CN111944011B - Multistage separation method for Mucuna pruriens biological components - Google Patents
Multistage separation method for Mucuna pruriens biological components Download PDFInfo
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Abstract
The invention relates to the technical field of leguminous plant processing and utilization, and particularly relates to a multistage separation method for a lathyrus lathyris biological component. The invention provides a multistage separation method of a Mucuna pruriens biological component, which comprises the following steps: pulverizing Mucuna pruriens, and leaching with water to obtain leaching solution; filtering the leaching solution to obtain cellulose and filtrate; performing gel chromatography on the filtrate to obtain a first eluent; concentrating the first eluent, and performing gel chromatography to obtain a second eluent; concentrating the second eluent, adding water after concentration, stirring, and performing centrifugal separation to obtain a protein solution and a starch precipitate; and adding an ammonium sulfate aqueous solution into the protein solution, filtering to obtain filter residues, and drying the filter residues to obtain the protein. The multistage separation method for the biological components of the lathus pruriens provided by the invention can realize efficient separation of cellulose, protein and starch components in the lathus pruriens, and realize classification and utilization of the lathus pruriens.
Description
Technical Field
The invention relates to the technical field of leguminous plant processing and utilization, and particularly relates to a multistage separation method for a lathyrus lathyris biological component.
Background
Mucuna pruriens is a plant of Mucuna genus of Leguminosae family, and is distributed in Korean, japan and Russian far east regions, and is generally distributed in northeast China, north China, shaanxi, southern Gansu, and eastern Qinghai in China. Mucuna pruriens grows in hillsides, forest borders, roadside, meadows and the like, can live in places with the elevation of 2500 m at most, and is rough and fond of warm and humid environments. The protein content of the mucuna pruriens seeds is about 25% -28%, the starch content is about 55% -61%, and the mucuna pruriens seeds are ideal high-protein leguminous feed crops and good plant starch resources. However, the leguma lathyris contains leguma lathyris toxin (ODAP) and the content of the neurotoxin in the leguma lathyris does not reach the safe edible level, so that the leguma lathyris is not widely applied, but the leguma lathyris cannot cause great waste of leguma lathyris resources, and therefore, how to effectively separate biological components in the leguma lathyris becomes a great research hotspot.
The existing separation method of biological components (such as starch, protein and the like) in the mucuna pruriens is mostly carried out by adopting a water extraction method, and then the separation of the biological components in the mucuna pruriens is realized by means of centrifugation, filtration and the like according to different specific gravities of the biological components in water, however, the existing separation method is low in separation efficiency, the extraction rate of the components such as starch, protein and the like is not high, and the waste of partial mucuna pruriens resources is caused.
Disclosure of Invention
The invention aims to overcome the defects that the extraction rate of components such as starch, protein and the like is not high and the biological components in the leguma mucronatum cannot be efficiently separated in the existing leguma mucronatum separation method, and further provides a multistage separation method for the biological components in the leguma mucronatum.
In order to achieve the purpose, the invention adopts the following technical scheme:
a multi-stage separation method for a Mucuna pruriens biological component comprises the following steps:
1) Pulverizing Mucuna pruriens, and leaching with water to obtain leaching solution;
2) Filtering the leaching solution to obtain cellulose and filtrate;
3) Performing gel chromatography on the filtrate to obtain a first eluent;
4) Concentrating the first eluent, and performing gel chromatography to obtain a second eluent;
5) Concentrating the second eluent, adding water after concentration, stirring, and performing centrifugal separation to obtain a protein solution and a starch precipitate;
6) And adding an ammonium sulfate aqueous solution into the protein solution, filtering to obtain filter residues, and drying the filter residues to obtain the protein.
Preferably, the eluent used in the gel chromatography treatment step in the step 3) is a mixed solution of n-butanol and an aqueous solution of acetic acid;
the eluent used in the gel chromatography treatment in the step 4) is a mixed solution of n-butyl alcohol and an acetic acid aqueous solution.
Preferably, the mass ratio of the n-butanol to the acetic acid aqueous solution is (1.5-3) to 1;
the concentration of the acetic acid aqueous solution is 30-40wt%.
Preferably, the eluent is a mixed solution of n-butanol, an aqueous acetic acid solution and 2-ethoxybutane.
Preferably, the mass ratio of the n-butanol to the acetic acid aqueous solution to the 2-ethoxybutane is (0.8-0.9) to 1 (0.2-0.3).
Preferably, the mass ratio of the n-butanol to the acetic acid aqueous solution to the 2-ethoxybutane is 0.9;
the concentration of the acetic acid aqueous solution is 35wt%.
Preferably, the filtrate is subjected to gel chromatography by using a sephadex column G-10 in the step 3);
and 4) performing gel chromatography on the concentrated first eluent by using a sephadex column G-25 in the step 4).
Preferably, the weight ratio of the lathyrus pruriens to the water in the step 1) is 1: (7-10);
the leaching temperature is 50-70 ℃, and the leaching time is 0.5-3 hours.
Preferably, the leaching liquor is filtered by a sieve with 100-120 meshes in the step 2);
the step 5) also comprises the step of adjusting the pH of the solution after adding water to 8-10;
the step 6) of adjusting the pH of the protein solution to 3-4 before adding the ammonium sulfate aqueous solution; the ammonium sulfate aqueous solution is saturated ammonium sulfate aqueous solution.
Preferably, the method further comprises the steps of washing the starch precipitate with water and then drying.
The sephadex column G-10 and the sephadex column G-25 used in the invention are conventional chromatographic columns in the field, can be obtained by self-filling corresponding solid phase carriers by the conventional method, and can also be obtained by the market. The separation range of the sephadex column G-10 used by the invention is less than 700Mr, and the separation range of the sephadex column G-25 is 1000-5000Mr. The mucuna pruriens disclosed by the invention is mucuna pruriens seeds.
The invention has the beneficial effects that:
1) According to the multistage separation method for the biological components of the lathyrium pruriens, provided by the invention, the water leaching is carried out on the lathyrium pruriens, the cellulose and the filtrate are obtained through filtering and separation, then the filtrate is subjected to gel chromatography twice, and the eluate is subjected to operations such as centrifugal separation and the like, so that the high-efficiency separation of the cellulose, the protein and the starch components in the lathyrium pruriens is realized. Meanwhile, the separation method disclosed by the invention can avoid the limitation of the leguma lathyris toxin (ODAP) contained in the leguma lathyris on the practical application of the leguma lathyris, and realizes the classification and utilization of the leguma lathyris.
2) The multistage separation method for the biological components of the mucuna pruriens provided by the invention further comprises the step 3) of carrying out gel chromatography on the mixture of n-butyl alcohol and acetic acid aqueous solution as eluent; the eluent used in the gel chromatography treatment in the step 4) is a mixed solution of n-butyl alcohol and an acetic acid aqueous solution. Preferably, the mass ratio of the n-butanol to the acetic acid aqueous solution is (1.5-3) to 1; the concentration of the acetic acid aqueous solution is 30-40wt%. According to the method, the mixed solution of the n-butyl alcohol and the acetic acid aqueous solution is used as the eluent, so that the separation of the biological components in the leguminous mucronatus is facilitated, the extraction rate of starch and protein in the leguminous mucronatus is improved, and the effective separation of the biological components in the leguminous mucronatus is further realized.
3) The multistage separation method of the biological components of the mucuna pruriens provided by the invention is further characterized in that the eluent is a mixed solution of n-butyl alcohol, an acetic acid aqueous solution and 2-ethoxybutane. Preferably, the mass ratio of the n-butanol to the acetic acid aqueous solution to the 2-ethoxybutane is (0.8-0.9) to 1 (0.2-0.3). According to the method, 2-ethoxybutane is added into the eluent, the mass ratio of n-butyl alcohol to acetic acid aqueous solution to 2-ethoxybutane is controlled to be (0.8-0.9) to 1 (0.2-0.3), and researches show that the extraction rate of starch and protein in the lathyrus sativus can be greatly improved.
4) The multistage separation method of the Mucuna pruriens biological components further comprises the step of performing gel chromatography on the filtrate by using a sephadex column G-10 in the step 3); and in the step 4), gel chromatography is carried out on the concentrated first eluent by using a sephadex column G-25. According to the method, the Mucuna pruriens is subjected to gel chromatography by using the sephadex column G-10 and the sephadex column G-25 in different steps, so that the separation of biological components in the Mucuna pruriens can be ensured to the greatest extent, and the effective separation of the biological components in the Mucuna pruriens is facilitated.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of a multi-stage separation process for the biological components of Mucuna pruriens according to the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
Example 1
The embodiment provides a multi-stage separation method for a Mucuna pruriens biological component, which comprises the following steps:
1) Crushing 5g of mucuna pruriens, and adding water with the weight 8 times that of the mucuna pruriens for leaching to obtain leaching liquor; the leaching temperature is 60 ℃, the leaching time is 1 hour, and then the leaching liquor is sieved by a 100-mesh sieve to obtain cellulose and filtrate;
2) Subjecting the filtrate obtained in the step 1) to gel chromatography by using a sephadex column G-10, after sample loading and adsorption, adding eluent for elution for 3 times (the dosage of the eluent is 50ml each time, the eluent is formed by mixing n-butanol and 35wt% of acetic acid aqueous solution, the mass ratio of the n-butanol to the 35wt% of acetic acid aqueous solution is 2:1), and combining 3 times of eluents to obtain a first eluent;
3) Concentrating the first eluent, performing gel chromatography on the concentrated first eluent by using a sephadex column G-25, performing sample adsorption, adding the eluent for elution for 3 times (the dosage of the eluent in each time is 50ml, the eluent is formed by mixing n-butanol and 35wt% of acetic acid aqueous solution, and the mass ratio of the n-butanol to the 35wt% of acetic acid aqueous solution is 2:1), and combining 3 times of eluents to obtain a second eluent;
4) Concentrating the second eluent, adding 100ml of water, adjusting the pH value of the solution to 9, stirring for 50 minutes at 40 ℃, then centrifuging by using a disc centrifuge, and separating to obtain an upper-layer protein solution and a lower-layer starch precipitate;
5) Washing the lower layer starch precipitate with water to neutrality, and drying at 40 deg.C for 3 hr to obtain dried starch; adjusting the pH value of the upper-layer protein solution obtained in the step 4) to 4, adding 100ml of saturated ammonium sulfate aqueous solution, heating to 40 ℃, filtering by a nanofiltration membrane (the molecular weight cut-off of the nanofiltration membrane is MWCO 300 Dalton), separating to obtain filter residue, and drying the filter residue at 40 ℃ for 5 hours to obtain the protein.
Through detection, the extraction rate of the obtained protein is 79%; the extraction rate of the obtained starch is 81 percent;
extraction rate of protein/% = mass of protein obtained in step 5)/mass of protein in leguma pruriens;
extraction rate of starch/% = mass of starch obtained in step 5)/mass of starch in leguma pruriens.
Example 2
The embodiment provides a multistage separation method for a Mucuna pruriens biological component, which comprises the following steps:
1) Crushing 5g of mucuna pruriens, and adding water 7 times of the weight of mucuna pruriens to leach to obtain a leaching solution; the leaching temperature is 70 ℃, the leaching time is 0.5 hour, and then the leaching liquor is sieved by a 200-mesh sieve to obtain cellulose and filtrate;
2) Subjecting the filtrate obtained in the step 1) to gel chromatography by using a sephadex column G-10, adding eluent for elution for 3 times (the dosage of the eluent in each time is 60ml, the eluent is formed by mixing n-butanol and 30wt% of acetic acid aqueous solution, the mass ratio of the n-butanol to the 30wt% of acetic acid aqueous solution is 1.5);
3) Concentrating the first eluent, performing gel chromatography on a sephadex column G-25, after sample adsorption, adding the eluent for elution for 3 times (the dosage of the eluent in each time is 60ml, the eluent is formed by mixing n-butanol and 30wt% acetic acid aqueous solution, and the mass ratio of the n-butanol to the 30wt% acetic acid aqueous solution is 1.5);
4) Concentrating the second eluent, adding 100ml of water, adjusting the pH value of the solution to 8, stirring for 50 minutes at 40 ℃, then centrifuging by using a disc centrifuge, and separating to obtain an upper-layer protein solution and a lower-layer starch precipitate;
5) Washing the lower layer starch precipitate with water to neutrality, and drying at 40 deg.C for 3 hr to obtain dried starch; adjusting the pH value of the upper-layer protein solution obtained in the step 4) to 3, adding 100ml of saturated ammonium sulfate aqueous solution, heating to 40 ℃, filtering by a nanofiltration membrane (the molecular weight cut-off of the nanofiltration membrane is MWCO 300 Dalton), separating to obtain filter residue, and drying the filter residue at 40 ℃ for 5 hours to obtain the protein.
The detection shows that the extraction rate of the obtained protein is 73 percent; the extraction rate of the obtained starch is 77%;
extraction rate of protein/% = mass of protein obtained in step 5)/mass of protein in leguma pruriens;
extraction rate of starch/% = mass of starch obtained in step 5)/mass of starch in leguma pruriens.
Example 3
The embodiment provides a multistage separation method for a Mucuna pruriens biological component, which comprises the following steps:
1) Crushing 5g of mucuna pruriens, and adding water with the weight 10 times that of mucuna pruriens for leaching to obtain leaching liquor; the leaching temperature is 50 ℃, the leaching time is 3 hours, and then the leaching liquor is sieved by a 200-mesh sieve to obtain cellulose and filtrate;
2) Subjecting the filtrate obtained in the step 1) to gel chromatography by using a sephadex column G-10, after sample loading and adsorption, adding eluent for elution for 3 times (the dosage of the eluent is 50ml each time, the eluent is formed by mixing n-butanol and 40wt% of acetic acid aqueous solution, the mass ratio of the n-butanol to the 40wt% of acetic acid aqueous solution is 3:1), and combining 3 times of eluents to obtain a first eluent;
3) Concentrating the first eluent, performing gel chromatography on the concentrated first eluent by using a sephadex column G-25, performing sample adsorption, adding the eluent for elution for 3 times (the dosage of the eluent in each time is 50ml, the eluent is formed by mixing n-butanol and 40wt% of acetic acid aqueous solution, and the mass ratio of the n-butanol to the 40wt% of acetic acid aqueous solution is 3:1), and combining 3 times of eluents to obtain a second eluent;
4) Concentrating the second eluent, adding 100ml of water, adjusting the pH value of the solution to 10, stirring for 60 minutes at 40 ℃, then centrifuging by using a disc centrifuge, and separating to obtain an upper-layer protein solution and a lower-layer starch precipitate;
5) Washing the lower layer starch precipitate with water to neutrality, and drying at 50 deg.C for 3 hr to obtain dried starch; adjusting the pH value of the upper-layer protein solution obtained in the step 4) to 4, adding 110ml of saturated ammonium sulfate aqueous solution, heating to 45 ℃, filtering by a nanofiltration membrane (the molecular weight cut-off of the nanofiltration membrane is MWCO 300 Dalton), separating to obtain filter residue, and drying the filter residue at 40 ℃ for 5 hours to obtain the protein.
Through detection, the extraction rate of the obtained protein is 75%; the extraction rate of the obtained starch is 78%;
extraction rate of protein/% = mass of protein obtained in step 5)/mass of protein in leguma pruriens;
extraction rate of starch/% = mass of starch obtained in step 5)/mass of starch in leguma pruriens.
Example 4
The embodiment provides a multistage separation method for a Mucuna pruriens biological component, which comprises the following steps:
1) Crushing 5g of mucuna pruriens, and adding water with the weight 9 times that of mucuna pruriens for leaching to obtain leaching liquor; leaching at 55 ℃ for 0.8 hour, and then sieving the leaching liquor with a 100-mesh sieve to obtain cellulose and filtrate;
2) Subjecting the filtrate obtained in the step 1) to gel chromatography by using a sephadex column G-10, after sample loading and adsorption, adding eluent to elute for 3 times (the dosage of the eluent in each time is 50ml, the eluent is formed by mixing n-butyl alcohol, 35wt% acetic acid aqueous solution and 2-ethoxybutane, the mass ratio of n-butyl alcohol, 35wt% acetic acid aqueous solution and 2-ethoxybutane is 0.8;
3) Concentrating the first eluent, then loading the concentrated first eluent into a sephadex column G-25 for gel chromatography, after sample loading and adsorption, adding eluent for elution for 3 times (the dosage of the eluent in each time is 50ml, the eluent is formed by mixing n-butyl alcohol, 35wt% acetic acid aqueous solution and 2-ethoxybutane, the mass ratio of the n-butyl alcohol to the 35wt% acetic acid aqueous solution to the 2-ethoxybutane is 0.8);
4) Concentrating the second eluent, adding 100ml of water, adjusting the pH of the solution to 9, stirring for 50 minutes at 40 ℃, then centrifuging by using a disc centrifuge, and separating to obtain an upper-layer protein solution and a lower-layer starch precipitate;
5) Washing the lower layer starch precipitate with water to neutrality, and drying at 40 deg.C for 3 hr to obtain dried starch; adjusting the pH value of the upper-layer protein solution obtained in the step 4) to 4, adding 120ml of saturated ammonium sulfate aqueous solution, heating to 40 ℃, filtering by a nanofiltration membrane (the molecular weight cut-off of the nanofiltration membrane is MWCO 300 Dalton), separating to obtain filter residue, and drying the filter residue at 40 ℃ for 5 hours to obtain the protein.
Through detection, the extraction rate of the obtained protein is 85%; the extraction rate of the obtained starch is 88%;
extraction rate of protein/% = mass of protein obtained in step 5)/mass of protein in leguma pruriens;
extraction rate of starch/% = mass of starch obtained in step 5)/mass of starch in leguma pruriens.
Example 5
The embodiment provides a multistage separation method for a Mucuna pruriens biological component, which comprises the following steps:
1) Crushing 5g of mucuna pruriens, and adding water with the weight 8 times that of the mucuna pruriens for leaching to obtain leaching liquor; the leaching temperature is 60 ℃, the leaching time is 1 hour, and then the leaching liquor is sieved by a 100-mesh sieve to obtain cellulose and filtrate;
2) Subjecting the filtrate obtained in the step 1) to gel chromatography by using a sephadex column G-10, after sample loading and adsorption, adding eluent to elute for 3 times (the dosage of the eluent is 50ml each time, the eluent is formed by mixing n-butyl alcohol, 35wt% acetic acid aqueous solution and 2-ethoxybutane, the mass ratio of the n-butyl alcohol to the 35wt% acetic acid aqueous solution to the 2-ethoxybutane is 0.9);
3) Concentrating the first eluent, then loading the concentrated first eluent into a sephadex column G-25 for gel chromatography, after sample loading and adsorption, adding eluent for elution for 3 times (the dosage of the eluent in each time is 50ml, the eluent is formed by mixing n-butyl alcohol, 35wt% acetic acid aqueous solution and 2-ethoxybutane, the mass ratio of the n-butyl alcohol to the 35wt% acetic acid aqueous solution to the 2-ethoxybutane is 0.9);
4) Concentrating the second eluent, adding 100ml of water, adjusting the pH of the solution to 9, stirring for 50 minutes at 40 ℃, then centrifuging by using a disc centrifuge, and separating to obtain an upper-layer protein solution and a lower-layer starch precipitate;
5) Washing the lower layer starch precipitate with water to neutrality, and drying at 40 deg.C for 3 hr to obtain dried starch; adjusting the pH value of the upper-layer protein solution obtained in the step 4) to 4, adding 100ml of saturated ammonium sulfate aqueous solution, heating to 40 ℃, filtering by a nanofiltration membrane (the molecular weight cut-off of the nanofiltration membrane is MWCO 300 Dalton), separating to obtain filter residue, and drying the filter residue at 40 ℃ for 5 hours to obtain the protein.
The detection shows that the extraction rate of the obtained protein is 87%; the extraction rate of the obtained starch is 91%;
extraction rate of protein/% = mass of protein obtained in step 5)/mass of protein in leguma pruriens;
extraction rate of starch/% = mass of starch obtained in step 5)/mass of starch in leguma pruriens.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (6)
1. A multistage separation method for a Mucuna pruriens biological component is characterized by comprising the following steps:
1) Pulverizing Mucuna pruriens, and leaching with water to obtain leaching solution;
2) Filtering the leaching solution to obtain cellulose and filtrate;
3) Performing gel chromatography on the filtrate to obtain a first eluent;
4) Concentrating the first eluent, and performing gel chromatography to obtain a second eluent;
5) Concentrating the second eluent, adding water after concentration, stirring, and performing centrifugal separation to obtain a protein solution and a starch precipitate;
6) Adding an ammonium sulfate aqueous solution into the protein solution, filtering to obtain filter residues, and drying the filter residues to obtain protein;
the eluent used in the gel chromatography treatment step in the step 3) is a mixed solution of n-butyl alcohol and an aqueous solution of acetic acid, the eluent used in the gel chromatography treatment step in the step 4) is a mixed solution of n-butyl alcohol and an aqueous solution of acetic acid, and the mass ratio of n-butyl alcohol to the aqueous solution of acetic acid is (1.5-3): 1;
or the eluent used in the gel chromatography treatment step is a mixed solution of n-butanol, an acetic acid aqueous solution and 2-ethoxybutane, and the mass ratio of the n-butanol to the acetic acid aqueous solution to the 2-ethoxybutane is (0.8-0.9) to 1 (0.2-0.3);
the concentration of the acetic acid aqueous solution is 30-40wt%.
2. The method of claim 1, wherein the multi-stage separation of the biological components of Mucuna pruriens is performed,
the mass ratio of the n-butanol to the acetic acid aqueous solution to the 2-ethoxybutane is 0.9;
the concentration of the acetic acid aqueous solution is 35wt%.
3. The method of claim 1, wherein the multi-stage separation of the biological components of Mucuna pruriens is performed,
gel chromatography is carried out on the filtrate by using a sephadex column G-10 in the step 3);
and 4) performing gel chromatography on the concentrated first eluent by using a sephadex column G-25 in the step 4).
4. The method of claim 1, wherein the multi-stage separation of the biological components of Mucuna pruriens is performed,
the weight ratio of the mucuna pruriens to the water in the step 1) is 1: (7-10);
the leaching temperature is 50-70 ℃, and the leaching time is 0.5-3 hours.
5. The method of claim 1, wherein the multi-stage separation of the biological components of Mucuna pruriens is performed,
filtering the leaching solution by using a 100-120-mesh sieve in the step 2);
the step 5) also comprises the step of adjusting the pH of the solution after adding water to 8-10;
the step 6) of adjusting the pH of the protein solution to 3-4 before adding the ammonium sulfate aqueous solution; the ammonium sulfate aqueous solution is saturated ammonium sulfate aqueous solution.
6. The method of claim 1, further comprising the step of washing the starch precipitate with water and then drying the starch precipitate.
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