A 3D printing bacteriocellulose-based lactobacillus rhamnosus / cottonseed peptide embedding material, a preparation method and application thereof

Abstract

The application discloses a 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material and a preparation method and application thereof. The method comprises the following steps: mixing sodium alginate and bacterial cellulose after shearing and homogenizing to obtain a sodium alginate-bacterial cellulose mixed solution, then adding Lactobacillus rhamnosus powder and cottonseed peptide to form a uniform suspension, and dropping the uniform suspension into a calcium chloride solution to solidify and form microspheres; another sodium alginate-bacterial cellulose mixed solution with high bacterial cellulose content is prepared as 3D printing ink, the microspheres are uniformly dispersed in the ink, and then 3D printing is performed, finally, a calcium chloride solution is sprayed on the surface of the printing product for secondary solidification, and a composite material with a double-layer embedding structure is obtained. The double-layer embedding system constructed by the bacterial cellulose and the sodium alginate can effectively protect and control the release of the Lactobacillus rhamnosus and the cottonseed peptide, and significantly improves the treatment effect of enteritis.

Description

Technical Field

[0001] This invention relates to the field of food packaging materials, specifically to a 3D-printed bacterial cellulose-based material for encapsulating probiotics and peptides, its preparation method, and its application. Background Technology

[0002] Enteritis, including ulcerative colitis and Crohn's disease, is a chronic, intractable, immune-mediated inflammatory bowel disease. Traditional treatments often have significant adverse reactions, potentially disrupting the gut microbiota, causing gastrointestinal discomfort, and causing hepatotoxicity and nephrotoxicity. Given the high relapse rate and difficulty in curing enteritis, continuous medication is required, placing a heavy financial burden on patients. Furthermore, recurrent flare-ups of ulcerative colitis can lead to increased drug resistance and poor treatment outcomes. Safe bioactive substances with high anti-inflammatory activity have garnered widespread attention. Common food-derived substances used to improve gastrointestinal disorders include probiotics, polyphenols, polysaccharides, and peptides. Probiotics can regulate gastrointestinal function and fat storage, but the clinical use of oral probiotics is hampered by poor bacterial bioactivity and insufficient preservation of the gastrointestinal tract. Peptides often exhibit potent biological activities, such as antihypertensive, antioxidant, immunomodulatory, anticancer, antibacterial, and lipid-lowering activities. However, the exposed hydrophobic amino acids in food-derived peptides can lead to a bitter taste. They are also affected by bioactive compounds in the food matrix; for example, the presence of proteins and reducing carbohydrates in food components can lead to the formation of Maillard compounds. Furthermore, they are influenced by oral and gastrointestinal enzymes. Therefore, there is a need to develop a simple and efficient probiotic / peptide encapsulation delivery method to manufacture novel encapsulation materials. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material, its preparation method, and its applications. This material possesses excellent biocompatibility, structural designability, gastric acid resistance, and intestinal targeted drug release performance, effectively improving the stability, survival rate, and bioavailability of *Lactobacillus rhamnosus* and cottonseed peptide.

[0004] The 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material provided by this invention is specifically a 3D-printed encapsulation system that is biocompatible, safe and non-toxic, and has gastric juice barrier protection, intestinal targeted release, and customizable structure, thereby achieving the purpose of protecting and delivering Lactobacillus rhamnosus and cottonseed peptide and improving their bioavailability.

[0005] The present invention also aims to provide a method for preparing a 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material. The preparation method uses bacterial cellulose as the matrix of the outer printing ink and sodium alginate as a rheology enhancer to prepare a bacterial cellulose-based 3D printing ink containing pre-crosslinked sodium alginate bacterial cellulose-*Lactobacillus rhamnosus* / cottonseed peptide microspheres. Then, a shell-core fiber material with a double-layer structure, namely the 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material, is prepared using 3D printing technology.

[0006] The present invention also aims to provide a 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material that can be used for the protective delivery of probiotics and peptides, and for application in the treatment research of enteritis.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material and its preparation method, the method comprising the following steps:

[0009] (1) Sodium alginate was dissolved in an aqueous solution under water bath stirring conditions to obtain a sodium alginate solution;

[0010] (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation.

[0011] (3) Add bacterial cellulose nanofibers to the sodium alginate solution in step (1), and perform shearing and homogenization to obtain sodium alginate and bacterial cellulose mixed solution 1;

[0012] (4) Under stirring conditions, add Lactobacillus rhamnosus powder and cottonseed peptide to the sodium alginate bacterial cellulose mixed solution 1 described in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension.

[0013] (5) The sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension described in step (4) is added dropwise to a calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres (sodium alginate undergoes a rapid and mild ionic cross-linking reaction upon encountering calcium ions, instantly forming a solidified gel film on the droplet surface. The bacterial cellulose is uniformly dispersed in the sodium alginate solution, and during the dropwise addition and cross-linking process, it is encapsulated within the "egg-box" gel network formed by sodium alginate, thus reinforcing the framework).

[0014] (6) Add bacterial cellulose nanofibers to the sodium alginate solution in step (1) and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2;

[0015] (7) Use the mixture 2 described in step (6) as 3D printing ink, and uniformly mix the sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing to obtain 3D printing products.

[0016] (8) Spray calcium chloride solution evenly onto the surface of the 3D printed product in step (7) and perform curing treatment to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

[0017] In the above preparation method, in step (1), the mass-to-volume ratio of the sodium alginate solution is 0.01-0.05 g / mL, and the water bath temperature is 45-65℃.

[0018] In the above preparation method, in step (3), the mass-to-volume ratio of bacterial cellulose nanofibers to sodium alginate solution in the mixture 1 is 0.01-0.05 g / mL.

[0019] In the above preparation method, in step (4), the total mass ratio of Lactobacillus rhamnosus powder and cottonseed peptide to sodium alginate bacterial cellulose mixed solution 1 is 1~10:100; wherein, the mass ratio of Lactobacillus rhamnosus powder to cottonseed peptide is 1~5:1~5.

[0020] Preferably, the total mass ratio of Lactobacillus rhamnosus powder and cottonseed peptide to sodium alginate bacterial cellulose mixed solution 1 is 1~5:100; wherein, the mass ratio of Lactobacillus rhamnosus powder to cottonseed peptide is 1~5:1~3.

[0021] In the above preparation method, the mass-volume ratio concentration of the calcium chloride solution in steps (5) and (8) is 0.01-0.1 g / mL; the volume ratio of the suspension and calcium chloride solution in step (5) is 1:10-1:50; the mass-volume ratio of the 3D printing product to the calcium chloride solution in step (8) is 1-5 g / mL; and the curing time in step (8) is 5-60 s.

[0022] In the above preparation method, in step (6), the mass-to-volume ratio of bacterial cellulose nanofibers to sodium alginate solution in the mixture 2 is 0.1-1 g / mL.

[0023] In the above preparation method, in step (7), the volume-to-mass ratio of the sodium alginate bacterial cellulose microspheres and the mixed solution 2 is 0.01-0.2 g / mL; the size of the 3D printing needle is 1.2-2.0 G; the printing speed is 5-45 mm / s; and the spray thickness is 0.6-1.2 mm.

[0024] A 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material was prepared by the above-described preparation method.

[0025] In this invention, the 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material prepared by the above method relates to applications in functional foods, food processing technology, and biomaterials. Furthermore, the material is also used for encapsulating probiotics and peptides.

[0026] The 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material provided by this invention exhibits excellent 3D printing performance and can effectively encapsulate and deliver Lactobacillus rhamnosus and peptides. This 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material is resistant to acidic conditions, enabling successful delivery of Lactobacillus rhamnosus and cottonseed peptides into the intestine.

[0027] This invention utilizes coaxial 3D printing technology to develop a probiotic delivery complex with a bilayer structure in a simple and direct way, which can slowly release Lactobacillus rhamnosus and peptides; and protect lactic acid bacteria and cottonseed peptides from adverse environmental effects during gastric digestion.

[0028] This invention uses bacterial cellulose as the wall material, which is biocompatible, safe and non-toxic, and has an ultrafine network and layered structure. Sodium alginate is used as a rheological property enhancer.

[0029] This invention uses bacterial cellulose (BC) as the core wall material. Its natural nanofiber network provides physical encapsulation and structural support for probiotics (Lactobacillus rhamnosus) and active peptides (cottonseed peptides). By introducing sodium alginate (SA) as a rheology enhancer and gel component, not only is the 3D printing suitability of the BC-based "bio-ink" improved, but the calcium ion crosslinking properties and pH responsiveness of SA are also utilized to synergistically construct a smart composite gel carrier with the BC network. This carrier maintains a dense structure in the harsh environment of the stomach, effectively isolating gastric acid and proteases, achieving dual protection for the contents. Upon entering the intestine, the SA gel swells and gradually dissociates in response to a neutral environment, while the BC network slowly degrades as dietary fiber, thereby precisely triggering the targeted release and long-lasting delivery of active ingredients. Ultimately, this system achieves integrated synergy between probiotics and peptides from gastric protection to efficient intestinal delivery through the organic combination of complementary material properties (the structural stability of BC and the environmental responsiveness of SA) and manufacturing processes (the structural controllability of 3D printing), aiming to improve their bioavailability and intestinal regulatory function.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) Advantages of microsphere encapsulation: By encapsulating Lactobacillus rhamnosus and cottonseed peptides in sodium alginate / bacterial cellulose composite microspheres, external humid heat, acid and alkali and enzymatic hydrolysis environment are effectively isolated, significantly improving the stability of active ingredients; the microsphere structure helps to mask the hydrophobic odor of cottonseed peptides and improve palatability.

[0032] (2) Advantages of 3D printing structure: 3D printing technology can achieve precise control of the macroscopic structure of materials, forming a composite system of porous scaffolds and microspheres evenly distributed inside. This not only provides a micro-oxygen environment for probiotics and enhances their survival ability, but also facilitates the controllable preparation of multi-layer and multi-scale structures, which is superior to traditional embedding methods.

[0033] (3) Synergistic protection of internal structure: Bacterial cellulose nanofibers adsorb cottonseed peptides through hydrophobic interactions, forming stable micelles, reducing peptide degradation and odor release; its three-dimensional network provides physical anchoring points for Lactobacillus rhamnosus, alleviating fluid shear damage and improving bacterial survival rate.

[0034] (4) Targeted delivery performance: Sodium alginate remains stable under gastric acid conditions and gradually dissolves and releases its contents in the neutral environment of the intestine, achieving targeted controlled release in the intestine and effectively improving the local concentration of probiotics and peptides and the therapeutic effect.

[0035] (5) Advantages of raw materials and processes: The bacterial cellulose and sodium alginate used are widely available, have good biocompatibility, and are inexpensive. The preparation process does not require complex equipment, has low energy consumption, and has the potential for large-scale production. Attached Figure Description

[0037] Figure 1 A represents the survival rate of encapsulated and unencapsulated Lactobacillus rhamnosus during simulated gastrointestinal digestion.

[0038] Figure 1 B is a scanning electron microscope image of the surface of the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material obtained in Example 1;

[0039] Figure 1 C is a cross-sectional scanning electron microscope image of the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedded material obtained in Example 1, showing the distribution of microspheres in the printed structure;

[0040] Figure 1 D represents the cross-sectional morphology of the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material obtained in Example 1 after curing a single layer of ink. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments and protection scope of the present invention are not limited thereto.

[0042] The sources of raw materials used in the examples and comparative examples are as follows:

[0043] Bacterial cellulose (produced by Guilin Qihong Technology Co., Ltd., brand name BC-1.8)

[0044] Lactobacillus rhamnosus (10 billion CFU / g, Shaanxi Guanchen Biotechnology Co., Ltd., Product No.: GC-01-399)

[0045] Cottonseed protein (Cottonseed protein 98, Shaanxi Haosen Biotechnology Co., Ltd., product number: HSSW-00548)

[0046] Example 1

[0047] The specific steps for 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material are as follows:

[0048] (1) Under the stirring conditions of a water bath at 60℃, 6g of sodium alginate powder was added to 300mL of aqueous solution and mixed evenly to obtain a sodium alginate solution with a concentration of 0.02g / mL.

[0049] (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation.

[0050] (3) Add 2g of bacterial cellulose nanofibers to the 100mL sodium alginate solution in step (1), and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 1;

[0051] (4) Under stirring conditions, add 0.5g of Lactobacillus rhamnosus powder and 1g of cottonseed peptide to the 100mL sodium alginate bacterial cellulose mixed solution in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension.

[0052] (5) Add the 10 mL sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension from step (4) to 200 mL of 0.02 g / mL calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres;

[0053] (6) Add 20g of bacterial cellulose nanofibers to the 200mL sodium alginate solution in step (1), and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2.

[0054] (7) Use the 20mL mixture 2 described in step (6) as 3D printing ink, and uniformly mix the 2g sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing. The printing needle size is 1.2G, the printing speed is 15mm / s, the spray thickness is 0.8mm, and the 3D printed product is obtained.

[0055] (8) A calcium chloride solution with a concentration of 0.02 g / mL is uniformly sprayed onto the surface of the 3D printed product in step (7). The mass-to-volume ratio of the 3D printed product (wet weight) to the calcium chloride solution is 1.5 g / mL. The product is then cured for 20 seconds to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

[0056] The bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material prepared in Example 1 has a tight outer layer. The bacterial cellulose and sodium alginate form a tight layered structure, which resists gastric acid penetration and has a good protective effect on Lactobacillus rhamnosus and cottonseed peptide. This was observed under scanning electron microscopy. Figure 1 The 3D printing ink and embedded microspheres are clearly visible on the C-axis, demonstrating the successful construction of the bilayer structure; the scanning electron microscope also shows this. Figure 1 The cross-section of the microsphere-containing 3D printing ink can be seen on the image, with the microspheres tightly connected to the ink. Numerous *Lactobacillus rhamnosus* and cottonseed peptides are embedded internally, causing no physical damage to the *Lactobacillus rhamnosus*. After simulated digestion in vitro, the embedded material resists the harsh environment of simulated gastric juice and is slowly released in simulated intestinal juice. Furthermore, the embedded *Lactobacillus rhamnosus* and peptides retain excellent activity. This 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide embedding material achieves both protection and sustained release of *Lactobacillus rhamnosus* and cottonseed peptides. In vitro anti-inflammatory experiments (see Table 1) further confirmed that the material obtained in this embodiment (corresponding to the BC-SA@CS-LC group) can significantly inhibit the pro-inflammatory factors TNF-α (274.21±28.63 pg / mL) and IL-6 (6.47±0.17 pg / mL), and increase the anti-inflammatory factor IL-10 (1.39±0.05 pg / mL), showing a synergistic anti-inflammatory effect superior to the single-component encapsulation groups (BC-SA@CS-L, BC-SA@CS-C).

[0057] Example 2

[0058] The specific steps for 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material are as follows:

[0059] (1) Under the stirring conditions of a water bath at 65℃, 4.5g of sodium alginate powder was added to 300mL of aqueous solution and mixed evenly to obtain a sodium alginate solution with a concentration of 0.015g / mL.

[0060] (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation.

[0061] (3) Add 3g of bacterial cellulose nanofibers to the 100mL sodium alginate solution in step (1), and shear and homogenize to obtain a sodium alginate and bacterial cellulose mixed solution.

[0062] (4) Under stirring conditions, add 1g of Lactobacillus rhamnosus powder and 0.5g of cottonseed peptide to the 100mL sodium alginate bacterial cellulose mixed solution in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension.

[0063] (5) Add the 5 mL sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension from step (4) to 100 mL of 0.02 g / mL calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres;

[0064] (6) Add 30g of bacterial cellulose nanofibers to the 200mL sodium alginate solution in step (1), and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2.

[0065] (7) Use the 20mL mixture 2 described in step (6) as 3D printing ink, and uniformly mix the 1g sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing. The printing needle size is 1.2G, the printing speed is 10mm / s, the spray thickness is 0.8mm, and the 3D printed product is obtained.

[0066] (8) A calcium chloride solution with a concentration of 0.02 g / mL is uniformly sprayed onto the surface of the 3D printed product in step (7). The mass-to-volume ratio of the 3D printed product (wet weight) to the calcium chloride solution is 1.5 g / mL. The product is then cured for 20 seconds to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

[0067] The bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material prepared in Example 2 has similar effects to that in Example 1. The outer layer of the bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material is tightly packed, with bacterial cellulose and sodium alginate forming a tight layered structure that resists gastric acid penetration and provides good protection for *Lactobacillus rhamnosus* and cottonseed peptide. This is evident under scanning electron microscopy. Figure 1 The 3D printing ink and embedded microspheres are clearly visible on the C-axis, demonstrating the successful construction of the bilayer structure; the scanning electron microscope also shows this. Figure 1The cross-section containing the microsphere-containing 3D printing ink can be seen on D, with the microspheres tightly connected to the ink. A large number of *Lactobacillus rhamnosus* and cottonseed peptides are embedded internally, causing no physical damage to the *Lactobacillus rhamnosus*. After simulated digestion in vitro, the embedded material resisted the harsh environment of simulated gastric juice and was slowly released in simulated intestinal juice. The embedded *Lactobacillus rhamnosus* and peptides retained good activity. The 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide embedding material can achieve both protection and sustained release of *Lactobacillus rhamnosus* and cottonseed peptides. The material in this embodiment also showed synergistic anti-inflammatory activity in the LPS-induced RAW264.7 cell inflammation model (see Table 1). Its TNF-α and IL-6 inhibition rates and IL-10 promotion rates were consistent with those in Example 1, demonstrating a superior synergistic anti-inflammatory effect compared to the single-component embedding group.

[0068] Example 3

[0069] The specific steps for 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material are as follows:

[0070] (1) Under the stirring conditions of a water bath at 60℃, 9g of sodium alginate powder was added to 300mL of aqueous solution and mixed evenly to obtain a sodium alginate solution with a concentration of 0.03g / mL.

[0071] (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation.

[0072] (3) Add 2g of bacterial cellulose nanofibers to the 100mL sodium alginate solution in step (1), and shear and homogenize to obtain a sodium alginate and bacterial cellulose mixed solution.

[0073] (4) Under stirring conditions, add 0.6g of Lactobacillus rhamnosus powder and 0.9g of cottonseed peptide to the 100mL sodium alginate bacterial cellulose mixed solution in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension.

[0074] (5) Add the 8 mL sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension from step (4) to 200 mL of 0.02 g / mL calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres;

[0075] (6) Add 30g of bacterial cellulose nanofibers to the 200mL sodium alginate solution in step (1), and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2.

[0076] (7) Use the 20mL mixture 2 described in step (6) as 3D printing ink, and uniformly mix the 1.5g sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing. The printing needle size is 1.2G, the printing speed is 10mm / s, the spray thickness is 1.0mm, and the 3D printed product is obtained.

[0077] (8) A calcium chloride solution with a concentration of 0.02 g / mL is uniformly sprayed onto the surface of the 3D printed product in step (7). The mass-to-volume ratio of the 3D printed product (wet weight) to the calcium chloride solution is 2 g / mL. The product is then cured for 30 seconds to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

[0078] The bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material prepared in Example 3 has similar effects to that in Example 2. The outer layer of the bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material is tightly packed, with bacterial cellulose and sodium alginate forming a tight layered structure that resists gastric acid penetration and provides good protection for *Lactobacillus rhamnosus* and cottonseed peptide. This is evident under scanning electron microscopy. Figure 1 The 3D printing ink and embedded microspheres are clearly visible on the C-axis, demonstrating the successful construction of the bilayer structure; the scanning electron microscope also shows this. Figure 1 The cross-section containing the microsphere-based 3D printing ink can be seen on D, with the microspheres tightly connected to the ink. A large number of *Lactobacillus rhamnosus* and cottonseed peptides are embedded internally, causing no physical damage to the *Lactobacillus rhamnosus*. After simulated digestion in vitro, the embedded material resisted the harsh environment of simulated gastric juice and was slowly released in simulated intestinal juice. The embedded *Lactobacillus rhamnosus* and peptides retained good activity. The 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide embedding material can achieve both protection and sustained release of *Lactobacillus rhamnosus* and cottonseed peptides. The material in this embodiment also showed synergistic anti-inflammatory activity in the LPS-induced RAW264.7 cell inflammation model (see Table 1). Its TNF-α and IL-6 inhibition rates and IL-10 promotion rates were consistent with those in Example 2, demonstrating a superior synergistic anti-inflammatory effect compared to the single-component embedding group.

[0079] Example 4

[0080] The specific steps for 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material are as follows:

[0081] (1) Under the stirring conditions of a water bath at 55℃, 9g of sodium alginate powder was added to 300mL of aqueous solution and mixed evenly to obtain a sodium alginate solution with a concentration of 0.03g / mL.

[0082] (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation.

[0083] (3) Add 5g of bacterial cellulose nanofibers to the 100mL sodium alginate solution in step (1), and shear and homogenize to obtain a sodium alginate and bacterial cellulose mixed solution.

[0084] (4) Under stirring conditions, add 1.2g of Lactobacillus rhamnosus powder and 0.3g of cottonseed peptide to the 100mL sodium alginate bacterial cellulose mixed solution in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension.

[0085] (5) Add the 15 mL sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension from step (4) to 200 mL of 0.02 g / mL calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres;

[0086] (6) Add 35g of bacterial cellulose nanofibers to the 200mL sodium alginate solution in step (1), and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2.

[0087] (7) Use the 20mL mixture 2 described in step (6) as 3D printing ink, and uniformly mix the 0.5g sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing. The printing needle size is 1.6G, the printing speed is 10mm / s, the spray thickness is 1.0mm, and the 3D printed product is obtained.

[0088] (8) A calcium chloride solution with a concentration of 0.02 g / mL is uniformly sprayed onto the surface of the 3D printed product in step (7). The mass-to-volume ratio of the 3D printed product (wet weight) to the calcium chloride solution is 1 g / mL. The product is then cured for 15 seconds to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

[0089] The bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material prepared in Example 4 has similar effects to that in Example 2. The outer layer of the bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide encapsulation material is tightly packed, with bacterial cellulose and sodium alginate forming a tight layered structure that resists gastric acid penetration and provides good protection for *Lactobacillus rhamnosus* and cottonseed peptide. This is evident under scanning electron microscopy. Figure 1 The 3D printing ink and embedded microspheres are clearly visible on the C-axis, demonstrating the successful construction of the bilayer structure; the scanning electron microscope also shows this. Figure 1The cross-section containing the microsphere-based 3D printing ink can be seen on D, with the microspheres tightly connected to the ink. A large number of *Lactobacillus rhamnosus* and cottonseed peptides are embedded internally, causing no physical damage to the *Lactobacillus rhamnosus*. After simulated digestion in vitro, the embedded material resisted the harsh environment of simulated gastric juice and was slowly released in simulated intestinal juice. The embedded *Lactobacillus rhamnosus* and peptides retained good activity. The 3D-printed bacterial cellulose-based *Lactobacillus rhamnosus* / cottonseed peptide embedding material can achieve both protection and sustained release of *Lactobacillus rhamnosus* and cottonseed peptides. The material in this embodiment also showed synergistic anti-inflammatory activity in the LPS-induced RAW264.7 cell inflammation model (see Table 1). Its TNF-α and IL-6 inhibition rates and IL-10 promotion rates were consistent with those in Example 2, demonstrating a superior synergistic anti-inflammatory effect compared to the single-component embedding group.

[0090] Table 1: Comparison of synergistic anti-inflammatory effects of combined use of Lactobacillus rhamnosus and cottonseed peptide

[0091] Group LC content (%) TNF-α (pg / mL) IL-6 (pg / mL) IL-10 (pg / mL) Model group 0 418.19±44.60 135.32±0.35 0.94±0.07 BC-SA@CS-L 2.16（L） 394.65±14.41 9.28±0.49 1.33±0.05 BC-SA@CS-C 2.16（C） 302.09±31.72 7.96±0.67 1.31±0.05 BC-SA@CS-LC 0.72 (L) + 1.44 (C) 274.21±28.63 6.47±0.17 1.39±0.05

[0092] Note: LC content (%) refers to the mass fraction of Lactobacillus rhamnosus powder and cottonseed peptide in the dry weight of the final 3D printed product; data are from the lipopolysaccharide-induced RAW264.7 cell inflammation model experiment, n=3 per group. TNF-α and IL-6 are pro-inflammatory factors, and IL-10 is an anti-inflammatory factor.

[0093] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material and its preparation method, characterized in that, Includes the following steps: (1) Sodium alginate was dissolved in an aqueous solution under water bath stirring conditions to obtain a sodium alginate solution; (2) The bacterial cellulose was purified, and after shearing and homogenization, bacterial cellulose nanofibers were obtained by centrifugation. (3) Add bacterial cellulose nanofibers to the sodium alginate solution in step (1), and perform shearing and homogenization to obtain sodium alginate and bacterial cellulose mixed solution 1; (4) Under stirring conditions, add Lactobacillus rhamnosus powder and cottonseed peptide to the sodium alginate bacterial cellulose mixed solution 1 described in step (3), mix evenly, and obtain sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension. (5) Add the sodium alginate bacterial cellulose-Lactobacillus rhamnosus / cottonseed peptide suspension obtained in step (4) to calcium chloride solution to obtain sodium alginate bacterial cellulose microspheres; (6) Add bacterial cellulose nanofibers to the sodium alginate solution in step (1) and shear and homogenize to obtain sodium alginate and bacterial cellulose mixed solution 2; (7) Use the mixture 2 described in step (6) as 3D printing ink, and uniformly mix the sodium alginate bacterial cellulose microspheres described in step (5) into the 3D printing ink, and perform 3D printing to obtain 3D printing products. (8) Spray calcium chloride solution evenly onto the surface of the 3D printed product in step (7) and perform curing treatment to obtain the 3D printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide embedding material.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the sodium alginate solution is 0.01-0.05 g / mL, and the water bath temperature is 45-65℃.

3. The preparation method according to claim 1, characterized in that, In step (3), the mass-to-volume ratio of bacterial cellulose nanofibers to sodium alginate solution in the mixture 1 is 0.01-0.05 g / mL.

4. The preparation method according to claim 1, characterized in that, In step (4), the total mass ratio of Lactobacillus rhamnosus powder and cottonseed peptide to sodium alginate bacterial cellulose mixed solution 1 is 1~10:100; wherein, the mass ratio of Lactobacillus rhamnosus powder to cottonseed peptide is 1~5:1~5. Preferably, the total mass ratio of Lactobacillus rhamnosus powder and cottonseed peptide to sodium alginate bacterial cellulose mixed solution 1 is 1~5:100; wherein, the mass ratio of Lactobacillus rhamnosus powder to cottonseed peptide is 1~5:1~3.

5. The preparation method according to claim 1, characterized in that, The mass-volume ratio concentration of the calcium chloride solution in steps (5) and (8) is 0.01-0.1 g / mL; the volume ratio of the suspension to the calcium chloride solution in step (5) is 1:10-1:50; the mass-volume ratio of the 3D printing product to the calcium chloride solution in step (8) is 1-5 g / mL; and the curing time in step (8) is 5-60 seconds.

6. The preparation method according to claim 1, characterized in that, In step (6), the mass-to-volume ratio of bacterial cellulose nanofibers to sodium alginate solution in the mixture 2 is 0.1-1 g / mL.

7. The preparation method according to claim 1, characterized in that, In step (7), the volume-to-mass ratio of the sodium alginate bacterial cellulose microspheres to the mixed solution 2 is 0.01-0.2 g / mL; the size of the 3D printing needle is 1.2-2.0 G; the printing speed is 5-45 mm / s; and the spray thickness is 0.6-1.2 mm.

8. A 3D printing bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material prepared by the preparation method according to any one of claims 1-7.

9. The 3D-printed bacterial cellulose-based Lactobacillus rhamnosus / cottonseed peptide encapsulation material prepared by the method of claim 1 is applicable to functional foods, food processing technology and biomaterials.

10. The application according to claim 9, characterized in that, The application of the described material in encapsulating probiotics and peptides.

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