CN113981030A - Method for preparing soybean oligopeptide by combining enzyme method with microbial fermentation and application thereof - Google Patents
Method for preparing soybean oligopeptide by combining enzyme method with microbial fermentation and application thereof Download PDFInfo
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- CN113981030A CN113981030A CN202111506751.7A CN202111506751A CN113981030A CN 113981030 A CN113981030 A CN 113981030A CN 202111506751 A CN202111506751 A CN 202111506751A CN 113981030 A CN113981030 A CN 113981030A
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- fermentation
- soybean
- enzymolysis
- powder
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Links
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The invention provides a method for preparing soybean oligopeptide by combining an enzymatic method and microbial fermentation, which comprises the steps of pretreatment of soybean protein isolate, enzymolysis of complex enzyme, microbial fermentation, separation and purification and the like. The disclosure also provides an application of the soybean oligopeptide, namely the soybean oligopeptide chewable tablet can be prepared.
Description
Technical Field
The disclosure relates to the technical field of food processing, in particular to a method for preparing soybean oligopeptide by combining an enzymatic method and microbial fermentation and application thereof.
Background
The soybean protein has high nutritive value, is rich in high-quality protein, and can provide sufficient 8 essential amino acids, vitamins, minerals, etc. required by human body. However, soybean protein has a complex molecular structure, a large relative molecular weight, poor solubility, and a digestion rate and biological value inferior to those of animal proteins such as cow milk. The soybean peptide is an oligopeptide mixture obtained by treating soybean protein through protease action or microbial technology and then carrying out special treatment such as separation and refining, and also comprises free amino acids, water, ash, a small amount of sugar and the like. The soybean peptide is generally a low molecular weight peptide consisting of 3 to 6 amino acids, the protein content is about 85%, the amino acid composition is substantially the same as that of soybean protein, and the essential amino acid content is rich and balanced. The average molecular weight of the soybean peptide is less than 1000 daltons, and the main peak position is 300-700 daltons. The soybean peptide has good biological activity, such as promoting fat metabolism, resisting oxidation, reducing blood fat, resisting thrombosis and the like; the soybean peptide can promote the growth, development and metabolism of microorganisms, can obviously improve the quality and nutritive value of products by adding the soybean peptide into vinegar, soy sauce, cheese and fermented food, enhances the taste and flavor of the products and the like, is used as a new generation of super protein nutrient and multifunctional physiological active substance, and has wide application and market prospect.
The prior art adopts an enzymatic method or a microbial fermentation method to prepare the soybean oligopeptide, and the process for preparing the soybean oligopeptide by adopting a single enzymatic method has the defects of long enzymolysis period, more used chemicals, large generated bitter taste, poor dissolving property, low product yield, expensive membrane separation equipment needing investment, low treatment efficiency and high maintenance cost. The microbial fermentation method has the problems of long fermentation period, severe fermentation conditions, low strain efficiency, limited productivity, high cost and the like.
Disclosure of Invention
In view of the problems of the conventional methods for preparing soybean oligopeptide, the present disclosure provides a method for preparing soybean oligopeptide by combining an enzymatic method and microbial fermentation, comprising:
step one, pretreatment of isolated soy protein: the soybean protein isolate is used as a raw material, and the ratio of the soybean protein isolate to the soybean protein isolate is 1: 8-1: adding purified water into the mixture at a material-liquid ratio (kg: L) of 10(w/v), and shearing the mixture at normal temperature for 30-60 min at a shearing speed of 600-1000 r/min; obtaining protein homogenate;
step two, compound enzyme enzymolysis: dropwise adding alkali liquor into the protein homogenate to adjust the pH of the system to 8.0-8.5, continuously enriching oligopeptide containing carboxyl groups under the condition of maintaining the pH value to be 8.0-8.5, positively improving the hydrolysis degree of the soybean protein isolate, and performing gradient enzymolysis by adopting a complex enzyme A and a complex enzyme B: firstly, adding a complex enzyme A, and performing enzymolysis for 2-3 hours at an enzymolysis temperature of 50-60 ℃ by controlling the pH of a system to be 8.0-8.5; adding the complex enzyme B, performing enzymolysis for 1-2 hours, performing high-temperature sterilization, and then cooling to 30-38 ℃; obtaining complex enzyme enzymolysis liquid;
step three, microbial fermentation: controlling the fermentation temperature to be 30-38 ℃; adding a carbon source and an inorganic salt ammonium chloride buffer system into the complex enzyme enzymolysis liquid, adding active dry yeast for fermentation and fermentation, wherein the adding mass of the active dry yeast is 0.05-0.4% (w/v) of the volume of fermentation liquid, fermenting for 3-5 h, and controlling the alcoholic strength to be 1-3 (% Vol); adding the acetobacter orientalis for fermentation, wherein the adding mass of the acetobacter orientalis is 0.1-1.0% (w/v) of the volume of the fermentation liquid, and fermenting for 1-3 hours, and controlling the amount of acetic acid produced to be 5-20 g/L; then adding bacillus subtilis and lactobacillus plantarum for fermentation, wherein the adding mass of the bacillus subtilis and the lactobacillus plantarum is 0.1-0.8% (w/v) of the volume of the fermentation liquor, and fermenting for 4-8 hours; acid production enrichment of the acetobacter orientalis, and stopping fermentation when the pH value of a fermentation system is 4.4-4.8;
step four, separation and purification: sterilizing after fermentation is finished, enriching acid produced by metabolism of acetobacter orientalis to denature and precipitate dispersed protein, separating by a centrifuge at 2000-4000 r/min to obtain fermented clear liquid and solid residues, sterilizing the solid residues at high temperature, and freeze-drying to obtain soybean fermented powder; and (3) carrying out processes of activated carbon decoloration and deodorization, plate-and-frame filtration, nanofiltration desalination and concentration, UHT sterilization and spray drying on the fermentation clear liquid to obtain the soybean oligopeptide.
In a preferable embodiment, in the second step, the addition amount of the complex enzyme A is 0.8-2.0% (w/w) of the mass of the isolated soy protein powder, and the addition amount of the complex enzyme B is 0.3-1.0% (w/w) of the mass of the isolated soy protein powder.
In a preferred embodiment of the second step, the complex enzyme a or the complex enzyme B is a complex enzyme of one of alkaline protease Alcalase 2.4L, pancreatin, collagenase, neutral protease, papain, and flavourzyme, and pectinase, and the compounding ratio is 5: 1.
as a preferable mode of the embodiment, in the third step, the mass component ratio of bacillus subtilis to lactobacillus plantarum is 2: 1.
the present disclosure also provides a composition, comprising the following components in parts by weight: 25-35 parts of soybean fermented powder, 15-25 parts of polyfructose, 15-25 parts of mulberry fermented powder, 10-15 parts of beta-cyclodextrin, 10-15 parts of maltodextrin, 5-10 parts of inulin and 1.5-2 parts of vitamin C.
The present disclosure also provides another composition, which comprises the following components in parts by weight: 25-35 parts of soybean oligopeptide, 15-20 parts of sorbitol, 15-20 parts of isomaltooligosaccharide, 10-15 parts of globin powder, 10-15 parts of oat, 1-5 parts of magnesium stearate and 1-5 parts of Na-CMC.
The method utilizes the mixed enzyme generated by the metabolic activity of the microorganism to catalyze the generation of bioactive peptide, the microorganism can improve the growth and enzyme production capacity by means of polypeptide hydrolysate, simultaneously, substances generated in the metabolic process of the microorganism have different modification functions in the processing process of raw materials, have certain synergistic effect and higher production efficiency, and the released small peptide is modified, transferred and recombined certain bitter groups through grafting and rearrangement and the microbial effect, so that the obtained soybean peptide has better solubility and no bitter taste, and the application value of the soybean peptide is enhanced.
After adopting above-mentioned technical scheme, this disclosure has following beneficial effect:
1. the soybean protein isolate is subjected to enzymolysis in an alkaline protease and neutral protease gradient composite system, oligopeptide containing carboxyl groups is continuously enriched under the condition of maintaining the pH value of 8.0-8.5, the hydrolysis degree of the soybean protein isolate is positively improved, and the hydrolysis degree of the soybean protein isolate is secondarily improved and the yield of a soybean oligopeptide product is greatly improved by utilizing the efficient synergistic fermentation of mixed floras of acetobacter orientalis, active dry yeast, bacillus subtilis and lactobacillus plantarum.
2. The method relies on the acid production effect of the enrichment of the acetic acid bacteria oriental, so that soybean protein isolate which is difficult to degrade generates flocculation precipitation under the condition that the pH value is 4.4-4.8, the soybean protein isolate has good dispersibility, the emulsification phenomenon of soybean fermentation liquor is easy to cause, the clarification treatment is difficult, the separation of production materials is difficult, the means such as chemical flocculation agents, clarifiers, ceramic membrane equipment and the like are needed, the acid production characteristic of the acetic acid bacteria oriental is used for further efficiently solving the clarification separation problem of the fermentation liquor, the energy consumption and the cost are reduced, and the edible safety of products is ensured.
3. The microbial fermentation disclosed by the invention is used for modifying, transferring and recombining bitter groups of soybean oligopeptide, and the characteristic modification has no bitter taste, good solubility and stability under strong acidity.
4. The soybean oligopeptide is added with the globin powder to inhibit the activity of the pancreatic lipase, so that the absorption of fat by small intestines is reduced, the health-care effect of reducing cholesterol is achieved, the taste is improved by isomaltooligosaccharide, and the magnesium stearate and Na-CMC are added for tabletting, so that the superior hardness of the product is ensured, and the biological activity function and the taste of the soybean oligopeptide are improved further.
5. The method improves the high added value of the isolated soy protein, comprehensively utilizes processing byproducts, does not discharge production waste, and realizes a high-yield, green, environment-friendly and pollution-free co-production method for preparing the soy baking powder and the soy oligopeptide product.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the disclosure and together with the description serve to explain the principles of the disclosure.
Fig. 1 is a process flow diagram of the present disclosure.
Fig. 2 is a graph of yield results for soy oligopeptides of the present disclosure.
Detailed Description
The present disclosure will be described in further detail with reference to the drawings and embodiments. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limitations of the present disclosure. It should be further noted that, for the convenience of description, only the portions relevant to the present disclosure are shown in the drawings.
It should be noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
As shown in fig. 1, the present disclosure provides a method for preparing soybean oligopeptide by combining an enzymatic method with microbial fermentation, comprising:
step one, pretreatment of isolated soy protein: the soybean protein isolate is used as a raw material, and the ratio of the soybean protein isolate to the soybean protein isolate is 1: 8-1: adding purified water at a material-liquid ratio of 10(w/v), and shearing at the normal temperature for 30-60 min at a shearing speed of 600-1000 r/min; obtaining protein homogenate;
step two, compound enzyme enzymolysis: dropwise adding alkali liquor into the protein homogenate liquid to adjust the pH of the system to 8.0-8.5, and performing gradient enzymolysis by adopting a complex enzyme A and a complex enzyme B: firstly, adding a complex enzyme A, and performing enzymolysis for 2-3 hours at an enzymolysis temperature of 50-60 ℃ by controlling the pH of a system to be 8.0-8.5; adding the complex enzyme B, performing enzymolysis for 1-2 hours, performing high-temperature sterilization, and then cooling to 30-38 ℃; obtaining complex enzyme enzymolysis liquid;
in a preferable embodiment, in the second step, the addition amount of the complex enzyme A is 0.8-2.0% (w/w) of the mass of the isolated soy protein powder, and the addition amount of the complex enzyme B is 0.3-1.0% (w/w) of the mass of the isolated soy protein powder.
In a preferred embodiment of the second step, the complex enzyme a or the complex enzyme B is a complex enzyme of one of alkaline protease Alcalase 2.4L, pancreatin, collagenase, neutral protease, papain, and flavourzyme, and pectinase, and the compounding ratio is 5: 1.
step three, microbial fermentation: controlling the fermentation temperature to be 30-38 ℃; adding a carbon source and an inorganic salt ammonium chloride buffer system into the complex enzyme enzymolysis liquid, adding active dry yeast for fermentation and fermentation, wherein the adding mass of the active dry yeast is 0.05-0.4% (w/v) of the volume of fermentation liquid, fermenting for 3-5 h, and controlling the alcoholic strength to be 1-3 (% Vol); adding the acetobacter orientalis for fermentation, wherein the adding mass of the acetobacter orientalis is 0.1-1.0% (w/v) of the volume of the fermentation liquid, and fermenting for 1-3 hours, and controlling the amount of acetic acid produced to be 5-20 g/L; then adding bacillus subtilis and lactobacillus plantarum for fermentation, wherein the adding mass of the bacillus subtilis and the lactobacillus plantarum is 0.1-0.8% (w/v) of the volume of the fermentation liquor, and fermenting for 4-8 hours; acid production enrichment of the acetobacter orientalis, and stopping fermentation when the pH value of a fermentation system is 4.4-4.8;
according to the method, the high-efficiency synergistic fermentation is carried out by utilizing the mixed flora of the acetobacter orientalis, the active dry yeast, the bacillus subtilis and the lactobacillus plantarum, the active dry yeast utilizes the sugar source to metabolize to generate ethanol, the alcohol-resistant acetobacter orientalis utilizes the ethanol to metabolize to generate organic acid, and then the bacillus subtilis and the lactobacillus plantarum improve the growth and endogenous protease production capacity by virtue of polypeptide hydrolysate, so that the synergistic effect is achieved, and the product yield can be improved. Depending on the acid production benefit of the oriental acetic acid bacteria enrichment, soybean protein isolate which is difficult to degrade generates flocculation precipitation under the condition that the pH value is 4.4-4.8, the soybean protein isolate has good dispersibility, the emulsification phenomenon of soybean fermentation liquor is easy to cause, the clarification treatment is difficult, the separation of production materials is difficult, the acid production characteristic of the oriental acetic acid bacteria is required to be assisted by means of chemical flocculation agents, clarifiers, ceramic membrane equipment and the like, the problem of clarification separation of the fermentation liquor is solved efficiently, the energy consumption and the cost are reduced, and the edible safety of products is ensured. Modifying, transferring and recombining bitter groups of soybean oligopeptide through microbial action, wherein the characteristic modification has no bitter taste, good solubility and stable acidity.
As a preferable mode of the embodiment, in the third step, the mass component ratio of bacillus subtilis to lactobacillus plantarum is 2: 1.
step four, separation and purification: sterilizing after fermentation is finished, enriching acid produced by metabolism of acetobacter orientalis to denature and precipitate dispersed protein, separating by a centrifuge at 2000-4000 r/min to obtain fermented clear liquid and solid residues, sterilizing the solid residues at high temperature, and freeze-drying to obtain soybean fermented powder; and (3) carrying out processes of activated carbon decoloration and deodorization, plate-and-frame filtration, nanofiltration desalination and concentration, UHT sterilization and spray drying on the fermentation clear liquid to obtain the soybean oligopeptide.
Specifically, the solid residues are made into soybean fermentation powder through high-temperature sterilization and freeze-drying technology, and the soybean fermentation powder is uniformly mixed through a square cone mixer, wherein the frequency is 20-30 Hz, and the time is 30-60 minutes. Adding activated carbon into the fermentation clear liquid for decolorization and deodorization according to 0.08-0.15% (w/v) of the fermentation liquid, wherein the temperature is 45-50 ℃, the time is 1-1.5 h, filling white diatomite and red diatomite into the plate frame uniformly according to 0.1-0.3% (w/v) of the fermentation liquid, and the weight ratio of the white clay to the red diatomite is as follows: red earth 1: 1, after filtering and clarifying, utilizing a nanofiltration membrane DK200, wherein the specification is a SUEZ roll type organic membrane, the molecular weight is 200Da, the pressure before controlling the membrane is 0.2-0.3 MPa, the pressure after controlling the membrane is 0.2-0.3 MPa, the initial concentration is 4-5% Brix, desalting and concentrating are carried out to 8-10% Brix, then adding pure water with the volume 0.2-0.5 times of that of fermentation liquor, carrying out water replenishing and desalting for 2-3 times on average, controlling the end point material to be 10% Brix, controlling the conductivity end point to be 6-8 ms/cm, feeding to full-automatic UHT sterilization equipment, and the sterilization parameters are as follows: 105-110 ℃ for 2-3 min; conveying the sterilized materials to a spray drying tower, wherein the drying parameters are as follows: and (3) the screw pump is 5-10 Hz, the air inlet temperature is 180-190 ℃, the air outlet temperature is 90-95 ℃, and the soybean oligopeptide powder with the water content lower than 7% is obtained.
The present disclosure also provides a composition, comprising the following components in parts by weight: 25-35 parts of soybean fermented powder, 15-25 parts of polyfructose, 15-25 parts of mulberry fermented powder, 10-15 parts of beta-cyclodextrin, 10-15 parts of maltodextrin, 5-10 parts of inulin and 1.5-2 parts of vitamin C.
The present disclosure also provides another composition, which comprises the following components in parts by weight: 25-35 parts of soybean oligopeptide, 15-20 parts of sorbitol, 15-20 parts of isomaltooligosaccharide, 10-15 parts of globin powder, 10-15 parts of oat, 1-5 parts of magnesium stearate and 1-5 parts of Na-CMC.
Example 1
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; and (2) dropwise adding 20% sodium hydroxide solution into the soybean protein homogenate to adjust the pH value of the system to be 8.5, performing gradient enzymolysis by adopting alkaline protease Alcalase 2.4L and neutral protease, wherein the alkaline protease Alcalase 2.4L is 16g, the pectinase is 3.2 g, the pH value of the system is maintained at 8.0-8.5, the enzymolysis temperature is 55 ℃, performing enzymolysis for 3 hours, adding neutral protease 5g and pectinase 1g into the natural pH value, performing enzymolysis for 1 hour, performing high-temperature enzyme deactivation at 90 ℃, and sterilizing for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multi-stage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 4h, controlling the alcoholic strength to be 3 (% Vol), adding 20g of acetobacter orientalis, fermenting for 2h, producing 10g/L of acetic acid, adding 20g of bacillus subtilis and 10g of lactobacillus plantarum respectively, fermenting for 6h, performing combined fermentation by using the acetobacter orientalis and the compound bacteria thereof which highly produce the acid, controlling the fermentation temperature to be 35 ℃, and fermenting for 12 h; enriching the acid produced by the acetic acid bacteria, stopping fermentation when the pH of a fermentation system is 4.5, performing denaturation precipitation on the dispersed protein by enriching the acid produced by the metabolism of the acetic acid bacteria, heating to 90 ℃, inactivating enzyme and sterilizing for 30 minutes; through pressing from both sides cover rapid cooling to normal atmospheric temperature, utilize disc centrifuge separation 3000r/min, separate out fermented clear liquid and solid sediment, the soybean baking powder is made into through high temperature sterilization to the solid sediment, freeze-drying technique, mixes through the square cone blendor, frequency 30Hz, time 30 minutes, and granule medicine to be taken after being mixed with boiling water product is according to the component content of part by weight: 30 parts of soybean fermented powder, 20 parts of polyfructose, 20 parts of mulberry fermented powder, 10 parts of beta-cyclodextrin, 10 parts of maltodextrin, 8 parts of inulin and 2 parts of vitamin C; adding 15g of HPM-05 type activated carbon of 100 meshes into clear liquid for decolorization and deodorization, adding 15g of white diatomite and red diatomite respectively after filling the plate frame, uniformly filling, filtering and clarifying, utilizing a nanofiltration membrane DK200 with the specification of a SUEZ roll type organic membrane with the molecular weight of 200Da, controlling the pressure before membrane control to be 0.25MPa, controlling the pressure after membrane to be 0.25MPa and the initial concentration to be 4% Brix, desalting and concentrating to be 10% Brix, adding 5kg of pure water, performing water replenishing and desalting for 2 times on average, controlling the end point material to be 10% Brix, controlling the conductivity end point to be 6.5ms/cm, feeding to a full-automatic UHT sterilization device, wherein the sterilization parameters are as follows: 110 ℃ for 3 min; conveying the sterilized materials to a spray drying tower, wherein the drying parameters are as follows: the screw pump is 5Hz, the air inlet temperature is 185 ℃, the air outlet temperature is 95 ℃, and the soybean oligopeptide powder with the water content of 4.8 percent is obtained; the chewable tablet product comprises the following components in parts by weight: 30 parts of soybean oligopeptide, 20 parts of sorbitol, 15 parts of isomaltooligosaccharide, 15 parts of globin powder, 10 parts of oat, 5 parts of magnesium stearate and 5 parts of Na-CMC (sodium carboxymethylcellulose) added in a proper amount.
Example 2
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; and (2) dropwise adding 20% sodium hydroxide solution into the soybean protein homogenate to adjust the pH value of the system to be 8.5, adding 15g of collagenase and 3 g of pectinase for enzymolysis, controlling the pH value of the system to be 8.0-8.5, carrying out enzymolysis at 55 ℃ for 3 hours, adding 6g of papain and 1.2g of pectinase into the natural pH value, carrying out enzymolysis for 1 hour, carrying out high-temperature enzyme deactivation at 90 ℃ and sterilizing for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multi-stage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 2h, controlling the alcoholic strength to be 1 (% Vol), adding 20g of acetobacter orientalis, fermenting for 4h, producing 10g/L of acetic acid, respectively adding 20g of bacillus subtilis and 10g of plant lactic acid bacteria, fermenting for 10h, performing combined fermentation by using the acetobacter orientalis and the compound bacteria thereof which highly produce the acid, controlling the fermentation temperature to be 35 ℃, and fermenting for 15 h; acid production enrichment of the acetic acid bacteria, namely the pH value of a fermentation system is 4.5, ending the fermentation, denaturing and precipitating the dispersed protein through the enrichment of acid production by the metabolism of the acetic acid bacteria, heating to 90 ℃, inactivating enzyme and sterilizing for 30 minutes; rapidly cooling to normal temperature through a jacket, separating 3000r/min by using a disc centrifuge, separating fermented clear liquid and solid residues, preparing soybean fermented powder from the solid residues through high-temperature sterilization and freeze-drying technology, and uniformly mixing the soybean fermented powder by using a square cone mixer at the frequency of 30Hz for 30 minutes; the granule product comprises the following components in parts by weight: 25 parts of soybean fermented powder, 20 parts of polyfructose, 20 parts of mulberry fermented powder, 12 parts of beta-cyclodextrin, 12 parts of maltodextrin, 9 parts of inulin and 2 parts of vitamin C; the chewable tablet product comprises the following components in parts by weight: 25 parts of soybean oligopeptide, 22 parts of sorbitol, 18 parts of isomaltooligosaccharide, 15 parts of globin powder, 10 parts of oat, 5 parts of magnesium stearate and 5 parts of proper amount of added Na-CMC;
example 3:
a method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; and (2) dropwise adding 20% sodium hydroxide solution into the soybean protein homogenate to adjust the pH value of the system to be 8.5, adding 15g of pancreatin and 3 g of pectinase for enzymolysis, controlling the pH value of the system to be 8.0-8.5, carrying out enzymolysis at 55 ℃ for 3 hours, adding 6g of flavourzyme and 1.2g of pectinase into the natural pH value, carrying out enzymolysis for 1 hour, carrying out high-temperature enzyme deactivation at 90 ℃ and sterilizing for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multistage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 6 hours, controlling the alcoholic strength to be 4 (% Vol), adding 20g of acetobacter orientalis, fermenting for 1.5 hours, producing 10g/L of acetic acid, respectively adding 20g of bacillus subtilis and 10g of plant lactic acid bacteria, fermenting for 4 hours, performing combined fermentation by using the acetobacter orientalis and the compound bacteria thereof which highly produce the acid, controlling the fermentation temperature to be 35 ℃, and fermenting for 11.5 hours; enriching the acid produced by the acetic acid bacteria, stopping fermentation when the pH of a fermentation system is 4.5, performing denaturation precipitation on the dispersed protein by enriching the acid produced by the metabolism of the acetic acid bacteria, heating to 90 ℃, inactivating enzyme and sterilizing for 30 minutes; rapidly cooling to normal temperature through a jacket, separating 3000r/min by using a disc centrifuge, separating fermented clear liquid and solid residues, preparing soybean fermented powder from the solid residues through high-temperature sterilization and freeze-drying technology, and uniformly mixing the soybean fermented powder by using a square cone mixer at the frequency of 30Hz for 30 minutes; the granule product comprises the following components in parts by weight: 22 parts of soybean fermented powder, 20 parts of polyfructose, 20 parts of mulberry fermented powder, 15 parts of beta-cyclodextrin, 12 parts of maltodextrin, 9 parts of inulin and 2 parts of vitamin C; the chewable tablet product comprises the following components in parts by weight: 32 parts of soybean oligopeptide, 20 parts of sorbitol, 13 parts of isomaltooligosaccharide, 15 parts of globin powder, 10 parts of oat, 5 parts of magnesium stearate and 5 parts of proper amount of added Na-CMC.
Example 4
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; adding 20% sodium hydroxide solution dropwise into the soybean protein homogenate to adjust the pH value of the system to 8.5, adding 2.4L 16g of alkaline protease Alcalase and 3.2 g of pectinase for enzymolysis, controlling the pH value of the system to be 8.0-8.5, carrying out enzymolysis at 55 ℃ for 3 hours, adding 5g of neutral protease and 1g of pectinase at natural pH, carrying out enzymolysis for 1 hour, and carrying out high-temperature enzyme deactivation and sterilization at 90 ℃ for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multi-stage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 4h, controlling the alcoholic strength to be 3 (% Vol), respectively adding 20g of bacillus subtilis and 10g of plant lactic acid bacteria, fermenting for 8h, controlling the fermentation temperature to be 35 ℃, and fermenting for 12 hours; stopping fermentation, and causing serious emulsification phenomenon in a fermentation system; high-speed centrifugation and plate-frame filtration cannot be clarified by means of chemical agents or membrane filtration equipment.
Example 5
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; adding 20% sodium hydroxide solution dropwise into the soybean protein homogenate to adjust the pH value of the system to 8.5, adding 2.4L 16g of alkaline protease Alcalase and 3.2 g of pectinase for enzymolysis, controlling the pH value of the system to be 8.0-8.5, carrying out enzymolysis at 55 ℃ for 3 hours, adding 5g of neutral protease and 1g of pectinase at natural pH, carrying out enzymolysis for 1 hour, and carrying out high-temperature enzyme deactivation and sterilization at 90 ℃ for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multi-stage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 4h, controlling the alcoholic strength to be 3 (% Vol), adding 20g of acetobacter orientalis, fermenting for 1.5h, producing 10g/L of acetic acid, fermenting for 8h, controlling the fermentation temperature to be 35 ℃, and fermenting for 12 h; and (3) stopping fermentation, wherein the pH value of a fermentation system is 4.5, flocculation occurs, the fermentation is stopped, and the soybean fermented powder is separated by a disc centrifuge, so that the yield of the soybean fermented powder is improved by 10 percent and the yield of the soybean oligopeptide product is reduced by 15 percent.
Comparative example 1
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; and (3) inactivating enzyme and sterilizing for 30 minutes at a high temperature of 90 ℃ in the soybean protein homogenate. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; transferring to a multi-stage temperature-controlled fermentation tank, adding 1kg of carbon source white granulated sugar, adding 50g of buffer system inorganic salt ammonium chloride, sequentially adding 15g of active dry yeast, fermenting for 6h, controlling the alcoholic strength to be 3 (% Vol), adding 20g of acetobacter orientalis, fermenting for 4h, producing 10g/L of acetic acid, adding 20g of bacillus subtilis and 10g of lactobacillus plantarum respectively, fermenting for 10h, performing combined fermentation by using the acetobacter orientalis and the compound bacteria thereof which highly produce the acid, controlling the fermentation temperature to be 35 ℃, and fermenting for 18 h; enriching the acid produced by the acetic acid bacteria, stopping fermentation when the pH of a fermentation system is 4.5, performing denaturation precipitation on the dispersed protein by enriching the acid produced by the metabolism of the acetic acid bacteria, and heating to 90 ℃ for high-temperature sterilization for 30 minutes; through pressing from both sides cover rapid cooling to normal atmospheric temperature, utilize disc centrifuge separation 3000r/min, separate out fermented clear liquid and solid sediment, the soybean baking powder is made into through high temperature sterilization to the solid sediment, freeze-drying technique, mixes through the square cone blendor, frequency 30Hz, time 30 minutes, and granule medicine to be taken after being mixed with boiling water product is according to the component content of part by weight: 30 parts of soybean fermented powder, 20 parts of polyfructose, 20 parts of mulberry fermented powder, 10 parts of beta-cyclodextrin, 10 parts of maltodextrin, 8 parts of inulin and 2 parts of vitamin C; adding 15g of HPM-05 type activated carbon of 100 meshes into clear liquid for decolorization and deodorization, adding 15g of white diatomite and red diatomite respectively after filling the plate frame, uniformly filling, filtering and clarifying, utilizing a nanofiltration membrane DK200 with the specification of a SUEZ roll type organic membrane with the molecular weight of 200Da, controlling the pressure before membrane control to be 0.25MPa, controlling the pressure after membrane to be 0.25MPa and the initial concentration to be 4% Brix, desalting and concentrating to be 10% Brix, adding 5kg of pure water, performing water replenishing and desalting for 2 times on average, controlling the end point material to be 10% Brix, controlling the conductivity end point to be 6.5ms/cm, feeding to a full-automatic UHT sterilization device, wherein the sterilization parameters are as follows: 110 ℃ for 3 min; conveying the sterilized materials to a spray drying tower, wherein the drying parameters are as follows: the screw pump is 5Hz, the air inlet temperature is 185 ℃, the air outlet temperature is 95 ℃, and the soybean oligopeptide powder with the water content of 4.8 percent is obtained; the chewable tablet product comprises the following components in parts by weight: 30 parts of soybean oligopeptide, 20 parts of sorbitol, 15 parts of isomaltooligosaccharide, 15 parts of globin powder, 10 parts of oat, 5 parts of magnesium stearate and 5 parts of Na-CMC (sodium carboxymethylcellulose) added in a proper amount.
Comparative example 2
A method for preparing soybean oligopeptide by enzyme method combined with microbial fermentation comprises taking soybean protein isolate powder as main raw material, adding 9kg of purified water and 1kg of soybean protein isolate into a shearing tank, and rapidly shearing at normal temperature for 30min at a shearing speed of 800 r/min; and (2) dropwise adding 20% sodium hydroxide solution into the soybean protein homogenate to adjust the pH value of the system to be 8.5, performing gradient enzymolysis by adopting alkaline protease Alcalase 2.4L and neutral protease, wherein the alkaline protease Alcalase 2.4L is 16g, the pectinase is 3.2 g, the pH value of the system is maintained at 8.0-8.5, the enzymolysis temperature is 55 ℃, performing enzymolysis for 3 hours, adding neutral protease 5g and pectinase 1g into the natural pH value, performing enzymolysis for 1 hour, and performing high-temperature enzyme deactivation at 90 ℃ for 30 minutes. Rapidly cooling to 35 ℃ by adopting plate type heat exchange; separating with a disc centrifuge at 3000r/min to obtain clear enzymolysis liquid and solid residue with poor separation effect; adding 15g of HPM-05 type activated carbon of 100 meshes into clear liquid for decolorization and deodorization, adding 15g of white diatomite and red diatomite respectively after filling the plate frame, uniformly filling, filtering and clarifying, utilizing a nanofiltration membrane DK200 with the specification of a SUEZ roll type organic membrane with the molecular weight of 200Da, controlling the pressure before membrane control to be 0.25MPa, controlling the pressure after membrane to be 0.25MPa and the initial concentration to be 4% Brix, desalting and concentrating to be 10% Brix, adding 5kg of pure water, performing water replenishing and desalting for 2 times on average, controlling the end point material to be 10% Brix, controlling the conductivity end point to be 6.5ms/cm, feeding to a full-automatic UHT sterilization device, wherein the sterilization parameters are as follows: 110 ℃ for 3 min; conveying the sterilized materials to a spray drying tower, wherein the drying parameters are as follows: the screw pump is 5Hz, the air inlet temperature is 185 ℃, the air outlet temperature is 95 ℃, and the soybean oligopeptide powder with the water content of 4.8 percent is obtained; the chewable tablet product comprises the following components in parts by weight: 30 parts of soybean oligopeptide, 20 parts of sorbitol, 15 parts of isomaltooligosaccharide, 15 parts of globin powder, 10 parts of oat, 5 parts of magnesium stearate and 5 parts of Na-CMC (sodium carboxymethylcellulose) added in a proper amount.
TABLE 1 Soybean oligopeptide yields in examples 1-5 and comparative examples 1-2 of the present disclosure
| Item | Yield/% |
| Example 1 | 80 |
| Example 2 | 72 |
| Example 3 | 68 |
| Example 4 | 45 |
| Example 5 | 65 |
| Comparative example 1 | 35 |
| Comparative example 2 | 40 |
The yields of soybean oligopeptide of examples 1-5 and comparative examples 1-2 are shown in fig. 2 and table 1, the yields of soybean oligopeptide of examples 1-3 are between 68% and 80% by adding mixed bacteria of Acetobacter orientalis, active dry yeast, Bacillus subtilis and Lactobacillus plantarum, while the yields of soybean oligopeptide of examples 4 are 45% by adding only active dry yeast, Bacillus subtilis and Lactobacillus plantarum, and the yields of soybean oligopeptide of examples 5 are 65% by adding Acetobacter orientalis and active dry yeast, which indicates that Acetobacter orientalis has an obvious effect on improving the yields of soybean oligopeptide, the comparative example 1 directly adopts microbial fermentation to find that the fermentation period is long, the yield of soybean oligopeptide is 35%, it is possible that the utilization rate of macromolecular protein to microorganism is low, and the case 2 directly adopts biological protease to degrade, the yield is 40 percent, is higher than that of a comparative example 6 and is lower than that of an embodiment 4, which shows that the soybean oligopeptide needs the combined fermentation of protease and microorganism, and the yield of the product can be obviously improved.
In the description herein, reference to the description of the terms "one embodiment/mode," "some embodiments/modes," "example," "specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment/mode or example is included in at least one embodiment/mode or example of the application. In this specification, the schematic representations of the terms used above are not necessarily intended to be the same embodiment/mode or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments/modes or examples. Furthermore, the various embodiments/aspects or examples and features of the various embodiments/aspects or examples described in this specification can be combined and combined by one skilled in the art without conflicting therewith.
It will be understood by those skilled in the art that the foregoing embodiments are merely for clarity of illustration of the disclosure and are not intended to limit the scope of the disclosure. Other variations or modifications may occur to those skilled in the art, based on the foregoing disclosure, and are still within the scope of the present disclosure.
Claims (6)
1. A method for preparing soybean oligopeptide by combining an enzymatic method and microbial fermentation is characterized by comprising the following steps:
step one, pretreatment of isolated soy protein: the method is characterized in that soybean protein isolate powder is used as a raw material, and the mass volume ratio of the soybean protein isolate is 1: 8-1: 10 adding purified water, and shearing at normal temperature for 30-60 min at the shearing speed of 600-1000 r/min; obtaining protein homogenate;
step two, compound enzyme enzymolysis: dropwise adding alkali liquor into the protein homogenate liquid to adjust the pH of the system to 8.0-8.5, and performing gradient enzymolysis by adopting a complex enzyme A and a complex enzyme B: firstly, adding a complex enzyme A, and performing enzymolysis for 2-3 hours at an enzymolysis temperature of 50-60 ℃ by controlling the pH of a system to be 8.0-8.5; adding the complex enzyme B, performing enzymolysis for 1-2 hours, performing high-temperature sterilization, and then cooling to 30-38 ℃; obtaining complex enzyme enzymolysis liquid;
step three, microbial fermentation: controlling the fermentation temperature to be 30-38 ℃; adding a carbon source and an inorganic salt ammonium chloride buffer system into the complex enzyme enzymolysis liquid, adding active dry yeast for fermentation and fermentation, wherein the adding mass of the active dry yeast is 0.05-0.4% of the volume of fermentation liquid, fermenting for 3-5 h, and controlling the alcoholic strength to be 1-3% Vol; adding the acetobacter orientalis for fermentation, wherein the adding mass of the acetobacter orientalis is 0.1-1.0% of the volume of the fermentation liquid, and fermenting for 1-3 hours, and controlling the amount of acetic acid produced to be 5-20 g/L; then adding bacillus subtilis and lactobacillus plantarum for fermentation, wherein the adding mass of the bacillus subtilis and the lactobacillus plantarum is 0.1-0.8% of the volume of the fermentation liquor, and the fermentation lasts for 4-8 h; acid production enrichment of the acetobacter orientalis, and stopping fermentation when the pH value of a fermentation system is 4.4-4.8;
step four, separation and purification: sterilizing after fermentation is finished, enriching acid produced by metabolism of acetobacter orientalis to denature and precipitate dispersed protein, separating by a centrifuge at 2000-4000 r/min to obtain fermented clear liquid and solid residues, sterilizing the solid residues at high temperature, and freeze-drying to obtain soybean fermented powder; and (3) carrying out processes of activated carbon decoloration and deodorization, plate-and-frame filtration, nanofiltration desalination and concentration, UHT sterilization and spray drying on the fermentation clear liquid to obtain the soybean oligopeptide.
2. The method as claimed in claim 1, wherein in the second step, the addition amount of the complex enzyme A is 0.8-2.0% of the mass of the soybean protein isolate powder, and the addition amount of the complex enzyme B is 0.3-1.0% of the mass of the soybean protein isolate powder.
3. The method according to claim 1, wherein in the second step, the compound enzyme A or the compound enzyme B is a compound enzyme of pectase and any one of alkaline protease Alcalase 2.4L, pancreatin, collagenase, neutral protease, papain and flavourzyme, and the compounding ratio is 5: 1.
4. the method of claim 1, wherein in the third step, the mass component ratio of the bacillus subtilis to the lactobacillus plantarum is 2: 1.
5. the composition is characterized by comprising the following components in parts by weight: the soybean fermented powder according to any one of claims 1 to 4, which comprises 25 to 35 parts of soybean fermented powder, 15 to 25 parts of polyfructose, 15 to 25 parts of mulberry fermented powder, 10 to 15 parts of beta-cyclodextrin, 10 to 15 parts of maltodextrin, 5 to 10 parts of inulin and 1.5 to 2 parts of vitamin C.
6. The composition is characterized by comprising the following components in parts by weight: the soybean oligopeptide of any one of claims 1 to 4, 25 to 35 parts of sorbitol, 15 to 20 parts of isomaltooligosaccharide, 10 to 15 parts of globin powder, 10 to 15 parts of oat, 1 to 5 parts of magnesium stearate, and 1 to 5 parts of Na-CMC.
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| CN100569951C (en) * | 2006-02-08 | 2009-12-16 | 沈阳农业大学 | Process for producing functional soybean peptide by two-step method |
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