CN116926054B

CN116926054B - Armoured probiotics with catalytic function and preparation method thereof

Info

Publication number: CN116926054B
Application number: CN202310668278.5A
Authority: CN
Inventors: 杨琼琼; 王芷轩; 林丽炫; 张岳霖; 黄琬淳; 张薄博; 李植书; 陈文婷
Original assignee: Guangdong Yichao Biological Co ltd; Shantou University
Current assignee: Guangdong Yichao Biological Co ltd; Shantou University
Priority date: 2023-06-06
Filing date: 2023-06-06
Publication date: 2024-01-23
Anticipated expiration: 2043-06-06
Also published as: CN116926054A

Abstract

The invention relates to the technical field of probiotics, and discloses a armoured probiotic with a catalytic function and a preparation method thereof, wherein catalase and superoxide dismutase solution are added into tannic acid aqueous solution, and then the mixed solution and Fe are added ³⁺ Aqueous solution addition of aqueous bacterial solution (OD ₆₀₀ In the process of the preparation method, =1), MOPS buffer is added into the oscillated solution, supernatant is removed by centrifugation, the solution is washed three times by sterilized water, centrifugation is carried out, thalli are collected, the thalli are resuspended in ice bath calcium phosphate solution containing calcium chloride, L100-55 solution is added, pH is adjusted to be below 4-5, and the armoured probiotics are obtained by centrifugation and collection.

Description

Armoured probiotics with catalytic function and preparation method thereof

Technical Field

The invention relates to the technical field of probiotics, in particular to a armoured probiotic with a catalytic function and a preparation method thereof.

Background

At present, most of the commonly used probiotics embedding and delivering technologies are large-volume encapsulation or multicellular encapsulation, namely, a large amount or a plurality of probiotics are encapsulated in hydrogel or a polymer film, and the volume after encapsulation is often more than tens of microliters. The encapsulation with excessive volume is unfavorable for the transmission of substances, and is easy to cause the problems of metabolite accumulation, insufficient nutrient supply and the like, thereby influencing the normal growth and functions of the encapsulated cells. Meanwhile, an excessively thick encapsulation layer is not beneficial for the encapsulated probiotics to respond quickly to external or host environmental changes. In addition, the number of probiotics packaged by the multicellular packaging technology is randomly distributed and is nonuniform in size, the nonuniform size and the number of probiotics are difficult to accurately control the number of transplanted probiotics in clinical application, and the stability is low. As reported in the paper 'study of nitrifying bacteria embedding immobilization conditions', polyethylene glycol and sodium alginate are adopted to embed and immobilize bacteria to form 2-3mm pellets, and each embedding condition has a certain influence on degradation of immobilized pellets. In order to solve the defects and shortcomings in the technology, the invention provides a preparation method of a plant polyphenol armor shell for encapsulating probiotics by single cells, the technology can realize independent encapsulation of single cells, and the encapsulated armor shell can protect the probiotics from oxidative stress damage and resist gastrointestinal tract digestive environment. In addition, the armor shell can be shelled by mild biomolecules, and the probiotics after shelled can immediately recover growth and maintain the original biological functions of the probiotics.

Disclosure of Invention

(one) solving the technical problems

Aiming at the defects of the prior art, the invention provides the armoured probiotics with the catalytic function and the preparation method thereof, which realize the independent encapsulation of single cells, protect the probiotics from oxidative stress damage, resist gastrointestinal tract digestion environment, and keep the original biological functions of the probiotics after the shell is opened.

(II) technical scheme

1. The preparation method of the armoured probiotics with the catalytic function comprises the following steps:

adding catalase and superoxide dismutase solution into tannic acid water solution, manually shaking for 10-30s, and mixing the above solution and Fe ³⁺ The aqueous solution was added sequentially with an aqueous bacterial solution (OD ₆₀₀ In=1), the manual shake is repeated for 10-30s. Adding MOPS buffer into the oscillated solution, oscillating for 10-30s manually, centrifuging, removing supernatant, washing precipitate with sterilized water for 3 times, and centrifuging. Resuspension it in ice bath calcium phosphate solution containing calcium chloride, adding L100-55 solution to adjust pH of the solution to 4-5, centrifuging and collecting to obtain the armoured probiotics.

Preferably, the concentration of the catalase, superoxide dismutase and tannic acid solution is 300-400U/ml, 1800-2500U/ml and 5-20mM.

Preferably, the Fe ³⁺ The concentration of the aqueous solution is 5-20mM.

Preferably, the MOPS buffer is at a concentration of 10-20mM and is adjusted to 7-8 using a pH meter.

Preferably, the two centrifugation parameters are 10000-12000rpm, 10-30min.

Preferably, the concentration of the calcium chloride is 8-15M.

Preferably, the concentration of the L100-55 solution is 1-5mg/mL.

Preferably, the concentration of the hydrochloric acid solution is 0.1-0.5M.

(III) beneficial technical effects

Adding catalase and superoxide dismutase solution into tannic acid water solution, and mixing the above solution and Fe ³⁺ The aqueous solution was added sequentially with an aqueous bacterial solution (OD ₆₀₀ In=1), MOPS buffer was added to the shaken solution, the supernatant was removed by centrifugation, and the solution was centrifuged with sterile water. Re-suspending in ice bath calcium phosphate solution containing calcium chloride, adding L100-55 solution and hydrochloric acid solution,regulating pH to 4-5, swirling, centrifuging and collecting to obtain the armoured probiotics.

The single-cell encapsulation of the probiotics can be realized quickly, and the encapsulation armor shell can protect the probiotics from oxidative stress damage and resist gastrointestinal tract digestion environment. In addition, the armor shell can be shelled by mild biomolecules, and the probiotics after shelled can immediately recover growth and maintain the original biological functions of the probiotics.

Drawings

FIG. 1 is Fe ³⁺ Cross-linking complexation and de-cross-linking patterns of tannic acid.

FIG. 2 is Fe ³⁺ -cross-linking complexation of tannic acid and de-cross-linking uv spectra. TA: tannic acid; AA: ascorbic acid

FIG. 3 is a scanning electron microscope topography. EcN: coli Nissle 1917 probiotic; coated EcN: armoured probiotics

Fig. 4 is a scanning electron microscope topography. EcN: coli Nissle 1917 probiotic; coated EcN: armoured probiotics

Figure 5 is a probiotic growth curve. EcN: coli Nissle 1917 probiotic; coated EcN: armoured probiotics

Figure 6 is a graph of probiotic bacteria against oxidative stress damage by hydrogen peroxide. EcN: coli Nissle 1917 probiotic; coated EcN: armoured probiotics

Figure 7 is the survival rate of probiotics following simulated gastric fluid digestion. EcN: coli Nissle 1917 probiotic; coated EcN: armoured probiotics

Figure 8 is the survival rate of probiotics following simulated intestinal fluid digestion. EcN: unpackaged probiotics; coated EcN: encapsulated probiotics

Detailed Description

Example 1

1 preparation of armoured probiotics

Preparing 350U/ml catalase and 2000U/ml superoxide dismutase solution, adding 6 μl catalase and 30 μl superoxide dismutase into 125 μl10 mM tannic acid water solution, manually shaking for 20s, and mixing the above solution and 125 μl10 mM Fe ³⁺ The aqueous solution was added sequentially with 250. Mu.L of an aqueous bacterial solution (OD ₆₀₀ In=1), the manual concussion is repeated for 20s. 10mM MOPS buffer was prepared and adjusted to 7.2 with a pH meter. Adding 0.5 mM OPS buffer solution into the above oscillated bacterial solution, manually oscillating for 20s, centrifuging at 12000rpm for 10min, removing supernatant, washing precipitate with sterilized water for 3 times, and centrifuging at 12000rpm for 10min.

Resuspension it in ice bath calcium phosphate solution containing 0.9mL of 8M calcium chloride, shake for 5min manually, add 1mL of 1mg/mL L100-55 solution, continue to add 80 μl of 0.1M hydrochloric acid solution, adjust pH of the solution to 4-5 using pH meter, and collect by centrifugation to obtain the armoured probiotics.

2 removing probiotics armor

The packed bacterial pellet was resuspended in 500. Mu.L of phosphate buffer, shaken well, centrifuged to remove the supernatant, the bacterial pellet was resuspended in 1mL of ascorbic acid, and allowed to stand for 10min to remove the bacterial armor shell.

3 determination of growth curve

1mL OD ₆₀₀ Bacterial liquids=1, divided into three groups: the unembossed group, the embedded group and the unshelling group were removed after 4 hours of embedding, the OD value of the bacterial liquid was measured with an ultraviolet spectrophotometer every 1 hour, and the data were recorded.

4 scanning electron microscope for observing the shape of the bacteria

Taking a cover glass, soaking the cover glass in 1M hydrochloric acid overnight, then flushing the cover glass with absolute ethyl alcohol for three times, carrying out ultrasonic treatment, and drying the cover glass for later use. The samples were fixed in 5mM PIPES buffer (fixative) pH 6.5 containing 2% glutaraldehyde and allowed to stand overnight. The next day was washed 4 times with 5mM PIPES buffer pH 6.5, centrifuged to remove the supernatant, and lyophilized to obtain a powder, which was placed on a cover glass and observed by a scanning electron microscope.

5 in vitro gastrointestinal tract simulated digestion experiment

All digestion processes were carried out at 37℃and the digests were prepared as follows:

simulated gastric fluid (simulated gastric fluid, SGF) sodium bicarbonate (2.1 g), sodium chloride (2.758 g), magnesium chloride (0.1 mM), potassium chloride (0.515 g), potassium dihydrogen phosphate (0.122 g) and ammonium carbonate (0.048 g) were dissolved in 1000ml deionized water and the pH was adjusted to 2.5 with hydrochloric acid. Pepsin (final 2000U/ml) was added to the solution before use. Samples from bacterial fluids were mixed with simulated gastric fluid at a volume ratio of 1:1 and the pH was adjusted to 2.0. The mixture was stirred continuously at 100rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

Simulated intestinal fluid (simulated intestinal fluid, SIF) sodium bicarbonate (7.138 g), sodium chloride (2.244 g), magnesium chloride (0.35 mM), potassium chloride (0.511 g) and potassium dihydrogen phosphate (0.112 g) were dissolved in 1000mL deionized water, and the pH was adjusted to 7.0. Prior to use, bile salts (10 mM) and pancreatin (1:3000) were added to the solution. The bacterial liquid sample and the simulated intestinal liquid were mixed in a volume ratio of 1:1, and the pH was adjusted to 7.0. The mixture was stirred continuously at 100rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

6 experiment against Hydrogen peroxide damage

Dispersing thallus in 250 μl of 0.1%, 0.2%, 0.5%, and 1% hydrogen peroxide solution, centrifuging for 30min and 1 hr, cleaning with sterile water for 3 times, and adding ascorbic acid to remove tannic acid-Fe ³⁺ Washing the shell with sterile water for 3 times, dispersing into 250 μl of culture medium, plating, and counting bacteria.

Example 2

1 preparation of armoured probiotics

Preparing 400U/ml catalase and 2500U/ml superoxide dismutase solution, adding 10 μl catalase and 50 μl superoxide dismutase into 150 μl 20mM tannic acid water solution, manually shaking for 30s, and mixing the above solution and 130 μl 20mM Fe ³⁺ The aqueous solution was added sequentially with 300. Mu.L of an aqueous bacterial solution (OD ₆₀₀ In=1), the manual shake is repeated for 30s. Preparing 20mM MOPS buffer, adjusting to 7.2 with pH meter, adding 0.5 mM OPS buffer to the well-shocked thallus solution, manually shocking for 30s, centrifuging at 12000rpm for 30min, removing supernatant, washing precipitate with sterilized water for 3 times, centrifuging at 12000rpm for 10min, and collecting thallus.

The strain is resuspended in ice-bath calcium phosphate solution containing 1mL of 12M calcium chloride, 1mL of 3mg/mL L100-55 solution is added, 60 mu L of 0.3M hydrochloric acid solution is continuously added, the pH value of the solution is adjusted to 4.8 by using a pH meter, and the armoured probiotics are obtained by centrifugal collection.

2 removing probiotics armor

The packed bacterial pellet was resuspended in 1000. Mu.L of phosphate buffer, shaken well, centrifuged to remove the supernatant, the bacterial pellet was resuspended in 4mL of ascorbic acid and allowed to stand for 10min to remove the bacterial armor shell.

3 determination of growth curve

Take 3mL OD ₆₀₀ Bacterial liquids=1, divided into three groups: the unembossed group, the embedded group and the unshelling group were removed 8h after embedding, the OD value of the bacterial liquid was measured with an ultraviolet spectrophotometer every 2h, and the data were recorded.

4 scanning electron microscope for observing the shape of the bacteria

Taking a cover glass, soaking the cover glass in 1.5M hydrochloric acid overnight, then flushing the cover glass with absolute ethyl alcohol for three times, carrying out ultrasonic treatment, and drying the cover glass for later use. The samples were fixed in 5mM PIPES buffer (fixative) pH 6.5 containing 4% glutaraldehyde and allowed to stand overnight. The next day was washed 8 times with 5mM PIPES buffer pH 6.5, centrifuged to remove the supernatant, and lyophilized to obtain a powder, which was placed on a cover glass and observed by a scanning electron microscope.

5 in vitro gastrointestinal tract simulated digestion experiment

simulated gastric fluid (simulated gastric fluid, SGF) sodium bicarbonate (2.3 g), sodium chloride (2.655 g), magnesium chloride (0.1 mM), potassium chloride (0.520 g), potassium dihydrogen phosphate (0.122 g) and ammonium carbonate (0.048 g) were dissolved in 1000ml deionized water and the pH was adjusted to 2.3 with hydrochloric acid. Pepsin (final 2000U/ml) was added to the solution before use. Samples from bacterial fluids were mixed with simulated gastric fluid at a volume ratio of 1:1 and the pH was adjusted to 2.0. The mixture was stirred continuously at 150rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

Simulated intestinal fluid (simulated intestinal fluid, SIF) sodium bicarbonate (7.247 g), sodium chloride (2.186 g), magnesium chloride (0.32 mM), potassium chloride (0.532 g) and potassium dihydrogen phosphate (0.119 g) were dissolved in 1000mL deionized water and the pH was adjusted to 7.0. Prior to use, bile salts (10 mM) and pancreatin (1:3000) were added to the solution. The bacterial liquid sample and the simulated intestinal liquid were mixed in a volume ratio of 1:1, and the pH was adjusted to 7.0. The mixture was stirred continuously at 120rpm for 4h at 37 ℃. The liquid was then diluted to plate and counted.

6 experiment against Hydrogen peroxide damage

Dispersing thallus in 300 μl of 0.1%, 0.2%, 0.5%, and 1% hydrogen peroxide solution, centrifuging for 30min and 1 hr respectively, cleaning with sterile water for 3 times, and adding ascorbic acid to remove tannic acid-Fe ³⁺ The shell is washed by sterile water for 6 times, finally dispersed into 300 mu L of culture medium, plated and counted.

Example 3

1 preparation of armoured probiotics

Preparing 300U/ml catalase and 1800U/ml superoxide dismutase solution, adding 6 μl catalase and 30 μl superoxide dismutase into 125 μl 5mM tannic acid water solution, manually shaking for 10s, and mixing the above solution and 125 μl 5mM Fe ³⁺ The aqueous solution was added sequentially with 250. Mu.L of an aqueous bacterial solution (OD ₆₀₀ In=1), the manual shake is repeated for 10s. Preparing 10mM MOPS buffer, adjusting pH to 7 with pH meter, adding into 0.5mL of the shake solution, manually shaking for 10s, centrifuging at 10000rpm for 10min, removing supernatant, washing precipitate with sterilized water for 2 times, centrifuging at 10000rpm for 10min, and repeating the above steps.

Resuspension it in ice bath calcium phosphate solution containing 0.9mL of 12.5M calcium chloride, shake for 10min manually, then add 1mL of 1mg/mL L100-55 solution, continue to add 80 μl of 0.1M hydrochloric acid solution, adjust the pH of the solution to 4.4 using a pH meter, and collect the armoured probiotics by centrifugation.

2 removing probiotics armor

The packed bacterial pellet was resuspended in 800. Mu.L of phosphate buffer, shaken well, centrifuged to remove the supernatant, the bacterial pellet was resuspended in 1mL of ascorbic acid and allowed to stand for 20min to remove the bacterial armor shell.

3 determination of growth curve

2mL of bacterial liquid with OD=1 is taken and divided into three groups: the unembossed group, the embedded group and the unshelling group were removed after 4 hours of embedding, the OD value of the bacterial liquid was measured with an ultraviolet spectrophotometer every 2 hours, and the data were recorded.

4 scanning electron microscope for observing the shape of the bacteria

Taking a cover glass, soaking the cover glass in 1M hydrochloric acid overnight, then flushing the cover glass with absolute ethyl alcohol for three times, carrying out ultrasonic treatment, and drying the cover glass for later use. The samples were fixed in 5mM PIPES buffer (fixative) pH 6.5 containing 2% glutaraldehyde and allowed to stand overnight. The next day was washed 6 times with 5mM PIPES buffer pH 6.5, centrifuged to remove the supernatant, and lyophilized to obtain a powder, which was placed on a cover glass and observed by a scanning electron microscope.

5 in vitro gastrointestinal tract simulated digestion experiment

simulated gastric fluid (simulated gastric fluid, SGF) sodium bicarbonate (2.056 g), sodium chloride (2.745 g), magnesium chloride (0.1 mM), potassium chloride (0.508 g), potassium dihydrogen phosphate (0.122 g) and ammonium carbonate (0.048 g) were dissolved in 1000ml deionized water and the pH was adjusted to 2.5 with hydrochloric acid. Pepsin (final 2000U/ml) was added to the solution before use. Samples from bacterial fluids were mixed with simulated gastric fluid at a volume ratio of 1:1 and the pH was adjusted to 2.0. The mixture was stirred continuously at 100rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

Simulated intestinal fluid (simulated intestinal fluid, SIF) sodium bicarbonate (7.138 g), sodium chloride (2.244 g), magnesium chloride (0.33 mM), potassium chloride (0.507 g) and potassium dihydrogen phosphate (0.109 g) were dissolved in 1000mL deionized water, and the pH was adjusted to 7.0. Prior to use, bile salts (10 mM) and pancreatin (1:3000) were added to the solution. The bacterial liquid sample and the simulated intestinal liquid were mixed in a volume ratio of 1:1, and the pH was adjusted to 7.0. The mixture was stirred continuously at 100rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

6 experiment against Hydrogen peroxide damage

Dispersing thallus in 250 μl of 0.1%, 0.2%, 0.5%, and 1% hydrogen peroxide solution, centrifuging for 1 hr and 2 hr respectively, cleaning with sterile water for 3 times, adding ascorbic acid to remove tannic acid-Fe ³⁺ Washing the shell with sterile water for 3 times, dispersing into 250 μl of culture medium, plating, and counting bacteria.

Example 4

1 preparation of armoured probiotics

Preparing 380U/ml catalase and 2200U/ml superoxide dismutase solution, adding 8 μl catalase and 25 μl superoxide dismutase into 150 μl 15mM tannic acid water solution, manually shaking for 15s, mixing the above solution and 130 μl 18mM Fe ³⁺ The aqueous solution was added sequentially with 250. Mu.L of an aqueous bacterial solution (OD ₆₀₀ In=1), the manual concussion is repeated for 20s. Preparing 15mM MOPS buffer, adjusting to 7.5 with pH meter, adding 1.5mL of the shaking solution, manually shaking for 10s, centrifuging at 12000rpm for 15min, removing supernatant, washing the precipitate with sterilized water for 4 times, centrifuging at 12000rpm for 15min, and repeating the above steps.

Resuspension it in ice bath calcium phosphate solution containing 1mL 15M calcium chloride, shake for 20min manually, add 3mL 5mg/mL L100-55 solution, continue to add 80 μL 0.5M hydrochloric acid solution, adjust pH of the solution to 4.2 with pH meter, centrifugate and collect to obtain the armoured probiotic.

2 removing probiotics armor

3 determination of growth curve

1mL of bacterial liquid with OD=1 is taken and divided into three groups: the unembossed group, the embedded group and the unshelling group were removed 4 hours after embedding, the OD value of the bacterial liquid was measured once every other hour by using an ultraviolet spectrophotometer, and the data were recorded.

4 scanning electron microscope for observing the shape of the bacteria

5 in vitro gastrointestinal tract simulated digestion experiment

6 experiment against Hydrogen peroxide damage

Dispersing thallus in 250 μl of 0.1%, 0.2%, 0.5%, and 1% hydrogen peroxide solution, centrifuging for 30min and 1 hr, cleaning with sterile water for 3 times, and adding ascorbic acid to remove tannic acid-Fe ³⁺ Washing the shell with sterile water for 3 times, dispersing into 300 μl of culture medium, plating, and counting bacteria.

Example 5

1 preparation of armoured probiotics

Preparing 380U/ml hydrogen peroxidase and 2000U/ml superoxide dismutase solution, adding 6 μl hydrogen peroxidase and 30 μl superoxide dismutase into 125 μl 15mM tannic acid water solution, manually shaking for 15s, mixing the above solution and 125 μl10 mM Fe ³⁺ The aqueous solution was added sequentially to 300 μl of aqueous bacterial solution (od600=1) and the shaking was repeated manually for 30s. Preparing 15mM MOPS buffer, adjusting to 7.2 with pH meter, adding into 0.5mL of the shake solution, manually shaking for 30s, centrifuging at 10000rpm for 10min, removing supernatant, washing the precipitate with sterilized water for 4 times, centrifuging at 10000rpm for 10min, and repeating the above steps.

Resuspension it in ice bath calcium phosphate solution containing 0.9mL of 10M calcium chloride, shake for 10min manually, add 1mL of 2mg/mL L100-55 solution, continue to add 70 μl of 0.1M hydrochloric acid solution, adjust pH of the solution to 4.0 with pH meter, and collect by centrifugation to obtain the armoured probiotics.

2 removing probiotics armor

The packed bacterial pellet was resuspended in 300. Mu.L of phosphate buffer, shaken well, centrifuged to remove the supernatant, the bacterial pellet was resuspended in 2mL of ascorbic acid, and allowed to stand for 30min to remove the bacterial armor shell.

3 determination of growth curve

2mL of bacterial liquid with OD=1 is taken and divided into three groups: the unembossed group, the embedded group and the unshelling group were removed 4 hours after embedding, the OD value of the bacterial liquid was measured once every other hour by using an ultraviolet spectrophotometer, and the data were recorded.

4 scanning electron microscope for observing the shape of the bacteria

Taking a cover glass, soaking the cover glass in 1M hydrochloric acid overnight, then flushing the cover glass with absolute ethyl alcohol for three times, carrying out ultrasonic treatment,

and (5) drying for later use. The samples were fixed in 5mM PIPES buffer (fixative) pH 6.5 containing 2% glutaraldehyde and allowed to stand overnight. The next day was washed 4 times with 5mM PIPES buffer pH 6.5, centrifuged to remove the supernatant, and lyophilized to obtain a powder, which was placed on a cover glass and observed by a scanning electron microscope.

5 in vitro gastrointestinal tract simulated digestion experiment

simulated gastric fluid (simulated gastric fluid, SGF) sodium bicarbonate (2.022 g), sodium chloride (2.813 g), magnesium chloride (0.12 mM), potassium chloride (0.535 g), potassium dihydrogen phosphate (0.125 g) and ammonium carbonate (0.052 g) were dissolved in 1000ml deionized water and the pH was adjusted to 2.5 with hydrochloric acid. Pepsin (final 2000U/ml) was added to the solution before use. Samples from bacterial fluids were mixed with simulated gastric fluid at a volume ratio of 1:1 and the pH was adjusted to 2.0. The mixture was stirred continuously at 100rpm for 4h at 37 ℃. The liquid was then diluted to plate and counted.

Simulated intestinal fluid (simulated intestinal fluid, SIF) sodium bicarbonate (7.146 g), sodium chloride (2.238 g), magnesium chloride (0.38 mM), potassium chloride (0.521 g) and potassium dihydrogen phosphate (0.115 g) were dissolved in 1000mL deionized water and the pH was adjusted to 7.0. Prior to use, bile salts (10 mM) and pancreatin (1:3000) were added to the solution. The bacterial liquid sample and the simulated intestinal liquid were mixed in a volume ratio of 1:1, and the pH was adjusted to 7.0. The mixture was stirred continuously at 120rpm for 2h at 37 ℃. The liquid was then diluted to plate and counted.

6 experiment against Hydrogen peroxide damage

Dispersing thallus in 280 μl of 0.1%, 0.2%, 0.5%, and 1% hydrogen peroxide solution, centrifuging for 30min and 1 hr, cleaning with sterile water for 5 times, and adding ascorbic acid to remove tannic acid-Fe ³⁺ Washing the shell with sterile water for 5 times, dispersing into 280 mu L of culture medium, plating, and counting bacteria.

Detection result

As shown in fig. 1, fe3+ and tannic acid are both pale yellow, and when both 1:1 are mixed to give a greenish black color, indicating that the complexing reaction takes place immediately after mixing. Upon addition of ascorbic acid, the greenish black in the solution turned pale yellow, indicating that the complex had been destroyed. The concentration of ascorbic acid used in the experiment is usually 5-10mM, preferably 5mM, to ensure that the probiotic is not killed by ascorbic acid, and not more than 20mM.

FIG. 2 is a graph showing the ultraviolet spectrum of the crosslinking complexation and decrosslinking of Fe3+ -tannin. In the ultraviolet spectrum shown in FIG. 2, fe3+ has no ultraviolet absorption, and tannic acid-Fe3+ have similar ultraviolet absorption, and bacteria have absorption peaks at 250nm and 280 nm. After addition of ascorbic acid, the UV absorption was reduced to be close to that of Fe3+, indicating that the complex had been destroyed. The ascorbic acid treatment time is usually 10-15min, and in order to ensure that the complex has been destroyed, it is preferably shaken during the ascorbic acid treatment.

Fig. 3 is a scanning electron microscope morphology of the probiotic bacteria and the armoured probiotic bacteria of the present invention. The surface of the probiotics is wrinkled, the edge is sharp, and the surface of the probiotics subjected to the armor treatment is provided with a snowflake-shaped coating, and the outer side of the coating is loose. Normally the armoured probiotic is slightly larger than the unarmed probiotic to the naked eye under an electron microscope.

Fig. 4 is a scanning electron microscope morphology of the probiotics and the armoured probiotics of the invention. The probiotics are elliptical, the surface is flat, the edge is sharp, the surface of the probiotics subjected to the armor treatment is provided with a shell with lighter color, and the thickness of the shell is about 10-20nm; the cells were completely embedded. Typically the edge of the armoured probiotic is smoother than the edge of the probiotic.

Figure 5 is a graph of the growth of probiotics, armoium probiotics of the present invention. The probiotics enter logarithmic phase growth after 1h, but the armoured probiotics do not grow within 0-8h, and resume growth after 8h, which indicates that the armoured shell is automatically released at this time, and the armoured shell makes the bacteria in dormant state in 8h before, and does not kill the probiotics; the addition of ascorbic acid at 4 hours removed the armoured shell and the immediate recovery of the growth of the probiotic was found, further proving that the armour only put the bacteria in a dormant state and did not have an effect on the growth of the probiotic. The concentration of ascorbic acid used should be slightly lower, so that the embedding is released without inhibiting the growth of the cells.

Figure 6 is a graph of the resistance of probiotics of the present invention to oxidative stress damage by hydrogen peroxide. The probiotics and the armoured probiotics are respectively treated in hydrogen peroxide solutions with different concentrations for 30min and 1h,0.1% of hydrogen peroxide does not cause bacterial death, 0.5% of hydrogen peroxide has serious killing effect on the probiotics without the armoured probiotics, the killing effect is gradually enhanced along with the extension of the treatment time, almost all the probiotics are killed, but the armoured probiotics can resist the damage of high-concentration hydrogen peroxide, the viable count is still up to 106CFU/ml after the treatment for 1h, and the effective concentration of the probiotics is 106CFU/ml under the normal condition.

Figure 7 shows the survival rate of probiotics of the present invention after digestion with simulated gastric fluid. After the non-armoured probiotics are digested by artificial simulated gastric juice, the survival rate is obviously reduced, and the viable count is less than 104CFU/ml; the survival rate of the probiotics subjected to the armour is reduced, but the survival rate is obviously improved compared with that of probiotics not subjected to the armour, and the viable count is still up to 108CFU/ml after 2 hours of digestion. Proved that the armoured probiotics have stronger capability of resisting gastric acid degradation, and can be maintained for 2 hours without reduction.

Figure 8 shows the survival rate of probiotics of the present invention after simulated intestinal fluid digestion. The survival rate of the probiotics which are not subjected to the armour is not affected after the probiotics are digested by artificial simulation intestinal juice; the probiotic bacteria subjected to the armor are digested and initially packaged by the shell, so that the bacteria are in a dormant state, the number of viable bacteria is less than that of the probiotic bacteria not subjected to the armor, the armor is gradually released along with the time extension, the number of viable bacteria is gradually increased, and the number of the viable bacteria is more than that of the probiotic bacteria not subjected to the armor until 4 hours. Proved that the armoured probiotics can recover the activity of the non-armoured probiotics in the intestinal environment, immediately recover the growth and maintain the original biological functions of the probiotics.

Claims

1. A armoured probiotic with catalytic function, characterized in that: the preparation method comprises the following steps:

adding catalase and superoxide dismutase solution into tannic acid water solution with concentration of 5-20mM, manually oscillating 10-30s, sequentially adding above mixed solution and 5-20mM Fe3+ water solution into OD ₆₀₀ In bacterial aqueous solution=1, repeated manual concussion is 10-30 s; adding MOPS buffer with pH of 7-8 into the oscillated solution, oscillating manually for 10-30s, centrifuging, removing supernatant, washing precipitate with sterilized water for 2-6 times, and centrifuging; resuspension it in ice bath calcium phosphate solution containing calcium chloride, wherein the concentration of calcium chloride is 8-15M, adding 1-5mg/mL L100-55 solution and hydrochloric acid solution, adjusting pH of the solution to 4-5, centrifuging and collecting to obtain the armoured probiotic.

2. A catalytic armoured probiotic bacterium according to claim 1, characterized in that: the concentration of the catalase and superoxide dismutase is 300-400U/ml and 1800-2500U/ml respectively.

3. A catalytic armoured probiotic bacterium according to claim 1, characterized in that: the concentration of the MOPS buffer is 10-20mM, and the MOPS buffer is adjusted to 7-8 by a pH meter.

4. A catalytic armoured probiotic bacterium according to claim 1, characterized in that: the two times of centrifugation parameters are 10000-12000rpm and 10-30min.

5. A catalytic armoured probiotic bacterium according to claim 1, characterized in that: the concentration of the hydrochloric acid solution is 0.1-0.5M.