Oil-water double-gel bio-ink as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food biological processing, and particularly relates to oil-water double-gel bio-ink, a preparation method thereof and application of the oil-water double-gel bio-ink in probiotic multi-dimensional printing. Protein and polysaccharide react with each other to form hydrogel with better thixotropic restorability, higher viscoelasticity, higher thermal stability and excellent shear thinning capacity, and a proper amount of oil gel is further added to form double gel, so that a gel network structure can be changed, better gel strength and a better protection effect are provided, and the hydrogel can be applied to the field of medical treatment. The method has unique advantages when being applied to 3D printing of probiotic living cells, and the preservation and freeze-drying stability of the probiotics can be remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of food bioprocessing, and particularly relates to an oil-water double gel bioink and a preparation method and application thereof. Background Art

[0002] 3D printing, also known as additive manufacturing technology, is a technology that uses adhesive materials to print layer by layer on the basis of a digital model file to construct a three-dimensional object. At present, it has been gradually applied to the food field, which can effectively solve the problems of low energy utilization efficiency, unsustainability, large greenhouse gas emissions, and large food waste or loss in traditional food processing methods.

[0003] As an important element for maintaining intestinal health, probiotics have significant effects and are recognized by consumers, and their market demand continues to climb. However, the processing methods of traditional probiotic foods often have problems such as unstable probiotic activity, single shape, poor taste, and uneven nutritional components, making it difficult to meet the needs of consumers for high-quality and personalized probiotic foods.

[0004] Through food 3D printing technology, consumers can design and manufacture foods with customized sensory functions and nutritional functions, which can meet the production requirements of intelligent, efficient, convenient, and personalized products, conform to the development direction of future foods, and inject new vitality into the customized development of probiotics.

[0005] The 3D printing probiotic wall material needs to have a certain mechanical strength, the internal gel structure has a certain spatial structure, and has biocompatibility. In addition, the metabolic activities and environmental adaptability of bacteria are diverse, and the materials suitable for 3D printing of probiotics need to simultaneously have the possibility of providing an ideal living environment for the bacteria and be suitable for the bacteria to grow and reproduce in the printing module.

[0006] Food-grade bioinks need to have good mechanical properties, be easy to obtain, easy to process, safe, have nutritional value, and have low cost. It is necessary to not only consider whether the printing is formed, but also consider whether the sensory and nutritional value of the printed material is required by consumers. Hydrogels are common food 3D printing ink materials, and among them, proteins are the most common type of food hydrogels, but the mechanical properties of hydrogels formed by proteins are usually not strong. Protein-polysaccharide type hydrogel inks are prepared by reacting or mixing proteins and polysaccharides to form high internal phase emulsions, which have better thixotropic recovery, higher viscoelasticity, higher thermal stability, and excellent shear thinning ability compared with single polysaccharides and proteins, and can better deliver active substances.

[0007] However, hydrogels have problems such as poor precision, low structural stability, and poor printing quality during the 3D printing process. Adding an appropriate amount of oleogel to the hydrogel to form a double gel can change the gel network structure, provide better gel strength and protection, and has unique advantages in the 3D printing of probiotics. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide an oil-water double gel bioink and its preparation method and application in view of the deficiencies of the prior art.

[0009] To solve the above technical problems, the present invention discloses an oil-water double gel bioink and its preparation method and application.

[0010] A preparation method of an oil-water double gel bioink includes the following steps:

[0011] (1) Prepare probiotic hydrogel: Mix a probiotic suspension with konjac gum to obtain probiotic hydrogel;

[0012] (2) Prepare oleogel: Mix soy protein isolate with vegetable oil to obtain oleogel;

[0013] (3) Mix and homogenize the probiotic hydrogel prepared in step (1) and the oleogel prepared in step (2) to obtain an oil-water double gel bioink. Preferably, the addition of the oleogel can improve the hydrophobicity, thermal stability, and crystallinity of the gel, promote the conversion of free water in the gel into bound water, enhance the water retention capacity of the gel, and limit the flow of water molecules to form a more stable gel, thereby improving the cell encapsulation rate.

[0014] Among them, in step (1), the probiotic is Lactobacillus paracasei, preferably Lactobacillus paracasei FM-LP-PC18, with a preservation number of CGMCC No. 25604, and is preserved in the China General Microbiological Culture Collection Center. The detailed information of Lactobacillus paracasei FM-LP-PC18 has been disclosed in Chinese Patent CN116064314A.

[0015] Among them, in step (1), the probiotic suspension is prepared as follows: After activating the probiotic and centrifuging and washing, resuspend it with physiological saline; the OD of the probiotic suspension 600is 0.9 to 1.1. Preferably, for the activation, the method is: thaw the probiotics stored frozen at -20°C or -80°C at room temperature, inoculate them into MRS medium, and culture and activate them at 36 - 38°C for 24 - 54 h. Preferably, the specific method for preparing the bacterial suspension is: centrifuge the probiotic bacterial liquid obtained by activation at 4000 - 5000 rpm and 4 - 6°C for 8 - 15 min, discard the supernatant, suspend and wash with sterile water or 0.75% physiological saline, repeat the above centrifugation process twice, and adjust the OD of the bacterial liquid with 0.75% physiological saline 600 is 0.9 to 1.1.

[0016] Among them, in step (1), in the probiotic hydrogel, the mass concentration of konjac gum is 2% - 6%. The mass concentration mentioned here represents the mass ratio of konjac gum to the probiotic bacterial suspension.

[0017] Among them, in step (2), the vegetable oil is soybean oil; in the oleogel, the mass concentration of soy protein isolate is 15% - 50%. The mass concentration mentioned here represents the mass ratio of soy protein isolate to the vegetable oil.

[0018] Among them, in step (3), the mixing mass ratio of the probiotic hydrogel and the oleogel is 2:8 to 6:4.

[0019] Among them, in step (3), in the oil-water double gel bioink, the mass concentration of konjac gum is 1.9% - 2.1%, and the mass concentration of soy protein isolate is 9% - 10%. The mass concentration of konjac gum or soy protein isolate mentioned here is the mass ratio of konjac gum or soy protein isolate to the total mass of the bacterial suspension and the vegetable oil.

[0020] Among them, in step (3), for the homogenization, homogenize at 8000 - 10000 rpm for 2 - 3 min. Preferably, homogenize at 10000 rpm for 3 min.

[0021] In a second aspect, the present invention provides an oil-water double gel bioink prepared by the preparation method described in the first aspect.

[0022] In a third aspect, the present invention provides the application of the oil-water double gel bioink described in the second aspect in 3D printing probiotic products. Preferably, load the bioink into a printing cartridge for printing. The specific method is: use software to design the printing model and its parameters, load the bioink into the material cartridge of an extrusion 3D printer, centrifuge at 2000 - 5000 rpm for 5 - 10 min to remove air bubbles, debug the discharging needle head, extrude the material until the ink can flow out smoothly, and calibrate the printing position (zero adjustment). Import the preset model into the printer and use an extrusion 3D printer for printing.

[0023] Beneficial effects:

[0024] The present invention provides a material and process suitable for 3D printing of probiotics. The rheological properties and internal structure of specific gel components ensure that probiotics maintain high activity during printing and consumption, realizing high-activity personalized customization of probiotics. This not only provides consumers with more diverse and personalized product choices to meet the growing demand for customized food in the current market, but also provides theoretical and practical guidance for the 3D printing of probiotics, promoting the application of 3D printing in the field of probiotics. The specific beneficial effects are as follows:

[0025] (1) The bioink materials used in the present invention are all food-grade and have no toxic effect on probiotics.

[0026] (2) The oil-water double gel used in the present invention has excellent thixotropic recovery, viscoelasticity, thermal stability and shear thinning ability, and can better deliver live probiotic cells.

[0027] (3) The oil-water double gel used in the present invention has a good embedding efficiency, acid and bile salt tolerance and storage stability for the embedded probiotics, and has a good protective effect.

[0028] (4) The oil-water double gel used in the present invention gives the loaded probiotics space for growth and reproduction, and can realize the further proliferation of probiotics.

[0029] (5) The bioink prepared by the present invention can be used for 3D printing of probiotics and further applied to the production of fermenting agents, bacterial preparations, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0031] Figure 1 For the preparation and printing process of bioink.

[0032] Figure 2 For the rheological properties of double gels with different KGM contents, Figure 2 A is the viscosity data of double gels with different KGM contents, Figure 2 B is the elastic modulus data of double gels with different KGM contents, Figure 2 C is the loss factor (tanδ) data of double gels with different KGM contents, Figure 2 D is the creep / recovery data of double gels with different KGM contents.

[0033] Figure 3 For the rheological properties of double gels with different SPI contents, Figure 3 A is the viscosity data of double gels with different SPI contents, Figure 3 B is the elastic modulus data of double gels with different SPI contents,Figure 3 C is the loss factor (tanδ) data of double gels with different SPI contents, Figure 3 D is the creep / recovery data of double gels with different SPI contents.

[0034] Figure 4 are the rheological properties of double gels with different oil / hydrogel ratios, Figure 4 A is the viscosity data of double gels, Figure 4 B is the elastic modulus data of double gels, Figure 4 C is the loss factor (tanδ) data of double gels, Figure 4 D is the creep / recovery data of double gels.

[0035] Figure 5 are the scanning electron microscope images of double gels, Figure 5 A are the scanning electron microscope images of double gels with different KGM contents; Figure 5 B are the scanning electron microscope images of double gels with different SPI contents; Figure 5 C are the scanning electron microscope images of double gels with different O:H ratios.

[0036] Figure 6 are the stained sections of double gels, Figure 6 A are different KGM contents; Figure 6 B are different SPI contents; Figure 6 C are different oil gel / hydrogel ratios.

[0037] Figure 7 are the transverse relaxation images with different oil contents, Figure 7 A is the relaxation decay curve; Figure 7 B is the single-component relaxation time (T 2W ) spectrum; Figure 7 C is the distribution of the transverse relaxation time (T2) spectrum; Figure 7 D is Figure 7 the enlarged image within 10 -2 -10 2 range in C.

[0038] Figure 8 are the effects of different double gel components on the entrapment rate of strains, Figure 8 A are different KGM contents; Figure 8 B are different SPI contents; Figure 8 C are different oil gel / hydrogel ratios.

[0039] Figure 9 are the effects of different double gel components on the low-temperature storage of strains, Figure 9 A are different KGM contents; Figure 9 B are different SPI contents; Figure 9 C are different oil gel / hydrogel ratios.

[0040] Figure 10 Effect of different double-gel components on freeze-drying storage of strains Figure 10 A is different KGM contents; Figure 10 B is different SPI contents; Figure 10 C is different oleogel / hydrogel ratios.

[0041] Figure 11 Effect of different double-gel components on acid tolerance stability of strains Figure 11 A is different KGM contents; Figure 11 B is different SPI contents; Figure 11 C is different oleogel / hydrogel ratios.

[0042] Figure 12 Effect of different double-gel components on bile salt tolerance stability of strains Figure 12 A is different KGM contents; Figure 12 B is different SPI contents; Figure 12 C is different oleogel / hydrogel ratios.

[0043] Figure 13 Effect of different double-gel components on gastric juice tolerance stability of strains Figure 13 A is the effect of different KGM contents on gastric juice stability; Figure 13 B is the effect of different SPI contents on gastric juice stability; Figure 13 C is the effect of different oleogel / hydrogel ratios on gastric juice stability.

[0044] Figure 14 Effect of different double-gel components on intestinal juice tolerance stability of strains Figure 14 A is the effect of different KGM contents on intestinal juice stability; Figure 14 B is the effect of different SPI contents on intestinal juice stability; Figure 14 C is the effect of different oleogel / hydrogel ratios on intestinal juice stability. Specific implementation mode

[0045] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0046] Example 1 Screening of rapid gelation of polysaccharide colloids

[0047] In this example, the following 6 different colloid materials were selected to prepare the mother liquor:

[0048] 6% konjac glucomannan mother liquor: Dissolve 6 g of konjac glucomannan powder in 100 mL of distilled water, and stir evenly at room temperature until completely dissolved;

[0049] 1% Polyoxyethylene-polypropylene ether copolymer (PF127) stock solution: Dissolve 1 g of PF127 powder in 100 mL of distilled water and stir evenly at room temperature;

[0050] 6% Gellan gum stock solution: Add 6 g of gellan gum powder to 100 mL of distilled water, place it on a magnetic stirrer and stir evenly at 90 °C. After stirring evenly, maintain the temperature at 65 °C;

[0051] 3% Gelatin stock solution: Dissolve 3 g of gelatin flakes in 100 mL of distilled water and stir magnetically at 60 °C until completely dissolved;

[0052] 5% Peach gum stock solution: Place 5 g of peach gum in 100 mL of distilled water and let it swell overnight, then break it up evenly in a blender, and then stir evenly at 8000 r / min in a homogenizer for standby;

[0053] 6% Sodium alginate stock solution: Dissolve 6 g of sodium alginate in 100 mL of distilled water and stir evenly at room temperature until dissolved. Arbitrarily mix any two of the above 6 colloid materials in a mixing mass ratio of 1:1, and use magnetic stirring until completely stirred evenly. The results show that after screening a variety of materials, the above materials and related compounding did not obtain relatively excellent bioink materials. The gels after mixing were all relatively poor and could not gel quickly. After mixing, the gel temperature was adjusted appropriately, but no effective gel was formed. However, konjac gum has good film-forming properties and can be further investigated later.

[0054] Example 2 Protein Screening

[0055] Soybean protein isolate (SPI) has good gelation properties, but using single soybean protein isolate as printing ink will limit the integrity of the printed structure and the complexity of the shape.

[0056] In this example, 1 g, 2 g, 3 g, 4 g, and 5 g of whey protein isolate or soybean protein isolate were weighed respectively, dissolved in 50 mL of distilled water at room temperature to obtain protein solutions. Adjust the pH of the protein solution to 3 with 1 mol / L HCl solution, stir and heat overnight at 90 °C to prepare acid-modified whey protein isolate and soybean protein isolate, aiming to transform the protein in solution state into solid gel and investigate its influence on 3D printing. The results show that the gel effect is poor, the texture is fragile and difficult to recover when encountering shear force; the forming time is long and it cannot be printed on a 3D printer. In order to improve the printability of SPI, physical cross-linking methods or cross-linking with other substances are usually used to improve the properties of SPI, such as polysaccharides. In addition, based on the easy availability and low cost of soybean protein isolate, soybean protein isolate was selected to cross-link with polysaccharides in subsequent experiments to improve its properties.

[0057] Example 3 Preparation of Protein-polysaccharide 3D Printing Bioink

[0058] The preparation process of the bioink and the printing process are as Figure 1 shown. In this embodiment, the mass percentage, unless otherwise specified, refers to the mass ratio of the mixed solid to the liquid.

[0059] 1. Preparation of gel samples with different konjac glucomannan (KGM) contents

[0060] (1) Weigh 1, 2, and 3 g of KGM powder respectively and mix them evenly with 50 g of water, and let them fully absorb water and swell to prepare hydrogels with KGM mass contents of 2%, 4%, and 6% respectively (here, % represents the mass ratio of KMG powder to water).

[0061] (2) Weigh 10 g of SPI powder and mix it evenly with 50 g of soybean oil to prepare an oleogel with an SPI mass content of 20% (here, % represents the mass ratio of SPI powder to soybean oil).

[0062] (3) Mix the hydrogels with different KGM contents and the oleogel at a ratio of 1:1 w / w, homogenize them at 10000 rpm for 3 min, load them into the printing cylinder, and centrifuge them at 2000 rpm for 10 min to degas, to make SPI / KGM double gels with an SPI mass content of 10% and KGM mass contents of 1%, 2%, and 3% respectively, denoted as K1, K2, and K3.

[0063] 2. Preparation of gel samples with different soy protein isolate (SPI) contents

[0064] (1) Weigh 2 g of KGM powder and mix it evenly with 50 g of water to prepare a hydrogel with a KGM mass content of 4%.

[0065] (2) Weigh 6, 8, 10, 12, and 14 g of SPI powder respectively and mix them evenly with 50 g of soybean oil to prepare oleogels with SPI mass contents of 12%, 16%, 20%, 24%, and 28% respectively.

[0066] (3) Mix the oleogels with different SPI contents and the hydrogel at a ratio of 1:1 w / w, homogenize them at 10000 rpm for 3 min, load them into the printing cylinder, and centrifuge them at 2000 rpm for 10 min to degas, to make SPI / KGM double gels with a KGM mass content of 2% and SPI mass contents of 6%, 8%, 10%, 12%, and 14% respectively, denoted as S6, S8, S10, S12, and S14.

[0067] 3. Preparation of samples with different oil / hydrogel ratios

[0068] Preparation method of the control group: Weigh 1.00 g of KGM powder and 5.00 g of SPI powder respectively and mix them with 50 g of water to prepare SPI / KGM gel. The mass concentration of KGM is 2.00% (mass ratio of KMG powder to water), and the mass concentration of SPI is 10% (mass ratio of SPI to water), denoted as O:H 0:10, serving as the oil-free group control.

[0069] Preparation method of the experimental group:

[0070] (1) Weigh 1.25, 1.43, 1.67, 2.00 and 2.50 g of KGM powder respectively and mix them with 50 g of water to prepare KGM hydrogels with mass concentrations of 2.50%, 2.86%, 3.33%, 4.00% and 5.00%.

[0071] (2) Weigh 20.00, 15.00, 12.50, 10.00 and 8.33 g of SPI powder respectively and mix them evenly with 50 g of soybean oil to prepare oleogels with SPI mass contents of 50%, 33.33%, 25%, 20% and 16.66% respectively.

[0072] (3) Mix according to the mass ratio of oleogel to hydrogel in Table 1, homogenize at 10000 rpm for 3 min, fill into a printing cylinder, and centrifuge at 2000 rpm for 10 min to degas, to prepare SPI / KGM double gels with oleogel / hydrogel ratios of 2:8, 3:7, 4:6, 5:5 and 6:4, denoted as O:H 2:8, O:H 3:7, O:H 4:6, O:H 5:5 and O:H 6:4 respectively.

[0073] Table 1 Mixing mass ratio of oleogel and hydrogel

[0074]

[0075] Example 4 Rheological analysis

[0076] Rheological detection was measured using a rheometer DiscoveryHR10. The rheometer was equipped with parallel plates with a diameter of 40 mm and a gap of 1000 μm. The apparent viscosity (η) was measured through a static rheological experiment in the range of shear rate from 0.01 to 100 s -1 range.

[0077] Before evaluating the flow properties of the ink using dynamic scanning tests, ensure that the samples are measured under the conditions of 1 Hz, 10 rad / s speed and 0.01% - 10% strain, and maintain the condition of 0.2% constant strain change to obtain the rheological test data of the samples in the linear viscoelastic region. The creep / recovery test uses a constant shear force of 10 Pa, and the creep time and recovery time are set to 180 s and 420 s respectively.

[0078] Unless otherwise specified, the temperature was maintained at 25 °C for all the above tests.

[0079] 1. Influence of different konjac glucomannan (KGM) contents on rheological properties

[0080] The rheological properties of the material are closely related to its printability and self - supportability. The viscosities of the prepared double - gel samples all decreased with the increase of the shear rate. When the shear rate was in the range of 0.01 - 100 (1 / s), the viscosities of K1, K2, and K3 decreased from 2.2×10 4 Pa·s, 2.7×10 4 Pa·s, 3.8×10 4 Pa·s to 9.8 Pa·s, 13.3 Pa·s, and 14.9 Pa·s respectively, as shown in Figure 2 A, indicating that the above three double - gels all have the property of shear thinning, which is a prerequisite for 3D printing inks. According to the height difference of the Figure 2 A curve, within the shear rate range of 0.01 - 100 (1 / s), the viscosity of K3 is always greater than that of K2 and K1. Increasing the KGM content can increase the viscosity of the 3D printing ink material, which is beneficial for the ink to pass through the printing nozzle and maintain its shape, reducing the occurrence of filament breakage.

[0081] From the study of the viscoelasticity of double - gels with different KGM contents, it can be seen that the G’ and G” values of the three samples all show an upward trend, and G’ is greater than G”, indicating that the three samples are stable solids ( Figure 2 B). Tanδ represents G” / G’. At an angular frequency of 0.1 - 100 (rad / s), the Tanδ values of K1, K2, and K3 decreased from 0.13, 0.20, and 0.18 to 0.12, 0.14, and 0.14 respectively ( Figure 2 C). Tanδ values less than 1 all indicate the potential for 3D printing. With the increase of the KGM content, both G’ and G” increase, indicating that KGM can effectively improve the viscoelasticity and mechanical strength of the gel. This may be because increasing the KGM content can increase the molecular interaction between SPI, forming a denser and stronger molecular network structure. The higher the KGM content, the stronger the network structure, and the more it shows solid - like behavior.

[0082] The creep - recovery experiment is often used to test the ability of a sample to recover its shape under a certain constant stress and after the stress is removed. Among them, the magnitude of the strain value (γ) is related to the recovery performance of the sample before and after the stress is generated and disappeared and the mechanical strength after being stressed. The γ values of K1, K2, and K3 are 0.98%, 0.84%, and 0.38% respectively ( Figure 2D), as the KGM content increases, γ gradually decreases, indicating that the addition of KGM can improve the creep recovery ability and mechanical strength of the gel. Therefore, appropriately increasing the KGM content can effectively improve the viscosity, mechanical strength, and creep recovery ability of the ink.

[0083] 2. Influence of different protein contents on rheological properties

[0084] As one of the main components of the double gel, SPI has a particularly important influence on its rheological properties. As Figure 3 shown in A, the viscosities of double gels with different SPI concentrations all gradually decrease as the shear rate increases, indicating that they all have shear-thinning properties. In the shear rate range of 0.01 s -1 to 100 s -1 , higher viscosities are exhibited with the increase of SPI content. Materials with lower apparent viscosities are more conducive to passing through the printing nozzle, which indicates that the double gel samples S6, S8, and S10 with low protein contents are more conducive to extrusion, while the high protein contents S14 and S12 are more conducive to maintaining the shape after extrusion.

[0085] In the range of angular frequency from 0.1 to 100 rad / s, the G' of double gel samples with different SPI is greater than G", showing a stable solid state ( Figure 3 B). At the same time, as the angular frequency increases, both G' and G" increase, indicating that the internal friction force of the double gel increases, the mechanical strength improves, and the ability to resist external forces and maintain shape increases. As Figure 3 shown in C, the Tanδ of double gel samples with different SPI is less than 1, and the Tanδ value continuously decreases as the angular frequency increases, and the Tanδ value decreases as the SPI content increases. The above research shows that the increase of SPI will promote the connection of the internal gel network structure of the material to be closer, which has an important regulatory effect on the stability of the double gel.

[0086] SPI also has an important influence on the creep recovery of the double gel. As the SPI increases, the strain size of the double gel decreases from 2.34% to 0.53%, as Figure 3 shown in D, which indicates that increasing the SPI content can reduce the deformation of the double gel. This may be due to the increase of the physical cross-linking between SPI and KGM, thus enhancing the self-supporting force of the double gel.

[0087] 3. Influence of different oil / water ratios on rheological properties

[0088] Different oil / water gel ratios may affect the molecular interaction between SPI and KGM, thus affecting the rheological properties. As Figure 4It can be obtained that in the shear rate range of 0.01 - 100 (1 / s), the viscosities of O:H 0:10, O:H 2:8, O:H 3:7, O:H 4:6, O:H 5:5, and O:H 6:4 decrease from 1.12×10 5 Pa·s, 7.63×10 4 Pa·s, 6.16×10 4 Pa·s, 6.18×10 4 Pa·s, 5.01×10 4 Pa·s, and 8.15×10 4 Pa·s to 3.78 Pa·s, 13.75 Pa·s, 15.87 Pa·s, 15.23 Pa·s, 15.86 Pa·s, and 16.12 Pa·s, respectively. As the proportion of oleogel increases, the viscosity of the double gel shows a downward trend. In the shear rate range of 10 - 100 (1 / s), the addition of oil has no significant effect on the viscosity between the double gels. However, compared with the gel without oil, in the shear rate range of 0.01 - 10 (1 / s), the viscosity of O:H 0:10 is greater than that of various double gels, while in the shear rate range of 10 - 100 (1 / s), the viscosity of O:H 0:10 is less than that of the double gels, indicating that the presence or absence of oil has an important impact on the rheological properties of the gel.

[0089] Analysis of the viscoelasticity of double gels with different O:H ratios shows that, as Figure 4 shown in B, when the proportion of oleogel is less than or equal to that of hydrogel, as the proportion of oleogel increases, G’ and G” of the double gel samples decrease, indicating that increasing the proportion of oleogel promotes the transition of the double gel to a more fluid state. This may be because the addition of oil makes the molecular chains of SPI and KGM have greater freedom, are more likely to move and rearrange, making the internal structure of the gel looser and more unstable, thus affecting the viscosity and elasticity of the material. When the proportion of oleogel is greater than that of hydrogel, the values of G’ and G” increase instead when the oleogel is further increased. It may be that O:H reaches a certain balance, causing a change in the arrangement of the two emulsion molecules in the emulsion to form a new gel network, thus the values of G’ and G” increase. The loss factor Tanδ values of double gels with different O:H ratios are in the range of 0.07 - 0.24 Pa, and the ratios are all less than 1 ( Figure 4 C).

[0090] Figure 4D analyzed the effect of different oleogel / hydrogel ratios on the creep recovery of double gels. The γ values of samples with O:H ratios of 0:10, O:H 2:8, O:H 3:7, O:H 4:6, O:H 5:5, and O:H 6:4 at an angular frequency of 0.1 - 100 (rad / s) were 0.42%, 0.55%, 0.78%, 0.96%, 0.84%, and 0.65% respectively. The order of strain magnitude was O:H 4:6 > O:H 5:5 > O:H 3:7 > O:H 6:4 > O:H 2:8 > O:H 0:10. When adding oil to the hydrogel, at low oil content, the emulsification in the hydrogel was unstable, and the attraction between oil droplets was not sufficient to form a stable network structure, resulting in a decrease in viscosity, self-supporting force (G’, G”), and creep recovery. When the O:H ratio was greater than 4:6, the oil droplets were still flocculated but trapped in the semi-solid network and could not flow quickly, resulting in an increase in viscosity, self-supporting force (G’, G”), and creep recovery.

[0091] 4. Scanning Electron Microscope

[0092] After the gel or printed product was freeze-dried, a tool knife was used to cut the freeze-dried block of bioink into slices of 1 - 2 mm. The samples were fixed on a sample holder with conductive double-sided adhesive tape using the tape method, and a conductive layer (gold spraying) was deposited using a vacuum coating instrument. Observation was carried out under an accelerating voltage of 5 kV, and clear images were taken at an appropriate magnification.

[0093] Observed by scanning electron microscopy ( Figure 5 A - C), all SPI / KGM double gels presented a continuous, loose, and porous cross-linked network structure. This structure was caused by the cross-linking and aggregation of proteins and polysaccharides and the interaction between oil and polysaccharides. The gel microstructure had a certain effect on its viscoelasticity, etc. K1 had a relatively large number of large pores. As the KGM content increased, the number of large pores decreased. The more large pores there were, the less tightly the gel was combined, and the viscoelasticity of the gel decreased, which was consistent with the results of viscoelasticity tests in rheology. As the SPI content increased, the large porosity decreased significantly, and the hardness increased accordingly. There were relatively large pores in S6, which not only damaged the structural integrity but also decreased the elastic modulus and loss modulus. Increasing the protein concentration and the cross-linking degree of the gel network led to a decrease in pore size, indicating the key role of SPI concentration in enhancing gel strength and overall 3D printing performance. Figure 5 It can be seen from C that as the O:H ratio increased, the gel pore size gradually decreased because SPI was amphiphilic, and its hydrophobic groups gradually combined with the oil, while the hydrophilic groups were connected to the water phase, making the gel combine more tightly and thus reducing the pores in the gel.

[0094] 5. Gel Section Staining

[0095] When using Oil Red O staining, fix and embed the fixed sections with OCT at -20 °C. Then immerse the frozen sections in the Oil Red O dye solution for 8 - 10 min (protected from light), and rinse twice with 60% isopropanol followed by rinsing with distilled water. Subsequently, stain the sections with hematoxylin for 3 - 5 min and rinse with distilled water for 5, 10, and 30 s respectively. Finally, mount the sections with glycerol gelatin.

[0096] When using glycogen - naphthol yellow S staining (PAS - NYS), embed the samples in paraffin and section them. Then, stain the sections in the PAS dye for 25 min protected from light. After washing with distilled water for 5 min, immerse the sections in naphthol yellow S for 5 min, quickly rinse with distilled water, dehydrate in absolute ethanol, and mount with glycerol gelatin.

[0097] The above - mentioned sections were observed and recorded by microscopy photography.

[0098] To further understand the interactions between proteins, polysaccharides, and oils in the SPI / KGM double - gel, Oil Red O staining and PAS / naphthol yellow S (PAS - NYS) staining were used to label protein / oil and protein / polysaccharide respectively. When stained with Oil Red O / hematoxylin, the proteins in the sample were stained blue and the oil droplets were stained red; while when stained with PAS / NYS, the proteins in the sample were stained orange and the polysaccharides were stained pink. According to Figure 6 As shown in A - C, in the Oil Red O staining, it can be observed that SPI formed irregular ring - shaped proteins and also some circular structures. These circular protein structures might be spherical before sectioning, and some of the spherical structures were filled with oil droplets in the middle and were stained red. Through PAS - NYS, pink reticular structures were observed in the interstitial regions between proteins. These regions might be occupied by KGM, indicating that the KGM - based hydrogel and the SPI - based oleogel might be mutually penetrating.

[0099] The staining of gels with different KGM contents is shown in Figure 6 A. When increasing the KGM content, the red - dotted area of the double - gel in the Oil Red O staining decreased, but the number increased, indicating that the oil formed finer structures; while in the PAS / NYS staining, it was found that the pink area in the section background increased significantly, and the orange protein part became finer, indicating that the addition of KGM interpenetrated with SPI and gradually occupied the gaps of SPI, making the relatively loose protein structure transform into a more uniform, continuous, and dense one, forming larger aggregates.

[0100] The staining of gels with different SPI contents is shown in Figure 6B. When the SPI concentration increases, the diameter of the red oil droplets in the double gel stained with Oil Red O significantly decreases. This may be because SPI has amphiphilicity and emulsifying ability. The hydrophobic groups of SPI can adsorb with the oil. The hydrophobic groups on the surface of SPI at low concentration are oversaturated and bound by the oil, and the remaining oil aggregates together, increasing the oil droplet diameter. Conversely, the oil droplets will show a uniform dispersion structure.

[0101] In the PAS / NYS staining, KGM and SPI still form an interlaced network structure. As the SPI content increases, the corresponding orange area increases.

[0102] The gel staining with different O:H ratios is as Figure 6 C. As the oleogel content increases, the red area stained with Oil Red O gradually increases. Proteins and oils may increase the gel network structure through some interaction force. In the PAS / NYS staining, there is no significant difference in the composition between proteoglycans, indicating that the increase in oil content within this range does not significantly affect the relationship between polysaccharides and proteins.

[0103] 6. Low-field NMR analysis

[0104] Accurately weigh 2 g of the sample and place it in a 15-mm diameter NMR tube. Select the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence to measure the transverse (T2) relaxation time of the sample. Sampling frequency (SW) = 250 kHz, number of repeated scans (NS) = 4 times, half echo time (DL1) = 0.5 ms, number of echoes (EchoCount) = 5000, and repetition time = 2000 ms. Measure the T2 transverse relaxation time.

[0105] As shown in Figure 7 A, the attenuation trends of the double gel samples with different oil / hydrogel ratios are relatively similar. The higher the oleogel ratio, the greater the curvature of the curve, indicating that increasing the oleogel ratio accelerates the attenuation of water molecules. Using single-component relaxation time (T 2W ) to analyze the overall relaxation characteristics of hydrogen ions in the printing ink material sample ( Figure 7 B), the T 2W values of O:H 3:7, 4:6, 5:5, and 6:4 are 133.7 ms, 121.7 ms, 100.8 ms, and 90.7 ms respectively. As the oil content increases, T 2W continually decreases, indicating that the oil makes the hydrogen protons in the gel subject to a stronger binding force. The hydrogen protons in the SPI / KGM double gel mainly come from the hydrogen protons in the aqueous phase and the lipid protons in the oil phase. And T22 is often considered semi-immobilized water, which is not tightly bound to macromolecules but is located near hydrophilic groups. The transverse relaxation time spectrum (T2) distribution shows that ( Figure 7(Figs. C-D and Table 2), as the oil content increases, the relaxation time of T2 shifts to the left as a whole, and the peak area also decreases accordingly, indicating an increase in bound water and a decrease in free water. Increasing the oil content will convert some of the water in the SPI / KGM double gel into tightly bound water, thereby promoting the formation of a more compact network structure in the gel and further hindering the flow of water molecules to improve the water-holding capacity of the ink. This phenomenon is consistent with the rheological results of different oil / water gel ratios before. Relevant research shows that the main component of soybean oil is triglyceride, whose structure is more complex than that of water molecules, and its viscosity is also significantly higher than that of water. Under the action of a complex molecular structure and a relatively high viscosity, the hydrogen protons in the molecule are in a relatively bound state, and the movement of hydrogen protons in the external magnetic field is also inhibited, resulting in a shorter relaxation time of T2. At the same time, T2 also changes with the change of the macromolecular structure. Therefore, the increase in oil will also cause changes in the internal structure of the sample, resulting in a certain degree of reduction in the fluidity of the printing ink.

[0106] Table 2 Transverse relaxation time of double gels with different oil-water ratios

[0107]

[0108] Note: Lowercase letters indicate significant differences between sample groups (p < 0.05)

[0109] Example 5 Preparation of probiotic gel

[0110] The seed culture solution of a probiotic Lactobacillus paracasei FM-LP-PC18 (preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 25604, hereinafter referred to as PC18, and the detailed information of this strain has been published in Chinese Patent CN116064314A) was added to MRS medium and cultured at 37 °C for 24 h to obtain an activated bacterial solution, which was centrifuged at 4 °C and 5000 rpm for 15 min. The cell precipitate was taken and washed twice with physiological saline (0.75% NaCl), and then centrifuged at 4 °C and 5000 rpm for 5 min to obtain the cell precipitate. The OD of the bacterial solution was adjusted with physiological saline (0.75% NaCl) 600 to 1, which was the bacterial suspension used later. According to the preparation method described in Example 3, KGM powder was weighed and added to the above-mentioned bacterial suspension according to the ratio and stirred to prepare a KGM hydrogel containing PC18; at the same time, SPI powder was weighed and evenly dispersed in soybean oil. The probiotic hydrogel based on KGM and the oil gel based on SPI were loaded into the printing cylinder under a high-speed homogenizer (10,000 rpm, 3 min) and centrifuged at 2000 rpm for 10 min to degas, so as to prepare 3D printing with different formulations. In addition, 15% skim milk powder (SMP) and 2% KGM were added to encapsulate probiotics as a double-gel encapsulation control.

[0111] The 15% skim milk powder control group (SMP group): It is obtained by mixing skim milk powder with the bacterial suspension, where the mass concentration of skim milk powder is 15%.

[0112] The 2% KGM control group (CK group): It is obtained by mixing KGM with the bacterial suspension, where the mass concentration of KMG is 2%.

[0113] 1. Study on entrapment efficiency

[0114] Take 1 g of the sample containing bacteria and place it in physiological saline, and disrupt it at 5000 rpm for 3 min. After disruption, take the mixed solution and spread it on a plate for viable cell counting. Entrapment rate (%) = (N / N1) × 100, where N is the number of viable cells entrapped CFU / mL, and N1 is the number of viable cells added before entrapment CFU / mL.

[0115] The entrapment rates of K1, K2, and K3 are 69.29% ± 4.19%, 72.02% ± 8.11%, and 74.09% ± 3.40% respectively, and the entrapment rate increases with the increase of KGM concentration ( Figure 8 A).

[0116] Protein has the characteristics of being healthy, non-toxic, and highly biocompatible, etc. It can bind to polysaccharides through non-covalent bonds to form a more stable, denser, and higher mechanical strength gel network, compensating for the disadvantages brought by a single wall material. The entrapment rates of S10, S12, and S14 are 68.21% ± 4.61%, 71.90% ± 5.34%, and 72.02% ± 8.11% respectively, and the entrapment rate increases with the increase of SPI concentration, but there is no significant difference in the entrapment rate from the commonly used bacterial strain packaging material SMP ( Figure 8 B).

[0117] Oil can be used for bacterial cell encapsulation, and to a certain extent, it can greatly improve the encapsulation efficiency of probiotics. As Figure 8 shown in C, after increasing the oil content, the entrapment rate increases from 70.15% ± 3.42% to 87.37% ± 3.11%. This may be because increasing the oil content gradually expands the gel dispersion space, reduces the intermolecular friction, forms a more stable gel, and thus improves the bacterial cell entrapment rate.

[0118] 2. Study on low-temperature storage stability

[0119] Samples containing PC18 are 3D printed, and each sample maintains 1 cm 3, stored in a sterile container, stored at 4 °C for 60 days, sampled every 5 days, 1 g was taken each time and put into 100 mL of 0.85% sterile normal saline (containing 0.5% Tween 80) for disruption, vortexed and mixed evenly, 1 mL of the mixed solution was taken for pour plate counting, the viable bacteria count was measured, and the viable bacteria rate was calculated.

[0120] The concentrations of polysaccharides, proteins and oils directly affect the stability of the gel structure and thus the storage stability of the bacteria. Through low-temperature storage research ( Figure 9 ), it was found that the survival rates of the unprotected CK group and the KGM control group decreased rapidly during low-temperature storage and there was no viable bacteria at 35 days; the SMP group protected by conventional strain packaging materials also had no viable bacteria at 50 days; while for the groups embedded with SPI / KGM double gels, there was still a high strain survival rate at 60 days. After 60 days of low-temperature storage, the survival rates of K1, K2, and K3 were 39.39% ± 2.15%, 51.58% ± 1.72%, and 54.76% ± 1.05% respectively. When the KGM content was less than 2%, increasing the KGM content could significantly improve the low-temperature storage survival rate, as shown in Figure 9 Figure A; the survival rates of S10, S12, and S14 were 51.58% ± 1.72%, 52.06% ± 5.140%, and 56.08% ± 2.06% respectively. The bacterial survival rate increased with the increase of SPI, but increasing the SPI content did not significantly improve the viable bacteria count during storage ( Figure 9 Figure B), while the survival rates of O:H 0:10, 2:8, 3:7, 4:6, 5:5, and 6:4 were 44.34% ± 1.05%, 43.39% ± 2.54%, 46.44% ± 3.78%, 50.14% ± 3.29%, 51.58% ± 1.72%, and 44.74% ± 1.06% respectively. Increasing the proportion of the oil gel in the oil gel / hydrogel ratio had a significant effect on the storage survival rate, and the bacterial survival rate increased with the increase of the oil gel ratio ( Figure 9 Figure C), but too high an oil gel ratio also began to affect the probiotics during low-temperature storage.

[0121] Probiotics encapsulated solely with KGM did not extend the storage time, indicating that the main role of KGM might be to provide a viable space for the bacterial cells. When probiotics were encapsulated with a double gel, an increasing KGM content would enhance the interaction with SPI, thereby improving the stability of the gel structure to achieve the effect of protecting the bacterial cells. Common proteins could form a protein film on the surface of the bacterial cells, protecting the strains from damage by the external environment, and at the same time, could also serve as an energy source for the survival of the bacterial cells. Oil gels had high hydrophobicity and emulsifying properties, could interact with water through hydrogen bonds to better isolate water, reduce the water activity of the bacterial cells in the gel, and when the water content decreased to the critical value, the impact on the storage of the bacterial cells was reduced, which could, to a certain extent, extend the storage time. Therefore, increasing the contents of KGM, SPI, and oil gels could, to a certain extent, improve the low-temperature storage stability of probiotics.

[0122] 3. Study on the freeze-drying storage stability

[0123] Samples containing PC18 were 3D printed, and each sample was maintained at 1 cm 3 , and the printed PC18 was freeze-dried, placed in a sterile container for a 60-day storage experiment. Every 5 days, it was taken out and diluted with 100 mL of sterile normal saline containing 0.5% Tween 80, vortexed and mixed evenly, 1 mL of the mixed solution was taken and poured onto a plate for counting, and the viable bacteria count was measured to calculate the viable bacteria rate to evaluate the storage stability.

[0124] Through the freeze-drying storage study ( Figure 10 ), it was found that the survival rate of the unprotected CK group and the KGM control group decreased more during freeze-drying storage than that of the SMP group. For the groups encapsulated with the SPI / KGM double gel, the survival rate of the bacterial cells decreased slowly during freeze-drying storage, and at 60 d, the survival rate of the strains was at least twice that of the unprotected CK group and the KGM control group, and was also significantly higher than that of the SMP group (63% ± 1.00%). After 60 d of freeze-drying storage, the survival rates of the bacterial cells of K1, K2, and K3 were 67.27% ± 1.03%, 70.15% ± 2.36%, and 75.90% ± 3.06% respectively, as Figure 10 shown in Figure 10 A; the survival rates of the bacterial cells of S10, S12, and S14 were 70.15% ± 2.36%, 73.06% ± 2.71%, and 79.51% ± 1.27% respectively, as Figure 10C). When the SPI content is increased within the range of 10% - 14% and the KGM content is increased within the range of 1% - 3%, the freeze-dried storage survival rate can be significantly improved. The O:H ratio has a significant impact on the freeze-dried storage survival rate. When the O:H ratio is lower than 4:6, the viable cell count can be significantly increased, while when it is greater than 4:6, the storage survival rate will decrease significantly.

[0125] Ordinary hydrogels will form larger pores after freeze-drying. As the storage time prolongs, the bacteria are more affected by the external adverse factors, resulting in a decrease in the bacterial activity. Adding KGM to SPI can reduce the gel porosity and provide certain binding sites for probiotics. However, when the KGM concentration reaches 2%, the improvement of the gel structure is not significant. Increasing the SPI content can reduce the pore size and porosity of the gel after freeze-drying, and reduce the contact time of some embedded bacteria with the external environment. To a certain extent, the impact of SPI on improving the storage of bacteria is greater than that of KGM. When the O:H ratio is less than 4:6, the storage stability of the bacteria gradually increases. However, when O:H is greater than 4:6, excessive oil gels have oil precipitation after freeze-drying. Due to the poor freeze-thaw stability of the colloid, a large number of bacterial survival rates decrease. Therefore, increasing the contents of KGM, SPI, and oil gels can improve the freeze-dried storage stability of probiotics to a certain extent.

[0126] 4. Acid resistance

[0127] Samples containing PC18 were 3D printed, and each sample was maintained at 1 cm 3 , and 1 g of the accurately weighed PC18 sample after printing was added to the MRS liquid medium with a pH of 2, and stirred at 37 °C for 4 h. Samples were taken at 0, 1, 2, and 4 h for gradient dilution, and three suitable dilution gradients were selected for colony counting and calculation of the survival rate.

[0128] The survival rate (%) = (A1 / A2) × 100, where: A1 is the viable cell count CFU / mL in the MRS liquid medium with a pH of 2 at 0 h; A2 is the viable cell count CFU / mL at 1, 2, and 4 h in the MRS liquid medium with different pH values of 2.

[0129] After 4 h of acid resistance investigation, the survival rate of probiotics in the unprotected CK group was 0%. The bacterial survival rates of K1, K2, and K3 were 74.27 ± 0.24%, 79.13 ± 0.34%, and 84.23 ± 0.62% respectively. The bacterial survival rate increased with the increase of KGM, and was at least 6.11 times the survival rate of the bacteria encapsulated by KGM gel alone and 2.52 times that of the SMP group. For example, Figure 11As shown in Figure A; the 4-hour viable cell rates of S10, S12, and S14 were 79.63% ± 0.62%, 84.27% ± 0.49%, and 86.24% ± 0.34% respectively. The viable cell rate increased with the increase of SPI, as Figure 11 shown in Figure B; the viable cell rates of bacteria with O:H of 0:10, 2:8, 3:7, 4:6, 5:5, and 6:4 were 82.13% ± 0.23%, 84.71% ± 0.34%, 84.11% ± 0.17%, 84.23% ± 0.62%, 84.66% ± 0.46%, and 83.14% ± 0.18% ( Figure 11 Figure C). Increasing the contents of SPI and KGM within the range of 10% - 14% SPI content and 1% - 3% KGM content can significantly improve the acid tolerance stability of probiotics. However, increasing the oil / water gel ratio has no significant effect on improving the acid tolerance stability of probiotics.

[0130] When conducting acid tolerance experiments on bacteria encapsulated with KGM, the viable cell rate was only 12.16% ± 0.82%. However, when KGM and SPI were combined to form a gel network structure, the viable cell rate of bacteria increased significantly, indicating that KGM may be a component that regulates the gel network structure. The network structure formed by using KGM alone cannot prevent acidic substances from entering, thus unable to effectively protect the bacteria. When SMP is in a solution state, it cannot completely encapsulate the bacteria, and the bacteria quickly inactivate when entering the acidic solution. However, the double-gel structure encapsulation can provide a certain enclosed space to protect the bacteria and reduce the probability of contact with the external adverse environment. Increasing the SPI concentration can improve the acid tolerance of bacteria. This may be because SPI increases to form a denser proteoglycan protective film structure, reducing the damage to bacteria caused by the entry of acidic substances, thereby improving the acid tolerance of bacteria. Increasing the oil gel ratio did not have much effect on the viable cell rate of bacteria. This may be because both the protein content and the polysaccharide content are relatively high, and the formed gel structure is sufficient to protect the bacteria from the influence of highly acidic solutions.

[0131] 5. Bile salt tolerance stability

[0132] Samples containing PC18 were 3D printed, and each sample was maintained at 1 cm 3 . After precisely weighing 1 g of the printed PC18 sample, it was added to MRS liquid medium without bile salt and containing 0.3% bile salt, and cultured at 37 °C for 5 h. Samples were taken at 0, 1, 3, and 5 h for disruption and vortexed to mix evenly. The mixed solution was serially diluted and poured onto plates for counting. After culturing at 37 °C for 48 h, colony counting was performed, and the survival rate was calculated. The survival rate (%) = (A1 / A2) × 100, where: A1 is the number of viable bacteria CFU / mL at 0 h of culture in bile salt MRS liquid medium; A2 is the number of viable bacteria CFU / mL at 1, 3, and 5 h of culture in bile salt MRS liquid medium.

[0133] After 5 h of treatment with bile salts, the survival rates of probiotics in the unprotected CK group and KGM group were 43.18% ± 2.9% and 45.32% ± 2.14% respectively, the survival rate of probiotics in the SMP group was 55.14 ± 0.43%, while the survival rates of K1, K2, and K3 were 62.39 ± 3.90%, 65.25 ± 2.50%, and 66.45 ± 4.40% respectively. As Figure 12 shown in A, increasing the KGM content did not significantly improve the bile salt tolerance of probiotics encapsulated in double gels. The survival rates of S10, S12, and S14 were 65.25 ± 2.50%, 67.15 ± 1.50%, and 67.40 ± 9.80% respectively. As Figure 12 shown in B, increasing the SPI content did not significantly improve the bile salt tolerance of probiotics encapsulated in double gels. The survival rates of cells with O:H ratios of 0:10, 2:8, 3:7, 5:5, and 6:4 were 58.01 ± 1.8%, 63.35 ± 3.40%, 67.15 ± 1.50%, 65.25 ± 2.50%, and 63.31% ± 3.10% respectively. As Figure 12 shown in C, the presence or absence of oil had a significant effect on the bile salt tolerance of probiotics, while increasing the oil gel ratio had no significant effect on improving the bile salt tolerance of probiotics.

[0134] The above research shows that encapsulating cells with a single polysaccharide and protein cannot effectively protect the cells from bile salt damage. The gel formed by the combination of protein and polysaccharide encapsulated by KGM alone is not easily disintegrated, and increasing the KGM content does not reduce the exchange of internal water in the gel and the bile salt solution, and the difference in the protection effect on the cells is not significant. After the protein and polysaccharide are combined by non-covalent bonds, the hydrophobicity increases and the gel pores decrease, thereby reducing the chance of contact between the bacterial cells and bile salts and enhancing the bile salt tolerance of the bacteria. When the gel does not contain oil, bile salts react with proteins preferentially. After adding the oil gel, bile salts can gradually eliminate micelles through lipid digestion, thereby accelerating the emulsification of lipid droplets and continuous digestion, thus protecting the bacterial cells from bile salt erosion.

[0135] 6. Gastrointestinal fluid stability

[0136] Samples containing PC18 were 3D printed, and each sample was maintained at 1 cm 3, accurately weigh 1 g of the printed PC18 sample and place it in artificial gastric juice (0.35 g of pepsin is dissolved in 100 mL of 0.2% NaCl, and the pH is adjusted to 2 with 1 mol / L HCl). Shake and disintegrate the sample at 200 rpm. After digestion for 2 h, centrifuge at 5000 rpm for 10 min, collect the precipitate, weigh it, crush, dilute, and pour it onto a plate for counting, and calculate the survival rate. Put the precipitate produced by the above gastric juice digestion into artificial intestinal juice (0.1 g of trypsin and 0.5 g of bile salt are dissolved in 100 mL of 0.5% NaCl, and the pH is adjusted to 8 with 1 mol / L NaOH) according to the ratio of sample to artificial intestinal juice of 1:9 (w / v). After vortex mixing, incubate at a constant temperature with shaking at 37 °C, shake and disintegrate at a speed of 200 rpm for 6 h, centrifuge, collect samples respectively, and perform viable count to calculate the survival rate. The survival rate (%) = (A1 / A2) × 100, where: A1 is the number of viable bacteria CFU / mL in the gastric juice MRS liquid medium after culturing for 0 h; A2 is the number of viable bacteria CFU / mL in the gastric juice / intestinal juice for 8 h.

[0137] After 2 h of gastric juice digestion, the survival rate of SMP-protected probiotics was 49.98% ± 0.36%, while the survival rates of K1, K2, and K3 were 67.42 ± 0.86%, 72.14 ± 0.44%, and 72.32 ± 0.68% respectively, as Figure 13 shown in Figure 13 A; The survival rates of probiotics of S10, S12, and S14 were 72.14% ± 0.44%, 77.48% ± 0.44%, and 79.22% ± 0.63% respectively, as Figure 13 shown in

[0138] B; The survival rates of probiotics with O:H of 0:10, 2:8, 3:7, 4:6, and 5:5 were 60.21% ± 0.24%, 72.13% ± 0.25%, 71.46% ± 0.26%, 70.34% ± 0.98%, and 72.14% ± 0.44% Figure 14 shown in Figure 14As shown in B; the survival rates of O:H 0:10, O:H 2:8, O:H 3:7, and O:H 5:5 were 40.11% ± 0.56%, 53.12% ± 0.33%, 54.08% ± 0.31%, and 54.76% ± 0.46% respectively ( Figure 14 C).

[0139] The present invention provides an oil-water double gel bioink, its preparation method, and the ideas and methods of application. There are many ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing an oil-water dual-gel biological ink, characterized in that: The steps include: (1) Preparing a probiotic hydrogel: mixing a probiotic suspension with konjac gum to obtain a probiotic hydrogel; (2) preparing oil gel: mixing soybean protein isolate with vegetable oil to obtain oil gel; (3) Mixing and homogenizing the probiotic hydrogel prepared in step (1) and the oil gel prepared in step (2) to obtain an oil-water dual-gel bio-ink.

2. The preparation method according to claim 1, characterized in that: In step (1), the probiotic is Lactobacillus paracasei.

3. The preparation method according to claim 1, characterized in that: In step (1), the probiotic suspension is prepared as follows: the probiotics are activated, washed by centrifugation, and resuspended with physiological saline; the OD of the probiotic suspension is 600 It is 0.9~1.

1.

4. The preparation method according to claim 1, characterized in that: In step (1), the mass concentration of konjac gum in the probiotic hydrogel is 2% to 6%.

5. The preparation method according to claim 1, characterized in that: In step (2), the vegetable oil is soybean oil; and in the oil gel, the mass concentration of soybean protein isolate is 15% to 50%.

6. The preparation method according to claim 1, characterized in that: In step (3), the mixing mass ratio of the probiotic hydrogel and the oil gel is 2:8 to 6:

4.

7. The preparation method according to claim 1, characterized in that: In step (3), the mass concentration of konjac gum in the oil-water dual-gel bio-ink is 1.9% to 2.1%, and the mass concentration of soy protein isolate is 9% to 10%.

8. The preparation method according to claim 1, characterized in that: In step (3), the homogenization is carried out at 8000-10000 rpm for 2-3 min.

9. The oil-water dual-gel biological ink prepared by the preparation method according to any one of claims 1 to 8.

10. Application of the oil-water dual gel bio-ink according to claim 9 in 3D printing of probiotic products.

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