A 3D printed probiotic chocolate and preparation method thereof

Abstract

This invention relates to the field of food additive manufacturing technology, and provides a 3D-printed probiotic chocolate and its preparation method. The invention involves sequentially subjecting a compound starch suspension to thermal gelatinization and alcohol precipitation to obtain a thermally gelatinized-alcohol-precipitated solution. This solution is then concentrated, cultured, centrifuged, and dried to obtain porous starch. The porous starch is sterilized and mixed with a culture medium solution and probiotics. Through microgelatinization, centrifugation, and freeze-drying, porous starch granules encapsulating probiotics are obtained. These encapsulated probiotic porous starch granules are then mixed with melted chocolate and 3D printed to obtain probiotic-printed chocolate. This invention utilizes porous starch to encapsulate probiotics, improving the retention rate of probiotics in complex environments. Simultaneously, 3D printing technology rapidly forms chocolate products with uniformly distributed probiotics, allowing for personalized design and the production of functional chocolates with high probiotic content based on individual needs.

Description

Technical Field

[0001] This invention relates to the field of food additive manufacturing technology, and in particular to a 3D printed probiotic chocolate and its preparation method. Background Technology

[0002] Probiotics are a class of live microorganisms that play a significant role in maintaining gut microbiota balance, preserving health, and preventing and treating diseases. Currently, probiotic products on the market are mainly available in powder, tablet, and capsule forms, and the survival rate of probiotics after digestion in the gastrointestinal tract is low. The high fat content in chocolate has a protective effect on probiotics, and chocolate is rich in polyphenols such as catechins (flavan-3-ols, or flavanols), epicatechin, and proanthocyanidins, which can act as antioxidants and promote human health. Adding probiotics can further enhance the nutritional quality of chocolate.

[0003] 3D printing technology, also known as food additive manufacturing, is a production process that creates products with a target model through layer-by-layer deposition. The advantages of food printing technology lie in personalized nutritional control, precise regulation of complex structures, digital production, optimized food supply chains, and reduced food waste. Chocolate is an ideal material for hot melt extrusion because its main structural component, cocoa butter, solidifies upon cooling, forming self-supporting layers. Most importantly, chocolate maintains its structure during layer-by-layer deposition; its "self-supporting" ability depends on thermal properties such as its glass transition temperature and melting point, which are crucial during the solidification process after the deposition of the layers.

[0004] Currently, in the preparation of probiotic chocolate, probiotics are typically added directly to melted chocolate, followed by processes such as casting, hardening, and demolding to obtain the probiotic chocolate. However, probiotics generally have high environmental requirements, especially being sensitive to acids and oxygen, and have poor tolerance to external factors. They are difficult to maintain high activity during long-term storage at room temperature. This method results in a high loss of live bacteria during processing, leading to a low number of live probiotics in the resulting chocolate. Summary of the Invention

[0005] In view of this, the present invention provides a 3D-printed probiotic chocolate and its preparation method. The present invention utilizes porous starch to encapsulate probiotics, improving the retention rate of probiotics in complex environments; simultaneously, it uses 3D printing technology to rapidly form chocolate products with uniformly distributed probiotics, combining this with personalized design to prepare functional chocolates with high probiotic content according to different individual needs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for preparing 3D-printed probiotic chocolate includes the following steps:

[0008] The compound starch suspension was subjected to thermal gelatinization and alcohol precipitation in sequence to obtain a thermal gelatinization-alcohol precipitation solution; the starch in the compound starch suspension included amylose and amylopectin.

[0009] The hot gelatinized-alcohol precipitation solution was concentrated and cultured, then subjected to solid-liquid separation and drying to obtain porous starch.

[0010] The porous starch was sterilized and then mixed with culture medium solution and probiotics to obtain a mixture.

[0011] After microgelatinization, the mixture was separated into solid and liquid components. The resulting precipitate was freeze-dried to obtain porous starch granules encapsulating probiotics.

[0012] The porous starch granules containing the encapsulated probiotics were mixed with melted chocolate and then 3D printed to obtain the 3D printed probiotic chocolate.

[0013] Preferably, the percentage of amylose in the compound starch suspension is 20-80% of the total mass of amylose and amylopectin; and the total concentration of amylose and amylopectin in the compound starch suspension is 2.5-15 wt%.

[0014] Preferably, the temperature for thermal gelatinization is 60–100°C, and the time for thermal gelatinization is 4–8 hours.

[0015] Preferably, the alcohol precipitation treatment includes: uniformly dripping anhydrous ethanol into the thermally gelatinized compound starch suspension until the volume ratio of anhydrous ethanol to the thermally gelatinized compound starch suspension is 1 to 3:1; the dripping rate of the anhydrous ethanol is 5 to 25 mL / min, and stirring is performed during the dripping process; the temperature of the alcohol precipitation treatment is above 50°C.

[0016] Preferably, the concentration culture is carried out under sealed and static conditions; the concentration culture temperature is 50-70°C and the time is 30-70 min; the drying temperature is 35-75°C.

[0017] Preferably, the culture medium solution is MRS broth; the ratio of porous starch to live probiotics is 1g:10g. 9 ~10 12 CFU / mL.

[0018] Preferably, the microgelatinization reaction is carried out at a temperature of 20–40°C for 1–2.5 h under oscillation conditions.

[0019] Preferably, the mass ratio of the porous starch granules containing the encapsulated probiotics to the chocolate is 1-5:10; and the melting temperature of the chocolate is 35-80°C.

[0020] Preferably, the 3D printing conditions include: air pump extrusion, nozzle diameter of 0.4-1 mm, extrusion speed of 0.1-1.5 mm / s, nozzle movement speed of 500-1500 mm / min, and printing temperature of 35-80℃.

[0021] The present invention also provides a 3D printed probiotic chocolate prepared by the preparation method described above, comprising chocolate and porous starch particles containing encapsulated probiotics dispersed in the chocolate.

[0022] This invention provides a method for preparing 3D-printed probiotic chocolate, comprising the following steps: subjecting a compound starch suspension to sequential thermal gelatinization and alcohol precipitation to obtain a thermal gelatinization-alcohol precipitation solution; wherein the starch in the compound starch suspension includes amylose and amylopectin; concentrating and cultivating the thermal gelatinization-alcohol precipitation solution, followed by centrifugation and drying to obtain porous starch; sterilizing the porous starch and mixing it with a culture medium solution and probiotics to obtain a mixture; subjecting the mixture to microgelatinization and centrifugation, and freeze-drying the resulting precipitate to obtain porous starch particles encapsulated with probiotics; mixing the porous starch particles encapsulated with probiotics with melted chocolate and then performing 3D printing to obtain the 3D-printed probiotic chocolate. Compared to traditional probiotic chocolates, this invention employs a thermal gelatinization-alcohol precipitation method and 3D printing. In the first processing stage, a thermal gelatinization field promotes the gelatinization and chain disintegration of linear-branched starch solutions. Alcohol precipitation occurs promptly in the resulting uniformly dispersed starch chain gelatinized solution, followed by slow drying to form perfectly round pores—the porous starch. In the second stage, the sterilized porous starch is homogenized and suspended in a culture medium solution. Upon introduction of probiotic cultures, the nanoscale pores of the porous starch adsorb the probiotics. The microgelatinization environment promotes structural transformation of the porous starch, encapsulating the probiotics within. The starch shell enhances the retention rate of probiotics in complex environments, providing effective protection. This invention also involves freeze-drying the probiotic-encapsulated porous starch granules and mixing them with melted chocolate for 3D printing. Through program control, a chocolate product with uniformly distributed probiotics is rapidly formed. This, combined with personalized design, allows for the production of functional chocolates with high probiotic content based on individual needs.

[0023] In summary, the preparation method provided by this invention can effectively protect probiotics by utilizing porous starch. Furthermore, the resulting product not only has a good taste and appeal but also possesses certain nutritional value, making it suitable as a functional food. Moreover, the preparation method of this invention is simple to operate, environmentally friendly, has a large production capacity, good material supply performance, and can be operated continuously, thus showing broad prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process of preparing probiotic chocolate by 3D printing in this invention;

[0025] Figure 2 Different shapes of probiotic chocolate products were prepared using the method of the present invention;

[0026] Figure 3 Scanning electron microscope (SEM) images of porous starch (left) prepared for Example 1 and porous starch granules (right) encapsulating probiotics;

[0027] Figure 4 The images show laser confocal microscopy (CLSM) images of the probiotics in the probiotic chocolates prepared in Example 1 (right) and Comparative Example 2 (left). Detailed Implementation

[0028] This invention provides a method for preparing 3D-printed probiotic chocolate, comprising the following steps:

[0029] The compound starch suspension was subjected to thermal gelatinization and alcohol precipitation in sequence to obtain a thermal gelatinization-alcohol precipitation solution; the starch in the compound starch suspension included amylose and amylopectin.

[0030] The hot gelatinized-alcohol precipitation solution was concentrated and cultured, then subjected to solid-liquid separation and drying to obtain porous starch.

[0031] The porous starch was sterilized and then mixed with culture medium solution and probiotics to obtain a mixture.

[0032] After microgelatinization, the mixture was separated into solid and liquid components. The resulting precipitate was freeze-dried to obtain porous starch granules encapsulating probiotics.

[0033] The porous starch granules containing the encapsulated probiotics were mixed with melted chocolate and then 3D printed to obtain the 3D printed probiotic chocolate.

[0034] This invention involves subjecting a compound starch suspension to sequential thermal gelatinization and alcohol precipitation to obtain a thermally gelatinized-alcohol-precipitated solution. In this invention, the starch in the compound starch suspension comprises amylose and amylopectin, with the percentage of amylose in the total mass of amylose and amylopectin preferably being 20-80%, more preferably 75%. The solvent in the compound starch suspension is water, and the total concentration of amylose and amylopectin in the compound starch suspension is 2.5-15 wt%, more preferably 5-10 wt%. The thermal gelatinization temperature is preferably 60-100°C, more preferably 70-80°C, and the thermal gelatinization time is preferably 4-8 hours, more preferably 5-6 hours. The preferred alcohol precipitation treatment includes: uniformly dripping anhydrous ethanol into the thermally gelatinized compound starch suspension until the volume ratio of anhydrous ethanol to the thermally gelatinized compound starch suspension is 1-3:1, more preferably 1.5-2.5:1. In a specific embodiment of the present invention, the alcohol precipitation treatment is considered complete once the volume ratio of anhydrous ethanol to the thermally gelatinized compound starch suspension reaches the above requirement. The preferred dripping rate of the anhydrous ethanol is 5-25 mL / min, more preferably 10-20 mL / min, and stirring is preferred during the dripping process. The preferred temperature of the alcohol precipitation treatment is above 50°C, more preferably 50-80°C.

[0035] After obtaining the thermal gelatinization-alcohol precipitation solution, the present invention concentrates and cultivates the solution, then performs solid-liquid separation and drying to obtain porous starch. In this invention, the concentration cultivation is preferably carried out under sealed and static conditions; the concentration cultivation temperature is preferably 50–70°C, more preferably 55–65°C; the concentration cultivation time is preferably 30–70 min, more preferably 40–60 min; the solid-liquid separation method is preferably centrifugation; the drying temperature is preferably 35–75°C, more preferably 40–70°C; and the drying method is preferably forced-air drying.

[0036] This invention utilizes a thermal gelatinization field to promote the gelatinization and chain dissolution of compound starch solutions. The resulting uniformly dispersed starch chains are promptly precipitated with alcohol, and after aggregation, the starch chains are slowly dried by forced air to form perfectly round pores, i.e., porous starch. The pores, channels, and cavities in the porous starch provide a space for the probiotic cells to be loaded. Simultaneously, this invention prepares porous starch with high probiotic encapsulation efficiency by controlling the ratio of amylose to amylopectin and the thermal gelatinization temperature.

[0037] After obtaining porous starch, the present invention sterilizes the porous starch and mixes it with a culture medium solution and probiotics to obtain a mixture. In this invention, the sterilization method is preferably ultraviolet sterilization; the culture medium solution is preferably MRS broth; the present invention preferably first adds the sterilized porous starch to the culture medium solution for homogenization to obtain a porous starch suspension, and then adds probiotics; the homogenization speed is preferably 5000 rpm, and the time is preferably 1 min; the concentration of the porous starch suspension is preferably 5-20 wt%, more preferably 10 wt%; the ratio of porous starch to viable probiotics is preferably 1 g: 10 g. 9 ~10 12 CFU / mL, more preferably 1g:10 11 CFU / mL; the probiotics are specifically added in the form of a probiotic culture.

[0038] After obtaining the mixture, the present invention performs a microgelatinization reaction followed by solid-liquid separation, and freeze-dries the resulting precipitate to obtain porous starch granules encapsulating probiotics. In this invention, the temperature of the microgelatinization reaction is preferably 20–40°C, more preferably 30–37°C; the time of the microgelatinization reaction is preferably 1–2.5 h, more preferably 1–2 h; and the microgelatinization reaction is preferably carried out under shaking conditions. The solid-liquid separation method is preferably centrifugation, with a preferred centrifugation speed of 5000 rpm and a preferred centrifugation time of 10 min. The freeze-drying temperature is preferably -48°C, and the preferred time is 48 h.

[0039] In this invention, the nanoscale pores of porous starch can adsorb probiotics, and the microgelatinization environment promotes the structural transformation of porous starch, encapsulating probiotics inside the porous starch. The starch shell can provide effective protection for probiotics, thereby improving the retention rate of probiotics in complex environments.

[0040] After obtaining porous starch granules encapsulating probiotics, this invention mixes the porous starch granules with melted chocolate and then performs 3D printing to obtain the 3D-printed probiotic chocolate. In this invention, the preferred mass ratio of the porous starch granules encapsulating probiotics to chocolate is 1–5:10, more preferably 1–3:10; the porous starch granules encapsulating probiotics are preferably sieved before use, with a preferred mesh size of 200 mesh, and the sieved material is collected; the preferred melting temperature of the chocolate is 35–80°C, more preferably 50–70°C; the preferred melting method for the chocolate is water bath melting.

[0041] In this invention, the preferred 3D printing conditions include: air pump extrusion, a nozzle diameter of 0.4–1 mm, more preferably 0.5–0.6 mm, an extrusion speed of 0.1–1.5 mm / s, more preferably 0.5–1 mm / s, a nozzle movement speed of 500–1500 mm / min, more preferably 600–800 mm / min, and a printing temperature of 35–80°C, more preferably 40–60°C. This invention, through 3D printing, can rapidly form chocolate products with uniformly distributed probiotics using programmable control. Combined with personalized design, it can produce functional chocolates with high probiotic content according to different individual needs.

[0042] Figure 1 This is a schematic diagram of the process of preparing probiotic chocolate by 3D printing in this invention. The mixture of porous starch particles encapsulating probiotics and chocolate is added to the barrel of a 3D printer, and 3D printing is performed through the nozzle. After condensation on a condensation tray, probiotic chocolate of a predetermined shape is obtained. Figure 2 These are probiotic chocolate products of different shapes prepared using the method of the present invention.

[0043] This invention also provides a 3D-printed probiotic chocolate prepared by the method described above, comprising chocolate and porous starch granules containing encapsulated probiotics dispersed within the chocolate. The 3D-printed probiotic chocolate provided by this invention has a high live bacteria content, uniform probiotic distribution, high nutritional value, good taste, and strong appeal, and has broad market prospects.

[0044] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0045] Example 1

[0046] (1) The compound starch suspension was thermally gelatinized by water bath heating at 100℃ for 6 hours. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50℃. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at 50℃ for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50℃ to obtain porous starch.

[0047] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of 1 g of porous starch to 10 g of live bacteria. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0048] (3) The reaction solution obtained in step (2) was centrifuged at 5000 rpm for 10 min, and then the supernatant was carefully removed. The resulting precipitate was freeze-dried for 48 h to obtain porous starch granules encapsulated with probiotics. Scanning electron microscopy (SEM) was performed on the porous starch and the porous starch granules encapsulated with probiotics. The results are as follows: Figure 3 As shown, Figure 3 The left side shows porous starch, and the right side shows porous starch granules encapsulating probiotics; according to Figure 3 It can be seen that porous starch prepared by thermal gelatinization-alcohol precipitation can encapsulate probiotics inside, and the starch shell can improve the retention rate of probiotics in complex environments, thus providing effective protection for probiotics.

[0049] (4) Take the porous starch particles of the encapsulated probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles of the encapsulated probiotics added is 5% of the mass of the chocolate.

[0050] (5) The probiotic chocolate liquid from step (4) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0051] Probiotic content determination: The viability of probiotics was assessed by agar plate assay. The sample was hydrolyzed with α-amylase (100 mg, 10000 U / g) in phosphate-buffered saline (PBS) (pH 7.2-7.4) to release the encapsulated probiotic cells. The diluted sample was plated onto MRS agar plates. The plates were incubated at 37°C for 48 h. Colony forming units (CFU) were counted.

[0052] In vitro simulated digestion: Preparation of simulated gastric juice (SGF): Weigh 0.32g pepsin and 0.2g sodium chloride and add to 100mL sterile aqueous solution. Adjust the pH to 2.0 with hydrochloric acid solution and stir well. Preparation of simulated intestinal juice (SIF): Weigh 0.1g pancreatin and 0.08g bile salts and add to 0.2M PBS. Adjust the pH to 7.0 with NaOH solution and stir well. Preheat SGF and SSF at 37℃ for 20min and then sterilize using a 0.22μm filter membrane. Add 1g probiotic chocolate to 9mL of SGF, mix well, and continuously shake in a constant temperature shaker (37℃). After 0.5h and 1h, remove the test tube and centrifuge at 5000xg for 5min. Remove the supernatant, add 1mL of saccharifying enzyme to the precipitate to release the encapsulated probiotic cells, and then count them by dilution and spread. After the simulated gastric digestion was completed, 10 mL of SSF was immediately added to simulate intestinal digestion. The mixture was stirred evenly. After 0.5 h and 1 h, the test tube was removed and centrifuged at 5000 x g for 5 min. The supernatant was removed, and 1 mL of saccharifying enzyme was added to the precipitate to release the encapsulated probiotic cells. The cells were then counted by dilution and spread.

[0053] Tests showed that the probiotic chocolate prepared in this embodiment contained 5.174 × 10⁻⁶ live bacteria. 8 CFU / g, the viable bacterial count after 30 minutes of simulated gastric digestion was 3.545 × 10⁻⁶. 8 CFU / g, the viable bacterial count after 1 hour of simulated gastric digestion was 9.800 × 10⁻⁶. 7 CFU / g; the viable bacterial count after 30 minutes of simulated intestinal digestion was 3.200 × 10⁻⁶. 7 CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 1.100 × 10⁻⁶. 7 CFU / g.

[0054] Example 2

[0055] (1) The compound starch suspension was thermally gelatinized by water bath heating at 50°C for 6 hours. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50°C. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at 50°C for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50°C to obtain porous starch.

[0056] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of starch mass to live bacteria count of 1 g: 10 g. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0057] (3) After centrifuging the reaction solution obtained in step (2) at 5000 rpm for 10 min, carefully remove the supernatant and freeze-dry the precipitate for 48 h to obtain porous starch particles encapsulated with probiotics.

[0058] (4) Take the porous starch particles containing probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles containing probiotics added is 10% of the mass of the chocolate.

[0059] (5) The probiotic chocolate liquid from step (4) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0060] The probiotic content was tested and an in vitro simulated digestion experiment was conducted according to the method in Example 1. The results showed that the probiotic chocolate prepared in this example contained 6.465 × 10⁻⁶ live bacteria. 8 CFU / g, the viable bacterial count after 30 minutes of simulated gastric digestion was 4.851 × 10⁻⁶. 8 CFU / g, the viable bacterial count after 1 hour of simulated gastric digestion was 2.500 × 10⁻⁶. 7 CFU / g; the viable bacterial count after 30 minutes of simulated intestinal digestion was 6.700 × 10⁻⁶. 7 CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 4.100 × 10⁻⁶. 7 CFU / g.

[0061] Example 3

[0062] (1) The compound starch suspension was thermally gelatinized by water bath heating at 100℃ for 6 hours. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50℃. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at 50℃ for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50℃ to obtain porous starch.

[0063] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of starch mass to live bacteria count of 1 g: 10 g. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0064] (3) After centrifuging the reaction solution obtained in step (2) at 5000 rpm for 10 min, carefully remove the supernatant and freeze-dry the precipitate for 48 h to obtain porous starch particles encapsulated with probiotics.

[0065] (4) Take the porous starch particles of the encapsulated probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles of the encapsulated probiotics added is 15% of the mass of the chocolate.

[0066] (5) The probiotic chocolate liquid from step (4) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0067] The probiotic content was tested and an in vitro simulated digestion experiment was conducted according to the method in Example 1. The results showed that the probiotic chocolate prepared in this example contained 7.50 × 10⁻⁶ live bacteria. 8 CFU / g, the viable bacterial count after 30 minutes of simulated gastric digestion was 5.831 × 10⁻⁶. 8 CFU / g, the number of viable bacteria contained in the simulated gastric digestion after 1 hour was 3.50 × 10⁻⁶. 8 CFU / g; the viable bacterial count after 30 minutes of simulated intestinal digestion was 7.00 × 10⁻⁶. 7CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 5.14 × 10⁻⁶. 7 CFU / g.

[0068] Example 4

[0069] (1) The compound starch suspension was thermally gelatinized by water bath heating at 100℃ for 6 hours. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50℃. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at 50℃ for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50℃ to obtain porous starch.

[0070] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of starch mass to live bacteria count of 1 g: 10 g. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0071] (3) After centrifuging the reaction solution obtained in step (2) at 5000 rpm for 10 min, carefully remove the supernatant and freeze-dry the precipitate for 48 h to obtain porous starch particles encapsulated with probiotics.

[0072] (4) Take the porous starch particles of the encapsulated probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles of the encapsulated probiotics added is 20% of the mass of the chocolate.

[0073] (5) The probiotic chocolate liquid from step (4) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0074] The probiotic content was tested and an in vitro simulated digestion experiment was conducted according to the method in Example 1. The results showed that the probiotic chocolate prepared in this example contained 8.15 × 10⁻⁶ live bacteria. 8 CFU / g, the viable bacterial count after 30 minutes of simulated gastric digestion was 6.80 × 10⁻⁶. 8CFU / g, the number of viable bacteria contained in the simulated gastric digestion after 1 hour was 5.15 × 10⁻⁶. 8 CFU / g; the viable bacterial count after 30 minutes of simulated intestinal digestion was 7.90 × 10⁻⁶. 7 CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 6.80 × 10⁻⁶. 7 CFU / g.

[0075] Example 5

[0076] (1) The compound starch suspension was thermally gelatinized by water bath heating at a temperature of 100℃ for 6 minutes. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50℃. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at a temperature of 50℃ for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50℃ to obtain porous starch.

[0077] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of starch mass to live bacteria count of 1 g: 10 g. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0078] (3) After centrifuging the reaction solution obtained in step (2) at 5000 rpm for 10 min, carefully remove the supernatant and freeze-dry the precipitate for 48 h.

[0079] (4) Take the porous starch particles of the encapsulated probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles of the encapsulated probiotics added is 25% of the mass of the chocolate.

[0080] (5) The probiotic chocolate liquid from step (4) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0081] The probiotic content was tested and an in vitro simulated digestion experiment was conducted according to the method in Example 1. The results showed that the probiotic chocolate prepared in this example contained 9.57 × 10⁻⁶ live bacteria. 8 CFU / g, the viable bacterial count after 30 minutes of simulated gastric digestion was 8.853 × 10⁻⁶. 8 CFU / g, the number of viable bacteria contained in the simulated gastric digestion after 1 hour was 7.00 × 10⁻⁶. 8 CFU / g; the viable bacterial count after 30 minutes of simulated intestinal digestion was 1.09 × 10⁻⁶. 8 CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 8.65 × 10⁻⁶. 7 CFU / g.

[0082] Comparative Example 1: 3D printing with probiotic powder added directly

[0083] (1) Add probiotic powder to the melted chocolate in a double boiler, wherein the amount of probiotic powder added is 10% of the weight of the chocolate.

[0084] (2) The probiotic chocolate liquid from step (1) is loaded into the 3D printer barrel for 3D printing to obtain probiotic chocolate. The nozzle diameter is 0.6 mm, the extrusion speed is 1 mm / s, the nozzle movement speed is 800 mm / min, the printing temperature is 40℃, and the printed model is 2×2×1 cm. 3 The mesh model.

[0085] The probiotic content was tested and an in vitro simulated digestion experiment was conducted according to the method in Example 1. The results showed that the probiotic chocolate prepared in this comparative example contained 3.01 × 10⁻⁶ live bacteria. 8 The CFU / g count, after 30 minutes of simulated gastric digestion, showed a viable bacterial count of 4.153 × 10⁻⁶ CFU / g. 7 CFU / g, the number of viable bacteria contained in the simulated gastric digestion after 1 hour was 2.00 × 10⁻⁶. 5 CFU / g; After 30 minutes of simulated intestinal digestion, the viable bacterial count was 9.70 × 10⁻⁶. 4 CFU / g, the viable bacterial count after 1 hour of simulated intestinal digestion was 1.10 × 10⁻⁶. 4 CFU / g. Significantly lower than the live bacteria count in chocolate containing porous starch granules with added encapsulated probiotics.

[0086] Comparative Example 2: Probiotic Chocolate Prepared Without 3D Printing

[0087] (1) The compound starch suspension was thermally gelatinized by water bath heating at 100℃ for 6 hours. The amylose in the compound starch suspension accounted for 75% of the total mass of amylose and amylopectin. Anhydrous ethanol was added dropwise to the thermally gelatinized compound starch suspension at a rate of 25 mL / min until the volume ratio of ethanol to thermally gelatinized compound starch suspension was 1.5:1. During the dropwise addition, the mixture was stirred continuously and the solution temperature was kept above 50℃. After the treatment, a thermally gelatinized-alcohol precipitation solution was obtained. The thermally gelatinized-alcohol precipitation solution was cultured under sealed and static conditions at 50℃ for 30 minutes. After the culture was completed, the mixture was centrifuged and the precipitate was dried at 50℃ to obtain porous starch.

[0088] (2) After ultraviolet sterilization, the porous starch obtained in step (1) is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension. Probiotic culture is added in a ratio of starch mass to live bacteria count of 1 g: 10 g. 11 CFU / mL, incubated in a microgelatinization environment at 37°C for 1 hour.

[0089] (3) After centrifuging the reaction solution obtained in step (2) at 5000 rpm for 10 min, carefully remove the supernatant and freeze-dry the precipitate for 48 h to obtain porous starch particles encapsulated with probiotics.

[0090] (4) Take the porous starch particles of the encapsulated probiotics after freeze-drying in step (3), pass them through a 200-mesh sieve, and add them to the chocolate after it has been melted in a water bath. The amount of porous starch particles of the encapsulated probiotics added is 15% of the mass of the chocolate. Pour it into a mold to solidify and shape it to obtain probiotic chocolate.

[0091] Laser confocal microscopy (CLSM) test: Frozen sections of the probiotic chocolate from Example 3 and the probiotic chocolate from this comparative example were prepared into 10 μm thin slices. The probiotics were stained using a LIVE / DEAD staining kit, and the distribution of the probiotics was observed using CLSM. The test results are as follows: Figure 4 As shown, Figure 4 The image on the left shows probiotic chocolate made without 3D printing, while the image on the right shows probiotic chocolate made using 3D printing.

[0092] according to Figure 4 It can be seen that the probiotics in the probiotic chocolate obtained in this comparative example are unevenly distributed, while the probiotic chocolate prepared by 3D printing has a more uniform distribution of probiotics due to the further shearing action of the printing needle.

[0093] In summary, this invention modifies starch granules into porous capsules (commonly used in applications such as drug delivery and flavor encapsulation). Utilizing the pores, channels, and cavities of porous starch, it provides a space for loading probiotic cells. Furthermore, by combining 3D printing technology with personalized design, it prepares functional chocolates with high probiotic content based on individual needs. Specifically, this invention prepares porous starch with high probiotic encapsulation efficiency by controlling the ratio of amylose to amylopectin and the thermal gelatinization temperature. By continuously optimizing the 3D printing parameters by controlling the ratio of the porous starch granules encapsulating the probiotics to the chocolate, the probiotic capsules are more evenly distributed within the chocolate, resulting in a better chocolate flavor.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing 3D-printed probiotic chocolate, characterized in that, Includes the following steps: The compound starch suspension was subjected to thermal gelatinization and alcohol precipitation in sequence to obtain a thermal gelatinization-alcohol precipitation solution; the starch in the compound starch suspension included amylose and amylopectin. The hot gelatinized-alcohol precipitation solution was concentrated and cultured, then subjected to solid-liquid separation and drying to obtain porous starch. The porous starch was sterilized and then mixed with culture medium solution and probiotics to obtain a mixture. After microgelatinization, the mixture was separated into solid and liquid components. The resulting precipitate was freeze-dried to obtain porous starch granules encapsulating probiotics. The porous starch granules containing the encapsulated probiotics are mixed with melted chocolate and then 3D printed to obtain the 3D printed probiotic chocolate. The 3D printing conditions include: air pump extrusion, nozzle diameter of 0.4-1 mm, extrusion speed of 0.1-1.5 mm / s, nozzle movement speed of 500-1500 mm / min, and printing temperature of 35-80℃.

2. The preparation method according to claim 1, characterized in that, The percentage of amylose in the compound starch suspension is 20-80% of the total mass of amylose and amylopectin; the total concentration of amylose and amylopectin in the compound starch suspension is 2.5-15 wt%.

3. The preparation method according to claim 1, characterized in that, The temperature for thermal gelatinization is 60–100°C, and the time for thermal gelatinization is 4–8 hours.

4. The preparation method according to claim 1, characterized in that, The alcohol precipitation process includes: adding anhydrous ethanol dropwise at a uniform rate to the thermally gelatinized compound starch suspension until the volume ratio of anhydrous ethanol to the thermally gelatinized compound starch suspension is 1 to 3:1; the dropping rate of the anhydrous ethanol is 5 to 25 mL / min, and stirring is performed during the dropping process; the temperature of the alcohol precipitation process is above 50°C.

5. The preparation method according to claim 1, characterized in that, The concentration culture is carried out under sealed and static conditions; the concentration culture temperature is 50-70℃ and the time is 30-70 min; the drying temperature is 35-75℃.

6. The preparation method according to claim 1, characterized in that, The culture medium solution was MRS broth; the ratio of porous starch to live probiotics was 1g:10g. 9 ~10 12 CFU / mL.

7. The preparation method according to claim 1, characterized in that, The microgelatinization reaction is carried out at a temperature of 20–40°C for 1–2.5 h under oscillation conditions.

8. The preparation method according to claim 1, characterized in that, The mass ratio of the porous starch granules containing the encapsulated probiotics to the chocolate is 1–5:10; the melting temperature of the chocolate is 35–80°C.

9. The 3D-printed probiotic chocolate prepared by the preparation method according to any one of claims 1 to 8, comprising chocolate and porous starch particles containing encapsulated probiotics dispersed in the chocolate.

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