Tea polyphenol gelatin and sodium alginate edible material probiotic coating method and product

Abstract

The invention provides a probiotic single cell coating method based on calcium-tea polyphenol network, gelatin and sodium alginate assembly and a product, firstly, tea polyphenol and calcium ions are subjected to coordination self-assembly to form a calcium-tea polyphenol network layer on the surfaces of probiotic cells, and then the gelatin is guided to be adsorbed to form a second coating on the surfaces of the probiotic cells. And finally, guiding and adsorbing sodium alginate to form a third coating on the surfaces of the probiotic cells. According to the probiotic coating method, an economical and safe food-grade coating material is provided, operation is easy, the coating time is shortened, the number of coating layers is small, but the coating layers are compact, cells can be protected against damage of the gastrointestinal tract stress environment so that the activity of the probiotics can be maintained, and the coated probiotics have good antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the technical fields of food, daily chemicals, and medical health care, and particularly relates to a probiotic single cell encapsulation method and product based on the assembly of a calcium-tea polyphenol network, gelatin, and sodium alginate. Background Art

[0002] Probiotics are a class of living microorganisms that, when ingested in sufficient amounts, can benefit the host's health. Probiotics are diverse, primarily classified into the genera Lactobacillus and Bifidobacterium, and oral probiotics hold great promise for the treatment of intestinal diseases. However, improving the stability of probiotics in extreme environments such as gastric and intestinal fluids and achieving their high bioavailability remain pressing challenges in probiotic applications. Recent studies have shown that probiotic single-cell encapsulation technology has become a promising strategy for providing personalized protection for each probiotic cell. This technology enhances probiotic stability, ensures targeted release in the gastrointestinal tract, and improves overall therapeutic efficacy.

[0003] In the previous patent document "Method for Encapsulating Probiotic Single Cells Based on Metal-Polyphenol Networks and Photo-Clicked Thiol-Ene Gelatin Layer-by-Layer Self-Assembly" (Application No.: 2023113176701), a cross-linked olefin-functionalized gelatin protective coating was formed based on the principle of thiol-ene photo-click reaction, simplifying the operation steps of the layer-by-layer self-assembly technology. However, the olefin-functionalized gelatin with photosensitive reactive groups used for photo-clicking was obtained by grafting and modifying edible gelatin and could not be directly used in edible products. Therefore, it is necessary to find a coating method that directly utilizes edible materials such as edible gelatin while providing superior protection to enhance the food application value of the encapsulation technology.

[0004] Tea polyphenols are a unique resource in my country. As natural polyphenolic compounds, they possess excellent coordination and self-assembly properties with metals, proteins, and other materials. Tea polyphenols are highly promising components of self-assembling nanocarriers. Numerous studies have demonstrated that metal-polyphenol networks can form stable self-assembled coatings at various interfaces, thus offering potential applications in the encapsulation of probiotics. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a probiotic single-cell encapsulation method based on the assembly of a calcium-tea polyphenol network, gelatin and sodium alginate. The method is simple, and the prepared encapsulated probiotics significantly enhance the viability and anti-oxidative damage ability of the probiotics in gastrointestinal fluid, thereby improving the protective effect on the probiotics.

[0006] Gelatin is a natural polymer material. Type A gelatin usually carries a positive charge, has good biocompatibility and low immunogenicity, and tea polyphenols and gelatin have coordination self-assembly properties. Sodium alginate is a natural polysaccharide that is usually negatively charged, safe, non-toxic, biocompatible, and acid-resistant, and is currently the most widely used encapsulation material. Based on this, the present invention first uses tea polyphenols to prepare the first layer of calcium-tea polyphenol network coating for probiotics. Then, the hydrogen bond adsorption effect of tea polyphenols on gelatin is utilized to guide the gelatin to the surface of the probiotic cells to obtain a second layer of coating. Finally, the electrostatic adsorption effect of gelatin and sodium alginate is utilized to guide the sodium alginate to the surface of the probiotic cells to obtain a third layer of coating, thereby achieving a simplified and efficient probiotic assembly coating.

[0007] A probiotic single cell encapsulation method based on the assembly of a calcium-tea polyphenol network, gelatin and sodium alginate comprises the following steps:

[0008] (1) Adding tea polyphenol solution dropwise to the probiotic solution and mixing evenly, adding metal ion solution thereto, stirring and reacting to obtain calcium-tea polyphenol network-coated probiotics;

[0009] (2) adding gelatin solution to the probiotics coated with the calcium-tea polyphenols network, stirring and reacting, and obtaining probiotics coated with the calcium-tea polyphenols network and gelatin;

[0010] (3) Adding sodium alginate solution to the calcium-tea polyphenols network and gelatin-coated probiotics, stirring and reacting, and obtaining calcium-tea polyphenols network, gelatin and sodium alginate-coated probiotics.

[0011] In the above-mentioned coating method, after the reaction in step (1) is completed, a first protective layer, namely a calcium-tea polyphenol network layer, is formed on the surface of the probiotic cells; after the reaction in step (2), a second protective layer, namely a gelatin layer, is formed outside the calcium-tea polyphenol network layer; after the reaction in step (3), a third protective layer, namely a sodium alginate layer, is formed outside the gelatin layer, and finally, assembled and coated probiotics are obtained. The above-mentioned probiotic single cell coating process is carried out at 10-40°C.

[0012] Preferably, the probiotics are Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus paracasei or Bifidobacterium.

[0013] As a preferred ratio of probiotics to tea polyphenols, 9 :1~1×10 9 :6 CFUs / mg

[0014] Preferably, the tea polyphenols are commercially available green tea extract.

[0015] Preferably, the metal ion solution is a calcium chloride solution.

[0016] Preferably, the mass ratio of tea polyphenols to metal ions is 1:20 to 20:1, more preferably (1 to 10):1, and more specifically, the mass ratio of tea polyphenols to metal ions is (2 to 5):1.

[0017] Preferably, the gelatin is type A gelatin.

[0018] Preferably, in step (3), the mass ratio of tea polyphenols to gelatin is 20:1 to 1:20. More preferably, it is 1:(1 to 10). Furthermore, the mass ratio of tea polyphenols to gelatin is 1:(1 to 2).

[0019] Preferably, in step (4), the mass ratio of tea polyphenols to sodium alginate is 20:1 to 1:20. More preferably, it is 1:(1 to 10). Furthermore, the mass ratio of tea polyphenols to sodium alginate is 1:(2 to 5).

[0020] The present invention's single-cell probiotic encapsulation method utilizes the metal chelating ability of tea polyphenols to coordinate and self-assemble tea polyphenols with calcium ions on the probiotic cell surface to form a calcium-tea polyphenol network layer. A gelatin layer is then assembled via the interaction between tea polyphenols and proteins. Finally, a sodium alginate layer is assembled using the electrostatic adsorption of gelatin and sodium alginate. This dense protective layer protects the probiotic cells from damage caused by adverse gastrointestinal environmental conditions, maintaining the activity of the probiotics. The present invention's single-cell probiotic encapsulation material has excellent safety, providing a simple, efficient, rapid, and safe method for encapsulating probiotics.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The probiotic encapsulation method of the present invention is a single-cell encapsulation technology, which is simple to prepare materials and has little pollution during the preparation process;

[0023] (2) The probiotic coating materials of the present invention are safe and are all food-grade raw materials;

[0024] (3) The probiotic coating material of the present invention can enhance the viability of probiotics in gastrointestinal fluid and improve the bioavailability of probiotics in the intestine, etc., and can be used as an enhanced therapy for the prevention and treatment of intestinal diseases.

[0025] In summary, the probiotic coating method of the present invention provides an economical and safe food-grade coating material, is simple to operate, shortens the encapsulation time, has a small but dense encapsulation layer, can protect cells from damage by the adverse gastrointestinal environment to maintain the activity of probiotics, and the coated probiotics have good antioxidant activity. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0027] Example 1 Preparation of Lactobacillus delbrueckii Assembly Coating

[0028] 3 mL of tea polyphenols (1 mg / mL) solution was added dropwise to 1 mL of Lactobacillus delbrueckii (1×10 9 CFUs) bacterial solution to obtain a mixed solution;

[0029] Then, 1 mL of calcium chloride (1 mg / mL) solution was added dropwise to the above mixed solution, and the mixture was stirred gently at 25°C for 30 min to obtain calcium-tea polyphenol-coated Lactobacillus delbrueckii slurry.

[0030] 4 mL of gelatin (type A, 2 mg / mL) solution was added to the calcium-tea polyphenols-coated Lactobacillus delbrueckii slurry, and the mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols- and gelatin-coated Lactobacillus delbrueckii slurry.

[0031] 4 mL of sodium alginate (2 mg / mL) solution was added to the calcium-tea polyphenols and gelatin-coated Lactobacillus delbrueckii slurry, and the mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols, gelatin and sodium alginate-coated Lactobacillus delbrueckii slurry.

[0032] Example 2 Preparation of Lactobacillus reuteri Assembly Coating

[0033] 3 mL of tea polyphenols (1 mg / mL) solution was added dropwise to 1 mL of Lactobacillus reuteri (1×10 9 CFUs) bacterial solution to obtain a mixed solution;

[0034] Then, 1 mL of calcium chloride (1 mg / mL) solution was added dropwise to the mixed solution, and the mixture was stirred gently at 25°C for 30 min to obtain calcium-tea polyphenol-coated Lactobacillus reuteri slurry.

[0035] 4 mL of gelatin (2 mg / mL) solution was added to the calcium-tea polyphenols-coated Lactobacillus reuteri slurry, and the mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols- and gelatin-coated Lactobacillus reuteri slurry.

[0036] 4 mL of sodium alginate (2 mg / mL) solution was added to the calcium-tea polyphenols and gelatin-coated Lactobacillus reuteri slurry. The mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols, gelatin and sodium alginate-coated Lactobacillus reuteri slurry.

[0037] Example 3 Preparation of Lactobacillus paracasei Assembly Coating

[0038] 3 mL of tea polyphenols (1 mg / mL) solution was added dropwise to 1 mL of Lactobacillus paracasei (1×10 9 CFUs) bacterial solution to obtain a mixed solution;

[0039] Then, 1 mL of calcium chloride (1 mg / mL) solution was added dropwise to the mixed solution, and the mixture was stirred gently at 25°C for 30 min to obtain calcium-tea polyphenol-coated Lactobacillus paracasei slurry.

[0040] 4 mL of gelatin (2 mg / mL) solution was added to the calcium-tea polyphenols-coated Lactobacillus paracasei slurry, and the mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols- and gelatin-coated Lactobacillus paracasei slurry.

[0041] 4 mL of sodium alginate (2 mg / mL) solution was added to the calcium-tea polyphenols and gelatin-coated Lactobacillus paracasei slurry. The mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols, gelatin and sodium alginate-coated Lactobacillus paracasei slurry.

[0042] Example 4 Preparation of Bifidobacterium Assembly Coating

[0043] 3 mL of tea polyphenols (1 mg / mL) solution was added dropwise to 1 mL of Bifidobacterium (1×10 9 CFUs) bacterial solution to obtain a mixed solution;

[0044] Then, 1 mL of calcium chloride (1 mg / mL) solution was added dropwise to the above mixed solution, and the mixture was stirred gently at 25°C for 30 min to obtain calcium-tea polyphenol-coated bifidobacterium slurry.

[0045] 4 mL of gelatin (2 mg / mL) solution was added to the calcium-tea polyphenols-coated bifidobacterium slurry, and the mixture was stirred gently at 25°C for 30 min to obtain calcium-tea polyphenols- and gelatin-coated bifidobacterium slurry.

[0046] 4 mL of sodium alginate (2 mg / mL) solution was added to the calcium-tea polyphenols and gelatin-coated bifidobacterium slurry, and the mixture was stirred gently at 25°C for 30 min to obtain the calcium-tea polyphenols, gelatin and sodium alginate-coated bifidobacterium slurry.

[0047] Stability test of encapsulated probiotics:

[0048] 5×10 8 Uncoated CFUs and the different probiotics coated in Examples 1 to 4 were placed in simulated gastric fluid (SGF) and bile salt solution containing pepsin for a period of time to evaluate the in vitro tolerance of different probiotic cells, and the number of surviving cells was detected by plate count method.

[0049] After being treated with SGF solution (pH 2.0) for 30 min, the survival numbers of the encapsulated Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus paracasei and Bifidobacterium were significantly increased by 2.1, 11.6, 4.2 and 3.5 times, respectively, compared with those of the unencapsulated group (P < 0.05), indicating that the assembled protective layer of the present invention can be used as a protective layer to prevent H + Entering the "armor" of the cell membrane, thereby improving the gastric acid tolerance of these four probiotics.

[0050] After being treated with 4% bile salt solution for 30 min, the survival numbers of the encapsulated Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus paracasei and Bifidobacterium were significantly increased by 80.3, 16.0, 1173.9 and 17.6 times respectively compared with the unencapsulated group (P < 0.05), indicating that the assembled protective layer of the present invention can effectively improve the bile salt tolerance of these four probiotics.

[0051] Antioxidant test of encapsulated probiotics:

[0052] 1×10 8 Uncoated CFUs and the bacterial suspensions of the various probiotics coated in Examples 1-4 were added to a 0.2 mM DPPH solution, mixed thoroughly, and reacted in the dark for 30 minutes. After the reaction, the sample was centrifuged at 12,000 rpm for 2 minutes, and the absorbance of the supernatant was measured at 517 nm to determine the DPPH free radical scavenging rate.

[0053] The test results showed that the scavenging rates of DPPH free radicals by encapsulated Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus paracasei and Bifidobacterium were significantly increased by 60.6%, 56.2%, 41.8% and 38.3% respectively compared with the unencapsulated group (P < 0.01), indicating that the encapsulated probiotics exhibited good antioxidant activity.

Claims

1. A method for coating probiotic single cells assembled with a calcium-tea polyphenol network, gelatin and sodium alginate, characterized in that: The following steps are involved: (1) Add the tea polyphenol solution dropwise to the probiotic solution and mix well, add the calcium ion solution thereto, stir and react, and obtain the calcium-tea polyphenol network-coated probiotics; (2) adding gelatin solution to the probiotics coated with the calcium-tea polyphenols network, stirring and reacting, and obtaining probiotics coated with the calcium-tea polyphenols network and gelatin; (3) Adding sodium alginate solution to the calcium-tea polyphenols network and gelatin-coated probiotics, stirring and reacting, and obtaining calcium-tea polyphenols network, gelatin and sodium alginate-coated probiotics.

2. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The tea polyphenol solution is a tea polyphenol aqueous solution, and the concentration of the tea polyphenol aqueous solution is 0.1-5 mg / mL.

3. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The calcium ion solution is a calcium chloride aqueous solution, and the concentration of the calcium chloride aqueous solution is 0.1~5 mg / mL.

4. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: In step (2), the mass ratio of tea polyphenols to metal ions is 1:20 to 20:

1.

5. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The gelatin is type A gelatin, the gelatin solution is a gelatin aqueous solution, and the concentration of the gelatin aqueous solution is 1-10 mg / mL; the sodium alginate solution is a sodium alginate aqueous solution, and the concentration of the sodium alginate aqueous solution is 1-10 mg / mL.

6. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The mass ratio of tea polyphenols to gelatin is 20:1~1:

20.

7. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The mass ratio of tea polyphenols to sodium alginate is 20:1~1:

20.

8. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The concentration of the tea polyphenol solution is 0.5~5 mg / mL; the concentration of the calcium ion solution is 0.5~5 mg / mL; the concentration of the gelatin solution is 2~5 mg / mL; the concentration of the sodium alginate solution is 2~5 mg / mL; the mass ratio of the tea polyphenols to the metal ions is (2~5):1; the mass ratio of the tea polyphenols to the gelatin is 1:(1~2); and the mass ratio of the tea polyphenols to the sodium alginate is 1:(2~5).

9. The probiotic single cell encapsulation method based on calcium-tea polyphenols network, gelatin and sodium alginate assembly according to claim 1, characterized in that: The probiotics are Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus paracasei or Bifidobacterium.

10. A probiotic single-cell encapsulated product, characterized in that: The coating is obtained by the coating method according to any one of claims 1 to 9.