Bacillus subtilis-loaded 3D printing porous polycaprolactone capsule and preparation method thereof

Abstract

The invention provides a 3D printing porous polycaprolactone capsule loaded with bacillus subtilis and a preparation method of the 3D printing porous polycaprolactone capsule. The capsule comprises a shell structure and a cavity which is surrounded by the shell structure and contains wrappage, the shell structure sequentially comprises a shell layer and a dialysis membrane layer from outside to inside, and the wrappage is bacillus subtilis; wherein the shell layer is prepared from the following raw materials: polycaprolactone, polyethylene, modified polypropylene and a heat stabilizer; the dialysis membrane layer is prepared from the following raw materials: polycaprolactone, a polyvinyl alcohol antioxidant, a plasticizer and a heat stabilizer; the bacillus subtilis-loaded 3D printing porous polycaprolactone capsule can uptake manganese ions in the intestinal tract, meanwhile, the bacillus subtilis-loaded porous polycaprolactone capsule is prevented from entering the intestinal tract, then the capsule carries the bacillus subtilis and the ions to be discharged out of the body, the content of the manganese ions in the human body can be effectively reduced, white fat can be prevented from being converted into beige fat, and cachexia formation is inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and particularly to a 3D printed porous polycaprolactone capsule loaded with Bacillus subtilis and a preparation method thereof. Background Art

[0002] Manganese, as an important trace element, is widely present in various foods, such as whole grains, nuts, green leafy vegetables, and some fruits. The intestine absorbs manganese from these foods through epithelial cells, enters the blood circulation, and supplies all organs and tissues of the body. Manganese ions play a key role in various physiological processes in the body, including serving as cofactors for some enzymes and participating in energy metabolism, bone development, and antioxidant reactions, etc.

[0003] In the process of adipocyte formation, manganese also plays a non-negligible role. Recent studies have shown that manganese ions are involved in the regulation of adipose tissue, especially in the formation process of beige adipocytes. Beige fat (also known as "brown fat") has a higher energy consumption capacity compared to ordinary white fat, so it is considered to have potential benefits for weight control and anti-obesity. The formation of beige fat is a process in which white fat is transformed into beige fat under specific conditions. This transformation process involves multiple biological signaling pathways, and manganese ions promote this process by affecting the metabolic pathways of adipose tissue.

[0004] However, excessive manganese ions may have a negative impact on the body. Especially when the manganese level in the body is too high, it may cause damage to the nervous system and is even associated with certain neurodegenerative diseases. In order to maintain the balance of manganese ions in the body, the absorption of manganese by the intestine needs to be precisely regulated. Bacillus subtilis, as a common probiotic, has the characteristic of adsorbing manganese ions. This enables Bacillus subtilis to play an important role in the intestine, helping to reduce the absorption of excessive manganese ions, thereby regulating the level of manganese in the body.

[0005] Bacillus subtilis can adsorb and bind manganese ions through its cell wall. This characteristic enables it to "capture" excess manganese ions in the intestine and prevent them from entering the blood circulation in excess. At the same time, Bacillus subtilis does not directly decompose or damage manganese ions in the intestine. It selectively adsorbs manganese ions through a special biological mechanism and transports these adsorbed manganese ions out of the body. When this bacterium enters the intestine through a capsule, it can effectively adsorb manganese ions in the intestinal lumen, control the absorption amount of manganese ions, and reduce their excessive accumulation in the body.

[0006] Therefore, a specific capsule can be designed that can not only remain stable in the intestine, but also effectively ensure that manganese ions pass through the capsule wall and are adsorbed by Bacillus subtilis. The material of the outer wall of the capsule should be resistant to acid and digestive enzymes to ensure that the capsule will not be decomposed prematurely in the intestine. At the same time, the microporous structure of the capsule wall allows manganese ions to pass through, while Bacillus subtilis can adsorb these manganese ions and take them away when they are excreted from the body. In this way, the capsule can not only regulate the concentration of manganese ions in the body, but also prevent excessive beige fat formation by reducing excessive accumulation of manganese, thereby achieving an inhibitory effect on diseases such as cachexia. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a 3D printed porous polycaprolactone capsule loaded with Bacillus subtilis and a preparation method thereof, which can effectively manage the level of manganese ions, not only without affecting the normal physiological functions of the human body, but also while avoiding excessive manganese ions, regulate the transformation process of adipose tissue and help maintain a healthy balance of metabolism in the body.

[0008] The technical solution of the present invention:

[0009] In a first aspect, the present invention provides a 3D printed porous polycaprolactone capsule carrying Bacillus subtilis, wherein the capsule comprises a shell structure and a cavity containing a package surrounded by the shell structure, wherein the shell structure comprises an outer shell layer and a dialysis membrane layer from the outside to the inside, and the package is Bacillus subtilis; wherein, in terms of mass fraction,

[0010] The raw materials of the outer shell layer include 80-100 parts of polycaprolactone, 20-30 parts of polyethylene, 50-80 parts of modified polypropylene, and 0.5-1.5 parts of heat stabilizer;

[0011] The raw materials of the dialysis membrane layer include 100-120 parts of polycaprolactone, 50-70 parts of polyvinyl alcohol, 5-15 parts of antioxidant, 1-20 parts of plasticizer and 0.5-1.5 parts of heat stabilizer.

[0012] In some embodiments, the modified polypropylene is copolymerized by monomer A, monomer B and monomer C; wherein the monomer A is a reaction product of pentaerythritol and allyl chloride in a molar ratio of 1:3; monomer B is propylene; and the structural formula of monomer C is as shown in Formula 1:

[0013]

[0014] In some embodiments, the method for preparing the monomer A comprises the following steps:

[0015] Pentaerythritol and allyl chloride were added to a reactor, and a solvent was added. The mixture was stirred until the reactants were completely dissolved. A catalyst was added, and the reaction was carried out with heating and stirring. After the reaction was completed, the reaction solution was cooled to room temperature, and monomer A was obtained after post-treatment.

[0016] In some embodiments, the method for preparing monomer C comprises the following steps:

[0017] 2-Oxabicyclo[2.2.1]heptane-4-methanol (OBHM) and a solvent were added to a reactor, stirred evenly, concentrated sulfuric acid was added, and the reaction was carried out with heating. Monomer C was obtained after the reaction was completed.

[0018] In some embodiments, the method for preparing the modified polypropylene comprises the following steps:

[0019] Monomer A, monomer C, and an inert solvent were injected into a reaction kettle, a catalyst and monomer B were added, an inert gas was introduced, and the reaction was carried out under pressurized and heated conditions. After the reaction was completed, the modified polypropylene was separated.

[0020] In some embodiments, the molar ratio of monomer A, monomer B, and monomer C is 2-3:6-8:1-2; further, the molar ratio of monomer A, monomer B, and monomer C is 3:8:1.

[0021] In some embodiments, there are no special regulations for the heat stabilizer. For example, it can be selected from one or a combination of sorbitol, amorphous sorbitol solution, sorbitan sorbitol solution, glycerol, mannitol, polyethylene glycol with a molecular weight of 400-6000, and xylitol.

[0022] In some embodiments, there are no special regulations for the type of polyvinyl alcohol to increase the hydrophilicity of the dialysis membrane. Mixing polyvinyl alcohol and polycaprolactone to prepare a dialysis membrane can increase the body fluid permeability of the dialysis membrane, enabling the body fluid to carry manganese ions into the interior of the capsule cavity for adsorption.

[0023] In some embodiments, there are no special regulations for the type of antioxidant. For example, it can be one or several of tert-butylhydroxyanisole, propyl gallate, α-tocopherol, vitamin E acetate, VC palmitate, or vitamin C.

[0024] In some embodiments, there are no special regulations for the type of plasticizer. For example, it can be selected from one or more of medium-chain triglycerides (MCT), ethyl acetate, ethyl benzoate, benzyl benzoate (BB), m-cresol, propofol, chlorobutanol, or 4-phenylbutyric acid.

[0025] In some embodiments, the shape of the capsule is substantially an ellipsoidal structure, and it can also be "cylindrical" or "microspherical"; the longest diameter of the capsule is 5-10 mm.

[0026] In some embodiments, the outer shell layer is divided into an upper shell layer and a lower shell layer, and the present invention does not limit the connection manner of the upper and lower shell layers; for example, it can be heat-sealed, ultrasonically sealed, adhered or connected through a mechanical structure; the mechanical structure can be to set a threaded structure on the upper and lower shell layers by 3D printing to connect the upper shell layer and the lower shell layer of the capsule.

[0027] In some embodiments, there are many micropores on the outer shell layer. Bacillus subtilis adsorbs manganese ions in body fluids through these micropores, and at the same time, the dialysis membrane can prevent Bacillus subtilis from detaching from the capsule and entering the body fluids. The diameter of the micropores is 10-30 μm. Further, the diameter of the micropores can be selected from one of 30 μm, 20 μm, and 10 μm.

[0028] In some embodiments, the content of Bacillus subtilis is 10 8 -10 10 cfu.

[0029] On the other hand, the present invention also provides a preparation method of the 3D printed porous polycaprolactone capsule loaded with Bacillus subtilis, which specifically includes the following steps:

[0030] S1: Add polycaprolactone, polyvinyl alcohol and a solvent to a reactor according to the formula of the dialysis membrane layer, dissolve them, then add an antioxidant, a plasticizer and a heat stabilizer, stir evenly and coat on a flat glass plate or steel plate, adjust the thickness to 5-20 μm, and dry the membrane by solvent evaporation to form a firm film and cut it to obtain a dialysis membrane;

[0031] S2: Culture Bacillus subtilis using an LB medium; centrifuge to collect the bacterial cells; load 10 8 -10 10 cfu of the bacterial cells into the dialysis membrane and seal it;

[0032] S3: According to the raw material formula of the outer shell layer, mix polycaprolactone, polyethylene, modified polypropylene and a heat stabilizer and heat them to melt, print a shell with a pore structure according to requirements, and perform post-treatment to obtain the upper shell layer and the lower shell layer of the outer shell layer;

[0033] S4: Put the dialysis membrane loaded with Bacillus subtilis obtained in S2 into the printed upper shell layer and lower shell layer, ensure that the inner and outer two-layer structures are completely wrapped, and connect the upper shell layer and the lower shell layer to obtain a 3D printed porous polycaprolactone capsule.

[0034] Advantageous effects:

[0035] The 3D-printed porous polycaprolactone capsule loaded with Bacillus subtilis can uptake manganese ions in the intestine. Polycaprolactone enables the capsule not to be decomposed in the intestine, and the cross-linked structure of modified polypropylene makes the dialysis bag restrict the entry of Bacillus subtilis into the intestine. Subsequently, the capsule carries Bacillus subtilis and ions out of the body, which can effectively reduce the manganese ion content in the human body, help prevent the transformation of white fat into beige fat, and inhibit the formation of cachexia.

[0036] By designing modified polypropylene, the capsule outer shell layer is copolymerized with polycaprolactone and polyethylene, which has excellent gastric acid resistance and excellent mechanical properties, ensuring that the capsule outer shell remains in its original state in the gastric juice environment. At the same time, this outer shell layer can be obtained by 3D printing, which is convenient for preparation. Brief Description of the Drawings

[0037] Figure 1 It is a conceptual diagram of the 3D-printed porous polycaprolactone capsule loaded with Bacillus subtilis provided by the present invention.

[0038] Among them, 1 represents the upper shell layer, 2 represents the lower shell layer, 3 represents the outer shell layer, 4 represents the dialysis membrane layer, and 5 represents Bacillus subtilis in the cavity. Detailed Embodiments

[0039] The following will describe the present invention in combination with specific implementation schemes. It should be noted that the following examples are examples of the present invention, only used to illustrate the present invention, and not used to limit the present invention. Without departing from the gist or scope of the present invention, other combinations and various improvements within the concept of the present invention can be made.

[0040] Unless otherwise specified, the chemical reagents used in the present invention are all ordinary commercially available analytical pure. The Bacillus subtilis used in the examples was purchased from the China Center for Industrial Culture Collection, and the strain model was CICC10164; the polycaprolactone was selected from the polycaprolactone with the model 6400 produced by Perstorp Company in Sweden; the polyethylene was purchased from Filamentive PE; the polyvinyl alcohol was purchased from BP-17 of Shanghai Huihu Industry Co., Ltd.; the antioxidant was propyl gallate; the plasticizer was ethyl acetate; the heat stabilizer was mannitol.

[0041] Preparation Example of Modified Polypropylene 1

[0042] Add 1 mol of pentaerythritol and 3 mol of allyl chloride to a reactor, add 50 ml of toluene, and stir until the reactants are completely dissolved; add 0.1 mol of sodium hydroxide, heat to 60 °C and stir for 4 h. After the reaction is completed, cool the reaction solution to room temperature, and obtain the structurally fixed monomer A through post-treatment.

[0043] Perform infrared spectrum analysis on monomer A. According to the infrared spectrum analysis, it can be obtained that at 3400 cm -1The peak at [frequency] is the O-H stretching vibration, usually from the alcoholic hydroxyl groups in pentaerythritol; at 1150 cm -1 The absorption peak at [frequency] is generated by the stretching vibration of the ether bond; at 1645 cm -1 The absorption peak at [frequency] is generated by C=C (alkene). From the above analysis, it can be obtained that the infrared spectrum data proves that allyl chloride is grafted onto the three alcoholic hydroxyl groups of pentaerythritol, and the pentaerythritol ether compound is successfully prepared.

[0044] Add 1 mol of OBHM and 50 ml of chloroform to the reactor, stir evenly, add 0.1 mol of concentrated sulfuric acid, heat to 60 °C and react for 3 h. After the reaction is completed, remove the solvent to obtain monomer C.

[0045] Inject 3 mol of monomer A, 1 mol of monomer C and 500 ml of chloroform into the reaction kettle, add 0.3 mol of catalyst (a mixture of TiCl4 and triethylaluminum with a molar ratio of 1:1) and 8 mol of monomer B propylene, introduce nitrogen, and react under the conditions of 60 °C and 1.5 MPa. After the reaction is completed, modified polypropylene 1 is obtained by centrifugal separation, washing and drying.

[0046] Preparation example of modified polypropylene 2

[0047] Inject 4 mol of monomer A and 500 ml of chloroform into the reaction kettle, add 0.3 mol of catalyst (a mixture of TiCl4 and triethylaluminum with a molar ratio of 1:1) and 8 mol of monomer B propylene, introduce nitrogen, and react under the conditions of 60 °C and 1.5 MPa. After the reaction is completed, modified polypropylene 2 is obtained by centrifugal separation, washing and drying.

[0048] Example 1

[0049] S1: Add 120 parts of polycaprolactone, 50 parts of polyvinyl alcohol and 200 parts of dichloromethane to the reactor for dissolution. Then add 7 parts of antioxidant, 5 parts of plasticizer and 1 part of tert-butyl-p-cresol, stir evenly and coat on a flat glass plate or steel plate. Adjust the thickness to 10 μm, dry at 60 °C for 6 h to volatilize the solvent, and dry the film to form a firm film and cut it into a length and width of 8 mm and 5 mm respectively to obtain a dialysis membrane.

[0050] S2: Culture Bacillus subtilis using LB medium; collect the bacterial cells by centrifugation; load 10 8 cfu of bacterial cells into the dialysis membrane obtained in S1 and seal it by radiation crosslinking, and the radiation dose is 4 Mard;

[0051] S3: According to the raw material formula of the outer shell layer, mix 100 parts of polycaprolactone, 20 parts of polyethylene, 70 parts of modified polypropylene 1 and 1 part of tert-butyl-p-cresol and heat to melting, and 3D print a capsule outer shell with a pore structure. The shape of the capsule outer shell is as Figure 1As shown, the long diameter is 10 mm, the short diameter is 4 mm, the micropore diameter is 20 μm, and the connecting part of the upper shell layer and the lower shell layer has threads; after printing is completed, post-treatment is carried out to obtain the upper shell layer and the lower shell layer of the outer shell layer;

[0052] S4: Put the dialysis membrane loaded with Bacillus subtilis obtained in S2 between the printed upper shell layer and the lower shell layer, ensure that the inner and outer two-layer structures are completely wrapped, and stably connect the upper shell layer and the lower shell layer through the threads to obtain a 3D printed porous polycaprolactone capsule.

[0053] Example 2

[0054] Basically the same as Example 1, the only difference is that in step S1, the length and width of the dialysis membrane are 4 mm and 1.5 mm respectively; in step S3, the shape of the capsule shell is as Figure 1 shown, the long diameter is 5 mm, the short diameter is 2 mm, and the micropore diameter is 10 μm.

[0055] Comparative Example 1

[0056] Basically the same as Example 1, the only difference is that in step S3, modified polypropylene 2 is used to replace modified polypropylene 1.

[0057] Performance Test

[0058] 1. Artificial gastric juice stability test

[0059] Prepare artificial gastric juice: Take 16.4 ml of dilute hydrochloric acid with a concentration of about 10%, add about 800 ml of water and 10 g of pepsin, shake well and then dilute with water to 1000 ml.

[0060] Take the 3D printed porous polycaprolactone capsules prepared in Example 1 and Comparative Example 1 and place them in artificial gastric juice respectively, culture them in a shaker at 37 °C and 180 rpm, sample and neutralize to neutral after 6 h of treatment, and measure the viable count of Bacillus subtilis in the capsules and calculate the survival rate.

[0061] 2. Manganese ion adsorption test

[0062] Using a graduated cylinder, 100 mL of a manganese adsorption solution with a concentration of 20 μg / mL was separately measured and placed into 2 conical flasks. The pH was adjusted to 2.0 with hydrochloric acid solution, and then the concentration of manganese ions was measured using an inductively coupled plasma-atomic emission (ICP-AES) spectrometer; 10 g of the capsules prepared in Example 1 and Comparative Example 1 were separately added to each conical flask. The mouths of each conical flask were sealed with plastic wrap, and then the conical flasks were placed in a constant temperature oscillator and oscillated at a speed of 120 r / min and an oscillation temperature of 25 °C; after oscillating for 8 h, the oscillation was stopped. The mixture in each conical flask was transferred into a centrifuge tube. After high-speed centrifugation, the supernatant and the lower layer adsorbent were obtained, and the concentration of manganese ions in the supernatant was measured again using an inductively coupled plasma-atomic emission (ICP-AES) spectrometer to calculate the adsorption percentage.

[0063] 3. Toxicity test

[0064] According to the determination standard of GB / T15193.17-2003 "Chronic Toxicity and Carcinogenicity Tests", 100 healthy weaned mice with almost the same age and body weight were taken, with half male and half female, and the female mice were non-pregnant, as a test group. A test group was divided into 2 groups. The first group was the normal dose group, and the second group was the control group. The first group was fed 1 3D printed porous polycaprolactone capsule prepared in Example 2 every day, and the other feeding conditions were the same as those of the control group. After feeding for 18 months, the organs and tissues of all mice were examined by biological microscopy, and those that died during the test were also examined. The mice were weighed and recorded weekly in the first three months and once a month after three months; and blood tests were carried out once every three months, six months and every six months thereafter during the test; the data were recorded and combined with data analysis to check whether there were poisoning, carcinogenic and anorexia symptoms in each group of mice, and the obtained phenomena were recorded.

[0065] The above test results are shown in Table 1.

[0066] Table 1 Test results

[0067]

[0068] Note: Among them, "-" means not tested.

[0069] The above test results show that the 3D printed porous polycaprolactone capsule provided by the present invention can stably exist in the gastric acid environment, will not be decomposed and digested, and can also protect the survival rate of Bacillus subtilis; at the same time, it has no toxic effect on mice and has safety.

[0070] Since the present invention has relatively high performance requirements for the capsule outer shell layer, it can be found by comparing the examples and comparative examples that the capsule outer shell layer made of modified polypropylene copolymerized with polycaprolactone and polyethylene through structural design has good acid resistance, excellent mechanical properties and can be 3D printed into the required capsule structure to form the required capsule.

[0071] Through the 3D printed porous polycaprolactone capsule provided by the present invention, the modified polypropylene has a cross-linked structure, which reduces the permeability of the dialysis membrane, can limit the entry of Bacillus subtilis into the intestine, but can still adsorb manganese ions in body fluids. Subsequently, the capsule carries the loaded Bacillus subtilis and ions out of the body, reducing the manganese ions in the body, helping to prevent the transformation of white fat into beige fat and inhibiting the formation of cachexia.

[0072] The present invention can also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes and deformations should all fall within the protection scope of the appended claims of the present invention.

Claims

1. A 3D-printed porous polycaprolactone capsule loaded with Bacillus subtilis, characterized in that, The capsule includes a shell structure and a cavity containing an inclusion enclosed by the shell structure. The shell structure includes an outer shell layer and a dialysis membrane layer from outside to inside. The inclusion is Bacillus subtilis, and the outer shell layer of the capsule has micropores. Among them, by mass fraction, the raw materials of the outer shell layer include 80-100 parts of polycaprolactone, 20-30 parts of polyethylene, 50-80 parts of modified polypropylene, and 0.5-1.5 parts of heat stabilizer; the raw materials of the dialysis membrane layer include 100-120 parts of polycaprolactone, 50-70 parts of polyvinyl alcohol, 5-15 parts of antioxidant, 1-20 parts of plasticizer, and 0.5-1.5 parts of heat stabilizer.

2. The 3D printed porous polycaprolactone capsule according to claim 1, wherein The modified polypropylene is copolymerized from monomer A, monomer B, and monomer C. Among them, monomer A is the reaction product of pentaerythritol and allyl chloride with a molar ratio of 1:3; monomer B is propylene; the structural formula of monomer C is shown in Formula 1:

3. The 3D printed porous polycaprolactone capsule according to claim 2, wherein The preparation method of monomer A includes the following steps: Add pentaerythritol and allyl chloride into a reactor, add a solvent, and stir until the reactants are completely dissolved; add a catalyst, heat and stir for reaction. After the reaction is completed, cool the reaction solution to room temperature, and obtain monomer A through post-treatment.

4. The 3D printed porous polycaprolactone capsule according to claim 2, wherein The preparation method of monomer C includes the following steps: Add 2-oxabicyclo[2.2.1]heptane-4-methanol and a solvent into a reactor, stir evenly, add concentrated sulfuric acid, heat for reaction, and obtain monomer C after the reaction is completed.

5. The 3D printed porous polycaprolactone capsule according to claim 1 or 2, characterized in that, The preparation method of the modified polypropylene includes the following steps: Inject monomer A, monomer C, and an inert solvent into a reaction kettle, add a catalyst and monomer B, introduce an inert gas, and carry out the reaction under pressurized and heated conditions. After the reaction is completed, separate to obtain the modified polypropylene.

6. The 3D printed porous polycaprolactone capsule according to claim 2, wherein, The molar ratio of monomer A, monomer B, and monomer C is 2-3:6-8:1-2.

7. The 3D printed porous polycaprolactone capsule according to claim 1, wherein The shape of the capsule is selected from one of an ellipsoid, a cylinder, or a microsphere.

8. The 3D printed porous polycaprolactone capsule according to claim 1, characterized in that, The diameter of the micropores is 10-30 μm.

9. The 3D printed porous polycaprolactone capsule according to claim 1, wherein, The content of the Bacillus subtilis is 10 8 -10 10 cfu.

10. The preparation method of the 3D printed porous polycaprolactone capsule according to any one of claims 1-9, characterized in that, Including the following steps: S1: Add polycaprolactone, polyvinyl alcohol, and a solvent into a reactor according to the formula of the dialysis membrane layer for dissolution, then add an antioxidant, a plasticizer, and a heat stabilizer, stir evenly and coat on a flat glass plate or steel plate, adjust the thickness to 5-20 μm, dry the film by solvent evaporation method to form a firm film and cut it to obtain a dialysis membrane; S2: Cultivate Bacillus subtilis using LB medium; centrifuge to collect the bacterial cells; load 10 8 -10 10 cfu of the bacterial cells into a dialysis membrane and seal it; S3: According to the raw material formula of the outer shell layer, mix polycaprolactone, polyethylene, modified polypropylene, and a heat stabilizer and heat them to melting to obtain a masterbatch material. Print a shell with a hole structure by 3D according to requirements, and carry out post-treatment to obtain the upper shell layer and the lower shell layer of the outer shell layer; S4: Put the dialysis membrane loaded with Bacillus subtilis obtained in S2 into the printed upper shell layer and lower shell layer, ensure that the inner and outer two-layer structures are completely wrapped, and connect the upper shell layer and the lower shell layer to obtain a 3D printed porous polycaprolactone capsule.

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