Probiotic-loaded starch-based emulsion gel and preparation method thereof

Abstract

The invention discloses a preparation method of probiotic-loaded starch-based emulsion gel, which comprises the following steps: (1) mixing water and corn starch to form a suspension, heating and gelatinizing, and cooling to obtain starch paste; (2) dispersing probiotics in vegetable oil to obtain an oil phase containing the probiotics; (3) mixing the starch paste with the probiotic-containing oil phase, adding Tween 80, and carrying out high-speed shearing dispersion to prepare a probiotic-loaded starch emulsion; and (4) standing the starch emulsion loaded with the probiotics in a low-temperature environment to obtain the starch-based emulsion gel loaded with the probiotics. The invention provides a probiotic loading method which simplifies the preparation process, reduces the production cost and meets the food-grade standard by optimizing the types of starch-based materials and gelation conditions thereof.

Description

Technical Field

[0001] This invention relates to the field of emulsion gel technology, and in particular to a starch-based emulsion gel loaded with probiotics and its preparation method. Background Technology

[0002] Probiotics are defined as live microorganisms that, when ingested in sufficient quantities, can provide one or more health benefits to the host. Numerous studies have shown that when the number of live bacteria ingested exceeds 10... 6 ~10 7 At colony-forming units (CFU / mL or CFU / g), probiotics can exhibit beneficial physiological effects. However, the strong corrosiveness of gastric acid (pH 1.5–3.5) and bile salts in the gastrointestinal tract, as well as the mechanical shearing forces generated by gastrointestinal motility, cause a large number of probiotics to become inactive before reaching specific sites in the gastrointestinal tract. This severely restricts the full realization of their beneficial functions and has become a major challenge that urgently needs to be overcome in the application of probiotics. Encapsulating probiotics through physical and chemical methods to isolate them from the external environment is currently an important means of reducing the impact of the harsh gastrointestinal environment on the bacteria. Currently, common encapsulation and delivery systems include microcapsule encapsulation, emulsion systems, and hydrogels. Although microcapsules can partially isolate the external environment, they are prone to disintegration under high temperature, low pH, or bile salt environments; traditional emulsions are prone to delamination and rupture under the action of gastric acid and shearing forces; although hydrogels are biocompatible, their molding is complex and their acid resistance is poor.

[0003] Emulsion gels are a type of gel matrix filled with emulsified oil droplets. Their structure is typically formed based on interactions between gel molecules, interactions between the gel (aqueous phase) and emulsified oil droplets (oil phase), and interactions between oil droplets. Emulsion gels constructed from natural biomacromolecules such as polysaccharides and proteins are also gradually becoming a research focus. Starch, as a commonly used polysaccharide, possesses certain encapsulation and loading capabilities due to its polyhydroxy groups and amylose single-helix structure. However, it faces numerous challenges in practical applications. For example, the gel network structure formed by ordinary starch during processing exhibits low strength, poor acid and heat resistance, and difficulty in effectively protecting the activity of probiotics in the harsh environment of the gastrointestinal tract. Furthermore, its molding process is complex, making it difficult to meet the needs of large-scale industrial production.

[0004] Currently disclosed technologies for preparing probiotic-loadable emulsion gels or gels have the following limitations: (1) A dual-gel composition, a high-temperature and high-moisture resistant gel bead that can protect lipophilic active ingredients and probiotics, and its preparation method and application (Patent Application No. 202411492475.7) achieves the protection of lipophilic active ingredients and probiotics by combining low-temperature responsive gel and high-temperature responsive gel, combined with cross-linking and pelleting processes. However, this method relies on the synergistic effect of multiple gelling agents (such as gellan gum, sodium alginate, konjac gum, etc.), and the formulation is complex and costly. In addition, the complex preparation process (such as cooling pelleting, cross-linking treatment, etc.) requires high-end equipment, which limits its application in large-scale industrial production.

[0005] (2) A water-in-oil emulsion gel loaded with probiotics and its preparation method (patent application number 202510303248.3) forms a water-in-oil emulsion gel by adding oleoglycan factors (such as β-sitosterol and γ-oryzanol) and lipophilic emulsifiers to the oil phase, which significantly improves the stability of probiotics and the storage time at room temperature. However, this system is highly dependent on the oil phase ratio and the concentration of oleoglycan factors. If the ratio is inappropriate, it may lead to emulsion instability or excessively large crystal structure, which may destroy the stability of the system.

[0006] In summary, existing technologies generally suffer from two major drawbacks: First, existing methods rely on a variety of exogenous gelling agents or complex oleogel factors, resulting in complex formulations, high costs, and strong sensitivity to process conditions, which limits their widespread industrial application. Second, there is a lack of development and utilization of natural, low-cost materials (such as high amylose corn starch), which fails to fully realize their potential in terms of probiotic loading, acid resistance, and heat resistance, and also fails to effectively simplify the preparation process to meet the needs of large-scale production. Summary of the Invention

[0007] To address the above problems, this invention provides a starch-based emulsion gel loaded with probiotics and its preparation method. This invention reduces dependence on complex exogenous gelling agents while optimizing the type of corn starch and its gelatinization and cold gelation degrees, achieving efficient loading and protection of probiotics. By selecting corn starches with different amylose contents and applying specific gelatinization procedures (pre-gelatinization at 60-65℃ followed by heating to 115-121℃) and cooling conditions (cold gelation at 4-10℃), a dense three-dimensional network structure is formed, providing a beneficial physical barrier for probiotics and significantly improving their survival rate in processing environments (low-temperature storage and heat processing) and in vivo digestive environments (gastrointestinal tract).

[0008] Currently available probiotic-loaded emulsion gelation technologies mostly rely on complex exogenous gelling agents (such as gellan gum and sodium alginate) or oleogelling factors (such as β-sitosterol), which suffer from problems such as complex formulations, high costs, and redundant processes. This invention provides a probiotic loading method that simplifies the preparation process, reduces production costs, and meets food-grade standards by optimizing the types of starch-based materials and their gelation conditions.

[0009] The technical solution of the present invention is as follows: The first objective of this invention is to provide a method for preparing a starch-based emulsion gel loaded with probiotics, comprising the following steps: (1) Mix water and corn starch to form a suspension, then heat to gelatinize, and then cool to obtain starch paste; (2) Disperse probiotics in vegetable oil to obtain an oil phase containing probiotics; (3) Mix the starch paste obtained in step (1) with the oil phase containing probiotics obtained in step (2), add Tween 80, and disperse by high-speed shearing to obtain a starch emulsion loaded with probiotics. (4) The starch emulsion loaded with probiotics obtained in step (3) is left to stand at low temperature to obtain starch-based emulsion gel loaded with probiotics.

[0010] In one embodiment of the present invention, in step (1), the corn starch is one or more of ordinary corn starch, high amylose corn starch, and waxy corn starch; The concentration of the suspension formed by mixing water and corn starch is 7-9 wt%.

[0011] In one embodiment of the present invention, the amylose content of ordinary corn starch is 22-28%; the high amylose corn starch is G50 or G70; the amylose content of G50 is 50-55%, and the amylose content of G70 is 70-75%.

[0012] In one embodiment of the present invention, in step (1), the heating and gelatinization method is as follows: first, heat and pregelatinize at 60-65°C for 15-30 min, then heat at 115-121°C for 20-30 min; cool to 60-65°C and maintain.

[0013] In one embodiment of the present invention, in step (2), the probiotics are one or more of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium, yeast, and Escherichia coli Nissle 1917; the vegetable oil is one or more of soybean oil, corn oil, peanut oil, olive oil, rapeseed oil, and sunflower seed oil.

[0014] The amount of probiotics added should be at least 10% of the total mass of the starch paste and oil phase. 9 -1011 CFU / g.

[0015] In one embodiment of the present invention, in step (3), the mass ratio of starch paste to probiotic oil phase is 7-9:3-1.

[0016] In one embodiment of the present invention, in step (3), the amount of Tween 80 added is 0.05-0.1 wt% of the total mass of starch paste and oil phase.

[0017] In one embodiment of the present invention, in step (3), the high-speed shear dispersion speed is 12000-15000 rpm, the time is 2-5 min, and the shear temperature is maintained at 60-65℃.

[0018] In one embodiment of the present invention, in step (4), the temperature of the static environment is 4-10°C and the time is 12-24 h.

[0019] The second objective of this invention is to provide a starch-based emulsion gel loaded with probiotics prepared by the above-described method.

[0020] A third objective of this invention is to provide the application of the above-mentioned starch-based emulsion gel loaded with probiotics in the preparation of food or health food.

[0021] The beneficial technical effects of this invention are as follows: This invention uses corn starch and vegetable oil as the main raw materials. By optimizing the type of corn starch and its gelatinization and cold gelation degree, while reducing dependence on complex exogenous gelling agents, and utilizing the thermal gelatinization and gelation properties of starch, a dense three-dimensional network structure is formed, endowing the emulsion gel with good physical stability and a physical protective barrier for probiotics. The starch-based emulsion gel loaded with probiotics of this invention maintains structural stability under in vitro digestion processes (simulating the oral-gastric-intestinal digestive environment) and pasteurization treatment (65°C, 30 minutes or 72°C, 15 seconds). Simultaneously, this invention simplifies the preparation process, reduces production costs, and reduces dependence on complex exogenous materials, providing a probiotic loading method that meets food-grade standards, and offering new technical support for the application of probiotics in the food industry. Attached Figure Description

[0022] Figure 1 The images show the appearance of the products obtained in Comparative Examples 1-3 and Examples 1-7 of Test Example 1.

[0023] Figure 2 The apparent viscosity and frequency scan analysis graphs are for Examples 1-3 in Test Example 2.

[0024] Figure 3The number of live probiotics loaded in the samples of Comparative Example 2 and Examples 1-5 in Test Example 3.

[0025] Figure 4 This test examines the morphology of the samples from Examples 1-3 in Test Example 4 and the number of viable probiotics during the storage period.

[0026] Figure 5 The number of live probiotics in the products of Examples 1-3 in Test Example 5 after simulated pasteurization treatment.

[0027] Figure 6 The number of live probiotics in the products of Examples 1-3 in Test Example 6 after simulated in vitro digestion treatment. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0029] Raw material sources: ordinary corn starch with an amylose content of 20-28%; high amylose corn starch G50 with an amylose content of 50-55%; high amylose corn starch G70 with an amylose content of 70-75%. Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available products.

[0030] Example 1 A method for preparing a starch-based emulsion gel loaded with probiotics includes the following steps: (1) Ordinary corn starch was prepared into a 9wt% suspension, pregelatinized at 65°C for 30 min, then transferred to 121°C for 30 min, and cooled at 65°C after heating to obtain starch paste.

[0031] (2) E. coli Nissle 1917 (EcN) was inoculated at a rate of 1% into 100 mL of LB broth medium and incubated at 37°C for 12 h. The resulting EcN bacterial culture was centrifuged twice (4000 rpm, 10 min), washed (with sterile PBS buffer), and the medium was removed to obtain EcN bacterial sludge.

[0032] (3) Disperse EcN bacterial mud in soybean oil and mix well to obtain an oil phase containing EcN. Mix starch paste and oil phase in a ratio of 7:3 (m:m) and add 0.1 wt% Tween 80. Shear it for 3 min at 15000 r / min using a high-speed shearing machine to obtain EcN-loaded starch emulsion.

[0033] (4) The starch emulsion was transferred to a low-temperature environment of 4℃ and allowed to stand for 24 h to obtain a starch-based emulsion gel loaded with EcN. The loading of EcN was higher than 1×10⁻⁶. 9CFU / g.

[0034] Example 2 Same as Example 1, except that in step (1), ordinary corn starch is replaced with high amylose corn starch G50.

[0035] Example 3 Same as Example 1, except that in step (1), ordinary corn starch is replaced with high amylose corn starch G70.

[0036] Example 4 Same as Example 1, except that the heating and gelatinization procedure in step (1) is replaced by heating and pregelatinizing at 65°C for 30 min, then transferring to 115°C for 30 min, and cooling at 65°C after heating to obtain starch paste.

[0037] Example 5 Same as Example 1, except that in step (1), the heating and gelatinization procedure is replaced by: heating and pregelatinizing at 65°C for 30 min, then transferring to 121°C for 30 min, and cooling at 60°C after heating to obtain starch paste.

[0038] Example 6 Same as Example 1, except that in step (3), the ratio of starch paste to oil phase is replaced with 9:1. Example 7 Same as Example 1, except that in step (3), the amount of Tween 80 added is 0.05 wt% of the total mass of starch paste and oil phase.

[0039] Comparative Example 1 Same as Example 2, except that in step (1), the heating and gelatinization procedure is replaced by: heating and pregelatinizing at 65°C for 30 min, then transferring to 90°C for 30 min, and cooling at 65°C after heating to obtain starch paste.

[0040] Comparative Example 2 Same as Example 1, except that in step (1), the heating and gelatinization procedure is replaced by heating and pregelatinizing at 65°C for 30 min, then transferring to 121°C for 30 min, and not cooling after heating to obtain starch paste.

[0041] Comparative Example 3 Same as Example 1, except that in step (3), the ratio of starch paste to oil phase is replaced with 3:7. Test Example 1: Appearance Stability of Emulsion Gel Experimental method: The prepared Examples 1-7 and Comparative Examples 1-3 were photographed and observed.

[0042] Experimental results: such as Figure 1 As shown, the starch-based emulsion gels prepared in all seven examples and Comparative Example 2 can form stable emulsion gels and maintain their intact morphology. The sample prepared under the heating and gelatinization procedure of Comparative Example 1 failed to form a stable starch-based emulsion gel with a fixed morphology. The sample prepared under the oil phase ratio conditions of Comparative Example 3 exhibited stratification, indicating that an ideal starch-based emulsion gel could not be prepared under these conditions.

[0043] Test Example 2: Rheological Properties of Emulsion Gels Experimental methods: The rheological properties of Examples 1-3 were measured using a rheometer. A 40 mm plate was used, with a plate gap of 1.0 mm, and the time was 0.01 s. -1 up to 100 s - The apparent viscosity of the three embodiments was measured by shear rate. Frequency sweep tests (0.1–100 rad / s) were performed at a constant strain of 1.0% to measure the storage modulus (G') and loss modulus (G'). Experimental results: such as Figure 2 As shown, the apparent viscosity of all three embodiments decreased with increasing shear rate, exhibiting obvious pseudoplastic fluid shear-thinning behavior. Furthermore, G' in all three embodiments was greater than G'', indicating that the prepared starch-based emulsion gel primarily exhibited elastic deformation and solid-like behavior.

[0044] Test Example 3: Probiotic Loading Capacity of Emulsion Gel Test method: The number of viable probiotics loaded in the samples of Comparative Example 2 and Examples 1-5 was determined by plate counting method.

[0045] Experimental results: such as Figure 3 As shown, the viable bacterial counts in all five examples exceeded 9 log CFU / g, while Comparative Example 2 had a viable bacterial count of less than 4 log CFU / g. This significant difference indicates that the cooling step is crucial for the probiotic loading capacity of starch-based emulsion gels. During the heating and gelatinization process, the absence of a cooling step will cause the high-temperature environment to destroy the activity of probiotics, resulting in a substantial reduction in their loading quantity.

[0046] Test Example 4: Storage stability of emulsion gel Experimental methods: The prepared Examples 1-3 were stored at 4°C in the dark. During the storage period, the morphology of the samples was observed on days 0, 5, 10, 15, 20, 25 and 30, and the viable number of probiotics loaded on the samples was determined by plate count method.

[0047] Experimental results: such as Figure 4As shown in Figure A, the emulsion gels prepared in the three examples did not exhibit stratification after being stored at 4°C in the dark for 30 days, indicating strong stability. Figure 4 As can be seen from B, after 30 days of storage in the dark at 4°C, the values ​​of Examples 1-3 decreased by 1.19, 0.41, and 0.38 log CFU / g, respectively, indicating that starch-based emulsion gels play a positive role in the loading and storage of probiotics, and the higher the content of amylose, the stronger the storage stability of the emulsion gels prepared.

[0048] Test Example 5: Heat sterilization stability of emulsion gel Experimental Methods: Two pasteurization methods were simulated. The prepared samples from Examples 1-3 were placed in environments where they were heated at 65°C for 30 min (Low Temperature Long Time Pasteurization, LTLT) and at 72°C for 15 s (High Temperature Short Time Pasteurization, HTST), and then placed in ice water to rapidly stop the reaction. Free EcN was used as a control, and the viable count of probiotics loaded on the samples was determined using the plate count method.

[0049] Experimental results: such as Figure 5 As shown, after heating at 72℃ for 15 s, free EcN decreased by 3.05 log CFU / g, while in Examples 1-3, the decreases were 1.18, 0.66, and 0.61 log CFU / g, respectively. After heating at 65℃ for 30 min, free EcN decreased by 5.99 log CFU / g, while in Examples 1-3, the decreases were 3.24, 2.85, and 2.99 log CFU / g, respectively. This indicates that high-temperature, short-time pasteurization is more suitable for processing probiotic products, and the probiotics loaded in the emulsion gel exhibit a higher survival rate after this pasteurization. Furthermore, the higher the amylose content, the greater the protective effect of the prepared emulsion gel on the heat resistance of the probiotics. These results demonstrate that starch-based emulsion gel systems play a significant positive role in improving the heat resistance of probiotics.

[0050] Test Example 6: Simulated in vitro digestion stability of emulsion gel Experimental Methods: Simulated saliva consisted of NaCl, KCl, KSCN, and KH₂PO₄, with a pH of 6.8; simulated gastric juice contained pepsin and NaCl, with a pH of 2.0; simulated intestinal juice consisted of bile salts, pancreatic enzymes, and pancreatic lipase, with a pH of 7.0. The prepared samples from Examples 1-3 were mixed with an equal volume of simulated saliva and incubated at 37°C in a water bath at 100 rpm for 10 min. After oral digestion, simulated gastric juice was added and the pH was adjusted to 2.0, and the mixture was incubated at 37°C in a water bath at 100 rpm for 2 h. After gastric digestion, simulated intestinal juice was added and the pH was adjusted to 7.0, and the mixture was incubated at 37°C in a water bath at 100 rpm for 2 h. The reaction was terminated by placing the solution in an ice-water bath. Free EcN was used as a control, and the viable count of probiotics loaded in the samples was determined using the plate count method.

[0051] Experimental results: such as Figure 6 As shown, after simulated oral digestion, the number of viable bacteria in free EcN decreased by 0.69 log CFU / g, while there was no significant decrease in Examples 1-3. After simulated gastric digestion, the number of viable bacteria in free EcN decreased by 5.90 log CFU / g, while the decreases in Examples 1-3 were 4.37, 2.33, and 2.13 log CFU / g, respectively. After simulated small intestinal digestion, the survival rate of free EcN was 0, while the number of viable bacteria in Examples 1-3 reached 4.63, 6.86, and 7.20 log CFU / g, respectively. The experimental results indicate that starch-based emulsion gels can improve the survival rate of probiotics during simulated in vitro digestion. Furthermore, starch-based emulsion gels prepared using high amylose corn starch G50 and G70 as raw materials have a stronger protective effect on probiotics.

[0052] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A process for the preparation of a starch-based emulsion gel loaded with probiotic bacteria, characterized in that, The method comprises the following steps: (1) mixing water and corn starch to form a suspension, then heating and gelatinizing, and cooling to obtain a starch paste; (2) dispersing probiotics in vegetable oil to obtain an oil phase containing probiotics; (3) mixing the starch paste obtained in step (1) with the oil phase containing probiotics obtained in step (2), adding Tween 80, and high-speed shearing and dispersing to obtain a starch emulsion loaded with probiotics; (4) placing the starch emulsion loaded with probiotics obtained in step (3) in a low-temperature environment to obtain a starch-based emulsion gel loaded with probiotics.

2. The production method according to claim 1, characterized by, In step (1), the corn starch is one or more of ordinary corn starch, high-amylose corn starch, and waxy corn starch. The concentration of the mixture of water and corn starch to form a suspension is 7-9 wt%.

3. The production method according to claim 1, characterized by, In step (1), the method of heating and gelatinization is: first heating and pre-gelatinizing at 60-65°C for 15-30 min, then heating at 115-121°C for 30-40 min, and cooling to 60-65°C and keeping.

4. The method of claim 1, wherein, In step (2), the probiotics are one or more of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium, yeast, and Escherichia coli Nissle1917; and the vegetable oil is one or more of soybean oil, corn oil, peanut oil, olive oil, rapeseed oil, and sunflower seed oil.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the starch paste to the oil phase containing probiotics is 7-9:3-1.

6. The method of claim 1, wherein, In step (3), the amount of Tween 80 added is 0.05-0.1 wt% of the total mass of the starch paste and the oil phase.

7. The production method according to claim 1, characterized by, In step (3), the high-speed shearing and dispersing is performed at a speed of 12000-15000 rpm for 2-5 min, and the shearing temperature is kept at 60-65°C.

8. The production method according to claim 1, characterized by, In step (4), the temperature of the low-temperature environment is 4-10°C, and the time is 12-24 h.

9. A starch-based emulsion gel loaded with probiotics prepared by the method of any one of claims 1-8.

10. Use of the starch-based emulsion gel loaded with probiotics of claim 9 in the preparation of food or health food.

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