Enteric lignin bacteria-embedded particles for treating ulcerative colitis and preparation thereof

Abstract

The present invention discloses an enteric-coated lignin bacterial embedding particle for treating ulcerative colitis and its preparation. Based on the principle of electrostatic interaction, the present invention uses anionic lignin to embed bacteria. Under neutral or weakly alkaline conditions, the surface of the bacteria carries a negative charge. Through electrostatic interaction, a layer of Ca<supgt;2+< / supgt; is adsorbed on the surface of the bacteria, and then Ca<supgt;2+< / supgt; adsorbs anionic lignin to form a nano-coating on the surface of the bacteria, realizing the embedding of the bacteria. The preparation process of the enteric-coated lignin bacterial embedding particle of the present invention is simple. It not only increases the viable count of bacteria in simulated gastric juice and is smoothly released in simulated intestinal juice, but also lignin and the embedded bacteria can play a synergistic therapeutic effect, effectively treating DSS-induced colitis in mice and increasing the colon length of diseased mice, providing new ideas for the development of new oral dosage forms of bacteria, and expanding the high-value application range of lignin, promoting the development and application of biomass resources.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and in particular relates to enteric lignin bacteria-embedded particles for treating ulcerative colitis and a preparation method thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic intestinal inflammatory disease with a high incidence in the United States and Western European countries. In recent years, with the improvement of the living standards of Chinese residents, the number of patients with ulcerative colitis in my country has increased year by year, and the patient population has shown a trend of younger age. The onset of ulcerative colitis is the lower colon and rectum, and the clinical manifestations are abdominal pain, diarrhea, and blood in the stool, which seriously affect the patient's daily life and may even develop into cancer. There are currently two main treatments for ulcerative colitis, namely drug therapy and surgical treatment. Drug therapy includes sulfonamides (5-aminosalicylic acid), corticosteroids, immunosuppressants, antibiotics, etc., but these drugs can only relieve the inflammation and cannot completely cure the inflammation. Surgical treatment refers to the surgical removal of part or all of the colon, but the operation has certain risks and complications. In summary, there is an urgent need to find a new treatment method. A large number of studies have found that the etiology of ulcerative colitis involves multiple factors, including intestinal flora imbalance, infection, immune response, and psychological factors. Among them, intestinal flora plays an important role in the pathogenesis of ulcerative colitis and may determine the severity of intestinal inflammation (Journal of Clinical Microbiology, 2006, 44 (11): 4136-4141). In response to intestinal flora imbalance, scientists have developed a new treatment for ulcerative colitis - fecal microbiota transplantation, which first extracts fecal microbiota from healthy individuals, and after certain treatment, delivers the fecal microbiota to the patient's gastrointestinal tract through oral administration to restore the balance of the patient's intestinal flora. However, when bacteria are taken orally, they will be attacked by gastric acid, bile salts, enzymes and excessive reactive oxygen species at the site of inflammation, resulting in a serious shortage of live bacteria reaching the site of inflammation, which cannot play a therapeutic role. To address this problem, two methods can be used to solve it: constructing engineered bacteria and embedding bacteria. For the engineered bacteria method, few people can prove that engineered bacteria can effectively prevent or treat humans or animals. For encapsulated bacteria, it refers to the use of emulsification, extrusion, electrostatic self-assembly, spray drying, freeze drying, ultrasonic vacuum spray drying and other methods to wrap a layer of shell on the surface of bacteria. Encapsulating bacteria can significantly increase the retention of bacteria in the gastrointestinal environment, which is an effective method. Among them, the use of emulsification, extrusion, spray drying, freeze drying, and ultrasonic vacuum spray drying to encapsulate bacteria is to encapsulate multiple bacteria, so the resulting product has a large particle size, uneven size, and bacteria are easy to leak. Liu et al. used liposomes, namely dioleyl phosphate (DOPA), to encapsulate bacteria based on the principle of electrostatic interaction. The preparation method is simple and efficient, significantly increasing the retention of bacteria in the gastrointestinal tract, and animal experiments have proved that the prepared encapsulated bacteria can treat ulcerative colitis (Nat. Commun, 2019, 10, 5783).In addition, the team also used enteric polymer-methacrylic acid copolymer type C (L100-55) and silk protein to encapsulate bacteria, and also achieved good encapsulation and treatment effects (Adv. Mater, 2020, 32, 2002406; Small, 2021, 17, 2101810). In short, the team proposed a new bacterial encapsulation method for the first time, which self-assembled on the surface of a single bacterium. The resulting product was pH-responsive, significantly increased the retention of bacteria in the gastrointestinal environment, and achieved targeted release of bacteria in the intestine. The disadvantage is that this method only encapsulates bacteria to increase bacterial retention, but the materials used to encapsulate bacteria, such as DOPA and L100-55, have no anti-inflammatory effect themselves and cannot synergistically treat ulcerative colitis. Although silk protein has certain anti-inflammatory effects, it is expensive. Similarly, some research teams have used tannic acid to encapsulate bacteria. Tannic acid also has certain anti-inflammatory effects, but tannic acid has low safety and is on the list of Class 3 carcinogens (ACS Nano, 2022, 16, 4041; Nat. Commun, 2022, 13, 1).

[0003] Lignin is a natural polymer compound with abundant reserves in nature and is the largest biomass resource besides cellulose. Lignin exists naturally in plants and gives plants rigidity and structure, so lignin is green and safe with good biocompatibility. At the same time, lignin dissolves under alkaline conditions and does not dissolve under acidic conditions, that is, lignin has pH responsiveness and can achieve targeted release. Lignin is also a crude fiber that can regulate intestinal health, and lignin is rich in phenolic hydroxyl groups, which can scavenge free radicals and play an anti-inflammatory role (Industrial crops and products, 2011, 33, 259-276). Therefore, using lignin to embed bacteria can not only increase the retention of bacteria in the gastrointestinal environment, but lignin itself can also play an anti-inflammatory role, achieving a dual therapeutic effect.

[0004] In summary, the existing bacterial encapsulation methods have the disadvantages of large product particle size, uneven size, easy leakage, complicated preparation process, lack of synergistic therapeutic effect, etc. Therefore, it is necessary to develop a new bacterial encapsulation method with small product particle size, high bacterial retention, simple and efficient, synergistic treatment, and low price. Summary of the invention

[0005] In order to solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing enteric lignin bacteria-embedded particles for treating ulcerative colitis.

[0006] This process uses the principle of electrostatic interaction and anionic lignin to embed bacteria, achieving the embedding of single bacteria, increasing the retention of bacteria in the gastrointestinal environment, and can effectively treat dextran sodium sulfate (DSS)-induced colitis in mice. Bacteria carry a negative charge under near-neutral or weakly alkaline conditions, and a layer of Ca2+ is adsorbed on the bacterial surface through electrostatic interaction. 2+ , then Ca 2+ Then, anionic lignin is adsorbed to form a nano-coating on the surface of bacteria, thereby obtaining lignin bacteria-embedded particles. This process is simple and efficient, uses green and natural lignin, is safer, and has lower costs. In addition, the phenolic hydroxyl groups rich in lignin can remove excessive free radicals in the inflammatory site, exert anti-inflammatory effects, and achieve a dual effect of treating inflammation.

[0007] Another object of the present invention is to provide enteric lignin bacteria-embedded particles prepared by the above preparation method for treating ulcerative colitis.

[0008] The enteric lignin bacteria-embedded particles of the present invention have a simple preparation process, which not only increases the number of live bacteria in simulated gastric fluid and is smoothly released in the simulated intestinal fluid, but also the lignin and the embedded bacteria can play a synergistic therapeutic role, effectively treat DSS-induced colitis in mice, and increase the colon length of diseased mice, providing new ideas for the development of new oral bacterial dosage forms, expanding the high-value application range of lignin, and promoting the development and application of biomass resources.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing enteric lignin bacteria-embedded particles comprises the following steps:

[0011] (1) Mix the bacterial solution with the CaCl2 solution, adsorb it for a period of time, and then wash it to obtain the adsorbed CaCl2 solution. 2+ of bacteria;

[0012] (2) Adsorb Ca 2+ The bacteria were added to the anionic lignin solution, adsorbed for a period of time, and then washed to obtain enteric lignin bacteria-embedded particles.

[0013] Preferably, in step (1), the volume ratio of the CaCl2 solution to the bacterial solution is 1:0.1 to 1:3; the OD value of the bacteria in the bacterial solution is 0.5 to 1; and the CaCl2 solution is 0.1 to 1.2 mol / L.

[0014] Preferably, in step (2), the volume ratio of the anionic lignin solution to the bacterial solution is 1:0.5 to 1:2, and the concentration of the anionic lignin solution is 0.1 to 3.0 g / L, more preferably 0.2 to 1.5 g / L.

[0015] Preferably, the bacteria in the bacterial solution of step (1) is at least one of Escherichia coli Nissle 1917, Escherichia coli, Staphylococcus aureus, lactic acid bacteria, Bifidobacterium, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Bacillus licheniformis and Bacillus subtilis.

[0016] Preferably, the adsorption time in step (1) is 15 to 90 minutes.

[0017] Preferably, the washing in step (1) is centrifugal washing, the speed of the centrifugal washing is 5000-10000 r / min, the time is 10-30 min, and the number of centrifugal washing is 2-5 times.

[0018] Preferably, in the anionic lignin solution of step (2), the anionic lignin is carboxylic acid-modified lignin; more preferably, it is carboxymethyl lignin.

[0019] Preferably, the adsorption time in step (2) is 0.25 to 3 h, more preferably 0.5 to 1.5 h.

[0020] Preferably, the washing in step (2) is centrifugal washing, the speed of the centrifugal washing is 5000-10000 r / min, the time is 10-30 min, and the number of centrifugal washing is 2-5 times.

[0021] The enteric lignin bacteria-embedded particles prepared by the preparation method.

[0022] The enteric lignin bacteria-embedded particles of the present invention can not only effectively increase the retention of bacteria in the gastrointestinal environment, but also play a synergistic therapeutic role, and have application prospects in the treatment of ulcerative colitis and other metabolic diseases.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) The present invention uses lignin as raw material, which comes from plants, has abundant reserves, is green and natural, and is therefore safer and cheaper.

[0025] (2) The lignin used in the present invention is pH responsive, that is, it precipitates under acidic conditions and dissolves under neutral or weakly alkaline conditions, which can protect the bacteria from passing through the highly acidic gastric juice smoothly and finally be released in the colon.

[0026] (3) The present invention performs self-assembly on the surface of a single bacterium to form a nano-coating to encapsulate the bacteria. The resulting product has a small particle size and uniform size, which can effectively increase the retention of bacteria in the gastrointestinal environment. In addition, the lignin is rich in phenolic hydroxyl groups, which can exert an anti-inflammatory effect and achieve a dual therapeutic effect.

[0027] (4) The preparation process is simple, the preparation technology is easy to master, and it is easy to put into actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The right picture shows the carboxymethyl lignin solution prepared in step (1) of Example 1. Figure 1 The left figure shows the dissolution of the sodium lignin sulfonate solution prepared in step (1) of comparative example 1 under the condition of pH = 1.2. It can be seen that under the condition of pH = 1.2, carboxymethyl lignin is in a precipitated state and sodium lignin sulfonate is in a dissolved state, while the normal range of pH value of human gastric juice is 0.9-1.5, so sodium lignin sulfonate cannot protect bacteria in the gastric juice environment.

[0029] Figure 2 The number of viable bacteria in the simulated gastric juice of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles prepared in step (3) of Example 1. After being treated with simulated gastric juice for the same time, the number of viable bacteria in the lignin Escherichia coli Nissle1917 embedded particles was higher than that of the unembedded Escherichia coli Nissle1917, indicating that the embedding method can effectively protect the bacteria from gastric juice and increase the retention of bacteria in the gastric juice environment.

[0030] Figure 3 The number of viable bacteria in the simulated intestinal fluid of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles prepared in step (3) of Example 1. After being treated with simulated intestinal fluid for the same time, both the lignin Escherichia coli Nissle1917 embedded particles and the unembedded Escherichia coli Nissle1917 were able to proliferate normally, indicating that the embedding method does not affect the growth ability of bacteria.

[0031] Figure 4 The scanning electron microscope images of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles, carboxymethyl lignin Escherichia coli embedded particles, and carboxymethyl lignin Staphylococcus aureus embedded particles prepared in step (3) of Example 1, Example 2, and Example 3, respectively. It can be seen from the figure that the surfaces of the three carboxymethyl lignin bacteria embedded particles are granular, and the particles are carboxymethyl lignin, indicating that the lignin shell is successfully embedded on the surface of the bacteria.

[0032] Figure 5This is a graph showing the change in colon length during the treatment of a mouse colitis model induced by the chemical drug dextran sulfate sodium (DSS) using carboxymethyl lignin-encapsulated Escherichia coli Nissle1917 prepared in step (3) of Example 1, wherein A is the colon of a normal mouse, B is the colon of a mouse fed with DSS but not treated with any drug, and C, D, and E are the colons of mice fed with DSS and treated with unencapsulated Escherichia coli Nissle1917, a mixture of unencapsulated Escherichia coli Nissle1917 and carboxymethyl lignin, and carboxymethyl lignin-encapsulated Escherichia coli Nissle1917 particles, respectively. As can be seen from the figure, after DSS-induced colitis in mice, the length of the mice's colon decreased. After treatment with Escherichia coli Nissle1917 and a mixture of Escherichia coli Nissle1917 and carboxymethyl lignin, the length of the mice's colon did not increase significantly, that is, the symptoms of colitis were not alleviated, because the Escherichia coli Nissle1917 delivered to the mice was killed by adverse environments such as simulated gastric juice, resulting in insufficient number of live bacteria reaching the site of colitis and unable to play a therapeutic role. After treatment with carboxymethyl lignin Escherichia coli Nissle1917-encapsulated particles, the length of the mice's colon increased significantly, indicating that encapsulation of Escherichia coli Nissle1917 can effectively protect Escherichia coli Nissle1917 and has a good effect on the treatment of colitis in mice.

[0033] Figure 6 The viable bacterial count of the sodium lignin sulfonate Escherichia coli Nissle1917 embedded particles prepared in step (3) of Comparative Example 1 in simulated gastric juice was shown in Table 1. After being treated with simulated gastric juice for the same period of time, the viable bacterial count of the sodium lignin sulfonate Escherichia coli Nissle1917 embedded particles did not increase compared with the unembedded Escherichia coli Nissle1917, indicating that embedding bacteria with sodium lignin sulfonate cannot effectively protect the bacteria from gastric juice because sodium lignin sulfonate is dissolved in the simulated gastric juice.

[0034] Figure 7 This is the scanning electron microscope image of comparative example 2. It can be seen that the surfaces of the unencapsulated Escherichia coli Nissle1917 and Escherichia coli are wrinkled, while the surface of the unencapsulated Staphylococcus aureus is smooth. The reason for the surface wrinkling is that the bacteria are treated with ethanol during the preparation of the scanning electron microscope sample, which causes the bacteria to dehydrate and shrink. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0036] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. All raw materials, reagents, etc., whose manufacturers are not specified, are conventional products that can be purchased commercially.

[0037] Example 1

[0038] (1) Dissolve 800 mg of carboxymethylated lignin (CML) in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0039] (2) After activating Escherichia coli Nissle 1917 (purchased from Mingzhou Biotechnology Co., Ltd.) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it three times with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0040] (3) Take 1 mL of bacterial liquid, add 1 mL of CaCl2 solution, adsorb for 30 minutes, centrifuge at 10,000 rpm for 15 minutes, remove excess CaCl2, and wash three times with sterile water. After washing, add 1 mL of carboxymethyl lignin solution, treat for 30 minutes, centrifuge at 10,000 rpm for 15 minutes, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Escherichia coli Nissle1917 embedded particles.

[0041] Figure 1 The right figure and the left figure respectively show the dissolution of the carboxymethyl lignin solution prepared in step (1) of Example 1 and the sodium lignin sulfonate solution prepared in step (1) of Comparative Example 1 under the condition of pH = 1.2. It can be seen that under the condition of pH = 1.2, the carboxymethyl lignin is in a precipitated state and the sodium lignin sulfonate is in a dissolved state, while the normal range of the pH value of human gastric juice is 0.9 to 1.5, so the sodium lignin sulfonate cannot protect bacteria in the gastric juice environment.

[0042] Figure 2 is the number of viable bacteria in the simulated gastric juice of the lignin Escherichia coli Nissle1917 embedded particles prepared in step (3) of Example 1. After being treated with simulated gastric juice for the same time, the number of viable bacteria in the lignin Escherichia coli Nissle1917 embedded particles is higher than that of the non-embedded Escherichia coli Nissle1917, indicating that the embedding method can effectively protect the bacteria from gastric juice, because the carboxymethyl lignin precipitates in the simulated gastric juice and tightly wraps the bacteria, thereby increasing the retention of bacteria in the gastric juice environment.

[0043] Figure 3It is the number of viable bacteria in the simulated intestinal fluid of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles prepared in step (3) of Example 1. After being treated with the simulated intestinal fluid for the same time, both the lignin Escherichia coli Nissle1917 embedded particles and the unembedded Escherichia coli Nissle1917 can proliferate normally, indicating that the embedding method will not affect the growth ability of bacteria, because carboxymethyl lignin is dissolved in the weakly alkaline intestinal fluid, and the embedded bacteria are smoothly released to exert treatment, and the antibacterial property of carboxymethyl lignin is weak and will not reduce the number of viable bacteria.

[0044] Figure 4 The leftmost figure is a scanning electron microscope image of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles prepared in step (3) of Example 1. As can be seen from the figure, the surface of the carboxymethyl lignin Escherichia coli Nissle1917 embedded particles is granular, and the particles are carboxymethyl lignin, indicating that the lignin shell is successfully embedded on the surface of the bacteria.

[0045] Figure 5 This is a graph showing the change in colon length during the treatment of a mouse colitis model induced by the chemical drug dextran sulfate sodium (DSS) using the lignin-encapsulated Escherichia coli Nissle1917 particles prepared in step (3) of Example 1, wherein A is the colon of a normal mouse, B is the colon of a mouse that was fed with DSS but not treated with any drug, and C, D, and E are the colons of mice that were fed with DSS and treated with unencapsulated Escherichia coli Nissle1917, a mixture of unencapsulated Escherichia coli Nissle1917 and carboxymethyl lignin, and carboxymethyl lignin-encapsulated Escherichia coli Nissle1917 particles, respectively. As can be seen from the figure, after DSS-induced colitis in mice, the length of the mice's colon decreased. After treatment with Escherichia coli Nissle1917 and a mixture of Escherichia coli Nissle1917 and carboxymethyl lignin, the length of the mice's colon did not increase significantly, that is, the symptoms of colitis were not alleviated, because the Escherichia coli Nissle1917 delivered to the mice was killed by adverse environments such as simulated gastric juice, resulting in insufficient number of live bacteria reaching the site of colitis and unable to play a therapeutic role. After treatment with carboxymethyl lignin Escherichia coli Nissle1917-encapsulated particles, the length of the mice's colon increased significantly, indicating that encapsulation of Escherichia coli Nissle1917 can effectively protect Escherichia coli Nissle1917 and has a good effect on the treatment of colitis in mice.

[0046] Example 2

[0047] (1) Dissolve 800 mg of carboxymethyl lignin in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0048] (2) After activating the purchased Escherichia coli strain (ATCC25922) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0049] (3) Take 1 mL of bacterial solution, add 1 mL of CaCl2 solution, adsorb for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess CaCl2, and wash three times with sterile water. After washing, add 1 mL of carboxymethyl lignin solution, treat for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Escherichia coli embedded particles.

[0050] Figure 4 The middle figure is a scanning electron microscope image of the carboxymethyl lignin Escherichia coli embedded particles in step (3) of Example 2. It can be seen that the surface of the bacteria is granular, indicating that the surface of the bacteria is coated with lignin.

[0051] Example 3

[0052] (1) Dissolve 800 mg of carboxymethyl lignin in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0053] (2) After activating the purchased Staphylococcus aureus strain (ATCC6538) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0054] (3) Take 1 mL of bacterial liquid, add 1 mL of CaCl2 solution, adsorb for 30 min, centrifuge at 10000 rpm for 15 min, remove excess CaCl2, and wash three times with sterile water. After washing, add 1 mL of carboxymethyl lignin solution, treat for 30 min, centrifuge at 10000 rpm for 15 min, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Staphylococcus aureus embedded particles.

[0055] Figure 4 The rightmost figure is a scanning electron microscope image of the carboxymethyl lignin Staphylococcus aureus embedded particles prepared in step (3) of Example 3. It can be seen that the surface of Staphylococcus aureus embedded with carboxymethyl lignin is granular, indicating that the embedding is successful.

[0056] Example 4

[0057] (1) Dissolve 600 mg of carboxymethyl lignin in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0058] (2) After activating the purchased Escherichia coli strain (ATCC25922) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0059] (3) Take 1 mL of bacterial solution, add 1 mL of CaCl2 solution, adsorb for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess CaCl2, and wash three times with sterile water. After washing, add 1 mL of carboxymethyl lignin solution, treat for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Escherichia coli embedded particles.

[0060] Example 5

[0061] (1) Dissolve 600 mg of carboxymethyl lignin in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0062] (2) After activating Escherichia coli Nissle1917 (purchased from Mingzhou Biotechnology Co., Ltd.) twice, culture it in LB liquid medium for 4-5 hours to allow the bacteria to be in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0063] (3) Take 1 mL of bacterial solution, add 1 mL of CaCl2 solution, adsorb for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess CaCl2, and wash three times with sterile water. After washing, add 4 mL of carboxymethyl lignin solution, treat for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Escherichia coli Nissle1917 embedded particles.

[0064] Example 6

[0065] (1) Dissolve 600 mg of carboxymethyl lignin in 1000 ml of deionized water, stir overnight to fully dissolve the lignin, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0066] (2) After activating the purchased Staphylococcus aureus strain (ATCC6538) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to about OD = 0.5 for use.

[0067] (3) Take 1 mL of bacterial solution, add 1 mL of CaCl2 solution, adsorb for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess CaCl2, and wash three times with sterile water. After washing, add 4 mL of carboxymethyl lignin solution, treat for 30 min, centrifuge at 10,000 rpm for 15 min, remove excess lignin, and wash three times with sterile water to obtain carboxymethyl lignin Staphylococcus aureus embedded particles.

[0068] Comparative Example 1

[0069] (1) Dissolve 800 mg of sodium lignin sulfonate (LS) in 1000 ml of deionized water, stir overnight to fully dissolve the sodium lignin sulfonate, and filter through a 0.22 μm filter to sterilize for later use; prepare a CaCl2 solution with a concentration of 0.4 mol / L, and also filter through a 0.22 μm filter to sterilize for later use.

[0070] (2) After activating Escherichia coli Nissle 1917 (purchased from Mingzhou Biotechnology Co., Ltd.) twice, culture it in LB liquid medium for 4-5 hours to make the bacteria in the logarithmic growth phase. Centrifuge the bacterial solution at 5000 rpm for 3 minutes, collect the precipitate, wash it three times with sterile water, and finally resuspend the bacteria in sterile water and adjust the bacterial concentration to OD = 0.50 for use.

[0071] (3) Take 1 mL of bacterial liquid, add 1 mL of CaCl2 solution, adsorb for 30 minutes, centrifuge at 10,000 rpm for 15 minutes, remove excess CaCl2, and wash three times with sterile water. After washing, add 1 mL of sodium lignin sulfonate solution, treat for 30 minutes, centrifuge at 10,000 rpm for 15 minutes, remove excess sodium lignin sulfonate, and wash three times with sterile water to obtain sodium lignin sulfonate Escherichia coli Nissle1917 embedded particles.

[0072] Comparative Example 2

[0073] (1) Prepare 2.5% (v / v) glutaraldehyde solution and 10%, 30%, 50%, 70%, 90%, 100% (v / v) ethanol solutions in a series of concentration gradients and set aside.

[0074] (2) After activating Escherichia coli Nissle 1917 (purchased from Mingzhou Biotechnology Co., Ltd.), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 6538) twice, they were cultured in LB liquid medium for 4 to 5 hours to allow the bacteria to be in the logarithmic growth phase. The bacterial solution was centrifuged at 10,000 rpm for 3 minutes, the precipitate was collected, and washed with sterile water for 3 times. Finally, the bacteria were resuspended in sterile water and the bacterial concentration was adjusted to about OD = 1.0 for use.

[0075] (3) Take 1 mL of the bacterial solution obtained in step (2), centrifuge at 10,000 rpm for 3 min, collect the precipitate, add 2 mL of 2.5% glutaraldehyde solution, and fix overnight.

[0076] (4) The bacterial solution fixed in step (3) was centrifuged at 10,000 rpm for 5 min to remove glutaraldehyde, and then 1 mL of the series of ethanol solutions of the concentration gradient described in step (1) was added to the precipitate after centrifugation, and each gradient was treated for 1 h. The solution was centrifuged at 10,000 rpm for 15 min, and the ethanol solution was removed. Then, the next gradient concentration of ethanol was added for 1 h. This process was repeated until the treatment with 90% ethanol was completed, and the solution was resuspended in 1 mL of 100% ethanol solution, mixed evenly, and set aside.

[0077] (5) Drop the sample obtained in step (4) on a silicon wafer and take a scanning electron microscope photo.

[0078] Figure 6 The viable bacterial count of the sodium lignin sulfonate Escherichia coli Nissle1917 embedded particles prepared in step (3) of Comparative Example 1 in simulated gastric juice was shown in Table 1. After being treated with simulated gastric juice for the same period of time, the viable bacterial count of the sodium lignin sulfonate Escherichia coli Nissle1917 embedded particles did not increase compared with the unembedded Escherichia coli Nissle1917, indicating that embedding bacteria with sodium lignin sulfonate cannot effectively protect the bacteria from gastric juice because sodium lignin sulfonate is dissolved in the simulated gastric juice.

[0079] Figure 7 This is the scanning electron microscope image of comparative example 2. It can be seen that the unencapsulated Escherichia coli Nissle1917 and Escherichia coli are wrinkled, and the unencapsulated Staphylococcus aureus has a smooth surface. This is because ethanol is used to dehydrate the bacteria during SEM sample preparation, and the bacteria lose water and shrink normally.

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing enteric lignin bacteria-embedded particles, characterized in that: The following steps are involved: (1) Mix the bacterial solution with the CaCl2 solution, adsorb it for a period of time, and then wash it to obtain the adsorbed CaCl2 solution. 2+ of bacteria; (2) Adsorb Ca 2+ The bacteria are added to anionic lignin solution, adsorbed for a period of time, and then washed to obtain enteric lignin bacteria-embedded particles; In the anionic lignin solution of step (2), the anionic lignin is carboxymethyl lignin; The bacteria in the bacterial solution in step (1) are at least one of Escherichia coli Nissle 1917, common Escherichia coli, and Staphylococcus aureus.

2. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: In step (1), the volume ratio of the CaCl2 solution to the bacterial solution is 1:0.1 to 1:3; the bacterial OD value in the bacterial solution is 0.5 to 1; and the CaCl2 solution is 0.1 to 1.2 mol / L.

3. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: In step (2), the volume ratio of the anionic lignin solution to the bacterial solution is 1:0.5-1:2; the bacterial OD value in the bacterial solution is 0.5-1; and the concentration of the anionic lignin solution is 0.1-3.0 g / L.

4. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: The concentration of the anionic lignin solution in step (2) is 0.2-1.5 g / L.

5. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: The adsorption time in step (1) is 15 to 90 minutes.

6. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: The adsorption time in step (2) is 0.25 to 3 hours.

7. The method for preparing enteric lignin bacteria-embedded particles according to claim 1, characterized in that: The washing in steps (1) and (2) is centrifugal washing, the speed of the centrifugal washing is 5000-10000 r / min, the time is 10-30 min, and the number of centrifugal washing is 2-5 times.

8. Enteric lignin bacteria-embedded particles obtained by the preparation method according to any one of claims 1 to 7.

Images