Process for extracting antimicrobial peptides and albumin from pea processing wastewater

Abstract

The present invention discloses a method for extracting antimicrobial peptides and albumin from pea processing wastewater, which comprises: to extract albumin, centrifuging as a raw material pea processing wastewater produced during pea protein processing, adjusting the temperature of the raw material by temperature control and heat exchange, and then successively performing microfiltration, nanofiltration, ultrafiltration and secondary nanofiltration to obtain an albumin suspension; performing multi-effect concentration on the albumin suspension, adding an alkaline substance to adjust the pH, and then sterilizing and drying to obtain albumin.It can extract low molecular weight albumin from pea processing wastewater in a targeted manner and avoid waste of resources, effectively recover low molecular weight albumin and antimicrobial peptides from pea processing wastewater, avoid environmental pollution caused by poor treatment on pea wastewater and realize resource reuse. no figure.

Description

Title of the invention: Process for extracting antimicrobial peptides and albumin from pea processing wastewater Technical field

[0001] The present invention relates to the field of protein extraction technology, in particular to a process for extracting antimicrobial peptides and albumin from pea processing wastewater.

[0002] TECHNICAL BACKGROUND

[0003] As a starch-rich crop, peas are often used as raw materials for fine and thick vermicelli. Pea processing wastewater is wastewater produced during the production of pea protein isolate. The production of one ton of protein isolate will produce 25 to 35 m3 of pea processing wastewater discharged. Pea protein isolate manufacturers in China produce more than 13 million tons of pea processing wastewater each year, and this number is relatively large.

[0004] The wastewater from pea processing is rich in ammonia nitrogen and COD, with ammonia nitrogen reaching 700; the COD (chemical oxygen demand) and BOB (biological oxygen demand) values ​​are very high, above 10,000, generally, a large volume of flocculant for example polyacrylamide and polyaluminum chloride is needed during the treatment to separate by air flotation, then it passes through a plate and frame filter press to remove water and can be used as solid waste; or it can be treated and used as fodder or industrial fermentation raw material. However, the above treatment methods have low technical added values, the products bring only small economic benefits and cause waste of resources, moreover, the addition of a large volume of flocculant causes safety hazards and potential safety risks.

[0005] At present, there is another method for treating pea processing wastewater, including recovering protein isolate and precipitating with acid at isoelectric points; but after recovering pea protein isolate, the pea processing wastewater still contains low molecular weight protein components which cannot be extracted and utilized effectively.

[0006] The low molecular weight proteins contained in the wastewater of pea processing have effects of improving immunity and anti-oxidation, which can be extracted and used as raw materials for health foods having high nutritional and beneficial effects. Less efficient extractions will result in waste of pea lactalbumin, loss of albumin, and environmental pollution.

[0007] However, due to the inherent physical properties of pea legumelin, it is difficult to efficiently recover low molecular weight albumin.At present, there are few studies on the recovery and processing methods of pea legumes in China, in the existing art, pea protein polypeptides are extracted by enzymatic hydrolysis technique, in which, firstly, the preparation target products are different, secondly, the enzymatic hydrolysis technology used for extracting albumin from pea legumes produces bitter flavor of the protein, affecting the taste, thirdly, extracted protein polypeptides are unfavorable to the absorption and utilization by the human body; generally, other methods for separating and purifying lactalbumin consist of extracting lactalbumin from the wastewater of soybean processing, it is impossible to directly use the technique because of the initial raw materials are different, and thus it is impossible to guarantee the yield and purity of pea albumin.

[0008] Antibacterial peptides are polypeptides with antibacterial activity. By acting on the bacterial cell membrane, they destroy its integrity and cause perforation, penetrate into the cells, destroy the organelles and cause metabolic disorders. Antibacterial peptides not only have a good bactericidal effect on bacteria and fungi, but also have antiviral activity and a wound healing-promoting effect. Although antimicrobial peptides have broad application prospects and huge development potential, the industrialization process of antimicrobial peptides is relatively slow. At present, the main production processes of antimicrobial peptides involve genetic engineering technology, with high costs, a risk of enzymatic hydrolysis, and limited industrialization scale.In addition, antimicrobial peptide products exhibit low activity, poor heat resistance, and poor thermal stability.

[0009] At present, there is no other method for treating wastewater from pea processing, except for the separation of various proteins, which also causes the loss of antimicrobial peptides.

[0010] DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a method for extracting antimicrobial peptides and albumin from pea processing wastewater, to solve the problems of the existing art, such as waste of resources, low utilization rate albumin processing and recovery, inability to extract target albumin, and loss of antimicrobial peptides due to inability to extract low molecular weight albumin from pea processing wastewater.

[0012] In particular, the technical solution is as follows:

[0013] A method for extracting antimicrobial peptides from pea processing wastewater is characterized in that it comprises: centrifuging as a raw material pea processing wastewater produced during pea protein processing, adjusting the temperature of the raw material by temperature control and heat exchange, and then successively performing microfiltration, nanofiltration, ultrafiltration and secondary nanofiltration to obtain an albumin suspension; performing multi-effect concentration on the albumin suspension, adding an alkaline substance to adjust the pH, and then sterilizing and drying to obtain albumin;

[0014] add water to albumin to obtain albumin suspension, pretreat the albumin suspension in a water bath, cool and then act in a magnetic stirrer at constant temperature, add papain and add alkaline solution drop by drop to keep the pH of the enzymatic hydrolysate at the constant level; boil to deactivate the enzyme after the enzymatic hydrolysis is complete, cool, adjust the pH to 7.0; centrifuge, concentrate the supernatant by rotary evaporation and sterilize with a 0.22 µm syringe filtration membrane to obtain an initial product of antimicrobial peptide;

[0015] passing the initial antimicrobial peptide product through an extractor which is previously washed and activated with an aqueous methanol solution and then washed with an aqueous trifluoroacetic acid solution and equilibrated; successively passing the aqueous methanol solution and the aqueous trifluoroacetic acid solution through said extractor, collecting the passing liquids, concentrating the passing liquids under vacuum on a rotary evaporator to obtain a concentrated antimicrobial peptide solution, freezing the concentrated antimicrobial peptide solution at -15°C, and then drying the frozen antimicrobial peptide solution in a vacuum freeze dryer to obtain a purified antimicrobial peptide product;

[0016] the antimicrobial peptide product is characterized in that: the pH value of the antimicrobial peptide product measured with an acidity meter is 2.56-2.78;

[0017] The antimicrobial peptide product contains the following molecules: glycine, cysteine, arginine, lysine, histidine, alanine, threonine, aspartic acid, leucine, phenylalanine, serine, glutamic acid, valine, methionine and tyrosine.

[0018] A method for extracting albumin from pea processing wastewater comprises: centrifuging as raw material the pea processing wastewater produced during pea protein processing, adjust the temperature of the raw material by temperature control and heat exchange, and then successively carry out microfiltration, nanofiltration, ultrafiltration and secondary nanofiltration to obtain albumin suspension; carry out multi-effect concentration on the albumin suspension, add alkaline substance to adjust the pH, and then sterilize and dry to obtain albumin.

[0019] Preferably, the steps are as follows:

[0020] 1) centrifugation: centrifuge the wastewater from pea processing as material first ;

[0021] 2) temperature control and heat exchange: subject the waste water from the treatment of centrifuged peas with heat exchange;

[0022] 3) microfiltration: carry out microfiltration on the wastewater from the pea treatment after heat exchange;

[0023] 4) nanofiltration: adjust the systematic pressure and filtration temperature with a group of nanofiltration modules to remove 93%-95% of water;

[0024] 5) ultrafiltration: elute and separate in 5 to 10 cycles on an ultrafiltration membrane to obtain a suspension of crude albumin;

[0025] 6) secondary nanofiltration: passing the crude albumin through a member of Anti-pollution nanofiltration, wash with clean water, and dispose of the wash water at the end;

[0026] 7) multi-effect concentration: add the washed albumin suspension into a evaporator and concentrate by evaporation;

[0027] 8) neutralization: adding an alkaline substance into a steel tank stainless steel to adjust pH;

[0028] 9) sterilization and drying.

[0029] Preferably, the temperature during temperature control and heat exchange is 40 to 50°C.

[0030] Preferably, the microfiltration membrane is a silicon carbide membrane or a ceramic membrane.

[0031] Preferably, the silicon carbide membrane has a pore size of 10 nanometers to 30 microns.

[0032] Preferably, during nanofiltration, the systematic pressure is adjusted to 18-25bar and the filtration temperature is adjusted to 40-65°C.

[0033] Preferably, during ultrafiltration, an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons will be used.

[0034] Preferably, during the secondary nanofiltration, the clean water for washing is 93 to 95% of the clean water obtained after the nanofiltration in step 4).

[0035] Preferably, during multi-effect concentration, the vapor pressure is 0.6 to 0.8 Mpa; the concentration of the product discharged after concentration is 25% to 50%; during neutralization, at a temperature of 40 to 65°C, an alkaline substance is added to the system to adjust the pH to 6.5-8.

[0036] BENEFICIAL EFFECTS

[0037] 1. The configuration of the overall process flow according to the present invention allows to extract low molecular weight albumin from pea processing wastewater in a targeted manner, in which the pea processing wastewater is centrifuged before temperature control and heat exchange and the high molecular weight proteins contained in the pea processing wastewater are removed, which ensures that the pea albumin and oligosaccharides entering the next phase do not clog the equipment and thus builds a basis for the subsequent relative homogenization of the molecular weight of the albumin.

[0038] 2. During ultrafiltration, an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons will be used, which can ensure the separation purity of albumin and the molecular weight range of 1000-5000 Daltons, albumins with molecular weight between 1000-5000 account for a proportion of 85%, the molecular weight distribution is relatively regular and the molecular weight is relatively low, making the molecules easy to be absorbed by the human body.

[0039] 3. Temperature control and heat exchange carried out before the microfiltration ensure temperature stability of the pea processing wastewater to reach the optimum operating temperature before moving on to the microfiltration stage and build a basis for subsequent effective separations; the microfiltration stage allows for further separation of large molecular weight proteins that have not been separated by the centrifuge in the pea processing wastewater and build a basis for subsequent nanofiltration; a group of nanofiltration modules is used during nanofiltration to further separate albumin and to further remove 93% to 95% of the water, with the albumin after nanofiltration still containing a small amount of oligosaccharides;at this time, an ultrafiltration step will be carried out to separate the albumin and oligosaccharides, but at this time the albumin still contains excess salts, resulting in low purity of the albumin, then secondary nanofiltration will be carried out, for this, use a health grade anti-pollution nanofiltration membrane, add the separated clean water during nanofiltration to elute the excess salt, and remove the above clean water at the end of washing to recycle. The above steps can effectively recover the low molecular weight albumin from the pea processing wastewater with high purity, avoid pollution; environmental damage caused by poor treatment of pea wastewater and to achieve resource reuse.

[0040] 4. The present invention does not relate to the enzymatic hydrolysis process conventional, it can ensure the taste of albumin to the maximum extent by ensuring product yield, without foreign odor or bitter taste and providing high-quality products; the solution of the present invention is achieved by physical extraction, no chemical reaction involved, which can completely retain amino acids contained in albumin, albumin containing a variety of amino acids, with contents similar to those of amino acids in pure albumin, and the products have good performance and high nutritional value.

[0041] 5. In this solution, the high molecular weight proteins contained in The wastewater from pea processing is removed by various water separation and washing processes, effectively reducing the interference of large molecular weight proteins on the subsequent albumin separation process and ensuring the purity of albumin; for the process technology, desalting is carried out, the salts contained in albumin will be removed by water washing and nanofiltration membrane separation, so as to eliminate the bitter taste of pea legume.

[0042] 6. In the present invention, a group of nanofiltration modules is used during nanofiltration to further separate albumin and to further remove 93% to 95% of the water, this part of water can be reused during secondary nanofiltration, thus reducing the dirty water treatment process and saving energy and water resources.

[0043] 7. The present invention uses wastewater from the treatment process of pea protein and provides two ways of recycling and reusing wastewater. It not only extracts antibacterial peptides, but also extracts low-molecular-weight albumin, thereby improving the recovery and reuse rate of soybeans from clean wastewater. In addition, the method for extracting antimicrobial peptides provided by the present invention not only uses pea processing wastewater as a raw material, but also uses the albumin extracted according to the present invention as a raw material for extracting antimicrobial peptides with good heat resistance and thermal stability.

[0044] 8. To extract antimicrobial peptides according to the present invention, add papain and add NaOH dropwise to keep the pH of the enzymatic hydrolysate at the constant level; hydrolyze well for 5-8 h, boil for 15 min to deactivate the enzyme, cool, adjust the pH to 7.0 with NaOH or HCl; centrifuge at 8500 rpm for 20 min to remove the precipitate, concentrate the supernatant by rotary evaporation, sterilize with 0.22 μm syringe filtration membrane, to effectively control the level of protein hydrolysis without destroying the amino acids of antimicrobial peptides, maintain a variety of amino acids of antimicrobial peptides, avoid deformation after being heated, and provide good heat resistance and thermal stability.

[0045] DETAILED DESCRIPTION OF EMBODIMENT

[0046] The present invention can be described in more detail below via the exemplary embodiments.

[0047] I. Extraction of antimicrobial peptides

[0048] The present invention utilizes the wastewater from the pea protein processing process and provides two ways of recycling and reusing the wastewater, consisting of centrifuging the pea processing wastewater, performing heat exchange on the pea processing wastewater after centrifugation, performing microfiltration on the pea processing wastewater after heat exchange, extracting antimicrobial peptides from the retained liquid, and extracting albumin from the suspension obtained after microfiltration, thereby not only extracting antibacterial peptides, but also extracting low molecular weight albumin, thereby improving the recovery and reuse rate of soybeans from the clean wastewater.In addition, the method for extracting antimicrobial peptides provided by the present invention not only makes it possible to use the wastewater of pea processing as a raw material, but also makes it possible to use the albumin extracted according to the present invention as a raw material for extracting antimicrobial peptides with good heat resistance and thermal stability. The present invention can be described in more detail below via the embodiments.

[0049] A method for extracting antimicrobial peptides from pea processing wastewater comprises: centrifuging as a raw material pea processing wastewater produced during pea protein processing, performing heat exchange on the pea processing wastewater after centrifugation, performing microfiltration on the pea processing wastewater after heat exchange; the microfiltration membrane is a silicon carbide membrane having a pore size of 10 nanometers to 30 microns, obtaining a retained liquid;

[0050] pretreat the retained liquid in a water bath at 85°C for 15 min, cool and then act in a magnetic stirrer at constant temperature, add papain, add alkaline solution drop by drop to keep the pH of the enzymatic hydrolysate at the constant level; hydrolyze well for 5-8h, boil for 15min to deactivate the enzyme, cool, adjust the pH to 7.0 with NaOH or HCl; centrifuge at 8500 rpm for 20 min to remove the precipitate, concentrate the supernatant by rotary evaporation and sterilize with a 0.22pm syringe filtration membrane to obtain an initial antimicrobial peptide product;

[0051] passing the initial antimicrobial peptide product through an extractor which is previously washed and activated with an aqueous methanol solution and then washed with an aqueous trifluoroacetic acid solution and equilibrated; successively passing the aqueous methanol solution and the aqueous trifluoroacetic acid solution through said extractor, collecting the passing liquids, concentrating the passing liquids under vacuum on a rotary evaporator to obtain a concentrated antimicrobial peptide solution, freezing the concentrated antimicrobial peptide solution at -15°C for 9-12h, and then drying the frozen antimicrobial peptide solution in a vacuum freeze dryer for 20-25h by controlling the vacuum degree at 30-50Pa to obtain a purified antimicrobial peptide product;

[0052] Example 1:

[0053] centrifuging as raw material the pea processing wastewater produced during the processing of pea protein, performing heat exchange on the pea processing wastewater after centrifuging, performing microfiltration on the pea processing wastewater after heat exchange; the microfiltration membrane is a silicon carbide membrane having a pore size of 20 microns, obtaining a retained liquid;

[0054] pretreat the retained liquid in a water bath at 85°C for 15 min, cool and then act in a magnetic stirrer at constant temperature, add papain, add alkaline solution drop by drop to keep the pH of the enzyme hydrolysate at the constant level; hydrolyze well for 7h, boil for 15min to deactivate the enzyme, cool, adjust the pH to 7.0 with NaOH or HCl; centrifuge at 8500 rpm for 20 min to remove the precipitate, concentrate the supernatant by rotary evaporation and sterilize with a 0.22pm syringe filtration membrane to obtain an initial product of antimicrobial peptide;

[0055] passing the initial antimicrobial peptide product through an extractor which is previously washed and activated with an aqueous methanol solution and then washed with an aqueous trifluoroacetic acid solution and equilibrated; successively passing the aqueous methanol solution and the aqueous trifluoroacetic acid solution through said extractor, collecting the passing liquids, concentrating the passing liquids under vacuum on a rotary evaporator to obtain a concentrated antimicrobial peptide solution, freezing the concentrated antimicrobial peptide solution at -15°C for 10h, and then drying the frozen antimicrobial peptide solution in a vacuum freeze dryer for 20h by controlling the vacuum degree at 40Pa to obtain a purified antimicrobial peptide product;

[0056] Example 2:

[0057] centrifuging as raw material the pea processing wastewater produced during the processing of pea protein, adjusting the temperature of the raw material by temperature control and heat exchange, and then successively performing microfiltration, nanofiltration, ultrafiltration and secondary nanofiltration to obtain an albumin suspension; performing multi-effect concentration on the albumin suspension, adding an alkaline substance to adjust the pH, and then sterilizing and drying to obtain albumin;

[0058] add water to albumin to obtain albumin suspension, pretreat the albumin suspension in a water bath at 85°C for 15min, cool and then act in a magnetic stirrer at constant temperature, add papain, add NaOH dropwise to keep the pH of the enzyme hydrolysate at the constant level; hydrolyze well for 5-8h, boil for 15min to deactivate the enzyme, cool, adjust the pH to 7.0 with NaOH or HCl; centrifuge at 8500 rpm for 20min to remove the precipitate, concentrate the supernatant by rotary evaporation and sterilize with a 0.22pm syringe filtration membrane to obtain an initial product of antimicrobial peptide;

[0059] passing the initial antimicrobial peptide product through an extractor which is previously washed and activated with an aqueous methanol solution and then washed with an aqueous trifluoroacetic acid solution and equilibrated; successively passing the aqueous methanol solution and the aqueous trifluoroacetic acid solution through said extractor, collecting the passing liquids, concentrating the passing liquids under vacuum on a rotary evaporator to obtain a concentrated antimicrobial peptide solution, freezing the concentrated antimicrobial peptide solution at -15°C for 9-12h, and then drying the frozen antimicrobial peptide solution in a vacuum freeze dryer for 20-25h by controlling the vacuum degree at 30-50Pa to obtain a purified antimicrobial peptide product;

[0060] Determination of amino acid types:

[0061] The amino acid types of the antimicrobial peptides obtained in Examples 1 and 2 above are determined by an automatic amino acid analyzer, which are as follows:

[0062] glycine, cysteine, arginine, lysine, histidine, alanine, threonine, aspartic acid, leucine, phenylalanine, serine, glutamic acid, valine, methionine and tyrosine.

[0063] PH: The pH values ​​of the antimicrobial peptides in Examples 1 and 2 are measured with an acidity meter, which are 2.63 and 2.77.

[0064] Testing of thermal stability of antimicrobial peptides:

[0065] By heating the antimicrobial peptides obtained in Examples 1 and 2 in a boiling water bath, the diameters of the inhibition zone were measured and the results are shown in Table 1 below. [Tables 1] Heating time (min) 0 10 20 30 40 Diameter of the inhibition zone of example 1 (mm) 18.33 18.32 18.25 18.22 18.11 Diameter of the inhibition zone of example 2 (mm) 18.25 18.24 18.20 18.20 18.02 The above table shows that the treatment at 100°C has almost no effect on the antibacterial activity of the antibacterial peptides, and the antibacterial activity remains at 98.79% and 98.74% after reheating for 40 minutes in the boiling water bath, which indicates that the antibacterial peptides prepared according to the present invention have a low tendency of denaturation upon reheating and have good heat resistance and thermal stability.

[0066] II. Extraction of albumin

[0067] In the present invention, pea processing wastewater produced during pea protein processing will be used, which mainly contains components such as albumin and oligosaccharides; a disc centrifuge, a horizontal centrifuge or a three-foot centrifuge can be used during centrifugation.

[0068] Example 1:

[0069] In the present invention, pea processing wastewater produced during pea protein processing will be used, which mainly contains components such as albumin and oligosaccharides, and in particular, the extraction comprises the following steps:

[0070] 1) centrifugation: centrifuge the wastewater from pea processing as material first to remove high molecular weight proteins from the wastewater from pea processing, which ensures that pea albumin and oligosaccharides entering the next phase do not clog the equipment and guarantees a molecular weight range of 1000-5000 Daltons;

[0071] 2) temperature control and heat exchange: subject the waste water from the centrifuged pea treatment at a heat exchange at a temperature of 45°C, in order to ensure stability of the temperature of the wastewater from the pea treatment for reach the optimum operating temperature before moving on to the separation phase with a membrane.

[0072] 3) microfiltration: carry out microfiltration on the wastewater from the treatment of peas after heat exchange, the microfiltration membrane is a silicon carbide membrane with a pore size of 10 nanometers to 30 microns; separate the large molecular weight proteins that have not been separated by the centrifuge in the wastewater from pea processing, remove the retained liquid and pass the albumin and oligosaccharides.

[0073] 4) nanofiltration: adjust the systematic pressure to 20bar and the temperature of filtration at 60°C with a group of nanofiltration modules to remove 95% of the water;

[0074] 5) ultrafiltration: elute and separate in 8 cycles on an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons for separating albumin and oligosaccharides and thus obtaining an albumin product with a dry protein content of 80% to 90% and oligosaccharides containing 7% to 30% dry substances.

[0075] 6) secondary nanofiltration: passing the crude albumin through a member of anti-pollution nanofiltration, wash with 95% clean water obtained in step 4), remove the wash water at the end and keep the retained liquid;

[0076] 7) multi-effect concentration: concentrate the washed albumin suspension by evaporation in a three-effect evaporator at a speed of 1800kg / h (water evaporation), whose loading concentration is 10%, the discharging concentration is 47%, and the working vapor pressure is 0.7Mpa.

[0077] 8) neutralization: adding an alkaline substance into a steel tank stainless steel to adjust the pH to 6.5, the temperature to 55°C and the dry substance content to 14%;

[0078] 9) sterilization and drying: sterilize for deodorization in equipment of flash by controlling the temperature at 140°C; then send the protein to a drying system for drying, the drying temperature being adjusted according to the products and times, the inlet air temperature being controlled at 143°C and the exhaust air temperature being controlled at 55°C.

[0079] Example 2:

[0080] In the present invention, pea processing wastewater produced during pea protein processing will be used, which mainly contains components such as albumin and oligosaccharides, and in particular, the extraction comprises the following steps:

[0081] 1) centrifugation: centrifuge the wastewater from pea processing as material first to remove large molecular weight proteins from water used from pea processing, which ensures that pea albumin and oligosaccharides entering the next phase do not clog the equipment and guarantees a molecular weight range of 1000-5000 Daltons;

[0082] 2) temperature control and heat exchange: subject the waste water of the Centrifuged pea treatment at a heat exchange temperature of 40°C, to ensure temperature stability of the pea treatment wastewater to reach the optimum operating temperature before moving on to the separation phase with a membrane.

[0083] 3) microfiltration: carry out microfiltration on the wastewater from the treatment of peas after heat exchange, the microfiltration membrane is a silicon carbide membrane with a pore size of 10 nanometers to 30 microns; separate the large molecular weight proteins that have not been separated by the centrifuge in the wastewater from pea processing, remove the retained liquid and pass the albumin and oligosaccharides.

[0084] 4) nanofiltration: adjust the systematic pressure to 20 bar and the temperature of filtration at 45°C with a group of nanofiltration modules to remove 95% of the water;

[0085] 5) ultrafiltration: elute and separate in 7 cycles on an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons for separating albumin and oligosaccharides and thus obtaining an albumin product with a dry protein content of 80% to 90% and oligosaccharides containing 7% to 30% dry substances.

[0086] 6) secondary nanofiltration: passing the crude albumin through a member of anti-pollution nanofiltration, wash with 95% clean water obtained in step 4), remove the wash water at the end and keep the retained liquid;

[0087] 7) multi-effect concentration: concentrate the washed albumin suspension by evaporation in a three-effect evaporator at a speed of 1800kg / h (water evaporation), whose loading concentration is 8%, the discharging concentration is 48%, and the working vapor pressure is 0.6 Mpa.

[0088] 8) neutralization: adding an alkaline substance into a steel tank stainless steel to adjust the pH to 7, the temperature to 45°C and the dry substance content to 15%;

[0089] 9) sterilization and drying: sterilize for deodorization in equipment of flash by controlling the temperature at 140°C; then send the protein to a drying system for drying, the drying temperature being adjusted according to the products and times, the inlet air temperature being controlled at 130°C and the exhaust air temperature being controlled at 48°C.

[0090] Example 3:

[0091] In the present invention, pea processing wastewater produced during pea protein processing will be used, which mainly contains components such as albumin and oligosaccharides, and in particular, the extraction comprises the following steps:

[0092] 1) centrifugation: centrifuge the wastewater from pea processing as a material first to remove high molecular weight proteins from the wastewater from pea processing, which ensures that pea albumin and oligosaccharides entering the next phase do not clog the equipment and guarantees a molecular weight range of 1000-5000 Daltons;

[0093] 2) temperature control and heat exchange: subject the waste water from the Centrifuged pea treatment at a heat exchange temperature of 44°C, to ensure temperature stability of the pea treatment wastewater to reach the optimum operating temperature before moving on to the separation phase with a membrane.

[0094] 3) microfiltration: carry out microfiltration on the wastewater from the pea treatment After heat exchange, the microfiltration membrane is a ceramic membrane; separate the large molecular weight proteins that have not been separated by the centrifuge in the pea processing wastewater, remove the retained liquid, and pass the albumin and oligosaccharides.

[0095] 4) nanofiltration: adjust the systematic pressure to 18bar and the temperature of filtration at 55°C with a group of nanofiltration modules to remove 94% of the water;

[0096] 5) ultrafiltration: elute and separate in 8 cycles on an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons for separating albumin and oligosaccharides and thus obtaining an albumin product with a dry protein content of 80% to 90% and oligosaccharides containing 7% to 30% dry substances.

[0097] 6) secondary nanofiltration: passing the crude albumin through a member of anti-pollution nanofiltration, wash with 94% clean water obtained in step 4), remove the wash water at the end and keep the retained liquid;

[0098] 7) multi-effect concentration: concentrate the washed albumin suspension by evaporation in a three-effect evaporator at a speed of 1800kg / h (water evaporation), whose loading concentration is 9%, the discharging concentration is 49%, and the working vapor pressure is 0.6 Mpa.

[0099] 8) neutralization: adding an alkaline substance into a steel tank stainless steel to adjust the pH to 7.8, the temperature to 42°C and the dry substance content to 16%;

[0100] 9) sterilization and drying: sterilize for deodorization in equipment of flash while controlling the temperature at 138 °C; then send the protein to a drying system for drying, the drying temperature being adjusted according to the products and times, the temperature of the inlet air being controlled at 145 °C and the temperature of the exhaust air being controlled at 50 °C.

[0101] Example 4:

[0102] In the present invention, the wastewater from the treatment of peas produced during the treatment of pea protein will be used, which mainly contains components such as albumin and oligosaccharides, and in particular, the extraction comprises the following steps:

[0103] 1) centrifugation: centrifuge the wastewater from the treatment of peas as the raw material first to remove high molecular weight proteins from the wastewater from pea processing, which ensures that pea albumin and oligosaccharides entering the next phase do not clog the equipment and guarantees a molecular weight range of 1000-5000 Daltons;

[0104] 2) temperature control and heat exchange: subject the waste water from the Centrifuged pea treatment at a heat exchange temperature of 41°C, to ensure temperature stability of the pea treatment wastewater to reach the optimum operating temperature before moving on to the separation phase with a membrane.

[0105] 3) microfiltration: carry out microfiltration on the wastewater from the treatment of peas after heat exchange, the microfiltration membrane is a silicon carbide membrane with a pore size of 10 nanometers to 30 microns; separate the large molecular weight proteins that have not been separated by the centrifuge in the wastewater from pea processing, remove the retained liquid and pass the albumin and oligosaccharides.

[0106] 4) nanofiltration: adjust the systematic pressure to 25 bars and the temperature of filtration at 58°C with a group of nanofiltration modules to remove 95% of the water;

[0107] 5) ultrafiltration: elute and separate in 10 cycles on an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons for separating albumin and oligosaccharides and thus obtaining an albumin product with a dry protein content of 80% to 90% and oligosaccharides containing 7% to 30% dry substances.

[0108] 6) secondary nanofiltration: passing the crude albumin through a member of anti-pollution nanofiltration, wash with 95% clean water obtained in step 4), remove the wash water at the end and keep the retained liquid;

[0109] 7) multi-effect concentration: concentrate the washed albumin suspension by evaporation in a three-effect evaporator at a speed of 1800kg / h (water evaporation), whose loading concentration is 12%, the discharging concentration is 50%, and the working vapor pressure is 0.8 Mpa.

[0110] 8) neutralization: adding an alkaline substance into a steel tank stainless steel to adjust the pH to 8, the temperature to 65°C and the dry substance content to 18%;

[0111] 9) sterilization and drying: sterilize for deodorization in equipment of flash by controlling the temperature at 137°C; then send the protein to a drying system for drying, the drying temperature being adjusted according to the products and times, the inlet air temperature being controlled at 135°C and the exhaust air temperature being controlled at 60°C.

[0112] Experimental data:

[0113] I. The yield and purity of albumin

[0114] The yield and purity of the albumin extracted in Examples 1 to 4 were determined, and the purity was determined by conventional Kjeldahl determination. The determined results are shown in Table 2 below. [Tables 2] Items Examples of achievement Results Yield in / % Example 1 89% Example 2 85% Example 3 89% Example 4 90% Purity in / % Example 1 93% Example 2 88% Example 3 91% Example 4 92%

[0115] The above table shows that the albumin extracted by the method of the present invention has a high purity of between 88% and 93%, and the yield of the albumin of the present invention is 85% to 90%, which shows that the extraction method of the present invention can effectively recover low molecular weight albumin from the wastewater of pea processing.

[0116] IL Detection of the molecular weight distribution range of peptides

[0117] The molecular weight distribution range of the peptides on the albumin obtained in Example 1 of the present invention and the pea protein peptides are detected. conventional according to GB / T22492-2008 and the results are shown in Table 3 below. [Tables 3] Molecular Weight Range Albumin prepared according to the present invention Conventional Pea Protein Peptides Peak Area in % (X=220nn) Number Average Molecular Weight Weight Average Molecular Weight Peak Area in % (X=220nn) Number Average Molecular Weight Weight Average Molecular Weight >10000 3.06 15437 16357 / / / 10000-500 0 5.1 6497 5814 / / / 5000-3000 39.65 3968 4042 0.44 3657 3765 3000-2000 28.19 2480 2517 1.08 2340 2372 2000-1000 17.16 1502 1557 8.76 1270 1313 1000-500 1.89 710 739 25.56 641 667 500-180 2.74 286 313 51.85 281 303 <180 2.21 / / 12.32 / /

[0118] The above table shows that for the albumin extracted by the method of the present invention, the components with a molecular weight of 1000-5000 Daltons account for a proportion of 85%, and the components with a molecular weight of 180-5000 Daltons and above 10000 Daltons account for a proportion of 15%; and for conventional pea protein peptides, the molecular weight distribution range is relatively wide, in which the components with a molecular weight of 1000-5000 Daltons account for only a proportion of 10.28%, and the components with a molecular weight of 180-1000 Daltons account for a proportion of 89.72%.Taking into account that the higher the molecular weight, the more difficult the molecules are to be absorbed by the human body, the albumin prepared by the method of the present invention is easy to be absorbed and utilized by the human body, because its molecular weight distribution is relatively regular and its molecular weight is lower.

[0119] III. Detection of amino acids

[0120] An amino acid assay of the albumin obtained in Example 1 of the present invention was carried out by HPLC, and the results are shown in Table 4 below. [Tables 4] Amino acids Albumin Amino acid content of pure albumin Standard mode W HO / FAO Cystine 4.97 5.7 3.5 Methionine Threonine 5.48 6.00 4 Valine 3.98 4.15 5 Isoleucine 2.56 2.93 4 Leucine 3.00 3.44 7 Tyrosine 6.63 7.61 6 Phenylalanine Lysine 8.85 10.16 5.5 Tryptophan 0.89 1.03 1

[0121] The above table shows that, the albumin obtained by the extraction method of the present invention contains a variety of amino acids, with contents similar to those of the amino acids of pure albumin, and compared with the WHO / FAO standard model, except that the content of some amino acids is lower than the content of the standard model due to the restriction of the structure of albumin, the content of other amino acids exceeds the content in the standard mode, which shows that the albumin product obtained by the extraction method of the present invention has good performance, high nutritional value and good product quality.

[0122] The technical solution of the present invention is based on an overall inventive concept, which is an indivisible whole and cannot be divided within the technical scope. The integrity of the invention will be described in more detail below and validated through experiments:

[0123] centrifugation on pea processing wastewater before temperature control and heat exchange can remove large molecular weight proteins contained in pea processing wastewater, ensures albumin separation purity and molecular weight range of 1000-5000 Daltons and relatively regular molecular weight distribution; temperature control and heat exchange performed before microfiltration ensure temperature stability of the pea processing wastewater to reach the optimum operating temperature before moving to the microfiltration stage and build a basis for subsequent effective separations; the microfiltration stage helps to further separate large molecular weight proteins that have not been separated by the centrifuge in the pea processing wastewater and build a basis for subsequent nanofiltration, if the microfiltration stage is removed, the subsequent nanofiltration membrane will be blocked soon after, the lifetime will be reduced and the flux will decrease, ultimately resulting in a decrease in the purity of the final albumin and a decrease in the yield rate;a group of nanofiltration modules is used during nanofiltration to further separate albumin and further remove 93%-95% of water, this part of water can be reused during secondary nanofiltration without dirty water treatment, saving energy and water resources, the albumin after nanofiltration still contains a small amount of oligosaccharides; at this time, an ultrafiltration step will be carried out to separate albumin and oligosaccharides, but at this time the albumin still contains excess salts, resulting in low purity of albumin, then secondary nanofiltration will be carried out, for this, use a health grade anti-pollution nanofiltration membrane, add the clean water separated during nanofiltration to elute the excess salt, remove the above clean water at the end of washing to recycle;in this solution, the large molecular weight proteins contained in the wastewater of pea processing are removed by various water separation and washing processes, effectively reducing the interference of large molecular weight proteins on the subsequent albumin separation process and ensuring the purity of albumin; for the process technology, desalting is carried out, the salts contained in albumin will be removed by water washing and nanofiltration membrane separation, so as to eliminate the bitter taste of pea legume. ;

[0124] Comparison Example A: The extraction process is similar to that of Example 1, and the difference is that the microfiltration step is omitted.

[0125] Comparison Example B: The extraction method is similar to that of Example 1, and the difference is that the nanofiltration step is omitted.

[0126] Comparison Example C: The extraction method is similar to that of Example 1, and the difference is that the ultrafiltration step is omitted.

[0127] Comparison Example D: The extraction method is similar to that of Example 1, and the difference is that the secondary nanofiltration step is omitted.

[0128] The yield and purity were determined on the albumins obtained in the above comparison examples, and the results are shown in Table 5 below. [Tables 5] Items Examples of achievement Results Yield in / % Example 1 89% Comparison example A 47% Comparison example B 45% Comparison example C 0% Comparison example D 77% Purity in / % Example 1 93% Comparison example A 64% Comparison example B 59% Comparison example C 0% Comparison example D 71%

[0129] The above results show that when the microfiltration and nanofiltration steps are removed, the albumin yield drops significantly to 45% and the purity also drops significantly to 59%, and when the ultrafiltration step is removed, the albumin yield drops to 0, which shows that low molecular weight albumin meeting the specifications cannot be separated without the ultrafiltration step and the albumin purity drops significantly without the secondary nanofiltration step.

[0130] Determination of albumin yield at different heat exchange temperatures: The steps of other methods tested are similar to those of Example 1, the difference is that the heat exchange temperatures are 30, 35, 40, 45, 50, 55, 60, 65 and 70°C respectively, the corresponding performances of the final albumin products will be tested, and the results are shown in Table 6 below. [Tableauxô] Items Temperature Results Yield in / % 30°C 35°C 0% 0% 40°C 84% 45°C 89% 50°C 82% 55°C 9% 60°C 8% 65°C 6% 70°C 6%

[0131] The above table shows that the extraction efficiency of albumin is 0 to 30-40°C, that is, when the heat exchange temperature is lower than 40°C, albumin cannot be extracted and recovered, in the heat exchange range of the present invention, that is, when the temperature is 40 to 50°C, the efficiency is 82 to 89% and when the temperature is 45°C, the efficiency reaches the maximum value; when the temperature is higher than 50°C, the albumin yield drops significantly and reaches below 10%, which shows that the reasonable temperature control and heat exchange ensure the temperature stability of the pea processing wastewater to reach the optimum operating temperature before moving to the microfiltration stage and build a basis for the following effective separations, thus increasing the albumin yield.

[0132] The above experimental data show that the technical solution of the present invention is an indivisible whole, the steps cooperate and link with each other to ensure the yield and purity of the final extracted albumin and a simple technical division is impossible. The above experimental data show that the present invention can effectively solve the problem of pore blockage and decrease in membrane flux when using ultrafiltration or nanofiltration alone to extract protein peptides by conventional techniques, effectively reducing investment costs and production costs, and is more suitable for industrial production.

[0133] Of course, the realization of the present invention is not limited to the above embodiments, any modifications which respect the spirits and the solution of the present invention and any direct applications of the spirits and the solution of the present invention to other situations must be included within the scope of the protection of the present invention.

Claims

Claims

1. A method for extracting albumin from pea processing wastewater, characterized in that it comprises: centrifuging as a raw material the pea processing wastewater produced during the processing of pea protein, adjusting the temperature of the raw material by temperature control and heat exchange, said temperature being 40-50°C, and then successively performing microfiltration, nanofiltration, ultrafiltration and secondary nanofiltration to obtain an albumin suspension; performing multi-effect concentration on the albumin suspension, adding an alkaline substance to adjust the pH, and then sterilizing and drying to obtain albumin.

2. A method for extracting albumin from pea processing wastewater according to claim 1, characterized in that the steps are as follows: 1) centrifugation: centrifuging the pea processing wastewater as a raw material; 2) temperature control and heat exchange: subjecting the centrifuged pea processing wastewater to heat exchange; 3) microfiltration: performing microfiltration on the pea processing wastewater after heat exchange; 4) nanofiltration: adjusting the systematic pressure and filtration temperature with a nanofiltration module group to remove 93%-95% of the water; 5) ultrafiltration: eluting and separating in 5-10 cycles on an ultrafiltration membrane to obtain a crude albumin suspension; 6) Secondary nanofiltration: Pass the crude albumin through an anti-pollution nanofiltration member, wash with clean water, and discard the washing water at the end;7) Multi-effect concentration: Add the washed albumin suspension into an evaporator and concentrate by evaporation; 8) Neutralization: Add an alkaline substance into a stainless steel tank to adjust the pH; 9) Sterilization and drying.;

3. A method for extracting albumin from pea processing wastewater according to claim 1 or 2, characterized in that, the Microfiltration membrane is a silicon carbide membrane or a ceramic membrane.

4. A method for extracting albumin from pea processing wastewater according to claim 3, characterized in that the silicon carbide membrane has a pore size of 10 nanometers to 30 microns.

5. A method for extracting albumin from pea processing wastewater according to claim 1 or 2, characterized in that during nanofiltration the systematic pressure is adjusted to 18-25 bar and the filtration temperature is adjusted to 40-65°C.

6. A method for extracting albumin from pea processing wastewater according to claim 1 or 2, characterized in that during ultrafiltration an ultrafiltration membrane having a retention threshold of 1000-5000 Daltons will be used.

7. A method for extracting albumin from pea processing wastewater according to claim 2, characterized in that during secondary nanofiltration the clean water for washing is 93-95% of the clean water obtained after nanofiltration in step 4).

8. A method for extracting albumin from pea processing wastewater according to claim 1 or 2, characterized in that, during multi-effect concentration, the vapor pressure is 0.6 to 0.8 Mpa; the concentration of the product discharged after concentration is 25% to 50%; during neutralization, at a temperature of 40 to 65°C, an alkaline substance is added to the system to adjust the pH to 6.5-8.