High-yield nutrient 3D printing biological scaffold prepared based on neurospora crassa spores and bean dregs

Abstract

The invention relates to the technical field of food processing, in particular to a high-yield nutrient 3D printing biological scaffold prepared on the basis of neurospora crassa spores and bean dregs. The biological scaffold prepared by the invention can form a closed-loop system of agricultural wastes, biological carriers and high-value products. The system has the characteristics of high efficiency, sustainability, flexibility, controllability and the like and the capabilities of modular design and dynamic regulation and control, programmable regulation and control of active compound synthesis can be realized through material-cell interaction, and a novel manufacturing mode is provided for synthesis and precise biosynthesis of nutrient substances such as fungal protein and carotenoid.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue. Background Technology

[0002] Fungi are rich in protein, dietary fiber, vitamins, and minerals, and their cultivation process boasts advantages such as low carbon footprint, low water consumption, and high space efficiency, making them a valuable supplement to traditional agriculture. Solid-state fermentation (SSF) technology, in particular, shows significant potential in areas such as functional food development, alternative protein production, and industrial enzyme synthesis. However, traditional SSF processes rely on planar substrate cultivation, which suffers from low space utilization, uneven nutrient distribution, and spore concentration leading to limited growth efficiency, thus restricting its large-scale application. Furthermore, the dependence of traditional processes on environmental conditions such as gas exchange and temperature and humidity control further increases operational complexity, necessitating the development of novel cultivation models to overcome these bottlenecks. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue. This invention combines Neurospora crassa with a 3D-printed scaffold, enabling precise control over mycelial spatial expansion and metabolite synthesis. This not only improves culture density and product uniformity but also allows for modular design to adapt to diverse production needs.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue, comprising the following steps: After drying the soybean residue, pulverize it and pass it through a 50-70 mesh sieve. Collect the sieve residue to obtain soybean residue powder. The soybean residue powder, soluble starch, agar, and water are mixed and then sterilized to obtain sterile ink; The sterile ink and Neurospora crassa ( Neurospora crassa The spore suspension was mixed to obtain spore-containing ink; The spore-containing ink is added to a 3D printer to print a porous three-dimensional scaffold. The porous three-dimensional scaffold was cultured in an environment of 28-30℃ and 50%-90% humidity to obtain a 3D-printed biological scaffold containing nutrients.

[0005] Preferably, the mass ratio of soybean residue powder to water is 20-24:100; the mass ratio of soluble starch to water is 0-4:100; and the mass ratio of agar to water is 1-1.5:100.

[0006] Preferably, the volume-to-mass ratio of the *Neurospora crassa* spore suspension to the sterile ink is 1 mL: 50 g, and the spore concentration in the *Neurospora crassa* spore suspension is 1.5 × 10⁻⁶. 7 -2.0×10 7 CFU / mL.

[0007] Preferably, the Neurospora crassa includes Neurospora crassa HJDF, and the preservation number of Neurospora crassa HJDF is CGMCC No. 41518.

[0008] Preferably, the printing parameters include: material barrel temperature 60℃, nozzle temperature 60℃, nozzle diameter 1.20-2.00mm, printing air pressure 15-35kPa, and printing speed 100-600mm / min.

[0009] Preferably, the porous three-dimensional scaffold has 2-16 layers; the shape of the holes in the porous three-dimensional scaffold includes circles and / or polygons; the side length of the polygon is 25-32mm.

[0010] Preferably, the drying includes low-temperature drying and / or freeze drying; the low-temperature drying temperature is 35-45°C.

[0011] Preferably, the soybean residue is the residue produced when soybeans are used to make soy milk.

[0012] Preferably, the nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids, and ergosterol.

[0013] This invention provides the application of the high-nutrient-yielding 3D-printed bioscaffold described above in the production of nutrients, wherein the nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids, and ergosterol.

[0014] Beneficial effects: (1) This invention transforms agricultural waste soybean residue into a 3D-printed biological scaffold with high nutrient yield, realizing the efficient conversion and reuse of waste. This not only reduces the pressure of waste on the environment, but also creates high-value-added products and promotes the development of the circular economy.

[0015] (2) This invention draws on the concept of efficient space utilization in vertical agriculture and uses 3D printing technology to construct a porous three-dimensional scaffold, which provides a larger growth space and a more optimized growth environment for Neurospora crassa, thereby significantly improving the production efficiency of bioactive compounds and realizing the high efficiency and intensification of fungal culture.

[0016] (3) The invention can precisely control the growth and metabolic processes of fungi by using specific 3D printing structural parameters, such as porosity and scaffold shape, thereby realizing the programmable production of bioactive compounds, meeting the needs of different application scenarios for product types and yields, and improving the flexibility and controllability of production.

[0017] Biological Preservation Instructions Neurospora crassa HJDF, classified as Neurospora crassa Neurospora crassa It was deposited on September 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41518. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a morphological diagram of Neurospora crassa HJDF growing on PDA medium; Figure 2 Phylogenetic tree of Neurospora crassa HJDF; Figure 3 Top view, side view, and front view of 3D printed scaffold samples with different ratios of spore-containing ink; Figure 4 Side view of 3D printed scaffold samples with different layers of spore-containing ink; Figure 5 A diagram of a culture device for a 3D soybean residue scaffold containing spores; Figure 6 Side view, top view and freeze-dried cross-section of 3D soybean residue printed scaffolds with different layers containing spores after 4 days of cultivation; Figure 7 The results of carotenoid content determination for 3D soybean residue scaffolds with different layers containing spores after 4 days of cultivation. Detailed Implementation

[0020] This invention provides a high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue, comprising the following steps: After drying the soybean residue, pulverize it and pass it through a 50-70 mesh sieve. Collect the sieve residue to obtain soybean residue powder. The soybean residue powder, soluble starch, agar, and water are mixed and then sterilized to obtain sterile ink; The sterile ink and Neurospora crassa ( Neurospora crassa The spore suspension was mixed to obtain spore-containing ink; The spore-containing ink is added to a 3D printer to print a porous three-dimensional scaffold. The porous three-dimensional scaffold was cultured in an environment of 28-30℃ and 50%-90% humidity to obtain a 3D-printed biological scaffold containing nutrients.

[0021] This invention involves drying and pulverizing soybean residue, passing it through a 50-70 mesh sieve, and collecting the sieve-passing material to obtain soybean residue powder. In one embodiment, the soybean residue is the residue produced during the preparation of soy milk from soybeans. In one embodiment, the drying includes low-temperature drying and / or freeze-drying; the low-temperature drying temperature is 35-45°C. In another embodiment, the low-temperature drying temperature is 40°C. In one embodiment, the pulverized soybean residue is passed through a 60-mesh sieve.

[0022] After obtaining soybean residue powder, the present invention mixes the soybean residue powder, soluble starch, agar, and water, and then sterilizes the mixture to obtain sterile ink. In one embodiment, the mass ratio of soybean residue powder to water is 20-24:100; the mass ratio of soluble starch to water is 0-4:100; and the mass ratio of agar to water is 1-1.5:100. In another embodiment, the mass ratio of soybean residue powder to water is 22-24:100; the mass ratio of soluble starch to water is 2-4:100; and the mass ratio of agar to water is 1.35:100. In one embodiment, the sterilization method includes high-pressure sterilization at 121°C for 20 minutes. The sterile ink formulated by the present invention exhibits optimal rheological properties through a specific formulation: its continuous extrudability ensures accurate reproduction of the printing path (forming accuracy error <5%), while possessing excellent self-supporting strength (overhanging structure deformation rate <3%), effectively maintaining the three-dimensional structural stability during the printing process.

[0023] After obtaining sterile ink, the present invention mixes the sterile ink with a suspension of *Neurospora crassa* spores to obtain spore-containing ink. As one embodiment, the volume-to-mass ratio of the *Neurospora crassa* spore suspension to the sterile ink is 1 mL: 50 g, and the spore concentration in the *Neurospora crassa* spore suspension is 1.5 × 10⁻⁶. 7 -2.0×10 7 CFU / mL. As one embodiment, the *Neurospora crassa* includes *Neurospora crassa* HJDF, whose preservation number is CGMCC No. 41518. The *Neurospora crassa* HJDF of this invention was extracted and purified from a traditional snack from Wuping, Fujian. This strain has the characteristics of safe source, high protein content, complete and balanced amino acid composition, and natural "umami" flavor, thus showing good potential as a meat substitute.

[0024] After obtaining the spore-containing ink, this invention adds the spore-containing ink to a 3D printer, prints it, and then forms it on a cooling plate at 10°C to obtain a porous three-dimensional scaffold. As one embodiment, the printing parameters include: barrel temperature 60°C, nozzle temperature 60°C, nozzle diameter 1.20-2.00 mm, printing air pressure 15-35 kPa, and printing speed 100-600 mm / min. As one embodiment, the porous three-dimensional scaffold has 2-16 layers; the shape of the holes in the porous three-dimensional scaffold includes circles and / or polygons; the side length of the polygons is 25-32 mm.

[0025] After obtaining the porous three-dimensional scaffold, the present invention places the porous three-dimensional scaffold in an environment of 28-30℃ and 50%-90% humidity to obtain a 3D-printed biological scaffold containing nutrients. As one embodiment, the culture time is 3-5 days. As one embodiment, the nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids, and ergosterol.

[0026] The bioscaffold prepared by this invention can form a closed-loop system of "agricultural waste-biological carrier-high-value products". This system has the characteristics of high efficiency, sustainability, flexibility and controllability, as well as modular design and dynamic regulation capabilities. It can achieve programmable regulation of active compound synthesis through material-cell interaction, providing a new manufacturing mode for the synthesis and precise biosynthesis of nutrients such as fungal proteins and carotenoids.

[0027] Based on the above advantages, the present invention provides the application of the high-nutrient-yielding 3D-printed bioscaffold described in the above technical solution in the production of nutrients, wherein the nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids and ergosterol.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Isolation and identification of Neurospora crassa HJDF 1. Source of Neurospora crassa HJDF strain: It was isolated, purified and cultured from a traditional fermented soybean product snack in Wuping County, Fujian Province, China.

[0030] 2. Colony characteristics: The colony morphology of Neurospora crassa HJDF on PDA plates is as follows: Figure 1As shown. The colonies of *Neurospora crassa* initially appear powdery, later developing into a fluffy texture, ranging in color from pink to light orange. The mycelium is loose, spreading outwards and septate. Conidia are spherical, smooth, with septa, forming chains, often clustered together. Asci are cylindrical, with a short stalk at the base and a ring at the apex. Ascospores are elliptical, initially olive green, later turning brownish-black.

[0031] 3. Growth characteristics: It grows best at a temperature of 28-30 ℃, with the highest and lowest initial growth pH being 8.0 and 3.0, respectively, and the optimal initial growth pH being 5.0; the mycelial germination stage lasts 12-24 hours.

[0032] 4. Method for preserving mycelial strains: Preserve them in PDA slant tubes at 4℃.

[0033] 5. Molecular identification: The liquid strain was inoculated into the fermentation medium at a 10% inoculum and cultured at 28-30℃ for 3-5 days. Mycelia were collected, and total DNA was extracted from the mycelia using the CTAB method. The tested DNA sequence was amplified by PCR using the designed universal primers ITS1 / ITS4 for the fungal ribosomal interstitial region (ITS).

[0034] Primer ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′, SEQ ID NO.1; Primer ITS4: 5′-TCCTCCGCTTATTGATATGC-3′, SEQ ID NO.2.

[0035] After sequencing the PCR product, the ITS sequence is shown in SEQ ID NO.3, as follows: 5′--3′.

[0036] The sequencing results were input into the GeneBank database for BLAST alignment analysis. Then, the neighbor-joining algorithm in MEGA software was used to construct the NJ phylogenetic tree. The results are as follows: Figure 2 As shown in the figure. It can be seen from the figure that the HJDF strain and... Neurospora crassa, Neurospora intermedia and Neurospora tetrasperma The HJDF strain is most closely related, and the results of molecular identification show that it is related to... Neurospora crassa Because of its high affinity, this strain was named Neurospora crassa HJDF.

[0037] Based on morphological characteristics, culture properties, and physiological and biochemical features, the above-mentioned strains were identified as belonging to the phylum Ascomycota (Ascomycota). Ascomycetes ), Conchiales ( Sordariales ), Dung Shell Family ( Sordariaceae Neurospora ( Neurospora Neurospora crassa Neurospora crassa And named it Neurospora crassa HJDF ( Neurospora crassaThe strain (HJDF) was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, with accession number CGMCC No. 41518. The viability of the culture was tested on September 23, 2023, and the result was positive.

[0038] Example 2: Adjustment of Printing Adaptability of Spore-Containing Soybean Residue Ink with Different Coefficients 1. Preparation of soybean residue powder: Soybean residue, the waste generated when making soybean milk, is dried at low temperature (40℃), then crushed by an ultra-fine pulverizer and passed through a 60-mesh sieve to obtain soybean residue for later use.

[0039] 2. Preparation of *Neurospora crassa* spore suspension: The preserved *Neurospora crassa* HJDF strain was inoculated onto PDA medium and cultured for 4-5 days to produce a large number of spores. Then, a small amount of sterile distilled water or physiological saline was added to the slant culture medium. Spores were scraped off with an inoculation loop or collected by rinsing with sterile distilled water. The spore suspension was transferred to an Erlenmeyer flask, sterile distilled water and glass beads were added, and the mixture was shaken to disperse the spores into clumps. The mycelium was then removed by filtration through cotton gauze or filter paper. The suspension was centrifuged three times to precipitate the spores and the supernatant was removed. Finally, the concentration of the spore suspension was adjusted with sterile distilled water, and hemocytometer counting was performed if necessary. The final concentration of the spore suspension was set at 1.5 × 10⁻⁶. 7 CFU / mL.

[0040] 3. Preparation of spore-containing inks with different ratios: First, according to the ratios given in Table 1, soybean residue powder, soluble starch (CAS No. 9005-84-9), agar, and water were mixed evenly using a homogenizer to ensure complete uniformity. Next, the mixture was autoclaved at 121℃ for 20 minutes to obtain sterile ink. Finally, 1 mL of *Neurospora crassa* spore suspension was mixed with 50 g of sterile ink and stirred thoroughly to disperse the spores, thus obtaining spore-containing printing ink.

[0041] Table 1 Composition of spore-containing ink

[0042] 4. Printing of spore-containing soybean residue scaffolds with different ratios: The spore-containing ink prepared in step 3 was introduced into a sterilized 3D printing barrel, and printing was performed according to the set printing parameters (printer purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., model EFL-BP-660). The specific parameters are as follows: barrel temperature 60℃, nozzle temperature 60℃, nozzle diameter 1.20mm, printing air pressure 25-35kPa, printing speed 600mm / min, and printing line spacing 4mm. After printing, it was shaped on a cooling plate at 10℃, and finally a porous 32mm×32mm×9-layer three-dimensional scaffold was produced.

[0043] Printing characteristics of different ink ratios, such as Figure 3 As shown in the figure. Experiments show that ink A has insufficient extrusion performance, resulting in distorted printed lines; ink D has extrusion continuity defects (top view); ink E exhibits insufficient structural self-support (side / head view), manifesting as linear collapse during the 3D forming process. In contrast, inks B, C, and F exhibit the best rheological properties: their continuous extrusion ensures accurate reproduction of the printing path (forming accuracy error <5%), while possessing excellent self-support strength (overhanging structure deformation rate <3%), effectively maintaining the stability of the 3D structure during the printing process.

[0044] Example 3: Evaluation of the growth characteristics of Neurospora crassa in 3D soybean residue printed scaffolds with different numbers of layers Printing of spore-containing bean residue scaffolds with different layers Following Example 1, the spore-containing ink C prepared in step (3) was introduced into a sterilized 3D printing barrel, and printing was performed according to the set printing parameters. The specific parameters were as follows: barrel temperature 60℃, nozzle temperature 60℃, nozzle diameter 1.55mm, printing air pressure 25-35kPa, printing speed 600mm / min, printing line spacing 4mm, and the number of printing layers were 2, 4, 8, and 16 respectively. After printing, the ink was shaped on a cooling plate at 10℃, ultimately producing a porous 25mm×25mm three-dimensional support. The printed products with different numbers of ink layers are shown below. Figure 4 As shown.

[0045] 2. Growth of Neurospora crassa in 3D soybean residue scaffolds with different number of layers The 3D soybean residue scaffolds containing spores, obtained after printing, were placed in a 60mm diameter sterile petri dish and covered with a disposable plastic cup with an opening, which was sterilized with 75% ethanol. This setup effectively controls ambient humidity while avoiding interference from the incubator fan (see [link to incubator setup]). Figure 5 Subsequently, the scaffold was incubated in a constant temperature and humidity incubator at 30°C and 70% humidity for 4 days.

[0046] The cultured scaffold, as Figure 6 The image shows a side view, a top view, and a cross-sectional view of the lyophilized sample. Based on the growth characteristics of *Neurospora crassa* and the side view of the sample, it can be observed that, as an aerobic fungus, its spores are mainly distributed on the surface of the scaffold. It is speculated that this is because the scaffold surface is rich in oxygen and has low humidity, which is conducive to spore formation and growth.

[0047] As can be seen from the bottom view and freeze-dried cross-section of the sample, the hyphae of *Neurospora crassa* grow intertwined between the scaffolds, but the number of spores produced inside the scaffolds is relatively small. Furthermore, the spore and hyphal yields across the entire scaffold vary with the number of scaffold layers. Specifically, the spore yield per unit of ink decreases with increasing layer count, while the hyphal yield increases with increasing layer count.

[0048] Based on the above observations, a multi-scale spatial structure control platform based on 3D printing technology can be used to achieve precise spatiotemporal control of the fungal solid-state culture process. By adjusting the number of scaffold layers and structural design, the growth pattern of fungi and the distribution of metabolites can be effectively controlled, thereby optimizing the production efficiency and quality of high-value fungal by-products.

[0049] Example 4: Analysis of carotenoid production after *Neurospora crassa* was cultured on 3D soybean residue scaffolds with different layer counts. Carotenoid production was evaluated using 3D soybean residue scaffolds with different layers of spores cultured in Example 3. In Neurospora crassa, carotenoids were mainly found in conidia and mycelia, with the number of pigment granules in conidia significantly higher than in mycelial tissue.

[0050] After culturing, the samples were first freeze-dried and then ground into powder under liquid nitrogen conditions to disrupt the spore walls. Next, 95% ethanol was added to the powder, and carotenoids were extracted using an ultrasonic disruptor under ice bath and light-protected conditions. After ultrasonic disruption, the solid-liquid mixture was separated by centrifugation (4°C, 3500 rpm, 15 minutes). Subsequently, the absorbance of the ethanol layer was measured at 440 nm using a UV-Vis spectrophotometer. The carotenoid content per unit mass of sample (dry weight) was calculated according to the following formula (1).

[0051] Formula (1): Carotenoid content (mg / g, dry weight) = OD 440 × V E × F ×1000÷( W × ε × d ); ε The molar absorptivity of carotenoids is 250 L / g·cm; d The optical path length of the cuvette is 1 cm. V E Extraction liquid volume: 25 mL for single-layer printing structure and 60 mL for multi-layer printing structure; 1000: Unit conversion factor, 1g = 1000mg; F The dilution factor for the extract is 1. W Sample mass (on dry weight).

[0052] The carotenoid content of 3D soybean residue scaffolds with different layers containing spores was determined as follows: Figure 7 As shown, the carotenoid content produced by *Neurospora crassa* ranged from 0.20 to 0.33 mg / g in scaffolds with 2 to 16 layers. There was no significant difference in carotenoid production per unit of ink in scaffolds with 8 layers or less; however, the carotenoid content decreased significantly when the number of layers increased to 16. This phenomenon may be directly related to the reduction in spore production in scaffolds with higher layer counts.

[0053] Example 5: Synergistic Regulation of Protein and Other Active Component Synthesis in *Neurospora crassa* within a Three-Dimensional Soybean Residue Scaffold by the Number of Scaffold Layers and Humidity According to Examples 2 and 3, 8-layer and 16-layer scaffolds containing spore-containing ink C were obtained and cultured at 50% and 90% humidity, respectively. Based on the number of scaffold layers, culture humidity, and culture days, they were divided into 4 groups (named according to "number of layers-humidity-days"): 8-50%-4d, 16-50%-4d, 8-90%-4d, and 16-90%-4d.

[0054] *Neurospora crassa* is not only an important source of carotenoids, but its hyphae are also rich in protein and fatty acids, and its cell membranes contain ergosterol. After culturing, samples were dried at 40°C, and the contents of protein, amino acids, fatty acids, and ergosterol in the dried samples were then determined. Protein content was determined using the Kjeldahl method, which includes digestion, distillation, and titration calculation steps. Amino acid content was quantitatively analyzed using an amino acid analyzer (S-433DUP, Sykam GmbH, Germany) combined with post-column derivatization ion exchange chromatography after acid hydrolysis. Liposome content was determined by chromatographic analysis. Ergosterol content was quantified by high-performance liquid chromatography (1260 Infinity II, Agilent Technologies, Germany). The quantitative results are shown in Table 2.

[0055] Table 2. Analysis of active ingredients (g / 100g, dry weight)

[0056] The results showed that by controlling the number of layers and the culture humidity of the three-dimensional soybean residue scaffold, the metabolic flow of *Neurospora crassa* could be effectively guided, achieving the targeted synthesis of active ingredients. Specifically, a high humidity (90%) environment significantly promoted the biosynthesis of proteins and amino acids, reaching its optimal level in an 8-layer scaffold. Under low humidity (50%) conditions, increasing the number of layers to 16 synergistically enhanced the accumulation of polyunsaturated fatty acids. Ergosterol synthesis was highly dependent on the matching of humidity and structure, reaching its peak only under a high humidity-thin-layer (8-90%) combination; increasing the number of layers severely inhibited its formation.

[0057] The aforementioned differences stem from humidity-driven selection of metabolic pathways, while the number of layers amplifies or inhibits these pathways by regulating oxygen and mass transport efficiency. Under high humidity conditions, thin-layer structures facilitate hyphal expansion and protein synthesis; under low humidity conditions, the diffusion restriction formed by thick-layer structures may enhance the stress response of lipid accumulation. Therefore, to achieve customized production of target products, it is recommended to use an 8-layer high-humidity combination to obtain high-protein, high-amino acid products, and a 16-layer low-humidity combination to enrich polyunsaturated fatty acids. These results provide important evidence for solid-state fermentation processes based on structure-environment synergistic regulation.

[0058] In the future, by comprehensively managing the structure of 3D-printed scaffolds (such as the number of printing layers and line spacing) and cultivation conditions (such as humidity, temperature, and light), it will be possible to precisely control the yield of high-value byproducts within the scaffold. This controllability provides high flexibility and customization potential for biosynthesis using Neurospora crassa.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high-nutrient-yielding 3D-printed bioscaffold prepared from Neurospora crassa spores and soybean residue, characterized in that, Includes the following steps: After drying the soybean residue, pulverize it and pass it through a 50-70 mesh sieve. Collect the sieve residue to obtain soybean residue powder. The soybean residue powder, soluble starch, agar, and water are mixed and then sterilized to obtain sterile ink; The sterile ink and Neurospora crassa ( Neurospora crassa The spore suspension was mixed to obtain spore-containing ink; The spore-containing ink is added to a 3D printer to print a porous three-dimensional scaffold. The porous three-dimensional scaffold was cultured in an environment of 28-30℃ and 50%-90% humidity to obtain a 3D-printed biological scaffold containing nutrients.

2. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The mass ratio of soybean residue powder to water is 20-24:100; the mass ratio of soluble starch to water is 0-4:100; and the mass ratio of agar to water is 1-1.5:

100.

3. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The volume-to-mass ratio of the *Neurospora crassa* spore suspension to the sterile ink was 1 mL: 50 g, and the spore concentration in the *Neurospora crassa* spore suspension was 1.5 × 10⁻⁶. 7 -2.0×10 7 CFU / mL.

4. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1 or 3, characterized in that, The Neurospora crassa includes Neurospora crassa HJDF, whose preservation number is CGMCC No. 41518.

5. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The printing parameters include: material barrel temperature 60℃, nozzle temperature 60℃, nozzle diameter 1.20-2.00mm, printing air pressure 15-35kPa, and printing speed 100-600mm / min.

6. The high-nutrient-yielding 3D-printed biological scaffold according to claim 1, characterized in that, The porous three-dimensional scaffold has 2-16 layers; the shape of the holes in the porous three-dimensional scaffold includes circles and / or polygons; the side length of the polygons is 25-32mm.

7. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The drying process includes low-temperature drying and / or freeze drying; the low-temperature drying temperature is 35-45°C.

8. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The soybean residue is the residue produced when soybeans are used to make soy milk.

9. The high-nutrient-yielding 3D-printed bio-scaffold according to claim 1, characterized in that, The nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids, and ergosterol.

10. The application of the high-nutrient-yielding 3D-printed bioscaffold according to any one of claims 1-9 in the production of nutrients, wherein the nutrients include one or more of fungal proteins, unsaturated fatty acids, dietary fiber, carotenoids and ergosterol.

Images