Co-production process for ultrasonic-assisted extraction of high-activity lectin from white hyacinth beans and enzymolysis detoxification protein powder

Abstract

The invention discloses a co-production process for ultrasonic-assisted extraction of high-activity lectin from white hyacinth beans and enzymolysis detoxified protein powder, and belongs to the technical field of deep processing of agricultural products. The process comprises the following steps: cleaning and drying a white hyacinth bean raw material, crushing and degreasing; extracting for 100-130 minutes by using an extraction buffer solution at the temperature of 40-50 DEG C under the ultrasonic assistance of 40 kHz; after centrifugal separation, carrying out ammonium sulfate salting-out and multi-stage chromatographic purification on supernate to obtain the high-activity lectin. And carrying out enzymolysis-thermal inactivation dual detoxification treatment on degreased bean dregs obtained by centrifugation, and then carrying out fine separation and freeze drying to obtain the high-quality protein powder. By optimizing ultrasonic extraction parameters, the problems of low lectin extraction efficiency and difficulty in activity maintenance are solved, anti-nutritional factors are removed by adopting an'enzymolysis-thermal inactivation double detoxification 'process without influencing the functional characteristics of protein, and a technical path for co-production of lectin and protein powder is established. The comprehensive utilization rate of the raw materials is increased to 98% or above, and an effective solution is provided for high-valued comprehensive utilization of white hyacinth bean resources.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product deep processing, bioseparation and food engineering technology. Specifically, it relates to a process for the co-production of highly active lectins and enzymatic detoxification protein powder from white hyacinth beans using ultrasound-assisted extraction. Background Technology

[0002] White hyacinth bean, a traditional agricultural product used for both medicinal and culinary purposes, is not only rich in nutrients but also contains a variety of substances with important biological activities. Among them, white hyacinth bean lectin, due to its unique sugar-binding specificity, shows promising application prospects in biomedical research and cell labeling. Meanwhile, white hyacinth bean protein possesses an excellent amino acid composition and good digestibility, making it a potential high-quality raw material for developing functional foods and foods for special medical purposes. With the increasing demand for health foods, how to efficiently utilize white hyacinth bean resources and develop high-value-added products has become an important research direction in the field of agricultural product processing.

[0003] In lectin extraction, current technologies primarily employ traditional low-temperature immersion extraction methods, which are not only time-consuming but also have limited extraction efficiency. While recent studies have attempted to introduce ultrasound-assisted extraction technology to improve efficiency, the lack of systematic optimization of ultrasound parameters often leads to unstable extraction efficiency and can easily result in the loss of lectin bioactivity. As a protein, lectin activity is highly sensitive to extraction conditions; even slight changes in parameters such as temperature and pH can affect the quality of the final product. Furthermore, single extraction methods are insufficient to meet the dual demands of efficiency and quality in industrial production, thus limiting the high-value utilization of lectins.

[0004] In the preparation of protein powder, the naturally occurring anti-nutritional factors in white hyacinth beans, such as lectins and trypsin inhibitors, have certain toxic or digestive inhibitory effects on the human body. Therefore, they must be effectively removed or inactivated before being used as food ingredients. Existing detoxification processes mainly include heat treatment and enzymatic hydrolysis, but each has significant shortcomings. Heat treatment alone requires high temperatures and long times to effectively remove anti-nutritional factors, but this process easily leads to protein denaturation, reducing its functional properties and nutritional value. While enzymatic hydrolysis is relatively mild, it often fails to completely remove anti-nutritional factors, and improper control of enzymatic hydrolysis conditions can lead to excessive protein hydrolysis, affecting product quality. In practical applications, neither of these methods can simultaneously meet the requirements of safety and functionality, limiting the application of white hyacinth bean protein powder in the food industry.

[0005] A more prominent problem is that existing processes are mostly single-product production models, focusing only on lectin extraction or protein powder preparation. This results in low comprehensive utilization of white hyacinth bean raw materials and generates a large amount of byproducts and waste. Furthermore, there is an inherent contradiction in the process conditions for lectin extraction and protein powder preparation: lectin extraction requires mild conditions to maintain its biological activity, while protein powder detoxification requires relatively harsh conditions to ensure the complete removal of anti-nutritional factors. This incoordination of process conditions makes it difficult to effectively combine the two in the same process route, leading to resource waste and increased production costs. In addition, existing technologies lack precise control over process parameters and a systematic quality evaluation system, resulting in insufficient product quality stability and difficulty in meeting the needs of industrial production.

[0006] In summary, current white hyacinth bean processing technology still faces numerous challenges in areas such as efficient lectin extraction, safe protein powder preparation, and comprehensive utilization of raw materials. There is an urgent need to develop a comprehensive process that can address these issues in a coordinated manner, achieving efficient and differentiated utilization of white hyacinth bean raw materials, simultaneously producing highly active lectins and high-quality detoxified protein powder, thereby improving resource utilization efficiency and product added value, and meeting market demand for high-quality processed agricultural products. Summary of the Invention

[0007] The purpose of this invention is to provide a process for the combined production of highly active lectin from white hyacinth bean using ultrasound-assisted extraction and enzymatic detoxification of protein powder, in order to solve the problems of difficulty in achieving efficient extraction of lectin and safe preparation of protein powder, and low comprehensive utilization rate of raw materials in the existing technology.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a process for the combined production of highly active lectin and enzymatically detoxified protein powder from white hyacinth bean using ultrasound-assisted extraction, comprising the following steps: (1) After washing and drying the white hyacinth bean raw material, it is pulverized to 40-60 mesh, degreased, and then vacuum dried until the residual ether content is ≤5 ppm to obtain defatted bean flour; (2) Mix the defatted soybean powder obtained in step (1) with the extraction buffer at a material-to-liquid ratio of 1:10, and extract for 100-130 minutes under ultrasonic assistance at 40 kHz at a temperature of 40-50℃ to obtain the extract; (3) Centrifuge the extract at 10,000-15,000 g for 20-30 minutes at 4°C, collect the supernatant as crude extract of lectin, and obtain defatted soybean residue at the same time; (4) Perform gradient ammonium sulfate precipitation on the crude extract of lectin: adjust the saturation to 40%, 60% and 80% in sequence, stir for 30 minutes and let stand at 4°C for 4 hours, centrifuge to collect the precipitates of each gradient; combine the precipitates of each gradient and reconstitute them, and put them into a dialysis bag and dialyze with the extraction buffer until there are no sulfate ions to obtain a preliminarily enriched lectin solution. (5) The preliminarily enriched lectin solution was subjected to DEAE ion exchange chromatography, CM ion exchange chromatography and Sephacryl S-300 gel filtration chromatography in sequence to obtain the purified lectin solution. (6) Freeze-dry the purified lectin solution to obtain lyophilized lectin powder; (7) The defatted soybean residue obtained in step (3) is slurried, the pH of the slurry is adjusted to 6.0-7.0, 3-4% neutral protease is added, and hydrolyzed at 45-50℃ for 1.5-2 hours to obtain the enzymatically hydrolyzed slurry; (8) Heat the enzymatically hydrolyzed slurry to 80°C and hold for 10 minutes to perform heat inactivation, and obtain the detoxified slurry; (9) The detoxified slurry is crushed at 38-40℃, the pH is adjusted to 4.3-4.5, and the fibers are separated by a pressure sieve to obtain the separated slurry; (10) Adjust the pH of the separated slurry to 5.5-6.0, separate the starch by hydrocyclone separator, collect the protein slurry by centrifugation, and freeze-dry to obtain white hyacinth bean protein powder.

[0009] Further, in step (1), the degreasing treatment is carried out twice at 4°C using anhydrous ether, with a material-to-liquid ratio of 1:4 for each degreasing process.

[0010] Further, in step (2), the extraction buffer is 0.02 M Tris-HCl and 0.15 M NaCl, with a pH of 7.8.

[0011] Further, in step (5), the DEAE ion exchange chromatography is equilibrated with Tris-HCl buffer at pH 8.0, and the CM ion exchange chromatography is equilibrated with acetate buffer at pH 5.0.

[0012] Further, in step (7), the amount of neutral protease added is 3.5% based on the dry soybean residue.

[0013] Furthermore, the pulverization described in step (9) is a two-stage pulverization.

[0014] Furthermore, in step (10), the centrifugation is carried out at 2000-2500 rpm and 40-45℃ for 40-45 minutes.

[0015] Furthermore, in step (6), the lyophilized lectin powder is stored at -80°C.

[0016] Furthermore, in step (9), the screen aperture size of the pressure curved screen is 0.2 mm.

[0017] Furthermore, in step (10), the feed pressure of the hydrocyclone separator is 0.2 MPa. Beneficial effects

[0018] (1) This invention achieves efficient extraction of lectins (extraction efficiency ≥92.1%) under mild conditions by using system-optimized ultrasonic-assisted extraction conditions (temperature 40-50℃, frequency 40kHz, extraction time 100-130 minutes) while maintaining high bioactivity (specific activity ≥8480 HU / mg), overcoming the problems of low efficiency or loss of activity in traditional extraction methods.

[0019] (2) The “enzymatic hydrolysis-thermal inactivation dual detoxification” process proposed in this invention achieves effective removal of anti-nutritional factors (lectin residue <2 HU / g, trypsin inhibition rate ≤5.0%) by hydrolyzing with 3-4% neutral protease at 45-50℃ for 1.5-2 hours and then inactivating with heating at 80℃ for 10 minutes. At the same time, it preserves the natural conformation and functional properties of the protein to the maximum extent (NSI index ≥86.2%, PER value ≥4.14), solving the problem that a single detoxification process cannot simultaneously meet the requirements of safety and functionality.

[0020] (3) This invention achieves full utilization of white hyacinth bean raw materials by using a targeted separation process of "extracting lectins first and then purifying proteins" combined with the fine recovery of starch and fiber. The comprehensive utilization rate of raw materials is increased to over 98.0%, which significantly reduces resource waste and improves economic benefits.

[0021] (4) The present invention adopts a “three-step chromatography combination purification” technology of DEAE, CM ion exchange and Sephacryl S-300 gel filtration. By finely controlling pH, ionic strength and molecular sieve effect, it achieves high efficiency and high selectivity of lectin purification, providing high-quality raw materials for subsequent high-value applications. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments. Example 1

[0023] 1. Raw material pretreatment: 50 kg of high-quality white hyacinth beans were selected, washed to remove surface impurities, and dried to constant weight in a 60℃ hot air circulating oven. The dried white hyacinth beans were then pulverized using a high-speed grinder and passed through a 50-mesh sieve to obtain white hyacinth bean powder.

[0024] White hyacinth bean powder and anhydrous ether pre-cooled to 4°C were mixed in a stirrer at a ratio of 1:4 (w / v) and defatted at 300 rpm for 30 minutes. The mixture was then centrifuged at 3000 rpm for 10 minutes. The defatted bean powder was collected, and the defatted bean powder was subjected to the same defatting process once more with anhydrous ether. The mixture was then placed in a vacuum drying oven and dried at -0.08 MPa and 40°C for 2 hours. After confirming that the residual ether content was ≤5 ppm, the defatted bean powder was collected for subsequent steps.

[0025] 2. High-efficiency extraction and crude separation of lectins: (1) Weigh 10 kg of defatted soybean flour obtained in step 1 and add 100 L of extraction buffer (prepared from 0.02 M Tris-HCl and 0.15 M NaCl, pH 7.8) preheated to 45°C. The material-to-liquid ratio is 1:10 (w / v). While maintaining 45°C, place the mixture in an industrial-grade 40 kHz ultrasonic reactor and extract for 115 minutes under continuous ultrasonic action. The ultrasonic power is set to 300 W, and the working mode is 2 seconds on and 1 second off.

[0026] (2) Transfer the extract to a centrifuge and centrifuge at 12500 g for 25 minutes at 4°C. Collect the supernatant (crude extract of lectin) and obtain defatted soybean residue containing protein.

[0027] (3) Under ice bath and electric stirring conditions, solid ammonium sulfate was added to the supernatant in stages: first, it was slowly added to 40% saturation, stirred at 150 rpm for 30 minutes in an ice bath, and then allowed to stand at 4°C for 4 hours. After collecting the precipitate by centrifugation at 10,000 g for 30 minutes, the supernatant was added to 60% saturation, and the operation was repeated; finally, the supernatant was added to 80% saturation to complete the third salting out. The collected precipitates containing lectins were combined and dissolved in a small amount of pH 7.8 Tris-HCl buffer, and then placed into a dialysis bag with a molecular weight cutoff of 12 kDa. The dialysis bag was then immersed in a large amount of buffer with the same composition as the extraction buffer (i.e., 0.02 M Tris-HCl + 0.15 M NaCl, pH 7.8) for dialysis to remove salt. The dialysis process continued until no sulfate ions were precipitated when the liquid outside the dialysis bag was tested with 10% BaCl2 solution. The dialysis solution was changed every 2 hours during this period to obtain a preliminarily enriched lectin solution.

[0028] 3. Precise multi-stage chromatography purification of lectins: After concentrating the initially enriched lectin solution, three-step chromatographic purification was performed sequentially: (1) DEAE ion exchange chromatography: The sample was loaded onto a pre-equilibrated DEAE-Sepharose fast flow column (2.5 cm × 30 cm), equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and then eluted linearly with a Tris-HCl buffer containing 0-0.5 M NaCl. The elution flow rate was set to 1.5 mL / min and the elution volume was 5 times the column volume.

[0029] (2) CM ion exchange chromatography: Adjust the pH of the lectin fraction collected in the previous step to 5.0, load it onto a pre-equilibrated CM-Sepharose fast flow column (2.5 cm × 30 cm), equilibrate with 50 mM acetate buffer (pH 5.0), and then perform linear gradient elution with acetate buffer containing 0-0.3 M NaCl. The elution flow rate is set to 1.0 mL / min, and the elution volume is 5 times the column volume.

[0030] (3) Sephacryl S-300 gel filtration chromatography: The sample purified by two ion exchange processes was concentrated to about 5 mL and loaded onto a pre-equilibrated Sephacryl S-300HR gel filter column (1.6 cm × 60 cm). It was eluted isocratically with PBS buffer (pH 7.4, containing 0.15 M NaCl) at a flow rate of 0.5 mL / min. The main peak was collected to obtain the purified lectin solution.

[0031] 4. Concentration and preservation of lectin products: The purified lectin solution was concentrated using a freeze dryer. The freeze-drying conditions were set as follows: pre-freezing temperature -40℃, pre-freezing time 4 hours; sublimation drying stage temperature -20℃, vacuum degree 10 Pa, time 10 hours; desorption drying stage temperature 25℃, vacuum degree 10 Pa, time 4 hours. The freeze-dried powder was aliquoted into sterile EP tubes and stored in a -80℃ ultra-low temperature freezer protected from light.

[0032] 5. High-quality protein powder preparation with high efficiency: (1) Enzymatic hydrolysis-thermal inactivation dual detoxification: The defatted soybean residue obtained in step 2 (2) was added to an appropriate amount of water to make a slurry (solid-liquid ratio 1:8 w / v). The pH of the slurry was monitored and adjusted to 6.5 using a pH meter. 3.5% (w / w, based on dry soybean residue) of neutral protease was added, and hydrolysis was carried out in a constant temperature reactor at 48°C for 1.8 hours, with the stirring speed maintained at 100 rpm during the hydrolysis process. After the hydrolysis was completed, the slurry was quickly transferred to a jacketed kettle, heated to 80°C and held for 10 minutes for thermal inactivation.

[0033] (2) Low-temperature grinding and separation of fine components: The detoxified slurry was ground at a constant temperature of 39℃ using a two-stage colloid mill (first stage gap 0.5 mm, second stage gap 0.2 mm). The pH of the slurry was adjusted to 4.4, and the fibers were separated and recovered by a pressure curved screen (screen aperture size 0.2 mm, working pressure 0.15 MPa). The pH of the separated slurry was adjusted to 5.8, and the starch was separated and recovered by a hydrocyclone separator (two-stage hydrocyclone separation, feed pressure 0.2 MPa).

[0034] (3) Protein precipitation and freeze drying: The starch-separated slurry was centrifuged at 2200 rpm and 42℃ for 42 minutes using a refrigerated high-speed centrifuge, and the precipitated protein slurry was collected. The protein slurry was reconstituted with a small amount of distilled water and then dried using a freeze dryer. The freeze drying conditions were set as follows: pre-freezing temperature -40℃, pre-freezing time 4 hours; sublimation drying stage temperature -20℃, vacuum degree 10 Pa, time 10 hours; desorption drying stage temperature 25℃, vacuum degree 10 Pa, time 4 hours, to obtain white fluffy white lentil protein powder. Example 2

[0035] 1. Raw material pretreatment: 50 kg of white hyacinth beans, the same type as in Example 1, were selected, washed, and dried as in Example 1. The dried white hyacinth beans were then pulverized using a high-speed grinder and passed through a 60-mesh sieve to obtain white hyacinth bean powder.

[0036] The degreasing and drying process is the same as in Example 1.

[0037] 2. High-efficiency extraction and crude separation of lectins: (1) Basically the same as Example 1, except that the extraction time under ultrasound was adjusted to 130 minutes.

[0038] (2) Basically the same as in Example 1, except that the centrifugation conditions are adjusted to centrifuge at 15000 g for 30 minutes.

[0039] (3) Same as Example 1.

[0040] 3. Precise multi-stage chromatography purification of lectins: Same as in Example 1.

[0041] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0042] 5. High-quality protein powder preparation with high efficiency: (1) Enzymatic hydrolysis-thermal inactivation dual detoxification: The defatted soybean residue obtained in step 2 (2) was added to an appropriate amount of water to make a slurry (solid-liquid ratio 1:8 w / v). The pH of the slurry was monitored and adjusted to 7.0 using a pH meter. 4% (w / w, based on dry soybean residue) of neutral protease was added, and hydrolysis was carried out in a constant temperature reactor at 50°C for 2 hours, with the stirring speed maintained at 100 rpm during the hydrolysis process. After the hydrolysis was completed, the slurry was quickly transferred to a jacketed kettle, heated to 80°C and held for 10 minutes for thermal inactivation.

[0043] (2) Low-temperature grinding and separation of fine components: The detoxified slurry was ground at a constant temperature of 40℃ using a two-stage colloid mill (first stage gap 0.5 mm, second stage gap 0.2 mm). The pH of the slurry was adjusted to 4.5, and the fibers were separated and recovered by a pressure curved screen (screen aperture size 0.2 mm, working pressure 0.15 MPa). The pH of the separated slurry was adjusted to 6.0, and the starch was separated and recovered by a hydrocyclone separator (two-stage hydrocyclone separation, feed pressure 0.2 MPa).

[0044] (3) Protein precipitation and freeze drying: The starch-separated slurry was centrifuged at 2500 rpm and 45°C for 45 minutes using a refrigerated high-speed centrifuge, and the precipitated protein slurry was collected. The protein slurry was reconstituted with a small amount of distilled water and then dried using a freeze dryer under the same conditions as in Example 1. Example 3

[0045] 1. Raw material pretreatment: 50 kg of white hyacinth beans, the same type as in Example 1, were selected, washed, and dried as in Example 1. The dried white hyacinth beans were then pulverized using a high-speed grinder and passed through a 40-mesh sieve to obtain white hyacinth bean powder.

[0046] The degreasing and drying process is the same as in Example 1.

[0047] 2. High-efficiency extraction and crude separation of lectins: (1) Basically the same as Example 1, except that the extraction time under ultrasound was adjusted to 100 minutes.

[0048] (2) Basically the same as in Example 1, except that the centrifugation conditions are adjusted to centrifuge at 10000 g for 20 minutes.

[0049] (3) Same as Example 1.

[0050] 3. Precise multi-stage chromatography purification of lectins: Same as in Example 1.

[0051] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0052] 5. High-quality protein powder preparation with high efficiency: (1) Enzymatic hydrolysis-thermal inactivation dual detoxification: The defatted soybean residue obtained in step 2 (2) was added to an appropriate amount of water to make a slurry (solid-liquid ratio 1:8 w / v). The pH of the slurry was monitored and adjusted to 6.0 using a pH meter. 3% (w / w, based on dry soybean residue) of neutral protease was added, and hydrolysis was carried out in a constant temperature reactor at 45°C for 1.5 hours, with the stirring speed maintained at 100 rpm during the hydrolysis process. After the hydrolysis was completed, the slurry was quickly transferred to a jacketed kettle, heated to 80°C and held for 10 minutes for thermal inactivation.

[0053] (2) Low-temperature grinding and separation of fine components: The detoxified slurry was ground at a constant temperature of 38℃ using a two-stage colloid mill (first stage gap 0.5 mm, second stage gap 0.2 mm). The pH of the slurry was adjusted to 4.3, and the fibers were separated and recovered using a pressure curved screen (screen aperture size 0.2 mm, working pressure 0.15 MPa). The pH of the separated slurry was adjusted to 5.5, and the starch was separated and recovered using a hydrocyclone separator (two-stage hydrocyclone separation, feed pressure 0.2 MPa).

[0054] (3) Protein precipitation and freeze drying: The starch-separated slurry was centrifuged at 2000 rpm and 40°C for 40 minutes using a refrigerated high-speed centrifuge, and the precipitated protein slurry was collected. The protein slurry was reconstituted with a small amount of distilled water and then dried using a freeze dryer under the same conditions as in Example 1. Comparative Example 1: Traditional process (low-temperature soaking to extract lectins + single heat treatment to detoxify protein powder)

[0055] 1. Raw material pretreatment: Same as in Example 1.

[0056] 2. High-efficiency extraction and crude separation of lectins: (1) Weigh 10 kg of defatted white hyacinth bean powder and add 100 L of extraction buffer (prepared from 0.02 M Tris-HCl and 0.15 M NaCl, pH 7.8) pre-cooled to 4°C. The material-to-liquid ratio is 1:10 (w / v). The mixture is extracted at 4°C for 240 minutes without the aid of ultrasound.

[0057] (2) Transfer the extract to a centrifuge and centrifuge at 12,500 g for 25 minutes at 4°C. Collect the supernatant (crude extract of lectin) and obtain defatted soybean residue containing protein.

[0058] (3) Same as Example 1.

[0059] 3. Precise multi-stage chromatography purification of lectins: Same as in Example 1.

[0060] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0061] 5. High-quality protein powder preparation with high efficiency: (1) Add the defatted soybean residue obtained in step 2 (2) to an appropriate amount of water to make a slurry (solid-liquid ratio 1:8 w / v), without enzymatic hydrolysis, and heat directly to 95°C for 30 minutes for heat inactivation.

[0062] (2) and (3) are the same as in Example 1. Comparative Example 2: Ultrasonic-assisted extraction only, but without parameter optimization (ultrasonic extraction at 55℃ for 150 minutes + protein powder process of this invention).

[0063] 1. Raw material pretreatment: Same as in Example 1.

[0064] 2. High-efficiency extraction and crude separation of lectins: (1) Weigh 10 kg of defatted white hyacinth bean powder and add 100 L of extraction buffer (prepared from 0.02 M Tris-HCl and 0.15 M NaCl, pH 7.8) preheated to 55°C, ensuring a material-to-liquid ratio of 1:10 (w / v). Place the mixture in an industrial-grade 40 kHz ultrasonic reactor and extract for 150 minutes under continuous ultrasonic action. Set the ultrasonic power to 300 W and the working mode to 2 seconds on and 1 second off.

[0065] (2) and (3) are the same as in Example 1.

[0066] 3. Precise multi-stage chromatography purification of lectins: Same as in Example 1.

[0067] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0068] 5. High-quality protein powder preparation: Same as Example 1. Comparative Example 3: Enzymatic detoxification without heat inactivation (the lectin extraction process of this invention + protein powder detoxified only by enzymes)

[0069] 1. Raw material pretreatment: Same as in Example 1.

[0070] 2. High-efficiency extraction and crude separation of lectins: (1) Weigh 10 kg of defatted white hyacinth bean powder and add 100 L of extraction buffer (prepared from 0.02 M Tris-HCl and 0.15 M NaCl, pH 7.8) pre-cooled to 4°C. The material-to-liquid ratio is 1:10 (w / v). The mixture is extracted at 4°C for 240 minutes without the aid of ultrasound.

[0071] (2) Transfer the extract to a centrifuge and centrifuge at 12,500 g for 25 minutes at 4°C. Collect the supernatant (crude extract of lectin) and obtain defatted soybean residue containing protein.

[0072] (3) Same as Example 1.

[0073] 3. Precise multi-stage chromatography purification of lectins: Same as in Example 1.

[0074] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0075] 5. High-quality protein powder preparation with high efficiency: (1) Add an appropriate amount of water to the defatted soybean residue obtained in step 2 (2) to make a slurry (solid-liquid ratio 1:8 w / v). Monitor and adjust the pH of the slurry to 6.5 using a pH meter. Add 3.5% (w / w, based on dry soybean residue) of neutral protease and hydrolyze it in a constant temperature reactor at 48°C for 1.8 hours, maintaining a stirring speed of 100 rpm during the hydrolysis process. After hydrolysis, do not perform heat inactivation and proceed directly to the next step.

[0076] (2) and (3) are the same as in Example 1. Comparative Example 4: No multi-stage chromatography purification (the lectin extraction process of this invention + single DEAE chromatography + the protein powder process of this invention)

[0077] 1. Raw material pretreatment: Same as in Example 1.

[0078] 2. High-efficiency extraction and crude separation of lectins: Same as in Example 1.

[0079] 3. Precise multi-stage chromatography purification of lectins: The pre-enriched lectin solution was subjected to DEAE ion exchange chromatography only: the sample was loaded onto a pre-equilibrated DEAE-Sepharose high-flow column (2.5 cm × 30 cm), equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and then eluted linearly with a gradient of Tris-HCl buffer containing 0–0.5 M NaCl at a flow rate of 1.5 mL / min for a volume of 5 column volumes. The target fraction was collected without subsequent CM ion exchange chromatography or Sephacryl S-300 gel filtration chromatography.

[0080] 4. Concentration and preservation of lectin products: Same as in Example 1.

[0081] 5. High-quality protein powder preparation: Same as Example 1. Data Analysis and Comparison

[0082] To objectively evaluate the technical effects of this invention, quantitative tests were conducted on the lectin and protein powder products obtained in Examples 1-3 and Comparative Examples 1-4, focusing on five core indicators: extraction efficiency, bioactivity, detoxification effect, functional characteristics, and raw material utilization rate. The details are as follows: (1) Lectin extraction efficiency: The protein concentration of the crude extract was determined by the Bradford method, and the hemagglutination titer of the crude extract was determined by the hemagglutination test. Lectin extraction efficiency = (Lectin content in crude extract / Total lectin content in raw material) × 100%. The total lectin content in the raw material was determined by the complete extraction method.

[0083] (2) Specific activity of lectin: The purified lectin solution was diluted to an appropriate concentration with extraction buffer and determined by the standard hemagglutination test. Specific activity = hemagglutination titer units (HU) / mg protein, where 1 hemagglutination unit (HU) is defined as the reciprocal of the lowest dilution factor that can cause complete agglutination.

[0084] (3) Detoxification effect of protein powder: The residual lectin in the protein powder was detected by hemagglutination test, and the result was expressed as HU / g. The control group was a mixture of trypsin and substrate BAPA, and the sample group was a mixture of trypsin and BAPA containing the protein powder to be tested. Both were reacted at 37℃ for 10 minutes and the absorbance at 410 nm was measured. The trypsin inhibition rate was determined by the BAPA method (N-α-benzoyl-DL-arginine p-nitroaniline hydrolysis method). The calculation formula was: Trypsin inhibition rate (%) = [(absorbance of control group - absorbance of sample group) / absorbance of control group] × 100%.

[0085] (4) Functional characteristics of protein powder: The protein content was determined by the Kjeldahl method; the nitrogen solubility index was evaluated by the NSI (nitrogen solubility index) determination method (AOCS Method Ba 11-65), NSI (%) = (dissolved nitrogen / total nitrogen) × 100%; the protein efficiency ratio (PER) was evaluated by the rat growth experiment, PER = animal weight gain (g) during the experimental period / protein intake (g) during the experimental period.

[0086] (5) Comprehensive utilization rate of raw materials: The calculation formula is: Comprehensive utilization rate of raw materials (%) = [(weight of lectin product × lectin content + weight of protein powder product × protein content + weight of recycled fiber + weight of recycled starch) / total weight of raw materials] × 100%. Among them, the lectin content is determined by the BCA method and the protein content is determined by the Kjeldahl method.

[0087] The test results are summarized in Table 1.

[0088] Table 1 Comparison of core indicators of white hyacinth bean lectin and protein powder products Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 lectin extraction efficiency (%) 92.5 92.2 92.1 63.2 85.6 90.4 82.7 % of lectin residue in soybean residue 14.0 14.1 14.4 36.8 18.4 16.1 23.3 lectin specific activity (HU / mg) 8520 8510 8480 7950 6820 8210 7540 Lectin extraction time (min) 115 130 100 240 150 115 115 Protein powder lectin residue (HU / g) <2 <2 <2 4.8 <2 3.5 <2 Trypsin inhibition rate (%) 4.7 4.9 5.0 15.3 5.1 11.2 5.2 Protein powder NSI (%) 86.8 86.3 86.2 72.5 85.9 80.2 85.8 Protein powder PER value 4.16 4.13 4.14 3.65 4.10 3.88 4.09 Comprehensive utilization rate of raw materials (%) 98.4 98.1 98.0 85.6 92.4 93.7 94.8 Table 1 shows that the lectin extraction efficiency and specific activity test data of Examples 1-3 are all ≥92.1% and ≥8480 HU / mg, which are significantly better than Comparative Example 1 (63.2%, 7950 HU / mg), Comparative Example 2 (85.6%, 6820 HU / mg), and Comparative Example 4 (82.7%, 7540 HU / mg). Comparative Example 1 used a traditional low-temperature soaking extraction method, which resulted in low extraction efficiency and long extraction time (240 minutes), 2.09 times longer than that of Example 1. Although Comparative Example 2 used ultrasound-assisted extraction, the excessively high temperature (55℃) and excessively long time (150 minutes) caused severe denaturation and inactivation of the lectin, resulting in a 19.5% decrease in specific activity. Comparative Example 4 did not undergo multi-stage chromatography purification, but only used single DEAE chromatography, resulting in poor purification effect and an 11% decrease in lectin specific activity. Data shows that the combination of process parameters of the present invention, namely, 40 kHz ultrasound-assisted extraction for 100-130 minutes at a temperature of 40-50℃, and the "three-step chromatography combination purification" technology of DEAE, CM ion exchange and Sephacryl S-300 gel filtration, can effectively improve the extraction efficiency of lectins and maintain their high biological activity.

[0089] Table 1 shows the test data on the detoxification effect and functional characteristics of the protein powder. The results indicate that the protein powders from Examples 1-3 all had lectin residues <2 HU / g, trypsin inhibition rates ≤5.0%, NSI indices ≥86.2%, and PER values ​​≥4.14, all significantly better than Comparative Example 1 (4.8 HU / g, 15.3%, 72.5%, 3.65), Comparative Example 2 (<2 HU / g, 5.1%, 85.9%, 4.10), and Comparative Example 3 (3.5 HU / g, 11.2%, 80.2%, 3.88). Comparative Example 1, due to its single heat treatment detoxification method, could not completely remove anti-nutritional factors, resulting in higher lectin residues and trypsin inhibition rates. Simultaneously, the prolonged high-temperature treatment caused severe protein denaturation, significantly reducing the NSI index and PER value. Although Comparative Example 3 used enzymatic detoxification, it lacked a heat inactivation step, leading to pH changes in the later stages of enzymatic hydrolysis that inactivated the enzyme, resulting in incomplete decomposition of residual lectins and thus higher lectin residues and trypsin inhibition rates. It is worth noting that Comparative Examples 2 and 4 were comparable to the Example in terms of protein powder lectin residue, but were still lower than Example 1 in terms of trypsin inhibition rate, NSI index, and PER value, indicating that their process parameters were not optimal. The data show that the "enzymatic hydrolysis-thermal inactivation dual detoxification" process of this invention (hydrolysis of 3-4% neutral protease at 45-50℃ for 1.5-2 hours, followed by inactivation at 80℃ for 10 minutes), combined with low-temperature grinding at 38-40℃ and freeze-drying technology, can effectively remove anti-nutritional factors while maximally preserving the natural conformation and functional properties of the protein.

[0090] The raw material utilization rate data in Table 1 shows that the comprehensive utilization rate of raw materials in Examples 1-3 is ≥98.0%, which is significantly better than that of Comparative Example 1 (85.6%), Comparative Example 2 (92.4%), Comparative Example 3 (93.7%), and Comparative Example 4 (94.8%). In Comparative Example 1, the extraction efficiency was low due to the use of low-temperature soaking to extract lectins, resulting in a large amount of lectins remaining in the soybean residue. Furthermore, the by-products were not systematically recovered during the protein powder preparation process, causing material loss. In Comparative Examples 2-4, although some of the process features of this invention were adopted, the systematic and differentiated utilization of each component of white hyacinth bean was not achieved due to differences in the process route, and some intermediate products were not fully recovered and utilized. Examples 1-3, through a directional separation process of "first extracting lectins, then purifying protein," used the soybean residue after lectin extraction as raw material for protein powder preparation. Fiber and starch were finely recovered using a pressure sieve and a hydrocyclone separator, achieving full utilization of the white hyacinth bean raw material, thus significantly improving the raw material utilization rate.

[0091] In summary, this invention successfully solves the problem of simultaneously achieving high-value utilization of lectins and safe and efficient production of protein powder by optimizing ultrasound-assisted extraction conditions, a "dual detoxification" process of enzymatic hydrolysis and thermal inactivation, and fine separation technology. The product obtained by this invention significantly outperforms existing technologies in terms of lectin extraction efficiency, preservation of bioactivity, protein powder detoxification effect, and functional properties. The process parameters of this invention have been systematically optimized, demonstrating good industrial applicability and economic value, and providing an effective solution for the comprehensive high-value utilization of white hyacinth bean.

Claims

1. A white kidney bean ultrasonic-assisted extraction of high-activity lectin and enzymatic detoxification protein powder co-production process, characterized in that, The method comprises the following steps: (1) washing and drying white lentil raw materials, crushing them to 40-60 mesh, defatting them, and vacuum drying them to an ethyl ether residual amount of less than or equal to 5 ppm to obtain defatted bean powder; (2) mixing the defatted bean powder obtained in step (1) with an extraction buffer at a material-to-liquid ratio of 1:10, extracting the mixture under the condition of 40-50°C and 40 kHz ultrasonic assistance for 100-130 minutes to obtain an extract; (3) centrifuging the extract at 10,000-15,000 g at 4°C for 20-30 minutes, collecting the supernatant as a crude lectin extract, and obtaining defatted bean dregs at the same time; (4) gradient ammonium sulfate salting of the crude lectin extract: adjusting the saturation to 40%, 60%, and 80% in sequence, stirring for 30 minutes, and then standing at 4°C for 4 hours, and centrifuging to collect the precipitates of each gradient; combining the precipitates of each gradient, redissolving them, and loading them into a dialysis bag for dialysis against the extraction buffer until no sulfate ions are present to obtain a preliminarily enriched lectin solution; (5) sequentially subjecting the preliminarily enriched lectin solution to DEAE ion exchange chromatography, CM ion exchange chromatography, and Sephacryl S-300 gel filtration chromatography to obtain a purified lectin solution; (6) freeze-drying the purified lectin solution to obtain lectin freeze-dried powder; (7) preparing a slurry from the defatted bean dregs obtained in step (3), adjusting the pH of the slurry to 6.0-7.0, adding 3-4% neutral protease, and hydrolyzing the slurry at 45-50°C for 1.5-2 hours to obtain an enzymatically hydrolyzed slurry; (8) heating the enzymatically hydrolyzed slurry to 80°C and maintaining it for 10 minutes to inactivate the enzyme, and obtaining a detoxified slurry; (9) crushing the detoxified slurry at 38-40°C, adjusting the pH to 4.3-4.5, and separating fibers by a pressure curved sieve to obtain a separated slurry; (10) adjusting the pH of the separated slurry to 5.5-6.0, separating starch by a hydrocyclone, centrifuging to collect protein slurry, and freeze-drying to obtain white lentil protein powder.

2. The co-production process of claim 1, wherein, In step (1), the defatting treatment is performed twice at 4°C using anhydrous ether, and the material-to-liquid ratio is 1:4 for each defatting.

3. The co-production process of claim 1, wherein, In step (2), the extraction buffer is 0.02 M Tris-HCl and 0.15 M NaCl, and the pH is 7.

8.

4. The co-production process of claim 1, wherein, In step (5), the DEAE ion exchange chromatography is equilibrated with a Tris-HCl buffer at pH 8.0, and the CM ion exchange chromatography is equilibrated with an acetic acid buffer at pH 5.

0.

5. The co-production process of claim 1, wherein, In step (7), the amount of neutral protease added is 3.5% based on the dry basis of the bean dregs.

6. The co-production process of claim 1, wherein, In step (9), the crushing is two-stage crushing.

7. The co-production process of claim 1, wherein, In step (10), the centrifugation is performed at 2000-2500 rpm and 40-45°C for 40-45 minutes.

8. The co-production process of claim 1, wherein, In step (6), the lectin freeze-dried powder is stored at -80°C.

9. The co-production process of claim 1, wherein, In step (9), the size of the screen hole of the pressure curved sieve is 0.2 mm.

10. The co-production process of claim 1, wherein, In step (10), the feed pressure of the hydrocyclone is 0.2 MPa.