3D bio-printing ink, preparation method and application thereof, and method for producing monascus pigment by 3D bio-printing monascus

Abstract

The invention provides 3D bio-printing ink, a preparation method and application thereof, and a method for producing monascus pigment by 3D bio-printing monascus, and belongs to the technical field of food. The 3D biological printing ink provided by the invention is prepared from the following components in percentage by mass: 15 to 18 percent of dry base material, 10 to 12 percent of food gum and 70 to 75 percent of water, the dry basis material comprises a nitrogen source and a carbon source, the nitrogen source is bean dregs, and the carbon source is corn sugar residues and / or soluble starch. The 3D biological printing ink provided by the invention can be used for 3D biological printing of monascus purpureus to produce monascus pigment, is expected to break through the limitation of traditional monascus fermentation, and realizes diversified development of functional foods and biological materials.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and in particular to 3D bioprinting ink, a preparation method and application thereof, and a method for producing monascus pigment using 3D bioprinting monascus. Background Art

[0002] Monascus spp., as a traditional fermentation microorganism, has a long history of application in East Asia, especially red yeast rice fermentation. Red yeast rice is a product obtained by inoculating Monascus spp. on steamed white rice for solid-state fermentation. It is often used for food coloring, brewing rice wine and traditional Chinese medicine preparations. Monascus spp. can synthesize a variety of secondary metabolites during the fermentation process, such as red yeast pigment, monacolin K and γ-aminobutyric acid (GABA), which have significant biological activities, including lipid-lowering, anti-oxidation and blood pressure regulation. Although the traditional red yeast rice fermentation process is mature, it is limited by the singleness of the fermentation matrix and the low controllability of the solid-state fermentation process, and it is difficult to meet the needs of modern industry for product consistency and functional diversity. In recent years, with the development of biotechnology, exploring new fermentation matrices and processes has become a technical problem that needs to be solved urgently. Summary of the invention

[0003] The purpose of the present invention is to provide a 3D bioprinting ink and a preparation method and application thereof, and a method for producing monascus pigment by 3D bioprinting Monascus. The 3D bioprinting ink provided by the present invention can be used for 3D bioprinting Monascus to produce monascus pigment, which is expected to break through the limitations of traditional monascus fermentation and realize the diversified development of functional foods and biomaterials.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a 3D bioprinting ink, comprising the following components in percentage by weight: 15-18% dry-base material, 10-12% food gum and 70-75% water; the dry-base material comprises a nitrogen source and a carbon source, the nitrogen source is bean dregs, and the carbon source is corn sugar residue and / or soluble starch.

[0006] Preferably, the mass ratio of the nitrogen source to the carbon source is 2 to 4:1.

[0007] Preferably, the food glue comprises gelatin and guar gum, and the mass ratio of the gelatin to the guar gum is 9:0.5-1.5.

[0008] Preferably, the particle sizes of the soybean dregs and corn sugar dregs are independently ≤0.25 mm.

[0009] The present invention provides a method for preparing the 3D bioprinting ink described in the above technical solution, comprising the following steps:

[0010] The components of the 3D bioprinting ink are mixed and homogenized to obtain the 3D bioprinting ink.

[0011] Preferably, the speed of the homogenization is 6000-13500 rpm, and the time is 10-20 min; sterilization is also included after the homogenization.

[0012] The present invention provides the use of the 3D bioprinting ink described in the above technical solution or the 3D bioprinting ink prepared by the preparation method described in the above technical solution in the production of monascus pigment by 3D bioprinting Monascus.

[0013] The present invention provides a method for producing monascus pigment by 3D bioprinting monascus, comprising the following steps:

[0014] Inoculating Monascus into 3D bioprinting ink to obtain Monascus-containing ink; the 3D bioprinting ink is the 3D bioprinting ink described in the above technical solution or the 3D bioprinting ink prepared by the preparation method described in the above technical solution;

[0015] The monascus pigment is obtained by 3D printing the monascus-containing ink, solidifying it into a shape, and then fermenting and culturing it.

[0016] Preferably, the concentration of Monascus spores in the Monascus ink is 0.5×10 7 ~1.5×10 7 Pieces / mL.

[0017] Preferably, coagulation molding is further included between the 3D printing and the fermentation culture, and the temperature of the coagulation molding is 10-15°C; the temperature of the fermentation culture is 28-32°C.

[0018] The present invention provides a 3D bioprinting ink, comprising the following components in percentage by mass: 15-18% dry base material, 10-12% food gum and 70-75% water; the dry base material comprises a nitrogen source and a carbon source, the nitrogen source is bean dregs, and the carbon source is corn dregs and / or soluble starch. The 3D bioprinting ink provided by the present invention uses bean dregs as a nitrogen source and corn dregs and / or soluble starch as a carbon source. Both of them are used as dry base materials, which can provide sufficient nutritional support for Monascus, meet the normal growth and metabolic needs of Monascus, and compound food gum can improve the rheological properties of the dry base material, which can not only improve the viscosity of the 3D bioprinting ink, so that it can be continuously extruded, but also improve its formability to better maintain the printed shape. In the examples of the present invention, the growth of Monascus on the 3D bioprinting ink was evaluated by the yield of Monascus pigment and the growth diameter of Monascus hyphae. The results showed that all 15 groups of 3D bioprinting inks provided by the present invention achieved the application of Monascus on the 3D printing matrix for cultivation, which is expected to break through the limitations of traditional Monascus fermentation and realize the diversified development of functional foods and biomaterials.

[0019] In addition, the raw material cost of the 3D bioprinting ink provided by the present invention is low, the preparation method is simple, and efficient utilization of food waste resources is achieved.

[0020] Furthermore, the 3D bioprinting ink provided by the present invention has good printability, and is convenient for solidifying the 3D bioprinting ink into a colloid by low-temperature cooling, which is beneficial to the molding of the 3D bioprinting ink and maintaining the printed shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a physical picture of simulating 3D printing using the 3D bioprinting ink in Examples 1 to 15;

[0022] Figure 2 The data graph of rheological properties (amplitude scanning) of 3D bioprinting ink in the embodiment with soluble starch content of 0 or corn sugar residue content of 0;

[0023] Figure 3 This is a graph showing the growth of the spore-containing ink in Application Examples 1 to 15 after 3D printing and fermentation at 30° C. for 7 days;

[0024] Figure 4 This is a graph showing the yield of red yeast rice pigments after 3D printing with different spore-containing inks and fermentation at 30°C for 7 days in Test Example 1;

[0025] Figure 5 This is a physical picture of the growth of Monascus mycelium on different ink plates in Test Example 2;

[0026] Figure 6This is a statistical chart of the growth diameter of Monascus mycelium on different ink plates in Test Example 2. DETAILED DESCRIPTION

[0027] The present invention provides a 3D bioprinting ink, comprising the following components in percentage by weight: 15-18% dry-base material, 10-12% food gum and 70-75% water; the dry-base material comprises a nitrogen source and a carbon source, the nitrogen source is bean dregs, and the carbon source is corn sugar residue and / or soluble starch.

[0028] In recent years, 3D printing technology has attracted widespread attention in the fields of food, medicine and biomanufacturing due to its advantages such as high design freedom, high material utilization efficiency and the ability to manufacture complex structures. Traditional 3D printing inks are mostly based on polymers, ceramics, metals or hydrogels, which have achieved significant applications in the industrial and biomedical fields. However, in the food and biological fields, the development of materials with natural sources, low costs and biocompatibility is still relatively lagging. The research on food-grade inks is still in its infancy, and current attempts are mostly focused on high-viscosity materials such as syrups and chocolate syrups, but the nutrition and functionality of these inks are relatively limited. In addition, there are few reports on 3D bioprinting inks that can support the growth of active microorganisms. The resource utilization of food waste is an important issue in the field of global sustainable development, especially in the food industry, where the generation of a large number of by-products and wastes has brought environmental pressure and resource waste problems. A large number of by-products are generated during food processing, such as soybean dregs and corn sugar dregs. Among them, soybean dregs, as a by-product of soybean processing, are rich in dietary fiber, protein and various nutrients. Corn sugar dregs are a by-product of corn starch production and contain high sugar and cellulose. These by-products all have high nutritional value, but due to the lack of efficient utilization technology, most of them are discarded or treated at a low value, and their maximum resource value cannot be realized. In this context, in view of the problems existing in the traditional red yeast rice fermentation process, the present invention applies Monascus to 3D printing substrate for cultivation, which is a brand-new attempt. Specifically, the present invention uses bean dregs as a nitrogen source, corn sugar residues and / or soluble starch as a carbon source, and compound food gum at the same time. On this basis, the obtained 3D bioprinting ink can be used as a culture medium, which can provide a rich source of nutrition for the growth and metabolism of Monascus. The present invention combines the advantages of food waste utilization and functional microbial development, not only solves the problem of low utilization of bean dregs and corn sugar residues, but also gives the Monascus fermentation system new flexibility and application scenarios. The 3D bioprinting ink provided by the present invention is described in detail below.

[0029] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0030] In terms of mass percentage, the 3D bioprinting ink of the present invention includes 15-18% dry base materials, specifically 15%, 15.5%, 16%, 16.5%, 17%, 17.5% or 18%. In the present invention, the dry base materials include nitrogen source and carbon source; the mass ratio of the nitrogen source to the carbon source can be 2-4:1, specifically 2:1, 2.5:1, 3:1, 3.5:1 or 4:1. In the embodiment of the present invention, the mass ratio of the nitrogen source to the carbon source is controlled within the above range. When the obtained 3D bioprinting ink is used for 3D bioprinting Monascus to produce Monascus pigment, the mycelium and Monascus pigment are both produced more; and the 3D bioprinting ink can be made to present a lighter color, which is conducive to the subsequent more intuitive observation of the growth of Monascus.

[0031] In the present invention, the nitrogen source is bean dregs; the carbon source is corn dregs and / or soluble starch, specifically corn dregs, soluble starch, or corn dregs and soluble starch; when the carbon source is corn dregs and soluble starch, the mass ratio of corn dregs and soluble starch can be 1-9:9-1, further 1:6-6:1, specifically 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 or 1:4. The present invention uses corn dregs as a carbon source, can directly use food waste, and convert it into easily usable sugars for microbial absorption, which is beneficial to reduce the cost of starch raw materials and reduce dependence on expensive carbon sources; using soluble starch as a carbon source can not only provide a stable carbon source, but also adjust the physical properties of the ink, which is beneficial to improve the viscoelasticity of the ink, thereby improving the stability of 3D printing. In the embodiment of the present invention, the mass ratio of corn dregs and soluble starch is limited to the above range, which is beneficial to ensure that the mycelium and red yeast pigment are more produced while having good printability.

[0032] As an embodiment of the present invention, the particle size of the bean dregs and corn sugar residue can be independently ≤0.25mm. As an embodiment of the present invention, the preparation method of the bean dregs can include the following steps: mixing soybeans with water and grinding them, filtering the obtained soy milk, collecting the filter cakes, drying and crushing them in sequence, and then passing them through a 60-mesh sieve, and collecting the sieve under the sieve for later use. As an embodiment of the present invention, the dosage ratio of the soybeans to water can be 100-180g:1-1.2L, specifically 150g:1L; the grinding can be specifically carried out in a soymilk machine, and the grinding time can be 15-30min, specifically 20min; the drying temperature can be 60°C, and the time can be 12h; the crushing can be specifically carried out in a grinder. As an embodiment of the present invention, the corn sugar residue is the residue with high sugar and cellulose remaining in the process of corn starch production. The corn sugar residue of the present invention is preferably washed, dried and crushed in sequence before use, and then passed through a 60-mesh sieve, and the sieve under is collected for standby use; the reagent used for the washing can be water; the drying temperature can be 60°C, and the time can be 12 hours; the crushing can be carried out in a grinder. The corn sugar residue in the embodiment of the present invention is specifically purchased from Linqu County Huamao Feed Co., Ltd., model YMTZ0375; the soluble starch is specifically purchased from Hangzhou Chentong Biochemical Technology Co., Ltd., model S817547-500g.

[0033] According to the percentage by mass, the 3D bioprinting ink of the present invention includes 10-12% food gelatin, which can be specifically 10%, 10.5%, 11%, 11.5% or 12%. As an embodiment of the present invention, the food gelatin can include gelatin and guar gum, and the mass ratio of the gelatin to the guar gum can be 9:0.5-1.5, further 9:0.8-1.2, and specifically 9:1. In the embodiment of the present invention, gelatin and guar gum are compounded and used. When the obtained 3D bioprinting ink is used for 3D bioprinting Monascus to produce Monascus pigment, the yield of mycelium and Monascus pigment is more; the inventor found in the research process that if only gelatin is used, Monascus produces more Monascus pigment in 3D bioprinting ink, but less mycelium; if only guar gum is used, the mycelium is more, but the Monascus pigment is less. In the embodiment of the present invention, the mass ratio of gelatin to guar gum is limited to the above range, which is conducive to ensuring that the yield of mycelium and Monascus pigment is more. The gelatin described in the embodiment of the present invention was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd., model number G6317-100g; the guar gum was specifically purchased from Hangzhou Chentong Biochemical Technology Co., Ltd., model number S30550-500g.

[0034] Calculated by mass percentage, the 3D bioprinting ink of the present invention includes 70-75% water, specifically 70%, 71%, 72%, 73%, 74% or 75%.

[0035] The present invention provides a method for preparing the 3D bioprinting ink described in the above technical solution, comprising the following steps:

[0036] The components of the 3D bioprinting ink are mixed and homogenized to obtain the 3D bioprinting ink.

[0037] As an embodiment of the present invention, the mixing may include: first mixing the food glue with water to obtain a colloidal solution (specifically a slightly yellow transparent colloidal solution); second mixing the colloidal solution with the dry material. As an embodiment of the present invention, the temperature of the first mixing may be 55 to 70°C, specifically 55°C, 60°C, 65°C or 70°C; the time may be 20 to 30 minutes, specifically 20 minutes, 25 minutes or 30 minutes; the first mixing is specifically carried out under stirring conditions. The present invention has no special limitation on the second mixing, and a method well known to those skilled in the art may be used.

[0038] As an embodiment of the present invention, the homogenization speed can be 6000-13500rpm, specifically 6000rpm, 8000rpm, 10000rpm, 12000rpm, 13000rpm or 13500rpm; the time can be 10-20min, specifically 10min, 15min or 20min; the homogenization can be specifically carried out in a homogenizer.

[0039] As an embodiment of the present invention, the homogenization further includes sterilization; the sterilization may be high temperature and high pressure sterilization; the sterilization temperature may be 121° C., the pressure may be 0.1 MPa, and the time may be 20 min.

[0040] The present invention provides the use of the 3D bioprinting ink described in the above technical solution or the 3D bioprinting ink prepared by the preparation method described in the above technical solution in the production of monascus pigment by 3D bioprinting Monascus.

[0041] The present invention provides a method for producing monascus pigment by 3D bioprinting monascus, comprising the following steps:

[0042] Inoculating Monascus into 3D bioprinting ink to obtain Monascus-containing ink; the 3D bioprinting ink is the 3D bioprinting ink described in the above technical solution or the 3D bioprinting ink prepared by the preparation method described in the above technical solution;

[0043] The monascus pigment is obtained by 3D printing the monascus-containing ink, solidifying it into a shape, and then fermenting and culturing it.

[0044] The present invention inoculates Monascus into 3D bioprinting ink to obtain Monascus-containing ink. As one embodiment of the present invention, specifically, sterile water is used to wash the pre-cultured Monascus PDA plate, a spore suspension is collected, and then the spore suspension is mixed with the 3D bioprinting ink to obtain the Monascus-containing ink. As one embodiment of the present invention, specifically, after the 3D bioprinting ink is prepared and sterilized according to the aforementioned method, when the sterilized 3D bioprinting ink is cooled to 45-50°C, the 3D bioprinting ink and the spore suspension are stirred and mixed to obtain the Monascus-containing ink. As one embodiment of the present invention, a blood cell counting plate can be used to adjust the concentration of the spore suspension so that the amount of spores inoculated into the 3D bioprinting ink meets the requirements; specifically, the concentration of Monascus spores in the Monascus-containing ink can be 0.5×10 7 ~1.5×10 7 / mL, specifically 1.0×10 7 / mL. As one embodiment of the present invention, the Monascus may come from the China Industrial Microbiological Culture Collection or the China Type Culture Collection, or may come from the Monascus extracted from Monascus products such as red yeast rice. The present invention does not specifically limit the specific type of the Monascus. As one embodiment of the present invention, the Monascus may come from the Red Yeast Factory in Gutian County, Fujian Province, specifically, it may be isolated from red yeast rice; the method for isolating the Monascus comprises the following steps: placing 3 to 5 grains of red yeast rice on a PDA plate, then culturing in a 30°C incubator for 5 to 7 days, marking the colony and transferring it to a new PDA plate, and culturing in a 30°C incubator for 5 to 7 days; then repeating the separation and purification until a single colony is obtained.

[0045] After obtaining the Monascus ink, the present invention performs fermentation and culture after 3D printing and solidification of the Monascus ink to obtain the Monascus pigment. As one embodiment of the present invention, the solidification temperature can be 10-15°C, and further can be 13-15°C, and the temperature required for solidification can be met by an ice box. As one embodiment of the present invention, the Monascus ink is filled into a syringe without a needle, and after exhausting the air in the syringe, the syringe is pushed at a uniform speed (the syringe is pushed slowly to ensure that a uniform and continuous strip is obtained after solidification), and the Monascus ink is extruded onto a sterile culture dish to simulate the 3D printing process; an ice box is placed at the bottom of the sterile culture dish in advance, and the sterile culture dish is precooled to 10-15°C, so that the Monascus ink can be quickly cooled and solidified after printing. As one embodiment of the present invention, the inner diameter of the needle mouth of the syringe can be 1.2-2.8mm, and can be 2mm. As an embodiment of the present invention, the fermentation temperature can be 28-32°C, specifically 30°C; the fermentation time can be determined according to actual needs. For example, in the embodiment, the monascus pigment content can reach up to 137.7U / g at 30°C for 7 days.

[0046] The present invention uses bean dregs as a nitrogen source, corn sugar residue and / or soluble starch as a carbon source, and compounded with food gum. On this basis, the obtained 3D bioprinting ink can be used as a culture medium, which can provide a rich source of nutrition for the growth and metabolism of Monascus. It is also possible to achieve structural optimization of microbial culture through 3D printing. Specifically, Monascus is an aerobic bacterium. It is mixed with 3D bioprinting ink and then 3D printed. By controlling the number of printing layers and spacing, etc., culture media with different shapes and spatial structures can be printed. Different printing structures may affect the growth of Monascus. For example, if the printing spacing is large, the oxygen flow rate increases, thereby achieving structural optimization of microbial culture, providing new possibilities for the modernization and customization of the Monascus fermentation system, and is expected to break through the limitations of traditional Monascus fermentation and achieve diversified development of functional foods and biomaterials.

[0047] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following embodiments and application examples are conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] The corn sugar residue used in the following examples was cleaned with water before use, dried in an oven at 60° C. for 12 h, then crushed with a grinder and passed through a 60-mesh sieve, and the residue under the sieve was collected for later use.

[0050] The preparation method of the okara used in the following examples comprises the following steps: adding 150 g of soybeans and 1 L of water into a soybean milk machine and grinding for 20 min, filtering the obtained soybean milk, placing the obtained okara in a 60° C. oven and drying for 12 h, then crushing it with a grinder and passing it through a 60-mesh sieve, and collecting the sieve material for later use.

[0051] The Monascus used in the following application examples is derived from the Monascus Factory in Gutian County, Fujian Province, and is specifically isolated from Monascus rice; the Monascus isolation method comprises the following steps: placing 5 grains of Monascus rice on a PDA plate, then culturing in a 30°C incubator for 7 days, marking the colonies and transferring them to a new PDA plate, and culturing in a 30°C incubator for 7 days; then repeating the separation and purification until a single colony is obtained.

[0052] Examples 1 to 15

[0053] Gelatin and guar gum were mixed with water, heated in a water bath at 70°C and stirred for 25 minutes to obtain a slightly yellowish transparent colloidal solution; bean dregs, corn sugar residue, and soluble starch were mixed with the colloidal solution, homogenized at a speed of 9500 rpm for 10 minutes, and then sterilized at a temperature of 121°C and a pressure of 0.1 MPa for 20 minutes to obtain a 3D bioprinting ink (the specific formula is shown in Table 1).

[0054] Table 1 Formula of 3D bioprinting ink in Examples 1 to 15 (unit: mass percentage %)

[0055]

[0056] In each embodiment, when the temperature of the 3D bioprinting ink after sterilization drops to 50°C, the 3D bioprinting ink is filled into a syringe without a needle (the inner diameter of the syringe needle nozzle is 2 mm), and after the air in the syringe is exhausted, the syringe is pushed at a uniform speed to extrude the 3D bioprinting ink onto a plastic flat plate. An ice box is placed on the bottom of the plastic flat plate in advance to cool to 13-15°C, so that the 3D bioprinting ink can be quickly cooled and solidified into a strip after being extruded onto the plastic flat plate to simulate the 3D printing process.

[0057] Figure 1 The actual pictures of simulating 3D printing using the 3D bioprinting inks in Examples 1 to 15 show that the 3D bioprinting inks prepared in Examples 1 to 15 all have certain printability, and after being squeezed out with a syringe, they are solidified at low temperature so that they can maintain their complete shape.

[0058] The rheological properties of 3D bioprinting inks prepared from three embodiments with 0 soluble starch content (group numbers 2-1, 3-1, and 4-1) and three embodiments with 0 corn sugar residue content (group numbers 2-5, 3-5, and 4-5) were tested. Figure 2 The data graph of the rheological properties (amplitude scanning) of the 3D bioprinting ink in the embodiment with 0 soluble starch content or 0 corn syrup content shows that in the linear viscoelastic range (strain range of 0.1-1%), the addition of soluble starch reduces the storage modulus of the 3D bioprinting ink. The higher the storage modulus, the better the support and stability of the 3D bioprinting ink, that is, when the soluble starch content is 0 (group numbers are 2-1, 3-1, and 4-1 respectively), the corresponding 3D bioprinting ink has the best printability; conversely, when the corn syrup content is 0 (group numbers are 2-5, 3-5, and 4-5 respectively), the printability of the corresponding 3D bioprinting ink is reduced.

[0059] Application Examples 1 to 15

[0060] Referring to Examples 1 to 15, 3D bioprinting ink was prepared and Monascus was inoculated and 3D printing was simulated simultaneously, comprising the following steps:

[0061] (1) Preparation of 3D bioprinting ink: Prepare 3D bioprinting ink with reference to Examples 1 to 15;

[0062] (2) Monascus inoculation: Wash the pre-cultured Monascus PDA plate with sterile water to collect the spore suspension; count the spores with a hemocytometer and adjust the concentration of the spore suspension so that the amount of spores inoculated into the 3D bioprinting ink is 10 7 When the temperature of the sterilized 3D bioprinting ink drops to 50°C, the spore suspension is inoculated into the 3D bioprinting ink to make the spore amount 10 7 / mL, and stir evenly to obtain spore-containing ink;

[0063] (3) Simulating 3D printing: The spore-containing ink was filled into a syringe without a needle (the inner diameter of the syringe needle tip was 2 mm), and after exhausting the air in the syringe, the syringe was pushed at a uniform speed to extrude the spore-containing ink onto a sterile culture dish. The bottom of the culture dish was placed in an ice box in advance and cooled to 13-15° C., so that the spore-containing ink could be quickly cooled and solidified into a strip after being extruded onto the sterile culture dish, thereby simulating the 3D printing process.

[0064] (4) Cultivation: Place the culture dish containing the strips in a 30°C incubator for fermentation.

[0065] After 2 days of fermentation at 30° C., a small amount of white hyphae began to grow in dots on the surface of the strips on the sterile culture dish. On the 4th day, the hyphae grew along the strips and produced a large amount of monascus pigment.

[0066] Figure 3 The growth diagram of the spore-containing ink in application examples 1 to 15 after 3D printing and fermentation at 30°C for 7 days shows that as the soluble starch content in the spore-containing ink increases, the amount of white mycelium and pigment content grown in the spore-containing ink also increase. It can be seen that this embodiment realizes the application of Monascus on a 3D printing substrate for cultivation, which is different from the traditional Monascus fermentation mode and provides new possibilities for the modernization and customization of the Monascus fermentation system.

[0067] Test Example 1

[0068] After the spore-containing inks in Application Examples 1 to 15 were 3D printed and fermented at 30° C. for 7 days, the monascus pigment produced in the obtained solid matrix was extracted and quantitatively analyzed, and the steps were as follows:

[0069] The solid matrix after fermentation and culture for 7 days was dried in an oven at 50°C, then crushed and ground, and the sample mass was weighed and recorded as m (g); an ethanol aqueous solution with a volume fraction of 70% was added to the obtained sample, and the added volume of the ethanol aqueous solution was recorded as V (mL), and ultrasonic treatment was performed at room temperature (23°C) for 2h, and then treated in a water bath at 60°C for 2h. After the water bath treatment, centrifugation was performed at 5000rpm for 20min, and the supernatant was taken and re-diluted to the scale (i.e., the volume was V) with an ethanol aqueous solution with a volume fraction of 70%, and the solution was allowed to stand for 10min to cool to room temperature, and then diluted with an ethanol aqueous solution with a volume fraction of 70% and shaken to obtain a sample dilution solution, and the dilution multiple was recorded as F. The ethanol aqueous solution with a volume fraction of 70% was used as a blank, and a 1cm colorimetric dish was used to measure the absorbance of the sample dilution solution at 410nm, 470nm, and 505nm, respectively, and the color value (unit: U / g) was calculated according to the measured absorbance according to the following formula:

[0070] Yellow pigment value (based on 1g sample) = F × A 410 ×V / m(U / g) Formula 1;

[0071] Orange pigment value (based on 1g sample) = F × A 470 ×V / m(U / g) Formula 2;

[0072] Red pigment value (based on 1g sample) = F × A 505 ×V / m(U / g) Formula 3;

[0073] Total color value of Monascus pigment = yellow pigment color value + orange pigment color value + red pigment color value (U / g) Formula 4;

[0074] In formulas 1 to 3, F is the dilution factor of the sample; m is the mass of the sample weighed (g); V is the volume of the 70% ethanol aqueous solution used for constant volume (V); A 410 A is the absorbance value of the sample dilution at 410 nm; 470 A is the absorbance value of the sample dilution at 470 nm; 505 It is the absorbance value of the sample dilution at 505nm.

[0075] Figure 4 The following is a graph showing the yield of red pigment after 3D printing with different spore-containing inks and fermentation at 30°C for 7 days in Test Example 1. The specific data are listed in Table 2. The results show that as the soluble starch content in the spore-containing ink increases, the yield of red pigment also increases. Among them, the red pigment content of group 2-5 is as high as 137.7U / g, followed by group 3-5 and group 4-5, which are 50.41U / g and 49.18U / g respectively.

[0076] Table 2 Color value of red yeast rice pigment per gram of solid matrix after 7 days of fermentation (unit: U / g)

[0077] Group No. Color value U / g Group No. Color value U / g Group No. Color value U / g 2-1 <![CDATA[16.33±0.83 d ]]> 3-1 <![CDATA[15±2.6 b ]]> 4-1 <![CDATA[13.78±1.13 d ]]> 2-2 <![CDATA[15.82±1.09 d ]]> 3-2 <![CDATA[15.06±1.49 b ]]> 4-2 <![CDATA[17.23±1.8 c ]]> 2-3 <![CDATA[34.01±3.23 c ]]> 3-3 <![CDATA[16.93±6.09 b ]]> 4-3 <![CDATA[11.89±2.62 c ]]> 2-4 <![CDATA[66.7±6.04 b ]]> 3-4 <![CDATA[35.88±7.16 a ]]> 4-4 <![CDATA[35.55±3.5 b ]]> 2-5 <![CDATA[137.7±15.49 a ]]> 3-5 <![CDATA[50.41±16.84 a ]]> 4-5 <![CDATA[49.18±1.7 a ]]>

[0078] Note: Different lowercase letters in the same column in Table 2 indicate significant differences (P<0.05).

[0079] Test Example 2

[0080] In this test example, 3D bioprinting ink was prepared with reference to Examples 1 to 15, and then the 3D bioprinting ink was made into a plate, and the growth of Monascus was quantitatively analyzed by inoculating Monascus cake, and the steps were as follows:

[0081] (1) Preparation of 3D bioprinting ink: Prepare 3D bioprinting ink with reference to Examples 1 to 15;

[0082] (2) Preparation of a plate: Use a culture dish with a diameter of 60 mm, take 8 g of the 3D bioprinting ink and pour it into the culture dish, gently tap the culture dish to remove air, and then use a scraper to evenly spread and smooth the 3D bioprinting ink, and leave it until the 3D bioprinting ink solidifies to obtain an ink plate;

[0083] (3) Inoculation and cultivation of the cake: Take a pre-cultured Monascus PDA plate, use a 1 mL pipette tip to punch holes in the center of the Monascus PDA plate and the ink plate, then inoculate the Monascus cake onto the ink plate, keeping the thickness of the Monascus cake consistent with that of the ink plate, and finally place it in a 30° C. incubator for fermentation and cultivation for 7 days; during the fermentation and cultivation process, measure the mycelium diameter with a vernier caliper every day and take photos and record.

[0084] Figure 5 The growth of Monascus mycelium on different ink plates in Test Example 2 (from left to right in each group are the growth pictures of 1 to 7 days), Figure 6 The results are statistical diagrams of the growth diameter of Monascus mycelium on different ink plates in Test Example 2; the results show that Monascus can grow on the 3D bioprinting inks prepared in Examples 1 to 15, and there is not much difference in the growth diameter, indicating that the growth rate of Monascus on different 3D bioprinting inks is similar. In the group with low soluble starch content, the mycelium grows sparsely, while as the soluble starch content increases, the white mycelium grows more vigorously and the Monascus pigment is more obvious.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A 3D bioprinting ink, comprising the following components in percentage by weight: 15-18% dry base material, 10-12% food gum and 70-75% water; the dry base material comprises a nitrogen source and a carbon source, the nitrogen source is bean dregs, and the carbon source is corn sugar residue and / or soluble starch.

2. The 3D bioprinting ink according to claim 1, characterized in that The mass ratio of the nitrogen source to the carbon source is 2 to 4:

1.

3. The 3D bioprinting ink according to claim 1, characterized in that The food glue comprises gelatin and guar gum, and the mass ratio of the gelatin to the guar gum is 9:0.5-1.

5.

4. The 3D bioprinting ink according to claim 1, characterized in that The particle sizes of the soybean dregs and corn sugar dregs are independently ≤0.25 mm.

5. The method for preparing the 3D bioprinting ink according to any one of claims 1 to 4, comprising the following steps: The components of the 3D bioprinting ink are mixed and homogenized to obtain the 3D bioprinting ink.

6. The preparation method according to claim 5, characterized in that: The speed of the homogenization is 6000-13500 rpm, and the time is 10-20 minutes; sterilization is also included after the homogenization.

7. Use of the 3D bioprinting ink according to any one of claims 1 to 4 or the 3D bioprinting ink prepared by the preparation method according to claim 5 or 6 in the production of monascus pigment by 3D bioprinting Monascus.

8. A method for producing monascus pigment by 3D bioprinting Monascus purpurogenum, comprising the following steps: Inoculating Monascus into 3D bioprinting ink to obtain Monascus-containing ink; the 3D bioprinting ink is the 3D bioprinting ink according to any one of claims 1 to 4 or the 3D bioprinting ink prepared by the preparation method according to claim 5 or 6; The monascus pigment is obtained by 3D printing the monascus-containing ink, solidifying it into a shape, and then fermenting and culturing it.

9. The method according to claim 7, characterized in that: The concentration of Monascus spores in the Monascus ink is 0.5×10 7 ~1.5×10 7 Pieces / mL.

10. The method according to claim 7 or 8, characterized in that: The temperature of the coagulation molding is 10-15°C; the temperature of the fermentation culture is 28-32°C.

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