CN113862324A

CN113862324A - Preparation method of mixed liquid of polypeptide and amino acid, amino acid foliar fertilizer and polypeptide water-soluble fertilizer

Info

Publication number: CN113862324A
Application number: CN202111158724.5A
Authority: CN
Inventors: 刘会丽; 赵佳宗; 罗冬贵; 郑若菁
Original assignee: Guangdong Lardmee Fertilizer Co ltd
Current assignee: Guangdong Lardmee Fertilizer Co ltd
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-31

Abstract

The invention provides a more environmentally friendly preparation method of a mixture of polypeptide amino acids, an amino acid foliar fertilizer and a polypeptide water-soluble fertilizer. The method for preparing a mixture of polypeptide amino acids provided by the present invention includes the following steps: S1, inoculating salt-tolerant bacteria into waste salted duck egg white liquid, fermenting for 20-50 h, and then performing centrifugal separation at 2000-4000 r/min; The supernatant and the precipitate are collected respectively; S2, the supernatant is concentrated to a flowing state after the ultrafiltration desalination treatment; S3, the precipitate and the concentrated supernatant are mixed according to a ratio of 1:(1-5) to obtain Mixed liquid, adjust the pH value of the mixed liquid to be between 5-6, add compound enzyme, and enzymatically hydrolyze for 2-8 h to obtain a mixed liquid of polypeptide amino acids, and the enzymatic hydrolysis temperature is 25°C-40°C.

Description

Preparation method of mixed liquid of polypeptide and amino acid, amino acid foliar fertilizer and polypeptide water-soluble fertilizer

Technical Field

The invention belongs to the technical field of preparation of amino acid mixed liquor, and particularly relates to a preparation method of polypeptide amino acid mixed liquor, an amino acid foliar fertilizer and a polypeptide water-soluble fertilizer.

Background

Salted duck eggs are one of the main egg products in China. Salted duck egg yolk is popular due to the unique texture and flavor, but salted duck egg white with no flavor is usually a waste in a dish. The fermented decomposition and putrefaction of the salted egg white cause serious pollution to the surrounding environment and water sources. The salted egg white contains various proteins such as ovalbumin, conalbumin, ovoglobulin, ovomucin-like protein and the like, the protein content is up to 12 percent, and the salted egg white is high-quality complete protein. The development and utilization value of the salted duck egg white is greatly limited due to the excessively high sodium content of the salted duck egg white. Except daily consumption of residents, the amount of wasted egg white in a food processing factory is hundreds of tons, so that protein resources are lost and the environment is polluted.

The egg white of the salted duck egg accounts for 85-88% of the total amount and is the substance with the largest content; the second is protein which accounts for 11-18% of the total amount, the main protein in the salted egg white is ovalbumin, ovotransferrin, ovomucin, ovomucoid, ovoglobulin and the like, and the molecular weight of the protein in the egg white is basically tens of thousands of the smallest and is also more than five thousand. In addition, it contains small amount of carbohydrate, vitamins, pigment, minerals, lipid, lysozyme, tributyrinesterase, peptidase, phosphatase, etc.

At present, the main direction for development and utilization of salted egg white is to use the salted egg white as a food additive in the production of biscuits and sausages so as to achieve the purpose of improving the quality of products. Egg white is a main raw material adopted in the prior lysozyme industrial production, and 0.3 percent of lysozyme contained in the salted eggs is not inactivated after the salted eggs are soaked in salt water. Therefore, it can still be used for extracting lysozyme. The salt content in the salted egg white reaches 4.97 percent, and the development and utilization of the egg white by people are greatly restricted. In recent years, many researchers have adopted methods such as ultrafiltration, dialysis, ion exchange, reverse osmosis, nanofiltration and the like to directly desalt salted egg white, certain research progress is achieved, the desalted salted egg white not only has obviously reduced salt content, but also effectively retains the original functional properties of egg white, and the salted egg white can be better applied to food industry. However, when desalination treatment is performed by membrane filtration methods such as ultrafiltration and nanofiltration, the watery salted egg white cannot completely pass through the filtration membrane, and part of egg white remains on the surface of the filtration membrane, so that membrane pores are blocked and difficult to clean.

In recent years, researchers have utilized protease preparations to carry out biological enzymolysis on salted egg white, so that protein molecular chains are broken, and then small-molecular polypeptide substances are obtained and applied to the food industry. One of the best methods for utilizing the salted duck egg white at present is to directly carry out enzymolysis on the salted duck egg white and then desalt the salted duck egg white to prepare albumin peptide with high nutritional value or prepare the albumin peptide into other substances, but the problems of complex process, higher cost and the like in the desalting process exist.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a more environment-friendly preparation method of a mixed liquid of polypeptide and amino acid, an amino acid foliar fertilizer and a polypeptide water-soluble fertilizer.

The invention provides a preparation method of a mixed solution of polypeptide amino acid, which comprises the following steps:

s1, inoculating the salt-tolerant strain into the waste salted duck egg clear liquid, fermenting for 20-50h, then carrying out 2000-4000r/min centrifugal separation, and respectively collecting the supernatant and the precipitate;

s2, concentrating the supernatant to be in a flowing state after ultrafiltration desalination treatment;

s3, precipitate and concentrated supernatant according to a 1: (1-5) to obtain a mixed solution, adjusting the pH value of the mixed solution to 5-6, adding a complex enzyme, performing enzymolysis for 2-8h to obtain a mixed solution of polypeptide amino acid, wherein the enzymolysis temperature is 25-40 ℃.

Preferably, the protein content of the waste salted duck egg white is 8% -9%, and the water content is 80% -90%.

Preferably, the halotolerant strain is micrococcus and/or staphylococcus, and the fermentation is performed for 24-48h in the step S1.

Preferably, in the step S3, the ratio of the precipitate to the concentrated supernatant is 1: and (1) mixing the components according to the proportion of (3) to obtain a mixed solution.

Preferably, in step S3, 0.5mol/L, HCl or NaOH is used to adjust the pH value.

Preferably, the addition amount of the complex enzyme is 0.1-0.5%.

Preferably, the enzyme composition is prepared by mixing any two or more of compound enzyme trypsin, acid protease, compound flavor protease and pepsin.

The embodiment of the invention also provides a preparation method of the leaf fertilizer containing amino acid, which comprises the following steps:

s11, weighing a first component according to a formula, adding the first component into water, and dissolving to prepare the nutrient solution, wherein the weight ratio of the first component to the water is 1: (2-3), wherein the first component comprises calcium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate and zinc sulfate, and the weight ratio of the calcium chloride to the magnesium sulfate to the ferrous sulfate to the manganese sulfate to the copper sulfate to the zinc sulfate is as follows: 2-3:1-2:0.5-1:0.2-0.5: 0.2-0.5: 0.2-0.5;

s12, weighing the second component according to the formula, adding the second component into water for dissolving to prepare the chelating solution, wherein the pH value of the chelating solution is 5-6, and the weight ratio of the second component to the water is 1: (2-3), wherein the second component comprises a polypeptide amino acid mixed solution prepared by a method for preparing a mixed solution of EDTA, citric acid and the polypeptide amino acid according to any one of claims 1-7, and the weight ratio of the EDTA, the citric acid and the polypeptide amino acid mixed solution is as follows: (0.2-0.5): (0.1-0.5): (1-2).

And S13, mixing the nutrient solution and the chelating agent solution, adjusting the pH value to 5-6, and carrying out chelating reaction.

S14, weighing a third component, adding the third component into the solution after the chelation reaction, wherein the weight ratio of the third component to the solution after the chelation reaction is (1-3): (1-3), wherein the third component comprises boric acid, ammonium molybdate, urea, monopotassium phosphate, potassium humate and a surfactant, and the weight ratio of the boric acid, the ammonium molybdate, the urea, the monopotassium phosphate, the potassium humate and the surfactant is as follows: (0.2-0.5): (0.2-0.5): (1-5): (1-5): (0.1-0.5): (0.02-0.05).

Preferably, in step S12, the chelating temperature is 50-80 ℃ and the chelating time is 0.3-0.5 h.

The embodiment of the invention also provides a preparation method of the polypeptide water-soluble fertilizer, which comprises the following steps:

s111, mixing the fertilizer components and water according to a weight ratio of 2: (2-3) mixing the fertilizer components, wherein the fertilizer components comprise urea, dipotassium hydrogen phosphate, potassium sulfate and polypeptide amino acid mixed liquor, and the polypeptide amino acid mixed liquor is prepared by the preparation method of any one of claims 1-7;

and S112, adding trace elements into the mixed solution obtained in the step S111, wherein the weight ratio of the mixed solution to the trace elements is as follows: 1: (0.02-0.1), wherein the trace elements comprise EDTA chelated trace elements and/or citric acid chelated trace elements.

The preparation method of the mixed liquid of the polypeptide amino acid provided by the invention adopts a method of combining microbial enzyme production with enzyme hydrolysis and combining enzymolysis with halotolerant bacteria fermentation, improves the desalting process treatment, can carry out enzymolysis on salted duck egg white into micromolecular polypeptide and amino acid, and contains a small amount of free amino acid molecules between 1000-5000 molecular weight of peptide contained in hydrolysate obtained after protein enzymolysis. The prepared polypeptide amino acid mixed solution can be applied to preparing fertilizers

Drawings

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings. Like reference numerals refer to like parts throughout the drawings, and the drawings are not intended to be drawn to scale in actual dimensions, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1 is a schematic diagram showing the effect of different reaction temperatures on the molecular weight distribution of a polypeptide.

FIG. 2 is a schematic diagram showing the effect of different enzymatic reaction times on the molecular weight distribution of a polypeptide.

Fig. 3 is a schematic diagram of the growth of 5/23 days of treated okra.

FIG. 4 is a graph showing the variation of plant height growth of okra treated by different treatments.

Fig. 5 is a schematic of the effect of different treatments on okra leaf count.

Fig. 6 is a graph comparing the length of leaves from different treatments of okra.

Detailed Description

The technical solutions of the present invention are further described in detail with reference to specific examples so that those skilled in the art can better understand the present invention and can implement the present invention, but the examples are not intended to limit the present invention.

Referring to fig. 1 to 6, an embodiment of the present invention provides a method for preparing a mixture of polypeptide amino acids, including the following steps:

s1, inoculating the salt-tolerant strain into the waste salted duck egg clear liquid, fermenting for 20-50h, then carrying out 2000-4000r/min centrifugal separation, and respectively collecting the supernatant and the precipitate. Acid is generated by the salt-tolerant strains in the fermentation process, the protease secreted by the salt-tolerant strains destroys the protein structure, the colloidal structure formed by the egg white protein and water is destroyed, and the water is analyzed to carry away partial salt; with the reduction of pH value, part of protein and peptide substances reach their isoelectric points and form precipitates, at this time, the fermentation liquor is obviously layered, the precipitates are extracted by centrifugation, and most of salt is still remained in the supernatant, thus achieving the aim of desalination. The protein source in the precipitate is roughly composed of three parts, namely thallus, protein precipitated when encountering acid and partially insoluble egg white protein.

S2, concentrating the supernatant to be in a flowing state after ultrafiltration desalting treatment. The supernatant is subjected to ultrafiltration desalination treatment by a 3KDa-5KDa ultrafiltration membrane, the viscosity of the supernatant is reduced due to fermentation of halotolerant bacteria, and when ultrafiltration desalination is adopted, the blockage and waste of the filtration membrane caused by overhigh viscosity can be avoided. The desalted supernatant contains proteins, polypeptides and other substances. The inventor finds that the unfermented salted egg white is sticky in texture, the problems of concentration polarization, membrane pollution and the like are often encountered by adopting a membrane filtration technology, and meanwhile, due to the fact that the salted egg is rich in nutrient substances and easy to breed microorganisms, the cleaning of a filtration membrane is difficult, and the fermented salted egg white is difficult to apply in actual production. Therefore, the invention can better avoid the problems of blockage and the like by fermenting the salted egg white and then performing ultrafiltration and desalination treatment.

S3, precipitate and concentrated supernatant according to a 1: (1-5) to obtain a mixed solution, adjusting the pH value of the mixed solution to 5-6 (the acidic environment is favorable for enzymolysis reaction), adding a complex enzyme, performing enzymolysis for 2-8h (the hydrolysis degree is about 43.6%) to obtain the mixed solution of polypeptide amino acid, wherein the enzymolysis temperature is 25-40 ℃. The egg white of the salted duck eggs has high salt concentration, and the activity of a plurality of proteases can be weakened or can not act under the high-salt concentration condition. In addition, the egg white contains protease inhibitors such as trypsin, chymotrypsin inhibitor and subtilisin inhibitor, so that the action efficacy of enzymes such as pancreatin is weakened or is difficult to act on egg white protein, the protein structure is changed after the fermentation of halotolerant bacteria, some nonpolar groups in original protein molecules are exposed, and new protein and polypeptide are generated after the degradation of protease.

According to the preparation method of the mixed liquid of the polypeptide amino acid, the prepared mixed liquid of the polypeptide amino acid can be used for preparing a fertilizer, the salted duck egg white can be applied to the preparation of the fertilizer, the waste of the salted duck egg white is reduced, the protein resource is lost, and the environment is polluted.

According to the preparation method of the mixed liquid of the polypeptide amino acid, provided by the embodiment of the invention, the method of combining microbial enzyme production with enzyme hydrolysis and combining enzymolysis with halotolerant bacteria fermentation is adopted, the desalting process is improved, the salted duck egg white can be subjected to enzymolysis to obtain small-molecular polypeptide and amino acid, and the peptide molecular weight of the hydrolysate obtained after the protein enzymolysis is between 1000-5000 and also contains a small amount of free amino acid molecules. Adding halotolerant bacteria for full fermentation to make it have light adaptability to crops. And adding a certain amount of medium and trace amount of the fertilizer to prepare the ammonia-containing water-soluble fertilizer. The reaction process can be carried out at normal temperature, no bad smell is generated in the reaction process, the energy consumption is low, no waste is generated in the reaction process, and no secondary pollution is generated.

The prior art hydrolysis of proteins is generally divided into acid hydrolysis and enzymatic hydrolysis. The method of acid hydrolysis of proteins is simple, easy to operate, and inexpensive, and can obtain desired substances in a short time, and thus is widely used in the industry. The hydrolysis of protein by enzyme is carried out under a mild condition, is safe and reliable, has low energy consumption, but is an incomplete and incomplete hydrolysis reaction, the hydrolysis product of the hydrolysis reaction is mainly peptide rather than amino acid, and the reaction degree of a reaction system is difficult to control, so that different batches of products have certain difference.

Compared with simple acidolysis or enzymolysis, the method has the scheme that halotolerant bacteria are fermented firstly, protease secreted by the halotolerant bacteria destroys a protein structure, some nonpolar groups in original protein molecules are exposed, the whole solution is controlled under an acidic condition, the enzymolysis reaction is facilitated, reaction products are more thorough, and new protein and polypeptide are generated after the protease degradation.

In a preferred embodiment, the protein content of the waste salted duck egg white is 8% -9%, preferably 8.65%, and the moisture content is 80% -90%, preferably 83.2%.

In a preferred embodiment, the salt-tolerant strain is micrococcus and/or staphylococcus, the salt-tolerant strain is separated and purified and then inoculated into the waste salted duck egg white liquid,

under the condition that other conditions of the preparation method in the embodiment are not changed, the inventor investigates the change conditions of different halotolerant bacteria fermentation time on the salt content of the supernatant, the viscosity and the salt content of the precipitate through a single-factor test. The specific test method is as follows:

collecting waste salted duck egg white (the protein content is 9.65 percent, and the water content is 86.2 percent); separating and purifying halotolerant bacteria (micrococcus and staphylococcus), inoculating the halotolerant bacteria into the mixed solution at the concentration of 0.1-0.5%, fermenting, then performing 2000-4000r/min centrifugal separation, and respectively collecting the supernatant and the precipitate, wherein the inoculation amount of the halotolerant bacteria is 3%.

The fermentation time is 0-60h, different fermentation times of 0h, 12h, 24h, 36h, 48h and 60h are respectively set, and then the change conditions of the salt content of the supernatant, the viscosity and the salt content of the precipitate are detected, and the specific numerical values are shown in table 1.

TABLE 1

Time of fermentation

0h

12h

24h

36h

48h

60h

Viscosity mp.s of supernatant

12.1

8.95

5.24

3.54

1.23

0.25

Content of salt in precipitate%

5.12％

4.13％

3.79％

3.46％

3.3％

3.21％

As can be seen from Table 1, the viscosity of the supernatant gradually decreases and the salt content of the precipitate gradually decreases with the increase of the fermentation time, but the decrease rate tends to decrease gradually, and in consideration of the energy consumption, the fermentation time is 24-48h in step S1, and in a more preferred embodiment, the fermentation time is 30-40h, and the reaction time is 36h, which is the most suitable fermentation time.

Under the condition that other conditions of the preparation method of the embodiment are not changed, the inventor investigates the change conditions of the temperature and the enzymolysis time of the compound enzyme to the crude protein content, the average nitrogen content and the total amino acid content of the product in the enzymolysis process of the mixture through a single-factor test.

The specific test method comprises the following steps:

collecting waste salted duck egg white (the protein content is 9.65 percent, and the water content is 86.2 percent);

separating and purifying halotolerant bacteria (micrococcus and staphylococcus), inoculating the halotolerant bacteria into the mixed solution, fermenting for 24-48h at the concentration of 0.1-0.5%, performing centrifugal separation at 2000-4000r/min, and collecting and treating the supernatant and the precipitate respectively. 30-

Desalting the supernatant with 3-5 KDa ultrafiltration membrane, and concentrating to flow state;

mixing the precipitate and the concentrated supernatant according to the proportion of 1:1-3, pretreating, adjusting the pH value of the mixed solution to 5-6 by using 0.5mol/L HCl or NaOH (the acidic environment is favorable for enzymolysis reaction), adding 0.1-0.5% of trypsin, acid protease, compound flavourzyme, pepsin and other compound enzymes, and keeping the enzymolysis temperature at a certain temperature for enzymolysis for a certain time.

(1) The enzymolysis reaction time is firstly fixed for 3h, and the reaction temperature is set to be different at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃.

The fixed enzymolysis time is 3h, the change conditions of different enzymolysis temperatures to the total amino acid amount of the mixed solution and the molecular weight of the polypeptide are studied, and the specific data are shown in tables 2 and 3.

TABLE 2

Temperature of enzymolysis

20℃

25℃

30℃

35℃

40℃

45℃

Crude protein content%

49.6％

51.4％

54.8％

56.7％

57.4％

58.1％

The total amount of amino acids%

53.1％

58.3％

60.7％

61.9％

62.2％

61.8％

The protein content and the total amino acid content are measured by an automatic Kjeldahl azotometer according to the national standard GB/T5009.5-2010.

TABLE 3

As can be seen from Table 2, the crude protein content in the mixture gradually increases with the increase of the enzymolysis temperature, and the increase rate decreases when the temperature exceeds 35 ℃; the total amount of amino acids increases and then decreases, and it is considered that an excessive temperature affects the enzymatic reaction. As can be seen from Table 3, referring to FIG. 1, different enzymolysis temperatures have little effect on the molecular weight distribution of the polypeptides in the mixed solution, the molecular weight of the polypeptides is mainly 1000-3000, and the polypeptides with molecular weights in this interval show a tendency of increasing gradually with the increase of the reaction temperature, and the enzymolysis selection is preferably 30-40 ℃.

(2) The fixed enzymolysis temperature is 30 ℃, and the fixed enzymolysis reaction time is 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8 h.

The fixed reaction temperature is 35 ℃, the change conditions of different enzymolysis time on the total amino acid amount of the mixed solution and the molecular weight of the polypeptide are researched, and the specific detection results are shown in tables 4 and 5.

TABLE 4

Time of enzymolysis

2h

3h

4h

5h

6h

7h

8h

Crude protein content%

52.6％

56.7％

58.8％

59.4％

60.1％

61.3％

62.4％

The total amount of amino acids%

56.3％

61.9％

62.7％

62.9％

63.5％

63.8％

63.4％

TABLE 5

As can be seen from Table 4, the crude protein content of the protein showed a tendency of increasing gradually with the time of the enzymatic reaction, but the increase was relatively reduced after 5 hours of the reaction; the total amount of amino acid shows a gradually increasing trend along with the prolonging of the reaction time, and the increasing range of the amino acid is gradually reduced after the reaction is carried out for 4 hours. As can be seen from Table 5, referring to FIG. 2, the ratio of the enzymolysis time to the molecular weight distribution of the polypeptide in the mixed solution is not greatly affected, and is mainly concentrated between 1000-3000, but the molecular weight of < 3000 shows a tendency of increasing first and then decreasing as the reaction time is prolonged. In general, the reaction time is preferably selected to be 3 to 6 hours, and more preferably 3 to 4 hours.

In a preferred embodiment, in step S3, the precipitate and the concentrated supernatant are mixed according to a ratio of 1: and (1) mixing the components according to the proportion of (3) to obtain a mixed solution.

In a preferred embodiment, 0.5mol/L, HCl or NaOH is used to adjust the pH in step S3.

In a preferred embodiment, the addition amount of the complex enzyme is 0.1-0.5%.

In a preferred embodiment, the protease inhibitor is obtained by mixing any two or more of compound enzyme trypsin, acid protease, compound flavor protease and pepsin.

s11, weighing the first component according to the formula, adding the first component into water, and dissolving to prepare the nutrient solution, wherein the weight ratio of the first component to the water is 1: (2-3), the first component comprises calcium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate and zinc sulfate, and the weight ratio of the calcium chloride to the magnesium sulfate to the ferrous sulfate to the manganese sulfate to the copper sulfate to the zinc sulfate is as follows: 2-3:1-2:0.5-1:0.2-0.5: 0.2-0.5: 0.2-0.5;

s12, weighing the second component according to the formula, adding the second component into water for dissolving to prepare the chelating solution, wherein the pH value of the chelating solution is 5-6, and the weight ratio of the second component to the water is 1: (2-3), the second component comprises EDTA, citric acid and polypeptide amino acid mixed solution, and the polypeptide amino acid mixed solution is prepared by the preparation method of any one of the polypeptide amino acid mixed solution. The weight ratio of the EDTA, the citric acid and the polypeptide amino acid mixed solution is as follows: (0.2-0.5): (0.1-0.5): (1-2). In the prior art, water-soluble phosphorus sources such as industrial ammonium phosphate, monopotassium phosphate and the like are almost selected in the production technology of the water-soluble fertilizer, but the raw materials are produced by purifying yellow phosphorus or wet-process phosphoric acid and are expensive, so that the production cost of the water-soluble fertilizer is high. In addition, when the traditional wet-process phosphoric acid is neutralized by ammonia, impurities such as iron, aluminum, magnesium, calcium and the like in the wet-process phosphoric acid can generate insoluble phosphoric acid and sulfuric acid double salt, and finally, the content of water-insoluble substances in the product exceeds the standard and cannot meet the national standard requirement of water-soluble fertilizers. In the step of the invention, the chelating agent is prepared firstly, which can avoid generating insoluble phosphoric acid and sulfuric acid double salt and improve water solubility.

S14, weighing a third component, adding the third component into the solution after the chelation reaction, wherein the weight ratio of the third component to the solution after the chelation reaction is (1-3): (1-3), the third component comprises boric acid, ammonium molybdate, urea, monopotassium phosphate, potassium humate and a surfactant, and the weight ratio of the boric acid, the ammonium molybdate, the urea, the monopotassium phosphate, the potassium humate and the surfactant is as follows: (0.2-0.5): (0.2-0.5): (1-5): (1-5): (0.1-0.5): (0.02-0.05).

The foliar fertilizer containing amino acid prepared by the embodiment of the invention reaches the standard of foliar fertilizer containing organic matters, the content of organic matters is more than or equal to 100g/L, the total nutrient is more than or equal to 80g/L, the content of trace elements is more than or equal to 20g/L, and the pH value is 2.0-9.0.

In a preferred embodiment, in step S12, the chelating temperature is 50-80 ℃ and the chelating time is 0.3-0.5 h.

s111, mixing the fertilizer components and water according to a weight ratio of 2: (2-3) mixing, wherein the fertilizer components comprise urea, dipotassium hydrogen phosphate, potassium sulfate and polypeptide amino acid mixed liquor, and the polypeptide amino acid mixed liquor is prepared by the preparation method of any one of claims 1-7;

The polypeptide water-soluble fertilizer prepared by the embodiment of the invention reaches the standard of amino acid-containing water-soluble fertilizers, and the content of medium elements, liquid and free amino acid is more than or equal to 100 g/l; the content of secondary elements is more than or equal to 30g/L, the content of water insoluble substances is less than or equal to 50g/L, and the pH value is 3.0-9.0.

In order to further understand and appreciate the technical solution of the present invention, the embodiments are now described in further detail.

Example 1 (preparation of polypeptide amino acid mixture solution)

2. separating and purifying halotolerant bacteria (micrococcus and staphylococcus), inoculating the halotolerant bacteria into the mixed solution, fermenting for 24-48h at the concentration of 0.1-0.5%, performing centrifugal separation at 2000-4000r/min, and collecting and treating the supernatant and the precipitate respectively.

3. Desalting the supernatant with 3-5 KDa ultrafiltration membrane, and concentrating to flow state;

4. mixing the precipitate and the concentrated supernatant according to the proportion of 1:1-3, pretreating, adjusting the pH value of the mixed solution to 5-6 by using 0.5mol/L HCl or NaOH (the acidic environment is favorable for enzymolysis reaction), adding 0.1-0.5% of trypsin, acid protease, compound flavourzyme, pepsin and other compound enzymes, and keeping the temperature at 20-45 ℃ for enzymolysis for 3-6 h;

5. after enzymolysis, mixed liquor containing various polypeptide amino acids is obtained.

Example 2 (preparation of leaf fertilizer containing amino acids)

(1) Weighing calcium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate and zinc sulfate according to the formula, sequentially adding the calcium chloride, the magnesium sulfate, the ferrous sulfate, the manganese sulfate, the copper sulfate and the zinc sulfate into water (1:2-3), stirring and dissolving to enable the solution to be clear and transparent, and preparing into the nutrient element solution.

(2) EDTA, citric acid and the polypeptide amino acid mixed solution obtained in example 1 are weighed according to the formula, the ratio of the EDTA to the citric acid to the polypeptide amino acid mixed solution is 0.2-0.5:0.1-0.5:1-2, the EDTA, the citric acid and the polypeptide amino acid mixed solution are added into water (1:2) and stirred to be dissolved, a chelating solution is prepared, the chelating temperature is 50-80 ℃, the chelating time is 0.3-0.5h, and the pH value of the solution is 5-6.

(3) Mixing the nutrient solution with the chelating agent solution, adjusting the pH value to 5-6, and stirring at a certain temperature for a period of time to perform a chelating reaction.

(4) According to the formula, boric acid, ammonium molybdate, urea, monopotassium phosphate, potassium humate and surfactant are weighed to be 0.2-0.5:0.2-0.5:1-5:1-5:0.1-0.5:0.02-0.05, added into the solution (1-3:1-3) after chelation reaction, and concentrated to obtain the leaf fertilizer containing amino acid.

Example 3 preparation of a polypeptide Water-soluble Fertilizer

(1) Urea, dipotassium hydrogen phosphate, potassium sulfate, the polypeptide-amino acid mixed solution prepared in example 1, and water were mixed in a ratio of 2:2-3, and then stirred and dissolved, wherein the polypeptide-amino acid mixed solution was 1-2:4-6:2-4: 1-3.

(2) Adding EDTA chelated trace elements and citric acid chelated trace elements (mixed solution: trace elements: 1:0.02-0.1), and stirring and dissolving in a reaction kettle.

(3) Obtaining the water-soluble fertilizer containing amino acid polypeptide.

Effects of the embodiment

1. Fertilizer efficiency test of example 2

Test site: the experimental soil is garden soil: peat soil: perlite was formulated at 4:2: 1. The pH value of the soil is between 6 and 7.

Test work: okra (2021 year 4 month 18 day seeding to 2021 year 5 month 23 day)

Fertilizer to be tested: the foliar fertilizer containing amino acid prepared from the polypeptide liquid and the foliar fertilizer containing amino acid purchased in the market.

And (3) experimental design: adopting a potting test, setting 3 treatments, repeating each treatment for 3 times, numbering the potted plants from 1 to 9, selecting the treatment I as the numbers 2,3 and 7, the treatment II as the numbers 4,8 and 9 and the treatment III as the numbers 1, 5 and 6 according to random numbers, wherein the treatment designs are as follows:

processing one: the foliar fertilizer containing amino acid prepared in the embodiment 2 is sprayed on 600 times of leaf surfaces and is sprayed once every 7 days;

and (5) processing: spraying 600 times of leaf surface of common leaf fertilizer containing amino acid once every 7 days;

and (3) treatment III: spraying with equal amount of clear water, wherein the spraying is carried out once every 7 days

Test and main cultivation management conditions: seeding is carried out by adopting a seedling raising plug tray in 18 months in 2021, planting is carried out in a 7cmx7cm black square pot in 25 days in 4 months, spraying is carried out for the first time in 2 days in 5 months, changing the pot to a pot with 20cmx20cm in 6 days in 5 months, spraying is carried out for the second time in 9 days in 5 months, spraying is carried out for the third time in 16 days in 5 months, the experiment is finished in 23 days in 5 months, and other pot culture management measures are consistent.

(1) Referring to fig. 3, it can be seen from the appearance that, by the end of the test, the okra of treatment one grew better than the other treatments, the leaves were big and the leaf color was darker green.

(2) The fixed plant height (from the surface of the substrate to the highest part of the plant) is measured every 7d, data statistics shows that the growth speed of the okra treated by different treatments gradually increases as shown in table 6 and as can be seen by referring to table 6 and fig. 4, the total shows that the treatment of the first growth speed is higher than the treatment of the second growth speed is higher than the treatment of the third growth speed, the treatment of the first plant height is higher than the treatment of the second growth speed is higher than the treatment of the third growth speed, and the treatment of the first plant height is higher than the treatment of the second growth speed.

TABLE 6

(3) The leaf number of the okra is periodically determined every 7 days, and through statistical analysis, as can be seen from fig. 5, the leaf numbers of different treatments are consistent in the first seven days; on the 2 nd seven days, the number of the leaves processed in the first step and the second step is equal and is larger than that of the leaves processed in the third step; starting from the 3 rd seven days, the leaf number is shown, treatment one is more than treatment two is more than treatment three, and the treatment one can effectively promote the growth of the okra leaves.

(4) The length of each plant leaf was investigated at 23 days 5 months, and as can be seen in fig. 6, the number of the whole plant leaves was treated one > treated two > treated three, and the length of each leaf was treated one > treated two > treated three, indicating that the treatment one can effectively promote the growth of okra leaves.

(5) The thickness of the stem and the thickness of the leaf of different okra are changed. As can be seen from table 7, the leaf thicknesses of the okra treated by the different treatments were about the same, and there was no significant difference, and the stem thickness showed a trend of treatment one > treatment two > treatment three, indicating that the treatment had an effect of promoting the growth of okra.

TABLE 7

In conclusion, the polypeptide amino acid foliar fertilizer has a certain promotion effect on the overall growth of the okra, and compared with the common amino acid foliar fertilizer, the polypeptide amino acid foliar fertilizer has certain differences in plant height, leaf number, leaf length and the like, and compared with a blank control, the polypeptide amino acid foliar fertilizer has significant differences in the agronomic traits of the okra.

2. Fertilizer efficiency test of example 3

Test site: the experimental soil is garden soil: peat soil: perlite is configured in a ratio of 1:1: 1. The pH value of the soil is between 6 and 7.

Test work: leaf of Chinese lettuce (3 months and 25 days in 2021 to 4 months and 19 days in 2021).

Fertilizer to be tested: polypeptide amino acid water-soluble fertilizer and amino acid water-soluble fertilizer (purchased in the market).

And (3) experimental design: the pot experiment was used, 3 treatments were set, each treatment was repeated 4 times, each treatment was designed as follows:

processing one: polypeptide amino acid water soluble fertilizer is diluted by 300 times and 200 ml/pot, and is fertilized once every 7 days for 2 times;

and (5) processing: diluting the conventional amino acid water-soluble fertilizer by 300 times, fertilizing once every 7 days at 200 ml/pot, and fertilizing for 2 times;

and (3) treatment III: blank control, equal amount of clear water irrigation.

Test and main cultivation management conditions: the seeds are transplanted into a plastic pot with the size of 15x15cm in 4 months and 4 days, the roots are irrigated for the first time in 6 days in 4 months, the roots are irrigated for the second time in 12 days in 4 months, and the harvesting test is finished in 19 days in 4 months. During the test period, other management measures of the pot culture are consistent.

Test index measurement: agronomic traits: the height and leaf number of leaf lettuce were measured during the test period, and the height, leaf number, weight, etc. of leaf lettuce were measured at the time of harvest.

(1) During collection, the whole growth vigor and leaf color of the leaf lettuce are respectively represented by treatment I, treatment II and blank control in appearance, and the length and root and stem thickness of the treatment I and the treatment II are approximately the same and are obviously superior to those of the blank control. See table 8.

TABLE 8

(2) As can be seen from Table 9, the fresh weight of each leaf of leaf lettuce exhibits that the first treatment is more than the second treatment and is more than the third treatment, the fresh weight increase rate of the first treatment reaches 11.8 percent compared with the second treatment, the fresh weight increase rate of the third treatment reaches 29.5 percent compared with the third treatment, and the fresh weight increase rate of the second treatment reaches 15.9 percent compared with the third treatment. The edible part and the edible rate of the leaf lettuce are respectively treated as one, two and three. The method shows that the treatment has obvious promotion effect on the yield increase of the leaf lettuce.

TABLE 9

Note: different lower case letters indicate that the difference is significant.

In conclusion, the polypeptide amino acid water-soluble fertilizer provided by the embodiment can achieve the effect of the conventional amino acid water-soluble fertilizer, has a certain promotion effect on the growth of the leaf lettuce, and has a promotion effect on the weight of each plant, the number of leaves and the like of the leaf lettuce. The promotion effect of the polypeptide amino acid water-soluble fertilizer on the leaf lettuce is superior to that of the conventional amino acid water-soluble fertilizer, and the polypeptide amino acid water-soluble fertilizer has remarkable advantages on the aspects of leaf number, leaf color and root growth, and also has remarkable advantages on the fresh weight of a single plant and an edible part.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. the preparation method of the mixed solution of a polypeptide amino acid, is characterized in that, comprises the steps:

S1, inoculate the salt-tolerant bacterial strain into the waste salted duck egg white liquid, ferment for 20-50h, and then carry out centrifugal separation at 2000-4000r/min, and collect the supernatant and the sediment respectively;

S2, the supernatant is concentrated to a flowing state after desalting treatment by ultrafiltration;

S3. Mix the precipitate and the concentrated supernatant according to the ratio of 1:(1-5) to obtain a mixed solution, adjust the pH value of the mixed solution to be between 5-6, add a compound enzyme, and enzymatically hydrolyze the polypeptide for 2-8 hours The mixture of amino acids, the enzymatic hydrolysis temperature is 25℃-40℃.

2 . The method for preparing a mixture of polypeptide amino acids according to claim 1 , wherein the protein content of the waste salted duck egg white liquid is 8%-9%, and the moisture content is 80%-90%. 3 .

3. The method for preparing a mixture of polypeptide amino acids according to claim 1, wherein the salt-tolerant bacterial species is Micrococcus and/or Staphylococcus, and the fermentation is carried out for 24-48h in the step S1.

4. the preparation method of the mixed solution of polypeptide amino acid as claimed in claim 1, is characterized in that, in described step S3, precipitate and concentrated supernatant are mixed according to the ratio of 1:(1-3) to obtain mixture.

5. The method for preparing a mixture of polypeptide amino acids as claimed in claim 1, wherein in the step S3, 0.5mol/L, HCl or NaOH adjusts its pH value.

6 . The method for preparing a mixture of polypeptide amino acids according to claim 1 , wherein the compound enzyme is added in an amount of 0.1%-0.5%. 7 .

7 . The method for preparing a mixture of polypeptide amino acids according to claim 1 , wherein any two or more of the complex enzymes trypsin, acid protease, complex flavor protease and pepsin are obtained by mixing. 8 .

8. a preparation method containing amino acid foliar fertilizer, is characterized in that, comprises the steps:

S11, the first component is weighed and dissolved in water to prepare a nutrient solution according to the formula, the weight ratio of the first component and water is 1: (2-3), and the first component includes calcium chloride, sulfuric acid Magnesium, ferrous sulfate, manganese sulfate, copper sulfate and zinc sulfate, the weight ratio of calcium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate and zinc sulfate is: 2-3:1-2:0.5 -1: 0.2-0.5: 0.2-0.5: 0.2-0.5;

S12, the second component is weighed and dissolved in water according to the formula to prepare a chelating solution, the pH value of the chelating solution is 5-6, and the weight ratio of the second component and water is 1: (2-3 ), the second component comprises the polypeptide amino acid mixed solution prepared by the preparation method of the mixed solution of EDTA, citric acid and the polypeptide amino acid described in any one of claims 1-7, the EDTA, citric acid and the polypeptide amino acid The weight ratio of the mixed solution is: (0.2-0.5): (0.1-0.5): (1-2).

S13, mix the nutrient solution with the chelating agent solution, adjust the corresponding pH value to 5-6, and carry out the chelation reaction.

S14, weigh the third component and add it to the solution after the chelation reaction, the weight ratio of the third component to the solution after the chelation reaction is (1-3): (1-3), the third component is The components include boric acid, ammonium molybdate, urea, potassium dihydrogen phosphate, potassium humate and surfactant, and the weight ratio of the boric acid, ammonium molybdate, urea, potassium dihydrogen phosphate, potassium humate and surfactant is : (0.2-0.5): (0.2-0.5): (1-5): (1-5): (0.1-0.5): (0.02-0.05).

9. The preparation method of amino acid-containing foliar fertilizer according to claim 8, wherein in step S12, the chelation temperature is 50-80°C, and the chelation time is 0.3-0.5h.

10. a preparation method of polypeptide water-soluble fertilizer, is characterized in that, comprises the steps:

S111, mixing fertilizer components and water in a ratio of 2:(2-3) by weight, the fertilizer components comprising urea, dipotassium hydrogen phosphate, potassium sulfate and a polypeptide amino acid mixed solution, the polypeptide amino acid mixed solution Prepared by the preparation method described in any one of claims 1-7;

S112, adding trace elements to the mixed solution obtained in the step S111, the weight ratio of the mixed solution and the trace elements is: 1: (0.02-0.1), and the trace elements include EDTA chelated trace elements and/or lemon Acid chelates trace elements.