CN111926049A - Soybean meal antibacterial peptide and preparation method and application thereof - Google Patents
Soybean meal antibacterial peptide and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a soybean meal antibacterial peptide and a preparation method and application thereof, wherein the method comprises the following steps: adding water into soybean meal, mixing, adding reducing sugar and yeast, oscillating, fermenting, inactivating enzyme, and centrifuging to obtain supernatant as soybean meal fermentation liquid; adding absolute ethyl alcohol into every 10-20% of the soybean meal antibacterial peptide in a concentration gradient manner, centrifuging, taking supernate, continuously adding absolute ethyl alcohol to enable the concentration of the ethanol in the supernate to reach 40-80%, centrifuging, taking supernate, and freeze-drying to obtain the soybean meal antibacterial peptide. According to the invention, an equal concentration gradient sequential alcohol precipitation method is adopted, and the weak antibacterial characteristic components are removed and the strong antibacterial characteristic components are enriched through ethanol treatment with different final concentrations, so that on one hand, the target peptide can be efficiently separated, the peptides with the molecular weight of between 1000-plus-5000 Da are enriched, the purpose of efficiently concentrating the target peptide is achieved, and the process flow is simplified.
Description
Technical Field
The invention belongs to the field of deep processing of soybean meal, and particularly relates to soybean meal antibacterial peptide and a preparation method and application thereof.
Background
The antibiotic is a drug which has a certain inhibiting or killing effect on bacteria, viruses or parasites, and is a secondary metabolite which is generated by microorganisms such as bacteria and fungi or other higher animals in the process of breeding and has an anti-pathogen effect. Since the discovery, the traditional Chinese medicine composition can effectively cure various infectious cases, is widely applied, and makes a great contribution in the field of medical treatment. Besides being used for human medical treatment, the antibiotic also has important contributions in the aspects of treating animal infectious diseases, improving animal immunity, increasing yield, improving economic value and the like.
With the increasing use amount of antibiotics and the lack of understanding of antibiotics by human beings, the problem of antibiotic abuse occurs, so that some pathogenic bacteria generate drug resistance, and the antibiotics lose the original sterilization and disease treatment functions. In addition, because the digestion and absorption rate of antibiotics is extremely low, most of the unabsorbed antibiotics are attached to urine or feces of people or animals and discharged out of the bodies after being ingested, so that the environment is greatly polluted, and even the antibiotics finally return to the bodies through natural circulation to be accumulated, thereby harming the health of human beings and other organisms.
The antibacterial peptide is a small molecular peptide substance which is carried by a certain organism or can be synthesized by the organism and has antibacterial activity, is a component of a nonspecific immune defense system in the organism and can resist the invasion of partial foreign pathogens. The antibacterial peptide can replace the medical effect of antibiotics, is very effective, can be synthesized by organisms to resist external pathogenic bacteria, and belongs to a small molecular protein of the internal components of a self-defense system. The antibacterial peptide has wide sources, and various antibacterial peptides with the function characteristics of resisting pathogenic bacteria (bacteria, fungi and the like) are found in animals, plants and bacteria at present.
Soybean is one of the important crops in China, and contains abundant protein and essential amino acid. The soybean meal is a byproduct of soybean oil production, and researches show that the soybean meal can be used for preparing and extracting soybean bioactive peptides after being fermented, can obviously increase the additional value of the soybean meal, can be applied to industries such as food, health care and the like, and has good development prospect.
Disclosure of Invention
The invention aims to provide a method for preparing antibacterial active polypeptide by using soybean meal. The method mainly utilizes the liquid state fermentation of the soybean meal by microorganisms to promote the soybean meal to release the antibacterial active peptide, and then regulates and controls the distribution condition of the antibacterial polypeptide in the soybean meal fermentation product through ethanol fractionation, so as to enrich the protein polypeptide with stronger antibacterial property.
Another object of the present invention is to provide an antibacterial polypeptide prepared by the above method.
Still another object of the present invention is to provide the use of the above-mentioned antibacterial polypeptide.
The purpose of the invention is realized by the following technical scheme:
a preparation method of soybean meal antibacterial peptide comprises the following steps:
(1) preparing soybean meal fermentation liquor: mixing soybean meal with water, adjusting pH to 4.5-5.5, adding reducing sugar, inoculating yeast, and fermenting at 32-37 deg.C under shaking for 36-48 hr; after fermentation, inactivating enzyme, and performing centrifugal separation to obtain supernatant as soybean meal fermentation liquor with antibacterial activity; based on the mass of the soybean meal, the addition amount of reducing sugar accounts for 1.5-2.5%, and the inoculation amount of the yeast essence accounts for 0.3-0.7 per mill.
(2) Ethanol separation and enrichment of soybean meal antibacterial peptide: properly concentrating soybean meal fermentation liquor, adding absolute ethyl alcohol in a concentration gradient of every 10-20% (by mass), stirring at constant temperature and constant speed for 1-2h after adding every concentration gradient of ethyl alcohol, centrifuging, taking supernate, continuously adding absolute ethyl alcohol in a concentration gradient to ensure that the concentration of ethyl alcohol in the supernate reaches 40-80% (by mass), stirring at constant temperature and constant speed for 1-2h, centrifuging to take supernate, removing residual ethyl alcohol in the supernate, and freeze-drying to obtain soybean meal antibacterial peptide;
the soybean meal selected in the step (1) is high-temperature or low-temperature soybean meal;
the reducing sugar in the step (1) is more than one of glucose, fructose or maltose, preferably glucose;
the yeast essence in the step (1) is special yeast essence for soy sauce fermentation, preferably yeast essence obtained by fermenting Aspergillus oryzae strain Huniang 3.042;
the enzyme deactivation in the step (1) is preferably carried out for 30min at the temperature of 90 ℃;
in the step (2), preferably, the ethanol concentration in the supernatant is made to be 80% (by mass);
in the step (2), preferably, the soybean meal fermentation liquor is concentrated in vacuum until the solid content is 30-45%;
the constant-temperature uniform stirring in the step (2) is uniform stirring at the rotating speed of 200 and 400r/min at normal temperature after the absolute ethyl alcohol is added;
and (3) removing the residual ethanol in the supernatant in the step (2), namely re-dissolving the ethanol by using deionized water and concentrating the ethanol under the vacuum condition, wherein the temperature is 50-60 ℃.
The centrifugation in the steps (1) and (2) adopts low-temperature centrifugation, generally at 4 ℃, and the temperature is kept at 5000-.
The preparation method of the soybean meal antibacterial peptide is a microorganism liquid state fermentation method, the microorganism can secrete enzymes which are consistent with the characteristics of raw materials and have higher specificity in the fermentation process, the soy sauce koji used in the invention is obtained by fermenting preferred Aspergillus oryzae 3.042 strain brewed in Shanghai, the amino acid conversion rate of a protein fermentation product is high, the Aspergillus oryzae mycelium is composed of multiple cells, and rich enzyme systems including protease, amylase, glucoamylase and cellulase can be produced, and compared with single commercial enzyme, the soybean meal can be decomposed more efficiently.
The method of the invention can achieve certain effect of enriching and separating the antibacterial peptide by ethanol, and the principle is as follows:
(1) the dielectric constant is changed; water is a high dielectric constant and ethanol is a low dielectric constant, so the addition of an organic solvent lowers the dielectric constant of an aqueous solution, which increases the attraction between two opposite charges, as is known from coulomb's law. Thus, the ionization degree of the dissociable group on the surface of the protein molecule is weakened, the hydration degree is reduced, the aggregation and precipitation of the macromolecular protein are promoted, and the small molecular protein peptide and the amino acid are remained in the supernatant.
(2) Probably like salting out, organic agents compete directly with proteinaceous substances for water of hydration, causing aggregation and precipitation of macromolecular proteins. Meanwhile, the ethanol has higher safety than other organic matters, has a boiling point of 78 ℃, is volatile, and can be widely applied to industrial production.
The molecular weight distribution of the polypeptide obtained by the method is mostly small molecular peptide fragments, the separation and enrichment by absolute ethyl alcohol are biased to be concentrated below 5000Da, and products obtained by the method which is not the method of the invention (such as soybean meal protein peptide A and soybean meal protein peptide B of a comparative example) mainly comprise peptides larger than 5000Da, so the antibacterial rate is lower.
The soybean meal antibacterial peptide prepared by the method can be applied to health food, health care products and animal feed.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention takes soybean meal as a fermentation substrate, and utilizes various enzymes secreted and produced by the koji during the fermentation process to hydrolyze macromolecular protein in the culture raw material. The enzyme which is consistent with the characteristics of the raw materials and has higher specificity can be obtained by utilizing a biological fermentation method, the original anti-nutritional factor components in the soybean meal can be reduced, and simultaneously more small molecular polypeptides and amino acids can be produced.
(2) The reducing sugar is added for fermentation, so that sufficient carbon source can be provided for the growth of aspergillus oryzae, and the residual reducing sugar can also perform Maillard reaction with peptide and amino acid in the fermentation liquid in the processes of fermentation and sterilization, so that the antibacterial activity of the fermentation liquid is further improved.
(3) According to the invention, an equal concentration gradient sequential alcohol precipitation method is adopted, and the weak antibacterial characteristic components are removed and the strong antibacterial characteristic components are enriched through ethanol treatment with different final concentrations, so that on one hand, the target peptide can be efficiently separated, the peptides with the molecular weight of between 1000-plus-5000 Da are enriched, the purpose of efficiently concentrating the target peptide is achieved, and the process flow is simplified.
(4) The ethanol used by the invention can be recycled, and is economical, safe and pollution-free.
(5) The method has the advantages of simple process operation, low production cost, no pollution, high safety and strong antibacterial activity of the obtained antibacterial peptide, and can be widely applied to the field of foods.
Drawings
FIG. 1 is a graph showing the molecular weight distribution of the polypeptides obtained in examples and comparative examples.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
In the following examples, the antibacterial effect was measured as follows:
and (3) determining the antibacterial rate by a tube-disc method:
(1) preparation of culture Medium
Preparation of beef extract peptone medium/gravy medium: weighing 5.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride and 20g of agar (not contained in a meat juice culture medium), heating and melting by using distilled water, fixing the volume to 1000mL, adjusting the pH value to 7.0-7.2, sterilizing at 121 ℃ for 15min, and standing for later use after sterilization.
Preparation of slant culture medium: preparing a plurality of test tubes (15mm multiplied by 150mm), heating and melting the beef extract peptone medium, subpackaging the heated beef extract peptone medium into the test tubes by using separating funnels, filling about 5mL of each test tube with a cotton plug, wrapping the cotton plug with newspaper, sterilizing for 15min at 121 ℃ in an autoclave, placing the test tubes on the inclined surface after completion, and cooling to room temperature for later use.
(2) Strain activation
Preparing two beef extract peptone culture medium flat plates; opening the ampule tube filled with the bacteria freeze-dried powder in a super-clean workbench, igniting the top by using an alcohol lamp, immediately dripping sterile water to break the tube opening, and then slightly breaking the tube opening by using tweezers; ③ sucking 0.5mL of meat juice culture medium, pumping into a freeze-drying tube, fully dissolving and uniformly mixing, sucking 200 μ L of bacterial suspension by each plate, uniformly coating the plates, and culturing at the constant temperature of 37 ℃ for 24 h.
(3) Preparation of the bacterial suspension
And washing the thallus on the subculture agar slant culture medium for 2-3 passages by using 85% normal saline which is cooled to room temperature after sterilization, centrifuging the eluate at 8000r/min and 4 ℃ for 15min, washing the precipitated thallus by using normal saline for 1-2 times, and diluting the thallus to a bacterial suspension with the light transmittance of 20% at 650nm for later use.
(4) Antibacterial experiments
Preparing a double-layer flat plate: taking a proper number of culture dishes (the diameter is 90mm, the height is 20mm, the required sizes are consistent, the bottoms of the culture dishes are flat), adding 20mL of hot melted beef extract peptone culture medium, uniformly spreading and solidifying the peptone culture medium to be used as a bottom layer, and waiting for solidification. And then melting and cooling to 50-60 ℃ beef extract peptone culture medium, adding 1mL of 50% glucose solution and 5mL of prepared bacterial suspension into each 100mL of beef extract peptone culture medium, fully and uniformly mixing, and uniformly spreading 5mL of prepared bacterial suspension on the solidified bottom culture medium to serve as an upper culture medium.
Sample adding: placing the sterilized Oxford cups (stainless steel tubes, the inner diameter is 6 +/-0.1 mm, the outer diameter is 8 +/-0.1 mm, and the height is 10 +/-0.1 mm) on the surface of a culture medium, the distance between every two Oxford cups is equal, placing and lightly pressing to ensure that the bottoms of the Oxford cups are contacted with the culture medium, adding 150 mu L of sample liquid (using normal saline to replace the sample liquid as a control) into each Oxford cup, and paralleling every sample for three times.
Culturing: after the sample adding is finished, a layer of sterile filter paper is attached to each flat plate cover, the culture dish cover is covered, and the flat plate cover is placed in a constant temperature incubator to be cultured for 24 hours at 37 ℃.
Measurement: after the culture was completed, the oxford cup was taken out, and the diameter of each antibacterial ring in the petri dish was measured with a vernier caliper.
(5) The calculation formula of the antibacterial rate is as follows:
example 1
A preparation method of soybean meal antibacterial peptide comprises the following steps:
(1) preparing soybean meal fermentation liquor: adding deionized water 7 times the weight of soybean meal, mixing, adjusting pH to 4.5, adding maltose 1.5% of the weight of soybean meal, mixing, placing in a conical flask, inoculating yeast 0.3 ‰ of the weight of soybean meal as fermentation strain, placing in a water bath constant temperature oscillator, and performing shake fermentation at 32 deg.C in 200r/min water bath for 36 hr. After fermentation, keeping the temperature at 90 ℃ for 30min to inactivate enzyme, centrifuging at 8000r/min and 4 ℃ for 20min to obtain supernatant as soybean meal fermentation liquor with antibacterial activity;
(2) ethanol separation and enrichment of soybean meal antibacterial peptide: vacuum concentrating soybean meal fermentation liquid to solid content of 30%, adding anhydrous ethanol until ethanol content in final system is 20% (by mass), stirring at 200r/min at constant speed for 1h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 1; and adding absolute ethyl alcohol into the supernatant 1 continuously until the content of the ethyl alcohol in the system reaches 40% (by mass), stirring at a constant speed of 200r/min for 2h at room temperature, centrifuging (8000r/min, 20min), taking the supernatant, removing residual ethyl alcohol in the supernatant, and freeze-drying to obtain the soybean meal antibacterial peptide 1.
Example 2
A preparation method of soybean meal antibacterial peptide comprises the following steps:
(1) preparing soybean meal fermentation liquor: adding 11 times of deionized water according to the mass of soybean meal, uniformly mixing, adjusting the pH value to 5.0, adding fructose accounting for 2.0% of the mass of the soybean meal, uniformly mixing, placing in a conical flask, inoculating yeast with 0.5 per mill of the mass of the soybean meal as a fermentation strain, placing in a water bath constant temperature oscillator, and carrying out shaking fermentation in a water bath of 200r/min at 35 ℃ for 42 hours. After fermentation, keeping the temperature at 90 ℃ for 30min to inactivate enzyme, centrifuging at 8000r/min and 4 ℃ for 20min to obtain supernatant as soybean meal fermentation liquor with antibacterial activity;
(2) ethanol separation and enrichment of soybean meal antibacterial peptide: vacuum concentrating soybean meal fermentation liquid to solid content of 40%, adding anhydrous ethanol until ethanol content in final system is 20% (by mass), stirring at 200r/min at constant speed for 1h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 1; adding anhydrous ethanol into the supernatant 1 until the ethanol content reaches 40% (by mass), stirring at 200r/min for 2h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 2; and adding absolute ethyl alcohol into the supernatant 2 continuously until the content of the ethyl alcohol in the system reaches 60% (by mass), stirring at the room temperature at a constant speed of 200r/min for 1h, centrifuging (8000r/min, 20min), taking the supernatant, removing residual ethyl alcohol in the supernatant, and freeze-drying to obtain the soybean meal antibacterial peptide 2.
Example 3
A preparation method of soybean meal antibacterial peptide comprises the following steps:
(1) preparing soybean meal fermentation liquor: adding deionized water 9 times the weight of soybean meal, mixing, adjusting pH to 5.5, adding glucose 2.5% of the weight of soybean meal, mixing, placing in a conical flask, inoculating yeast 0.7 ‰ of the weight of soybean meal as fermentation strain, placing in a water bath constant temperature oscillator, and performing shaking fermentation at 37 deg.C in 200r/min water bath for 48 hr. After fermentation, keeping the temperature at 90 ℃ for 30min to inactivate enzyme, centrifuging at 8000r/min and 4 ℃ for 20min to obtain supernatant as soybean meal fermentation liquor with antibacterial activity;
(2) ethanol separation and enrichment of soybean meal antibacterial peptide: vacuum concentrating soybean meal fermentation liquid to solid content of 45%, adding anhydrous ethanol until ethanol content in final system is 20% (by mass), stirring at 200r/min at constant speed for 1.5h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 1; adding anhydrous ethanol into the supernatant 1 until the ethanol content reaches 40% (by mass), stirring at 200r/min for 1h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 2; adding anhydrous ethanol into the supernatant 2 until the ethanol content reaches 60% (by mass), stirring at 200r/min for 1.5h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 3; and adding absolute ethyl alcohol into the supernatant 3 continuously until the content of the ethyl alcohol in the system reaches 80% (by mass), stirring at the room temperature at a constant speed of 200r/min for 1h, centrifuging (8000r/min, 20min), taking the supernatant, removing residual ethyl alcohol in the supernatant, and freeze-drying to obtain the soybean meal antibacterial peptide 3.
Comparative example 1
A preparation method of soybean meal protein peptide comprises the following steps:
(1) weighing 1 part of soybean meal and 9 parts of deionized water according to the weight, uniformly mixing, homogenizing for 10min at 5000r/min by using a homogenizing and dispersing machine, adding 0.15% of compound plant hydrolase and 0.35% of alkaline protease by mass of the soybean meal, and then carrying out enzymolysis for 12h at the constant temperature of 200r/min at 55 ℃. After enzymolysis, keeping the temperature at 90 ℃ for 30min to inactivate enzyme, centrifuging at 8000r/min and 4 ℃ for 20min, and taking supernatant to obtain soybean meal enzymolysis liquid;
(2) vacuum concentrating soybean meal enzymolysis liquid to solid content of 45%, adding anhydrous ethanol until ethanol content in final system is 20% (by mass), stirring at room temperature at 200r/min for 1h at constant speed, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 1; adding anhydrous ethanol into the supernatant 1 until the ethanol content reaches 40% (by mass), stirring at 200r/min for 1h at room temperature, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 2; adding anhydrous ethanol into the supernatant 2 until the ethanol content reaches 60% (by mass), stirring at room temperature at 200r/min for 1h, centrifuging (8000r/min, 20min), and collecting supernatant to obtain supernatant 3; and adding absolute ethyl alcohol into the supernatant 3 continuously until the content of the ethyl alcohol in the system reaches 80% (by mass), stirring at the room temperature at a constant speed of 200r/min for 1h, centrifuging (8000r/min, 20min), taking the supernatant, removing residual ethyl alcohol in the supernatant, and freeze-drying to obtain the soybean meal protein peptide A.
Comparative example 2
A preparation method of soybean meal protein peptide comprises the following steps:
adding 9 times of deionized water according to the mass of soybean meal, mixing, homogenizing for 15min at 5000r/min by a homogenizing and dispersing machine, adjusting pH to 5.5 after mixing uniformly, adding glucose accounting for 2.5% of the mass of the soybean meal, mixing uniformly, placing in a conical flask, inoculating 0.7 per mill of yeast extract of the mass of the soybean meal as a fermentation strain, placing in a water bath constant temperature oscillator, and fermenting for 48h at 37 ℃ in a water bath oscillation of 200 r/min. After fermentation, keeping the temperature at 90 ℃ for 30min to inactivate enzyme, centrifuging at 8000r/min and 4 ℃ for 20min, taking supernatant, and freeze-drying to obtain soybean meal protein peptide B.
The antibacterial ratio of the polypeptides obtained in each example and comparative example is shown in table 1:
TABLE 1 antibacterial Rate of the polypeptides*
*: the measurement was performed at a sample concentration of 60 mg/ml.
From table 1, it can be found that, after the liquid fermentation and ethanol grade separation of the microorganisms of the present invention are used in examples 1, 2 and 3, the obtained antimicrobial peptides 1, 2 and 3 all have high inhibitory effects on escherichia coli and staphylococcus aureus, and when the final ethanol concentration reaches 80% (by mass), the inhibitory rates of the antimicrobial peptide 3 on escherichia coli and staphylococcus aureus reach 69.98% and 54.11%, respectively.
In comparative examples 1 and 2, in comparative example 1, a common commercial enzyme preparation is adopted for carrying out enzymolysis on soybean meal, and then the ethanol separation process of the patent technology is adopted for preparing soybean meal protein peptide, the inhibition effect of the obtained soybean meal protein peptide A on two bacteria is far inferior to that of the soybean meal antibacterial peptide prepared by adopting a microorganism liquid fermentation method and ethanol separation and enrichment, and the inhibition rate on escherichia coli and staphylococcus aureus is only 25.02% and 14.80%.
In comparative example 2, the liquid fermentation method of the present invention was used, but ethanol fractionation was not performed, and the obtained soybean meal protein peptide B had inhibition rates of 31.58% and 30.89% for escherichia coli and staphylococcus aureus, respectively, which were different from the inhibition rates in the examples, indicating that ethanol fractionation can effectively enrich soybean meal antimicrobial peptides.
Fig. 1 shows the peptide molecular weight distribution of the soybean meal antibacterial peptide in the comparative examples of the examples, and it can be seen from the figure that the soybean meal antibacterial peptide obtained by the method of the present invention (examples 1, 2 and 3) has a higher proportion of peptide fragments with a molecular weight of <5000 Da, while the soybean meal protein peptide a obtained by the common commercial enzymatic hydrolysis technology has more macromolecular peptide fragments (the ratio of peptide fragments >5000Da is up to 60.4%), and the soybean meal protein peptide B separated only by liquid fermentation without ethanol contains 43.1% of peptide fragments with a molecular weight of >5000 Da.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of soybean meal antibacterial peptide is characterized by comprising the following steps:
(1) preparing soybean meal fermentation liquor: mixing soybean meal with water, adjusting pH to 4.5-5.5, adding reducing sugar, inoculating yeast, and fermenting at 32-37 deg.C under shaking for 36-48 hr; after fermentation, inactivating enzyme, and performing centrifugal separation to obtain supernatant as soybean meal fermentation liquor with antibacterial activity; taking the mass of the soybean meal as a calculation reference, the addition amount of reducing sugar accounts for 1.5-2.5%, and the inoculation amount of the yeast essence accounts for 0.3-0.7 per mill;
(2) ethanol separation and enrichment of soybean meal antibacterial peptide: properly concentrating the soybean meal fermentation liquor, adding absolute ethyl alcohol in a concentration gradient of every 10-20% (by mass), stirring at constant temperature and constant speed for 1-2h after adding every concentration gradient of ethyl alcohol, centrifuging, taking supernatant, continuously adding absolute ethyl alcohol in a concentration gradient to ensure that the concentration of ethyl alcohol in the supernatant reaches 40-80% (by mass), stirring at constant temperature and constant speed for 1-2h, centrifuging to take supernatant, removing residual ethyl alcohol in the supernatant, and freeze-drying to obtain the soybean meal antibacterial peptide.
2. The method of claim 1, wherein: the ethanol concentration in the supernatant in the step (2) is 80% (by mass).
3. The method of claim 1, wherein: the koji essence in the step (1) is a koji essence special for soy sauce fermentation.
4. The method of claim 1, wherein: the yeast essence in the step (1) is obtained by fermenting Aspergillus oryzae strain 3.042 brewed in Shanghai province.
5. The method of claim 1, wherein: the reducing sugar in the step (1) is more than one of glucose, fructose or maltose.
6. The method of claim 1, wherein: the soybean meal selected in the step (1) is high-temperature or low-temperature soybean meal.
7. The method of claim 1, wherein: in the step (2), the soybean meal fermentation liquor is concentrated in vacuum until the solid content is 30-45%.
8. The method of claim 1, wherein: and (3) removing the residual ethanol in the supernatant in the step (2), namely re-dissolving the ethanol by using deionized water and concentrating the ethanol under the vacuum condition, wherein the temperature is 50-60 ℃.
9. A soybean meal antimicrobial peptide characterized by being produced by the method according to any one of claims 1 to 8.
10. The use of the soybean meal antimicrobial peptide of claim 9 in food, health products and feed.
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| CN102321182A (en) * | 2011-09-22 | 2012-01-18 | 广东海洋大学 | Composite antibiosis peptide and working method thereof that bacillus natto NT-6 bacterial strain is produced |
| CN103283961A (en) * | 2013-05-10 | 2013-09-11 | 浙江工业大学 | Method for preparing health-benefiting fermented soybean meal |
| CN107280015A (en) * | 2017-06-22 | 2017-10-24 | 新昌县南翔食品科技有限公司 | Compound based on large yellow croaker fish scale activity extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102321182A (en) * | 2011-09-22 | 2012-01-18 | 广东海洋大学 | Composite antibiosis peptide and working method thereof that bacillus natto NT-6 bacterial strain is produced |
| CN103283961A (en) * | 2013-05-10 | 2013-09-11 | 浙江工业大学 | Method for preparing health-benefiting fermented soybean meal |
| CN107280015A (en) * | 2017-06-22 | 2017-10-24 | 新昌县南翔食品科技有限公司 | Compound based on large yellow croaker fish scale activity extract |
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| CN112273553A (en) * | 2020-11-27 | 2021-01-29 | 广东海奕生物饲料有限公司 | Liquid suckling pig feed and preparation method thereof |
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Application publication date: 20201113 |