A 3D printing-based kombucha starter culture, and a preparation method and application thereof

Abstract

The present application belongs to the field of biotechnology engineering, and particularly relates to a kombucha fermentation agent based on 3D printing and a preparation method and application thereof. Based on the 3D printing technology, a hydrogel type kombucha fermentation agent is developed, which can be directly put into the kombucha water for fermentation, and through supplementing the dominant yeast bacteria lacking in the later fermentation stage, continuous inoculation for multiple times can be realized. The 3D printing design model is redesigned and improved, the kombucha fermentation agent containing yeast bacteria is prepared, the service life of the kombucha fermentation agent for continuous use is prolonged, and the good fermentation performance is maintained. The present application can form a microbial consortium in the hydrogel inside the microbial population of kombucha, can solve the problems of loss of bacterial film bacterial liquid, lack of standardized inoculation amount and the like in the traditional kombucha inoculation mode. The present application has the advantages of high fermentation efficiency, reusability, freeze-drying long-term preservation, easy storage and low cost, and has great application prospect for the commercialization of kombucha.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology engineering, and particularly relates to a kombucha fermentation agent based on 3D printing and a preparation method and application thereof. BACKGROUND

[0002] Kombucha is a traditional beverage, which is fermented by green tea, black tea water or a mixture of the two with sugar, and its chemical composition includes sugar, organic acids, alcohol, vitamins, amino acids, minerals, and particularly contains antioxidant molecules such as catechins. Due to its potential health benefits, kombucha has become increasingly popular in recent years, and the development of kombucha drinks at home and abroad is rapid. With the increasing public concern about the health benefits of kombucha, the market demand for kombucha is also increasing year by year.

[0003] At present, the industrialization level of kombucha fermentation is relatively low, and the fermentation mode mainly uses bacterial film and bacterial liquid for inoculation. Kombucha bacterial liquid is the main product, and the main component of bacterial film is bacterial cellulose, which has good application prospect. The traditional fermentation mode needs to consume bacterial liquid and bacterial film, so the actual yield is low. Based on this situation, some researchers have developed a new type of kombucha direct-vat fermentation agent, the main component of which is freeze-dried bacterial slurry. Although this mode improves the concentration of live bacteria in the fermentation agent, it has the disadvantage of complex preparation process and cannot be reused. In addition, kombucha is fermented by a complex microbial community, and the growth and reproduction of microorganisms is not easy to control in liquid fermentation. If there is no complex genetic control system or specific nutrient conditions, repeated batch liquid co-culture will usually fail due to changes in the structure of the microbial community over time. Traditional fermentation of kombucha requires bacterial film and bacterial liquid, which means that this production method is not easy to carry and transport. Therefore, it is very important to develop a kombucha fermentation agent that does not consume bacterial film and bacterial liquid, is efficient and low-cost, can be reused, and is convenient to carry, transport and store, which will help to improve the production efficiency of kombucha, increase by-products, and provide a certain theoretical basis for its industrial production.

[0004] At present, 3D printing technology is mainly applied in the medical and biological fields. Its application in the food field is less, and mainly focuses on direct printing of food such as chocolate, meat, milk protein and cheese. Therefore, it may be a feasible new idea to embed kombucha microorganisms in biological ink and then use 3D printing technology to make fermentation agents. The key point is how to associate the design of 3D printing model with the fermentation performance of kombucha. The present application aims to improve the fermentation performance of hydrogel by exploring the interaction between hydrogel and the microorganisms contained therein and designing a 3D model. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a kombucha fermentation agent based on 3D printing and a preparation method and application thereof to solve the problems of the prior art.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of a kombucha fermentation agent based on 3D printing, characterized in that it comprises the steps of preparing a kombucha fermentation agent I and preparing a yeast fermentation agent.

[0008] The step of preparing the kombucha fermentation agent I comprises:

[0009] (I1) After the kombucha fermentation, the precipitate is obtained by centrifugation, and the kombucha cell body is diluted to prepare a kombucha cell suspension;

[0010] (I2) In the aqueous solution of the modified polyether, the photoinitiator and the kombucha cell suspension prepared in step (I1) are added to prepare a biological ink A;

[0011] (I3) The biological ink A prepared in step (I2) is added to the 3D printer ink tank at -20℃ to 4℃, heated to convert the biological ink A from a liquid state to a gel state, and then 3D printing is performed according to the grid model. The printed model is cured under the absorption wavelength of the photoinitiator to prepare a kombucha hydrogel, i.e., the kombucha fermentation agent I;

[0012] The step of preparing the yeast fermentation agent comprises:

[0013] (I4) After the yeast fermentation, the yeast cell body is obtained by washing the colony with sterile water, and the yeast cell body is diluted to prepare a yeast cell suspension;

[0014] (I5) In the aqueous solution of the modified polyether, the photoinitiator and the yeast cell suspension prepared in step (I4) are added to prepare a biological ink B;

[0015] (I6) The biological ink B prepared in step (I5) is added to the 3D printer ink tank at -20℃ to 4℃, heated to convert the biological ink B from a liquid state to a gel state, and then 3D printing is performed according to the grid model. The printed model is cured under the absorption wavelength of the photoinitiator to prepare a yeast hydrogel, i.e., the yeast fermentation agent.

[0016] A preparation method of a kombucha fermentation agent based on 3D printing, characterized in that it comprises the steps of preparing a kombucha fermentation agent II.

[0017] The step of preparing the kombucha fermentation agent II comprises:

[0018] (II1) after the fermentation of the black tea fungus, the precipitate is obtained by centrifugation, and the black tea fungus is obtained by diluting the black tea fungus; after the fermentation of the yeast, the yeast is obtained by washing the colony with sterile water, and the yeast suspension is prepared by diluting the yeast;

[0019] (II2) in the aqueous solution of the modified polyether, the photoinitiator and the black tea fungus suspension prepared in step (II1) are added to prepare the bio-ink C;

[0020] (II3) in the aqueous solution of the modified polyether, the photoinitiator and the yeast suspension prepared in step (II1) are added to prepare the bio-ink D;

[0021] (II4) the bio-ink C prepared in step (II2) and the bio-ink D prepared in step (II3) are added to the two ink tanks of a double-nozzle 3D printer at-20℃-4℃, the bio-ink C and the bio-ink D are heated to change from liquid to gel state, then the 3D printing is carried out according to the grid model, the printed model is cured under the absorption wavelength of the photoinitiator to prepare the black tea fungus-yeast hydrogel, i.e. the black tea fungus starter II.

[0022] The black tea fungus is not limited to a specific classification of black tea fungus, and the black tea fungus in the prior art is suitable for the present application. In some embodiments of the present application, the black tea fungus is purchased from the Taobao store: Linzhi Natural Saussurea.

[0023] The yeast is not limited to a specific classification of yeast, and the yeast capable of food fermentation in the prior art is suitable for the present application, such as any one or combination of several of the yeast in Zygosaccharomyces, Pichia, Brettanomyces, Torulopsis, Saccharomycodes, Candida, Torulaspora, Dekkera, Schizosaccharomyces, Saccharomyces and Lachancea. In some embodiments of the present application, the yeast is the dominant yeast Yeast2 separated from the black tea fungus stock solution, which is identified as Lachancea fermentati and belongs to Lachancea.

[0024] In steps (I1) and (II1), the dilution ratio of the black tea fungus to water is 1:4-16, preferably 1:8; the viable count of the black tea fungus suspension is ≥1×10 7CFU / ml.

[0025] In step (I2) and step (II2), the mass ratio of the red tea fungus suspension to the modified polyether aqueous solution is 5-40%, preferably 10%.

[0026] In step (I2), step (I5), step (II2) and step (II3), the modified polyether aqueous solution is prepared by dissolving modified polyether in sterile water, and the mass percentage of modified polyether is 20-35wt%, preferably 30wt%; the volume ratio or mass ratio of the photoinitiator to the modified polyether aqueous solution is 0.1%-1%, preferably 0.1%.

[0027] Specifically, the modified polyether is F127-B, which is modified from F127 with an average molar mass of 12600g / mol.

[0028] Specifically, the photoinitiator includes ultraviolet light initiator or visible light initiator, preferably 2-hydroxy-2-methyl benzene propyl ketone in ultraviolet light initiator.

[0029] In step (I4) and step (II1), the dilution ratio of the yeast cell to water is 1:4-16, preferably 1:8; the viable cell count of the yeast suspension is ≥1×10 7 CFU / ml.

[0030] In step (I5) and step (II3), the mass ratio of the yeast suspension to the modified polyether aqueous solution is 5-40%, preferably 10%.

[0031] In step (I3), the grid model is a double-layer grid structure, a plurality of parallel cuboids are designed first, and the cuboids are connected by short cuboids to form the first layer of the double-layer grid structure, then the first layer is copied and rotated 90° clockwise or counterclockwise to be stacked on the first layer as the second layer; wherein the first layer and the second layer both embed the red tea fungus.

[0032] In step (II4), the grid model is a four-layer grid structure. A plurality of parallel cuboids are designed first, and the cuboids are connected by short cuboids to form a first layer of the four-layer grid structure. Then the first layer is copied, and the copied layer is stacked on the first layer as a second layer after being rotated 90° clockwise or counterclockwise. Then the second layer is copied, and the copied layer is stacked on the second layer as a third layer after being rotated 90° in the same direction. Finally, the third layer is copied, and the copied layer is stacked on the third layer as a fourth layer after being rotated 90° in the same direction. The first layer and the fourth layer are used to embed the yeast, and the second layer and the third layer are used to embed the red tea bacteria, so that the yeast is located at the outermost side of the model, thereby avoiding the model being blocked by the produced bacterial cellulose and the channels for releasing the microorganisms being blocked.

[0033] The red tea bacteria starter I prepared by the above method is also within the scope of the present application.

[0034] The application of the prepared red tea bacteria starter I and yeast starter in the fermentation preparation of red tea bacteria is also within the scope of the present application.

[0035] Specifically, the red tea bacteria starter I is directly inoculated into red tea water at a mass ratio of 1% to 10%, and after fermentation at 25°C to 30°C for 8 to 10 days, the red tea bacteria starter I is taken out, washed with sterile water, and stored in a red tea bacteria stock solution with pH≤3, to complete the first round of fermentation of the red tea bacteria. The red tea bacteria starter I taken out in the first round of fermentation is inoculated again, and after fermentation at 25°C to 30°C for 8 to 10 days, the red tea bacteria starter I is taken out, washed with sterile water, and stored in a red tea bacteria stock solution with pH≤3, to complete the second round of fermentation of the red tea bacteria. The second round of fermentation is repeated for 4 to 8 times for continuous fermentation. When the total acid content of the red tea bacteria after the continuous fermentation is less than 2g / L for two rounds, the yeast starter is inoculated at a mass ratio of 1% to 10%, and after reaction at 25°C to 30°C for 1 to 2 hours, the red tea bacteria starter I is taken out, inoculated again, and the above continuous fermentation process is repeated. When the total acid content of the red tea bacteria after the continuous inoculation and fermentation is less than 2g / L for two rounds, the fermentation is ended.

[0036] The red tea bacteria starter II prepared by the above method is also within the scope of the present application.

[0037] The application of the prepared red tea bacteria starter II in the fermentation preparation of red tea bacteria is also within the scope of the present application.

[0038] Specifically, the black tea starter II is inoculated into black tea water at a mass ratio of 1% to 10%, and after fermentation at 25 DEG C to 30 DEG C for 8 to 10 days, the black tea starter II is taken out and stored in the black tea starter stock solution with pH less than or equal to 3, and the first round of fermentation of the black tea is completed, and the black tea starter II taken out in the first round of fermentation is inoculated continuously, and after fermentation at 25 DEG C to 30 DEG C for 8 to 10 days, the black tea starter II is taken out and stored in the black tea starter stock solution with pH less than or equal to 3, and the second round of fermentation of the black tea is completed, and the second round of fermentation is repeated to perform continuous fermentation, and when the total acid content of the fermented black tea is less than 2g / L for two consecutive rounds, the fermentation is completed.

[0039] Beneficial effects:

[0040] 1. The present application is based on 3D printing technology, and a hydrogel type black tea starter is developed, and the fermentation process and parameters of the fermentation for preparing the black tea are optimized, the problems of the current traditional fermentation of the black tea, such as the loss of the bacterial liquid and bacterial membrane caused by inoculation, high transportation cost and the like are solved, and better fermentation effect is obtained, and the application prospect of the commercial market of the black tea is great.

[0041] 2. The hydrogel type black tea starter prepared in the present application is resistant to the low pH environment of the black tea, has good mechanical strength and biocompatibility, and can be used for continuous inoculation for at least 13 times.

[0042] 3. The present application prolongs the service life of the black tea starter for continuous use and maintains good fermentation performance by supplementing the yeast bacteria lacking in the late fermentation period in time or by improving the 3D printing design model to prepare the black tea starter containing the yeast bacteria.

[0043] 4. Compared with the traditional fermentation, the present application not only can be repeatedly used to reduce the loss and improve the yield, but also can utilize the variability of the 3D printing technology and the structural complexity of the hydrogel preparation to control the morphological structure of the starter and artificially control the distribution of the microbial community in space, so that the customized production effect is achieved.

[0044] 5. The grid-shaped hydrogel prepared by 3D printing has better live bacteria releasing capacity, and can complete the continuous inoculation of the black tea faster. BRIEF DESCRIPTION OF DRAWINGS

[0045] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0046] Figure 1 F127-B synthesis flow chart.

[0047] Figure 2F127-B shear thinning results

[0048] Figure 3 F127-B recovery test

[0049] Figure 4 F127-B cytotoxicity test

[0050] Figure 5 F127-B hydrogel extrusion test

[0051] Figure 6 F127-B hydrogel tensile recovery test

[0052] Figure 7 F127-B hydrogel tensile fracture test

[0053] Figure 8 Stress comparison at tensile fracture of different types of hydrogels

[0054] Figure 9 Acid resistance test of different types of hydrogels (significance difference represented by different letters, P<0.05)

[0055] Figure 10 3D model and slice results of hydrogels (a: CAD model; b: slice results)

[0056] Figure 11 Hydrogels prepared in different ways (a: spherical shape made by mold; b: cylindrical shape made by mold; c: 3D printed mesh)

[0057] Figure 12 Live bacteria number released by different shape hydrogels (Note: significant difference represented by different letters, P<0.05)

[0058] Figure 13 Shear thinning characteristics under different proportions of Kombucha suspension

[0059] Figure 14 Recovery test under different proportions of Kombucha suspension

[0060] Figure 15 pH of fermented Kombucha under different proportions of Kombucha suspension

[0061] Figure 16 Total acid and brix of fermented Kombucha under different proportions of Kombucha suspension

[0062] Figure 17 Changes of corresponding indicators of Kombucha hydrogel continuously fermented Kombucha (a: pH; b: total acid; c: brix)

[0063] Figure 18 Changes of pH of Kombucha hydrogel continuously fermented to the limit life

[0064] Figure 19 Changes in total acid content of kombucha hydrogel during continuous fermentation to the limit of life

[0065] Figure 20 Changes in sugar content of kombucha hydrogel during continuous fermentation to the limit of life

[0066] Figure 21 State of kombucha hydrogel fermented for different times

[0067] Figure 22 Results of live bacteria released by hydrogel at different stages(**indicates extremely significant difference, P < 0.01)

[0068] Figure 23 Cross-sectional view of kombucha hydrogel after 8 rounds of continuous use(a: 100 μm scale; b: 20 μm scale)

[0069] Figure 24 Laser confocal imaging of bacterial population in kombucha hydrogel at different stages(a: after 4 rounds of use; b: after 8 rounds of use)

[0070] Figure 25 Swelling capacity test of kombucha hydrogel at different stages

[0071] Figure 26 pH and total acid content after continuous fermentation with added yeast bacteria(different letters indicate significant difference, P < 0.05)

[0072] Figure 27 Sugar content and live bacteria count after continuous fermentation with added yeast bacteria(different letters indicate significant difference, P < 0.05)

[0073] Figure 28 Schematic diagram of four-layer grid model(a: CAD model; b: sliced model; c: printed physical object)

[0074] Figure 29 Changes in pH and total acid content after continuous fermentation with improved model

[0075] Figure 30 Changes in sugar content after continuous fermentation with improved model DETAILED DESCRIPTION

[0076] In the following examples, the experimental methods described are routine methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0077] In the following examples, the kombucha described is commercially available, and the kombucha used in the present application is purchased from a Taobao store: Linzhi Natural Saussurea Mushroom.

[0078] In the following examples, the Kombucha fermentation broth is obtained by inoculating commercially available Kombucha into black tea water (obtained by adding 0.9% m / v of black tea and 10% m / v of sucrose to water, boiling and removing the tea residue, and then cooling to room temperature) at a fermentation temperature of 30°C for 7-8 days, with an inoculation amount of 3% v / v of Kombucha broth and 5% v / v of Kombucha film.

[0079] In the following examples, the PCA medium and the Malachite Green medium are purchased from Guangdong Huan Kai Microbial Science and Technology Stock Company Limited.

[0080] Preparation and evaluation test of modified polyether (F127-B) of Example 1

[0081] 1.1 Preparation of modified polyether (F127-B)

[0082] The glassware required for synthesis was dried in a drying oven for 24 h to ensure water-free. 60 g of F127 polymer (average molar mass of 12600 g / mol, purchased from Sigma Aldrich, item number P2443) was weighed and transferred to a 1 L round-bottom flask. 550 ml of dichloromethane was added. After waiting for the F127 to completely dissolve, 12 drops of dibutyltin dilaurate were added with a Pasteur pipette. 50 ml of dichloromethane was measured with a graduated cylinder and poured into a beaker. 3.5 ml of isocyanatoethyl methacrylate was taken with a pipette and added to the beaker. After mixing well, the mixture was added dropwise to the F127 solution with a dropper. The final mixed solution was flushed with N2 for protection. The flask was placed in a 30°C water bath shaker and reacted at a speed of 100 r / min for 2 d. After the reaction was completed, 60 ml of methanol was added to terminate the reaction. A rotary evaporator was used to concentrate the solution at a temperature of 30°C, and the solution was concentrated to about 20 ml. 2000 ml of diethyl ether was poured into the concentrated solution, and a magnetic stirrer was used to stir for 15 min to mix well. The mixed solution was centrifuged at a speed of 5000 rpm / min, and the supernatant was removed and the precipitate was washed twice with diethyl ether. The final precipitate was placed in a fume hood to volatilize the diethyl ether, and the final polymer powder (F127-B) was obtained, dried and stored in a 4°C refrigerator. The preparation process is shown in the schematic diagram Figure 1 .

[0083] 1.2 Printability test

[0084] F127-B was added to deionized water at specific proportions (20 wt%, 25 wt%, 30 wt%, 35 wt%) and placed in a 4°C refrigerator overnight to completely dissolve. A hybrid rheometer was used to test the rheological properties, and all tests used rotary shear. The shear thinning characteristics used a speed range of 0.01 s -1 -1000 s -1, test the viscosity change under different shear rate; test the recovery of the material by shear rate and viscosity change, first use 0.01 s -1 shear rate to extrude the sample for 200 s, then use 895 s -1 shear rate to process for 200 s, finally use 0.01 s -1 shear rate to process for 200 s, detect the viscosity change during the process.

[0085] The experimental results of shear thinning property test are shown in Figure 2 . The experimental results show that the bio-ink prepared by dissolving F127-B powder in different proportions all exhibit obvious shear thinning property, the viscosity is 10 4 Pa·s-10 5 Pa·s at low shear rate, the viscosity decreases by one order of magnitude while the shear rate increases by one order of magnitude, and the material can exhibit this property in the shear rate range of 10 -2 -10 3 s -1 , which shows that the shear thinning adaptation range of the material is wide, which meets the requirements of 3D printing.

[0086] After the bio-ink material has shear thinning property, it also needs to have certain recovery ability. The experimental results are shown in Figure 3 . The results show that the viscosity of the 30wt% treatment group is the largest at low shear rate, after the shear rate increases, the viscosity of all treatment groups decreases rapidly, after the shear rate decreases, all treatment groups quickly recover to high viscosity state, except for the 25wt% treatment group, the viscosity of other treatment groups decreases to a certain extent compared with that before high shear rate treatment. The viscosity of 25wt%, 30wt% and 35wt% treatment groups is relatively larger, and the recovery ability of 20wt% treatment group is poor. Because the bacterial solution needs to be added when preparing hydrogel later, although the recovery of 25wt% treatment group is good, the printability may be reduced after dilution; some studies show that high concentration of bio-ink will make the prepared hydrogel highly cross-linked, which will lead to lower cell survival rate, therefore, the 30wt% treatment group is selected as the F127-B dissolving formula because its viscosity is the largest and the concentration is moderate.

[0087] 1.3 Cytotoxicity test

[0088] To test the toxicity of F127-B to kombucha microorganisms, three treatments were administered: (1) A (30 wt% F127-B aqueous solution) + B (0.1% photoinitiator) + 10% kombucha fermentation broth; (2) A (30 wt% F127-B aqueous solution) + B (0.1% photoinitiator) + 10% kombucha fermentation broth + UV irradiation for 1 min; (3) kombucha fermentation broth as the control group. The three treatments were diluted and viable cells were counted using PCA medium and Red Tiger Broth (purchased from Guangdong Huankai Microbial Technology Co., Ltd.).

[0089] F127-B aqueous solution was prepared according to the formula determined in section 1.2, and treated with photoinitiator and kombucha fermentation broth for 1 day. The experimental results are shown in [Figure 1]. Figure 4 The results showed that there was no significant difference between the treatment with F127-B and the control group. After UV irradiation to form hydrogel, the number of viable bacteria in the hydrogel was also not significantly different from the control group. Therefore, it can be concluded that F127-B has no biological toxicity to kombucha.

[0090] 1.4F127-B Hydrogel Strength Test

[0091] F127-B powder was prepared into an aqueous solution at a ratio of 30 wt%. 0.1% 2-hydroxy-2-methylphenylacetone was added as a photoinitiator. The mixture was thoroughly mixed at 4°C and poured into a cylindrical mold. The solution was allowed to solidify at room temperature and then irradiated with 365 nm UV light for 1 min to form a hydrogel. The compressive properties were tested using a property analyzer at a constant compression rate of 5 mm / min. The compressive strength was calculated using the following formula: σ = P / πr 2 (Where: P is the maximum load in N; r is the cylinder radius in mm). F127-B solid powder was dissolved in water (30 wt%), a photoinitiator was added, and the solution was poured into a corresponding mold. The film was then irradiated with 365 nm UV light for 1 min to create a film with a thickness of 1 mm, a width of 1 cm, and a length of 2 cm. The film was fixed using a TMS-Pro texture analyzer (Food Technology Corporation, USA) with the following parameters: stretching rate of 25 mm / min and initial force of 0.049 N. The first experiment measured the hydrogel's ductility and recovery of deformation, with a stretching range of 150%. The second experiment tested the absolute strength of the hydrogel at fracture.

[0092] The experimental results are shown in Figures 5 to 8 . Figure 5 The results showed that the F127-B hydrogel reached a stress of 0.76 MPa under 90% deformation, and no obvious damage was observed by the naked eye. The hydrogel could recover its deformation after the pressure was released, which indicates that it has excellent compressive strength. Figure 6The results show that with the increase of tensile deformation, the stress increases, and when the deformation reaches 150%, the stress reaches 200KPa + ; in the cycle of restoring deformation, with the decrease of strain, the stress decreases, but compared with the tensile time, the stress of the same deformation is smaller, which shows that the tensile in the ductility range will cause the loss of energy. The Young's modulus of F127 hydrogel is higher than that of general protein cross-linked muscular hydrogel, that is, it has good strength and ductility. From Figures 3-7 It can be seen that the fracture occurs when the deformation is stretched to 220%. The ultimate stress is 285kPa. Figures 3-8 The comparison of the stress and strain of F127 hydrogel fracture with other kinds of hydrogels shows that the greater the fracture stress, the smaller the strain, that is, the harder and more brittle it is; F127 has a larger strain and a smaller fracture stress, which shows that the material has strong toughness and can cope with a certain degree of stretching after being made into a leavening agent. In summary, the F127-B material has good mechanical properties, mainly reflected in the compression resistance, large tensile deformation range and deformation recovery.

[0093] 1.5 F127-B hydrogel acid resistance test

[0094] The preparation method of F127-B hydrogel is the same as 1.4. Common sodium alginate and chitosan hydrogel are used as controls. Prepare sodium alginate-calcium chloride hydrogel: prepare 1% sodium alginate solution and 2% calcium chloride solution, and mix them to prepare. Prepare chitosan-glutaraldehyde hydrogel: weigh 1g chitosan and dissolve it in 40ml, 2% concentration acetic acid solution, add 16ml, 2% concentration glutaraldehyde, stir, and heat at 50℃ for 1h to prepare. Put F127 hydrogel, sodium alginate-calcium chloride hydrogel, and chitosan-glutaraldehyde hydrogel into the original liquid of black tea bacteria (pH=2.55) for immersion, weigh the remaining mass of the hydrogel at intervals, and calculate the mass ratio.

[0095] The prepared hydrogel needs to be used as a direct inoculation of tea soup, and the pH of the black tea bacteria will decrease to about 3 during the fermentation process. Strong acidity may damage the hydrogel, so the hydrogel needs to be tested. The experimental results are shown in Figure 9 , the results show that the F127-B hydrogel has no significant change in mass after being immersed in an environment with pH=2.55 for 60min; the mass of the sodium alginate and chitosan prepared hydrogel decreases continuously and will be destroyed in a strong acidic environment. In summary, the F127-B hydrogel is relatively stable in the acidic environment of black tea bacteria and will not be easily degraded, meeting the requirements of a leavening agent.

[0096] 1.6 F127-B hydrogel model preparation and live bacteria release capacity test

[0097] Based on the concept, a CAD model was designed using 123Ddesign software. The model was then sliced ​​using Slic3r software and converted into G-code commands recognizable by the 3D printer to generate the printing path. Printing parameters were set as follows: a 1mm nozzle, a printing temperature of 25℃, and a printing speed of 20mm / s. F127-B bio-ink (30wt% F127-B aqueous solution + 0.1% photoinitiator + 10% kombucha fermentation broth) was prepared and added to the ink cartridge of a FOODBOT-D1 3D printer (purchased from Hangzhou Shiyin Technology Co., Ltd.). Printing was then performed according to the set model. The printed model was irradiated with 365nm UV light for 1 minute, ultimately forming a hydrogel. Hemispherical (0.75cm high, 1.5cm diameter) and cylindrical (0.6cm high, 1.5cm diameter) hydrogel models were prepared using molds and bio-ink. A double-layer mesh-like hydrogel model was fabricated using 3D printing technology. The model consisted of seven parallel cuboids connected by shorter cuboids, forming the first layer of the double-layer mesh structure. This first layer was then copied, rotated 90° to the right, and superimposed on top of the first layer to form the second layer. The mesh dimensions were 15.6 mm in length and width, and 2 mm in height. Figure 10 After cleaning the 3D printed model and the model prepared by the mold with sterile water, they were placed in sterile water and placed in a shaker at 30°C and 100 r / min for 1 day. The live bacteria released by different models were counted.

[0098] Physical samples of the three models are available. Figure 11 The viable bacterial release capacity of three different hydrogel shapes was tested by immersing them in sterile water. The experimental results are shown below. Figures 3-12 The results showed that the mesh-like hydrogel had a significantly higher ability to release live bacteria than the other two groups, increasing the release by one order of magnitude, with 7.91 × 10⁻⁶ fungi released. 6 CFU / g, bacteria count reached 6.76 × 10⁻⁶. 6 CFU / g. For the same volume, the mesh-like structure has a larger surface area than the regular shape. The experimental results demonstrate the advantages of 3D printing technology in creating complex structures. Therefore, this model was selected as the hydrogel template for subsequent experiments.

[0099] Example 2: Optimization of kombucha cell dilution ratio and formulation

[0100] 2.1 Rheological tests of kombucha hydrogels with different formulations

[0101] Using the kombucha fermentation broth after 8 days of fermentation, centrifuged at 10000 rpm for 8 minutes at pH ≤ 3, the supernatant was removed and the precipitate was collected. The kombucha cells were then diluted with water at a ratio of 1:8 to prepare a kombucha suspension (viable count ≥ 1 × 10⁻⁶). 7F127-B bio-inks were prepared according to formulations of 30 wt% F127-B aqueous solution + 0.1% photoinitiator (2-hydroxy-2-methylphenylacetone) + 10% / 20% / 30% / 40% kombucha suspension, respectively. Their rheological properties and printability were tested using the same methods as in section 1.2. The optimal mixing ratio was selected based on the rheological properties.

[0102] The results of shear thinning are shown in Figure 13 The results showed that all treated bio-inks exhibited significant shear-thinning behavior, with the control group without bacterial solution showing the largest viscosity change, reaching a viscosity of 10 at low shear rates. 4 -10 5 For every order of magnitude increase in shear rate (Pa·s), the viscosity decreases by an order of magnitude. Experimental results for the resilience test are shown below. Figure 14 The results showed that the control group had the highest viscosity at low shear rates, while the other treatment groups had lower and similar viscosities. When the shear rate suddenly increased, the viscosity of all treatment groups decreased. When the shear rate recovered, except for the treatment group with 10% added amount, the other treatment groups did not recover to the initial viscosity. Therefore, 10% kombucha suspension was selected as the final ratio.

[0103] 2.2 Fermentation capacity test of kombucha suspension at different dilution concentrations for preparing hydrogels

[0104] Using the kombucha fermentation broth after 8 days of fermentation, centrifuged at 10000 rpm for 8 minutes at pH ≤ 3, the supernatant was removed, the precipitate was collected, and the kombucha cells were diluted with water at ratios of 1:4, 1:8, and 1:16 to prepare kombucha suspensions (viable count ≥ 1 × 10⁻⁶). 7 (CFU / ml). A bio-ink was prepared by mixing 30 wt% F127-B aqueous solution, 0.1% photoinitiator (2-hydroxy-2-methylphenylacetone), and 10% kombucha suspension. A hydrogel starter was prepared based on the model determined in section 1.6. The inoculum was inoculated at a 2% v / v rate into black tea water (0.9% m / v black tea, 10% m / v sucrose, boiled, tea residue removed, and cooled to room temperature). The mixture was placed in a 30℃ incubator for 8 days of fermentation. During this period, pH (measured using a handheld pH meter), total acid (measured using NaOH titration), and sugar content (measured using a VBR32 saccharimeter) were determined, and the optimal dilution concentration was selected based on the results.

[0105] The results show that after printing, the 1:4 dilution treatment group cannot be normally coagulated and shaped, the 1:8 and 1:16 dilution treatment groups can be shaped, because F127-B depends on UV irradiation crosslinking to coagulate into hydrogel, the 1:4 treatment group may fail to crosslink because the bacterial concentration is too high, resulting in poor light transmittance, therefore the 1:8 and 1:16 treatment groups are subjected to fermentation test. The indicators during fermentation are shown in Figure 15 and Figure 16 The results show that the black tea fungus hydrogel prepared by dilution at 1:8 has a lower relative pH and reaches 2.88 after 8 days of fermentation; a higher total acid content, reaching 3.8274 g / L; and a lower sugar content, reaching 8.3 after 8 days. In summary, 1:8 is selected as the dilution ratio of the bacterial body after centrifugation.

[0106] Example 3: Continuous fermentation test of black tea fungus hydrogel (and black tea fungus starter) and evaluation test of various indicators

[0107] 3.1 Test of continuous fermentation capacity of black tea fungus hydrogel

[0108] The black tea fungus fermentation liquid after 8 days of fermentation is centrifuged at a speed of 10000 rpm / min for 8 min, the supernatant is removed, the precipitate is taken out, the bacterial body is collected, and then diluted according to the water ratio of 1:8 to prepare a black tea fungus suspension (viable bacterial count ≥ 1 × 10 7 CFU / ml). The bio-ink is prepared according to the ratio of 30wt% F127-B aqueous solution + 0.1% photoinitiator (2-hydroxy-2-methylpropiophenone) + 10% black tea fungus suspension, and the black tea fungus hydrogel is prepared according to the double-layer grid model determined in 1.6 as the black tea fungus starter. The inoculation amount is 2% v / v, which is inoculated into the black tea water and placed in a 30°C incubator for 8 days of fermentation, which is regarded as 1 round of fermentation, then the hydrogel is washed with sterile water, and the tea soup is continuously inoculated, and this is repeated for 6 rounds. The measured pH (determined by using a handheld pH meter), total acid (determined by using NaOH titration method) and sugar content (determined by using VBR32 sugar content meter) during fermentation are detected.

[0109] The continuous fermentation is carried out for 6 rounds, and the corresponding indicators are shown in Figure 17 The results show that the pH is lower, the total acid content is relatively higher, and the sugar content is lower in the continuous fermentation compared with the traditional fermentation, and there is no obvious sign of decline in the continuous fermentation process, which indicates that the hydrogel as the starter has a certain reuse ability.

[0110] 3.2 Service life test of black tea fungus hydrogel

[0111] The fermented liquid of red tea bacteria after 8 days of fermentation was used, centrifuged at 10000 rpm / min for 8 min at pH≤3, the supernatant was removed, the precipitate was collected, and the bacterial suspension (viable bacteria≥1×10 7 CFU / ml) was prepared according to 1:8 water ratio. According to the ratio of 30wt% F127-B aqueous solution+0.1% photoinitiator (2-hydroxy-2-methylpropiophenone)+10% red tea bacteria suspension, the bio-ink was prepared, and the red tea bacteria hydrogel was prepared according to the double-layer grid model determined in 1.6 as the red tea bacteria starter. Inoculated in red tea water at an inoculation amount of 2% v / v, placed in a 30℃ incubator for fermentation for 8d, regarded as 1 round of fermentation, then the red tea bacteria hydrogel was washed with sterile water to remove the microorganisms attached to the surface, and was continuously put into the red tea water for fermentation. When the total acid content of the red tea bacteria after fermentation was less than 2.0g / L for two consecutive rounds, it was considered to have reached the service life. During continuous use, the pH (determined by a handheld pH meter), total acid (determined by NaOH titration method) and brix (determined by VBR32 brix meter) were measured.

[0112] The corresponding indicators of the whole stage are shown in Figures 18 to 20 , and the state of the red tea bacteria hydrogel at different stages is shown in Figure 21 . The results show that the normal service life of the red tea bacteria hydrogel is 6 rounds of continuous fermentation. After 6 rounds of continuous fermentation, the pH increases significantly and the total acid content decreases significantly. After 6 rounds of fermentation, the pH is 3.05, and after 7 rounds and 8 rounds of fermentation, the pH is 3.35 and 3.36 respectively. After 6 rounds of fermentation, the total acid content is 2.6511g / L, and after 7 rounds and 8 rounds of fermentation, the total acid content is 1.6207g / L and 1.6204g / L respectively. The brix also increases significantly to 8.9 and 8.8 respectively after 7 rounds and 8 rounds of fermentation. With the increase of fermentation times, the color of the red tea bacteria hydrogel gradually deepens from light yellow to brown after 4 rounds of fermentation, and to black after 8 rounds of fermentation. Moreover, the volume of the hydrogel significantly decreases after 8 rounds of fermentation.

[0113] 3.3 Release of viable bacteria by red tea bacteria hydrogel at different stages

[0114] The number of fungi and bacteria in the red tea bacteria hydrogel at the 0th round, 2nd round, 4th round and 8th round in the service life test of the red tea bacteria hydrogel in 3.2 was determined by colony counting method.

[0115] The experimental results are shown in Figure 22 . The results show that the ability of the red tea bacteria hydrogel to release bacteria is continuously increasing, from the initial 7.55×10 5 CFU / g to 1.32×10 12 CFU / g; the ability of the red tea bacteria hydrogel to release fungi reaches the highest after two rounds of use, which is 1.91×10 7CFU / g, then began to decrease, and the final release value was 1.23 x 10 3 CFU / g; there was no significant difference in the release of fungi and bacteria between the freshly prepared and the 4th cycle Kombucha hydrogel, but there was a significant difference in the release of live bacteria between the 6th and 8th cycle Kombucha hydrogel. In summary, after continuous fermentation, the release of fungi by Kombucha hydrogel decreased significantly, and the release of bacteria increased.

[0116] 3.4 Scanning electron microscope observation of Kombucha hydrogel structure reaching the service life

[0117] The Kombucha hydrogel reaching the service life was placed in liquid nitrogen for 10 min, then cut open and placed in a vacuum freeze dryer for 2 days. Finally, the surface and internal structure were observed using an electron scanning microscope.

[0118] The experimental results are shown in Figure 23 . The results show that there are a large number of fibrous debris in the Kombucha hydrogel, which is speculated to be bacterial cellulose produced by acetic acid bacteria in Kombucha. The outer layer of the Kombucha hydrogel presents a foam-like state, still maintaining a porous state, and the internal structure appears more compact, with a large number of pores being blocked. The densification of the structure of the hydrogel is directly related to the decrease in the ability to release live bacteria.

[0119] 3.5 Observation of the internal microbial population of the hydrogel using a laser confocal microscope

[0120] The 4th and 8th cycle Kombucha hydrogels were taken out and washed with sterile water, then cut into thin slices of about 1 mm for staining. Syto Green dye was diluted to 5 x 10 -6 mol / L, and PI dye was diluted to 2 x 10 -4 mol / L. The Kombucha hydrogels were soaked in the above two staining solutions for 20 min, then washed with pure water and observed using a laser confocal microscope.

[0121] The experimental results are shown in Figure 24 . After staining of live and dead cells, live cells will appear green, dead cells will appear red, and cells close to apoptosis will have both green and red. The results show that there are many yeast cells in the 4th cycle Kombucha hydrogel, and they appear green or yellow-green, indicating that the yeast cells in this period are live cells or cells close to apoptosis; there are very dense yeast cells in the 8th cycle Kombucha hydrogel, all of which are dead cells, and yeast cells can be seen being blocked at the edges of the Kombucha hydrogel. Combined with the experimental results of 3.4, the internal space available in the Kombucha hydrogel decreases after the internal structure changes to be dense, resulting in limited microbial proliferation and a large number of cell deaths. Moreover, the dense structure limits the internal yeast cells, which continue to accumulate dead cells.

[0122] 3.6 Swelling capacity test of kombucha hydrogel in different periods

[0123] The kombucha hydrogel of the 0th, 4th, and 8th rounds was taken out, washed with sterile water, and placed in a -20°C refrigerator overnight. The frozen kombucha hydrogel was placed in a vacuum freeze dryer for 2 days. The freeze-dried hydrogel was placed in pure water at room temperature. Every time, the surface water was absorbed with an absorbent paper, and the mass was measured.

[0124] The experimental results are shown in Table 3. Figure 25 The results show that the mass of the freeze-dried kombucha hydrogel of the 8th round is 0.2011 g, that of the 4th round is 0.1655 g, and that of the freshly prepared kombucha hydrogel is 0.0845 g. Because the kombucha hydrogel used multiple times contains microorganisms and metabolites, and the carbon source is concentrated and crystallized, the more times it is used, the greater the mass. After the kombucha hydrogel is soaked for 250 min, it reaches equilibrium, and the mass of the freshly prepared kombucha hydrogel after water absorption and swelling is 1.0323 g, that of the kombucha hydrogel after 4 rounds is 1.0242 g, and that of the kombucha hydrogel after 8 rounds is 0.6542 g. The more times it is used, the worse the swelling capacity. Studies have found that the swelling capacity in hydrogel is positively correlated with the release of internal drugs. By analogy, the kombucha hydrogel releases microorganisms, and with the increase in the number of uses, the swelling capacity decreases, and the ability to release yeast bacteria also decreases.

[0125] Example 4: Preparation of kombucha starter based on 3D printing

[0126] In the fermentation process of kombucha, it mainly relies on two types of microorganisms, yeast and acetic acid bacteria. Through the research of the above examples, it is found that after embedding the kombucha microbial community in the hydrogel, because the kombucha contains acetic acid bacteria, insoluble bacterial cellulose is produced in the growth and metabolism process, which blocks the original porous structure. In addition, the kombucha hydrogel exceeds the service life, which leads to the weakening of the ability to release live bacteria, and the internal yeast bacteria are limited in the internal and die in large quantities because of the overcrowding of the living space. Therefore, in order to improve the fermentation times and prolong the service life of the kombucha hydrogel, this embodiment will optimize it through two means: one is to supplement the yeast bacteria lacking in the later fermentation stage; the other is to improve the original design model and prepare a new kombucha hydrogel.

[0127] 4.1 Screening, isolation, and identification of yeast bacteria

[0128] The mother liquor of the black tea starter culture was inoculated on a Malonigrow medium (purchased from Guangdong Huan Kai Microbial Science and Technology Stock Company Limited) and incubated in a 30°C incubator for 2 days. Colonies with different morphologies were picked and streaked on plates. The above steps were repeated for more than 3 times to ensure that the plates contained single colonies. The samples were sent to Shanghai Generay Biotech for strain identification. The results showed that 6 strains of yeast, Yeast 1-6, were screened. Yeast 2 was most closely related to the dominant yeast strain LF (Lachancea fermentati), and the similarity reached 99.8% according to the results of the NCBI database. Therefore, Yeast 2 was LF.

[0129] 4.2 Preparation of yeast hydrogel using the dominant yeast strain LF

[0130] The dominant yeast strain LF was inoculated on a Malonigrow solid medium slope for expansion culture. After incubation at 30°C for 2 days, the colonies were washed off with sterile water to obtain yeast cells. The yeast cells were diluted 1:8 to prepare a yeast cell suspension (the concentration was more than 1×10 7 CFU / ml). The 30wt% modified polyether was dissolved in sterile water to form a modified polyether aqueous solution. Then, 0.1% of a photoinitiator and 10% of the yeast cell suspension prepared in step (1) were added to prepare a bio-ink B. The bio-ink B was added to a 3D printer ink tank below 4°C, and was heated to convert from a liquid state to a gel state. Then, the model was 3D printed according to a grid model (double-layer grid structure). The printed model was cured at the absorption wavelength of the photoinitiator to prepare a yeast hydrogel, i.e., a yeast starter culture.

[0131] 4.3 Preparation of black tea starter culture I and its application in fermented black tea

[0132] (1) A commercially available black tea starter culture was inoculated in black tea water containing 10% m / v sucrose and 0.9% m / v black tea at a inoculation amount of 3% v / v black tea starter culture liquid and 5% v / v black tea starter culture film. After fermentation at 30°C for 7 days, the pH was less than or equal to 3. The precipitate was obtained by centrifugation at 10,000 rpm / min for 8 min. The black tea starter culture was diluted 8 times to prepare a black tea starter culture suspension (the number of viable bacteria was more than or equal to 1×10 7 CFU / ml).

[0133] (2) The 30wt% modified polyether was dissolved in sterile water to form a modified polyether aqueous solution. Then, 0.1% of a photoinitiator (2-hydroxy-2-methylpropiophenone) and 10% of the black tea starter culture suspension prepared in step (1) were added to prepare a bio-ink A.

[0134] (3) The biological ink A prepared in step (2) is added to a 3D printer ink tank below 4°C, heated to convert the biological ink A from a liquid state to a gel state, and then 3D printing is performed according to a grid model (a double-layer grid structure). The printed model is cured under the absorption wavelength of a photoinitiator to prepare a kombucha hydrogel, i.e., kombucha starter I;

[0135] (4) The kombucha starter I is directly inoculated into black tea water at a mass ratio of 2%, and after 8 days of fermentation at 30°C, the kombucha starter I is taken out, washed with sterile water, and stored in a kombucha stock solution with pH≤3 to complete the first round of fermentation of the kombucha. The kombucha starter I taken out after the first round of fermentation is inoculated again, and after 8 days of fermentation at 30°C, the kombucha starter I is taken out, washed with sterile water, and stored in a kombucha stock solution with pH≤3 to complete the second round of fermentation of the kombucha. The step (4) of the second round of fermentation is repeated 4 times, and a total of 6 rounds of continuous fermentation are performed. At this time, the kombucha starter I reaches the service life;

[0136] (5) The yeast starter containing yeast is inoculated at a mass ratio of 1%, shaken at 30°C and 100 r / min for 1 h, and then the kombucha starter I is inoculated again. The step (4) is repeated for 7 rounds of continuous fermentation, and the pH (determined by a handheld pH meter), total acid (determined by NaOH titration), and sugar content (determined by a VBR32 sugar meter) are measured to achieve the red tea kombucha hydrogel with a service life as a control group.

[0137] The results are shown in Figure 26 and Figure 27 . The results show that the pH of the fermented kombucha after supplementing yeast decreases significantly, the pH of the control group is 3.35, and the average pH after 7 rounds of fermentation after supplementing yeast is 2.99. The total acid yield increases significantly after supplementing yeast, the total acid content of the control group is 1.6205 g / L, and the average total acid content after 7 rounds of fermentation after supplementing yeast is 4.9594 g / L. The sugar content decreases after supplementing yeast, the sugar content of the control group is 9.2, and the average sugar content after 7 rounds of fermentation after supplementing yeast is 8.32. In summary, supplementing the yeast starter to the kombucha starter I that has reached the service life can significantly improve the fermentation efficiency and prolong the use of the kombucha starter I. The kombucha starter I can be fermented for 7 more rounds based on the original fermentation.

[0138] 4.4 Preparation of kombucha starter II and its application in fermented kombucha

[0139] (a) The commercially available black tea fungus is inoculated into black tea water containing 10% m / v sucrose and 0.9% m / v black tea at an inoculation amount of 3% v / v black tea fungus liquid and 5% v / v black tea fungus film, and then fermented at a fermentation temperature of 30℃ for 7d, after which the pH is ≤3, the precipitate is obtained by centrifugation at 10000rpm / min for 8min, and the black tea fungus body is obtained, and then the black tea fungus body is diluted 8 times to prepare a black tea fungus suspension (viable bacterial count ≥1×10 7 CFU / ml); 7 CFU / ml);

[0140] (b) After dissolving 30wt% modified polyether in sterile water to form a modified polyether aqueous solution, 0.1% photoinitiator (2-hydroxy-2-methylpropiophenone) and 10% black tea fungus suspension prepared in step (1) are added to prepare a bio-ink C;

[0141] (c) After dissolving 30wt% modified polyether in sterile water to form a modified polyether aqueous solution, 0.1% photoinitiator (2-hydroxy-2-methylpropiophenone) and 10% yeast fungus suspension are added to prepare a bio-ink D;

[0142] (d) The bio-ink C prepared in step (b) and the bio-ink D prepared in step (c) are added to two ink tanks of a double-nozzle 3D printer below 4℃, and heating is performed to convert the bio-ink C and the bio-ink D from a liquid state to a gel state, then 3D printing is performed according to a grid model (four-layer grid structure, the first layer and the fourth layer embedding yeast fungus, and the second layer and the third layer embedding black tea fungus), and the printed model is cured at the absorption wavelength of the photoinitiator to prepare a black tea fungus-yeast fungus hydrogel (improved black tea fungus hydrogel), i.e. black tea fungus starter Ⅱ.

[0143] The four-layer grid structure is designed as follows: first, seven parallel cuboids are designed, and the cuboids are connected by short cuboids to form the first layer of the four-layer grid structure, then the first layer is copied and stacked on the first layer as the second layer after rotating 90° to the right, then the second layer is copied and stacked on the second layer as the third layer after rotating 90° to the right, and finally the third layer is copied and stacked on the third layer as the fourth layer after rotating 90° to the right. Figure 28

[0144] ​(e) the red tea starter II is inoculated into the red tea water at a mass ratio of 2%, and after fermentation at 30 DEG C for 8 days, the red tea starter II is taken out and stored in the red tea starter stock solution with pH less than or equal to 3, to complete the first round of fermentation of the red tea starter II, and the red tea starter II taken out from the first round of fermentation is inoculated again, and after fermentation at 30 DEG C for 8 days, the red tea starter II is taken out and stored in the red tea starter stock solution with pH less than or equal to 3, to complete the second round of fermentation of the red tea starter II, and the second round of fermentation is repeated, and when the total acid content of the red tea starter II after fermentation is less than 2g / L for two consecutive rounds, the fermentation is ended.

[0145] The experimental results are shown in Figure 29 and Figure 30 The results show that, compared with the red tea starter II before the improvement, the red tea starter II after the improvement (i.e., the red tea starter II) is used for 13 rounds in succession, and the pH is always stable at about 3 without obvious increase; the total acid content is significantly higher than that of the control group, although there is a certain fluctuation, but the average acid production after fermentation is 5.4883g / L, and the sugar content is basically maintained at 8-10. In summary, the service life of the red tea starter II is significantly improved after the structural improvement, and can be used for at least 13 times. And compared with the 4.3 unimproved printing model, the advantage is that the yeast does not need to be supplemented during use, and the use is more simple and convenient.

[0146] The present application provides a kind of red tea starter based on 3D printing and its preparation method and application ideas and methods, the method and approach for specifically realizing this technical scheme are many, above-mentioned only is preferred embodiment of the present application, it should be pointed out, for the ordinary skilled in the art, on the premise of not departing from the principle of the present application, can make a number of improvements and refinements, these improvements and refinements also should be regarded as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.

Claims

1. A method for preparing a 3D printing-based kombucha starter, characterized by, The step of preparing the black tea fungus starter Ⅱ comprises the following steps: The step of the black tea fungus starter Ⅱ comprises the following steps: (Ⅱ1) After the black tea fungus is fermented, the precipitate is obtained by centrifugation, and the black tea fungus body is obtained, and then the black tea fungus body is diluted to prepare a black tea fungus suspension; after the yeast fungus is fermented, the yeast fungus body is obtained by washing the colonies with sterile water, and then the yeast fungus body is diluted to prepare a yeast fungus suspension; (Ⅱ2) In the aqueous solution of modified polyether, a photoinitiator and the black tea fungus suspension prepared in step (Ⅱ1) are added to prepare a bio-ink C; (Ⅱ3) In the aqueous solution of modified polyether, a photoinitiator and the yeast fungus suspension prepared in step (Ⅱ1) are added to prepare a bio-ink D; (Ⅱ4) The bio-ink C prepared in step (Ⅱ2) and the bio-ink D prepared in step (Ⅱ3) are respectively added to two ink tanks of a double-nozzle 3D printer at -20℃~4℃, and heating is performed to convert the bio-ink C and the bio-ink D from a liquid state to a gel state, then 3D printing is performed according to a grid model, and the printed model is cured under the absorption wavelength of the photoinitiator to prepare a black tea fungus-yeast fungus hydrogel, i.e., the black tea fungus starter Ⅱ. In step (Ⅱ4), the grid model is a four-layer grid structure, a plurality of parallel cuboids are designed, short cuboids are used to connect the cuboids, a first layer of the four-layer grid structure is formed, the first layer is copied, and then is stacked on the first layer as a second layer after being rotated by 90° clockwise or counterclockwise, the second layer is copied, and then is stacked on the second layer as a third layer after being rotated by 90° in the same direction, and finally the third layer is copied, and then is stacked on the third layer as a fourth layer after being rotated by 90° in the same direction. The first layer and the fourth layer embed the yeast fungus, and the second layer and the third layer embed the black tea fungus. In the step (II1), the viable cell count of the red tea fungus suspension is ≥1×10 7 CFU / ml; and the viable cell count of the yeast fungus suspension is ≥1×10 7 CFU / ml. In step (Ⅱ1), the dilution ratio of the black tea fungus body to water is 1:8~16, and the dilution ratio of the yeast fungus body to water is 1:8~16.

2. The production method according to claim 1, characterized by, In step (Ⅱ2), the mass ratio of the black tea fungus suspension to the aqueous solution of modified polyether is 5~40%.

3. The preparation method according to claim 1, characterized in that, In steps (Ⅱ2) and (Ⅱ3), the aqueous solution of modified polyether is prepared by dissolving modified polyether in sterile water, and the mass percentage of the modified polyether is 20~35wt%.

4. The method of claim 1, wherein, In steps (Ⅱ2) and (Ⅱ3), the volume ratio or mass ratio of the photoinitiator to the aqueous solution of modified polyether is 0.1%~1%.

5. The preparation method according to claim 1, characterized in that, In step (Ⅱ3), the mass ratio of the yeast fungus suspension to the aqueous solution of modified polyether is 5~40%.

6. The black tea fungus starter Ⅱ prepared by the preparation method of the 3D printing-based black tea fungus starter according to claim 1.

7. The application of the black tea fungus starter Ⅱ according to claim 6 in the fermentation preparation of black tea fungus.

8. Use according to claim 7, characterized in that, The black tea starter II is inoculated into black tea water directly at a mass ratio of 1%-10%, and after fermentation at 25-30 ℃ for 8-10 days, the black tea starter II is taken out, and the first round of fermentation of the black tea starter II is completed. The black tea starter II taken out in the first round of fermentation is inoculated continuously, and after fermentation at 25-30 ℃ for 8-10 days, the black tea starter II is taken out, and the second round of fermentation of the black tea starter II is completed. The second round of fermentation is repeated to perform continuous fermentation, and when the total acid content of the fermented black tea starter II is less than 2 g / L for two rounds of fermentation continuously, the fermentation is ended.

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