CN109762862B - Preparation method of high-purity lotus seed protein oligopeptide - Google Patents
Preparation method of high-purity lotus seed protein oligopeptide Download PDFInfo
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Abstract
The invention provides a lotus seed protein oligopeptide and a preparation method thereof, wherein the method comprises the steps of (1) soaking and grinding; (2) salt leaching: according to the material-liquid ratio of 1: adding NaCl to 0.2-0.5 mol/L from 10 to 20, adjusting the pH value of the solution to 8-10, filtering and centrifuging to obtain supernatant; (3) acid precipitation: adding HCl with the concentration of 0.25-0.5 mol/L to adjust the pH value of the supernatant to 4.3-4.7, standing for precipitation, and centrifuging to obtain a lotus seed protein precipitate; (4) enzymolysis: adding water to adjust the concentration of a substrate to be 8-16%, setting the temperature to be 35-45 ℃, adjusting the pH to be 3-8, adding protease, wherein the protease is neutral protease and papain with the mass ratio of 1.5-2.5, and performing enzymolysis for 2-4 h; enzyme deactivation; dialyzing; membrane filtration; and (5) drying. The yield of the lotus seed protein oligopeptide can reach 78.6% at most, and the lotus seed protein oligopeptide has good appearance, color and taste quality.
Description
Technical Field
The invention relates to the technical field of product processing, in particular to lotus seed protein oligopeptide and a preparation method thereof.
Background
Lotus seeds are seeds of Nymphaeaceae plant lotus, are produced in Hunan, hubei, jiangxi, fujian and other places, and are abundant in resources. The lotus seed has high protein content, contains 18 amino acids, has the content and the proportion of the essential amino acids close to the FAO/WHO recommended mode, has relatively balanced amino acid content proportion, can reduce the contents of cholesterol and saturated fatty acid in diet, and keeps balanced nutrition. Compared with the bean protein which lacks methionine, the lotus seed protein reaches the WHO specified standard. The lotus seed protein is rich in lysine which is the first restrictive amino acid in common food, and can improve the condition of insufficient intake of people lacking the lotus seed protein. The lotus seed protein oligopeptide keeps the reasonable proportion of amino acid, the micromolecule peptide is absorbed more quickly, the lotus seed protein oligopeptide also has a remarkable immunoregulation function, and has an auxiliary curative effect on liver injury caused by chemical sources and can remove free radicals. In addition, the lotus seed protein oligopeptide can be applied to multiple industries, and has a very wide market prospect.
Comparison document 1: the application number is CN201310407100.1, the name of lotus seed protein powder and the production method thereof comprises the following production steps: (1) and (4) checking and accepting the lotus seeds: picking fresh lotus seeds of seven to eight ripens; (2) crushing; (3) salt extraction, acid precipitation and centrifugation: adjusting the pH value of the extracted mixed solution to 4.2 by using HCl and NaOH. (4) Stirring and centrifugally filtering; (5) and (3) ultrafiltration: carrying out ultrafiltration concentration on the filtrate to obtain high-concentration protein; (6) and (3) drying: drying with a spray dryer to obtain finished protein powder; (7) packaging: and (7) vacuum packaging. Compared with the prior art, the lotus seed protein powder and the preparation method thereof have the beneficial effects that: 1) The lotus seed protein powder fully maintains the faint scent of lotus seeds, is white to light yellow, is uniform paste after being mixed with hot water, and has fine taste. 2) The lotus seed protein powder has simple production process, and is convenient for large-scale production, packaging, transportation and sale. 3) Is convenient for storage, and has a shelf life of 12 months at normal temperature. However, the extraction rate of the lotus seed protein extraction by adopting a salt reduction, extraction and acid precipitation method is not high; and the extracted protein is not desalted and has poor taste.
Comparison document 2: literature "lotus seed protein extraction process research-zhangyu: food science 2007,28 (9): 144-147' this craft adopts alkali extraction acid precipitation method, the feed-liquid ratio 125 (lotus seed powder: water), pH11 (alkali extraction), soak extract 3 hours under 40 deg.C, the extraction rate of protein can reach 86.15%, however this method adopts alkali extraction, the protein color and luster obtained is darker, the mouthfeel is bad and produces a large amount of waste liquids, the after-treatment cost is high.
The extraction process of lotus seed protein of CN101215316A and CN101215318A adopts microwave and ultrasonic wave to assist salt to extract CO 2 Or HCl acid precipitation to obtain lotus seed protein, the processes have short extraction time, low energy consumption and high yield, but the industrial production is difficult at present, the requirement on equipment is high, and the investment is large. The preparation method of lotus seed protein peptide of CN101497913A adopts Ca (OH) 2 The alkaline extraction process replacing NaOH lacks of process steps for removing impurity ions, and CaCl can be remained in the obtained protein peptide 2 It will affect the quality and taste of the product.
In summary, at present, domestic reports and patents/patent applications on the preparation process of the lotus seed protein oligopeptide are few, and most of the lotus seed protein oligopeptide products have the problems of low yield and more impurities.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the lotus seed protein oligopeptide and the preparation method thereof, the yield of the lotus seed protein oligopeptide prepared by the invention can reach 78.6 percent, and the lotus seed protein oligopeptide has good appearance, color and taste quality.
The invention is realized in the following way:
the invention provides a preparation method of lotus seed protein oligopeptide, which comprises the following steps:
step 1, soaking and grinding: soaking the lotus seeds without the cores in warm water, adding water, uniformly mixing, carrying out wet grinding in a grinding machine, and sieving after grinding;
step 2, salt leaching: according to the mass volume ratio of the feed liquid of 1: adding NaCl to 0.2-0.5 mol/L at 10-20 ℃, adjusting the pH value of the solution to 8-10, extracting the lotus seed powder for 2-4 h at the extraction temperature of 30-45 ℃, filtering and centrifuging to obtain supernatant;
step 3, acid precipitation: adding HCl with the concentration of 0.25-0.5 mol/L to adjust the PH value of the supernatant to 4.3-4.7, standing and precipitating the solution, and centrifuging to obtain lotus seed protein precipitate;
step 4, enzymolysis: adding water into the lotus seed protein precipitate to adjust the concentration of a substrate to be 8-16%, setting the temperature to be 35-45 ℃, adjusting the pH to be 3-8, adding protease, wherein the protease is neutral protease and papain, the total addition amount of the protease is 1-5% of the mass of the lotus seed protein precipitate, the mass ratio of the neutral protease to the papain is 1.5-2.5, and carrying out enzymolysis for 2-4 h to obtain lotus seed protein peptide liquid;
step 5, enzyme deactivation: heating the lotus seed protein polypeptide liquid, and inactivating enzyme;
step 6, dialysis: dialyzing the lotus seed protein polypeptide liquid to obtain desalted lotus seed protein peptide liquid;
and 7, membrane filtration: the retention degree is set at 1500Da, protein peptide liquid is obtained by filtration and concentrated to obtain concentrated solution, and the concentration of the obtained concentrated solution is 25-45%;
and 8, drying the concentrated solution to obtain the lotus seed protein oligopeptide. Obtaining the lotus seed protein oligopeptide with the purity of more than 97 percent and the relative molecular mass of less than 1500 Da.
The invention has the following beneficial effects:
1. according to the preparation method of the lotus seed protein oligopeptide, the lotus seed protein is leached by adding NaCl and adjusting the pH value of the solution to be 8-10, so that the influence of strong alkali on the lotus seed protein is greatly reduced, the degradation and agglomeration of the protein are reduced, the denaturation of lotus seed starch in the process is reduced, the obtained protein is good in appearance color and luster and taste quality, and meanwhile, the residual starch can be used for continuously developing products with higher added values; specifically, the method comprises the following steps:
the applicant discovers through a great deal of innovative research that the extraction rate of the lotus seed protein is in an increasing trend along with the change of the pH value within the range of 5.0-13.0. From the influence of pH on the extraction rate, although the extraction rate is highest when the pH is 13.0, lotus seed protein is more easily separated out along with the increase of the alkalinity of the extraction rate, the protein is easily denatured and degraded due to the overhigh alkalinity, and the quality of the obtained lotus seed protein is poor. Considering the application in food industry, the application of the method is limited due to the overhigh pH value (equipment and operation), the extraction pH value is controlled to be 8-10, the extraction rate is higher and more stable in the interval, the equipment requirement is reduced, and the use of strong base is reduced.
2. According to the preparation method of the lotus seed protein oligopeptide, a great amount of innovative research by the applicant discovers that enzymolysis is carried out by using two enzymes, namely neutral protease and papain, at the same time, the mass ratio of the neutral protease to the papain is controlled to be 1.5-2.5, and the total addition amount of the protease is 1% -5% of the mass of the lotus seed protein precipitate; first, it is also the most important, and the degree of hydrolysis of protein and the yield of polypeptide can be improved to the utmost extent. Secondly, the application range of the neutral protease and the papain is wide, the pH value does not need to be adjusted in the enzymolysis process, the hydrolysis process condition is simplified, the production cost is reduced, and the neutralization step is omitted. Thirdly, the concentration of the substrate is further set to be 8-16%, the temperature is set to be 35-45 ℃, and the PH is adjusted to be 3-8, so that the enzymolysis condition is further accurately controlled, the hydrolysis reaches the required degree, the micromolecule bitter peptides cannot be generated by over hydrolysis, and the quality of the finished product can be improved. The molecular weight of the product is averagely controlled to be about 800D (the content of the molecular weight above 5000D is detected to be less than or equal to 2.9%), and the input-output ratio of the protein reaches more than 97%.
3. The invention can remove NaCl added in the extraction and can also remove other ions brought in the raw materials through dialysis, thereby improving the taste of the product; meanwhile, protein which does not meet the hydrolysis requirement is intercepted through membrane filtration (the interception degree is set at 1500 Da), macromolecular protein which is not easy to digest and absorb by a human body is removed, and the purity of the target protein peptide is improved.
4. The yield of the lotus seed protein oligopeptide prepared by the invention can reach as high as 78.6%, and the lotus seed protein oligopeptide has good appearance, color and taste quality.
Drawings
FIG. 1 shows the extraction rate of lotus seed protein under different pH conditions.
Detailed Description
Example 1
Soaking the lotus seeds without the cores for 3 hours in water at the temperature of 35 ℃, uniformly adding the lotus seeds and the water in a mass ratio of 1; leaching lotus seed powder by using 0.3mol/LNaCl solution, adjusting the pH value to 10, leaching for 2.5h, filtering to remove filtrate, centrifuging the filtrate, centrifuging at the speed of 2500rpm, and centrifuging for 25min to obtain supernatant, wherein the material-liquid ratio is 1; adjusting the pH of the supernatant to 4.5 with 0.25mol/L HCl, standing and precipitating for 1.0h, centrifuging at the speed of 2500rpm for 15min to obtain lotus seed protein precipitate; adding water into the lotus seed protein precipitate to adjust the concentration to 10%, adjusting the pH to 6.0, wherein the adding amount of neutral protease and papain is 3.5% of the weight of the protein, the mass ratio of the neutral protease to the papain is 1; heating the protein polypeptide liquid to 85 ℃, keeping for 15min, and inactivating the protease; putting the protein polypeptide liquid into a circulating dialysis tank, dialyzing for 6 hours at the set temperature of 35 ℃, and obtaining desalted lotus seed protein peptide, wherein the flow rate of the dialysate is 100 mL/min; the retention degree of a filter membrane is 1500Da, the filtering pressure is 0.1MPa, the set temperature is 40 ℃, and the obtained protein peptide liquid is concentrated to the concentration of 30%; the temperature of the feed liquid is 55 ℃, the temperature of a spraying inlet is 125 ℃, the temperature in the tower is 75 ℃, and the temperature of an air outlet is 80 ℃, spray drying is carried out, and the lotus seed protein oligopeptide powder with the purity of 96% is obtained. The yield of the prepared lotus seed protein oligopeptide (oligopeptide with the molecular weight less than or equal to 1500 Da) is 78.6%, and the appearance color and the taste quality are both good.
Example 2
Soaking the lotus seeds without the cores for 3.5 hours by using water with the temperature of 40 ℃, uniformly adding the lotus seeds and the water into a pulping machine for wet grinding, wherein the mass ratio of the lotus seeds to the water is 1; leaching lotus seed powder by using 0.4mol/LNaCl solution, adjusting the material-liquid ratio to be 1; adjusting the pH of the supernatant to 4.4 with 0.3mol/L HCl, standing and precipitating for 1.0h, centrifuging at a speed of 3000rpm for 10min to obtain a lotus seed protein precipitate; adding water into the lotus seed protein precipitate to adjust the concentration to 12%, adjusting the pH to 5.5, wherein the adding amount of neutral protease and papain is 4.5% of the weight of the protein, the mass ratio of the neutral protease to the papain is 1.5, the enzymolysis temperature is 42 ℃, and the enzymolysis time is 3.5h; heating the protein polypeptide liquid to 90 ℃, keeping the temperature for 10min, and inactivating protease; putting the protein polypeptide liquid into a circulating dialysis tank, dialyzing for 5 hours at the set temperature of 40 ℃ and the flow rate of the dialysate of 150mL/min to obtain the desalted lotus seed protein peptide; the retention degree of a filter membrane is 1500Da, the filtering pressure is 0.15MPa, the set temperature is 35 ℃, and the obtained protein peptide liquid is concentrated to the concentration of 40%; the temperature of the feed liquid is 60 ℃, the temperature of a spraying inlet is 130 ℃, the temperature in the tower is 70 ℃, and the temperature of an air outlet is 75 ℃, spray drying is carried out, and the lotus seed protein oligopeptide powder with the purity of 97 percent is obtained. The yield of the prepared lotus seed protein oligopeptide (oligopeptide with the molecular weight less than or equal to 1500 Da) is 78.2%, and the appearance color and the taste quality are both good.
Example 3
Soaking the lotus seeds without the cores for 3 hours in water at the temperature of 45 ℃, uniformly adding the lotus seeds and the water into a pulping machine for wet grinding, wherein the mass ratio of the lotus seeds to the water is 1; leaching lotus seed powder by using 0.5mol/LNaCl solution, adjusting the material-liquid ratio to be 1; adjusting the pH of the supernatant to 4.7 with 0.5mol/L HCl, standing and precipitating for 1.0h, centrifuging at a speed of 3000rpm for 10min to obtain a lotus seed protein precipitate; adding water into the lotus seed protein precipitate to adjust the concentration to 16%, adjusting the pH to 6.0, wherein the adding amount of neutral protease and papain is 5% of the weight of the protein, the mass ratio of the neutral protease to the papain is 1; heating the protein polypeptide liquid to 90 ℃, keeping the temperature for 10min, and inactivating protease; putting the protein polypeptide liquid into a circulating dialysis tank, dialyzing for 5h at the set temperature of 40 ℃ and the flow rate of the dialysate of 150mL/min to obtain the desalted lotus seed protein peptide; the retention degree of a filter membrane is 1500Da, the filtering pressure is 0.15MPa, the set temperature is 35 ℃, and the obtained protein peptide liquid is concentrated to the concentration of 40%; the temperature of the feed liquid is 60 ℃, the temperature of a spraying inlet is 130 ℃, the temperature in the tower is 70 ℃, and the temperature of an air outlet is 75 ℃, spray drying is carried out, and the lotus seed protein oligopeptide powder with the purity of 98 percent is obtained. The yield of the prepared lotus seed protein oligopeptide (oligopeptide with the molecular weight less than or equal to 1500 Da) is 77.8%, and the appearance color and the taste quality are both good.
Comparative example 1
The enzymolysis removing process is replaced by: adding water into the lotus seed protein precipitate to adjust the concentration to 10%, adjusting the pH to 6.0, wherein the adding amount of neutral protease and papain is 3.5% of the weight of the protein, the mass ratio of the neutral protease to the papain is 1; the other steps are the same as in example 1.
Comparative example 2
The enzymolysis removing process is replaced by: adding water into the lotus seed protein precipitate to adjust the concentration to 10%, adjusting the pH to 6.0, wherein the adding amount of neutral protease and papain is 3.5% of the weight of the protein, the mass ratio of the neutral protease to the papain is 1; the other steps are the same as in example 1.
Experimental example 1
As can be seen from the attached figure 1, under the condition of unchanged extraction temperature, time and salt concentration, the extraction rate of the lotus seed protein is in an increasing trend along with the change of the pH value within the range of 5.0-13.0. The extraction rate is the lowest pH value of 5.0, the pH value is predicted to be closer to the isoelectric point of the lotus seed protein, the extraction rate is continuously increased when the pH value is higher than 5.0, and the extraction rate is close to 80% when the pH value is 13.0, which shows that the lotus seed protein is more easily separated out under the alkaline condition and the extraction rate is higher.
From the influence of pH on the extraction rate, although the extraction rate is highest when the pH is 13.0, lotus seed protein is more easily separated out along with the increase of the alkalinity of the extraction rate, the protein is easily denatured and degraded due to the overhigh alkalinity, and the quality of the obtained lotus seed protein is poor. Too high a pH, considering its application in the food industry, would make its use limited (equipment and operation). The extraction pH is controlled to be 8-10, the extraction rate is higher and more stable in the interval, the equipment requirement is reduced, and the use of strong alkali is reduced.
Experimental example 2
The hydrolysis degree and the yield of the finally obtained lotus seed protein oligopeptide of the examples 1 to 3 and the comparative examples 1 to 2 are counted as shown in the following table 1.
TABLE 1
| Group of | Degree of hydrolysis (%) | Oligopeptide yield (%) | Taste of food |
| Comparative example 1 | 10.8 | 30.8 | Has no bitter taste |
| Comparative example 2 | 9.3 | 36.2 | Has no bitter taste |
| Example 1 | 60.1 | 78.6 | Has no bitter taste |
As shown in table 1 above, a great deal of innovative research by the applicant discovers that the hydrolysis is performed by using two enzymes, namely neutral protease and papain, and the mass ratio of the neutral protease to the papain is controlled to be 1.5-2.5, so that the hydrolysis degree of protein and the yield of polypeptide are improved to the maximum extent; the invention further sets the concentration of the substrate to be 8-16%, the temperature to be 35-45 ℃, and the PH to be 3-8, so as to further accurately control the enzymolysis condition, the hydrolysis reaches the required degree, the micromolecule bitter peptides can not be generated by over hydrolysis, and the quality of the finished product can be improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of lotus seed protein oligopeptide is characterized by comprising the following steps:
step 1, soaking and grinding: soaking the lotus seeds without the cores in warm water, adding water, uniformly mixing, carrying out wet grinding in a grinding machine, and sieving after grinding;
step 2, salt leaching: according to the mass volume ratio of the feed liquid of 1: adding NaCl to 0.2-0.5 mol/L at 10-20, adjusting the pH value of the solution to 8-10, extracting the lotus seed powder at 30-45 ℃ for 2-4 h, filtering and centrifuging to obtain supernatant;
step 3, acid precipitation: adding HCl with the concentration of 0.25-0.5 mol/L to adjust the PH value of the supernatant to 4.3-4.7, standing and precipitating the solution, and centrifuging to obtain a lotus seed protein precipitate;
step 4, enzymolysis: adding water into the lotus seed protein precipitate to adjust the substrate concentration to 10%, setting the temperature at 38 ℃, adjusting the pH to 6, adding protease, wherein the protease is neutral protease and papain, the total addition amount of the protease is 3.5% of the lotus seed protein precipitate by mass, and the mass ratio of the neutral protease to the papain is 1;
step 5, enzyme deactivation: heating the lotus seed protein peptide liquid, and inactivating enzyme;
step 6, dialysis: dialyzing the lotus seed protein peptide liquid to obtain desalted lotus seed protein peptide liquid;
and 7, membrane filtration: the retention degree is set at 1500Da, protein peptide liquid is obtained by filtration and concentrated to obtain concentrated solution, and the concentration of the obtained concentrated solution is 25-45%;
and 8, drying the concentrated solution to obtain the lotus seed protein oligopeptide.
2. The preparation method of lotus seed protein oligopeptide according to claim 1, wherein in the step 1, the temperature for soaking in warm water is 25-45 ℃, and the soaking time is 2-4 h; the lotus seeds and water are uniformly mixed according to the mass ratio of 1.
3. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 2, the centrifugation speed is 1500rpm to 5000rpm, and the centrifugation time is 20min to 45min.
4. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 3, the standing and precipitating time is 0.5-2 h; the centrifugation speed is 2000 rpm-4000 rpm, and the centrifugation time is 15 min-45 min.
5. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 5, the heating temperature is 75-95 ℃ and the heating time is 10-20 min.
6. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 6, the dialysis comprises the following specific steps: the lotus seed protein polypeptide liquid is placed in a circulating dialysis tank, the temperature is set at 25-45 ℃, the dialysis time is 4-8 h, and the flow rate of the dialysate is set at 100-200 mL/min.
7. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 7, the pressure of membrane filtration is 0.05MPa to 0.25MPa, and the temperature is 25 ℃ to 45 ℃.
8. The method for preparing lotus seed protein oligopeptide according to claim 1, wherein in the step 8, the drying is spray drying, and the conditions of the spray drying are as follows: the temperature of the feed liquid is 45-75 ℃, the temperature of a spraying inlet is 125-130 ℃, the temperature in the tower is 65-85 ℃, and the temperature of an air outlet is 70-85 ℃.
9. A lotus seed protein oligopeptide prepared by the method of any one of claims 1 to 8.
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