ultimately also prepared a kind of gel beads. Example 4

[0062] The embodiment adopts the same technical scheme as that of Embodiment 3, the only difference being that the lactic acid bacteria protective agent comprises skimmed milk powder, inulin and glycerol, the proportion of the skimmed milk powder relative to the pure lactic acid bacteria liquid is 10wt%, the proportion of the inulin relative to the pure lactic acid bacteria liquid is 10wt%, and the proportion of the glycerol relative to the pure lactic acid bacteria liquid is 3wt%. In this embodiment, 20 g (about 20 mL) of the lactic acid bacteria liquid, 2 g of the skimmed milk powder, 2 g of the inulin and 0.6 g of the glycerol are put in.

[0063] Embodiment 4 finally also prepares a gel bead. Embodiment 5

[0064] The embodiment adopts the same technical scheme as that of Embodiment 3, the only difference being that the lactic acid bacteria protective agent comprises skimmed milk powder, inulin and glycerol, the proportion of the skimmed milk powder relative to the pure lactic acid bacteria liquid is 5wt%, the proportion of the inulin relative to the pure lactic acid bacteria liquid is 5wt%, and the proportion of the glycerol relative to the pure lactic acid bacteria liquid is 1wt%. In this embodiment, 20 g (about 20 mL) of the lactic acid bacteria liquid, 1 g of the skimmed milk powder, 1 g of the inulin and 0.2 g of the glycerol are put in.

[0065] Embodiment 5 finally also prepares a gel bead.

[0066] Embodiments 4-5 and Embodiment 3 have similar results, a film layer is formed on the surface of the lactic acid bacteria liquid, which can effectively protect the lactic acid bacteria.

[0067] Comparative Example 1

[0068] The comparative example adopts the same technical scheme as that of Embodiment 3, the only difference being that in the preparation of the outer water phase, the weight ratio of carrageenan to sodium caseinate is 1 / 9, and the total amount is 10 g. The obtained outer water phase is relatively thin, and the wrapping effect on the oil phase is poor. Finally, the gel bead prepared is not spherical in shape and is irregular in shape. This is because the sodium caseinate is excessive, and more sodium caseinate is not grafted, resulting in a relatively thin outer water phase and poor emulsification effect.

[0069] Comparative Example 2

[0070] The comparative example adopts the same technical scheme as that of Embodiment 3, the only difference being that in the preparation of the outer water phase, the weight ratio of carrageenan to sodium caseinate is 9 / 1, and the total amount is 10 g. The comparative example finally also prepares a gel bead. The obtained outer water phase is too viscous, which is not conducive to the transfer of β-carotene to the oil phase, and the gel bead obtained is not spherical in shape and is irregular in shape. This is because the carrageenan is excessive, which makes the outer water phase too viscous.

[0071] Comparative Example 3

[0072] The comparative example adopts the basically same technical scheme as example 3, the only difference is that when preparing the primary emulsion, W1 phase (2 g) is mixed with O phase (20 g) at a volume ratio of 2:20 and homogenized to obtain the primary emulsion (W1 / O). When preparing the secondary emulsion, 20 mL of the primary emulsion (W1 / O) is added to 30 mL of the external water phase (W2), 100 mg of β-carotene is added, the solution is mixed uniformly and homogenized using a homogenizer at 25°C at a speed of 11,000 rpm for 3 min to obtain the secondary emulsion (W1 / O / W2 double emulsion), in which the size of the internal W1 / O layer is uneven, the particle size is uneven, and the size of the finally prepared gel beads is uneven, which is caused by the over-thick O phase layer.

[0073] Comparative example 4

[0074] The comparative example adopts the basically same technical scheme as example 3, the only difference is that when preparing the secondary emulsion, the volume ratio of the primary emulsion and the external water phase is 1:3, specifically 20 mL of the primary emulsion (W1 / O) is added to 60 mL of the external water phase (W2), and the finally prepared gel beads are irregular in shape, not spherical, and uneven in size.

[0075] Comparative example 5

[0076] The comparative example adopts the basically same technical scheme as example 3, the only difference is that when preparing the secondary emulsion, the addition ratio of β-carotene to the primary emulsion is 10 mg / mL, specifically when preparing the secondary emulsion, 20 mL of the primary emulsion (W1 / O) is added to 30 mL of the external water phase (W2), 200 mg of β-carotene is added, the solution is mixed uniformly and homogenized using a homogenizer at 25°C at a speed of 11,000 rpm for 3 min to obtain the secondary emulsion (W1 / O / W2 double emulsion). Through color tracking and laser confocal microscope images, it is shown that the external water phase (W2) layer contains more β-carotene, indicating that β-carotene is not completely dissolved into the oil phase, and β-carotene is prone to oxidation and deterioration.

[0077] Comparative example 6

[0078] This comparative example uses essentially the same technical solution as Example 3, the only difference being that in preparing the gel beads, sodium alginate was dissolved in water to achieve a sodium alginate concentration of 2 wt%. This sodium alginate aqueous solution was mixed evenly with the prepared secondary emulsion at a volume ratio of 1:2 to obtain a mixed emulsion. This mixed emulsion was then added dropwise to a 5 wt% calcium chloride solution to form gel beads dispersed in water. These gel beads were irregular in shape, not spherical, and relatively loose. In contrast, the gel beads prepared in Example 3 were regularly spherical. The difference lies in the different volume ratio of sodium alginate aqueous solution to the secondary emulsion; Comparative Example 6 added too much secondary emulsion, resulting in poor gel bead formation.

[0079] Experimental Example 1

[0080] The reaction products generated by Maillard grafting of carrageenan (κ-Car) and sodium caseinate (NaCas) in different proportions in Examples 1-3 were screened according to the following method:

[0081] Particle size and PDI

[0082] The PDI (Polymer Dispersibility Index) and particle size of fresh suspensions of NaCas-κCar grafts prepared in water at different proportions were determined using a Nano ZS90 particle size analyzer (Brookhaven Instruments, Holtsville, UK). The results are as follows: Figure 1 As shown in the statistical data P <0.05. Example 1 used sodium caseinate and carrageenan at a mass ratio of 1 / 2 to prepare gel beads with particle size and PDI as shown below. Figure 1 The bar chart marked "1:2" shows a particle size of 525 nm and a PDI of 0.37. Example 2 used sodium caseinate and carrageenan at a mass ratio of 1:1, and the resulting gel beads had the following particle size and PDI: Figure 1 The bar chart marked "1:1" shows a particle size of 470 nm and a PDI of 0.33. Example 3 used sodium caseinate to carrageenan at a mass ratio of 2:1, and the resulting gel beads had the following particle size and PDI: Figure 1 The bar chart marked "2:1" shows a particle size of 370 nm and a PDI of 0.22. It can be seen that within the ratio range of Examples 1-3, as the proportion of sodium caseinate increases, the particle size of the gel beads decreases, and the dispersion index decreases. A smaller dispersion index indicates more uniform particle size, and a smaller dispersion coefficient indicates a more uniform and stable solution.

[0083] Experimental Example 2

[0084] Determination of grafting degree of carrageenan-sodium caseinate reaction product by OPA method:

[0085] Preparation of OPA (o-phthaldialdehyde) reagent: 40 mg of OPA was added into 1 mL of methanol, and then 25 mL (0.1 mol / L) of borax, 2.5 mL of 20 wt% SDS (sodium dodecyl sulfate) solution and 100 μL of mercaptoethanol were added, and the mixture was mixed thoroughly, and then the volume was made up to 50 mL to obtain the OPA reagent.

[0086] The reaction products (glycosylation products) of carrageenan and sodium caseinate prepared in Examples 1-3 were dissolved in phosphate buffer at pH 7.0, and the same mass of each was added. After centrifugation at 7000 r / min for 10 min, 200 μL of the supernatant was added to 4 mL of OPA reagent, and the mixture was mixed thoroughly and then placed in a water bath at 35 °C for 5 min in the dark. The absorbance was measured at 340 nm, and the calculation formula was as follows:

[0087] Drafting degree (%) = (A0-A1) / A1*100%

[0088] In the formula, A0 is the absorbance of the physical mixture of unreacted sodium caseinate and carrageenan, and A1 is the absorbance of the glycosylation product.

[0089] Figure 2 The grafting degree of the reaction product of sodium caseinate and carrageenan prepared in Examples 1-3 was 73.45%, which was the highest when the ratio of sodium caseinate to carrageenan was 2 / 1. Generally, the higher the grafting degree, the better the emulsifying effect. Therefore, the ratio of 2 / 1 of sodium caseinate to carrageenan was selected as the external aqueous phase, which had good emulsifying effect.

[0090] Test Example 3

[0091] SDS-PAGE (electrophoresis)

[0092] The conditions of the separation gel and the concentration gel for electrophoresis were as follows: the concentration of the separation gel was 12%, the concentration of the concentration gel was 5%, the protein concentration was 1 mg / mL, the electrophoresis sample volume was 12 μL, the electrophoresis voltage was 120 V, and the electrophoresis was stopped when the dye front was 1 cm from the bottom edge of the rubber frame.

[0093] After being fixed in the fixing solution for 30 min, the gel was stained with Coomassie brilliant blue R250 for 1 h and then decolorized until the bands were clearly visible. The gel was scanned using a chemiluminescence / fluorescence gel imaging system (G:BOX Chemi XT4, UK).

[0094] The samples were sodium caseinate (NaCas), the physical mixture of unreacted sodium caseinate and carrageenan (W1 / 2, W1 / 1, W2 / 1) and the reaction products (F1 / 2, F1 / 1, F2 / 1), respectively.

[0095] Figure 3 Figure 2 is SDS-PAGE diagram of carrageenan before and after reacting with sodium caseinate, the molecular weight of the reaction product is larger than that of the physical mixture, which can prove that carrageenan is successfully grafted with sodium caseinate.

[0096] Test Example 4

[0097] The W1 / O / W2 double emulsion prepared in Example 3 was characterized in terms of physicochemical properties and microstructure according to the following methods.

[0098] Emulsion particle size

[0099] The emulsion particle size was determined using a laser particle size analyzer, with distilled water as the dispersant. A small amount of sample (previously diluted 1:1 with distilled water) was injected into the flow system with a pipette, dispersed in distilled water at 2000 rpm, and a 6% ~ 10% obscuration was generated. The experiment used d 4,3 , which is the volume average diameter representing the size of the droplet particle size. Each sample was measured 3 times, and the average value was taken.

[0100] Figure 4 Figure 4 is a W1 / O / W2 double emulsion particle size distribution diagram, which can be seen that the particle size is distributed most widely around 10 μm, with small particle size and uniform distribution.

[0101] Test Example 5

[0102] The microstructure of the W1 / O / W2 double emulsion prepared in Example 3 was evaluated by optical microscopy. 10 μL of W1 / O / W2 double emulsion was dropped onto a glass slide, and after covering with a cover glass, it was placed on the stage. The size, shape and distribution of the emulsion droplets after magnification were observed and recorded by a computer-installed camera. Figure 5 Figure 5 is an image of W1 / O / W2 under an optical microscope, which can be directly seen that the structure of water-in-oil-in-water, proving that the W1 / O / W2 double emulsion is successfully prepared.

[0103] Test Example 6

[0104] The image of the W1 / O / W2 double emulsion prepared in Example 3 was collected by laser scanning confocal microscopy. 40 μL of mixed staining solution (containing 0.1% Nile blue and 0.1% Nile red) was added to 1 mL of W1 / O / W2 double emulsion, the excitation wavelengths of Nile red and Nile blue were 488 nm and 633 nm respectively, after complete staining, a drop of emulsion was prepared and placed on the stage, and CLSM (laser scanning confocal microscopy) image collection was performed using laser confocal microscope LSM 980 (Carl Zeiss LSM980, Germany). Figure 6are three images of W1 / O / W2 under laser confocal microscope, including left, right and bottom three subgraphs. In the color background of the left subgraph, the structure of the outer water phase is highlighted, the right subgraph highlights the structure of the primary oil phase, and the bottom subgraph highlights the structure of the W1 / O / W2 double emulsion. From Figure 6 The three subgraphs can be seen directly from the water-in-oil-in-water structure, which proves that the W1 / O / W2 double emulsion is successfully prepared.

[0105] Test Example 7

[0106] The double emulsion coated gel beads prepared in Example 3 were characterized according to the following method

[0107] Gel bead swelling experiment

[0108] Under different pH conditions, the swelling behavior of the gel beads was evaluated by the swelling rate. The appropriate amount of freeze-dried beads was incubated in PBS solution (phosphate buffered saline) (pH 3.0 or pH 7.4) at 37°C. At a certain time point, the swollen beads were taken out, the surface liquid was removed with filter paper, and then weighed. The calculation formula of the swelling ratio is as follows:

[0109] Swelling rate = (M1-M2) / M2*100%

[0110] In the formula, M1 refers to the mass of the swollen gel beads; M2 refers to the mass of the freeze-dried gel beads before swelling.

[0111] Figure 7 is the swelling rate under different pH conditions, it can be seen that the swelling rate of the gel beads under acidic conditions at pH 3.0 is much lower than that under weak alkaline environment at pH 7.4, because calcium ions in the weak alkaline environment can react with OH - , the internal network structure is loose, and it swells to a large extent. In the gel beads of the present application, the cross-linked network structure of the outer calcium alginate gel shell fills the micropore gaps of the shell with the outer water phase, making the internal structure more dense, and the shell will swell slightly under gastric acid conditions without dissolving, and the hydrogel beads can maintain the initial form.

[0112] Test Example 8

[0113] SEM image of the gel beads prepared in Example 3

[0114] The internal structure of the freeze-dried gel beads was observed by scanning electron microscope. After the sample was sprayed with gold, it was observed using a field emission scanning electron microscope (Hitachi SU1510, Japan). Figure 8 are two SEM images of freeze-dried gel beads at different magnifications, it can be seen that the internal structure of the gel beads is a dense network cross-linked structure, which is suitable for embedding probiotics as a carrier.

[0115] Test Example 9

[0116] In vitro simulated release experiment of the gel beads prepared in Example 3

[0117] Simulated gastric fluid (SGF) was prepared with 85 mM sodium chloride and 3 g / L pepsin, and then the pH was adjusted to 2.5 with hydrochloric acid. The gel beads were soaked in the SGF at 37 ℃ for 2 h. Subsequently, simulated intestinal fluid (SIF) containing 85 mM sodium chloride, 1 g / L bile salts, and 1 g / L trypsin was prepared, and then the pH was adjusted to 7.0, and the gel beads were soaked at 37 ℃ for 5 h. A small amount of solution was taken every hour for determination to determine the release amount of β-carotene and probiotics.

[0118] The probiotics were determined by plate counting method. The probiotics used in this test example were lactic acid bacteria LGG.

[0119] The detection method of β-carotene was as follows: a β-carotene standard sample was accurately weighed, dissolved in n-hexane, and diluted to a constant volume in a volumetric flask to prepare a standard solution with gradient concentrations. The absorbance at 450 nm was determined by a UV-visible spectrophotometer, and a standard curve was prepared. 1 mL of the solution to be determined was taken, and ethanol and n-hexane (1:2, v / v) were added. The extraction was repeated 3 times, and the upper layer extract was combined. The absorbance at 450 nm was determined. The content of β-carotene in the solution was calculated according to the standard curve of carotene.

[0120] Figure 9 refers to a β-carotene release rate graph in the in vitro gastrointestinal simulation experiment, Figure 10 refers to a probiotic release amount graph in the in vitro gastrointestinal simulation experiment. After the in vitro simulation of the stomach and intestine digestion, the release rate of β-carotene reached 89.81%, and the amount of probiotics embedded in the double-emulsion gel beads could reach 1.53×10 8 CFU / mL, which proved that the double-emulsion hydrogel bead structure had good protection effect on natural active ingredients and probiotics and good gastrointestinal sustained-release effect.

[0121] In the gel beads of the present embodiment, the probiotics survive in the inner water phase, and the oil phase wraps the inner water phase to embed the probiotics in a micro-aqueous environment. When the outer water phase wraps the oil phase, the β-carotene is embedded in the oil phase due to its liposoluble property, thereby achieving the dual protection of probiotics and liposoluble natural active ingredients.

[0122] The above is only a preferred embodiment of the present application, and the protection scope of the present application is not limited to the above-described embodiments. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application should also be considered to fall within the protection scope of the present application.

Claims

1. A double emulsion gel bead embedding β-carotene and probiotic bacteria, characterized in that, The composite layer, the oil phase and the inner water phase from outside to inside; The composite layer comprises the outer water phase and the calcium alginate which are fused with each other; the outer water phase comprises the product after the Maillard reaction of carrageenan and sodium caseinate; The oil phase comprises beta-carotene, vegetable oil and food emulsifier; The inner water phase comprises probiotics; In the outer water phase, the mass ratio of the sodium caseinate and the carrageenan is (1.8~2.2):

1.

2. The double emulsion gel bead embedding β-carotene and probiotic bacteria according to claim 1, characterized by, The inner water phase comprises bacterial solution which comprises the probiotics; The inner water phase further comprises probiotic protectant; the probiotic protectant comprises skimmed milk powder, inulin and glycerol; the mass of the skimmed milk powder is 5~10% of the mass of the bacterial solution; the mass of the inulin is 5~10% of the mass of the bacterial solution; the mass of the glycerol is 1~3% of the mass of the bacterial solution.

3. A method of preparing a double emulsion gel bead embedding β-carotene and probiotic bacteria, characterized by, The preparation method comprises: Preparation of primary emulsion: mixing probiotic bacterial solution and probiotic protectant to obtain inner water phase; dispersing the inner water phase in the oil phase to obtain the primary emulsion in which the inner water phase is wrapped by the oil phase; Preparation of outer water phase: mixing the product after the Maillard reaction of carrageenan and sodium caseinate and water to obtain the outer water phase; Preparation of secondary emulsion: dispersing the primary emulsion in the outer water phase and adding beta-carotene to obtain the secondary emulsion; Preparation of gel beads: mixing sodium alginate and the secondary emulsion to obtain mixed emulsion, and adding the mixed emulsion into calcium ion solution by dripping method or microfluidic technology to obtain the gel beads; The preparation of the outer water phase specifically comprises: dissolving sodium caseinate and carrageenan in water at a mass ratio of (2 ± 0.2):1, then freeze-drying, grinding into powder, placing the powder in saturated potassium bromide solution, controlling the solution temperature to be 50~70 ℃, reacting for 16~32 h, drying and cooling to obtain dry powder, and mixing the dry powder with water to obtain the outer water phase.

4. The method for preparing a double emulsion gel bead embedding β-carotene and probiotic bacteria according to claim 3, characterized by, The dispersing of the inner water phase in the oil phase to obtain the primary emulsion in which the inner water phase is wrapped by the oil phase specifically comprises: Mixing the inner water phase and the oil phase at a volume ratio of 2:(7~9) and homogenizing treatment at a speed of 10000~20000 rpm for 5~7 min to obtain the primary emulsion.

5. The method for preparing the double emulsion gel bead embedding β-carotene and probiotic bacteria according to claim 3, characterized in that, In the preparation of the secondary emulsion, the volume ratio of the primary emulsion and the outer water phase added is 1:(1.4~1.6).

6. The method for preparing a double emulsion gel bead embedding β-carotene and probiotic bacteria according to claim 5, characterized by, The preparation of the secondary emulsion specifically comprises: Adding the primary emulsion to the outer water phase, adding beta-carotene at a proportion of (4~6) mg / mL relative to the primary emulsion, and then homogenizing to obtain the secondary emulsion.

7. The method for preparing the double emulsion gel beads encapsulating β-carotene and probiotics according to claim 3, characterized in that, The preparation of the gel beads specifically comprises: Mixing 1.5~2.5 wt% sodium alginate aqueous solution and the secondary emulsion at a volume ratio of 1:(0.9~1.1) to obtain mixed emulsion, and adding the mixed emulsion into 4~6 wt% calcium chloride solution by dripping method or microfluidic technology to obtain the gel beads.

Images