Application of oil tea cake protein in improving 3D printing performance of plant meat

Abstract

The invention discloses application of oil tea cake protein in improving 3D printing performance of plant meat, and belongs to the technical field of food processing. According to the application, part of plant protein in the polysaccharide / protein composite system ink adopted for plant meat 3D printing is replaced by oil tea cake protein. The camellia oleifera cake protein is successfully developed and applied to preparation of the 3D printing plant meat, the apparent viscosity and storage modulus of the ink can be improved by adding the camellia oleifera cake protein, formation of a compact structure and change of moisture distribution of the ink are promoted, and the camellia oleifera cake protein shows excellent rheological adaptability; a printing sample has good self-supporting performance and printing precision. In addition, after the oil-tea cake protein is added, the thermal stability and the oxidation resistance of a printing sample are remarkably enhanced, and the protein digestibility is also improved to a certain extent. The oil-tea cake protein is extracted from the plant waste oil-tea cake, a sustainable protein raw material is provided for 3D printing in the food industry, and the additional value of the tea seed cake is increased.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to the application of camellia seed cake protein in improving the performance of plant-based meat 3D printing. Background Technology

[0002] Animal-derived protein, primarily derived from meat, eggs, and milk from poultry, livestock, and fish, is a vital food source for humans due to its rich nutritional value, delicious taste, and wide consumption. However, greenhouse gas emissions from livestock farming significantly impact global climate change, and global challenges such as climate change and population growth are placing increasing pressure on food systems, threatening food security. Furthermore, research indicates that high meat consumption is associated with an increased risk of cardiovascular disease, type 2 diabetes, and various cancers. Therefore, there is an urgent need to find sustainable and healthy alternatives to animal-derived protein.

[0003] To alleviate environmental and resource pressures, increasing research focuses on plant-based protein alternatives, using scientific formulations and technological innovation to produce plant-based foods similar to meat products. Many raw materials can be used to make plant-based meat, such as soybeans, peas, wheat, and edible fungi. Compared to traditional meat products, plant-based meat alternatives offer numerous significant advantages. For example, plant-based meat contains a large amount of protein, making it a suitable protein supplement to address malnutrition and meet specific high-protein needs; moreover, it is sustainable, has a lower carbon footprint, and is environmentally friendly.

[0004] With advancements in food processing technology, plant proteins, through techniques such as extrusion texturing, enzymatic modification, and flavor control, can now simulate fibrous structures, colors, aromas, and textures similar to animal proteins, with increasingly higher levels of realism. 3D printing technology, with its inherent layer-by-layer stacking innovation method, can create complex structures impossible to achieve with traditional manufacturing processes. Compared to other food processing methods, 3D printing offers irreplaceable advantages, such as flexible combinations of food ingredients, diverse model shapes, small production space per unit area, and ease of operation.

[0005] Printability is one of the key characteristics that printing materials must possess in 3D printing technology. It requires not only that the printing material can be smoothly extruded from the nozzle, but also that the printed product can be well supported on the printing platform. Plant protein-polysaccharide composites are attractive materials for producing edible inks for 3D food printing due to their advantages in stability, gelling properties, and mechanical properties. Proteins contribute quality attributes to food matrices, such as texture, taste, flavor, and color. Polysaccharides contain numerous active groups and different carbohydrate chain lengths, giving them different structures and properties. They can interact with proteins through their functional groups to form gels. Currently, research on food 3D printing still faces significant challenges, particularly in printing accuracy and the structural support of printing ink materials. Improving the self-support capability and printing accuracy of 3D printed products remains a hot research topic in this field.

[0006] Furthermore, the availability of suitable raw materials for 3D printing is crucial for the sustainable development of the 3D food printing industry. Developing widely available, inexpensive, low-allergenic (compared to soybean and peanut proteins), and nutrient-rich raw materials holds immense potential for research on polysaccharide / protein composite inks, and is expected to drive the development of high-performance food 3D printing inks. Camellia oil cake meal, as a protein raw material extracted from plant waste, has not yet been fully utilized. Therefore, researching the application of camellia oil cake meal protein in the field of 3D printed food processing is of great significance for the full utilization of plant resources. Summary of the Invention

[0007] The purpose of this invention is to provide a raw material suitable for 3D printing, which improves the self-support ability and printing accuracy of plant-based meat 3D printed samples by improving the rheological properties of edible printing ink.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of camellia seed cake protein in improving the performance of plant-based meat 3D printing. The application includes replacing part of the plant protein in the polysaccharide / protein composite ink used in plant-based meat 3D printing with camellia seed cake protein.

[0009] The 3D printing performance includes the self-supporting ability and printing accuracy of the printed sample. This invention improves the rheological properties of the 3D printing ink by adjusting the composition of the polysaccharide / protein composite ink and adding camellia seed cake protein, resulting in 3D printed plant-based meat products with excellent self-supporting ability and printing accuracy.

[0010] The polysaccharide / protein composite ink provided by this invention is an edible printing ink prepared by mixing plant protein and polysaccharide, adding water and vegetable oil and stirring evenly.

[0011] The plant protein is a mixture of camellia seed cake protein, pea protein, and wheat protein. The camellia seed cake protein is extracted from defatted camellia seed cake using an alkali-soluble acid-precipitation method. The pea protein is extracted from peas and has advantages such as wide availability, low price, low allergenicity, and rich nutrition. The wheat protein is extracted from wheat and has excellent viscoelasticity, extensibility, and thermal coagulation properties. The polysaccharide is a mixture of carrageenan and sodium alginate. Carrageenan is a highly soluble soluble polysaccharide with gelling properties; sodium alginate is a commonly used hydrophilic colloid with good binding ability to proteins. The vegetable oil is camellia oil. Adding vegetable oil makes the ink spray smoother during printing and also mimics fat in plant-based meat, making it closer to real meat. All the above-mentioned plant protein, polysaccharide, and vegetable oil raw materials are commercially available.

[0012] This invention demonstrates that, compared to polysaccharide / protein composite inks prepared without the addition of camellia seed cake protein, replacing some pea protein with camellia seed cake protein improves the printing quality of the ink. While meeting the requirements for extrudability, appropriate viscoelasticity, thixotropy, and gelation properties in 3D printing, it further enhances the self-supporting ability and printing accuracy of the printed products.

[0013] Preferably, the preparation method of the polysaccharide / protein composite ink includes: mixing pea protein and camellia seed cake protein mixture, wheat protein, carrageenan, and sodium alginate in a mass ratio of 12:10:1.4:1 to obtain a solid powder; then adding water in a material-to-liquid ratio of 0.4~0.5 g:1 mL and adding camellia oil in a material-to-liquid ratio of 2~3 g:1 mL, and stirring to obtain a uniform edible printing ink, wherein the camellia seed cake protein accounts for 8~25% of the mass of the pea protein and camellia seed cake protein mixture.

[0014] Studies have shown that, under the aforementioned raw material ratios, increasing the amount of camellia seed cake protein improved the apparent viscosity and storage modulus of the prepared edible printing ink, and also promoted the formation of its dense structure and changes in moisture distribution. The printed 3D samples exhibited good self-supporting ability, tight layering, and good overall integrity.

[0015] More preferably, the mass ratio of pea protein to camellia seed cake protein in the mixture of pea protein and camellia seed cake protein is 1:3~5.

[0016] The present invention also provides an edible 3D printing ink, which comprises an aqueous phase and an oil phase. The preparation method of the aqueous phase includes: mixing pea protein and camellia seed cake protein mixture, wheat protein, carrageenan, and sodium alginate in a mass ratio of 12:10:1.4:1 to obtain a solid powder, wherein the camellia seed cake protein accounts for 8%~25% of the mass of the pea protein and camellia seed cake protein mixture, and then dispersing it in water in a material-to-liquid ratio of 0.4~0.5 g:1 mL to obtain the aqueous phase; the oil phase is camellia oil, and the volume ratio of camellia oil to water in the aqueous phase is 1:6.

[0017] Furthermore, the edible 3D printing ink is prepared by mixing the aqueous phase and the oil phase evenly.

[0018] Furthermore, the preparation method of the camellia seed cake protein includes: adding defatted camellia seed cake powder to deionized water at a material-to-liquid ratio of 1g:30mL, adjusting the pH to 11.0±0.2, stirring at 40℃ for 1 h, centrifuging the extract at 5000×g for 10 minutes, collecting the supernatant, adjusting the pH to 3.0±0.2 at room temperature, allowing it to stand, centrifuging at 5000×g for 10 minutes, collecting the precipitate, washing with anhydrous ethanol, dissolving it in water, adjusting the pH to 7.0, and drying to obtain the camellia seed cake protein.

[0019] Furthermore, petroleum ether is used to degrease the camellia oil cake. Specifically, after crushing the camellia oil cake, petroleum ether is added at a material-to-liquid ratio of 1g:10mL. The degreasing is carried out for 2-3 hours, and the operation is repeated twice. The degreased camellia oil cake is collected and dried in a ventilated place to remove petroleum ether residue.

[0020] This invention also provides the application of the edible 3D printing ink in the 3D printing preparation of plant-based meat.

[0021] Furthermore, the application includes: transferring the edible 3D printing ink into the injection barrel of a 3D printer to prepare plant-based meat through 3D printing, wherein the 3D printing parameters are set as follows: nozzle temperature 25 ℃, nozzle diameter 0.84 mm, nozzle printing speed 15 mm / s, layer height 1.1 mm, and infill density 80%.

[0022] Studies have shown that the ink provided by this invention is suitable for printing at room temperature and does not require additional heating to consume energy.

[0023] The beneficial effects of this invention are as follows: (1) This invention successfully developed the application of camellia seed cake protein in the preparation of 3D printed plant-based meat. Adding camellia seed cake protein can improve the apparent viscosity and storage modulus of the ink, and promote the formation of its dense structure and changes in moisture distribution, exhibiting excellent rheological adaptability. The printed samples have good self-supporting properties and printing accuracy. In addition, after adding camellia seed cake protein, the thermal stability and antioxidant properties of the printed samples are significantly enhanced, and the protein digestibility is also improved to a certain extent.

[0024] (2) This invention extracts camellia seed cake protein from plant waste camellia seed cake, providing sustainable protein raw materials for 3D printing in the food industry and increasing the added value of camellia seed cake.

[0025] (3) The preparation method of 3D printed plant protein meat provided by the present invention has a simple and easy process route, short preparation time, easy industrial operation, and broad application prospects. Attached Figure Description

[0026] Figure 1 The 3D samples printed with edible inks of different SCP contents for the control group and Examples 1-5 are shown from left to right as side and top views of a hollow cylinder and a cuboid, respectively.

[0027] Figure 2 The rheological properties of edible inks with different SCP contents in Examples 1-5 are shown in the diagrams, where A is the apparent viscosity, B is the strain scan, C is the frequency scan, D is the shear recovery test, and E is the temperature scan.

[0028] Figure 3 These are scanning electron microscope images of edible inks with different SCP contents from Examples 1 to 5.

[0029] Figure 4 The distribution of transverse relaxation time (T2) spectra of edible inks with different SCP contents in Examples 1-5 is shown. B is an enlarged view of A in the range of 100 to 1000.

[0030] Figure 5 DSC images of 3D samples printed with edible inks of different SCP contents from Examples 1 to 5.

[0031] Figure 6 The DPPH free radical scavenging activity and ABTS free radical scavenging activity of 3D samples printed with edible inks of different SCP contents in Examples 1-5 are shown.

[0032] Figure 7 The results of protein digestibility at different times for 3D printed samples with different SCP contents printed with edible inks in Examples 1-5 in simulated in vitro digestion experiments. Detailed Implementation

[0033] This invention uses pea protein, wheat protein, sodium alginate, and carrageenan as the raw materials for basic edible ink. Protein and polysaccharides are two essential nutrients in the human diet. Pea protein is widely used in the food industry due to its wide availability, low price, low allergenicity, and rich nutritional content. Wheat protein is usually a byproduct of starch separation from wheat flour and possesses excellent viscoelasticity, extensibility, and thermal coagulation properties. Sodium alginate is a commonly used hydrophilic colloid with good protein binding capacity. Carrageenan is a highly soluble polysaccharide with gelling properties. Therefore, we selected these substances as the raw materials for the basic edible ink. However, we found that the precision of the 3D printed samples of this edible ink is not yet high enough, and there is still considerable room for improvement.

[0034] Based on previous experimental results regarding the physicochemical properties and functional characteristics of camellia seed cake protein (SCP), pea protein (PPI), and wheat protein (WG), we attempted to partially replace PPI with SCP. We used a 3D printer to fabricate hollow cylinders and cuboids. The effect of adding SCP on the printability of protein-polysaccharide-based edible inks was evaluated.

[0035] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0037] The camellia seed cake and camellia oil used in the following examples were purchased from Zhejiang Jiusheng Camellia Oil Technology Co., Ltd., pea protein powder (PPI) was purchased from Linyi Yuwang Plant Protein Co., Ltd., wheat protein (WG) was purchased from Fengqiu Huafeng Flour Industry Co., Ltd., soybean protein powder (SPC) was purchased from Linyi Shansong Biological Products Co., Ltd., peanut protein powder (PP) was purchased from Anyang Tianxiang Food Technology Co., Ltd., and carrageenan and sodium alginate were purchased from Shanghai Aladdin Co., Ltd.

[0038] The method for extracting camellia seed cake protein (SCP) from camellia seed cake is as follows: (1) Crush the camellia seed cake and defatt it with petroleum ether at a ratio of 1:10 (w / v) for 2 hours. Repeat the operation twice. Then dry it in a fume hood. (2) Add deionized water to the defatted powder obtained in step (1) at a ratio of 1:30 (w / v), adjust the pH to 11.0 with 1 mol / L sodium hydroxide solution, and stir at 40°C for 1 hour. Centrifuge the extract at 5000×g for 10 minutes, collect the supernatant, adjust the pH of the supernatant to 3.0 with 1 mol / L hydrochloric acid at room temperature, let it stand for 30 minutes, and centrifuge again under the same conditions to collect the precipitate. (3) Wash the precipitate obtained in step (2) three times with anhydrous ethanol, then dissolve it in water and adjust the pH to 7.0. Freeze-dry the obtained protein solution under vacuum. Store the freeze-dried sample at 4°C for subsequent use.

[0039] Example 1 Take 6 g of pea protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the powdered substances together and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as IW.

[0040] Example 2 Take 0.5g of camellia seed cake protein, 5.5g of pea protein, 5g of wheat protein, 0.7g of carrageenan and 0.5g of sodium alginate. Mix the above powdered substances and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as SIW0.5.

[0041] Example 3 Take 1 g of camellia seed cake protein, 5 g of pea protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the above powdered substances and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, which is denoted as SIW1.

[0042] Example 4 Take 1.5 g of camellia seed cake protein, 4.5 g of pea protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the above powdered substances and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as SIW1.5.

[0043] Example 5 Take 2 g of camellia seed cake protein, 4 g of pea protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the above powdered substances and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as SIW2.

[0044] Compare with Example 1 Take 6 g of soybean protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the powdered substances together and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as SW.

[0045] Compare with Example 2 Take 6 g of peanut protein, 5 g of wheat protein, 0.7 g of carrageenan and 0.5 g of sodium alginate. Mix the powdered substances together and stir for 5 minutes to disperse evenly. Then add 30 mL of deionized water and 5 mL of camellia oil, and stir again for 3 minutes to obtain a uniform edible ink, denoted as PW.

[0046] Test case I. Preparation of Printing Ink and Its Performance in 3D Printing The edible printing inks prepared in Examples 1-5 and Comparative Examples 1-2 were transferred into the printer's injection cylinder and printed using a FoodBot-D1 food 3D printer (Hangzhou Shiyin Technology Co., Ltd.). At the same printing ambient temperature (25℃), two preset models were printed: one a cuboid (20 × 20 × 12 mm), and the other a hollow cylinder with a height, outer diameter, and inner diameter of 15 mm, 30 mm, and 15 mm, respectively. The main printing parameters were set as follows: nozzle temperature 25℃, nozzle diameter 0.84 mm, nozzle printing speed 15 mm / s, layer height 1.1 mm, and infill density 80%.

[0047] The results are as follows Figure 1As shown, the control groups SW and PW exhibited extremely low printing accuracy and self-supporting ability, while IW ink showed poor deformation resistance and support. The diameter of the printed lines failed to reach the set height for each layer, resulting in insufficient overall height of the printed samples and collapse. This may be due to its loose network structure and low viscosity. With the increase of SCP content, the printing quality of the ink improved, and the printed object gradually became more complete and clear. 3D samples printed by SIW1 and SIW1.5 had good self-supporting ability, tight layering, and good overall integrity. The final printed shape highly matched the model design. However, SIW2 ink had excessively high viscosity, resulting in poor material flow and inability to be smoothly and evenly extruded through the nozzle. Each layer of ink failed to adhere together, and the lines were too stiff and easily broken.

[0048] II. Rheological Properties Rheological analysis was performed using a rheometer (MCR302e, Anton Paar, Austria) with PP-25 test plates (25 mm in diameter) spaced 1 mm apart. All experiments, except for temperature scans, were conducted at room temperature. Figure 2 The rheological results of edible inks with different SCP (expanded concentration) levels are presented. The rheological properties of these inks directly affect their printing performance in 3D printing based on extruded samples, and appropriate apparent viscosity is a key parameter in extrusion 3D printing. On the one hand, it should be low enough to pass through the nozzle; on the other hand, it must be high enough to adhere to the previous layer.

[0049] 1. Apparent viscosity test Test method: Tests were conducted from 0.01 to 100 seconds. -1 Steady-state shear tests were conducted to obtain the viscosity profile of the ink.

[0050] The results are as follows Figure 2 As shown in Figure A, all samples exhibited viscosity changes with decreasing shear rate, indicating typical non-Newtonian pseudoplastic shear-thinning behavior. This is due to the disruption and rearrangement of the original sample structure. This inherent characteristic of shear-thinning generally facilitates successful ink extrusion from the nozzle and deposition onto the platform in the designed structure. The ink viscosity increases with increasing SCP content. This is likely because SCP enhances cross-linking between inks, promoting the formation of a dense network. However, excessively high viscosity can hinder the extrusion process, and its excessive hardness and susceptibility to cracking can reduce adhesion between printed layers and decrease printing accuracy, as is the case with materials like SIW2.

[0051] 2. Strain scanning test Test method: The linear viscoelastic region (LVR) was determined by strain scanning test. The test was conducted within the strain range of 0.1% to 100% at a constant frequency of 10 rad / s.

[0052] The results are as follows Figure 2 As shown in Figure B, the linear viscoelastic region (LVR) ranges from approximately 0.1% to 2% for all inks. Within this region, the G' value of all edible inks is higher than their G'' value. Above the LVR, the G' and G'' values ​​exhibit a significant decreasing trend due to deformation of the ink network. Under sufficiently large strains, the G' value is lower than the G'' value, indicating that the disruption of the network structure promotes flow. This rheological behavior may be beneficial for 3D printing, as the material tends to soften under relatively high stress during extrusion and solidify after stress relief. With continued addition of SCP, the G' and G'' values ​​of the ink gradually increase, and the yield stress rises slightly, indicating an enhancement in its strength.

[0053] 3. Frequency scanning test To ensure the structural stability of printed products and enable them to withstand shape changes caused by gravity and pressure from the extruded material during the printing process, thereby achieving high-precision printing, a relatively high storage modulus value is crucial. G' represents the elasticity of the ink, i.e., the deformation energy stored during dynamic oscillation. G'' represents dissipated energy, i.e., the energy lost due to viscous deformation (irreversible).

[0054] Test method: The viscoelasticity of the ink was investigated by frequency scanning test. The test range was 0.1 to 100 rad / s, and the strain amplitude was 0.1%.

[0055] The results are as follows Figure 2 As shown in Figure C, all samples were tested, and within a specific frequency range, the storage modulus (G') was significantly greater than the loss modulus (G"), indicating that it exhibits predominantly elastic semi-solid behavior. The IW ink had a significantly lower G', indicating a lack of self-supporting capacity required for 3D printing. Inks containing more SCP (strain coefficient of mass) showed a significantly increased G' value, suggesting a higher cross-linking density in the protein-polysaccharide system, resulting in better self-supporting properties and resistance to deformation. However, excessively high viscoelasticity may adversely affect 3D printing, as the resulting high shear forces may hinder the ink extrusion process or cause ink breakage after extrusion.

[0056] 4. Shear recovery test Shear recovery testing is an effective method that can simulate instantaneous deformation to mimic the effects of extrusion shear forces during printing and the processing steps of 3D printed objects. The three stages—low shear rate, high shear rate, and low shear rate—can simulate the three stages of 3D printing: loading, printing, and ink leveling.

[0057] Test method: First, the ink is poured at a speed of 1 second. -1 The shearing rate was increased to 100 s for 250 s. -1 Hold for 100 seconds. Finally, perform a third scan under the same conditions as the first round.

[0058] The shear recovery test results for all inks are as follows: Figure 2 As shown in Figure D, in the first low-shear stage (0–250 s), the apparent viscosity of the ink was positively correlated with the amount of SCP added. In the subsequent high-shear stage (250–350 s), the viscosity of all inks decreased, confirming the shear-thinning behavior. In the final recovery stage (350–600 s), each system exhibited rapid structural recovery, and the final viscosity of the ink increased significantly with increasing SCP content. A recovery rate higher than 70% was considered an indicator of excellent thixotropic recovery performance of the ink. The results showed that the thixotropic recovery rate of the ink increased from 65.51% (IW) to a maximum of 70.33% (SIW1) due to the addition of SCP. This indicates that the addition of SCP helps to enhance the rapid recovery capability of ink viscosity, which is important for ensuring the clarity and shaping of the printed structure.

[0059] 5. Temperature scanning test Temperature scanning using a rheometer can simulate the effect of temperature on the modulus of a sample during processing or transportation. It can demonstrate the influence of extrusion temperature on the behavior and state of printed materials during printing, clarifying the extrudability of materials under suitable temperature conditions.

[0060] Test Method: Temperature scanning tests were conducted under conditions of constant strain rate of 0.1% and frequency of 10 rad / s. The ink was heated from 25 ℃ to 90 ℃ at a heating rate of 5 ℃ / min. The changes of G' and G'' with temperature were recorded during the heating process. Before the test, excess ink was removed, and a thin layer of light silicone oil was applied to the surface to prevent moisture evaporation, thus simulating the heating state of the ink in the injection cartridge.

[0061] The results are as follows Figure 2As shown in Figure E, the G' and G" values ​​of all inks exhibit the same trend, gradually decreasing with increasing temperature. Adding SCP only affects the increase in initial modulus. The decrease in modulus may be due to the fact that lower temperatures help maintain the stability of the food structure within hydrated particles. At high temperatures, the polysaccharides in the ink melt, hindering cross-linking between protein molecules, and the hydrogen bonds and other intermolecular forces between ink molecules are also broken by heat. Therefore, this type of formulated ink is suitable for printing at room temperature, requiring no additional heating to consume energy. Furthermore, when choosing a curing method, methods such as frying can be considered to achieve rapid curing.

[0062] III. Observation of the microstructure of ink using scanning electron microscopy The microstructure of the ink was observed using a scanning electron microscope (SU8100, Hitachi, Japan). The ink was first freeze-dried, and then the samples were observed at different magnifications (50x, 200x, 500x, and 1000x) under an accelerating voltage of 3 kV.

[0063] Figure 3 This study showcases the internal morphology of edible 3D printing inks with varying SCP (saccharide superoxide dismutase) substitution levels. All food-grade inks exhibit a non-uniform microstructure composed of aggregates of proteoglycan particles. Different ink formulations exhibit varying degrees of cavity densification and orientation, suggesting the presence of larger, denser protein aggregates, particularly noticeable with the addition of SCP. IW inks show a relatively low cohesive structure with large pores, resulting in a loose structure that fails to support the height of the product. As the SCP content increases, the cross-linked structure of the ink becomes denser, with a reduction in the number and size of pores, leading to the formation of larger aggregates. The internal structure of the printed product exhibits a more compact arrangement and fewer pores, consistent with the fact that the printed sample maintains a more precise center height on a macroscopic scale. This dense network structure provides excellent mechanical properties to the ink, improving the accuracy of the printed shape and helping to maintain the shape of the 3D printed product. However, the internal morphology of SIW2 ink is too dense, forming large clumps with a typical sheet-like structure. These larger sheet-like structures clog the nozzle tip, making extrusion difficult. Furthermore, overly dense network structures require greater extrusion force and are prone to extrusion line breakage during the 3D printing deposition process.

[0064] IV. Low-field nuclear magnetic resonance imaging (NMR) analysis of moisture distribution Low-field nuclear magnetic resonance (LF-NMR, MesoMR23 / 12-060H-I, NUMBER, Suzhou, China) was used to determine the moisture distribution in the printing material. The ink test parameters were as follows: number of echoes (NECH) = 9000, waiting time (TW) = 4500 ms, number of scans (NS) = 4, and echo time (TE) = 0.15 ms.

[0065] The distribution of transverse relaxation time (T2) was used to assess the relaxation time of hydrogen protons in water and oil. A key characteristic of proteins is their ability to interact with water, a property that alters the fluidity of water molecules in ink. The fluidity of water molecules in ink is closely related to its network structure and rheological properties; the denser the internal structure, the lower the fluidity of the water. For example... Figure 4 As shown in Figure A, two or three peaks were observed in the spectrum, labeled T21, T22, and T23, respectively. T21 mainly refers to water tightly bound to large protein molecules, while T22 represents water that is not easily mobile within the ink network. With increasing SCP addition, the T21 and T22 values ​​of the inks (SIW0.5, SIW1, SIW1.5, and SIW2) showed a significant decreasing trend compared to IW inks. The numerical differences between SIW0.5, SIW1, and SIW1.5 were relatively small, while the T21 and T22 values ​​of SIW2 showed a more significant and pronounced decrease. This phenomenon indicates that the introduction of SCP can effectively inhibit the migration of water molecules in the system, especially when the added SCP amount reaches 2 g, its three-dimensional network structure significantly enhances the binding effect on water, which is consistent with the printing results. Overall, the results indicate that SCP has excellent water retention performance. Furthermore, a new T23 peak was observed in the spectra of all inks with added SCP. This may be due to the enhanced binding of SCP with poorly bound water molecules, thereby promoting the inhomogeneity of the ink system. This leads to the separation of the oil-water interface, resulting in the characteristic relaxation peak of camellia oil. As the amount of SCP added increases, more oil phase components are released, and the proportion of hydrogen protons provided by the oil increases, leading to a shortening of the T23 relaxation process.

[0066] V. Differential Scanning Calorimetry for Evaluating the Thermal Properties of 3D Printed Samples The thermal properties of the 3D-printed samples were evaluated using a differential scanning calorimeter (TA. DISCOVERY, DSC25, USA). Samples (20 mg) were accurately weighed and placed into the DSC apparatus. Samples were analyzed at a scan rate of 10 °C / min over a range of 20 °C to 190 °C, with a nitrogen flow rate of 50 mL / min. A sealed empty dish was used as a blank control.

[0067] Since the properties and structure of most substances change with temperature, these changes are usually accompanied by energy variations. Information related to the structural stability of a sample can be observed from changes in DSC spectra. Figure 5 As shown, the SCP content significantly affects the thermal stability of the 3D printed samples. All samples exhibit a single endothermic peak in the temperature range of 115–140°C. Notably, with increasing SCP content, the thermal denaturation temperatures of IW, SIW0.5, SIW1, and SIW1.5 are 119.65±1.59°C, 121.86±2.74°C, 125.41±1.86°C, and 133.10±3.15°C, respectively. The increase in the thermal denaturation temperature of the 3D printed samples is likely due to SCP promoting interactions between ink molecules, resulting in a more compact three-dimensional structure, consistent with SEM observations.

[0068] VI. Antioxidant Activity The antioxidant activity of 3D-printed samples was determined using a DPPH and ABTS free radical scavenging activity assay. When DPPH and ABTS free radicals come into contact with proton-donating molecules (such as antioxidants), these free radicals are effectively scavenged, resulting in a decrease in absorbance.

[0069] DPPH method: 0.5 mL (20 mg / mL) of sample solution was reacted with 4 mL (0.1 mol / L, methanol) of DPPH solution at 25 °C in the dark for 30 minutes, and the absorbance at 517 nm was measured.

[0070] ABTS+ method: React 0.2 mL of sample solution and 4 mL of ABTS reagent solution in the dark for 7 minutes, then measure the absorbance at 734 nm. Mix equal volumes of ABTS solution (7 mmol / L) and potassium persulfate solution (2.45 mmol / L), react in the dark for 16 hours, and then dilute to an absorbance value of 0.7 ± 0.02 to obtain the ABTS reagent solution.

[0071] The formula for calculating the DPPH / ABTS free radical scavenging rate is as follows:

[0072] Where Ai is the absorbance value of the sample after mixing DPPH / ABTS solution, Aj is the absorbance value of the 3D printed sample, and Ac is the absorbance value of DPPH / ABTS solution.

[0073] Figure 6The free radical scavenging activity of 3D-printed samples with different SCP additions was demonstrated. The addition of SCP significantly enhanced the antioxidant activity of the 3D-printed samples. Compared with the DPPH free radical scavenging rate of IW (17.77±3.18%) and the ABTS free radical scavenging rate (27.88±3.18%), SIW1.5 showed the largest increases of 81.89±1.59% and 43.80±1.02%, respectively. The experimental results indicate that SCP possesses significant antioxidant activity, which may be due to its high content of antioxidant amino acids, such as aspartic acid and glutamic acid, which can effectively neutralize free radicals and thus exert a powerful antioxidant effect. In conclusion, SCP can also serve as a high-quality antioxidant when added to 3D printing systems for food.

[0074] VII. Texture Feature Analysis (TPA) Taste is a primary factor determining the acceptability of printed products. The TPA test predicts acceptability during chewing by simulating human sensations, thus determining the chewing characteristics of food. Hardness is crucial for describing the firmness or toughness during biting or chewing, representing the most direct response to taste sensations. Adhesion, on the other hand, arises from the negative force generated by the attraction between the probe and the sample surface during retraction; it is considered a sticky oral sensation closely related to the force of the tongue driving food through the pharynx.

[0075] The TPA experiments used a texture analyzer (TA. XT Plus 100C, StableMicro Systems, USA) equipped with a P0.5 probe to measure the texture features of the 3D printed samples. Two compression cycle tests were performed on products printed in a (20×20×12 mm) cuboid shape. The test parameters were set as follows: probe speed 1 ms / s; contact force 5 g; compression amount 50% of the original height; interval between two compressions 5 s; measurement temperature 25°C. The test position for each sample was exactly at its center, and three tests were performed to ensure repeatability.

[0076] Table 1

[0077] As shown in Table 1, with the increase of SCP content, the hardness of the printed samples significantly increased from 45.67±1.90 g to 54.69±2.26 g, while the adhesion of the samples also increased. These results indicate that adding SCP can enhance the mechanical strength of printed samples, improve ink texture, and facilitate the formation of sample structures during extrusion and layer-by-layer assembly printing, which is consistent with the rheological properties of the ink and the 3D printing results. Resilience is considered to be the ability of food to return to its original state after chewing, while cohesion refers to the connections within the network and the integrity of the structure. The data show that although the elasticity and adhesion of the samples increased slightly with the increase of SCP content, the difference was not significant.

[0078] 8. In vitro digestion of proteins Protein digestion is a complex, multi-stage process: chewing in the mouth facilitates enzyme access to the substrate in subsequent stages. Gastric acid proteases initiate the initial hydrolysis in the stomach, but the main digestion occurs in the small intestine. In the duodenum, trypsin, chymotrypsin, and carboxypeptidase work in conjunction with the intestinal brush border peptidase system to break down proteins into absorbable oligopeptides, dipeptides, tripeptides, and amino acids.

[0079] All simulated digestive solutions were freshly prepared and preheated at 37 °C for 10 min before the digestion experiments. The digestive environment was simulated by stirring at 37 °C at a speed of 200 rpm.

[0080] Simulated oral cavity stage: 2 g of 3D printed sample was mixed with 2 mL of simulated saliva containing α-amylase (75 U / mL), the pH was adjusted to 6.8, and then incubated for 2 min.

[0081] Simulated gastric digestion: The mixture after oral digestion was mixed with 4 mL of simulated gastric juice (SGF, consisting of 3.2 mg / mL pepsin and 2.0 mg / mL sodium chloride). The pH was adjusted to 1.2. The supernatant was then collected after 2 h.

[0082] Simulated intestinal digestion: The mixture from gastric digestion was mixed with 8 mL of simulated intestinal fluid (SIF, containing 8 mg / mL trypsin, 5 mg / mL bile salts, 6.8 mg / mL sodium dihydrogen phosphate, and 8.8 mg / mL sodium chloride), and the pH was adjusted to 7.5. The supernatant was then collected at 1 h, 2 h, 3 h, and 4 h. The digestibility of proteins in the 3D-printed sample was calculated using the following formula:

[0083] M0 represents the total protein content in the sample, and M1 represents the protein content in the supernatant.

[0084] Protein digestibility, as a key indicator for assessing protein bioavailability, can reflect the nutritional value of a sample. Figure 7 This study demonstrates the in vitro protein digestibility of 3D-printed samples with different SCPs added during simulated gastrointestinal digestion. Experimental data show no significant difference in digestibility between samples during the gastric digestion phase (2 hours) and the initial intestinal digestion phase (0-3 hours), with overall digestibility at the gastric stage being low (<10%). Notably, all samples showed a significant increase in digestibility (50%-58%) after 1 hour of intestinal digestion, followed by a gradual increase. By the final intestinal digestion phase (4 hours), a positive correlation was observed between the level of SCP addition and digestibility: the digestibility of the IW group was 72.31±0.72%, while the SIW1.5 group, with the highest SCP addition, reached 81.30±2.62%. This phenomenon may be attributed to the following factors: First, PPIs contain various anti-nutritional factors, such as trypsin inhibitors, tannins, and phytic acid. In particular, trypsin inhibitors have been shown to inhibit the activity of key pancreatic enzymes (trypsin and chymotrypsin), hindering protein digestion and absorption even with sufficient digestive enzymes. On the other hand, in the early intestinal stages (0-3 hours), there was no significant difference in protein digestibility among the IW, SIW0.5, and SIW1 samples. The decrease in some values ​​for SIW0.5 and SIW1 may be due to the increased viscosity of the samples caused by the addition of SCP. The samples failed to disperse in the digestive fluid in a timely manner, thus reducing their contact with digestive enzymes. At this point, the increased digestibility due to the reduction in anti-nutritional factors could not offset the decrease caused by increased viscosity. In conclusion, in terms of protein digestibility, SCP can be considered an ideal alternative protein source to PPIs.

[0085] In summary, this study successfully utilized SCP as a protein feedstock for 3D printing plant-based meat. The rheological behavior, moisture distribution, and microstructure results of the ink showed that increasing SCP content improved the apparent viscosity and storage modulus of the ink, and also promoted the formation of a dense structure and changes in moisture distribution. However, the extrudability of the SIW2 printing ink was significantly limited due to its high viscosity and the formation of large flake aggregates. In contrast, the SIW1.5 formulation exhibited excellent rheological adaptability, with printed samples demonstrating good self-supporting properties and printing accuracy, better conforming to the designed 3D model. Furthermore, the addition of SCP significantly enhanced the thermal stability and antioxidant properties of the printed samples, and also improved the in vitro digestibility of the protein to some extent. Overall, our research provides a reference for the application of SCP in 3D printing plant-based meat, which will contribute to the development of new sustainable proteins from plant waste, increase the added value of tea seed cake, and provide further insights into the application of 3D printing technology in the food industry.

[0086] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of camellia seed cake protein in improving the performance of plant-based meat 3D printing, characterized in that, The applications include: in the polysaccharide / protein composite ink used for plant-based meat 3D printing, some of the plant protein is replaced by camellia seed cake protein.

2. The application as described in claim 1, characterized in that, The preparation method of the polysaccharide / protein composite ink includes: mixing pea protein and camellia seed cake protein mixture, wheat protein, carrageenan, and sodium alginate in a mass ratio of 12:10:1.4:1 to obtain a solid powder; then adding water in a material-to-liquid ratio of 0.4~0.5 g:1 mL and adding camellia oil in a material-to-liquid ratio of 2~3 g:1 mL, and stirring to obtain a uniform edible printing ink, wherein the camellia seed cake protein accounts for 8%~25% of the mass of the pea protein and camellia seed cake protein mixture.

3. The application as described in claim 2, characterized in that, The mass ratio of pea protein to camellia seed cake protein in the mixture of pea protein and camellia seed cake protein is 1:3~5.

4. The application as described in any one of claims 1-3, characterized in that, The protein in the camellia oil cake meal was extracted from defatted camellia oil cake meal using an alkali dissolution and acid precipitation method.

5. An edible 3D printing ink, characterized in that, The edible 3D printing ink comprises an aqueous phase and an oil phase. The preparation method of the aqueous phase includes: mixing pea protein and camellia seed cake protein mixture, wheat protein, carrageenan, and sodium alginate in a mass ratio of 12:10:1.4:1 to obtain a solid powder, wherein the camellia seed cake protein accounts for 8%~25% of the mass of the pea protein and camellia seed cake protein mixture, and then dispersing it in water at a material-to-liquid ratio of 0.4~0.5 g:1 mL to obtain the aqueous phase; the oil phase is camellia oil, and the volume ratio of camellia oil to water in the aqueous phase is 1:

6.

6. The edible 3D printing ink as described in claim 5, characterized in that, The edible 3D printing ink is prepared by mixing the aqueous phase and the oil phase evenly.

7. The edible 3D printing ink as described in claim 5, characterized in that, The preparation method of the camellia oil cake protein includes: adding defatted camellia oil cake powder to deionized water at a ratio of 1g:30mL, adjusting the pH to 11.0±0.2, stirring at 40℃ for 1 h, centrifuging the extract at 5000×g for 10 minutes, collecting the supernatant, adjusting the pH to 3.0±0.2 at room temperature, centrifuging at 5000×g for 10 minutes after standing, collecting the precipitate, washing with anhydrous ethanol, dissolving in water, adjusting the pH to 7.0, and drying to obtain the camellia oil cake protein.

8. The application of the edible 3D printing ink as described in any one of claims 5-7 in the 3D printing preparation of plant-based meat.

9. The application as described in claim 8, characterized in that, The 3D printing parameters are: nozzle temperature 25℃, nozzle diameter 0.84 mm, nozzle printing speed 15 mm / s, layer height 1.1 mm, and infill density 80%.

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