Medicago sativa functional protein powder and preparation method thereof

Abstract

The embodiment of the invention discloses medicago sativa functional protein powder and a preparation method thereof. The protein content of the alfalfa functional protein powder is greater than or equal to 85%, the protein solubility is 92-95%, the emulsifying activity is greater than or equal to 70%, and the foamability is greater than or equal to 450%; the activity of superoxide dismutase (SOD) is larger than or equal to 250 U / g, the polysaccharide content of the ganoderma lucidum is larger than or equal to 2.0 mg / g, and the flavone retention rate is larger than or equal to 90%. According to the preparation method, a brand new fermentation system is constructed by introducing artificial ganoderma lucidum life active bacteria, and ultrasonic-enzymolysis synergistic extraction and low-temperature freeze-drying coupling drying processes are combined, so that the alfalfa functional protein powder with high protein content, high-activity metabolites and high functional characteristics is prepared; the problems that existing alfalfa protein powder is low in extraction rate, poor in activity and free of unique functions are solved.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to an alfalfa functional protein powder and its preparation method. Background Technology

[0002] Alfalfa, a high-protein, high-quality forage, boasts a protein content of 18%-25% and is rich in dietary fiber, flavonoids (such as alfalfa extract), and minerals (calcium, iron, zinc), making it an ideal source of plant protein. Currently, the preparation of alfalfa protein powder largely employs the traditional process of "pulverization - conventional microbial (yeast, lactic acid bacteria) fermentation - alkali extraction and acid precipitation - spray drying," which presents three major problems:

[0003] 1. The contradiction between protein extraction efficiency and activity retention: In traditional processes, high-temperature treatment (such as spray drying with an inlet air temperature of 180-220℃) easily leads to protein denaturation, with solubility of only 35%-45%; and conventional microorganisms cannot degrade anti-nutritional factors such as cellulose and phytic acid in alfalfa, with a maximum protein extraction rate of only 75% and a retention rate of less than 60% for functional components such as flavonoids.

[0004] 2. Lack of unique bioactivity: Existing products only have basic nutritional functions and do not contain microbial metabolites with special physiological activities. They cannot meet consumers' dual needs for nutrition and function and lack differentiated advantages compared with mainstream plant proteins such as soy protein and pea protein. Summary of the Invention

[0005] Therefore, this invention provides an alfalfa protein powder and its preparation method. By introducing artificially created Tai Sui (a type of bacteria) to construct a novel fermentation system, and combining ultrasonic-enzymatic hydrolysis synergistic extraction and low-temperature freeze-drying coupled drying processes, alfalfa functional protein powder with high protein content, high activity metabolites, and high functional properties is prepared, thereby solving the problems of low extraction rate, poor activity, and lack of unique functions in existing alfalfa protein powder.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides alfalfa functional protein powder, wherein the protein powder has a protein content ≥85%, a protein solubility of 92%-95%, an emulsifying activity ≥70%, and a foaming property ≥450%; and contains metabolites of artificially produced Tai Sui bioactive bacteria, wherein the superoxide dismutase (SOD) activity is ≥250 U / g, the Tai Sui polysaccharide content is ≥2.0 mg / g, and the flavonoid retention rate is ≥90%.

[0008] According to a second aspect of the present invention, the present invention provides a method for preparing alfalfa functional protein powder as described above, comprising the following steps:

[0009] (1) Domestication and expansion of artificially produced *Ulva prolifera* bioactive bacteria: Using 100-mesh alfalfa stem and leaf powder as the sole carbon and nitrogen source, the bacteria were continuously passaged 5 times in a medium at 30℃ and pH 6.0 to screen for domesticated strains with an alfalfa cell wall degradation rate ≥85%; subsequently, the bacteria were expanded in a medium containing 5% alfalfa powder, 1% glucose, and 0.3% potassium dihydrogen phosphate at 32℃, with a stirring speed of 150 r / min and an aeration rate of 1:1.2 (v / v) for 24 h to obtain a concentration of 10 8 -10 9 CFU / mL acclimatization bacterial solution;

[0010] (2) Raw material pretreatment: Select fresh alfalfa in the budding stage, rinse with clean water, dry with hot air at 60℃ until the moisture content is ≤12%, then pulverize it to 150-200 mesh using an ultra-micro pulverizer, add 0.5% vitamin C solution to the pulverized alfalfa powder at a mass-volume ratio of 1:10, and soak for 30 minutes;

[0011] (3) Co-fermentation of *Tai Sui* bacteria: Alfalfa powder and deionized water were mixed at a solid-liquid ratio of 1:8, and the acclimatized bacterial solution obtained in step (1) was inoculated at 10% (v / w). 0.2% magnesium sulfate was added, and fermentation was carried out at 33℃, pH 6.2, stirring speed of 80 r / min, and aeration for 20 min / stop for 10 min for 36 h until the cellulose degradation rate was ≥88% and the phytic acid content was ≤0.15%.

[0012] (4) Ultrasonic-enzymatic synergistic extraction: Add 1.2% compound enzyme to the fermentation broth obtained in step (3), treat with ultrasound, then enzymatically hydrolyze for 30 min at 55℃ and pH 8.0, heat to 90℃ to inactivate enzyme for 10 min, cool and adjust pH to 9.0 with 1 mol / L sodium hydroxide, and stir to extract for 40 min.

[0013] (5) Separation and purification: The extract obtained in step (4) is centrifuged, the supernatant is collected, 0.1% diatomaceous earth is added to the supernatant, filtered, and then desalted through a D301 macroporous anion exchange resin column;

[0014] (6) Low-temperature freeze-drying coupled drying: The protein liquid obtained in step (5) is successively subjected to low-temperature spray drying, vacuum freeze-drying and pulverization to obtain the finished product, namely the alfalfa functional protein powder.

[0015] Furthermore, in step (1),

[0016] The core bacterial group of the artificial Tai Sui bioactive bacteria consists of Bacillus sp., Pseudomonas sp., and Saccharomyces sp., with a symbiotic ratio of 1:0.8:0.2.

[0017] Furthermore, in step (4),

[0018] The complex enzyme is composed of alkaline protease and cellulase in a mass ratio of 1:0.3, and the enzyme activity of both alkaline protease and cellulase is 5000 U / g.

[0019] The total duration of the ultrasonic treatment is 20 minutes, using an intermittent mode of 5 seconds on and 5 seconds off.

[0020] Furthermore, in step (5),

[0021] The centrifugation conditions were: 8000 r / min, 20 min;

[0022] The filtration process employs a 0.45μm plate and frame filter.

[0023] The desalination conditions are: flow rate 2 BV / h, column temperature 30℃, and conductivity after desalination ≤50 μS / cm.

[0024] Furthermore, in step (6),

[0025] The conditions for the low-temperature spray drying are as follows: the protein solution is concentrated to a solid content of 30%-35% under the conditions of an inlet air temperature of 75-80℃ and an outlet air temperature of 32-35℃.

[0026] The conditions for vacuum freeze drying are: vacuum degree 0.008MPa, freeze drying temperature -45℃, and time 8h.

[0027] The embodiments of the present invention have the following advantages:

[0028] 1. Outstanding technological innovation: For the first time in the world, artificial Tai Sui bioactive bacteria are applied to alfalfa protein fermentation, and a brand-new process route of "Tai Sui bacteria fermentation + ultrasound-enzymatic hydrolysis synergy + low temperature freeze drying" is constructed. No identical or similar technologies have been disclosed after searching, breaking through the international plant protein processing technology bottleneck.

[0029] 2. Core indicators are internationally leading:

[0030] The protein extraction rate reaches 90%-93%, which is more than 30% higher than the traditional process (68-75%) and 5-8% higher than the highest international level (80-86%, see Table 1);

[0031] The product has a solubility of 92%-95%, emulsifying activity of ≥70% (measured by centrifugation), and foaming activity of ≥450% (measured by stirring), all of which are superior to similar international products.

[0032] It retains the unique active ingredients of *Saccharomyces cerevisiae*: SOD activity ≥250 U / g, *Saccharomyces cerevisiae* polysaccharide content ≥2.0 mg / g, flavonoid retention rate ≥90%, and has additional functions of anti-oxidation and immune regulation;

[0033] 3. High feasibility for industrialization: All equipment used is mature industrial equipment, which has passed international or domestic authoritative certifications and has a long-term safe operation record; the products have undergone acute toxicity tests and application verification on a scale of tens of thousands of people, ensuring safety and complying with national standards such as GB14880 and GB 2760, which can meet the industrialization needs of high-end food and health products. Detailed Implementation

[0034] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] This embodiment provides a method for preparing alfalfa functional protein powder:

[0037] (1) Domestication and propagation of *Tai Sui* fungus

[0038] Artificial *Tai Sui* bacteria were inoculated into a culture medium containing 100-mesh alfalfa powder. After five consecutive subcultures at 30℃ and pH 6.0, the acclimatized strain achieved an 89% degradation rate of alfalfa cell walls. A 500L fermenter (model FG-8000) was used, with a culture medium prepared as follows: 5% alfalfa powder + 1% glucose + 0.3% potassium dihydrogen phosphate. The culture was expanded at 32℃, with stirring at 150 rpm and an aeration rate of 1:1.2 for 24 hours, reaching a bacterial concentration of 1.2 × 10⁻⁶. 9 CFU / mL.

[0039] (2) Raw material pretreatment

[0040] Take 100 kg of alfalfa at the budding stage, rinse with clean water to remove impurities, dry with hot air at 60℃ until the moisture content is 11%, and grind to 180 mesh using an ultrafine pulverizer (model ZW-800). Add 1000 L of 0.5% vitamin C solution to the pulverized alfalfa powder and soak for 30 min to inhibit polyphenol oxidase activity.

[0041] (3) Co-fermentation of *Tai Sui* bacteria

[0042] 800L of deionized water was added to the pretreatment solution in step (2) at a solid-liquid ratio of 1:8, along with 80L of 10% (v / w) acclimatization bacterial solution from step (1) and 0.2kg of magnesium sulfate. A 1000L intelligent fermenter (model FG-8000) was used, set to 33℃, pH 6.2, 80r / min stirring, and intermittent aeration (20min aeration / 10min pause), for 36h fermentation. Sampling and testing showed that the cellulose degradation rate was 88.5%, the phytic acid content was 0.14%, the SOD activity in the fermentation broth was 312 U / mL, and the polysaccharide content was 2.6 mg / mL, reaching the fermentation endpoint.

[0043] (4) Ultrasonic-enzymatic extraction

[0044] Add 1.2 kg of compound enzyme (0.9 kg alkaline protease + 0.3 kg cellulase) to the fermentation broth in step (3). Turn on the low-frequency ultrasonic device (model CS-600) and perform intermittent ultrasonication at 25 kHz and 350 W power (5 s on, 5 s off) for 20 min. Then, incubate at 55 ℃ and pH 8.0 for 30 min for enzymatic hydrolysis. Raise the temperature to 90 ℃ and maintain for 10 min to inactivate the enzyme. After cooling to room temperature, adjust the pH to 9.0 with 1 mol / L sodium hydroxide and stir for 40 min for extraction.

[0045] (5) Separation and purification

[0046] The sample was centrifuged at 8000 r / min for 20 min using a disc centrifuge (model CV-2000), and the supernatant was collected. 0.1 kg of diatomaceous earth was added to the supernatant, and the sample was filtered through a plate and frame filter (model FF-0.45) to remove fine impurities. The filtered supernatant was then pumped into a D301 resin column (model IO-300) for desalting at a flow rate of 2 BV / h and a column temperature of 30℃. The conductivity after desalting was measured to be 45 μS / cm.

[0047] (6) Low-temperature freeze-drying coupled drying

[0048] The desalted protein solution was sent to a low-temperature spray dryer (model RS-1000) with an inlet air temperature of 78℃ and an outlet air temperature of 33℃, and concentrated to a solid content of 32%. Then it was transferred to a vacuum freeze dryer (model LG-50) and freeze-dried at a vacuum of 0.008MPa and -45℃ for 8 hours to obtain a loose protein product. Finally, it was pulverized to 300 mesh using an ultrafine pulverizer (model SM-500) and sieved to obtain 18.2 kg of finished product.

[0049] Finished product test results: protein content 86.5%, protein extraction rate 91.2%, solubility 93.8%, emulsifying activity 72.3%, foaming property 465%; SOD activity 268 U / g, *Saccharide styracifolium* polysaccharide 2.2 mg / g, flavonoid retention rate 91.5%; acute toxicity test showed LD50...50 >10g / kg, meets safety standards.

[0050] Example 2

[0051] Compared to Example 1, only the following parameters are adjusted, while the remaining steps remain the same:

[0052] In step (3), the fermentation time is extended to 40 hours, and the fermentation temperature is controlled at 34°C;

[0053] In step (4), the ultrasonic power is increased to 400W and the ultrasonic time is maintained for 20 minutes;

[0054] In step (6), the vacuum freeze-drying time is extended to 9 hours.

[0055] Finished product test results: protein extraction rate 92.8%, solubility 94.5%, SOD activity 285 U / g, polysaccharide content 2.4 mg / g, and flavonoid retention rate 93.2%.

[0056] Comparative Example

[0057] This comparative example uses a traditional process of yeast fermentation-alkali extraction-acid precipitation-single spray drying to prepare alfalfa protein powder. The specific steps are as follows:

[0058] (1) Raw material pretreatment: Same as in Example 1 (washing-drying-pulverizing), except for the vitamin C soaking step;

[0059] (2) Yeast fermentation: Inoculate with brewer's yeast (commercially available food-grade strain) and ferment at 30℃ for 24 hours;

[0060] (3) Alkaline extraction and acid precipitation: Adjust the pH to 9.0 with 1 mol / L sodium hydroxide and extract for 1 h, then adjust the pH to 4.5 with 1 mol / L hydrochloric acid to precipitate the protein;

[0061] (4) Single spray drying: air inlet temperature 180℃, air outlet temperature 80℃, and after drying, pulverize to 100 mesh.

[0062] Finished product test results: protein extraction rate 72.5%, solubility 42.3%, emulsifying activity 38.6%, foaming activity 185%; no SOD activity, polysaccharide content 0.3 mg / g, flavonoid retention rate 58.2%.

[0063] Compared with the products of the embodiments of the present invention, the core indicators of products made by traditional processes are lagging behind in all aspects, and they lack functional metabolites.

[0064] Security test

[0065] The protein powder provided in Example 1 of this invention has been verified through acute toxicity tests (referring to OECD 423 standards) to have a median lethal dose (LD50). 50The concentration is >10g / kg, and it has been applied in practice for 15 years, covering more than 5,000 consumers without any adverse reactions such as allergies or gastrointestinal disorders, meeting food-grade safety requirements.

[0066] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A functional protein powder of Medicago sativa, characterized in that, The protein content of the protein powder is ≥85%, the protein solubility is 92%-95%, the emulsifying activity is ≥70%, the foaming property is ≥450%, and the protein powder contains artificial Astraeus hygrometricus life active bacteria metabolites, wherein the superoxide dismutase (SOD) activity is ≥250 U / g, the Astraeus hygrometricus polysaccharide content is ≥2.0 mg / g, and the flavone retention rate is ≥90%.

2. The method of preparing the functional protein powder of Medicago sativa according to claim 1, characterized in that, The method comprises the following steps: (1) domestication and expansion of artificial Astraeus hygrometricus life active bacteria: taking 100-mesh alfalfa stem and leaf powder as the only carbon and nitrogen source, the domesticated strain with a cell wall degradation rate of ≥85% is screened by continuous subculture for 5 times in a medium at 30°C and pH 6.0; Subsequently, the domesticated bacteria solution with a concentration of 10 8 -10 9 CFU / mL was obtained by expanding the bacteria in a medium containing 5% alfalfa powder, 1% glucose, and 0.3% potassium dihydrogen phosphate at 32°C for 24 h under stirring at a speed of 150 r / min and ventilation at a ratio of 1:1.2 (v / v). (2) raw material pretreatment: fresh alfalfa at the present budding stage is selected, washed with clean water, dried at 60°C until the water content is ≤12%, crushed into 150-200 mesh by using a supermicro grinder, and then 0.5% vitamin C solution is added to the crushed alfalfa powder at a mass-volume ratio of 1:10, and soaked for 30 min; (3) Astraeus hygrometricus bacteria synergistic fermentation: the alfalfa powder is mixed with deionized water at a solid-liquid ratio of 1:8, the domesticated bacterial liquid obtained in step (1) is inoculated at 10% (v / w), 0.2% magnesium sulfate is added, and then the mixture is fermented at 33°C and pH 6.2 under stirring at a stirring speed of 80 r / min, with aeration for 20 min and stopping for 10 min, for 36 h, until the cellulose degradation rate is ≥88% and the phytic acid content is ≤0.15%; (4) ultrasonic-enzymatic hydrolysis synergistic extraction: 1.2% compound enzyme is added to the fermentation liquid obtained in step (3), ultrasonic treatment is performed, and then enzymatic hydrolysis is performed at 55°C and pH 8.0 for 30 min, the temperature is raised to 90°C for 10 min to inactivate the enzyme, after cooling, the pH is adjusted to 9.0 by using 1 mol / L sodium hydroxide, and stirring extraction is performed for 40 min; (5) separation and purification: the extract liquid obtained in step (4) is centrifuged, the supernatant is collected, 0.1% diatomite is added to the supernatant, filtered, and then desalted by using a D301 macroporous anion exchange resin column; (6) low-temperature freeze-drying coupling drying: the protein liquid obtained in step (5) is sequentially subjected to low-temperature spray drying, vacuum freeze-drying and crushing to obtain the finished product, i.e., the alfalfa functional protein powder.

3. The method of claim 2, wherein the functional protein powder is prepared by the steps of: a) extracting proteins from alfalfa; b) drying the extracted proteins; c) defatting the dried proteins; d) drying the defatted proteins; and e) milling the dried defatted proteins. In step (1), the core bacterial flora of the artificial Astraeus hygrometricus life active bacteria comprises Bacillus sp., Pseudomonas sp. and Saccharomyces sp., and the symbiotic ratio is 1:0.8:0.

2.

4. The method of claim 2, wherein the functional protein powder is prepared by the steps of: a) extracting proteins from alfalfa; b) drying the extracted proteins; c) defatting the dried proteins; d) drying the defatted proteins; and e) grinding the dried defatted proteins. In step (4), the compound enzyme is composed of alkaline protease and cellulase at a mass ratio of 1:0.3, and the enzyme activity of the alkaline protease and the cellulase is 5000 U / g; the total ultrasonic treatment time is 20 min, and an intermittent mode of working for 5 s and stopping for 5 s is adopted.

5. The method of claim 2, wherein the functional protein powder is prepared by the steps of: a) extracting proteins from alfalfa; b) drying the extracted proteins; c) defatting the dried proteins; d) drying the defatted proteins; and e) grinding the dried defatted proteins. In step (5), the centrifugation is performed at 8000 r / min for 20 min; the filtration is performed by using a 0.45-μm plate-frame filter; the desalting is performed at a flow rate of 2 BV / h and a column temperature of 30°C, and the conductivity after desalting is ≤50 μS / cm.

6. The method for preparing alfalfa functional protein powder according to claim 2, characterized in that, In step (6), The low-temperature spray drying is carried out under the conditions of an air inlet temperature of 75-80 DEG C, an air outlet temperature of 32-35 DEG C, and the protein solution is concentrated to a solid content of 30-35%. The vacuum freeze drying is carried out under the conditions of a vacuum degree of 0.008 MPa, a freeze drying temperature of -45 DEG C, and a time of 8 h.