Organic wall-broken chlorella pyrenoidosa composite solid beverage and preparation method thereof

Abstract

The invention relates to the technical field of fermentation engineering, and discloses an organic wall-broken chlorella pyrenoidosa composite solid beverage and a preparation method thereof, the organic wall-broken chlorella pyrenoidosa composite solid beverage comprises the following raw materials by weight: 10-20 parts of wall-broken chlorella pyrenoidosa powder, 5-15 parts of fermentation auxiliary material dry powder, 3-8 parts of fructo-oligosaccharide, 5-10 parts of soybean protein isolate, 3-6 parts of coconut milk powder, 1-3 parts of lemon powder, and 0.001-0.002 part of selenomethionine. According to the technology, a complete technical chain is formed from raw material treatment to finished product packaging, efficient reservation of active ingredients, synergistic enhancement of functions, sensory quality optimization and application performance improvement are achieved, and the requirements for functional solid beverages in different scenes are met.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, specifically to an organic cell wall-breaking protein Chlorella complex solid beverage and its preparation method. Background Technology

[0002] With the development of the functional food industry, Chlorella proteinifera, rich in chlorophyll, chlorella growth factor (CGF), protein, and other active ingredients, has become an important raw material for solid beverages. However, the cell wall structure of Chlorella is dense, and conventional cell wall breaking processes suffer from insufficient cell wall breaking and high loss of active ingredients. Although mechanical grinding can improve the cell wall breaking rate, the frictional heat generated at room temperature can easily degrade heat-sensitive components, and the powder is prone to agglomeration, affecting subsequent reconstitution and solubility.

[0003] Meanwhile, existing chlorella solid beverages often include oats, peas, and other ingredients to enrich their nutritional value. However, these ingredients are usually directly ground and added, and their large starch and protein molecules are not converted. This results in low digestibility and absorption rates, and the product's palatability is reduced due to its rough texture and astringent taste. Some technologies attempt to improve the flavor of these ingredients through single-strain fermentation, but single-strain fermentation cannot efficiently degrade multiple large molecules, making it difficult to simultaneously achieve the dual goals of increasing the content of functional components and optimizing flavor.

[0004] Therefore, developing a compound solid beverage preparation technology that can achieve efficient cell wall disruption of Chlorella, precise retention of active ingredients, and enhanced functionality and flavor through deep conversion of excipients has become an urgent need in the industry. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides an organic cell wall-breaking protein Chlorella complex solid beverage and its preparation method.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: an organic cell wall-breaking protein Chlorella complex solid beverage, composed of the following raw materials in parts by weight:

[0009] 10-20 parts of broken cell wall protein Chlorella powder, 5-15 parts of fermentation auxiliary powder, 3-8 parts of fructooligosaccharides, 5-10 parts of soy protein isolate, 3-6 parts of coconut milk powder, 1-3 parts of lemon powder, and 0.001-0.002 parts of selenomethionine.

[0010] The preparation method of the cell wall-broken Chlorella powder includes the following steps:

[0011] A1. Mix Chlorella proteoglycans with deionized water at a weight ratio of 1:2-3, put the mixture into a high-pressure homogenizer, and circulate it three times at 0-4℃ and 280-300MPa pressure, holding the pressure for 30s each time. Then transfer it to an enzymatic hydrolysis reactor and stir it for 20-30 minutes at 30-35℃ and 150-200r / min to prepare a uniform Chlorella proteoglycans slurry. Adjust the pH of the slurry to 4.5-5.0 with citric acid and set aside.

[0012] A2. First, add 60% of the total enzyme amount of Trichoderma reesei cellulase, stir at 35-37℃ and 100-120 r / min for 1-2 hours for enzymatic hydrolysis; then add 30% of the total enzyme amount of Aspergillus niger pectinase, raise the temperature to 39-42℃ and continue the reaction for 2-3 hours; finally, add 10% of the total enzyme amount of Aspergillus oryzae protease, maintain the reaction at 35-37℃ for 1-1.5 hours, and immediately after the enzymatic hydrolysis is completed, pass saturated steam for 15-20 seconds and keep it for 10-15 minutes to inactivate the enzyme.

[0013] A3. The inactivated Chlorella protein hydrolysate was transferred to a vacuum freeze dryer for drying to obtain Chlorella protein powder with broken cell walls.

[0014] Furthermore, the preparation method of the fermentation auxiliary powder includes the following steps:

[0015] B1. Peel and grind oats, add 3 times the volume of deionized water, and soak in a water bath at 60-80℃ for 1.5-2 hours; dice carrots, blanch in hot water at 90-95℃ for 3-5 minutes, cool, and then blend into a smooth carrot puree using a blender; peel and grind peas into powder, pass through a 60-mesh sieve, add 2 times the volume of deionized water, and stir at 50-60℃ for 15-30 minutes; dry and grind chicory roots, add 4 times the volume of hot water at 80℃, keep warm and extract for 1 hour, filter and keep the filtrate; retain the residue.

[0016] B2. Mix the above-treated oats, pea flour, carrot pulp, and chicory root residue, add chicory root extract, and then add deionized water. The total weight of the materials to the weight of the water is 1:1.5-2. Put the mixture into a fermentation tank and stir for 30-40 minutes at 40-45℃ and 200-250 r / min to prepare the auxiliary fermentation substrate.

[0017] B3. Pasteurize the fermentation substrate by heating it to 68-72℃ and holding it at that temperature for 20-30 minutes, then rapidly cooling it to 30-32℃ for later use.

[0018] B4. Weigh out Lactobacillus casei, Saccharomyces cerevisiae, and Aspergillus niger in a weight ratio of 3:2:1. After activation, dissolve them separately in sterile physiological saline. The weight ratio of bacterial strain to physiological saline is 1:10 to prepare bacterial suspensions. Mix the three bacterial suspensions after activation to obtain a compound fermentation inoculum for later use.

[0019] B5. Inoculate the cooled auxiliary material fermentation substrate with 5-8% of the compound fermentation inoculum, maintain the temperature at 28-30℃, and the aeration rate at 0.5-1L / (L·min) for 18-24h; adjust the aeration rate of the fermenter to 0.1-0.2L / (L·min), raise the temperature to 32-35℃, and continue fermentation for 24-30h.

[0020] B6. Cool the fermentation broth to 20-25℃, first coarsely filter it with a 200-mesh nylon filter, then finely filter it through a plate and frame filter press with a filter cloth pore size of 10μm. Wash the filter residue twice with deionized water, and combine the washing liquid and filtrate. Use a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3kDa to perform ultrafiltration on the washing liquid and filtrate under the conditions of operating pressure of 0.2MPa and temperature of 30℃, and collect the permeate.

[0021] B7. Pump the permeate into the ion exchange resin column at a flow rate of 1-2 BV / h; pass it through the food-grade granular activated carbon column to adsorb the dark impurities produced by the oxidation of carotene.

[0022] B8. The purified liquid is transferred to a vacuum concentrator for concentration. After concentration, it is freeze-dried under vacuum to obtain fermentation auxiliary powder.

[0023] Furthermore, in step A2, the activity of Trichoderma reesei cellulase is 5000 U / g, the activity of Aspergillus niger pectinase is 3000 U / g, and the activity of Aspergillus oryzae protease is 2000 U / g. The total amount of enzymes added is 1.0 to 1.2% of the dry matter weight of Chlorella pyrenoidosa.

[0024] Furthermore, the vacuum freeze-drying process in step A3 is carried out in three stages: the first stage is pre-freezing at -35 to -38℃ for 3 hours; the second stage is vacuum drying at -30 to -35℃ for 8 to 10 hours; and the third stage is vacuum drying at -20 to -30℃ for 2 to 4 hours, ensuring that the looseness of the dry powder is ≥0.8 g / cm³. 3 .

[0025] Furthermore, in step B1, the mass ratio of peeled oats, carrots, peeled peas, and dried chicory root is 2:0.8–1:1–1.2:0.3–0.6.

[0026] Further, in step B4, *Lactobacillus casei* was activated using MRS medium containing 0.5% chicory root extract, under anaerobic conditions at 37°C for 4–6 hours; *Saccharomyces cerevisiae* was activated using YPD medium containing 0.3% carrot pulp, under aerobic conditions at 30°C for 3–4 hours; and *Aspergillus niger* was activated using PDA medium containing 0.2% oat gelatinized liquid, under aerobic conditions at 28°C for 6–8 hours. The total viable count of the activated compound fermentation culture was ≥10⁻⁶. 8 CFU / mL.

[0027] Furthermore, in step B7, the ion exchange resin column is a mixture of cation resin 001×7 and anion resin D301 in a volume ratio of 1:1.

[0028] Further, in step B8, the vacuum concentration is carried out at 50-55°C and a vacuum degree of -0.08 to -0.09 MPa until the solid content is 45-50%. The vacuum freeze-drying process is carried out at -20 to -40°C, 10-20 Pa, for 10-12 hours.

[0029] Furthermore, a method for preparing an organic cell wall-breaking protein Chlorella complex solid beverage includes the following steps:

[0030] S1. Weigh the dry powder of fermentation auxiliary materials, put it into a three-dimensional mixer, add fructooligosaccharides, soy protein isolate, coconut milk powder, lemon powder, and selenomethionine, and mix for 25 to 30 minutes at a speed of 180 to 220 r / min. Add 0.5 to 0.8% of food-grade silica by weight of the total materials to the mixture and continue mixing for 10 to 15 minutes.

[0031] S2. Transfer the mixed material from S1 into a vacuum drying oven and dry it for 2-3 hours at 40-50℃ and a vacuum of -0.08 to -0.09 MPa, controlling the moisture content of the material to ≤2%.

[0032] S3. Put the dried material into a low-temperature ultrafine pulverizer, control the temperature inside the machine to ≤25℃, pulverize for 15 to 20 minutes at a speed of 30000 to 35000 r / min, pass through a 400 to 600 mesh sieve, add broken cell wall protein Chlorella powder, and stir evenly to obtain a composite solid beverage powder.

[0033] S4. In a Class 100,000 cleanroom, seal the product in 5g / bag or 10g / bag specifications to obtain the compound solid beverage.

[0034] (III) Beneficial Technical Effects

[0035] This invention relates to an organic, cell-wall-broken Chlorella-based compound solid beverage that addresses key issues such as insufficient cell wall disruption of Chlorella, easy loss of active ingredients, limited excipient function, and poor product solubility through a multi-process synergistic approach. Low-temperature, high-pressure homogenization creates microcracks in the cell walls, followed by stepwise enzymatic hydrolysis to achieve cell wall disruption. This ensures a high disruption rate, releasing internal active substances such as CGF and chlorophyll, while avoiding component degradation caused by mechanical friction and heat generation. Simultaneously, it refines the powder particle size to improve solubility. The excipient preparation employs a multi-strain, staged fermentation process. Aspergillus niger first degrades the large molecules of the raw material to generate small-molecule carbon sources, followed by synergistic metabolism by lactic acid bacteria and yeast to produce functional components such as small-molecule peptides and fructooligosaccharides. This not only enhances the prebiotic effect to regulate intestinal flora but also improves the product flavor by degrading astringent substances in the raw materials. Subsequent purification processes further remove impurities and improve the compatibility between the excipients and Chlorella powder. Optimized mixing and low-temperature drying processes, combined with anti-caking agents, ensure uniform powder that is not prone to clumping and dissolves quickly without sediment during reconstitution.

[0036] The process of this invention forms a complete technology chain from raw material processing to finished product packaging, achieving efficient retention of active ingredients, enhanced functional synergy, optimized sensory quality, and improved application performance, thus meeting the needs of functional solid beverages in different scenarios. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, all components of the organic cell wall-breaking protein Chlorella complex solid beverage formula of this invention are commercially available.

[0039] All parts used in this invention are by weight; percentages, unless otherwise specified, are by mass percentage.

[0040] Example 1

[0041] An organic, cell-wall-broken protein Chlorella complex solid beverage is composed of the following ingredients in parts by weight:

[0042] 10 parts of broken cell wall protein Chlorella powder, 5 parts of fermentation auxiliary powder, 3 parts of fructooligosaccharide, 5 parts of soy protein isolate, 3 parts of coconut milk powder, 1 part of lemon powder, and 0.001 parts of selenomethionine.

[0043] The preparation method of the cell wall-broken Chlorella powder includes the following steps:

[0044] A1. Mix Chlorella proteoglycans with deionized water at a weight ratio of 1:2, put the mixture into a high-pressure homogenizer, and circulate it three times at 0℃ and 280MPa pressure, holding the pressure for 30s each time. Then transfer it to an enzymatic hydrolysis reactor and stir it for 20min at 30℃ and 150r / min to prepare a uniform Chlorella proteoglycans slurry. Adjust the pH of the slurry to 4.5 with citric acid and set aside.

[0045] A2. First, add 60% of the total enzyme amount of Trichoderma reesei cellulase, stir at 35℃ and 100r / min for 1 hour for enzymatic hydrolysis; then add 30% of the total enzyme amount of Aspergillus niger pectinase, raise the temperature to 39℃ and continue the reaction for 2 hours; finally, add 10% of the total enzyme amount of Aspergillus oryzae protease, maintain the reaction at 35℃ for 1 hour, and immediately after the enzymatic hydrolysis is completed, pass saturated steam for 15 seconds and keep it for 10 minutes to inactivate the enzyme.

[0046] A3. The inactivated Chlorella protein hydrolysate was transferred to a vacuum freeze dryer for drying to obtain Chlorella protein powder with broken cell walls.

[0047] In step A2, the activity of cellulase from Trichoderma reesei is 5000 U / g, the activity of pectinase from Aspergillus niger is 3000 U / g, and the activity of protease from Aspergillus oryzae is 2000 U / g. The total amount of enzymes added is 1.0% of the dry matter weight of Chlorella pyrenoidosa.

[0048] Step A3 involves a three-stage vacuum freeze-drying process: the first stage is pre-freezing at -35℃ for 3 hours, the second stage is vacuum drying at -30℃ for 8 hours, and the third stage is vacuum drying at -20℃ for 2 hours, ensuring the dry powder has a porosity ≥0.8 g / cm³. 3 .

[0049] The preparation method of fermentation auxiliary powder includes the following steps:

[0050] B1. Peel and grind oats, add 3 times the volume of deionized water, and soak in a 60℃ water bath for 1.5 hours; dice carrots, blanch in 90℃ hot water for 3 minutes, cool, and then blend into a smooth carrot puree using a blender; peel and grind peas into powder, pass through a 60-mesh sieve, add 2 times the volume of deionized water, and stir at 50℃ for 15 minutes; dry and grind chicory roots, add 4 times the volume of 80℃ hot water, keep warm and extract for 1 hour, filter and keep the filtrate for later use, and retain the residue;

[0051] B2. Mix the above-treated oats, pea flour, carrot pulp, and chicory root residue, add chicory root extract, and then add deionized water. The total weight of the materials to the weight of the water is 1:1.5. Put the mixture into a fermentation tank and stir for 30 minutes at 40°C and 200 r / min to prepare the auxiliary material fermentation substrate.

[0052] B3. Pasteurize the fermentation substrate, heat it to 68℃ and keep it at that temperature for 20 minutes, then rapidly cool it to 30℃ and set it aside.

[0053] B4. Weigh out Lactobacillus casei, Saccharomyces cerevisiae, and Aspergillus niger in a weight ratio of 3:2:1. After activation, dissolve them separately in sterile physiological saline. The weight ratio of bacterial strain to physiological saline is 1:10 to prepare bacterial suspensions. Mix the three bacterial suspensions after activation to obtain a compound fermentation inoculum for later use.

[0054] B5. Inoculate the cooled auxiliary material fermentation substrate with 5% compound fermentation inoculum solution by volume, maintain the temperature at 28℃, and the aeration rate at 0.5L / (L·min) for 18h; adjust the aeration rate of the fermenter to 0.1L / (L·min), raise the temperature to 32℃, and continue fermentation for 24h.

[0055] B6. Cool the fermentation broth to 20°C, first coarsely filter it using a 200-mesh nylon filter, then finely filter it using a plate and frame filter press with a filter cloth pore size of 10μm. Wash the filter residue twice with deionized water, and combine the washing liquid and filtrate. Use a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3kDa to perform ultrafiltration on the washing liquid and filtrate under the conditions of operating pressure of 0.2MPa and temperature of 30°C, and collect the permeate.

[0056] B7. Pump the permeate into the ion exchange resin column at a flow rate of 1 BV / h; pass it through the food-grade granular activated carbon column to adsorb the dark impurities produced by the oxidation of carotene.

[0057] B8. The purified liquid is transferred to a vacuum concentrator for concentration. After concentration, it is freeze-dried under vacuum to obtain fermentation auxiliary powder.

[0058] In step B1, the mass ratio of peeled oats, carrots, peeled peas, and dried chicory root is 2:0.8:1:0.3.

[0059] In step B4, *Lactobacillus casei* was activated using MRS medium containing 0.5% chicory root extract under anaerobic conditions at 37°C for 4 hours; *Saccharomyces cerevisiae* was activated using YPD medium containing 0.3% carrot pulp under aerobic conditions at 30°C for 3 hours; and *Aspergillus niger* was activated using PDA medium containing 0.2% oat gelatinized liquid under aerobic conditions at 28°C for 6 hours. The total viable count of the activated compound fermentation culture was ≥10⁻⁶. 8 CFU / mL.

[0060] In step B7, the ion exchange resin column is filled with a mixture of cation resin 001×7 and anion resin D301 in a volume ratio of 1:1.

[0061] In step B8, vacuum concentration is carried out at 50°C and a vacuum degree of -0.08MPa until the solid content is 45%. The vacuum freeze-drying process is carried out at -20°C, 10Pa, for 10 hours.

[0062] A method for preparing an organic cell wall-breaking protein Chlorella complex solid beverage includes the following steps:

[0063] S1. Weigh the fermentation auxiliary powder, put it into a three-dimensional mixer, add fructooligosaccharides, soy protein isolate, coconut milk powder, lemon powder, and selenomethionine, mix for 25 minutes at a speed of 180 r / min, add 0.5% food-grade silica by weight of the total material to the mixture, and continue mixing for 10 minutes.

[0064] S2. Transfer the material mixed in S1 into a vacuum drying oven and dry it at 40℃ and a vacuum of -0.08MPa for 2 hours, controlling the moisture content of the material to be ≤2%.

[0065] S3. Put the dried material into a low-temperature ultrafine pulverizer, control the temperature inside the machine to ≤25℃, pulverize for 15 minutes at a speed of 30000r / min, pass through a 400-mesh sieve, add broken cell wall protein Chlorella powder, and stir evenly to obtain composite solid beverage powder.

[0066] S4. In a Class 100,000 cleanroom, seal the product in 5g / bag or 10g / bag specifications to obtain the compound solid beverage.

[0067] Example 2

[0068] An organic, cell-wall-broken protein Chlorella complex solid beverage is composed of the following ingredients in parts by weight:

[0069] 15 parts of broken cell wall protein Chlorella powder, 10 parts of fermentation auxiliary powder, 5 parts of fructooligosaccharide, 8 parts of soy protein isolate, 4 parts of coconut milk powder, 2 parts of lemon powder, and 0.001 parts of selenomethionine.

[0070] The preparation method of the cell wall-broken Chlorella powder includes the following steps:

[0071] A1. Mix Chlorella proteoglycans with deionized water at a weight ratio of 1:2.5, put the mixture into a high-pressure homogenizer, and circulate it three times at 2℃ and 290MPa pressure, holding the pressure for 30s each time. Then transfer it to an enzymatic hydrolysis reactor and stir it at 32℃ and 180r / min for 25min to prepare a uniform Chlorella proteoglycans slurry. Adjust the pH of the slurry to 5.0 with citric acid and set aside.

[0072] A2. First, add 60% of the total enzyme amount of Trichoderma reesei cellulase, stir at 36℃ and 110 r / min for 1.5 h for enzymatic hydrolysis; then add 30% of the total enzyme amount of Aspergillus niger pectinase, heat to 40℃ and continue the reaction for 2.5 h; finally, add 10% of the total enzyme amount of Aspergillus oryzae protease, maintain the reaction at 36℃ for 1 h, and immediately after the enzymatic hydrolysis is completed, pass saturated steam for 20 s and keep for 15 min to inactivate the enzyme.

[0073] A3. The inactivated Chlorella protein hydrolysate was transferred to a vacuum freeze dryer for drying to obtain Chlorella protein powder with broken cell walls.

[0074] In step A2, the activity of cellulase from Trichoderma reesei was 5000 U / g, the activity of pectinase from Aspergillus niger was 3000 U / g, and the activity of protease from Aspergillus oryzae was 2000 U / g. The total amount of enzymes added was 1.1% of the dry matter weight of Chlorella pyrenoidosa.

[0075] Step A3 involves a three-stage vacuum freeze-drying process: the first stage is pre-freezing at -36℃ for 3 hours, the second stage is vacuum drying at -32℃ for 9 hours, and the third stage is vacuum drying at -25℃ for 3 hours, ensuring the dry powder has a porosity ≥0.8 g / cm³. 3 .

[0076] The preparation method of fermentation auxiliary powder includes the following steps:

[0077] B1. Peel and grind oats, add 3 times the volume of deionized water, and soak in a 70℃ water bath for 2 hours; dice carrots, blanch in 95℃ hot water for 4 minutes, cool, and then blend into a smooth carrot puree using a blender; peel and grind peas into powder, pass through a 60-mesh sieve, add 2 times the volume of deionized water, and stir at 55℃ for 20 minutes; dry and grind chicory roots, add 4 times the volume of 80℃ hot water, keep warm and extract for 1 hour, filter and keep the filtrate; retain the residue.

[0078] B2. Mix the above-treated oats, pea flour, carrot pulp, and chicory root residue, add chicory root extract, and then add deionized water. The total weight of the materials to the weight of the water is 1:1.8. Put the mixture into a fermentation tank and stir for 35 minutes at 42℃ and 220r / min to prepare the auxiliary material fermentation substrate.

[0079] B3. Pasteurize the fermentation substrate, heat it to 70°C and keep it at that temperature for 25 minutes, then rapidly cool it to 32°C and set it aside.

[0080] B4. Weigh out Lactobacillus casei, Saccharomyces cerevisiae, and Aspergillus niger in a weight ratio of 3:2:1. After activation, dissolve them separately in sterile physiological saline. The weight ratio of bacterial strain to physiological saline is 1:10 to prepare bacterial suspensions. Mix the three bacterial suspensions after activation to obtain a compound fermentation inoculum for later use.

[0081] B5. Inoculate the cooled auxiliary material fermentation substrate with 6% compound fermentation inoculum solution by volume, maintain the temperature at 30℃, and the aeration rate at 0.6L / (L·min) for 20h; adjust the aeration rate of the fermenter to 0.1L / (L·min), raise the temperature to 35℃, and continue fermentation for 24h.

[0082] B6. Cool the fermentation broth to 20°C, first coarsely filter it using a 200-mesh nylon filter, then finely filter it using a plate and frame filter press with a filter cloth pore size of 10μm. Wash the filter residue twice with deionized water, and combine the washing liquid and filtrate. Use a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3kDa to perform ultrafiltration on the washing liquid and filtrate under the conditions of operating pressure of 0.2MPa and temperature of 30°C, and collect the permeate.

[0083] B7. Pump the permeate into the ion exchange resin column at a flow rate of 2 BV / h; pass it through the food-grade granular activated carbon column to adsorb the dark impurities produced by the oxidation of carotene.

[0084] B8. The purified liquid is transferred to a vacuum concentrator for concentration. After concentration, it is freeze-dried under vacuum to obtain fermentation auxiliary powder.

[0085] In step B1, the mass ratio of peeled oats, carrots, peeled peas, and dried chicory root is 2:0.9:1.1:0.5.

[0086] In step B4, *Lactobacillus casei* was activated using MRS medium containing 0.5% chicory root extract under anaerobic conditions at 37°C for 5 hours; *Saccharomyces cerevisiae* was activated using YPD medium containing 0.3% carrot pulp under aerobic conditions at 30°C for 3.5 hours; and *Aspergillus niger* was activated using PDA medium containing 0.2% oat gelatinized liquid under aerobic conditions at 28°C for 7 hours. The total viable count of the activated compound fermentation culture was ≥10⁻⁶. 8 CFU / mL.

[0087] In step B7, the ion exchange resin column is filled with a mixture of cation resin 001×7 and anion resin D301 in a volume ratio of 1:1.

[0088] In step B8, vacuum concentration is carried out at 55°C and a vacuum degree of -0.08MPa until the solid content is 45%. The vacuum freeze-drying process is carried out at -30°C, 15Pa, for 11 hours.

[0089] A method for preparing an organic cell wall-breaking protein Chlorella complex solid beverage includes the following steps:

[0090] S1. Weigh the fermentation auxiliary powder, put it into a three-dimensional mixer, add fructooligosaccharides, soy protein isolate, coconut milk powder, lemon powder, and selenomethionine, mix for 30 minutes at a speed of 200 r / min, add 0.6% food-grade silica by weight of the total material to the mixture, and continue mixing for 15 minutes.

[0091] S2. Transfer the material mixed in S1 into a vacuum drying oven and dry it at 45℃ and a vacuum of -0.08MPa for 3 hours, controlling the moisture content of the material to be ≤2%.

[0092] S3. Put the dried material into a low-temperature ultrafine pulverizer, control the temperature inside the machine to ≤25℃, pulverize for 20 minutes at a speed of 32000r / min, pass through a 500-mesh sieve, add broken cell wall protein Chlorella powder, and stir evenly to obtain composite solid beverage powder.

[0093] S4. In a Class 100,000 cleanroom, seal the product in 5g / bag or 10g / bag specifications to obtain the compound solid beverage.

[0094] Example 3

[0095] An organic, cell-wall-broken protein Chlorella complex solid beverage is composed of the following ingredients in parts by weight:

[0096] 20 parts of broken cell wall protein Chlorella powder, 15 parts of fermentation auxiliary powder, 8 parts of fructooligosaccharides, 10 parts of soy protein isolate, 6 parts of coconut milk powder, 3 parts of lemon powder, and 0.002 parts of selenomethionine.

[0097] The preparation method of the cell wall-broken Chlorella powder includes the following steps:

[0098] A1. Mix Chlorella proteoglycans with deionized water at a weight ratio of 1:3, put the mixture into a high-pressure homogenizer, and circulate it three times at 4℃ and 300MPa pressure, holding the pressure for 30s each time. Then transfer it to an enzymatic hydrolysis tank and stir it at 35℃ and 200r / min for 30min to prepare a uniform Chlorella proteoglycans slurry. Adjust the pH of the slurry to 5.0 with citric acid and set aside.

[0099] A2. First, add 60% of the total enzyme amount of Trichoderma reesei cellulase, stir at 37℃ and 120r / min for 2 hours for enzymatic hydrolysis; then add 30% of the total enzyme amount of Aspergillus niger pectinase, raise the temperature to 42℃ and continue the reaction for 3 hours; finally, add 10% of the total enzyme amount of Aspergillus oryzae protease, maintain the reaction at 37℃ for 1.5 hours, and immediately after the enzymatic hydrolysis is completed, pass saturated steam for 20 seconds and keep it for 15 minutes to inactivate the enzyme.

[0100] A3. The inactivated Chlorella protein hydrolysate was transferred to a vacuum freeze dryer for drying to obtain Chlorella protein powder with broken cell walls.

[0101] In step A2, the activity of cellulase from Trichoderma reesei was 5000 U / g, the activity of pectinase from Aspergillus niger was 3000 U / g, and the activity of protease from Aspergillus oryzae was 2000 U / g. The total amount of enzymes added was 1.2% of the dry matter weight of Chlorella pyrenoidosa.

[0102] Step A3 involves a three-stage vacuum freeze-drying process: the first stage is pre-freezing at -38℃ for 3 hours, the second stage is vacuum drying at -35℃ for 10 hours, and the third stage is vacuum drying at -30℃ for 4 hours, ensuring the dry powder has a porosity ≥0.8 g / cm³. 3 .

[0103] The preparation method of fermentation auxiliary powder includes the following steps:

[0104] B1. Peel and grind oats, add 3 times the volume of deionized water, and soak in an 80℃ water bath for 2 hours; dice carrots, blanch in 95℃ hot water for 5 minutes, cool, and then blend into a smooth carrot puree using a blender; peel and grind peas into powder, pass through a 60-mesh sieve, add 2 times the volume of deionized water, and stir at 60℃ for 30 minutes; dry and grind chicory roots, add 4 times the volume of 80℃ hot water, keep warm and extract for 1 hour, filter and keep the filtrate; retain the residue.

[0105] B2. Mix the above-treated oats, pea flour, carrot pulp, and chicory root residue, add chicory root extract, and then add deionized water. The total weight of the materials to the weight of the water is 1:2. Put the mixture into a fermentation tank and stir for 40 minutes at 45°C and 250 r / min to prepare the auxiliary material fermentation substrate.

[0106] B3. Pasteurize the fermentation substrate, heat it to 72°C and keep it at that temperature for 30 minutes, then rapidly cool it to 32°C and set it aside.

[0107] B4. Weigh out Lactobacillus casei, Saccharomyces cerevisiae, and Aspergillus niger in a weight ratio of 3:2:1. After activation, dissolve them separately in sterile physiological saline. The weight ratio of bacterial strain to physiological saline is 1:10 to prepare bacterial suspensions. Mix the three bacterial suspensions after activation to obtain a compound fermentation inoculum for later use.

[0108] B5. Inoculate the cooled auxiliary material fermentation substrate with 8% compound fermentation inoculum solution by volume, maintain the temperature at 30℃, and the aeration rate at 1L / (L·min) for 24h; adjust the aeration rate of the fermenter to 0.2L / (L·min), raise the temperature to 35℃, and continue fermentation for 30h.

[0109] B6. Cool the fermentation broth to 25°C, first coarsely filter it using a 200-mesh nylon filter, then finely filter it using a plate and frame filter press with a filter cloth pore size of 10μm. Wash the filter residue twice with deionized water, and combine the washing liquid and filtrate. Use a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3kDa to perform ultrafiltration on the washing liquid and filtrate under the conditions of operating pressure of 0.2MPa and temperature of 30°C, and collect the permeate.

[0110] B7. Pump the permeate into the ion exchange resin column at a flow rate of 2 BV / h; pass it through the food-grade granular activated carbon column to adsorb the dark impurities produced by the oxidation of carotene.

[0111] B8. The purified liquid is transferred to a vacuum concentrator for concentration. After concentration, it is freeze-dried under vacuum to obtain fermentation auxiliary powder.

[0112] In step B1, the mass ratio of peeled oats, carrots, peeled peas, and dried chicory root is 2:1:1.2:0.6.

[0113] In step B4, *Lactobacillus casei* was activated using MRS medium containing 0.5% chicory root extract under anaerobic conditions at 37°C for 6 hours; *Saccharomyces cerevisiae* was activated using YPD medium containing 0.3% carrot pulp under aerobic conditions at 30°C for 4 hours; and *Aspergillus niger* was activated using PDA medium containing 0.2% oat gelatinized liquid under aerobic conditions at 28°C for 8 hours. The total viable count of the activated compound fermentation culture was ≥10⁻⁶. 8 CFU / mL.

[0114] In step B7, the ion exchange resin column is filled with a mixture of cation resin 001×7 and anion resin D301 in a volume ratio of 1:1.

[0115] In step B8, vacuum concentration is carried out at 55°C and a vacuum degree of -0.09MPa until the solid content is 50%. The vacuum freeze-drying process is carried out at -40°C, 20Pa, for 12 hours.

[0116] A method for preparing an organic cell wall-breaking protein Chlorella complex solid beverage includes the following steps:

[0117] S1. Weigh the fermentation auxiliary powder, put it into a three-dimensional mixer, add fructooligosaccharides, soy protein isolate, coconut milk powder, lemon powder, and selenomethionine, mix for 30 minutes at a speed of 220 r / min, add 0.8% food-grade silica by weight of the total material to the mixture, and continue mixing for 15 minutes.

[0118] S2. Transfer the mixed material from S1 into a vacuum drying oven and dry it at 40℃ and a vacuum of -0.09MPa for 3 hours, controlling the moisture content of the material to be ≤2%.

[0119] S3. Put the dried material into a low-temperature ultrafine pulverizer, control the temperature inside the machine to ≤25℃, pulverize for 20 minutes at a speed of 35000r / min, pass through a 600-mesh sieve, add broken cell wall protein Chlorella powder, and stir evenly to obtain composite solid beverage powder.

[0120] S4. In a Class 100,000 cleanroom, seal the product in 5g / bag or 10g / bag specifications to obtain the compound solid beverage.

[0121] Comparative Example 1: Without performing the high-pressure homogenization in step A1, the mixture was directly mixed with deionized water and transferred to an enzymatic fermentation tank. The remaining processes were the same as in Example 1.

[0122] Comparative Example 2: The broken cell wall protein Chlorella powder was mechanically broken down by putting it into a mechanical grinder at room temperature and a speed of 15,000 r / min for 20 min, and then passing it through a 900 mesh sieve. The remaining processes were the same as in Example 1.

[0123] Comparative Example 3: The auxiliary materials were not fermented. Peeled oats, carrots, peeled peas, and dried chicory roots were directly mixed according to the mass ratio of Example 1, freeze-dried and pulverized, and passed through a 400-mesh sieve. The remaining processes were the same as in Example 1.

[0124] Performance testing:

[0125] 1. Cell wall breakage rate: Microscopic counting method: Take the broken cell wall protein Chlorella powder to make a suspension, stain it and observe the number of intact cells and broken cells. Cell wall breakage rate = number of broken cells / total number of cells × 100%;

[0126] 2. Retention rate of active ingredients: Chlorophyll: Spectrophotometry (measure absorbance at wavelengths of 663nm and 645nm, and calculate chlorophyll content);

[0127] CGF (Chlorella growth factor): High performance liquid chromatography (HPLC, mobile phase: methanol-water = 30:70, detection wavelength 280 nm);

[0128] 3. Solubility: Dissolution time: the time required for 20g powder to completely dissolve in 200mL of 60℃ warm water;

[0129] Precipitation rate: After dissolution, let stand at room temperature for 2 hours, then centrifuge (3000 r / min, 10 min). Precipitation rate = (precipitate mass / total sample mass) × 100%.

[0130] 4. Intestinal tolerance (in vitro): Simulating the intestinal environment (pH 7.2, 37℃), adding Bifidobacterium (10... 7 (CFU / mL), after culturing for 24 hours, the number of viable bacteria was measured, and the fold increase of the bacterial community was calculated;

[0131] 5. Sensory evaluation: A professional judging panel of 10 people will score the sensory evaluation based on three dimensions: color (1-5 points, light green is excellent), flavor (1-5 points, no algae or fishy smell, with a light fruity aroma is excellent), and texture (1-5 points, smooth and without powdery feel is excellent). The average value will be taken.

[0132] Table 1. Test results of Chlorella cell wall disruption rate and active ingredient retention rate

[0133] Group Cell wall breakage rate (%) Chlorophyll retention rate (%) CGF retention rate (%) Example 1 96.5 91.2 87.6 Example 2 98.2 93.5 90.3 Example 3 98.1 95.8 92.5 Comparative Example 1 82.3 88.2 81.5 Comparative Example 2 95.1 85.7 78.9 Comparative Example 3 97.8 92.9 89.7

[0134] Table 2. Results of solubility test

[0135]

[0136]

[0137] Table 3 Results of intestinal tolerance and sensory evaluation tests

[0138]

[0139] As can be seen from the above, the cell wall breakage rate and active ingredient retention rate of Examples 1-3 are higher than those of Comparative Example 1, indicating that high-pressure homogenization and enzymatic hydrolysis can effectively improve the cell wall breakage rate. High-pressure homogenization can improve the efficiency of enzymatic hydrolysis and effectively retain active ingredients. The dissolution time and precipitation rate are lower than those of Comparative Example 2. Mechanical shearing can easily cause powder agglomeration and reduce the solubility. The scores for Bifidobacteria, color, flavor, and taste are higher than those of Comparative Example 3. The unfermented excipients lack small molecule peptides produced by bacterial metabolism, which reduces their effect on the proliferation of intestinal Bifidobacteria. In addition, the astringent substances contained in the unfermented peas and oats are not degraded, resulting in a decrease in flavor score. This shows that the fermentation of excipients can not only enhance the prebiotic function of the product, but also improve the sensory quality.

[0140] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An organic, cell-wall-broken protein-based Chlorella complex solid beverage, characterized in that, Composed of the following raw materials in parts by weight: 10-20 parts of broken cell wall protein Chlorella powder, 5-15 parts of fermentation auxiliary powder, 3-8 parts of fructooligosaccharides, 5-10 parts of soy protein isolate, 3-6 parts of coconut milk powder, 1-3 parts of lemon powder, and 0.001-0.002 parts of selenomethionine. The preparation method of the cell wall-broken Chlorella powder includes the following steps: A1. Mix Chlorella proteoglycans with deionized water at a weight ratio of 1:2-3, put the mixture into a high-pressure homogenizer, and circulate it three times at 0-4℃ and 280-300MPa pressure, holding the pressure for 30s each time. Then transfer it to an enzymatic hydrolysis reactor and stir it for 20-30 minutes at 30-35℃ and 150-200r / min to prepare a uniform Chlorella proteoglycans slurry. Adjust the pH of the slurry to 4.5-5.0 with citric acid and set aside. A2. First, add 60% of the total enzyme amount of Trichoderma reesei cellulase, stir at 35-37℃ and 100-120 r / min for 1-2 hours for enzymatic hydrolysis; then add 30% of the total enzyme amount of Aspergillus niger pectinase, raise the temperature to 39-42℃ and continue the reaction for 2-3 hours; finally, add 10% of the total enzyme amount of Aspergillus oryzae protease, maintain the reaction at 35-37℃ for 1-1.5 hours, and immediately after the enzymatic hydrolysis is completed, pass saturated steam for 15-20 seconds and keep it for 10-15 minutes to inactivate the enzyme. A3. The inactivated Chlorella protein hydrolysate was transferred to a vacuum freeze dryer for drying to obtain Chlorella protein powder with broken cell walls.

2. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 1, characterized in that, The preparation method of fermentation auxiliary powder includes the following steps: B1. Peel and grind oats, add 3 times the volume of deionized water, and soak in a water bath at 60-80℃ for 1.5-2 hours; dice carrots, blanch in hot water at 90-95℃ for 3-5 minutes, cool, and then blend into a smooth carrot puree using a blender; peel and grind peas into powder, pass through a 60-mesh sieve, add 2 times the volume of deionized water, and stir at 50-60℃ for 15-30 minutes; dry and grind chicory roots, add 4 times the volume of hot water at 80℃, keep warm and extract for 1 hour, filter and keep the filtrate; retain the residue. B2. Mix the above-treated oats, pea flour, carrot pulp, and chicory root residue, add chicory root extract, and then add deionized water. The total weight of the materials to the weight of the water is 1:1.5-2. Put the mixture into a fermentation tank and stir for 30-40 minutes at 40-45℃ and 200-250 r / min to prepare the auxiliary fermentation substrate. B3. Pasteurize the fermentation substrate by heating it to 68-72℃ and holding it at that temperature for 20-30 minutes, then rapidly cooling it to 30-32℃ for later use. B4. Weigh out Lactobacillus casei, Saccharomyces cerevisiae, and Aspergillus niger in a weight ratio of 3:2:

1. After activation, dissolve them separately in sterile physiological saline. The weight ratio of bacterial strain to physiological saline is 1:10 to prepare bacterial suspensions. Mix the three bacterial suspensions after activation to obtain a compound fermentation inoculum for later use. B5. Inoculate the cooled auxiliary material fermentation substrate with 5-8% of the compound fermentation inoculum, maintain the temperature at 28-30℃, and the aeration rate at 0.5-1L / (L·min) for 18-24h; adjust the aeration rate of the fermenter to 0.1-0.2L / (L·min), raise the temperature to 32-35℃, and continue fermentation for 24-30h. B6. Cool the fermentation broth to 20-25℃, first coarsely filter it with a 200-mesh nylon filter, then finely filter it through a plate and frame filter press with a filter cloth pore size of 10μm. Wash the filter residue twice with deionized water, and combine the washing liquid and filtrate. Use a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3kDa to perform ultrafiltration on the washing liquid and filtrate under the conditions of operating pressure of 0.2MPa and temperature of 30℃, and collect the permeate. B7. Pump the permeate into the ion exchange resin column at a flow rate of 1-2 BV / h; pass it through the food-grade granular activated carbon column to adsorb the dark impurities produced by the oxidation of carotene. B8. The purified liquid is transferred to a vacuum concentrator for concentration. After concentration, it is freeze-dried under vacuum to obtain fermentation auxiliary powder.

3. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 1, characterized in that, In step A2, the activity of cellulase from Trichoderma reesei is 5000 U / g, the activity of pectinase from Aspergillus niger is 3000 U / g, and the activity of protease from Aspergillus oryzae is 2000 U / g. The total amount of enzymes added is 1.0 to 1.2% of the dry weight of Chlorella pyrenoidosa.

4. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 1, characterized in that, Step A3 involves a three-stage vacuum freeze-drying process: the first stage is pre-freezing at -35 to -38°C for 3 hours; the second stage is vacuum drying at -30 to -35°C for 8 to 10 hours; and the third stage is vacuum drying at -20 to -30°C for 2 to 4 hours, ensuring the dry powder has a porosity ≥0.8 g / cm³. 3 .

5. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 2, characterized in that, In step B1, the mass ratio of peeled oats, carrots, peeled peas, and dried chicory root is 2:0.8-1:1-1.2:0.3-0.

6.

6. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 2, characterized in that, In step B4, *Lactobacillus casei* was activated using MRS medium containing 0.5% chicory root extract under anaerobic conditions at 37°C for 4–6 hours; *Saccharomyces cerevisiae* was activated using YPD medium containing 0.3% carrot pulp under aerobic conditions at 30°C for 3–4 hours; and *Aspergillus niger* was activated using PDA medium containing 0.2% oat gelatinized liquid under aerobic conditions at 28°C for 6–8 hours. The total viable count of the activated compound fermentation culture was ≥10⁻⁶. 8 CFU / mL.

7. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 2, characterized in that, In step B7, the ion exchange resin column is filled with a mixture of cation resin 001×7 and anion resin D301 in a volume ratio of 1:

1.

8. The organic cell wall-breaking protein Chlorella complex solid beverage according to claim 2, characterized in that, In step B8, vacuum concentration is carried out at 50-55℃ and a vacuum degree of -0.08--0.09MPa until the solid content is 45-50%. The vacuum freeze-drying process is carried out at -20--40℃, 10-20Pa, for 10-12 hours.

9. A method for preparing an organic cell wall-breaking protein Chlorella complex solid beverage according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Weigh the dry powder of fermentation auxiliary materials, put it into a three-dimensional mixer, add fructooligosaccharides, soy protein isolate, coconut milk powder, lemon powder, and selenomethionine, and mix for 25 to 30 minutes at a speed of 180 to 220 r / min. Add 0.5 to 0.8% of food-grade silica by weight of the total materials to the mixture and continue mixing for 10 to 15 minutes. S2. Transfer the material mixed in S1 into a vacuum drying oven and dry it for 2 to 3 hours at 40 to 50°C and a vacuum of -0.08 to -0.09 MPa, controlling the moisture content of the material to be ≤2%. S3. Put the dried material into a low-temperature ultrafine pulverizer, control the temperature inside the machine to ≤25℃, pulverize for 15 to 20 minutes at a speed of 30000 to 35000 r / min, pass through a 400 to 600 mesh sieve, add broken cell wall protein Chlorella powder, and stir evenly to obtain a composite solid beverage powder. S4. In a Class 100,000 cleanroom, seal and package the product in 5g / bag or 10g / bag specifications to obtain the compound solid beverage.

Images