Lutein-loaded Pickering emulsion for 3D printing and preparation method thereof

Abstract

The invention relates to the technical field of food industry, in particular to a lutein-loaded Pickering emulsion for 3D printing and a preparation method of the lutein-loaded Pickering emulsion. The preparation method comprises the following steps: extracting chlamydomonas reinhardtii protein from chlamydomonas reinhardtii powder as a raw material, preparing a chlamydomonas reinhardtii protein-xanthan gum compound from the chlamydomonas reinhardtii protein and xanthan gum with different contents, mixing the chlamydomonas reinhardtii protein-xanthan gum compound with lutein-loaded edible oil, and uniformly shearing to obtain stable lutein-loaded Pickering emulsion. And the xanthan gum is utilized to improve the stabilizing effect of the chlamydomonas reinhardtii protein on the Pickering emulsion and the protecting effect of the chlamydomonas reinhardtii protein on the xanthophyll. No chemical surfactant is added in the preparation process, so that the food safety is high; according to the present invention, the preparation operation is simple, the stability is good, the storage stability of xanthophyll can be significantly improved, and the xanthophyll nano-emulsion has characteristics of excellent viscoelasticity and excellent thixotropic recovery, can be used for 3D printing food, and has wide application prospects in the application of novel nutrient substance delivery systems and food-grade 3D printing materials.

Description

Technical Field

[0001] The invention relates to the technical field of food industry, and in particular to a Pickering emulsion loaded with lutein for 3D printing and a preparation method thereof. Background Art

[0002] Lutein is a carotenoid widely found in fruits, vegetables, flowers, and algae. It has a variety of biologically active effects. In addition to its excellent antioxidant capacity, lutein has great potential in anti-inflammatory and anti-cancer aspects. As a component of the retinal macular pigment, lutein can protect the macula from photooxidative damage and improve visual function. It has the effect of alleviating and intervening in various eye diseases such as cataracts, glaucoma, dry eyes, and age-related macular degeneration. It is a good eye protection factor. However, due to its low solubility in water, high sensitivity to light, heat, and oxygen, and low oral bioavailability, lutein is limited in its application in the food industry. Pickering emulsions are widely used in food, pharmaceuticals, and cosmetics. Pickering emulsions are emulsions stabilized by solid particles rather than traditional surfactants. Solid particles have a stronger ability to resist aggregation, Ostwald ripening, and phase separation as a stabilizer, giving the emulsion better stability. For example, the invention patent with application number CN118844627A provides a method of using whey protein isolate, sodium alginate and tea polyphenols complex as an emulsifier to stabilize Pickering emulsion and load lutein. The oil phase fraction of this method is 25%-45%, and the loaded lutein content is low. High internal phase Pickering emulsion has a higher internal phase volume fraction (≥74%), so it has a stronger ability to load nutrients, making it have great potential in encapsulating and delivering hydrophobic active substances. 3D printed food has attracted widespread attention because it can meet the specific needs of different consumers. However, traditional 3D printed products may have biosafety risks, which limits its development in the food industry. Therefore, it is urgent to develop a high internal phase Pickering emulsion loaded with special nutrients as a 3D printed food. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention proposes a Pickering emulsion loaded with lutein for 3D printing and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions: A Pickering emulsion loaded with lutein for 3D printing, wherein the preparation raw materials include the following components: Chlamydomonas reinhardtii protein, xanthan gum, lutein and edible oil.

[0005] Furthermore, the edible oil is one or a combination of corn oil, peanut oil, soybean oil, sunflower oil and olive oil, preferably soybean oil.

[0006] Furthermore, the method for preparing the Chlamydomonas reinhardtii protein comprises the following steps: (1) Mix Chlamydomonas reinhardtii powder and deionized water in a ratio of 1 mg: 5-20 mL, and stir magnetically at 800 rpm for 30-40 min to obtain a powder solution; (2) treating the algae powder solution obtained in step (1) with ultrasound at 500-700W for 10-20 min, adjusting the pH to 9-11 with 1 mol / L NaOH solution, stirring at 200-300 rpm for 50-70 min, centrifuging at 8000 rpm for 10-15 min, and collecting the supernatant; (3) The pH of the supernatant obtained in step (2) was adjusted to 3-5 with 1 mol / L HCl solution, and the mixture was centrifuged at 8000 rpm for 10-15 min. The precipitate was collected to obtain Chlamydomonas reinhardtii protein.

[0007] Furthermore, in step (1), the method for preparing Chlamydomonas reinhardtii algae powder comprises the following steps: (a) Dry the sea lettuce at 80-90°C, crush it through an 80-mesh sieve, add 10 times its weight of distilled water, heat it at 90-100°C for 3-4 hours, filter and sterilize the filtrate at 121°C for 30 minutes to obtain a fermentation substrate, and add the activated Lactobacillus casei at 10 7 The inoculum of CFU / mL was inoculated into the fermentation substrate and cultured at 37°C and 120 rpm in a shaking incubator for 48 h; (b) After the cultivation in step (a), the mixture was centrifuged at 10,000 rpm for 7-10 min, the supernatant was concentrated to 50% of the original volume under reduced pressure, 50% volume of anhydrous ethanol was added, the mixture was centrifuged at 8,000 rpm for 10-15 min, the precipitate was mixed with an equal amount of distilled water, and freeze-dried at -40°C to obtain a fermented extract of Sargassum fusiformis; (d) The fermented extract of Sargassum fusiformis obtained in step (b) was added to TAP liquid medium at an amount of 10 g / L, and 1×10 6 cells / mL, inoculated with Chlamydomonas reinhardtii in the logarithmic growth phase, 25°C, 120-130 rpm, and a light intensity of 120 μmol / (m 2 ·s), 16 h:8 h light-dark alternating culture, 5 days later, centrifugation at 11000-13000 rpm for 10 min, collection of precipitates, washing with deionized water, and vacuum freeze-drying at -60°C to obtain Chlamydomonas reinhardtii powder.

[0008] Furthermore, the Lactobacillus casei described in step (a) is purchased from China General Microbiological Culture Collection Center and is numbered as CGMCC 1.8727.

[0009] Furthermore, the present invention provides a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex, the preparation method of which comprises the following steps: S1: Dissolve xanthan gum in deionized water, heat at 60-90°C for 5-7 h to completely dissolve it, and obtain a xanthan gum solution; dissolve Chlamydomonas reinhardtii protein in deionized water, adjust the pH value to 7, and obtain a Chlamydomonas reinhardtii protein solution with a concentration of 3wt%; add different amounts of xanthan gum solution to the Chlamydomonas reinhardtii protein solution, so that the concentrations of xanthan gum in the solution system are 0.4wt%, 0.6wt%, 0.8wt%, 1wt% and 1.5wt%, respectively; and stir magnetically at 700-900 rpm for 20-25 h to obtain a Chlamydomonas reinhardtii protein-xanthan gum complex solution; S2: The Chlamydomonas reinhardtii protein-xanthan gum complex solution obtained in step S1 is mixed with edible oil in a volume ratio of 3:7, and high-speed shearing is performed at 9000-11000 rpm for 3-5 min to obtain a Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex.

[0010] Furthermore, the present invention also provides a Pickering emulsion loaded with lutein, and the preparation method comprises the following steps: dissolving 5wt% of lutein in edible oil, stirring at 200-300 rpm for 20-30 h in a dark environment, mixing the edible oil and the Chlamydomonas reinhardtii protein-xanthan gum complex solution in a volume ratio of 7:3, and high-speed shearing at 9000-11000 rpm for 3-5 min to obtain the Pickering emulsion loaded with lutein.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses Chlamydomonas reinhardtii as a raw material, and the Pickering emulsion prepared without adding any inorganic materials has high biological safety. Chlamydomonas reinhardtii is a unicellular eukaryotic green algae, which is rich in nutrients, wherein the protein content is greater than 40%, and the amino acid composition is reasonable, containing eight kinds of amino acids necessary for the human body. Xanthan gum is an anionic polysaccharide with a cellulose-like skeleton, which is secreted by microorganisms. Its aqueous solution can improve the physical stability of the emulsion due to its significant thickening effect and weak gel structure. The present invention uses Chlamydomonas reinhardtii protein-xanthan gum complex as a stabilizer to prepare the Pickering emulsion, which has strong stability, excellent rheological properties and self-supporting properties, can be used as a 3D printing material, and can effectively protect hydrophobic active substances. Specifically, the present invention uses Chlamydomonas reinhardtii protein-xanthan gum complex as a stabilizer to prepare Pickering emulsion, which still has self-supporting force after inversion, and remains stable after being stored at room temperature for 30 days, subjected to high-speed centrifugation or heated at 90°C, and is very suitable for 3D printing, and has good prospects in the application of new hydrophobic substance delivery systems and food-grade 3D printing materials; the storage modulus is higher than the loss modulus, and has the typical characteristics of a gel-like emulsion. The Pickering emulsion prepared by the present invention can effectively embed hydrophobic functional active substances. When used to load lutein, the stability and bioaccessibility of lutein can be effectively improved. After storage for 15 days under room temperature and light, the retention rate of lutein can be close to 90%; in the simulated digestion experiment, the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex should have a higher lipolysis degree and bioaccessibility. The invention adopts Lactobacillus casei to ferment Sargassum fusiformis to prepare Sargassum fusiformis fermented extract, and the extract is added into the culture process of Chlamydomonas reinhardtii. The active ingredients of the extract can effectively increase the protein content of Chlamydomonas reinhardtii, which is beneficial to improving the extraction efficiency of Chlamydomonas reinhardtii protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 This is a cold field scanning electron microscope image of the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 1 of the present invention; Figure 2 This is an appearance diagram of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 4 of the present invention; Figure 3This is a cold field scanning electron micrograph of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 5 of the present invention; Figure 4 This is an appearance diagram of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 6 of the present invention after centrifugation at 9000 rpm for 3 min; Figure 5 This is an appearance diagram of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 4 of the present invention after being heated at 90° C. for 30 min; Figure 6 This is an appearance diagram of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 4 of the present invention after being stored at room temperature for 30 days; Figure 7 The measurement results of the storage modulus and loss modulus of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex in Example 4 of the present invention as a function of frequency; Figure 8 The measurement results of the viscosity of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex as a function of shear rate in Example 4 of the present invention; Fig. 9 is the encapsulation efficiency of lutein in the lutein-loaded Pickering emulsion of Example 7 of the present invention; Fig.10 The retention rate of lutein in the Pickering emulsion loaded with lutein after storage for 15 days in Example 8 of the present invention; Fig.11 The free fatty acid release rate of the Pickering emulsion loaded with lutein obtained by in vitro simulated digestion experiment in Example 9 of the present invention; Fig.12 The bioaccessibility data of the Pickering emulsion loaded with lutein in Example 7 of the present invention obtained through an in vitro simulated digestion experiment; Fig.13 This is the appearance of the lutein-loaded Pickering emulsion of Example 7 of the present invention after 3D printing. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0015] Example 1: This example provides a method for preparing a Chlamydomonas reinhardtii protein-xanthan gum complex solution, comprising the following steps: (1) The sea lettuce was dried at 90°C, crushed and passed through an 80-mesh sieve, and 10 times its weight of distilled water was added and heated at 100°C for 3 h. The filtrate was filtered and sterilized at 121°C for 30 min to obtain a fermentation substrate. The activated Lactobacillus casei was added at 10 7 The inoculum of CFU / mL was inoculated into the fermentation substrate and cultured at 37°C and 120 rpm in a shaking incubator for 48 h; (2) After the cultivation in step (a), the mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was concentrated to 50% of the original volume under reduced pressure, 50% anhydrous ethanol was added, the mixture was centrifuged at 8,000 rpm for 15 min, the precipitate was mixed with an equal amount of distilled water, and freeze-dried at -40°C to obtain a fermented extract of Sargassum fusiformis; (3) The fermented extract of Sargassum fusiformis obtained in step (b) was added to TAP liquid medium at an amount of 10 g / L, and 1×10 6 cells / mL, inoculated with Chlamydomonas reinhardtii in the logarithmic growth phase, 25°C, 130 rpm, and a light intensity of 120 μmol / (m 2 ·s), 16 h:8 h light-dark alternating culture, 5 days later, centrifugation at 13000 rpm for 10 min, collecting the precipitate, washing with deionized water, and vacuum freeze-drying at -60°C to obtain Chlamydomonas reinhardtii powder; (4) 10 mg of Chlamydomonas reinhardtii powder was mixed with 100 mL of deionized water, and the powder solution was obtained after magnetic stirring at 800 rpm for 30 min; the powder solution was ultrasonically treated at 600 W for 15 min, the pH value was adjusted to 10 with 1 mol / L NaOH solution, stirred at 300 rpm for 50 min, centrifuged at 8000 rpm for 10 min, and the supernatant was collected; the pH value of the supernatant obtained in step (2) was adjusted to 4 with 1 mol / L HCl solution, and centrifuged at 8000 rpm for 10 min, and the precipitate was collected to obtain Chlamydomonas reinhardtii protein; (5) Xanthan gum was dissolved in deionized water and heated at 70 °C for 6 h to completely dissolve it to obtain a xanthan gum solution. Chlamydomonas reinhardtii protein was dissolved in deionized water and the pH value was adjusted to 7 to obtain a 3 wt% Chlamydomonas reinhardtii protein solution. Different amounts of xanthan gum solution were added to the Chlamydomonas reinhardtii protein solution to make the concentrations of xanthan gum in the solution system 0.4 wt%, 0.6 wt%, 0.8 wt%, 1 wt% and 1.5 wt%, respectively. The mixture was magnetically stirred at 800 rpm for 24 h to obtain Chlamydomonas reinhardtii protein-xanthan gum complex solutions with different xanthan gum concentrations.

[0016] Example 2: This example provides a method for preparing a Chlamydomonas reinhardtii protein-xanthan gum complex solution, comprising the following steps: (1) The sea lettuce was dried at 80°C, crushed and passed through an 80-mesh sieve, and 10 times its weight of distilled water was added and heated at 90°C for 4 h. The filtrate was filtered and sterilized at 121°C for 30 min to obtain a fermentation substrate. The activated Lactobacillus casei was added at 10 7 The inoculum of CFU / mL was inoculated into the fermentation substrate and cultured at 37°C and 120 rpm in a shaking incubator for 48 h; (2) After the cultivation in step (a), the mixture was centrifuged at 10,000 rpm for 7 min, the supernatant was concentrated under reduced pressure to 50% of the original volume, 50% volume of anhydrous ethanol was added, the mixture was centrifuged at 8,000 rpm for 10 min, the precipitate was mixed with an equal amount of distilled water, and freeze-dried at -40°C to obtain a fermented extract of Sargassum fusiformis; (3) The fermented extract of Sargassum fusiformis obtained in step (b) was added to TAP liquid medium at an amount of 10 g / L, and 1×10 6 cells / mL, inoculated with Chlamydomonas reinhardtii in the logarithmic growth phase, 25°C, 120 rpm, and a light intensity of 120 μmol / (m 2 ·s), 16 h:8 h light-dark alternating culture, 5 days later centrifugation at 11000 rpm for 10 min, collecting the precipitate, washing with deionized water, and vacuum freeze-drying at -60°C to obtain Chlamydomonas reinhardtii powder; (4) 10 mg of Chlamydomonas reinhardtii powder was mixed with 50 mL of deionized water, and magnetically stirred at 800 rpm for 40 min to obtain a powder solution; the powder solution was ultrasonically treated at 500 W for 20 min, the pH was adjusted to 11 with 1 mol / L NaOH solution, stirred at 200 rpm for 70 min, centrifuged at 8000 rpm for 15 min, and the supernatant was collected; the pH of the supernatant obtained in step (2) was adjusted to 5 with 1 mol / L HCl solution, and centrifuged at 8000 rpm for 15 min to collect the precipitate to obtain Chlamydomonas reinhardtii protein; (5) Xanthan gum was dissolved in deionized water and heated at 90 °C for 5 h to completely dissolve it to obtain a xanthan gum solution. Chlamydomonas reinhardtii protein was dissolved in deionized water and the pH value was adjusted to 7 to obtain a 3 wt% Chlamydomonas reinhardtii protein solution. Different amounts of xanthan gum solution were added to the Chlamydomonas reinhardtii protein solution to make the concentrations of xanthan gum in the solution system 0.4 wt%, 0.6 wt%, 0.8 wt%, 1 wt% and 1.5 wt%, respectively. The mixture was magnetically stirred at 900 rpm for 20 h to obtain Chlamydomonas reinhardtii protein-xanthan gum complex solutions with different xanthan gum concentrations.

[0017] Example 3: This example provides a method for preparing a Chlamydomonas reinhardtii protein-xanthan gum complex solution, comprising the following steps: (1) The sea lettuce was dried at 85°C, crushed and passed through an 80-mesh sieve, and 10 times its weight of distilled water was added and heated at 95°C for 3.5 h. The filtrate was filtered and sterilized at 121°C for 30 min to obtain a fermentation substrate. The activated Lactobacillus casei was added at 10 7 The inoculum of CFU / mL was inoculated into the fermentation substrate and cultured at 37°C and 120 rpm in a shaking incubator for 48 h; (2) After the cultivation in step (a), the mixture was centrifuged at 10,000 rpm for 8 min, the supernatant was concentrated to 50% of the original volume under reduced pressure, 50% volume of anhydrous ethanol was added, the mixture was centrifuged at 8,000 rpm for 12 min, the precipitate was mixed with an equal amount of distilled water, and freeze-dried at -40°C to obtain a fermented extract of Sargassum fusiformis; (3) The fermented extract of Sargassum fusiformis obtained in step (b) was added to TAP liquid medium at an amount of 10 g / L, and 1×10 6 cells / mL, inoculated with Chlamydomonas reinhardtii in the logarithmic growth phase, 25°C, 125 rpm, and a light intensity of 120 μmol / (m 2 ·s), 16 h:8 h light-dark alternating culture, 5 days later centrifugation at 12000 rpm for 10 min, collecting the precipitate, washing with deionized water, and vacuum freeze-drying at -60°C to obtain Chlamydomonas reinhardtii powder; (4) 10 mg of Chlamydomonas reinhardtii powder was mixed with 200 mL of deionized water, and magnetically stirred at 800 rpm for 35 min to obtain a powder solution; the powder solution was ultrasonically treated at 700 W for 10 min, the pH was adjusted to 9 with 1 mol / L NaOH solution, stirred at 250 rpm for 60 min, centrifuged at 8000 rpm for 12 min, and the supernatant was collected; the pH of the supernatant obtained in step (2) was adjusted to 3 with 1 mol / L HCl solution, centrifuged at 8000 rpm for 12 min, and the precipitate was collected to obtain Chlamydomonas reinhardtii protein; (5) Xanthan gum was dissolved in deionized water and heated at 60 °C for 7 h to completely dissolve it to obtain a xanthan gum solution. Chlamydomonas reinhardtii protein was dissolved in deionized water and the pH value was adjusted to 7 to obtain a 3 wt% Chlamydomonas reinhardtii protein solution. Different amounts of xanthan gum solution were added to the Chlamydomonas reinhardtii protein solution to make the concentrations of xanthan gum in the solution system 0.4 wt%, 0.6 wt%, 0.8 wt%, 1 wt% and 1.5 wt%, respectively. The solution was stirred magnetically at 700 rpm for 25 h to obtain Chlamydomonas reinhardtii protein-xanthan gum complex solutions with different xanthan gum concentrations.

[0018] Example 4: This example provides a method for preparing a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex, comprising the following steps: mixing the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations obtained in Example 1 with soybean oil in a volume ratio of 3:7, and high-speed shearing at 10,000 rpm for 4 min to obtain a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex.

[0019] Example 5: This example provides a method for preparing a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex, comprising the following steps: mixing the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations obtained in Example 1 with soybean oil in a volume ratio of 3:7, and high-speed shearing at 11,000 rpm for 3 min to obtain a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex.

[0020] Example 6: This example provides a method for preparing a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex, comprising the following steps: mixing the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations obtained in Example 1 with soybean oil in a volume ratio of 3:7, and high-speed shearing at 9000 rpm for 5 min to obtain a Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex.

[0021] Experimental Example 7: This example provides a method for preparing a Pickering emulsion loaded with lutein, comprising the following steps: dissolving 5 wt % lutein in soybean oil, stirring at 250 rpm for 24 h in a dark environment, mixing soybean oil with a Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations prepared in Example 1 in a volume ratio of 7:3, and high-speed shearing at 10,000 rpm for 4 min to obtain a Pickering emulsion loaded with lutein.

[0022] Experimental Example 8: This embodiment provides a method for preparing a Pickering emulsion loaded with lutein, comprising the following steps: dissolving 5 wt % lutein in edible oil, stirring at 300 rpm for 20 h in a dark environment, mixing soybean oil with the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations prepared in Example 1 in a volume ratio of 7:3, and high-speed shearing at 11000 rpm for 3 min to obtain a Pickering emulsion loaded with lutein.

[0023] Experimental Example 9: This example provides a method for preparing a Pickering emulsion loaded with lutein, comprising the following steps: dissolving 5 wt % lutein in edible oil, stirring at 200 rpm for 30 h in a dark environment, mixing soybean oil with the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations prepared in Example 1 in a volume ratio of 7:3, and high-speed shearing at 9000 rpm for 5 min to obtain a Pickering emulsion loaded with lutein.

[0024] Experimental Example 1: The microstructure of the Chlamydomonas reinhardtii protein-xanthan gum complex solution with different xanthan gum concentrations prepared in Example 1 was observed. The cold field scanning electron microscopy results are as follows: Figure 1 shown.

[0025] Figure 1 The results showed that the detailed structure of Chlamydomonas reinhardtii protein and Chlamydomonas reinhardtii protein-xanthan gum complexes with different xanthan gum concentrations was displayed by cold field scanning electron microscopy. The results showed that Chlamydomonas reinhardtii protein was spherical. With the addition of xanthan gum, the morphology of Chlamydomonas reinhardtii protein-xanthan gum complex changed significantly, forming a unique gel network structure inlaid with Chlamydomonas reinhardtii protein, which is beneficial to stabilize the high internal phase Pickering emulsion.

[0026] Experimental Example 2: The appearance of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex with different xanthan gum concentrations in Example 4 is shown in FIG. Figure 2 shown.

[0027] Figure 2 The results showed that compared with the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein alone, the Pickering emulsions stabilized by Chlamydomonas reinhardtii protein-xanthan gum complexes with different xanthan gum concentrations all had a uniform gel state, and all samples still had self-supporting ability after inversion, indicating that the Pickering emulsion can be used for 3D printing products.

[0028] Experimental Example 3: Example 5 The cold field scanning electron microscopy results of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex with different xanthan gum concentrations are as follows Figure 3 shown.

[0029] Figure 3The results showed that cold-field scanning electron microscopy can be used to observe the droplet state of Pickering emulsions. Compared with single algae protein and Chlamydomonas reinhardtii protein-xanthan gum complex with low xanthan gum concentration, Pickering emulsions prepared at higher xanthan gum concentrations showed smaller droplet size, smaller droplet spacing and more compact structure, which helped to improve the viscosity and stability of Pickering emulsions. With the increase of xanthan gum concentration, the particle coverage rate on the surface of emulsion droplets and the filling rate of droplet gaps increased, making the droplets fixed in the gel network structure of Pickering emulsions.

[0030] Experimental Example 4: The appearance of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex of Example 6 after centrifugation at 9000 rpm for 10 min is shown in the figure below: Figure 4 shown.

[0031] Figure 4 The results showed that the visual appearance results of the centrifugation test were consistent with the results of the storage experiment. An oil layer was observed on the top of the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein without xanthan gum after centrifugation, while the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex remained stable.

[0032] Experimental Example 5: The appearance of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex of Example 4 after heating at 90°C for 30 min is shown in the figure below: Figure 5 shown.

[0033] Figure 5 The results showed that the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein alone showed phase separation under heating, but the heat treatment did not cause any significant changes in the visual appearance of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex with different xanthan gum concentrations, and it could still remain self-supporting at the bottom of the bottle, which proves that the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex has good heat resistance and excellent high-temperature sterilization potential.

[0034] Experimental Example 6: The appearance of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex of Example 4 after being stored at room temperature for 30 days is shown in the figure below. Figure 6 shown.

[0035] Figure 6 The results showed that after 30 days of storage at room temperature / visible light, the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein alone showed phase separation, and the Pickering emulsion stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex with different xanthan gum concentrations exhibited gel-like properties and could remain self-supporting at the bottom of the bottle even when the sample bottle was inverted.

[0036] Experimental Example 7: The storage modulus and loss modulus of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex of Example 4 were tested by a rheometer at 25°C in the range of 0.1-10 Hz, and the fixed strain during the test was 0.5%. The measurement results of the storage modulus and loss modulus of the Pickering emulsion as a function of frequency are shown in Figure 7 shown.

[0037] Figure 7 The results show that the storage modulus of all samples is higher than the loss modulus in the entire frequency range, indicating the solid viscoelastic behavior of the Pickering emulsion. As the content of xanthan gum in the Chlamydomonas protein-xanthan gum composite system increases, the storage modulus of the Pickering emulsion also increases. In addition, the storage modulus of all emulsions is almost an order of magnitude higher than the loss modulus, indicating a strong network structure.

[0038] Experimental Example 8: Tested by rheometer at 25°C, at 0.1-100s -1 The shear viscosity of the Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex of Example 4 was characterized within the shear rate range. The measurement results of the viscosity of the Pickering emulsion as a function of shear rate are shown in FIG. Figure 8 shown.

[0039] Figure 8 The results showed that all Pickering emulsions exhibited typical shear thinning behavior. This shear thinning behavior of Pickering emulsions may support their application in 3D printing. In addition, the viscosity of Pickering emulsions was positively correlated with the content of xanthan gum in the Chlamydomonas reinhardtii protein-xanthan gum composite system.

[0040] Experimental Example 9: The Pickering emulsion loaded with lutein just prepared in Example 7 was centrifuged at 5000 rpm for 3 min, the supernatant containing free lutein was taken out and mixed with 4.8 mL of chloroform / methanol (2:1, v / v), the sample was vortexed for 2 min and centrifuged at 8000 rpm for 3 min, and the supernatant was collected. The absorbance of the obtained supernatant was measured at 450 nm by an ultraviolet spectrophotometer, and the concentration of lutein was calculated by the standard curve of lutein. Lutein encapsulation efficiency (%) = (the total amount of lutein initially added - the amount of free lutein) / the total amount of lutein initially added × 100%. The results are as follows: Fig. 9 shown.

[0041] Fig. 9The results showed that the encapsulation efficiency of lutein in Pickering emulsions was 80.087% (0wt%), 83.77% (0.4wt%), 85.47% (0.6wt%), 86.33% (0.8wt%), 89.50% (1wt%) and 92.19% (1.5wt%), respectively. All Pickering emulsions had high encapsulation efficiency. The content of xanthan gum in the Chlamydomonas reinhardtii protein-xanthan gum complex system can improve the encapsulation efficiency of lutein in the emulsion.

[0042] Experimental Example 10: After the Pickering emulsion loaded with lutein in Example 8 was stored at room temperature / visible light for 15 days, the retention rate of lutein in the Pickering emulsion was measured as follows: Fig.10 shown.

[0043] Fig.10 The results showed that after 15 days of storage, the retention of lutein was listed in the following order: 38.33% (soybean oil) < 74.54% (0.4wt%) < 78.54% (0.6wt%) < 83.701% (0.8wt%) < 85.60% (1wt%) < 89.52% (1.5wt%). This result shows that the Pickering emulsion delivery system stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex can increase the retention level of lutein. In addition, increasing the content of xanthan gum in the Chlamydomonas reinhardtii protein-xanthan gum complex can improve the protective effect of the emulsion on lutein.

[0044] Experimental Example 11: In vitro simulated digestion experiment process of Pickering emulsion loaded with lutein in Example 9: (1) All emulsion samples to be tested and simulated digestion solutions were incubated at 37°C for 10 min before the experiment; (2) Simulated oral stage: Before the simulated digestion experiment, the Pickering emulsion was diluted with deionized water to the same concentration as soybean oil. 7.5 mL of sample was mixed with an equal volume of simulated saliva, and the pH value was adjusted to 6.8. The simulated saliva mixture was stirred at 100 rpm at 37°C for 10 min; (3) Simulated gastric fluid stage: 15 mL of simulated gastric fluid was added to the sample digested in the oral stage, and the pH of the sample was adjusted to 2. The simulated gastric fluid was stirred at 100 rpm at 37°C for 120 min; (4) Simulated intestinal fluid stage: 7.5 mL of simulated intestinal fluid was added to the sample digested in the gastric fluid stage, and the pH value of the sample was adjusted to 7. The simulated intestinal fluid mixture was stirred at 100 rpm at 37°C for 120 min. After the Pickering emulsion loaded with lutein was subjected to simulated digestion in vitro, the fatty acid release rate of the Pickering emulsion was detected. The calculation method was as follows: During the small intestinal digestion stage, the pH value of the small intestinal stage was maintained at 7 by 0.02 mol / L NaOH solution, and the consumption of NaOH was recorded within 2 hours. The percentage of free fatty acids (FFA) released during the entire small intestinal stage was calculated based on the consumption of NaOH. FFA (%) = [(C NaOH ×V NaOH ×M oil ) / (2×m oil )]×100%, where C NaOH is the molar concentration of NaOH (mol / L), V NaOH is the volume of NaOH consumed (L), M oil is the average molecular weight of soybean oil (g / mol), m oil is the total mass of soybean oil (g). The fatty acid release rate of Pickering emulsion is shown in Fig.11 shown.

[0045] Fig.11The results showed that the lipids of all samples were rapidly digested within the first 30 min, and then the lipolysis rate decreased in the next 30 min. The free fatty acid release curve approached the horizontal line and reached the highest value in the last 60 min. During the whole intestinal digestion stage, all Pickering emulsions stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex showed higher free fatty acid release than soybean oil, indicating that Pickering emulsions stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex can promote lipid decomposition. After 120 min of digestion, the lipolysis degrees of the samples were 31.08% (soybean oil), 41.23% (0.4wt%), 45.99% (0.6wt%), 52.24% (0.8wt%), 66.44% (1wt%) and 73.28% (1.5wt%), respectively. The results showed that increasing the content of xanthan gum in the Chlamydomonas reinhardtii protein-xanthan gum complex can make the lipolysis degree of Pickering emulsion higher.

[0046] Experimental Example 12: After the Pickering emulsion loaded with lutein in Example 7 was subjected to simulated in vitro digestion, the conversion rate, bioaccessibility index and bioaccessibility of lutein were determined. The calculation method is as follows: The original undiluted emulsion was used in the oral digestion stage. The above three-stage digestion process was repeated, and the digestion liquid was centrifuged at 10,000 rpm for 60 min, and the middle layer of the lutein micelle layer of the centrifuge was collected. The lutein content was extracted and determined from the original digestion liquid and the micelles after centrifugation. The conversion rate, bioaccessibility index and bioaccessibility of lutein were calculated according to the following formulas: Conversion rate (%) = lutein concentration in the total digestate / initial lutein concentration × 100%; Bioaccessibility index (%) = lutein concentration in micelles / lutein concentration in the total digestate × 100%; Bioaccessibility (%) = lutein concentration in micelles / initial lutein concentration × 100%. The results are as follows. Fig.12 shown.

[0047] Fig.12The results showed that the Pickering emulsion-based delivery system significantly enhanced the conversion rate, bioaccessibility index and bioaccessibility of lutein. The bioaccessibility of lutein in unencapsulated soybean oil was only 16.72%, while the bioaccessibility of lutein in the Pickering emulsion stabilized by 0.4wt% C. reinhardtii protein-xanthan gum complex was significantly increased to 43.78%. The bioaccessibility of lutein in the Pickering emulsion stabilized by C. reinhardtii protein-xanthan gum complex with different xanthan gum contents was 43.78% (0.4wt%) < 44.17% (0.6wt%) < 45.71% (0.8wt%) < 48.00% (1wt%) < 52.21% (1.5wt%). The bioaccessibility of lutein in the Pickering emulsion was positively correlated with the content of xanthan gum in the C. reinhardtii protein-xanthan gum complex. The Pickering emulsion stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex can be used as an effective delivery system for hydrophobic substances to improve the bioaccessibility of lutein.

[0048] Experimental Example 13: The Pickering emulsion loaded with lutein in Example 7 was 3D printed. The emulsion was put into the material barrel of the 3D printer. The machine was controlled by a computer program. The shape of the 3D printed food in the present invention was "H". The results were as follows: Fig.13 shown.

[0049] Fig.13 The results show that based on the above analysis results of the environmental stability and rheological properties of Pickering emulsions, the potential of Pickering emulsions loaded with lutein stabilized by Chlamydomonas reinhardtii protein-xanthan gum complexes with different xanthan gum contents in 3D printed food was studied. The results show that the Pickering emulsion stabilized by 0.4wt% Chlamydomonas reinhardtii protein-xanthan gum complex has a relatively poor self-supporting "H" shape, and the "H" shape printed by the Pickering emulsion stabilized by 1.5wt% Chlamydomonas reinhardtii protein-xanthan gum complex has a complete shape, clear outline and high definition, and can retain the printed structure. Therefore, the Pickering emulsion loaded with lutein stabilized by Chlamydomonas reinhardtii protein-xanthan gum complex has a wide range of application value and prospects in 3D printing.

[0050] Experimental Example 14: The protein content of the Chlamydomonas reinhardtii powder prepared in Example 1-3 was determined by the Kjeldahl method. A control group without the addition of the fermented extract of Sargassum fusiformis was set up. The results are shown in Table 1.

[0051] Table 1:

[0052] The results in Table 1 show that the protein content of Chlamydomonas reinhardtii in Examples 1-3 groups is significantly higher than that in the control group, indicating that adding the fermented extract of Sargassum fusiformis of the present invention can increase the protein content of Chlamydomonas reinhardtii, thereby improving the extraction efficiency and facilitating the expansion of production.

[0053] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A Pickering emulsion for 3D printing loaded with lutein, characterized in that: The preparation raw materials include the following components: Chlamydomonas reinhardtii protein, xanthan gum, lutein, and edible oil; The method for preparing the Chlamydomonas reinhardtii protein comprises the following steps: (1) Mix Chlamydomonas reinhardtii powder and deionized water in a ratio of 1 mg: 5-20 mL, and stir magnetically at 800 rpm for 30-40 min to obtain a powder solution; (2) treating the algae powder solution obtained in step (1) with ultrasound at 500-700W for 10-20 min, adjusting the pH to 9-11 with 1 mol / L NaOH solution, stirring at 200-300 rpm for 50-70 min, centrifuging at 8000 rpm for 10-15 min, and collecting the supernatant; (3) adjusting the pH of the supernatant obtained in step (2) to 3-5 with 1 mol / L HCl solution, centrifuging at 8000 rpm for 10-15 min, collecting the precipitate, and obtaining Chlamydomonas reinhardtii protein; The method for preparing Chlamydomonas reinhardtii algae powder comprises the following steps: (a) drying and crushing Sargassum fusiformis, adding 10 times the weight of distilled water and heating, filtering and obtaining the filtrate, sterilizing, obtaining a fermentation substrate, inoculating activated Lactobacillus casei into the fermentation substrate, and culturing; (b) After the cultivation in step (a), centrifugation is performed, the supernatant is concentrated under reduced pressure, anhydrous ethanol is added, centrifugation is performed, the precipitate is mixed with distilled water, and freeze-dried to obtain a fermented extract of Sargassum fusiformis; (d) adding the fermented extract of Sargassum fusiformis obtained in step (b) in an amount of 10 g / L into TAP liquid culture medium, inoculating Chlamydomonas reinhardtii in the logarithmic growth phase, culturing, centrifuging, washing the precipitate, and freeze-drying to obtain Chlamydomonas reinhardtii algae powder.

2. The Pickering emulsion for 3D printing loaded with lutein according to claim 1, characterized in that: The edible oil is one or a combination of corn oil, peanut oil, soybean oil, sunflower oil and olive oil.

3. A Pickering emulsion stabilized by a Chlamydomonas reinhardtii protein-xanthan gum complex, characterized in that: The preparation method comprises the following steps: S1: Dissolve xanthan gum in deionized water, heat at 60-90°C for 5-7 h to completely dissolve it, and obtain a xanthan gum solution; dissolve Chlamydomonas reinhardtii protein in deionized water, adjust the pH value to 7, and obtain a Chlamydomonas reinhardtii protein solution with a concentration of 3wt%; add different amounts of xanthan gum solution to the Chlamydomonas reinhardtii protein solution, so that the concentrations of xanthan gum in the solution system are 0.4wt%, 0.6wt%, 0.8wt%, 1wt% and 1.5wt%, respectively; and stir magnetically at 700-900 rpm for 20-25 h to obtain a Chlamydomonas reinhardtii protein-xanthan gum complex solution; S2: The Chlamydomonas reinhardtii protein-xanthan gum complex solution obtained in step S1 is mixed with edible oil in a volume ratio of 3:7, and high-speed shearing is performed at 9000-11000 rpm for 3-5 min to obtain a Pickering emulsion stabilized by the Chlamydomonas reinhardtii protein-xanthan gum complex.

4. A Pickering emulsion loaded with lutein, characterized in that: The preparation method comprises the following steps: dissolving 5 wt% lutein in edible oil, stirring at 200-300 rpm for 20-30 h in a dark environment, mixing the edible oil and the Chlamydomonas reinhardtii protein-xanthan gum complex solution in a volume ratio of 7:3, and high-speed shearing at 9000-11000 rpm for 3-5 min to obtain a Pickering emulsion loaded with lutein.

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