Preparation method and application of lactic acid bacteria microcapsule with broad-spectrum antibacterial function

Abstract

The invention belongs to the field of microorganisms and application thereof, and discloses a preparation method and application of a lactic acid bacteria microcapsule with a broad-spectrum antibacterial function. On the basis of selecting excellent microcapsule core materials and wall materials, lactobacillus microcapsules of lactobacillus reuteri HLRE13, sodium alginate and skimmed milk powder are prepared, and the microbial starter is obtained. The core material of the microcapsule is lactobacillus reuteri HLRE13 (the preservation number is CCTCC M 20232505), and the wall material of the microcapsule is sodium alginate and skimmed milk powder. Compared with lactobacillus reuteri HLRE13 naked bacteria, the microcapsule has the advantages that the survival rate of the lactobacillus reuteri HLRE13 in a gastrointestinal tract environment is greatly improved, and a broad-spectrum antibacterial effect is well shown. Therefore, the developed lactobacillus microcapsule leavening agent is clear in performance and can be used for fermenting dairy products, medicines, feeds and the like; the product can also be used as a dietary supplement to improve the intestinal environment, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and their applications, and particularly relates to a preparation method and application of lactic acid bacteria microcapsules with antagonistic pathogenic bacteria. Background Art

[0002] Antibiotics and other drugs have long been considered the best treatments for and control of pathogenic infections. However, the continued overuse of antibiotics has led to the development of bacterial resistance, resulting in significant side effects. Lactic acid bacteria, due to their ability to inhibit the growth and reproduction of pathogenic bacteria, are considered potential "green antibiotics." Lactobacillus reuteri, a common lactobacillus colonizing the spinal cord, possesses multiple beneficial properties and specifically produces the broad-spectrum antimicrobial substance reuterin. Therefore, its application in the control and prevention of pathogenic infections is of great practical significance.

[0003] It is worth noting that an important prerequisite for lactic acid bacteria to exert its probiotic function is to have a certain number of viable bacteria. However, due to the influence of processing conditions, storage temperature and gastrointestinal environment, the number of viable bacteria will be greatly reduced. The review of probiotic health foods stipulates that the number of viable bacteria in probiotic health foods should not be less than 10 during their shelf life. 6 CFU / mL, and the resistance of naked bacteria to extreme environments is ultimately limited. Therefore, taking necessary measures to improve the ability of lactic acid bacteria to resist the gastrointestinal environment and the stability is of great significance for the application of lactic acid bacteria in the food industry. Summary of the Invention

[0004] In order to provide another effective measure for preventing and controlling pathogen infection and to solve the problem of poor tolerance of lactic acid bacteria to adverse environments, the present invention provides a lactic acid bacteria microcapsule with broad-spectrum antibacterial function and a preparation method thereof. The specific embodiments are as follows:

[0005] A microcapsule with an antagonistic effect on pathogenic bacteria, the microcapsule being composed of a core material made of lactic acid bacteria and a wall material made of a variety of materials;

[0006] Furthermore, the core material is Lactobacillus reuteri HLRE13, which has been deposited in the China Center for Type Culture Collection on December 8, 2023, at Wuhan University, Wuhan, China, with a deposit number of CCTCC M 20232505;

[0007] Furthermore, Lactobacillus reuteri HLRE13 fermentation can specifically produce a broad-spectrum antibacterial substance, reuterin, which has antagonistic effects on a variety of pathogenic microorganisms;

[0008] Furthermore, the wall material is composed of sodium alginate and skim milk powder;

[0009] Furthermore, the mass fraction (or concentration) ratio of sodium alginate to skim milk powder is: (1.5%-2.5%): (5%-15%).

[0010] In addition, the present invention also provides a method for preparing lactic acid bacteria microcapsules with broad-spectrum antibacterial function, wherein the concentration of live bacteria reaches 10 9 A lactic acid bacteria suspension with a CFU / mL content was mixed with a sodium alginate solution at a ratio of 1:4 to obtain a mixed solution I. The mixed solution I was mixed with skim milk powder at a ratio of 1:(5%-15%) to obtain a mixed solution II. The mixed solution II was added dropwise to a calcium chloride solution, allowed to stand at room temperature for 30 minutes, washed three times with a peptone solution, then immersed in a chitosan solution for 40 minutes, and then washed twice with a peptone solution to obtain wet microcapsules. The wet capsules were dried to obtain lactic acid bacteria microcapsules.

[0011] Furthermore, the preparation process of the calcium chloride solution includes: using distilled water as a solvent, adding calcium chloride to prepare a calcium chloride solution with a molar concentration of 0.3M, and sterilizing it under high pressure at 121°C; the preparation process of the peptone solution includes: using deionized water as a solvent, adding peptone to prepare a peptone solution with a mass percentage of 0.1%, and sterilizing it under high pressure at 121°C; the preparation process of the chitosan solution includes: using distilled water as a solvent, adding chitosan to prepare a solution with a mass percentage of 0.04%, then adding 2% glacial acetic acid, and then adjusting the pH value of the solution to 5.7-6.0 with sodium hydroxide solution.

[0012] The present invention also provides application of the lactic acid bacteria microcapsules in food, medicine and health care products.

[0013] In summary, the beneficial effects of the present invention are as follows: the method for preparing lactic acid bacteria microcapsules provided by the present invention is to encapsulate lactic acid bacteria using a variety of wall materials and then dry them to obtain lactic acid bacteria microcapsules. This method is simple to operate, has mild conditions, a short cycle, and high efficiency. At the same time, the specific proportion of wall materials in the present invention can also form a layer of insoluble substance on the surface of the core material, which can significantly improve the environmental tolerance of the lactic acid bacteria microcapsules. Therefore, the lactic acid bacteria microcapsules with broad-spectrum antibacterial effects prepared by the present invention have high lactic acid bacteria activity after entering the intestine, which is conducive to exerting the probiotic function of lactic acid bacteria and preventing and controlling the excessive proliferation of pathogenic microorganisms in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown is a flow chart of the preparation of lactic acid bacteria microcapsules in Example 1 of the present application;

[0015] Figure 2 Shown is a sample image of the lactic acid bacteria microcapsules in Example 1 of the present application;

[0016] Figure 3 The figure shows the test results of the tolerance of lactic acid bacteria microcapsules and naked bacteria to gastric juice in Example 2 of the present application;

[0017] Figure 4 The figure shows the results of the bile salt tolerance test of lactic acid bacteria microcapsules and naked bacteria in Example 3 of the present application;

[0018] Figure 5 Shown is a graph showing the intestinal release results of lactic acid bacteria microcapsules in Example 4 of the present application.

[0019] Figure 6 Shown is a graph showing the inhibitory ability of the lactic acid bacteria microcapsules in Example 5 of the present application against common foodborne pathogens. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments and drawings to fully understand the purpose, scheme and effects of the present invention.

[0021] Example 1:

[0022] Preparation method of lactic acid bacteria microcapsules with antibacterial function:

[0023] (1) Preparation of core material: Take out cryopreserved Lactobacillus reuteri HLRE13, streak it onto an MRS plate with an inoculation loop, and incubate it anaerobically at 37°C for 24 hours. Pick a single colony and inoculate it into 5 mL of MRS liquid medium. Incubate it anaerobically at 37°C for 20 hours, and subculture it twice with an inoculum size of 1%. Centrifuge the activated bacterial suspension at 4°C and 5000 rpm for 5 minutes. Resuspend the obtained bacteria in sterile saline to obtain a bacterial suspension.

[0024] (2) Preparation of wall material: The mass fractions of sodium alginate and skim milk powder are 1.5%-2.5% and 5%-15% respectively, and the concentration of calcium chloride is 0.1M-0.5M;

[0025] (3) Preparation of lactic acid bacteria microcapsules: The lactic acid bacteria suspension and sodium alginate solution were mixed at a ratio of 1:4 to obtain a mixture I, and the mixture I was mixed with skim milk powder at a ratio of 1: (5%-15%) to obtain a mixture II. A 1 mL disposable syringe was used to add the mixture II dropwise into the calcium chloride solution. After standing at room temperature for 30 minutes, the mixture was washed three times with a peptone solution, then immersed in a chitosan solution for 40 minutes, and then washed twice with a peptone solution to obtain wet microcapsules. The wet capsules were vacuum freeze-dried to prepare lactic acid bacteria microcapsules.

[0026] The preparation process of the calcium chloride solution includes: using distilled water as a solvent, adding calcium chloride to prepare a calcium chloride solution with a molar concentration of 0.3M, and sterilizing it under high pressure at 121°C; the preparation process of the peptone solution includes: using deionized water as a solvent, adding peptone to prepare a peptone solution with a mass percentage of 0.1%, and sterilizing it under high pressure at 121°C; and the preparation process of the chitosan solution includes: using distilled water as a solvent, adding chitosan to prepare a solution with a mass percentage of 0.04%, then adding 2% glacial acetic acid, and then adjusting the pH value of the solution to 5.7-6.0 with a sodium hydroxide solution.

[0027] The specific flow chart of lactic acid bacteria microcapsule preparation is as follows Figure 1 As shown, the lactic acid bacteria microcapsule samples are Figure 2 shown.

[0028] Example 2:

[0029] Detection of the tolerance of lactic acid bacteria microcapsules and naked bacteria to gastric juice:

[0030] Preparation of gastric juice: Weigh 1 g of pepsin, pre-dissolve it in distilled water, then add 1.64 mL of hydrochloric acid solution (0.1 mol / L), stir evenly and make up to 100 mL, filter with a 0.22 μm sterile filter membrane, and store at 4°C until use.

[0031] Weigh 0.1g of microcapsules into a centrifuge tube, add 1mL of gastric juice, and place in a shaker at 37°C and 180r / min. Samples were taken at 0, 30, 60, 90, 120, 150 and 180min to determine the number of viable bacteria, and then the survival rate was calculated. At the same time, unencapsulated lactic acid bacteria (the core material of the present invention) were used as a blank control. The method was the same as above to determine the number of viable bacteria and calculate the survival rate. The results are as follows: Figure 3 shown.

[0032] Survival rate = number of viable cells measured at the t min / number of viable cells measured at the 0 min × 100%.

[0033] Depend on Figure 2 It can be seen that the lactic acid bacteria microcapsules of the present invention have strong tolerance to gastric juice.

[0034] Example 3:

[0035] Detection of the tolerance of lactic acid bacteria microcapsules and naked bacteria to bile salts:

[0036] Preparation of 0.3% bile salt: Weigh 0.68 g potassium dihydrogen phosphate and 0.3 g ox bile salt, dissolve in 100 mL distilled water, adjust the pH to 6.8 with hydrochloric acid solution, filter with a 0.22 μm sterile filter membrane, and store at 4°C until use.

[0037] Weigh 0.1g of microcapsules into a centrifuge tube, add 1mL of 0.3% bile salt solution, and place in a shaker at 37°C and 180r / min. Samples were taken at 0, 1, 2, and 3 hours to determine the number of viable bacteria, and then the survival rate was calculated. At the same time, unencapsulated lactic acid bacteria (the core material of the present invention) were used as a blank control. The method was the same as above to determine the number of viable bacteria and calculate the survival rate. The results are shown in Figure 2. Figure 3 shown.

[0038] Survival rate = number of viable cells measured at the th hour / number of viable cells measured at the 0th hour × 100%.

[0039] Depend on Figure 4 It can be seen that the lactic acid bacteria microcapsules of the present invention have a strong tolerance to 0.3% bile salt solution.

[0040] Example 4:

[0041] Testing the release of lactic acid bacteria microcapsules in the intestine:

[0042] Preparation of intestinal juice: Weigh 0.68 g of potassium dihydrogen phosphate and dissolve it in 50 mL of distilled water. Adjust the pH to 6.8 with sodium hydroxide solution. Then weigh 1 g of trypsin, mix the two and make up to 100 mL. Filter the prepared solution through a 0.22 μm sterile filter membrane and store at 4°C until use.

[0043] Weigh 0.1g of microcapsules into a centrifuge tube, add 1mL of intestinal fluid, and place in a shaker at 37℃ and 180r / min. Aspirate intestinal fluid at 0, 1, 2, and 3 hours and measure its absorbance at 600nm. The results are as follows: Figure 5 shown.

[0044] Depend on Figure 5 It can be seen that the lactic acid bacteria microcapsules of the present invention almost completely disintegrated in the intestinal fluid within 2 hours, and a large amount of Lactobacillus reuteri HLRE13 was released from the microcapsules.

[0045] Example 5:

[0046] Testing the inhibitory ability of lactic acid bacteria microcapsules against common foodborne pathogens:

[0047] Preparation of cystolysis solution: Weigh 3.58 g of disodium hydrogen phosphate and 1.05 g of citric acid, dissolve in 100 mL of deionized water, adjust the pH to 7.25 with sodium hydroxide solution, sterilize the prepared solution by autoclaving at 121°C, and store at 4°C until use.

[0048] Indicator strains (Enterobacter sakazakii, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus) were inoculated into LB liquid medium for overnight culture. The bacteria were then evenly spread on LB solid plates. 0.1 g of microcapsules were weighed into a centrifuge tube, 1 mL of encapsulation solution was added, and the microcapsules were shaken for 10 minutes until the capsules were completely encapsulated. 100 μL of the liquid was taken into an Oxford cup and incubated at 37°C for 12 hours. The diameter of the inhibition zone was measured. The results were as follows: Figure 6 shown.

[0049] Depend on Figure 6 It can be seen that the lactic acid bacteria microcapsules of the present invention have a good inhibitory effect on common foodborne pathogens.

[0050] The specific embodiments of the present invention are merely explanations of the present invention and are not intended to limit the present invention. After reading the present specification, those skilled in the art may make various modifications and improvements to the present embodiment as needed, all of which should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A lactic acid bacteria microcapsule, characterized in that: The microcapsule comprises a core material made of Lactobacillus reuteri HLRE13 and a wall material made of sodium alginate and skim milk powder; The Lactobacillus reuteri HLRE13 was deposited in the China Center for Type Culture Collection on December 8, 2023, with the deposit address being Wuhan University, Wuhan, China, with the number being CCTCC M20231208.

2. The lactic acid bacteria microcapsule according to claim 1, characterized in that The concentration of live bacteria of Lactobacillus reuteri HLRE13 in the microcapsule reaches 10 9 CFU / g, while the mass fractions (concentrations) of sodium alginate and skim milk powder are 1.5%-2.5% and 5%-15% respectively.

3. A microbial fermentation agent, characterized in that The invention comprises the lactic acid bacteria microcapsule according to claim 1.

4. The microbial fermentation agent according to claim 3, characterized in that The fermentation agent can be used in dairy products, beverages, medicines and health products. The lactic acid bacteria microcapsule according to claim 1 , which has a wide application in the preparation of broad-spectrum antibacterial drugs.

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