CN108504709B - Corn oligopeptide and industrial production method thereof - Google Patents
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Abstract
The invention provides a corn oligopeptide and an industrial production method thereof, wherein the corn oligopeptide at least comprises peptide sections pEL, VL and LL. The industrial production method of the corn oligopeptide comprises the following steps: carrying out enzymolysis on the corn gluten meal by adopting protease to obtain an enzymolysis liquid; and sequentially carrying out centrifugation, filtration, activated carbon adsorption, cation exchange resin purification and anion exchange resin purification on the enzymolysis solution. The industrial production method provided by the invention can retain pEL with a plurality of physiological functions of liver protection, anti-inflammation, anti-depression and the like, and VL and LL three functional peptide segments as good branched chain amino acid supplements.
Description
Technical Field
The invention relates to a corn oligopeptide and an industrial production method thereof, in particular to a corn oligopeptide with pyroglutamyl leucine, valyl leucine and leucyl leucine peptide segments and an industrial production process thereof.
Background
Corn is one of three grain crops in the world and is also a traditional crop in China. One of the major uses of corn is in the production of corn starch, the major by-product of the wet milling process for producing corn starch is Corn Gluten Meal (CGM), also known as corn gluten meal. Corn gluten meal generally contains about 60% of protein, and the main components are zein, gluten and globulin. The corn protein powder contains hydrophobic amino acids such as leucine, isoleucine, valine and alanine, proline, glutamic acid and the like in high proportion, but the content of essential amino acids such as lysine and tryptophan is low, and the unique amino acid composition causes the corn protein to have poor water solubility and low nutritional value.
The corn protein powder is treated by protease, and can be completely dissolved in water after being hydrolyzed into micromolecular corn oligopeptide, so that the application of the corn protein powder in food becomes possible. Researches find that the corn oligopeptide is rich in branched chain amino acid, has the functions of promoting protein synthesis and inhibiting protein decomposition in muscle, and can be used for athlete foods and clinical nourishments; the corn oligopeptide contains a large amount of alanine, can slow down the speed of the body to absorb the ethanol, can promote the alcohol metabolism, reduce the toxicity of the alcohol, and can greatly reduce acute alcoholism caused by drinking. In addition, the corn oligopeptide has the physiological activity effects of reducing blood pressure and blood fat and assisting in treating liver diseases and breast cancer.
The corn oligopeptide is generally prepared by taking corn protein powder as a raw material and carrying out processes such as size mixing, enzymolysis, separation, purification, drying and the like. In the current production process, the purity and the oligopeptide content of the corn oligopeptide are emphasized in multiple ways, and dipeptide and tripeptide are kept as much as possible, so that the corn oligopeptide is more beneficial to the absorption of a human body. For example, in patent CN201410357708.2, alkaline protease and neutral protease are used to hydrolyze defatted and decolorized corn protein powder, and then a series of separation and purification treatments are performed to obtain oligopeptides as much as possible; patent CN200810084992.5 describes a process for preparing corn oligopeptide, which makes the molecular weight of oligopeptide distributed below 2000 Da.
However, in the production process of corn oligopeptide, in order to retain more oligopeptides such as dipeptide and tripeptide or pursue the purity of the oligopeptides, some important functional peptide segments, such as pyroglutamyl leucine (pyroGlu-Leu, pEL), valylleucine (Vla-Leu, VL), leucylleucine (Leu-Leu, LL) and the like, are often lost in the purification process of the enzymolysis liquid. While Yukako Yamamoto et al, published in 2015 on neuropeps, showed that pEL has a liver-protecting effect, and other studies showed that pEL also has a variety of physiological functions of anti-inflammation and anti-depression; VL and LL have also been shown to be good branched chain amino acid supplements. However, the functional peptide fragment cannot be basically detected in the existing corn oligopeptide product.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a corn oligopeptide, which retains three functional peptide fragments of pyroglutamyl leucine (pyroGlu-Leu, pEL), valyl leucine (Vla-Leu, VL) and leucyl leucine (Leu-Leu, LL).
The invention also provides an industrial production method of the corn oligopeptide, and the specific purification process is implemented, so that three functional peptide segments of pEL with various physiological functions of liver protection, anti-inflammation, depression resistance and the like and VL and LL serving as good branched chain amino acid supplements are reserved.
In order to achieve the above object, the present invention provides a corn oligopeptide, which at least comprises peptide fragments pEL, VL and LL.
Specifically, in the corn oligopeptide provided by the invention, based on the total mass (dry basis) of the corn oligopeptide, the content of pyroglutamyl leucine (pEL) is more than or equal to 0.85%, the content of Valyl Leucine (VL) is more than or equal to 0.15%, and the content of Leucyl Leucine (LL) is more than or equal to 1.15%.
The corn oligopeptide provided by the invention is prepared by carrying out protease enzymolysis on corn gluten meal and then separating and purifying,
wherein, the separation and purification sequentially comprises the steps of centrifuging, filtering, adsorbing by active carbon, purifying by cation exchange resin and purifying by anion exchange resin.
The protease can be specifically two-step enzymolysis of combination of alkaline protease and neutral protease, generally, the corn gluten meal is subjected to enzymolysis by the alkaline protease, the neutral protease is adopted for continuous enzymolysis after the enzymolysis is finished, the enzyme is deactivated after the enzymolysis is finished, and then the subsequent separation and purification treatment is carried out on the obtained enzymolysis liquid.
In the practice of the invention, the corn gluten meal is mixed with water, for example, the ratio of corn gluten meal to deionized water is 1: (5-20), mixing, pulping, adding alkaline protease for enzymolysis, wherein the amount of alkaline protease added per 100g of the maize yellow powder is 1.85-2.95AU, and then adding neutral protease for enzymolysis, wherein the amount of neutral protease added per 100g of the maize yellow powder is 0.45-0.90 AU.
The specific addition amount of the alkaline protease and the neutral protease can be reasonably adjusted according to the activity, for example, in the specific implementation process of the invention, the used alkaline protease is purchased from Novoxin (China) biotechnology limited, specifically Alcalase 2.4L type alkaline protease, and about 0.9g of alkaline protease is added per 100g of corn gluten meal; the neutral protease is obtained from Novoxin (China) Biotechnology Inc., and is neutral protease Neutrase 0.8L, and is added in an amount of about 0.72g per 100g of zeaxanthin.
The enzymolysis temperature and the enzymolysis time can be determined according to the actual conditions of the used alkaline protease and neutral protease, for example, the enzymolysis temperature of the alkaline protease can be controlled at 50-65 ℃, the pH value is adjusted to 8-10, the enzymolysis time is more than 20min, so that effective enzymolysis can be realized, the special peptide segment cannot be greatly influenced by overlong enzymolysis time, and the enzymolysis time of the general alkaline protease is 20-60min, for example, 30 min. The enzymolysis temperature of the neutral protease can be controlled at 50-65 deg.C, and the enzymolysis time can be controlled at above 150min, generally at 150-240min, such as 210 min.
After the enzymolysis is completed, the enzyme may be deactivated by conventional means in the art, such as heating to 100 ℃ and maintaining for about 30 min.
The enzymolysis liquid contains part of solid impurities which can not be subjected to enzymolysis, and can be removed by centrifugation, and in the actual industrial production, a horizontal spiral centrifuge (horizontal spiral sedimentation centrifuge) can be generally adopted to remove the solid impurities in the enzymolysis liquid; in pilot plant production or small-scale production, a bench centrifuge is generally used.
After centrifugal treatment, the obtained centrifugate is basically clarified, then macromolecular substances in the centrifugate can be further removed by filtration, microfiltration can be generally carried out by adopting a ceramic membrane with the aperture of 50-200nm, then activated carbon adsorption is adopted for decoloring, debitterizing and removing aromatic amino acids, and charged substances such as free amino acids and the like are removed by desalting through cation exchange resin and anion exchange resin in sequence, and three functional peptide sections of pEL, VL and LL in the enzymolysis liquid are retained through separation and purification.
Drying the separated and purified product to obtain powdered corn oligopeptide, namely corn oligopeptide powder.
The invention also provides an industrial production method of the corn oligopeptide, which comprises the following steps:
carrying out enzymolysis on the corn gluten meal by adopting protease to obtain an enzymolysis liquid;
and sequentially carrying out centrifugation, filtration, activated carbon adsorption, cation exchange resin purification and anion exchange resin purification on the enzymolysis solution.
According to the industrial production method of the corn oligopeptide, in the purification process, centrifugation is used for removing solid impurities in enzymolysis liquid, filtering can be used for removing macromolecular substances in the enzymolysis liquid, activated carbon adsorption is used for decoloring, debitterizing and removing aromatic amino acids, anion and cation exchange resin desalination and removal of charged substances such as free amino acids and the like, and in the process, three functional peptide sections of pEL, VL and LL are reserved.
The raw materials of the maize yellow powder used in the invention are not particularly limited, and the maize yellow powder commonly used in the current market can be adopted, wherein the protein content is not less than 50 percent, and is generally concentrated in 50 to 60 percent, such as 60 type maize yellow powder purchased from Qinhuang island Li Ye starch corporation.
The specific enzymolysis process is not particularly limited, and the enzymolysis process commonly used in the field for preparing the corn oligopeptide is adopted, so that the protein in the corn oligopeptide is hydrolyzed into the oligopeptide and peptide fragments pEL, VL and LL are obtained as many as possible. In the specific implementation process of the invention, the specific enzymolysis process comprises the following steps:
mixing the corn gluten meal with water, sequentially adding alkaline protease and neutral protease for enzymolysis, and finally performing enzyme deactivation treatment to obtain the enzymolysis liquid, wherein:
the mass ratio of the corn gluten meal to the water is 1: (5-20); the alkaline protease and neutral protease are added in an amount of 1.85-2.95AU and 0.45-0.90AU per 100g of corn gluten meal.
Specifically, the corn gluten meal can be added into a reaction tank, deionized water is added according to a certain proportion for size mixing, and sodium hydroxide is added for adjusting the pH value to 8-10; heating to 50-65 ℃, adding alkaline protease for enzymolysis, and continuing adding neutral protease for enzymolysis after the enzymolysis is finished; after the enzymolysis is finished, enzyme deactivation treatment is carried out, for example, the temperature is raised to 100 ℃ for enzyme deactivation, and the enzyme deactivation is maintained for about 30min, so as to obtain an enzymolysis liquid.
In order to remove solid impurities in the enzymatic hydrolysate, centrifugal separation may be performed on the enzymatic hydrolysate first. The specific means of centrifugation is not particularly limited, and for example, a horizontal decanter centrifuge or a desk centrifuge can be used to primarily separate and purify the enzymatic hydrolysate.
The filtration treatment of the enzymolysis solution after the centrifugal separation can be specifically performed by using a filter membrane with a pore size of 50-200nm, and a ceramic membrane with a pore size of 50-200nm is generally selected, such as a ceramic membrane with a pore size of 100-200nm, and further such as a ceramic membrane with a pore size of 200 nm. After filtration, a relatively clear enzymolysis liquid can be obtained.
The filtered enzymolysis liquid can be firstly treated by active carbon adsorption, specifically, in the active carbon adsorption process, the mass ratio of the active carbon to the maize yellow powder is (4-25): 100, the particle size of the active carbon is less than 0.0750 mm;
the temperature of the activated carbon adsorption is 40-60 deg.C, and the time is not less than 10 min.
It is understood that the activated carbon should be food grade activated carbon, such as food grade wood powdered activated carbon, with 100% fineness through a 200 mesh screen, which is commercially available.
And (3) allowing the enzymatic hydrolysate adsorbed by the activated carbon to pass through a cation exchange resin column at a linear flow rate of 1-10mL/min, starting to collect effluent when the ultraviolet detection value reaches 100mAu, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
Specifically, the cation exchange resin column is a hydrogen type cation exchange resin column with good pre-equilibrium, and the cation exchange resin is food grade cation exchange resin, specifically acrylic resin, and has particle size of 0.5-1.0 mm.
Further, in order to further improve the separation and purification effect, the cation exchange resin is continuously washed by deionized water after the sample is loaded.
And then the enzymolysis liquid purified by the cation exchange resin passes through an anion exchange resin column at a linear flow rate of 1-10mL/min, the effluent liquid is collected when the ultraviolet detection value reaches 100mAu, and the effluent liquid is stopped collecting when the ultraviolet detection value is lower than 200 mAu.
Specifically, the anion exchange resin column is a pre-balanced hydroxide-type anion exchange resin column, and the anion exchange resin is food grade anion exchange resin, specifically acrylic resin, and has a particle size of 0.5-1.0 mm.
Further, in order to further improve the separation and purification effect, after the sample is loaded, the anion exchange resin is continuously washed by deionized water.
The corn oligopeptide obtained after separation and purification can be further processed by any acceptable mode in the prior art so as to meet the corresponding application requirements, for example, the corn oligopeptide can be further dried to obtain a corn oligopeptide product, or called corn oligopeptide powder. Or before the drying treatment, the drying treatment is firstly carried out, so as to shorten the drying time and cost.
The concentration treatment can be carried out by conventional concentration means in the art, such as evaporation concentration. In the specific implementation process of the invention, the separated and purified enzymolysis liquid is concentrated to the Baume value of 20% by using a three-way vacuum concentrator (the content of soluble solid in the solution is rapidly detected by adopting a handheld refractometer, and the directly read value is the Baume value and is reflected by percentage).
The above-mentioned drying treatment can adopt the conventional drying means in the field, the invention is not limited in particular, in the concrete implementation process of the invention, the spray drying method is adopted to dry the purified product, and the spray drying conditions are specifically as follows: the inlet temperature is 160-180 ℃, and the outlet temperature is 60-80 ℃. Collecting the spray-dried product to obtain the corn oligopeptide powder.
In order to identify pEL, VL and LL in the corn oligopeptide powder, the invention uses size exclusion chromatography (Superdex Peptide 10/30GL, GE Healthcare) to pre-separate the corn oligopeptide, and then uses reversed phase high performance liquid chromatography to identify 3 main characteristic Peptide segments in the corn oligopeptide powder by combining mass spectrum: pEL, VL and LL.
Moreover, by adopting the enzymolysis process and the separation and purification process in a matching way, the content of pEL in the obtained corn oligopeptide is more than or equal to 0.85 percent and even can reach more than 1.00 percent; the VL content is more than or equal to 0.15 percent, even can reach more than 0.25 percent, and the LL content is more than or equal to 1.15 percent, even can reach more than 1.25 percent.
The corn oligopeptide provided by the invention contains pEL peptide segments with a plurality of physiological functions of liver protection, anti-inflammation, depression resistance and the like, and VL and LL peptide segments which can be used as good branched chain amino acid supplements, thereby providing a wider application prospect for corn oligopeptide products.
According to the industrialized production method of the corn oligopeptide, a specific separation and purification process is adopted, so that pEL peptide segments with multiple physiological functions of liver protection, anti-inflammation, anti-depression and the like and VL and LL peptide segments capable of serving as good branched chain amino acid supplements are reserved, wherein the content of the pEL is more than or equal to 0.85 percent and even can reach more than 1.00 percent; the VL content is more than or equal to 0.15 percent, even can reach more than 0.25 percent, and the LL content is more than or equal to 1.15 percent, even can reach more than 1.25 percent. Not only avoids the loss of functional peptide fragments, but also widens the application range of the corn oligopeptide product.
Drawings
FIG. 1 is a map of a standard used in the identification of pEL in an example of the present invention;
FIG. 2 is a map of a standard used in identifying VL in an example of the invention;
FIG. 3 is a map of a standard used to identify LL in an example of the present invention;
FIG. 4 is a pEL content detection map of corn oligopeptide prepared in example 1 of the present invention;
FIG. 5 is a VL content detection profile of corn oligopeptide prepared in example 1 of the present invention;
FIG. 6 is a detection spectrum of LL content in corn oligopeptide prepared in example 1 of the present invention;
FIG. 7 is a pEL content detection map of corn oligopeptide prepared in example 2 of the present invention;
FIG. 8 is a VL content detection profile of corn oligopeptide prepared in example 2 of the present invention;
FIG. 9 is a detection spectrum of LL content in corn oligopeptide prepared in example 2 of the present invention;
FIG. 10 is a graph showing a pEL content assay pattern in corn oligopeptide prepared in example 3 of the present invention;
FIG. 11 is a VL content detection profile of corn oligopeptide prepared in example 3 of the present invention;
FIG. 12 is a detection map of LL content in corn oligopeptide prepared in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples and comparative examples, the functional peptide fragments in the maize oligopeptide were detected by reversed phase high performance liquid chromatography Inertsil ODS-3(5 μm,2.1 mm. times.250 mm, GL Science, Tokyo, Japan) combined with mass spectrometry (LCMS-8040, Shimadzu, Kyoto, Japan): content of pEL, VL and LL.
In the following examples and comparative examples, the corn gluten meal used was purchased from type 60 corn gluten meal, from li-ju, qinhuang, li-ju, inc; the alkaline protease is purchased from Alcalase 2.4L type alkaline protease, Novoxin (China) Biotechnology Co., Ltd; neutral proteases were all purchased from Novoxin (China) Biotechnology Inc., Neutrase type 0.8L neutral protease.
Example 1
1. Preparing corn oligopeptide:
1) adding 500g of corn protein powder into a reaction tank, using 5L of deionized water for size mixing, adding NaOH to adjust the pH value to 9.0, heating to 50 ℃, adding 4.5g of alkaline protease, performing enzymolysis for 30min, then adding 3.6g of neutral protease, continuing enzymolysis for 3.5h, heating to 100 ℃ for enzyme deactivation, and maintaining for 30 min.
2) And (3) centrifuging the enzymolysis liquid by using a desk centrifuge, collecting clear liquid, and filtering by using a ceramic membrane with the aperture of 200nm to further clarify the enzymolysis liquid.
3) Adding 20g of food-grade wood powdery active carbon (with fineness of 100% passing through 200 mesh sieve), adsorbing at 50 deg.C for about 30min, and removing active carbon.
4) And (3) enabling the enzymatic hydrolysate adsorbed by the activated carbon to pass through a pre-balanced hydrogen type cation exchange resin column at a linear flow rate of 1mL/min, starting to collect effluent liquid when the ultraviolet detection value is higher than 100mAu, continuing to flush the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent liquid when the ultraviolet detection value is lower than 200 mAu.
5) And (3) enabling the collected effluent to pass through a pre-balanced hydroxide type anion exchange resin column at a linear flow rate of 1mL/min, starting to collect the effluent when the ultraviolet detection value is higher than 100mAu, continuously washing the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
6) Concentrating the effluent liquid by an evaporation concentration system until the Baume value is 20%, and then sending the effluent liquid into a spray drying tower, wherein the inlet temperature is 160 ℃, and the outlet temperature is 60 ℃ to obtain the corn oligopeptide powder product.
2. Detecting the content of functional peptide fragments pEL, VL and LL in the corn oligopeptide product:
pre-separating the corn oligopeptide by using molecular exclusion chromatography (Superdex Peptide 10/30GL, GE Healthcare), and identifying whether the corn oligopeptide contains a characteristic Peptide segment by using a reversed phase high performance liquid chromatography combined mass spectrum: pEL, VL and LL.
The structural identification maps of pEL, VL and LL are respectively shown in fig. 1 to fig. 3 (standard samples), and fig. 1 comprises a primary mass spectrum of pEL (m/z is 243.1) and a secondary fragment spectrum thereof under the condition that CE is-35.0; fig. 2 includes a primary mass spectrum of VL (m/z 231.2) and its secondary fragment spectrum at CE-35.0; fig. 3 includes the primary mass spectrum of LL (m/z-245.2) and its secondary fragment spectrum at CE-35.0.
The alignment with the standard sample (fig. 1 to fig. 3) proves that the peptide fragments pEL, VL and LL are simultaneously present in the corn oligopeptide. Fig. 4 to fig. 6 are the content detection maps of pEL, VL and LL in the corn oligopeptide product prepared in this example, and through detection, the content of the functional peptide pEL in the corn oligopeptide product is 0.88% ± 0.01%, the content of VL is 0.27% ± 0.00%, and the content of LL is 1.20% ± 0.02% (based on the total mass of the corn oligopeptide product, the same applies below).
The method provided by the embodiment is adopted to retain pEL peptide segments with a plurality of physiological functions of liver protection, anti-inflammation, depression resistance and the like, VL peptide segments and LL peptide segments which can be used as good branched chain amino acid supplements.
Example 2
1. Preparing corn oligopeptide:
1) adding 500g corn protein powder into a reaction tank, mixing with 5L deionized water, adding NaOH to adjust pH to 9.0, heating to 50 deg.C, adding alkaline protease 4.5g, performing enzymolysis for 30min, adding neutral protease 3.6g, performing enzymolysis for 3.5hr, heating to 100 deg.C, inactivating enzyme, and maintaining for 30 min.
2) And (3) centrifuging the enzymolysis liquid by using a desk centrifuge, collecting clear liquid, and filtering by using a ceramic membrane with the aperture of 200nm to further clarify the enzymolysis liquid.
3) Adding 100g food-grade wood powdered activated carbon (with fineness of 100% passing through 200 mesh sieve) into the enzymolysis solution, adsorbing at 50 deg.C for about 12min, and removing the activated carbon.
4) And (3) enabling the enzymatic hydrolysate adsorbed by the activated carbon to pass through a pre-balanced hydrogen type cation exchange resin column at a linear flow rate of 10mL/min, starting to collect effluent liquid when the ultraviolet detection value is higher than 100mAu, continuing to flush the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent liquid when the ultraviolet detection value is lower than 200 mAu.
5) And (3) enabling the collected effluent to pass through a pre-balanced hydroxide type anion exchange resin column at a linear flow rate of 10mL/min, starting to collect the effluent when the ultraviolet detection value is higher than 100mAu, continuously washing the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
6) Concentrating the effluent liquid by an evaporation concentration system until the Baume value is 20%, and then sending the effluent liquid into a spray drying tower, wherein the inlet temperature is 180 ℃, and the outlet temperature is 80 ℃ to obtain the corn oligopeptide powder product.
2. Detecting the content of functional peptide fragments pEL, VL and LL in the corn oligopeptide product:
the corn oligopeptide product of this example was evaluated for the presence and corresponding amounts of pEL, VL and LL by reference to the procedure of example 1. FIGS. 7 to 9 are the detection maps of pEL, VL and LL content in the maize oligopeptide products prepared in this example, respectively. Through detection, peptide sections pEL, VL and LL exist in the corn oligopeptide at the same time, wherein the content of the functional peptide section pEL is 0.97% +/-0.01%, the content of VL is 0.17% +/-0.00%, and the content of LL is 1.19% +/-0.02%.
The method provided by the embodiment is adopted to retain pEL peptide segments with a plurality of physiological functions of liver protection, anti-inflammation, depression resistance and the like, VL peptide segments and LL peptide segments which can be used as good branched chain amino acid supplements.
Example 3
1. Preparing corn oligopeptide:
1) adding 500g corn protein powder into a reaction tank, mixing with 5L deionized water, adding NaOH to adjust pH to 9.0, heating to 50 deg.C, adding alkaline protease 4.5g, performing enzymolysis for 30min, adding neutral protease 3.6g, performing enzymolysis for 3.5hr, heating to 100 deg.C, inactivating enzyme, and maintaining for 30 min.
2) And (3) centrifuging the enzymolysis liquid by using a desk centrifuge, collecting clear liquid, and filtering by using a ceramic membrane with the aperture of 200nm to further clarify the enzymolysis liquid.
3) Adding food-grade wood powdered activated carbon 50g (with fineness of 100% passing through 200 mesh sieve), adsorbing at 50 deg.C for about 30min, and removing activated carbon.
4) And (3) enabling the enzymatic hydrolysate adsorbed by the activated carbon to pass through a pre-balanced hydrogen type cation exchange resin column at a linear flow rate of 5mL/min, starting to collect effluent liquid when the ultraviolet detection value is higher than 100mAu, continuing to flush the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent liquid when the ultraviolet detection value is lower than 200 mAu.
5) And (3) enabling the collected effluent to pass through a pre-balanced hydroxide type anion exchange resin column at a linear flow rate of 5mL/min, starting to collect the effluent when the ultraviolet detection value is higher than 100mAu, continuously washing the cation exchange resin column with deionized water after the sample is loaded, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
6) Concentrating the effluent liquid by an evaporation concentration system until the Baume value is 20%, and then sending the effluent liquid into a spray drying tower, wherein the inlet temperature is 170 ℃, and the outlet temperature is 60 ℃ to obtain the corn oligopeptide powder product.
2. Detecting the content of functional peptide fragments pEL, VL and LL in the corn peptide:
the corn oligopeptide product of this example was evaluated for the presence and corresponding amounts of pEL, VL and LL by reference to the procedure of example 1. FIGS. 10 to 12 are the detection maps of pEL, VL and LL content in the maize oligopeptide products prepared in this example, respectively. Through detection, peptide sections pEL, VL and LL exist in the corn oligopeptide at the same time, wherein the content of the functional peptide section pEL is 1.02% +/-0.01%, the content of VL is 0.24% +/-0.00%, and the content of LL is 1.26% +/-0.02%.
The method provided by the embodiment is adopted to retain pEL peptide segments with a plurality of physiological functions of liver protection, anti-inflammation, depression resistance and the like, VL peptide segments and LL peptide segments which can be used as good branched chain amino acid supplements.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (3)
1. A corn oligopeptide, which is characterized in that the composition of the corn oligopeptide at least comprises peptide segments pEL, VL and LL;
based on the mass of the corn oligopeptide, the content of pEL is more than or equal to 0.85 percent, the content of VL is more than or equal to 0.15 percent, and the content of LL is more than or equal to 1.15 percent;
the corn oligopeptide is prepared by performing protease enzymolysis on corn gluten meal and then separating and purifying,
the enzymolysis step comprises the following steps:
mixing the corn gluten meal with water, sequentially adding alkaline protease and neutral protease for enzymolysis, and performing enzyme deactivation treatment to obtain an enzymolysis solution, wherein:
the mass ratio of the corn gluten meal to the water is 1: (5-20); the alkaline protease and neutral protease added per 100g of corn gluten meal are respectively 1.85-2.95AU and 0.45-0.90 AU;
wherein, the separation and purification sequentially comprises the steps of centrifuging, filtering, adsorbing by active carbon, purifying by cation exchange resin and purifying by anion exchange resin;
the filtration is carried out by adopting a filter membrane with the aperture of 50-200 nm;
in the active carbon adsorption process, the mass ratio of the active carbon to the maize yellow powder is (4-25): 100, the particle size of the activated carbon is less than 0.0750 mm;
the temperature of the activated carbon adsorption is 40-60 ℃, and the time is not less than 10 min;
the cation exchange resin purification specifically comprises:
allowing the enzymolysis solution separated and purified by macroporous adsorption resin to pass through cation exchange resin column at linear flow rate of 1-10mL/min, collecting effluent when ultraviolet detection value reaches 100mAu, and stopping collecting effluent when ultraviolet detection value is lower than 200 mAu;
the anion exchange resin purification specifically comprises:
and (3) allowing the enzymolysis solution purified by the cation exchange resin to pass through an anion exchange resin column at a linear flow rate of 1-10mL/min, starting to collect the effluent when the ultraviolet detection value reaches 100mAu, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
2. An industrial process for producing the corn oligopeptide according to claim 1, wherein the process comprises the following steps:
carrying out enzymolysis on the corn gluten meal by adopting protease to obtain an enzymolysis liquid;
the enzymolysis step comprises the following steps:
mixing the corn gluten meal with water, sequentially adding alkaline protease and neutral protease for enzymolysis, and performing enzyme deactivation treatment to obtain an enzymolysis solution, wherein:
the mass ratio of the corn gluten meal to the water is 1: (5-20); the alkaline protease and neutral protease added per 100g of corn gluten meal are respectively 1.85-2.95AU and 0.45-0.90 AU;
sequentially carrying out centrifugation, filtration, activated carbon adsorption, cation exchange resin purification and anion exchange resin purification on the enzymolysis solution;
the filtration is carried out by adopting a filter membrane with the aperture of 50-200 nm;
in the active carbon adsorption process, the mass ratio of the active carbon to the maize yellow powder is (4-25): 100, the particle size of the activated carbon is less than 0.0750 mm;
the temperature of the activated carbon adsorption is 40-60 ℃, and the time is not less than 10 min;
the cation exchange resin purification specifically comprises:
allowing the enzymolysis solution separated and purified by macroporous adsorption resin to pass through cation exchange resin column at linear flow rate of 1-10mL/min, collecting effluent when ultraviolet detection value reaches 100mAu, and stopping collecting effluent when ultraviolet detection value is lower than 200 mAu;
the anion exchange resin purification specifically comprises:
and (3) allowing the enzymolysis solution purified by the cation exchange resin to pass through an anion exchange resin column at a linear flow rate of 1-10mL/min, starting to collect the effluent when the ultraviolet detection value reaches 100mAu, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu.
3. The industrial process according to claim 2, further comprising subjecting the purified product to a spray-drying treatment under the conditions: the inlet temperature is 160-180 ℃, and the outlet temperature is 60-80 ℃.
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| CN104187924A (en) * | 2014-07-24 | 2014-12-10 | 江苏伟楼生物科技有限公司 | Preparation method of corn oligopeptide powder |
| JP2014234384A (en) * | 2013-06-05 | 2014-12-15 | 日本食品ペプチド研究所株式会社 | Alcohol metabolism accelerator |
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| CN102174626B (en) * | 2011-01-12 | 2013-05-01 | 武汉百信食品有限公司 | Method for preparing corn peptide |
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| CN104187924A (en) * | 2014-07-24 | 2014-12-10 | 江苏伟楼生物科技有限公司 | Preparation method of corn oligopeptide powder |
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| The Effects of Corn Peptide Ingestion on Facilitating Alcohol Metabolism in Healthy Men;Magoichi Yamaguchi 等;《Bioscience Biotechnology and Biochemistry》;19971031;第61卷(第9期);第1474-1481页 * |
| 玉米蛋白粉酶解工艺技术研究;陆启明 等;《粮食加工》;20171001;第42卷(第5期);第48-52页 * |
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