Emulsion gel for improving tolerance of probiotics and preparation method thereof

Abstract

The invention provides a preparation method of emulsion gel for improving tolerance of probiotics, and belongs to the field of biological medicine and functional food. The preparation method comprises the following steps: firstly dispersing probiotics in gallic acid, then dispersing a probiotic-gallic acid solution in rice oil, adopting a milk fat globule membrane and pectin as emulsifiers, mixing a water phase and an oil phase, and performing homogenization treatment to finally obtain the probiotic emulsion gel. The preparation method disclosed by the invention is simple, the obtained emulsion gel has excellent storage stability, the oxidation resistance is excellent, the emulsion gel has an excellent embedding effect on probiotics, and the activity of the probiotics during long-term storage and the protection capability of the probiotics under simulated gastrointestinal tract conditions are remarkably improved. The invention provides a new thought for designing and creating a more effective probiotic embedding carrier, and widens the application range of the probiotics in the fields of food and medicine.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and functional food, and in particular to an emulsion gel capable of improving the tolerance of probiotics and a preparation method thereof. Background Art

[0002] Probiotics are defined as "live microorganisms that, when administered in adequate doses, can modulate host biological functions and have beneficial effects on health." Research has shown that probiotics can enhance the nutritional value of food, boost the immune system, regulate intestinal inflammation, treat antibiotic-induced diarrhea, and reduce the risk of colon cancer. However, most probiotics have poor resistance to various adverse environmental conditions encountered during processing or after ingestion, limiting their bioavailability. Therefore, developing efficient delivery systems to improve the survival rate of probiotics during production, transportation, and gastrointestinal digestion is of great significance in the field of probiotics.

[0003] Encapsulation technologies for probiotics have been used as carriers or targeted delivery systems, including freeze-drying and extrusion. These methods have been shown to have the potential to improve the survival rate and bioactivity of probiotics. However, these technologies have some limitations. For example, the freeze-drying process requires a long period of freezing and drying, resulting in a longer production cycle. In addition, the process requires a large amount of energy to maintain a low temperature environment, which increases production costs and energy consumption. On the other hand, the extrusion method has low production efficiency and difficulty in preparing uniform microcapsules, making it unsuitable for large-scale production. In contrast, the emulsion gels formed by the emulsification method are easy to handle, have mild conditions and are low in cost, so they can be produced in large quantities. Because they are thermodynamically stable under specific conditions, they have a long shelf life.

[0004] The milk fat globule membrane (FFM) is secreted by mammary gland cells and has a unique three-layer structure. It contains a variety of bioactive components, including phospholipids and glycoproteins. Research has shown that the FFM can improve the tolerance of probiotics in the intestines, ensuring their high stability once they reach the intestine. Furthermore, the FFM has certain immunomodulatory effects, amplifying the beneficial effects of probiotics, helping to enhance the body's defenses against pathogens and reduce the incidence of intestinal infections. Certain components of the FFM can also promote intestinal motility and mucus secretion, thereby facilitating the spread and colonization of probiotics in the intestine. Furthermore, these components provide a rich source of nutrients for probiotics, promoting their growth and reproduction.

[0005] Pectin is a naturally occurring polysaccharide widely used in food as a thickener, emulsifier, and stabilizer. Due to the lack of pectinase in the human body, pectin cannot be hydrolyzed in the stomach and small intestine. It remains stable in the presence of gastric acid and high bile salts. Only in the colon is pectin broken down by enzymes secreted by bacteria. Pectin also has a beneficial laxative effect. When used in combination with probiotics, pectin can soften stool and promote intestinal motility. It can also absorb heavy metal ions in the intestine, reducing their toxicity and promoting their elimination.

[0006] Gallic acid is a natural polyphenol compound found widely in plants. As a potent natural antioxidant, it can create a more favorable microenvironment for probiotics by reducing oxidative stress, alleviating oxidative damage to probiotic cell membranes and DNA, and indirectly improving the overall stability and activity of encapsulated probiotics during processing, storage, and transit through the gastrointestinal tract.

[0007] Therefore, we speculate that the combined use of milk fat globule membrane, pectin, and gallic acid to encapsulate probiotics may produce a synergistic effect. This combination can fully utilize the advantages of all three and has great application potential in the encapsulation and delivery of bioactive substances. Summary of the Invention

[0008] The present invention aims to provide a method for preparing an emulsion gel that improves the tolerance of probiotics. The method involves dispersing probiotics in gallic acid, then dispersing the probiotic-gallic acid solution in rice oil, and using milk fat globule membranes and pectin as emulsifiers. In this way, oil droplets are fixed in the gel network structure formed by the milk fat globule membranes and pectin, thereby obtaining an emulsion gel. The present invention provides a method for preparing a highly stable emulsion gel for those skilled in the art, which helps to improve the bioavailability of probiotics during digestion.

[0009] To achieve the above purpose, the following steps are included:

[0010] (1) preparing milk fat globule membrane solutions of different mass fractions, stirring them at 800 rpm for 2 h at room temperature to completely dissolve them, and storing them at 4°C for 12 h to fully hydrate the protein to obtain milk fat globule membrane solutions; preparing pectin solutions of different mass fractions, stirring them until they are completely dissolved to obtain pectin solutions; (2) dispersing probiotics in gallic acid to obtain probiotic gallic acid solutions; (3) dispersing the probiotic-gallic acid solution in rice oil to obtain oil-in-water solutions; (4) mixing the milk fat globule membrane solution obtained in step (1) with the obtained pectin solution, then adding hydrochloric acid solution to adjust the pH to 4.0, stirring for 30 min, and then mixing them with the oil-in-water solutions obtained in step (3) in different proportions to obtain probiotic emulsion gels.

[0011] The present invention also includes the following features:

[0012] The mass fraction of the milk fat globule membrane solution in (1) is 0.5-8%, and the mass fraction of the pectin solution is 0.5-4%.

[0013] The probiotics in (2) are intestinal probiotics, further comprising Lactobacillus rhamnosus GG and Bifidobacterium longum subspecies infantis Y46.

[0014] The content of gallic acid in the oil-in-water solution in (3) is 0.05-0.10 wt%.

[0015] The volume ratio of the milk fat globule membrane solution and the pectin solution added in (4) is 1-4:1-4.

[0016] The concentration of hydrochloric acid used to adjust pH in (4) is 0.1-1 mol / L.

[0017] The oil-in-water solution in (4) accounts for 10-30%.

[0018] The homogenization conditions in (4) are a speed of 12000-15000 rpm and a time of 3-5 min.

[0019] The practical performance of the present invention is characterized by the storage stability of the emulsion gel, the activity of the probiotics during long-term storage, the survival rate under simulated gastrointestinal fluid conditions, and the antioxidant activity of the emulsion gel.

[0020] From the above steps, the advantages and beneficial effects of the present invention can be seen: First, the emulsifier pectin used in the emulsion gel prepared by the present invention is a naturally derived polysaccharide that is stable in gastric acid and high bile salt environments and is only decomposed by pectinase secreted by bacteria in the human colon. Second, the emulsifier milk fat globule membrane used in the emulsion gel prepared by the present invention contains a variety of bioactive molecules, which can improve the tolerance of probiotics to bile salts and has multiple functions such as anti-inflammatory, improving intestinal health, and regulating intestinal flora. Third, the gallic acid in the oil-in-water solution used in the emulsion gel prepared by the present invention provides an "antioxidant shield" for the encapsulated probiotics due to its excellent antioxidant activity, thereby improving the stability of the probiotics. Fourth, the preparation method provided by the present invention uses an emulsification method to prepare the probiotic emulsion gel. The preparation method is simple and easy to achieve large-scale production. Fifth, the emulsion gel provided by the present invention exhibits excellent storage stability, has a good encapsulation effect on probiotics, significantly improves the vitality of probiotics during long-term storage, and the ability to survive under simulated gastrointestinal conditions, so that the probiotics can effectively play a probiotic role. The present invention provides a new approach for designing and creating a more effective carrier for embedding probiotics, thereby expanding the application scope of probiotics in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the probiotic emulsion gel prepared by the method of the present invention; the names of the parts indicated by the numbers in the figure are as follows: 1. probiotics; 2. gallic acid; 3. oil droplets; 4. milk fat globule membrane; 5. pectin.

[0022] Figure 2 This is a visual observation diagram of the probiotic emulsion gel obtained in Examples 1 to 6 after storage at 4° C. for 1 month.

[0023] Figure 3 The figure shows the survival rate of the probiotic emulsion gels obtained in Examples 1 to 3 during storage at 4° C. Free Lactobacillus rhamnosus GG was used as a control.

[0024] Figure 4 The following is a statistical graph showing the survival rates of the probiotic emulsion gels obtained in Examples 1 to 3 in simulated gastric fluid digestion, with free Lactobacillus rhamnosus GG as a control.

[0025] Figure 5 The following is a statistical graph showing the survival rates of the probiotic emulsion gels obtained in Examples 1 to 3 in simulated intestinal digestion, with free Lactobacillus rhamnosus GG as a control.

[0026] Figure 6 The following is a statistical graph showing the survival rates of the probiotic emulsion gels obtained in Examples 4 to 6 during storage at 4° C. Free Bifidobacterium longum subsp. infantis Y46 was used as a control.

[0027] Figure 7 The following is a statistical graph showing the survival rates of the probiotic emulsion gels obtained in Examples 4 to 6 in simulated gastric fluid digestion, with free Bifidobacterium longum subsp. infantis Y46 as a control.

[0028] Figure 8 The following is a statistical graph showing the survival rates of the probiotic emulsion gels obtained in Examples 4 to 6 in simulated intestinal fluid digestion, with free Bifidobacterium longum subsp. infantis Y46 as a control.

[0029] Figure 9 Graph showing the DPPH and ABTS free radical scavenging abilities of the probiotic emulsion gels obtained in Examples 1 to 6. DETAILED DESCRIPTION

[0030] The present invention will be described in further detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0031] The milk fat globule membrane (MFGM-10, 69-76% protein, 16-22% fat) described in the present invention was purchased from Arla Food Ingredients Group, (Viby, Denmark). Pectin (citrus origin, esterification degree 65%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Gallic acid was purchased from Zhejiang Tianhe Food Biological Co., Ltd. (Zhejiang, China). Rice oil was purchased from Shanghai Kerry Food Industry Co., Ltd. (Shanghai, China). Lactobacillus rhamnosus GG (LGG) was purchased from China Industrial Microbiological Culture Collection Administration Center (Beijing, China). Bifidobacterium longum subsp. infantis Y46 (BI_Y46) Bifidobacterium longum subsp. infantis Y46 was provided by this laboratory and deposited in the China Center for Type Culture Collection, Wuhan, with the deposit number CCTCC M 20221253 and the deposit date of August 9, 2022.

[0032] The present invention relates to a method for preparing the required solution as follows:

[0033] MRS liquid medium: 10 g peptone, 5 g beef powder, 4 g yeast extract powder, 20 g glucose, 5 g anhydrous sodium acetate, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 2 g triammonium citrate, 0.05 g manganese sulfate, 1 mL Tween 80, and 1 L distilled water. Dissolve the solution in aliquots into 1 L wide-mouth bottles and autoclave at 118°C for 15 min.

[0034] MRS solid medium: agar (15 g / L) was added to the MRS liquid medium.

[0035] MRSC liquid culture medium: 0.05% (v / v) L-cysteine ​​hydrochloride was added to the MRS liquid culture medium.

[0036] MRSC solid culture medium: agar (15 g / L) was added to the MRSC liquid culture medium.

[0037] Simulated gastric fluid: simulated gastric fluid was prepared with 85 mM NaCl and 3 g / L pepsin, and then the pH of the solution was adjusted to 1.5 with 1.0 M HCl.

[0038] Simulated intestinal fluid: Prepare simulated intestinal fluid with 85 mM NaCl, 10 mM CaCl2, 1 g / L bile salt, and 1 g / L pancreatic enzyme, and adjust the pH to 7.0 with 1.0 M NaOH.

[0039] Example 1

[0040] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof, comprising the following steps:

[0041] Step (1) Preparation of the aqueous phase: dissolving 4 g of milk fat globule membrane powder in 100 mL of deionized water to obtain a 4% milk fat globule membrane solution, then stirring at 800 rpm at 25° C. for 2 h and maintaining at 4° C. for 12 h to fully hydrate the protein; dissolving 4 g of pectin powder in 100 mL of deionized water and stirring until completely dissolved to obtain a 4% pectin solution; mixing the obtained 4% milk fat globule membrane solution and 4% pectin solution (1:1, v / v) to form an aqueous phase; finally, the milk fat globule membrane concentration in the aqueous phase is 2% (w / v) and the pectin concentration is 2% (w / v).

[0042] Step (2) Cultivation and preparation of probiotics: Lactobacillus rhamnosus GG frozen at -80°C was cultured in MRS medium in a 37°C constant temperature incubator for 24 hours, and then cultured in MRS medium with a 1% inoculum size for 18 hours and passaged twice until Lactobacillus rhamnosus GG reached the logarithmic growth phase. 30 mL of the cultured bacterial solution was centrifuged at 8000 g for 5 minutes, and the supernatant was discarded to obtain a Lactobacillus rhamnosus GG bacterial slurry. The bacterial slurry was washed twice with 0.85% physiological saline to obtain a Lactobacillus rhamnosus GG bacterial slurry.

[0043] Step (3) Preparation of oil-in-water solution: Prepare 0.10 wt% gallic acid solution, add the obtained Lactobacillus rhamnosus GG bacterial slurry to the gallic acid solution and vortex to uniformly suspend it in gallic acid, then add the probiotic-gallic acid solution to rice oil to prepare 9 mL of oil-in-water solution at a ratio of 2:8 (v / v), and vortex to make it uniform.

[0044] Step (4) Preparation of probiotic emulsion gel: 21 mL of the aqueous phase obtained in (1) was mixed with 9 mL of the oil-in-water solution obtained in (3), and sheared for 3 min at 15,000 rpm using a high shear homogenizer to obtain a probiotic emulsion gel.

[0045] Step (5) Storage Experiment: The resulting probiotic emulsion gel was stored in a refrigerator at 4°C for one month. The number of viable probiotic bacteria in the emulsion gel was measured every seven days using the plate count method (MRS solid medium). The release of viable bacteria was determined by centrifuging the sample at 15,800 g for 10 minutes. Free Lactobacillus rhamnosus GG suspended in sterile water was used as a control.

[0046] Step (6) Simulated gastric fluid digestion experiment: The obtained probiotic emulsion gel was mixed with simulated gastric fluid (1:1) and incubated at 37°C in a shaker at 150 rpm for 3 h. Samples were taken every hour, serially diluted, and incubated on MRS solid medium for 24 h before counting the viable bacteria. Free Lactobacillus rhamnosus GG suspended in sterile water was used as a control.

[0047] Step (7) Simulated intestinal fluid digestion experiment: The obtained probiotic emulsion gel was mixed with simulated intestinal fluid (1:1) and incubated at 37°C in a shaker at 150 rpm for 3 h. Samples were taken every hour, serially diluted, and incubated on MRS solid medium for 24 h before counting the viable bacteria. Free Lactobacillus rhamnosus GG suspended in sterile water was used as a control.

[0048] Step (8) Antioxidant capacity test of probiotic emulsion gel

[0049] DPPH free radical scavenging ability assay: 1 mg / mL probiotic emulsion gel was mixed with DPPH (0.2 mmol / L ethanol solution) at a ratio of 1:1 (v / v), stored in the dark for 30 minutes, and the absorbance (A t ) was recorded at 517 nm. The scavenging activity (%) was calculated by the following formula:

[0050]

[0051] Among them A b is the absorbance of ethanol and emulsion, A c is the absorbance of a mixed solution of DPPH-ethanol and deionized water.

[0052] ABTS free radical scavenging ability assay: ABTS solution (7.0 mmol / L) and potassium persulfate solution (4.9 mmol / L) were mixed at a ratio of 1:1 (v / v) and stored in the dark at 20°C for 16 h. The mixture was diluted until its absorbance at 734 nm reached 0.70 ± 0.02. 0.1 mg / mL probiotic emulsion gel was then mixed with the diluted ABTS solution at a ratio of 1:1 and incubated for 30 minutes. The absorbance at 734 nm was measured. t The scavenging activity (%) was calculated by the following formula:

[0053]

[0054] Among them A b is the absorbance of PBS and emulsion, A c is the absorbance of the mixed solution of PBS and ABTS.

[0055] Example 2

[0056] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof. The specific preparation steps are referred to Example 1, except that in step (3), 9 mL of oil-in-water solution is replaced with 6 mL of oil-in-water solution, and in step (4), 21 mL of aqueous phase and 9 mL of oil-in-water solution are mixed instead of 24 mL of aqueous phase and 6 mL of oil-in-water solution.

[0057] Example 3

[0058] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof. The specific preparation steps are referred to Example 1, except that in step (3), 9 mL of oil-in-water solution is replaced by 3 mL of oil-in-water solution, and in step (4), 21 mL of aqueous phase and 9 mL of oil-in-water solution are mixed instead of 27 mL of aqueous phase and 3 mL of oil-in-water solution.

[0059] Example 4

[0060] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof, comprising the following steps:

[0061] Step (1) Preparation of the aqueous phase: dissolving 4 g of milk fat globule membrane powder in 100 mL of deionized water to obtain a 4% milk fat globule membrane solution, then stirring at 800 rpm at 25° C. for 2 h and maintaining at 4° C. for 12 h to fully hydrate the protein; dissolving 4 g of pectin powder in 100 mL of deionized water and stirring until completely dissolved to obtain a 4% pectin solution; mixing the obtained 4% milk fat globule membrane solution and 4% pectin solution (1:1, v / v) to form an aqueous phase; finally, the milk fat globule membrane concentration in the aqueous phase is 2% (w / v) and the pectin concentration is 2% (w / v).

[0062] Step (2) Cultivation and preparation of probiotics: -80°C frozen Bifidobacterium longum subsp. infantis Y46 was anaerobically cultured in MRSC medium in a 37°C constant temperature incubator for 24 hours, and then anaerobically cultured in MRSC medium with a 3% inoculum size for 18 hours and passaged twice until Bifidobacterium longum subsp. infantis Y46 reached the logarithmic growth phase. 30 mL of the cultured bacterial solution was centrifuged at 8000 g for 5 minutes, and the supernatant was discarded to obtain a Bifidobacterium longum subsp. infantis Y46 bacterial slurry. The bacterial slurry was washed twice with 0.85% normal saline to obtain a Bifidobacterium longum subsp. infantis Y46 bacterial slurry.

[0063] Step (3) Preparation of oil-in-water solution: Prepare 0.10 wt% gallic acid solution, add the obtained Bifidobacterium longum subsp. infantis Y46 bacterial mud to the gallic acid solution and vortex to uniformly suspend it in gallic acid, then add the probiotic-gallic acid solution to rice oil to prepare 9 mL of oil-in-water solution at a ratio of 2:8 (v / v), vortex to uniformly suspend it in oil to form an oil-in-water solution.

[0064] Step (4) Preparation of probiotic emulsion gel: 21 mL of the aqueous phase obtained in (1) was mixed with 9 mL of the oil-in-water solution obtained in (3), and sheared for 3 min at 15,000 rpm using a high shear homogenizer to obtain a probiotic emulsion gel.

[0065] Step (5) Storage Experiment: The resulting probiotic emulsion gel was stored in a refrigerator at 4°C for one month. The number of viable probiotics in the emulsion gel was measured every seven days using the plate count method (MRSC solid culture medium). The release of viable probiotics was determined by centrifuging the sample at 15,800 g for 10 minutes. Free Bifidobacterium longum subsp. infantis Y46 suspended in sterile water was used as a control.

[0066] Step (6) Simulated gastric fluid digestion experiment: The obtained probiotic emulsion gel was mixed with simulated gastric fluid (1:1) and incubated at 37°C in a shaker at 150 rpm for 3 h. Samples were taken every hour, serially diluted, and anaerobically incubated on MRSC solid medium for 48 h before counting the viable bacteria. Free Bifidobacterium longum subsp. infantis Y46 suspended in sterile water was used as a control.

[0067] Step (7) Simulated intestinal fluid digestion experiment: The obtained probiotic emulsion gel was mixed with simulated intestinal fluid (1:1) and incubated at 37°C in a shaker at 150 rpm for 3 h. Samples were taken every hour, serially diluted, and anaerobically incubated on MRSC solid medium for 48 h before counting the viable bacteria. Free Bifidobacterium longum subsp. infantis Y46 suspended in sterile water was used as a control.

[0068] Step (8) Antioxidant capacity test of probiotic emulsion gel

[0069] DPPH free radical scavenging ability assay: 1 mg / mL probiotic emulsion gel was mixed with DPPH (0.2 mmol / L ethanol solution) at a ratio of 1:1 (v / v), stored in the dark for 30 minutes, and the absorbance (A t ) was recorded at 517 nm. The scavenging activity (%) was calculated by the following formula:

[0070]

[0071] Among them A b is the absorbance of ethanol and emulsion, A c is the absorbance of a mixed solution of DPPH-ethanol and deionized water.

[0072] ABTS free radical scavenging ability assay: ABTS solution (7.0 mmol / L) and potassium persulfate solution (4.9 mmol / L) were mixed at a ratio of 1:1 (v / v) and stored in the dark at 20°C for 16 h. The mixture was diluted until its absorbance at 734 nm reached 0.70 ± 0.02. 0.1 mg / mL probiotic emulsion gel was then mixed with the diluted ABTS solution at a ratio of 1:1 and incubated for 30 minutes. The absorbance at 734 nm was measured. t The scavenging activity (%) was calculated by the following formula:

[0073]

[0074] Among them A b is the absorbance of PBS and emulsion, A c is the absorbance of the mixed solution of PBS and ABTS.

[0075] Example 5

[0076] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof. The specific preparation steps are referred to Example 4, except that the 9 mL oil-in-water solution in step (3) is replaced by 6 mL oil-in-water solution, and the 21 mL aqueous phase and 9 mL oil-in-water solution mixed in step (4) is replaced by 24 mL aqueous phase and 6 mL oil-in-water solution mixed.

[0077] Example 6

[0078] This embodiment provides an emulsion gel for improving probiotic tolerance and a preparation method thereof. The specific preparation steps are referred to Example 4, except that in step (3), 9 mL of oil-in-water solution is replaced with 3 mL of oil-in-water solution, and in step (4), 21 mL of aqueous phase and 9 mL of oil-in-water solution are mixed instead of 27 mL of aqueous phase and 3 mL of oil-in-water solution.

[0079] Result analysis:

[0080] Figure 1 The emulsion gel structure constructed using the present invention's technology. The network structure composed of milk fat globule membranes and pectin immobilizes oil droplets within it, significantly improving their stability. Gallic acid interacts with probiotics, dispersing them within the oil and providing dual protection for them.

[0081] Figure 2 The following are visual observation images of the probiotic emulsion gels obtained in Examples 1 to 6 after one month of storage. All samples appeared as white emulsion gels, and no delamination was observed after one month of storage, indicating good stability of the probiotic emulsion gels.

[0082] Figure 3The following is a statistical chart of the survival rates of the probiotic emulsion gels and free Lactobacillus rhamnosus GG obtained in Examples 1-3 during storage at 4°C. During the 28-day storage period, the colony counts of Lactobacillus rhamnosus GG in the probiotic emulsion gels of Examples 1-3 decreased by 1.31, 1.26, and 1.34 log CFU / mL, respectively, and the free Lactobacillus rhamnosus GG decreased by 5.49 log CFU / mL. The results show that the emulsion gel can form a protective barrier on the surface of the probiotics to effectively block the external environment from invading the probiotics, thereby maintaining a stable living environment for the probiotics. In addition, the milk fat globule membrane and pectin can act as prebiotics to provide the probiotics with the nutrients they need to survive, thereby extending the survival time of Lactobacillus rhamnosus GG in the emulsion.

[0083] Figure 4 The survival rate statistics of the probiotic emulsion gel obtained in Examples 1-3 and free Lactobacillus rhamnosus GG in simulated gastric juice digestion are shown. In the simulated gastric juice digestion process, free Lactobacillus rhamnosus GG all died within 1 hour. In the probiotic emulsion gels of Examples 1-3, the colony count dropped from 8.89 ± 0.035, 8.90 ± 0.054, and 8.86 ± 0.050 log CFU / mL to 8.09 ± 0.028, 7.90 ± 0.101, and 7.36 ± 0.137 log CFU / mL, respectively. This proves that the emulsion gel structure can better withstand the influence of extremely unfavorable conditions, thereby improving the survival rate of Lactobacillus rhamnosus GG, and the higher the oil-in-water solution content, the higher the survival rate of Lactobacillus rhamnosus GG.

[0084] Figure 5 Statistical graphs of the survival rates of the probiotic emulsion gels obtained in Examples 1-3 and free Lactobacillus rhamnosus GG in simulated intestinal fluid digestion are shown. During the simulated intestinal fluid digestion process, all free Lactobacillus rhamnosus GG died within 1 hour, and the viable counts in the probiotic emulsion gels in Examples 1-3 decreased from 8.89 ± 0.035, 8.90 ± 0.054, and 8.86 ± 0.050 log CFU / mL to 8.85 ± 0.042, 8.61 ± 0.051, and 8.34 ± 0.043 log CFU / mL, respectively. This indicates that the emulsion gels are not easily decomposed by digestive enzymes, resulting in a reduced digestion rate. Consequently, the survival rate of the embedded Lactobacillus rhamnosus GG increases, and the higher the content of the oil-in-water solution, the higher the survival rate of the Lactobacillus rhamnosus GG.

[0085] Figure 6The following is a statistical graph showing the survival rates of the probiotic emulsion gels and free Bifidobacterium longum subsp. infantis Y46 obtained in Examples 4-6 during storage at 4°C. During the 28-day storage period, the colony counts of Bifidobacterium longum subsp. infantis Y46 in the probiotic emulsion gels of Examples 4-6 decreased by 2.73, 2.73, and 2.86 log CFU / mL, respectively, and the free Bifidobacterium longum subsp. infantis Y46 decreased by 6.19 log CFU / mL. These results demonstrate that the emulsion gel can provide a stable living environment for probiotics. Furthermore, the milk fat globule membrane and pectin act as prebiotics, providing the probiotics with the nutrients they need to survive and extending the survival time of Bifidobacterium longum subsp. infantis Y46 in the emulsion.

[0086] Figure 7 Survival rate statistics of the probiotic emulsion gel obtained by embodiment 4-example 6 and free Bifidobacterium longum infants subspecies Y46 in simulated gastric juice digestion.In simulated gastric juice digestion process, free Bifidobacterium longum infants subspecies Y46 all died in 1h, and in embodiment 4-example 6 probiotic emulsion gels, colony count dropped to 7.66 ± 0.102,7.42 ± 0.086,7.00 ± 0.058log CFU / mL from 8.70 ± 0.022,8.71 ± 0.079,8.74 ± 0.040log CFU / mL respectively.This proves that emulsion gel structure can better resist the influence of extreme unfavorable conditions, thereby improves the survival rate of Bifidobacterium longum infants subspecies Y46, and oil-in-water solution content is higher, and the survival rate of Bifidobacterium longum infants subspecies Y46 is higher.

[0087] Figure 8 Be the probiotic emulsion gel of embodiment 4-example 6 gained and the survival rate statistical graph of free Bifidobacterium longum infants subspecies Y46 in simulated intestinal fluid digestion.In simulated intestinal fluid digestion process, free Bifidobacterium longum infants subspecies Y46 all died in 2h, and the viable count in embodiment 4-example 6 probiotic emulsion gels dropped to 8.43 ± 0.090,8.25 ± 0.068,8.08 ± 0.051log CFU / mL from 8.70 ± 0.022,8.71 ± 0.079,8.74 ± 0.040log CFU / mLlog CFU / mL respectively.Illustrate that emulsion gel is not easily decomposed by digestive enzyme, and then digestion rate reduces, and therefore the survival rate of embedded Bifidobacterium longum infants subspecies Y46 increases, and oil-in-water solution content is higher, and the survival rate of Bifidobacterium longum infants subspecies Y46 is higher.

[0088] Figure 9Figure 1 is the scavenging ability graph of DPPH and ABTS free radicals of the probiotic emulsion gel obtained from Example 1-Example 6. As the oil-in-water ratio decreases, the DPPH free radical scavenging ability of rhamnosus lactobacillus GG and bifidobacterium longum infant subspecies Y46 probiotic emulsion gel decreases from (85.07 ± 1.401)%, (85.83 ± 1.537)% to (72.53 ± 1.762)%, (72.73 ± 1.762)%. This shows that the higher the oil-in-water ratio, the better the antioxidant activity. The ABTS free radical scavenging ability of rhamnosus lactobacillus GG and bifidobacterium longum infant subspecies Y46 probiotic emulsion gel decreases from (74.90 ± 2.152)%, (74.96 ± 2.303)% to (57.40 ± 1.931)%, (57.50 ± 3.753)%. It shows that both depend on the content of oil-in-water solution, and the higher the content of oil-in-water solution, the stronger the antioxidant capacity of probiotic emulsion gel.

Claims

1. A method for preparing an emulsion gel for improving probiotic tolerance, characterized in that: The following steps are involved: (1) preparing milk fat globule membrane solutions with different mass fractions, stirring at 800 rpm for 2 h to completely dissolve the milk fat globule membrane solutions at room temperature, and storing at 4°C for 12 h to allow the protein to fully hydrate, thereby obtaining milk fat globule membrane solutions; preparing pectin solutions with different mass fractions, stirring until the milk fat globule membrane solutions are completely dissolved, thereby obtaining pectin solutions; Prepare gallic acid solution and stir until completely dissolved to obtain gallic acid solution; (2) dispersing the probiotics in gallic acid to obtain a probiotic-gallic acid solution; (3) dispersing the probiotic-gallic acid solution in rice oil to obtain an oil-in-water solution; (4) The milk fat globule membrane solution obtained in step (1) is mixed with the obtained pectin solution, and then hydrochloric acid solution is added to adjust the pH of the solution to 4.

0. After stirring for 30 minutes, the mixture is homogenized with the oil-in-water solution obtained in step (3) in different proportions to obtain a probiotic emulsion gel.

2. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: In the step (1), the mass fraction of the milk fat globule membrane solution is 0.5-8%; the mass fraction of the pectin solution is 0.5-4%.

3. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: The probiotics in step (2) are intestinal probiotics, further comprising Lactobacillus rhamnosus GG and Bifidobacterium longum subsp. infantis Y46.

4. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: The content of gallic acid in the oil-in-water solution in step (3) is 0.05-0.10 wt %.

5. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, wherein: The volume ratio of the milk fat globule membrane solution to the pectin solution added in step (4) is 1-4:1-4.

6. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: The concentration of hydrochloric acid used to adjust the pH in step (4) is 0.1-1 mol / L.

7. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: In the step (4), the proportion of the oil-in-water solution is 10-30%.

8. The method for preparing an emulsion gel for improving probiotic tolerance according to claim 1, characterized in that: The homogenization conditions in step (4) are a rate of 12000-15000 rpm and a time of 3-5 min.

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