3D printing-based cell culture fish meat and preparation method thereof

Abstract

This invention belongs to the field of stem cell and animal cell-cultured meat technology, specifically relating to a 3D-printed cell-cultured fish meat and its preparation method. The method includes the following steps: first, stem cells are expanded through suspension culture using edible microcarriers; next, the cell-laden microcarriers are combined with 3D printing technology to perform cell stacking, forming a 3D-printed cultured fish meat construct; finally, the 3D-printed cultured fish meat is shaped to obtain the finished product. This invention provides a 3D-printed cell-cultured fish meat and its preparation method, which effectively simulates the contents of the diaphragm and sarcomeres by utilizing surface-differentiated myotubes and fat microcarriers as the main raw materials for bio-ink, greatly improving the quality of the 3D-printed cell-cultured fish meat.

Description

Technical fields:

[0001] This invention belongs to the field of stem cell and animal cell cultured meat technology, specifically relating to a 3D-printed cell cultured fish meat and its preparation method. Background technology:

[0002] In today's society, with the pursuit of healthy lifestyles, the demand for high-fat, high-cholesterol foods is gradually decreasing. Furthermore, due to the continuous growth of the global population and the depletion of marine resources, the supply of seafood is declining year by year, while prices are constantly rising. Therefore, developing a healthy and sustainable alternative to meet people's demand for seafood has become a hot topic. Cell engineering technology is one potential solution.

[0003] Cell-cultured meat is an emerging technology that utilizes cell engineering to produce fish meat by culturing cells in vitro. This method can eliminate the environmental impact of wild fisheries and aquaculture, and can also improve the quality and safety of fish meat. However, it lacks an effective method to produce products with the texture and taste of fish meat. With the development of 3D printing technology, people have begun to try using 3D printing to create food with complex shapes and structures. This technology has broad application prospects in the preparation of cell-cultured fish meat. However, existing 3D printing technologies can often only print a single type of cell-cultured meat. When printing muscle cells and fat cells simultaneously, they cannot effectively fuse them, resulting in separation of fat and muscle, which is significantly different from traditional fish meat and of poor quality. New methods are needed to improve the quality of 3D-printed cell-cultured fish meat. Summary of the Invention:

[0004] The technical problem this invention aims to solve is that existing 3D printing technology cannot effectively simulate the contents of the diaphragm and myosarcophagus of fish meat, and further optimization is needed.

[0005] To address the aforementioned issues, this invention provides a 3D-printed cell-cultured fish meat and its preparation method. By utilizing differentiated myotubes and fat microcarriers on their surfaces as the main raw materials for bio-ink, the invention effectively simulates the contents of the myodiaphragm and myosarcomere, significantly improving the quality of 3D-printed cell-cultured fish meat.

[0006] To achieve the above objectives, the present invention provides a method for preparing cell-cultured fish meat based on 3D printing, comprising the following steps:

[0007] (1) Collect cell-covered microcarriers, let them stand so that they can sink to the bottom, discard the supernatant, remove excess water from the microcarriers, add low-enzyme transglutaminase, stir thoroughly at 0-15℃ to prepare bio-ink.

[0008] (2) Import the bio-ink from step (1) into the feed tube of the 3D printer, select the printing model for printing, and then shape the fish fillet into a three-dimensional shape. Combine the cell-rich microcarrier with 3D printing technology to perform cell stacking and form a 3D printed cultured fish meat structure. The 3D printer can be an extrusion 3D printer. Extrusion 3D printers require appropriate viscosity and moisture content of the printing raw materials. Otherwise, the food material will easily collapse after extrusion or be difficult to extrude due to excessive hardness.

[0009] Furthermore, in step (1), the ratio of transglutaminase to microcarrier is 1–100:1000 (w / w), and the transglutaminase activity is 10–1500 U. The addition of transglutaminase can crosslink the 3D printed polymer, preventing subsequent collapse, and increasing chewability, creating a meat-like texture. Meanwhile, because excessively high transglutaminase activity will reduce the fluidity of the bio-ink, thus decreasing printability, using transglutaminase with low activity can prevent loss of printability during 3D printing; low-temperature bio-ink can also reduce transglutaminase activity, preventing loss of fluidity during reprinting and meeting printing requirements.

[0010] Furthermore, in step (2), the 3D printer is set with a nozzle diameter of 0.5–2.00 mm, a printing speed of 10–40 mm / s, and a printing temperature of 4–15°C. A nozzle diameter of 0.5–2 mm and a printing speed of 10–40 mm / s ensure the printing accuracy of the 3D printer; excessively high nozzle diameters and printing speeds will lead to a significant decrease in printing accuracy. The printing temperature of 4–15°C is to inhibit the activity of transglutaminase to ensure printability; higher temperatures will cause a cross-linking reaction of transglutaminase, reducing the fluidity of the bio-ink and significantly decreasing printability.

[0011] Furthermore, the printed fish fillets in step (2) are left to stand at 4℃ for 0.5-20 hours to set. Low-temperature setting can prevent the 3D printed samples from collapsing. At higher temperatures, the fluidity of the 3D printed samples increases, making them more prone to collapse.

[0012] Furthermore, in step (1), the microcarrier, the enzyme-activated transglutaminase, and natural edible proteins or polysaccharides are thoroughly mixed to prepare the bio-ink. Examples include, but are not limited to, soy protein isolate, pea protein, pumpkin protein, peanut protein, oat protein, whey protein isolate, xanthan gum, carrageenan, and gellan gum. Both polysaccharides and proteins possess natural adhesiveness and viscosity, which can increase the tension and viscosity of the ink, making it easier for the ink to be positioned and fixed during printing, thereby improving the stability and printability of the bio-ink.

[0013] Furthermore, in step (1), the ratio of muscle tissue microcarriers to adipose tissue microcarriers in the microcarriers is 1:1 to 1:9 by mass. This ratio can be customized to the ratio of cell cultured myofibril protein to fat in fish meat.

[0014] Furthermore, the preparation method of the microcarrier in step (1) includes the following steps:

[0015] (0-1) Stem cells were seeded into edible microcarriers in the first-stage bioreactor at a density of 50,000-400,000 / mL and a density of 0.5-10 mg / mL for the microcarriers.

[0016] (0-2) After the cells are inoculated in the first-stage bioreactor, they are cultured for 5-7 days with constant stirring at 10-60 rpm. After the cells are covered with microcarriers, the bioreactor is allowed to stand, the supernatant is removed, the microcarriers are washed with PBS buffer or D-Hanks buffer, and then the cells are digested with enzyme digestion solution. After centrifugation and removal of supernatant, an appropriate amount of culture medium is added to resuspend the cells and transfer them to the second-stage bioreactor to continue expansion according to the method in step (0-1).

[0017] (0-3) After the second-stage bioreactor expansion is completed, continue the scale-up to the third-stage bioreactor using the method in step (0-2). After the third-stage bioreactor culture is completed, continue the scale-up culture; or perform cell maturation and harvesting, i.e., perform myogenesis or adipogenesis in myoblastic or adipogenic differentiation medium, and obtain the microcarriers that are filled with cells as described in step (1). By using microcarriers filled with cells, uniform distribution of cells in the construct can be achieved. This is very important for the consistency of texture and taste of cell-cultured meat.

[0018] Furthermore, in step (0-1), the first-stage bioreactor is a stirred bioreactor with a volume of 50-150 mL. The culture medium volume can also be 50-150 mL. The inoculation conditions are stirring at 10-120 rpm for 1-60 min, followed by stabilization for 30-300 min, and repeated 1-15 times. Stem cells can be derived from self-isolated and cultured cells or commercially available cell lines such as zebrafish muscle satellite cell lines, rainbow trout muscle satellite cell lines, and zebrafish fat-derived stem cell lines. Edible microcarriers can be derived from self-prepared or commercially available edible microcarriers, such as the 3D-Edi.MIC microcarrier from the Qingdao Marine Food and Applied Health Innovation Research Institute.

[0019] Furthermore, in step (0-2), the enzyme digestion solution is 0.1% collagenase digestion solution or 0.25% EDTA-trypsin digestion solution by mass to volume to digest the cells, wherein the ratio of microcarrier mass to digestion solution volume is 1 mg: 0.01-5 mL, and the cells are incubated at 25-40℃ for 5-30 min to digest the cells.

[0020] Furthermore, in step (0-2), after digestion, the supernatant is removed by centrifugation at 1000-2000 rpm for 5 min.

[0021] Furthermore, the volume of the second-stage bioreactor in step (0-2) is 500-1000 mL.

[0022] Furthermore, the volume of the third-stage bioreactor in steps (0-3) is 5000-10000 mL.

[0023] In this invention, all raw materials used in the microcarrier suspension culture technology are commercially available products. The microcarrier scaffold itself must be food-grade and edible. Otherwise, separating the cells from the microcarrier would require a significant amount of additional work.

[0024] The present invention also provides cell-cultured fish meat prepared by the above method, which has a good shape and obvious and smooth appearance boundaries.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) This invention uses microcarrier suspension culture technology to expand stem cells on a large scale, and then adds the expanded microcarriers to bio-ink. The surface-differentiated myotubes and fat microcarriers are used as the main raw materials of bio-ink, which effectively realizes the simulation of the contents of myodiaphragm and myopod, and greatly improves the quality of 3D printed cell cultured fish meat.

[0027] (2) The method of the present invention utilizes 3D printing technology combined with cell engineering technology to produce high-quality, nutritious, and harmless fish products, thus replacing traditional fishery.

[0028] (3) A high-quality 3D-printed cell-cultured fish meat is provided. Compared with traditional meat products, the cell-cultured fish meat of the present invention is not only free from pollution and drug residues, but also healthier and more nutritious. It does not require traditional fishing, which can greatly reduce the pressure on marine resources and has high economic and environmental benefits. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Cell expansion curves of large yellow croaker muscle satellite cells on edible microcarriers

[0031] Figure 2 Image of live cell staining of large yellow croaker muscle satellite cells on edible microcarriers, with bright spots indicating live cells.

[0032] Figure 3 Image of 3D-printed cell-cultured fish meat in Example 2.

[0033] Figure 4 Image of 3D-printed cell-cultured fish meat in Example 3.

[0034] Figure 5 Image of 3D-printed cell-cultured fish meat in Example 4.

[0035] Figure 6 Image of 3D-printed cell-cultured fish meat in Example 5. Detailed implementation method:

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] 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.

[0042] Example 1: Large-scale preparation of stem cells based on microcarrier suspension culture

[0043] (1) Large yellow croaker muscle satellite cells were inoculated into edible microcarriers in the first-stage bioreactor. The inoculation density of stem cells was 50,000 / mL, and the density of microcarriers was 2 mg / mL. The first-stage bioreactor could be a stirred bioreactor with a volume of 125 mL and a culture medium volume of 100 mL. The inoculation conditions were: stirring at 40 rpm for 10 min, followed by 60 min of incubation, and repeated 10 times. The large yellow croaker muscle satellite cells were obtained from the cell bank of the Qingdao Marine Food and Applied Health Innovation Research Institute, etc. The edible microcarriers could be obtained from the 3D-Edi.MIC microcarriers of the Qingdao Marine Food and Applied Health Innovation Research Institute.

[0044] (2) After the cells are inoculated in the bioreactor, they are cultured for 5 days under constant stirring at 40 rpm. After the cells are covered with microcarriers, the bioreactor is left to stand for 2 min, the supernatant is removed, and the microcarriers are washed once with PBS buffer. Then, the cells are digested with 0.25% EDTA-trypsin digestion solution, where the ratio of the mass of the microcarriers to the volume of the digestion solution can be 1 mg: 0.1 mL. The cells are incubated at 37°C for 5 min to digest them. Then, the cells are centrifuged at 1500 rpm for 5 min to remove the supernatant. The cells are resuspended in an appropriate amount of culture medium and transferred to the next stage bioreactor to continue expansion according to the method in step 1. The volume of the second stage bioreactor is 500 mL.

[0045] (3) After the second-stage bioreactor expansion is completed, continue to scale up to the next-stage bioreactor using the method in step 2. The volume can be 5000 mL. After the third-stage bioreactor culture is completed, the culture medium is converted to myogenic differentiation medium for myogenesis.

[0046] The results are as follows Figure 1 As shown, the muscle satellite cells of the large yellow croaker expanded to 2.5 × 10⁻⁶ cells in 16 days. 9 Each cell, in Figure 2 As a result, the live cells of the large yellow croaker muscle satellite cells could cover the entire surface of the microcarrier after suspension culture on the microcarrier.

[0047] Example 2: 3D printing of cell-cultured fish meat

[0048] Using a food 3D printer (model LuckyBot ONE) from Nanjing Weibu 3D Technology Co., Ltd., the following method was used to prepare the bio-ink: First, collect cell-rich microcarriers. After standing for 1-2 minutes, the microcarriers will sink to the bottom, removing excess water. The ratio of muscle tissue microcarriers to adipose tissue microcarriers was 1:1 (w / w). Pea protein isolate was added to the microcarriers at a ratio of 20:1 (w / w). Transglutaminase was added at a ratio of 1:1000 (w / w) to the microcarriers, with an enzyme activity of 300U. The above materials were thoroughly stirred at 3000 rpm and 4℃ to prepare the bio-ink. The bio-ink was introduced into the feed hopper of the 3D printer. The printing model was selected, and printing was performed with a nozzle diameter of 0.5 mm, a printing speed of 20 mm / s, and a printing temperature of 15℃. After printing, the three-dimensional fish fillet was placed at 4℃ for 12 hours to set. exist Figure 3 The results show that the 3D-printed cell-cultured fish meat has a good shape and a clear and smooth appearance.

[0049] Example 3: 3D printing of cell-cultured fish meat

[0050] Cell-rich microcarriers were collected and allowed to settle to the bottom for 1-2 minutes to remove excess water. The ratio of muscle tissue microcarriers to adipose tissue microcarriers was 1:9 (w / w). Pea protein isolate was added to the microcarriers at a ratio of 20:5 (w / w), and transglutaminase was added at a ratio of 1:1000 (w / w) to microcarriers, with an enzyme activity of 300 U. The materials were thoroughly mixed at 3000 rpm and 4°C to prepare bio-ink. The bio-ink was fed into the feed hopper of a 3D printer, and the printing model was selected. The nozzle diameter was set to 0.5 mm, the printing speed to 20 mm / s, and the printing temperature to 15°C. After printing, the three-dimensional fish fillet was placed at 4°C for 12 hours to set. Figure 4 The results show that the 3D-printed cell-cultured fish meat has a good shape and a clear and smooth appearance.

[0051] Example 4: 3D printing of cell-cultured fish meat

[0052] Cell-rich microcarriers were collected and allowed to settle to the bottom for 1-2 minutes to remove excess water. The ratio of muscle tissue microcarriers to adipose tissue microcarriers was 1:1 (w / w). Pea protein isolate was added to the microcarriers at a ratio of 20:1 (w / w), and transglutaminase was added at a ratio of 1:1000 (w / w) to microcarriers, with an enzyme activity of 300 U. The materials were thoroughly stirred at 3000 rpm and 4°C to prepare bio-ink. The bio-ink was fed into the feed hopper of a 3D printer, and the printing model was selected. The nozzle diameter was set to 2 mm, the printing speed to 20 mm / s, and the printing temperature to 15°C. After printing, the three-dimensional fish fillet was placed at 4°C for 12 hours to set. Figure 5 The results show that the 3D-printed cell-cultured fish meat has a good shape and a clear and smooth appearance.

[0053] Example 5: 3D printing of cell-cultured fish meat

[0054] Cell-rich microcarriers were collected and allowed to settle to the bottom for 1-2 minutes to remove excess water. The ratio of muscle tissue microcarriers to adipose tissue microcarriers was 1:1 (w / w). Pea protein isolate was added to the microcarriers at a ratio of 20:1 (w / w), and transglutaminase was added at a ratio of 10:1000 (w / w) to microcarriers, with an enzyme activity of 150 U. The materials were thoroughly stirred at 3000 rpm and 4°C to prepare bio-ink. The bio-ink was fed into the feed hopper of a 3D printer, and the printing model was selected. The nozzle diameter was set to 0.5 mm, the printing speed to 20 mm / s, and the printing temperature to 4°C. After printing, the three-dimensional fish fillet was placed at 4°C for 5 hours to set. Figure 6 The results show that the 3D-printed cell-cultured fish meat has a good shape and a clear and smooth appearance.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing cell-cultured fish meat based on 3D printing, characterized in that... Includes the following steps: (1) Collect cell-filled microcarriers, let them stand, add low-activity transglutaminase, and stir thoroughly at 0-15°C to prepare bio-ink; the mass ratio of muscle tissue microcarriers to adipose tissue microcarriers in the cell-filled microcarriers is 1:1 to 1:9; the ratio of transglutaminase to microcarriers is 1-100:1000 by mass, and the enzyme activity of transglutaminase is 10-1500U; The method for preparing the cell-filled microcarrier includes the following steps: (0-1) Stem cells were seeded into edible microcarriers in the first-stage bioreactor at a density of 50,000-400,000 / mL and a density of 0.5-10 mg / mL for the microcarriers. (0-2) After the cells are inoculated in the first-stage bioreactor, they are cultured for 5-7 days under constant stirring at 10-60 rpm. After the cells are covered with microcarriers, the bioreactor is allowed to stand, the supernatant is removed, the microcarriers are washed with buffer, and then enzyme digestion solution is added to digest the cells. After centrifugation and removal of supernatant, an appropriate amount of culture medium is added to resuspend the cells and transfer them to the second-stage bioreactor to continue expansion according to the method in step (0-1). (0-3) After the second-stage bioreactor expansion is completed, continue to scale up to the third-stage bioreactor according to the method of step (0-2). After the third-stage bioreactor culture is completed, continue to carry out scale-up culture or cell maturation and harvesting. Cell maturation and harvesting are carried out in myoblastic or adipogenic differentiation medium for myogenic or adipogenic differentiation. (2) Import the bio-ink from step (1) into the feed tube of the 3D printer, select the printing model for printing, and after printing, the fish fillet with a three-dimensional shape will be formed.

2. The preparation method according to claim 1, characterized in that: In step (2), the 3D printer is set with a nozzle diameter of 0.5 to 2.00 mm, a printing speed of 10 to 40 mm / s, and a printing temperature of 4 to 15℃.

3. The preparation method according to claim 1, characterized in that: The printed fish fillets in step (2) are left to stand at 4℃ for 0.5-20 hours to set.

4. The preparation method according to claim 1, characterized in that: In step (0-1), the first-stage bioreactor is a stirred bioreactor with a volume of 50-150 mL and a culture medium volume of 50-150 mL. The inoculation conditions are stirring at 10-120 rpm for 1-60 min, followed by standing for 30-300 min, and repeated 1-15 times.

5. The preparation method according to claim 1, characterized in that: The enzyme digestion solution in step (0-2) is 0.1% collagenase digestion solution or 0.25% EDTA-trypsin digestion solution by mass-to-volume ratio, wherein the ratio of microcarrier mass to digestion solution volume is 1 mg: 0.01-5 mL, and incubate at 25-40℃ for 5-30 min; after digestion, centrifuge at 1000-2000 rpm for 5 min to remove the supernatant.

6. The preparation method according to claim 1, characterized in that: The volume of the second-stage bioreactor in step (0-2) is 500-1000 mL; the volume of the third-stage bioreactor in step (0-3) is 5000-10000 mL.

7. A cell-cultured fish meat prepared by any one of claims 1-6.

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