CN112674350A - Millet bran SDF-Cr (III) complex and preparation method and application thereof - Google Patents
Millet bran SDF-Cr (III) complex and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of grain processing, and provides a preparation method of a millet bran SDF-Cr (III) complex, wherein the millet bran SDF is used as a raw material of an organic ligand, a weak alkaline reagent is used for adjusting the pH value of a mixed solution obtained after the millet bran SDF is mixed with water, so that the mixed solution is suitable for an alkaline environment required by a subsequent reaction, then a strong alkaline reagent is added, and the strong alkaline reagent is used for inducing the millet bran SDF to deprotonate so as to form the organic ligand; the formed organic ligand and chromium chloride are subjected to coordination reaction under the catalytic action of catalyst isopropanol to obtain the millet bran SDF-Cr (III) complex. Experimental results show that the millet bran SDF-Cr (III) complex obtained by the preparation method provided by the invention has high chelating rate, can control the content of chromium in organic chromium, and can be used as a raw material of chromium-rich food.
Description
Technical Field
The invention relates to the technical field of grain processing, in particular to a millet bran SDF-Cr (III) complex and a preparation method and application thereof.
Background
The millet bran contains 50-60% of dietary fiber, and the dietary fiber can be divided into water-Soluble Dietary Fiber (SDF) and Insoluble Dietary Fiber (IDF) according to the carbohydrate solubility. SDF, such as pectin, partial hemicellulose and vegetable gums, are non-starch polysaccharides that are soluble in hot water and are not digested and decomposed by human digestive enzymes. However, the research shows that the millet bran SDF contains a plurality of lone electron groups, such as C-O-C, C-O-H, O-H, is an excellent metal ion chelating organic ligand and has excellent stability and a plurality of biological activities.
Chromium is an essential trace element of the human body and plays an important role in regulating blood sugar levels. The inorganic chromium exists in nature mostly, but the inorganic chromium is difficult to be utilized by the organism and has the disadvantage of toxic and side effect on the organism. Therefore, the chromium content in the product is increased by adding organic chromium raw materials in the existing chromium-rich food, and the organic chromium in the raw materials is mostly prepared into the chromium-rich food by biotransformation, such as fermentation or planting by using chromium-rich yeast. SDF is one of food raw materials, and can be combined with inorganic chromium to be converted into organic chromium, so that the defects that the inorganic chromium is difficult to be utilized by the body and has toxic and side effects on the body can be overcome. However, the current processes for converting SDF into organic chromium by combining SDF with inorganic chromium have low chelating rates of SDF-cr (iii) complexes and unstable chromium content in the product. Therefore, a preparation method of the SDF-Cr (III) complex, which has high chelating rate and can control the chromium content in organic chromium, is needed.
Disclosure of Invention
The invention aims to provide a millet bran SDF-Cr (III) complex, and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a millet bran SDF-Cr (III) complex, which comprises the following steps:
(1) mixing the millet bran SDF with water to obtain a mixed solution;
(2) and (2) sequentially adding chromium chloride, a weakly alkaline reagent, a strongly alkaline reagent and isopropanol into the mixed solution obtained in the step (1) for coordination reaction to obtain the millet bran SDF-Cr (III) complex.
Preferably, the millet bran SDF in step (1) is obtained by enzymatic hydrolysis of defatted millet bran powder.
Preferably, the preparation of the millet bran SDF in the step (1) comprises the following steps:
(a) mixing the defatted millet bran powder with water to obtain a defatted millet bran powder suspension;
(b) adjusting the pH value of the defatted millet bran powder suspension obtained in the step (a) to 6.0-8.0, and then adding high-temperature-resistant alpha-amylase to perform a first enzymolysis reaction to obtain a first reaction system;
(c) adjusting the pH value of the first reaction system obtained in the step (b) to 6.0-8.0, and then adding neutral protease to perform a second enzymolysis reaction to obtain a second reaction system;
(d) adjusting the pH value of the second reaction system obtained in the step (c) to 4.0-5.0, and then adding amyloglucosidase to perform a third enzymolysis reaction to obtain a third reaction system;
(e) and (d) carrying out post-treatment on the third reaction system obtained in the step (d) to obtain the millet bran SDF.
Preferably, the temperature of the first enzymolysis reaction in the step (b) is 90-100 ℃, and the time of the first enzymolysis reaction is 15-25 min; the temperature of the second enzymolysis reaction in the step (c) and the temperature of the third enzymolysis reaction in the step (d) are respectively 55-65 ℃, and the time of the second enzymolysis reaction in the step (c) and the time of the third enzymolysis reaction in the step (d) are respectively 25-35 min.
Preferably, the mass ratio of the millet bran SDF in the step (1) to the isopropanol and the chromium chloride in the step (2) is 1: (0.05-0.1): (0.45-2).
Preferably, the weakly alkaline reagent in the step (2) is ammonia water, and the mass concentration of the ammonia water is 20-40%.
Preferably, the strong alkaline reagent in the step (2) is sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 2-4 mol/L.
Preferably, the temperature of the coordination reaction in the step (2) is 60-80 ℃, and the time of the coordination reaction is 2-4 h.
The invention also provides the millet bran SDF-Cr (III) complex prepared by the preparation method in the technical scheme.
The invention also provides application of the millet bran SDF-Cr (III) complex in the technical scheme in chromium-rich food.
The invention provides a preparation method of a millet bran SDF-Cr (III) complex, which comprises the following steps: mixing the millet bran SDF with water, and sequentially adding chromium chloride, a weakly alkaline reagent, a strongly alkaline reagent and isopropanol into a mixed solution obtained by mixing the millet bran SDF with the water for coordination reaction to obtain the millet bran SDF-Cr (III) complex. The method comprises the steps of taking millet bran SDF as a raw material of an organic ligand, adjusting the pH value of a mixed solution obtained after the millet bran SDF is mixed with water by using a weakly alkaline reagent to enable the mixed solution to be suitable for an alkaline environment required by subsequent reaction, then adding a strongly alkaline reagent, and inducing the millet bran SDF to deprotonate by using the strongly alkaline reagent to form the organic ligand; the formed organic ligand and chromium chloride are subjected to coordination reaction under the catalytic action of catalyst isopropanol to obtain the millet bran SDF-Cr (III) complex. Experimental results show that the chelating rate of the millet bran SDF-Cr (III) complex prepared by the preparation method provided by the invention can reach 35.179%, and the content of Cr (III) in the millet bran SDF-Cr (III) complex can reach 4.08 mg/g. The millet bran SDF-Cr (III) complex obtained by the preparation method provided by the invention has high chelating rate, can control the content of chromium in organic chromium, and can be used as a raw material of chromium-rich food.
Detailed Description
The invention provides a preparation method of a millet bran SDF-Cr (III) complex, which comprises the following steps:
(1) mixing the millet bran SDF with water to obtain a mixed solution;
(2) and (2) sequentially adding chromium chloride, a weakly alkaline reagent, a strongly alkaline reagent and isopropanol into the mixed solution obtained in the step (1) for coordination reaction to obtain the millet bran SDF-Cr (III) complex.
The millet bran SDF is mixed with water to obtain a mixed solution. The operation mode of mixing the millet bran SDF and the water is not particularly limited, and a solid-liquid mixing mode known by the technical personnel in the field can be adopted.
In the invention, the feed-liquid ratio of the millet bran SDF to water is preferably (1: 40-1: 60) g/mL, and more preferably (1: 50-1: 60) g/mL. In the invention, when the feed-liquid ratio of the millet bran SDF to water is within the range, the obtained mixed liquid has moderate concentration, and is more beneficial to the subsequent reaction.
The source of the millet bran SDF is not particularly specified in the invention, and the millet bran SDF can be prepared by adopting a commercial product or a preparation method which is well known by the technical personnel in the field.
In the present invention, the preparation of the millet bran SDF preferably comprises the following steps:
(a) mixing the defatted millet bran powder with water to obtain a defatted millet bran powder suspension;
(b) adjusting the pH value of the defatted millet bran powder suspension obtained in the step (a) to 6.0-8.0, and then adding high-temperature-resistant alpha-amylase to perform a first enzymolysis reaction to obtain a first reaction system;
(c) adjusting the pH value of the first reaction system obtained in the step (b) to 6.0-8.0, and then adding neutral protease to perform a second enzymolysis reaction to obtain a second reaction system;
(d) adjusting the pH value of the second reaction system obtained in the step (c) to 4.0-5.0, and then adding amyloglucosidase to perform a third enzymolysis reaction to obtain a third reaction system;
(e) and (d) carrying out post-treatment on the third reaction system obtained in the step (d) to obtain the millet bran SDF.
The invention preferably mixes the degreased millet bran powder with water to obtain the degreased millet bran powder suspension.
In the invention, the feed-to-liquid ratio of the defatted millet bran powder to water is preferably (1: 40-1: 60) g/mL, and more preferably (1: 50-1: 60) g/mL.
The source of the defatted millet bran powder is not particularly limited, and the defatted millet bran powder prepared by a commercial product or a preparation method which is well known by the technical personnel in the field can be used. In the invention, the defatted millet bran powder can eliminate the interference of fat on the extraction of the water-soluble dietary fiber and improve the yield of the water-soluble dietary fiber.
In the present invention, the method for preparing the defatted millet bran powder preferably comprises the following steps: and mixing the pretreated millet bran powder with petroleum ether, and extracting to obtain the defatted millet bran powder. The operation mode of mixing the pretreated millet bran powder and the petroleum ether is not particularly limited, and the mixing mode known by the technical personnel in the field can be adopted.
The source of the millet bran powder is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used. In the present invention, the millet bran powder is preferably millet bran powder that is not mildewed.
In the present invention, the pretreatment preferably includes drying and pulverization which are sequentially performed. The drying temperature and time are not specially limited, and the water mass fraction of the millet bran powder can be 5-8%. In the invention, when the water mass fraction of the millet bran powder is within the above range, the subsequent crushing and degreasing effects can be improved.
In the invention, the drying temperature is preferably 45-65 ℃, and the drying time is preferably 12-16 h. The drying apparatus of the present invention is not particularly limited, and any drying apparatus known to those skilled in the art may be used. In the present invention, the drying device is preferably a drying oven.
In the invention, the grinding can reduce the particle size of the millet bran powder, and is more beneficial to the dissolution of the millet bran SDF. The device for pulverizing is not particularly limited in the present invention, and a pulverizing device known to those skilled in the art may be used. In the present invention, the pulverization device is preferably an ultrafine pulverizer. The invention has no special limitation on the crushing parameters, and the crushing parameters can be adjusted according to the crushing effect of the millet bran powder. The time for the pulverization in the present invention is preferably 20 min.
The invention preferably screens the crushed millet bran powder to control the particle size of the millet bran powder. In the invention, the particle size of the millet bran powder is preferably 40-80 meshes, more preferably 50-60 meshes, and most preferably 60 meshes. In the invention, when the particle size of the sieve is in the above range, the obtained millet bran is more favorable for the dissolution of SDF.
In the present invention, the extraction is preferably an evaporative reflux. In the invention, the extraction temperature is preferably 45-60 ℃, and more preferably 55-60 ℃; the extraction time is preferably 2-5 h, and more preferably 3.5 h. The extraction apparatus of the present invention is not particularly limited, and an extraction apparatus known to those skilled in the art may be used. In the present invention, the extraction apparatus is preferably a soxhlet extractor.
After the defatted millet bran powder suspension is obtained, the pH value of the defatted millet bran powder suspension is preferably adjusted to 6.0-8.0, and then high-temperature-resistant alpha-amylase is added to perform a first enzymolysis reaction, so that a first reaction system is obtained.
In the invention, the pH value of the defatted millet bran powder suspension is preferably adjusted to 7.0-8.0. In the invention, when the pH value of the defatted millet bran powder suspension is within the above range, the first enzymolysis reaction is favorably carried out. The reagent for adjusting the pH value of the defatted millet bran powder suspension is not particularly limited, and the pH value of the defatted millet bran powder suspension can be adjusted to the range. In the present invention, the agent for adjusting the pH of the suspension of defatted millet bran powder is preferably a phosphate buffer solution. The addition amount of the phosphate buffer solution is not particularly limited, and the pH of the defatted millet bran powder suspension can be adjusted to the above range.
In the present invention, the ratio of the mass of the defatted millet bran to the volume of the high temperature resistant α -amylase is preferably 1g: 100 uL-10 g: 100uL, more preferably 5g: 100 uL. In the invention, when the ratio of the mass of the defatted millet bran to the volume of the high-temperature resistant alpha-amylase is in the above range, the starch in the defatted millet bran can be subjected to an enzymolysis reaction fully.
In the invention, the temperature of the first enzymolysis reaction is preferably 90-100 ℃, and more preferably 95-100 ℃; the time of the first enzymolysis reaction is preferably 15-25 min, and more preferably 20-25 min. In the present invention, when the temperature and time of the first enzymatic hydrolysis reaction are within the above ranges, the α -amylase can have an optimal activity, and complete performance of the first enzymatic hydrolysis reaction can be ensured.
After the first reaction system is obtained, the pH value of the first reaction system is preferably adjusted to 6.0-8.0, and then neutral protease is added for a second enzymolysis reaction to obtain a second reaction system.
In the invention, the pH value of the first reaction system is preferably adjusted to 7.0-8.0. In the present invention, when the pH of the first reaction system is within the above range, the second enzymatic reaction is favorably carried out. In the present invention, the reagent for adjusting the pH of the first reaction system is preferably a phosphate buffer solution. In the present invention, the amount of the phosphate buffer solution to be added is not particularly limited, and the pH of the first reaction system may be adjusted to the above range.
In the present invention, the ratio of the mass of the defatted millet bran to the volume of the neutral protease solution is preferably (1: 100) to (10: 100) g/uL, more preferably 5g: 100 uL. In the present invention, when the ratio of the mass of the defatted millet bran to the volume of the neutral protease solution is in the above range, the second enzymatic hydrolysis reaction can be sufficiently performed.
In the invention, the temperature of the second enzymolysis reaction is preferably 55-65 ℃, and more preferably 55-65 ℃; the time of the second enzymolysis reaction is preferably 25-35 min, and more preferably 25-30 min. In the present invention, when the temperature and time of the second enzymatic reaction are within the above ranges, the neutral protease can have an optimal activity, and the complete progress of the second enzymatic reaction can be ensured.
After the second reaction system is obtained, the pH value of the second reaction system is preferably adjusted to 4.0-5.0, and then amyloglucosidase is added to perform a third enzymolysis reaction to obtain a third reaction system.
In the invention, the pH value of the second reaction system is preferably adjusted to 4.5-5.0. In the present invention, when the pH of the second reaction system is within the above range, the third enzymatic reaction is favorably performed. In the present invention, the reagent for adjusting the pH of the second reaction system is not particularly limited, and the pH of the second reaction system may be adjusted to the above range.
In the present invention, the ratio of the mass of the defatted millet bran to the volume of amyloglucosidase is preferably (1: 100) to (10: 100) g/uL, more preferably 5g: 100 uL. In the present invention, when the ratio of the mass of the defatted millet bran to the volume of amyloglucosidase is in the above range, the third enzymatic hydrolysis reaction can be sufficiently performed.
In the invention, the temperature of the third enzymolysis reaction is preferably 55-65 ℃, and more preferably 55-65 ℃; the time of the second enzymolysis reaction is preferably 25-35 min, and more preferably 25-30 min. In the present invention, when the temperature and time of the second enzymatic reaction are within the above ranges, the neutral protease can have an optimal activity, and the complete progress of the second enzymatic reaction can be ensured.
The device for the first, second and third enzymatic hydrolysis reactions is not particularly limited, and any device for enzymatic hydrolysis reactions known to those skilled in the art may be used. In the present invention, the device for enzymatic hydrolysis reaction is preferably a GDE enzyme culture digester.
After the third reaction system is obtained, the third reaction system is preferably subjected to post-treatment to obtain the millet bran SDF.
In the present invention, the post-treatment preferably comprises enzyme inactivation, filtration, centrifugation, concentration, alcohol precipitation and refining, which are sequentially performed. The operation of enzyme inactivation, filtration, centrifugation, concentration and alcohol precipitation is not particularly limited in the invention, and the operation modes of enzyme inactivation, filtration, centrifugation, concentration and alcohol precipitation which are well known to those skilled in the art can be adopted.
In the present invention, the temperature of the enzyme deactivation is preferably more than 100 ℃, and the time of the enzyme deactivation is preferably 10 min.
In the present invention, the filtration device is preferably a CSF6 filtration device.
In the invention, the rotation speed of the centrifugation is preferably 3000 rmp-5000 rmp, more preferably 4000-4800 rpm, and the time of the centrifugation is preferably 15 min-25 min, more preferably 18 min-20 min.
The centrifugal product is preferably concentrated to 1/4-1/5 of the volume of the stock solution to obtain a concentrated liquid, and then alcohol precipitation is carried out. In the invention, the alcohol precipitation reagent is preferably 90-99.8% ethanol, and more preferably 95% ethanol. In the invention, the adding amount of the ethanol is preferably 4-6 times of the volume of the concentrated solution, and more preferably 4-5 times. In the invention, because SDF is insoluble in ethanol, and the ethanol is safe and non-toxic to use, pollution-free and easy to recover, the ethanol is used for carrying out alcohol precipitation, and the prepared product is safe and non-toxic and has high yield.
The invention preferably dries the precipitated solid. In the invention, the drying is preferably freeze drying, and the temperature of the freeze drying is preferably-99 to-108 ℃, and more preferably-100 to 105 ℃; the freeze drying time is preferably 6-8 h, and more preferably 6-7 h; the degree of vacuum of the freeze drying is preferably 4 to 5.5Pa, and more preferably 4.5 to 5.0 Pa.
In the present invention, the purification method is preferably deproteinization by the Sevag method. The method for deproteinizing by the Sevag method is not particularly limited, and a method known to those skilled in the art may be used. In the present invention, the Sevag deproteinization preferably comprises: mixing the dried product with water to form a first mixed slurry; mixing the first mixed slurry with a Sevag reagent, and centrifuging to obtain a second mixed slurry; decoloring the second mixed slurry to obtain crude SDF; and carrying out alcohol precipitation on the coarse SDF to obtain the millet bran SDF.
In the present invention, the ratio of the mass of the dried product to the volume of water is preferably 0.5g: 100 mL.
In the invention, the Sevag reagent is preferably a mixed solution of n-butanol and trichloromethane, and the volume ratio of n-butanol to trichloromethane in the mixed solution of n-butanol and trichloromethane is preferably 1: 4-1: 6, and more preferably 1: 5-1: 6. In the present invention, when the Sevag reagent is of the above type, it is more advantageous to remove the protein in the first mixed slurry.
In the present invention, when the mass to water volume ratio of the dried product is preferably 0.5g: and when the volume ratio of the n-butanol to the trichloromethane is preferably 1:5, the volume ratio of the first mixed slurry to the Sevag reagent is preferably 4: 1. in the present invention, the volume ratio of the first mixed slurry to the Sevag reagent is in the above range, which is more favorable for the Sevag reagent to react with the protein in the SDF.
In the present invention, the mixing of the first mixed slurry with Sevag reagent is preferably shaking, and the time period of the shaking is preferably 10 min. In the present invention, the shaking is useful to promote the reaction of Sevag reagent with the protein in SDF.
In the invention, the first mixed slurry and the Sevag reagent are preferably shaken and then centrifuged to obtain a second mixed slurry. In the present invention, the centrifugation enables removal of the protein layer. The present invention does not specifically limit the rotation speed and time of the centrifugation, and the effect of separating the protein layer can be achieved by using the rotation speed and time of the centrifugation, which are well known to those skilled in the art.
In the present invention, it is preferable to mix the product obtained after centrifugation with Sevag reagent and repeat the above operations of shaking and centrifugation to sufficiently remove the protein in SDF. In the present invention, the number of repetitions is preferably 10 or more.
After the second mixed slurry is obtained, the second mixed slurry is preferably decolorized to obtain crude SDF.
In the present invention, the step of decoloring is preferably: and adjusting the pH value of the second mixed slurry to 8-10 by using ammonia water, then adding hydrogen peroxide into the second mixed slurry after the pH value is adjusted, and preserving the obtained system at the temperature of 35-45 ℃ for 55-65 min to finish the decoloring process. In the invention, the hydrogen peroxide is preferably hydrogen peroxide with the mass concentration of 25-35%; the mass ratio of the volume of the hydrogen peroxide to the mass of the SDF obtained after alcohol precipitation is preferably (45-55) mL: 0.5 g. In the present invention, the decoloring step is of the above type so that the decoloring can be sufficiently completed.
The invention preferably carries out rotary evaporation on the millet bran SDF solution obtained after decolorization to remove the solvent. In the present invention, the removal of the solvent facilitates the subsequent dialysis process.
In the present invention, it is preferable that the solid obtained by removing the solvent is dialyzed to obtain a dialyzed solution. The operation of the dialysis is not particularly limited in the present invention, and a method of the dialysis known to those skilled in the art may be used. In the invention, the dialysis bag adopted by the dialysis is preferably a 14000Da dialysis bag; the agent for dialysis is preferably distilled water or tap water. In the present invention, when the agent for dialysis is preferably distilled water, the dialysis time is preferably 24 hours; when the dialysis agent is tap water, the dialysis time is preferably 48 hours. In the present invention, the dialysis can further remove impurities.
After the dialyzed solution is obtained, the dialyzed solution is sequentially subjected to alcohol precipitation and drying. In the present invention, the operation manner of alcohol precipitation and drying is the same as that of the above technical scheme, and is not described herein again.
The millet bran SDF prepared by the method can fully remove protein and starch in the millet bran, eliminate the interference of fat on the extraction of the millet bran SDF, and improve the yield of the millet bran SDF.
After the mixed solution is obtained, the invention preferably adds chromium chloride, a weakly alkaline reagent, a strongly alkaline reagent and isopropanol into the mixed solution in sequence for coordination reaction to obtain the millet bran SDF-Cr (III) complex.
In the invention, the weak alkaline reagent is preferably ammonia water, and the mass concentration of the ammonia water is preferably 20-40%, and more preferably 25-35%. In the invention, the ammonia water can adjust the pH value of a mixed solution obtained by mixing the millet bran SDF and water into a weakly alkaline environment. In the invention, under a weakly alkaline environment, the SDF structure of the millet bran contains a large number of C-O-C, C-O-H, O-H groups, and a large number of hydrogen ions can be dissociated from the groups, so that a large number of lone electron groups are formed, and active trivalent chromium ions are subjected to coordination reaction with the lone electron groups.
In the invention, the pH value of the weak alkaline environment is preferably 8-10, and more preferably 8-9. When the pH value of the weakly alkaline environment is adjusted to the above range, the chelating rate can be prevented from being reduced due to an excessively high pH value.
In the present invention, the strongly basic reagent is preferably a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is preferably 2 to 4mol/L, and more preferably 3 to 4 mol/L. In the invention, the strong alkaline reagent can induce the SDF structure of the millet bran to be deprotonated to form a ligand, so that the coordination reaction can be ensured to be carried out.
In the invention, when the concentration of the sodium hydroxide solution is preferably 2-4 mol/L, the ratio of the mass of the millet bran SDF to the volume of the sodium hydroxide solution is preferably 0.5g (20-40) mL, and more preferably 0.5g (30-40) mL. In the invention, when the ratio of the mass of the millet bran SDF to the volume of the sodium hydroxide solution is in the above range, the coordination reaction is promoted.
The source of the isopropyl alcohol is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used. In the present invention, the isopropanol serves as a catalyst and is capable of catalyzing the addition of a complex ion to a lone electron group of an organic compound.
In the invention, the mass of the isopropanol is preferably 5-10% of that of the SDF of the millet bran, and more preferably 6-8%. In the invention, when the mass ratio of the millet bran SDF to the isopropanol is in the range, the catalysis effect of the isopropanol on the coordination reaction can be ensured, and the waste caused by excessive consumption can be prevented.
The source of the chromium chloride is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used. In the present invention, the chromium chloride is preferably chromium chloride hexahydrate. In the invention, the mass ratio of the millet bran SDF to the chromium chloride hexahydrate is preferably 2.0: 1-2.2: 1, and more preferably 2.1: 1-2.2: 1. In the present invention, an excess of chromium chloride may cause a decrease in the reaction pH, disrupting the equilibrium between the SDF molecule and the inorganic chromium (III); when the dosage of the chromium chloride is in the range, the coordination reaction can be carried out fully, and the chelating rate is ensured to be higher.
In the invention, the temperature of the coordination reaction is preferably 60-80 ℃, and more preferably 65-75 ℃; the time of the coordination reaction is preferably 2-4 h, and more preferably 3-4 h. In the present invention, the higher reaction temperature may cause the SDF molecules and chromium ions in the solution system to move too fast to combine with each other, or change the chemical groups between or within the SDF molecules; when the temperature and time of the coordination reaction are within the above ranges, it is more advantageous for the coordination reaction to proceed sufficiently.
In the present invention, the coordination reaction is preferably carried out under stirring. In the present invention, the stirring rate is preferably 500 to 600rmp/min, more preferably 550 to 600 rmp/min. In the present invention, too slow a stirring rate may result in insufficient reaction and insufficient binding of the complex ion to the ligand; too fast a stirring rate will result in high velocity movement of free reactive ions in the reaction system, losing a portion of the enthalpy change capability required for binding. In the present invention, the stirring rate is in the above range, which is more advantageous for the coordination reaction to proceed sufficiently.
After the coordination reaction is finished, the pH value of the system after the coordination reaction is preferably adjusted, dialyzed and dried in sequence to obtain the millet bran SDF-Cr (III) complex.
In the invention, the pH value of the system after the coordination reaction is preferably adjusted to 7.35-7.45. In the invention, because the normal pH value of the human body is 7.35-7.45, when the pH value is more than 7.45, alkalosis occurs; less than 7.35, acidosis; the pH value of the system after the coordination reaction is preferably adjusted to the range, so that the obtained millet bran SDF-Cr (III) complex is suitable for the pH value range of human immune cells.
The reagent used for adjusting the pH of the system after the coordination reaction is not particularly limited, and the pH of the system after the coordination reaction can be adjusted to the above range. In the present invention, the reagent used for adjusting the pH of the system after the coordination reaction is preferably ammonia water. In the present invention, the mass concentration of the ammonia water is preferably 20 to 40%, and more preferably 20 to 30%. In the invention, the ammonia water does not introduce new impurities, and in an alkaline environment, excessive ammonium ions react to generate ammonia water to remove interference factors.
In the present invention, the system after the coordination reaction is preferably cooled to 25 ℃ or lower, more preferably 22 ℃ or lower before the pH is adjusted. In the invention, because the change of temperature has obvious influence on the pH, the system after the coordination reaction is cooled, so that the influence of the temperature change on the pH can be prevented, and the accurate stability in the preparation process is ensured.
After the pH value of the system after the coordination reaction is adjusted to 7.35-7.45, the system obtained after the pH value is adjusted is preferably dialyzed to obtain dialysate. In the present invention, the dialysis bag used for dialysis is preferably a 4000Da dialysis bag.
The present invention is not particularly limited to the operation of the dialysis, and the dialysis method known to those skilled in the art may be used. In the present invention, the dialysis is preferably performed in a manner including: dialyzing the system obtained after the pH value is adjusted for 48 hours by using running tap water, and dialyzing the dialyzate for 24 hours by using deionized water after rotary evaporation and concentration; during the first dialysis, 0.5-1.0 mL of dialysate is taken every 4-6 h, diphenylcarbonyldihydrazide is added, the dialysis is stopped when no mauve is observed, and preferably 0.5mL of dialysate is taken every 4h, and diphenylcarbonyldihydrazide is added for observation; and taking 1-2 mL of dialysate every 4-6 h during the second dialysis, detecting whether free chromium trichloride residues exist in the solution, and preferably taking 1mL of dialysate every 6h for detection.
After the dialysate is obtained, the dialysate is preferably dried by the invention to obtain the millet bran SDF-Cr (III) complex.
In the present invention, the drying is preferably freeze drying, and the operation of the freeze drying is the same as above, and will not be described herein again.
The method comprises the steps of taking millet bran SDF as a raw material of an organic ligand, adjusting the pH value of a mixed solution obtained after the millet bran SDF is mixed with water by using a weakly alkaline reagent to enable the mixed solution to be suitable for an alkaline environment required by subsequent reaction, then adding a strongly alkaline reagent, and inducing the millet bran SDF to deprotonate by using the strongly alkaline reagent to form the organic ligand; the formed organic ligand and chromium chloride are subjected to coordination reaction under the catalytic action of catalyst isopropanol to obtain the millet bran SDF-Cr (III) complex.
The invention provides a millet bran SDF-Cr (III) complex prepared by the preparation method in the technical scheme.
The chelating rate of the millet bran SDF-Cr (III) complex can reach 35.179%, and the content of Cr (III) in the millet bran SDF-Cr (III) complex can reach 4.08 mg/g.
The invention also provides application of the millet bran SDF-Cr (III) complex in the technical scheme in chromium-rich food.
The application method of the millet bran SDF-Cr (III) complex in the chromium-rich food is not particularly limited, and the application method of organic chromium in the chromium-rich food, which is well known to a person skilled in the art, is adopted.
The millet bran SDF-Cr (III) complex provided by the invention combines trivalent chromium with bioactive macromolecules, can improve the physiological activity of water-soluble dietary fiber, and converts inorganic chromium into organic chromium, thereby reducing the toxicity of inorganic chromium, improving the property of inorganic chromium not easy to be absorbed, improving the absorption rate, facilitating the utilization of people and animals, and further being used for chromium-rich food.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the 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.
Example 1
Preparing millet bran SDF:
step 1, performing superfine grinding pretreatment, namely selecting millet bran which does not mildew, drying the millet bran for 12 hours in a drying oven at the temperature of 45 ℃, weighing the millet bran, and controlling the water content to be within 6 percent. After drying, crushing by using an ultrafine crusher for the crushing time: and 20 min. Sieving the pulverized millet bran powder with a 60-mesh sieve. Placing the sieved millet bran powder into a Soxhlet extractor, adding petroleum ether, and distilling and refluxing at 55 ℃ for 3.5h to obtain the degreased millet bran powder.
Step 2, weighing 5.0g of defatted millet bran, adding distilled water according to a feed-to-liquid ratio of 1g to 40mL, adding 250mL of phosphate buffer solution, adjusting the pH value to 6.0, adding 100 mu L of high-temperature-resistant alpha-amylase to the GDE enzyme culture digester, performing water bath culture for 15min, and keeping the sample temperature at 90 ℃. The pH was adjusted to 6.0 and 100uL of neutral protease solution was added and incubated in GDE at 55 ℃ for 25 min. Adjusting pH to 4, adding 100uL amyloglucosidase, culturing in GDE at 55 deg.C for 25min, and detecting with iodine solution until no blue color is formed. Inactivating enzyme (more than 100 ℃ for 10min), transferring the reactant after enzymolysis to a CSF6 filter device, centrifuging the filtrate (4000rpm, 20min), concentrating to 1/4-1/5 of the stock solution, then precipitating with four times of volume of 95% ethanol for 12h (4 ℃), centrifuging at 4000rpm for 20min, and freeze-drying at-108 ℃ for 8h to obtain the SDF, wherein the content of the water-soluble dietary fiber of the processed millet bran reaches 11.49%.
Step 3, accurately weighing 0.5g of millet bran SDF, adding distilled water to dissolve the SDF to 100mL, deproteinizing by adopting a Sevag method according to the proportion of n-butyl alcohol: preparing Sevag reagent with chloroform at a ratio of 1:4, adding the Sevag reagent into 1/4 of the volume of the concentrated solution, shaking for 10min, centrifuging to remove protein layer, and repeating for more than 10 times. Adjusting the pH value of the millet bran SDF solution to about 8 by using ammonia water, adding 45mL of 25% hydrogen peroxide, preserving the temperature at 35 ℃ for 55min, removing organic reagents from the decolorized millet bran SDF solution by using a rotary evaporator, filling the decolorized millet bran SDF solution into 14000Da dialysis bags, dialyzing the decolorized millet bran SDF solution for 48h by using tap water, and dialyzing the decolorized millet bran SDF solution for 24h by using distilled water. Concentrating to a certain volume after dialysis, adding absolute ethyl alcohol to ensure that the alcohol precipitation concentration reaches 80%, carrying out alcohol precipitation in a refrigerator at 4 ℃ for 6h, carrying out centrifugal separation (9000r/min, 5min), sequentially washing precipitates twice by using the absolute ethyl alcohol, acetone and diethyl ether, and carrying out freeze drying (drying temperature is-99 ℃, time is 6h, vacuum degree is 0.095MPa) to constant weight to obtain refined millet bran SDF for later use.
Preparing a millet bran SDF-Cr (III) complex:
(1) mixing 5g of millet bran SDF with 200mL of distilled water (the feed-liquid ratio of the millet bran SDF to the water is 1:40g/mL) to obtain a mixed solution;
(2) and (2) adding a 6mg/mL chromium chloride solution into the mixed solution obtained in the step (1), controlling the mass ratio of the SDF to the chromium chloride of the millet bran to be 1:1, adding ammonia water to enable the pH value to be 8.0, then adding 4mL of 2mol/LNaOH solution, adding 1.5mL of isopropanol, and continuing to react for 2 hours at the temperature of 60 ℃. Cooling, centrifuging (10 min at 5000 rpm), concentrating the supernatant to 1/4, adding 90% ethanol with volume three times of the original volume, precipitating with ethanol at 4 deg.C for 6h, centrifuging, adding a small amount of distilled water into the precipitate, dialyzing, and freeze-drying to obtain the millet bran SDF-Cr (III) complex with chelating rate of 28.436%.
The prepared millet bran SDF-Cr (III) complex is detected by spectrophotometry by dissolving 1.5g of the millet bran SDF-Cr (III) complex in 50mL of distilled water, and the content of the chromium (III) is 2.06 mg/g. (wherein the wavelength in the spectrophotometric measurement was 600 nm.)
Example 2
Millet bran SDF was prepared using the method of example 1.
(1) Mixing 4g of millet bran SDF with 200mL of distilled water (the feed-liquid ratio of the millet bran SDF to the water is 1: 50) to obtain a mixed solution;
(2) and (2) adding a 6mg/mL chromium chloride solution into the mixed solution obtained in the step (1), controlling the mass ratio of the SDF to the chromium chloride of the millet bran to be 2:1, adding ammonia water to enable the pH value to be 9.0, then adding 6mL of a 2mol/LNaOH solution, adding 2mL of isopropanol, and continuing to react for 3 hours at 70 ℃. Cooling, centrifuging (10 min at 5000 rpm), concentrating the supernatant to 1/4, adding 95% ethanol with four times volume, precipitating with ethanol at 16 deg.C for 7h, centrifuging, adding a small amount of distilled water into the precipitate, dialyzing, and freeze-drying to obtain semen Setariae bran SDF-Cr (III) complex with chelating rate of 35.179%.
2.0g of the millet bran SDF-Cr (III) complex is dissolved in 50mL of distilled water, and the prepared millet bran SDF-Cr (III) complex is detected by a spectrophotometry method, so that the content of the chromium (III) is 4.08 mg/g. (wherein the wavelength in the spectrophotometric measurement was 600 nm.)
Example 3
Millet bran SDF was prepared using the method of example 1.
(1) Mixing 3.3g of millet bran SDF with 200mL of distilled water (the feed-liquid ratio of the millet bran SDF to the water is 1:60) to obtain a mixed solution;
(2) and (2) adding a 6mg/mL chromium chloride solution into the mixed solution obtained in the step (1), controlling the mass ratio of the SDF to the chromium chloride of the millet bran to be 2:1, adding ammonia water to adjust the pH value to be 10.0, then adding 8mL of 2mol/LNaOH solution, adding 2mL of isopropanol, and continuing to react for 4 hours at the temperature of 75 ℃. Cooling, centrifuging (10 min at 5000 rpm), concentrating the supernatant to 1/4, adding 99.8% ethanol with four times volume, precipitating with ethanol at 25 deg.C for 8h, centrifuging, adding a small amount of distilled water into the precipitate, dialyzing, and freeze-drying to obtain semen Setariae bran SDF-Cr (III) complex with chelating rate of 29.284%.
2.5g of the millet bran SDF-Cr (III) complex is dissolved in 50mL of distilled water, and the prepared millet bran SDF-Cr (III) complex is detected by a spectrophotometric method, so that the content of the chromium (III) is 1.89 mg/g. (wherein the wavelength in the spectrophotometric measurement was 600 nm.)
From the examples 1 to 3, the millet bran SDF-Cr (III) complex prepared by the method provided by the invention has high chelating rate, can control the content of chromium in organic chromium, and can be used as a raw material of chromium-rich food.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a millet bran SDF-Cr (III) complex comprises the following steps:
(1) mixing the millet bran SDF with water to obtain a mixed solution;
(2) and (2) sequentially adding chromium chloride, a weakly alkaline reagent, a strongly alkaline reagent and isopropanol into the mixed solution obtained in the step (1) for coordination reaction to obtain the millet bran SDF-Cr (III) complex.
2. The method according to claim 1, wherein the testa Tritici SDF obtained in step (1) is obtained by enzymolysis of defatted semen Setariae bran powder.
3. The method according to claim 2, wherein the preparation of the testa Tritici SDF in step (1) comprises the following steps:
(a) mixing the defatted millet bran powder with water to obtain a defatted millet bran powder suspension;
(b) adjusting the pH value of the defatted millet bran powder suspension obtained in the step (a) to 6.0-8.0, and then adding high-temperature-resistant alpha-amylase to perform a first enzymolysis reaction to obtain a first reaction system;
(c) adjusting the pH value of the first reaction system obtained in the step (b) to 6.0-8.0, and then adding neutral protease to perform a second enzymolysis reaction to obtain a second reaction system;
(d) adjusting the pH value of the second reaction system obtained in the step (c) to 4.0-5.0, and then adding amyloglucosidase to perform a third enzymolysis reaction to obtain a third reaction system;
(e) and (d) carrying out post-treatment on the third reaction system obtained in the step (d) to obtain the millet bran SDF.
4. The preparation method according to claim 3, wherein the temperature of the first enzymolysis reaction in the step (b) is 90-100 ℃, and the time of the first enzymolysis reaction is 15-25 min; the temperature of the second enzymolysis reaction in the step (c) and the temperature of the third enzymolysis reaction in the step (d) are respectively 55-65 ℃, and the time of the second enzymolysis reaction in the step (c) and the time of the third enzymolysis reaction in the step (d) are respectively 25-35 min.
5. The preparation method according to claim 1, wherein the mass ratio of the millet bran SDF in the step (1) to the isopropanol to the chromium chloride in the step (2) is 1: (0.05-0.1): (0.45-2).
6. The preparation method according to claim 1, wherein the weakly basic reagent in the step (2) is ammonia water, and the mass concentration of the ammonia water is 20-40%.
7. The method according to claim 1, wherein the strongly basic reagent in the step (2) is a sodium hydroxide solution having a concentration of 2 to 4 mol/L.
8. The preparation method according to claim 1, wherein the temperature of the coordination reaction in the step (2) is 60 to 80 ℃, and the time of the coordination reaction is 2 to 4 hours.
9. The millet bran SDF-Cr (III) complex prepared by the preparation method of any one of claims 1 to 8.
10. Use of the millet bran SDF-cr (iii) complex of claim 9 in chromium-rich food products.
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