CN103397068A - Preparation method of fly maggot peptides - Google Patents
Preparation method of fly maggot peptides Download PDFInfo
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- CN103397068A CN103397068A CN2013103550210A CN201310355021A CN103397068A CN 103397068 A CN103397068 A CN 103397068A CN 2013103550210 A CN2013103550210 A CN 2013103550210A CN 201310355021 A CN201310355021 A CN 201310355021A CN 103397068 A CN103397068 A CN 103397068A
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Abstract
The invention provides a preparation method of fly maggot peptides. The preparation method comprises the following steps of a, inoculating microbial strains for producing protease in a solid culture medium, and performing aerobic culture to obtain microbial spores; b, inoculating the microbial spores into a culture medium taking fly maggot proteins as a unique nitrogen source, performing fermentation culture and solid-liquid separation, and collecting a supernatant; and c, concentrating the supernatant, and drying at a low temperature to obtain the fly maggot peptides. The method provided by the invention is simple in steps, low in cost and high in product yield.
Description
Technical field
The invention belongs to microbial technology field, specifically, relate to a kind of method by fly maggot protein fermentative production antioxidation active peptides.
Background technology
Fly maggot is the larva of housefly (Musca domestica Linnaeus), and in its body, (dry-matter) contains protein 59-65 %, fatty 10-14 %, and chitin 8-10 %, in addition, also contain the nutritive ingredients such as multivitamin, trace element.Antibiotic, the antiviral and effect of removing free radical that fly maggot protein has, but be subjected to the impact of traditional concept, market is lower to the acceptance of fly maggot product, so fly maggot powder uses usually used as animal feedstuff additive.In order further effectively to utilize fly maggot protein, the researchist is arranged with the fly maggot protein enzymolysis, obtain thus the fly maggot peptide.The fly maggot peptide has good anti-oxidant, the activity that improves immunologic function, and it has huge market potential in industries such as medicine, makeup.But existing enzymolysis preparation technique is loaded down with trivial details, low conversion rate, cost expensive, is not suitable for industrial applications.
Summary of the invention
The preparation method who the purpose of this invention is to provide the fly maggot peptide that a kind of technique is simple, with low cost and transformation efficiency is high.
For realizing the object of the invention, the preparation method of fly maggot peptide provided by the present invention comprises the following steps:
A, the safe microorganisms bacterial classification that can produce proteolytic enzyme are inoculated in aseptic culture medium, and aerated culture, obtain microbial spore or thalline;
B, microbial spore or thalline are inoculated in substratum take fly maggot protein as only nitrogen source, fermentation culture 2 ~ 4 days, remove mycelium pellet or thalline, obtains the fly maggot peptide solution;
C, with the fly maggot peptide solution, cryodrying, obtain the fly maggot peptide.
A in the inventive method described solid medium of step will keep sterile state before inoculation, thus can sterilizing 20 min of 0.1 MPa, and bevel is standby.
The described safe microorganisms bacterial classification that produces proteolytic enzyme be in aspergillus oryzae (Aspergillus oryzae), aspergillus niger (Aspergillus niger), subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), actinomycetes (Actinomycete) any one.
Solid medium described in the inventive method can be any nutrient media that is suitable for the mentioned microorganism growth, as czapek's solution, PDA substratum, wheat bran dregs of beans substratum, beef-protein medium, Gause I substratum etc.Wherein take wheat bran dregs of beans substratum as preferred.
The formula of wheat bran dregs of beans substratum is by percentage to the quality:
Dregs of beans 30-40%, wheat bran 10-_15_%, fermented bean dregs extractive substance 0.1-1.0_%, water 40-50_%.
Compound method is: take the fermented bean dregs extractive substance of certain mass, be added to the water and stir, add dregs of beans, soaking at room temperature, to thoroughly, adds wheat bran finally, mixes sterilizing 20 min under 0.1 Mpa condition thoroughly.
The described substratum take fly maggot protein as only nitrogen source of the inventive method, to add carbohydrate (as any one of sucrose, glucose, molasses or starch Nulomoline in the fly maggot protein vat liquor, also can add again some other microbe carbon source), at 20-40 ℃, 10-400r/min shaking flask oscillarity ventilation, cultivate 12-100 hour.Its preparation method is in fresh fly maggot or the dry fly maggot adds damping fluid or the physiological saline of pH6.5-8.0 after fully soaking, under 4-10 ℃, homogenate, grinding, lixiviate 10-20h, centrifugal or filtration, remove solid insoluble, collect liquid, being adjusted to protein concn is 20-30mg/mL, adds wherein carbohydrate, and the mass concentration that makes carbohydrate is 2-8%, preferred 5%, obtain the substratum take fly maggot protein as only nitrogen source.
Above-mentioned solution for the lixiviate fly maggot protein can be physiological saline or pH6.5-8.0 phosphate buffered saline buffer any one, preferred 0.2 mol/L pH6.9 Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution.
In the inventive method more preferred culture medium prescription take fly maggot protein as only nitrogen source as: with the quality percentage composition, fly maggot protein 2-3%, glucose or other sugared 1-3%, Sodium phosphate dibasic 1.0-2.0%, SODIUM PHOSPHATE, MONOBASIC 0.5-1.5%, all the other are water.
The preparation method: take Sodium phosphate dibasic and the SODIUM PHOSPHATE, MONOBASIC of certain mass, water-soluble, add fresh fly maggot or dry fly maggot to soak and thoroughly add, homogenate, remove solid substance, measure protein concn, make it at 2-3%, add 1-3% glucose or other sugar, stir, sterilizing 20 min of 0.1 Mpa.
The more preferred condition of the inventive method is:
The described aerated culture of a step, its temperature is preferably 20-40 ℃, and the time is preferably 30 ~ 48h.
The described fermentation culture of b step of the present invention, its condition is preferably under 20-40 ℃, the concussion of 10-400r/min shaking flask, aerated culture 12-100h.
The present invention adopts Production by Microorganism Fermentation to prepare the fly maggot peptide, and its method is simple, low cost of manufacture, transformation efficiency high (greater than 30%).
The fly maggot protein of existing method preparation is generally that molecular weight is that the high molecular weight protein of 91.8 kD is main, and the fly maggot peptide content of all the other molecular weights is extremely low.The inventive method is the fly maggot peptide with fly maggot protein through microbial transformation, fly maggot peptide molecular weight scope is 65.9-22.4kD in the 10%SDS-PAGE testing product, detect through Tricine-SDS-PAGE, also contain the multiple fly maggot peptide of molecular weight ranges 20.2-5.9kD in product.The fly maggot peptide of preparation has good anti-oxidant activity and anti-aging effects.
Description of drawings
Fig. 1 is that 10% separation gel SDS-PAGE detects fly maggot protein that the present invention produces and the electrophoretogram of fly maggot peptide.Wherein a, fly maggot protein band; B, antioxidation active peptides band;
Fig. 2 be Tricine-SDS-PAGE detect the fly maggot peptide electrophoretogram, concentrated glue 4% wherein, squeegee 10%, fine and close glue 16.5%; 1 is: small molecular weight Marker, 2-5 is for transforming the fly maggot peptide of different time.
Fig. 3 is the influence curve figure of the fly maggot peptide for preparing of the present invention to the female fruit bat survival time.
Fig. 4 is the influence curve figure of the fly maggot peptide for preparing of the present invention to the male drosophila survival time.
Embodiment
Below with specific embodiment, further illustrate content of the present invention, but and mean and limit the invention never in any form.In the following example, method therefor is ordinary method if no special instructions.
The PDA substratum, at sterilizing 20 min of 0.1 MPa, is paved into inclined-plane standby.
The preparation of the substratum take fly maggot protein as only nitrogen source: with the quality percentage composition, fly maggot protein 2-3%, glucose or other sugared 1-3%, Sodium phosphate dibasic 1.0-2.0%, SODIUM PHOSPHATE, MONOBASIC 0.5-1.5%, all the other are water.
The preparation method: take Sodium phosphate dibasic and the SODIUM PHOSPHATE, MONOBASIC of certain mass, water-soluble, add fresh fly maggot or dry fly maggot to soak and thoroughly add, homogenate, remove solid substance, measure protein concn, make it at 2-3%, add 1-3% glucose or other sugar, stir, sterilizing 20 min of 0.1 Mpa.
The preparation of fly maggot peptide:
A, aspergillus oryzae (Aspergillus oryzae) 3042 is inoculated in above-mentioned PDA substratum, under 32 ℃, aerated culture 48-72h, obtain the aspergillus oryzae spore;
Aspergillus oryzae (Aspergillus oryzae) 3042 is buied from the market sale channel.
B, to above-mentioned fly maggot protein, be in the substratum of only nitrogen source with the aspergillus oryzae spore inoculating, under 20 ℃, the concussion of 10-400r/min shaking flask, aerated culture 96h, the centrifugal 20min of 6000r/min, removes insoluble solid mycelium pellet, acquisition fly maggot peptide solution;
C, with above-mentioned fly maggot peptide solution, cryodrying, obtain pulverous fly maggot peptide.
Through two kinds of electrophoresis technique determinings, the present embodiment gained fly maggot peptide molecular weight scope is that 65.9-5.9kD(is as figure, shown in Figure 2).The albumen transformation efficiency is 30.6%.
A, the appropriate czapek's solution of packing in vitro, sterilizing 20 min of 0.1 MPa, bevel is standby; With the Aspergillus oryzae(3.042 that preserves) the access inclined-plane, be cultured to and cover with spore in the constant incubator of 32 ℃.
B, with the Aspergillus oryzae 3.042 that has grown, access is appropriate in the triangular flask of the substratum take fly maggot protein as only nitrogen source, 32 ℃, 180 r/min concussions were cultivated 96 hours.The centrifugal 20min of 6000r/min, remove mycelium, and supernatant liquor is the fly maggot peptide solution;
The preparation method of above-mentioned substratum take fly maggot protein as only nitrogen source: after fully being soaked, fresh fly maggot or dry fly maggot add the pH6.5 damping fluid, homogenate, not centrifugal, adjusting its protein concn is 2.5%, add 2% glucose, every 500mL triangular flask 50mL that packs into, sterilizing 30 min of 0.1 MPa.
The peptide concentration that c, biuret method record supernatant liquor is 7.89mg/mL, and cryodrying obtains the fly maggot Gly-His-Lys.
Through two kinds of electrophoresis technique determinings, the present embodiment gained fly maggot peptide molecular weight scope is that 65.9-5.9kD(is as figure, shown in Figure 2).The transformation efficiency that the microbial transformation fly maggot protein forms the fly maggot peptide is 31.6%.
A, aspergillus niger (Aspergillus niger) 3350 is inoculated in the Cha Shi slant medium, under 30 ℃, aerated culture 4 days, obtain aspergillus niger spore;
B, aspergillus niger spore is inoculated in the substratum that fly maggot protein is only nitrogen source, under 30-32 ℃, the concussion of 200r/min shaking flask, aerated culture 80-100h, the centrifugal 20min of 6000r/min, remove insoluble solid mycelia, collects supernatant liquor (being the fly maggot peptide solution);
The preparation method of above-mentioned substratum take fly maggot protein as only nitrogen source is:
After fully being soaked, fresh fly maggot or dry fly maggot add tap water, homogenate, and not centrifugal, make soluble protein concentration in the 2.0-4.5% scope, directly add sucrose or the molasses of 1-3%, salt acid for adjusting pH to 5.5 left and right, the bottled liquid 50mL of every 500mL triangle, sterilizing 30 min of 0.1 MPa.
C, the present embodiment gained antioxidation active peptides, measuring the albumen transformation efficiency through biuret method is 31.1%.
, with supernatant concentration, cryodrying, can obtain pulverous fly maggot peptide.
Through two kinds of electrophoresis technique determinings, the present embodiment gained fly maggot peptide molecular weight scope is that 65.9-5.9kD(is as figure, shown in Figure 2).
A, bacterial strain activation: the appropriate beef-protein medium of in vitro packing into, sterilizing 20 min of 0.1 MPa, bevel is standby; , with subtilis (Bacillus subtilis) the bacterial strain access inclined-plane of preserving, cultivate in the constant incubator of 32 ℃.
B, cultivation seed: 8% Semen Maydis powder, 12% soybean cake powder, potassium primary phosphate 1.2 grams, Sodium phosphate dibasic .12H
2O, 1.8 grams, zinc chloride 0.5 gram, all the other are tap water, regulate pH8.0.Sterilizing 30 min of above-mentioned seed culture medium 0.1 MPa.The subtilis of inoculation 2 ring activation, 37 ℃, 200r/min shake-flask culture 24-48 hour.
C, fly maggot protein transform: add tap water after fresh fly maggot or dry fly maggot are fully soaked, homogenate, not centrifugal, make soluble protein concentration in the 2.0-4.5% scope, the sucrose or the molasses that directly add 1-3%, regulate initial pH to pH7.5-9.0, the bottled liquid 50mL of every 500mL triangle, sterilizing 30 min of 0.1 MPa.Press the 1-3% inoculation, at temperature 40-50 ℃, the concussion of 200r/min shaking flask was cultivated 3 days.
D, results fly maggot peptide:, with the centrifugal 20min of fermenting bacillus subtilis liquid 6000r/min, remove thalline, collect supernatant liquor (being the fly maggot peptide solution); Can, with fly maggot peptide solution low-temperature freeze drying, obtain the fly maggot Gly-His-Lys.
Through two kinds of electrophoresis technique determinings, the present embodiment gained fly maggot peptide molecular weight scope is that 65.9-5.9kD(is as figure, shown in Figure 2).The yield that biuret method is measured the fly maggot peptide is 29.8%.
Embodiment 5
A, bacterial strain activation: the appropriate beef extract-peptone solid medium of in vitro packing into, sterilizing 20 min of 0.1 MPa, bevel is standby; , with Bacillus licheniformis (Bacillus licheniformis) the 2709 access inclined-planes of preserving, cultivate in the constant incubator of 32 ℃.
B, seed culture:, from the Bacillus licheniformis inclined-plane of activation, get 2 rings and be inoculated in seed culture medium, 37 ℃, 200r/min shake-flask culture 24-48 hour.The formula of seed culture medium adopts the beef extract-peptone liquid nutrient medium.
C, fly maggot protein are converted into the fly maggot peptide: be in the liquid nutrient medium of only nitrogen source to fly maggot protein by 2% inoculum size access seed, 37 ℃, 48h is cultivated in the concussion of 200r/min shaking flask.Fly maggot protein is the preparation method of the liquid nutrient medium of only nitrogen source: add tap water after fresh fly maggot or dry fly maggot are fully soaked, homogenate, not centrifugal, make soluble protein concentration in the 2.0-4.5% scope, the sucrose or the molasses that directly add 1-3%, regulate initial pH to pH7.59.0, the bottled liquid 50mL of every 500mL triangle, sterilizing 30 min of 0.1 MPa.The centrifugal 20min of fermented liquid 6000r/min, remove thalline, collects supernatant liquor (being the fly maggot peptide solution); Can, with fly maggot peptide solution low-temperature freeze drying, obtain the fly maggot Gly-His-Lys.Through two kinds of electrophoresis technique determinings, the present embodiment gained fly maggot peptide molecular weight scope be 65.9-5.9kD(as shown in Figure 1 and Figure 2).The yield that biuret method is measured the fly maggot peptide is 33.4%.
Embodiment 6
Aspergillus oryzae Aspergillus oryzae 3042 transforms the experiment of the fly maggot peptide raising Life span of Drosophila melanogaster of fly maggot protein formation.
Choose in 24 h each 400 of the female male drosophilas of the not mating of sprouting wings, after etherization by the sex random packet.Female fruit bat is numbered: ♀, the 96 high ♀ of h in contrast ♀, 96 h low ♀, 96 h; Male Drosophila is numbered: ♂, the 96 high ♂ of h in contrast ♂, 96 h low ♂, 96 h.The fly maggot peptide is added in basic medium, and dosage accounts for respectively 1 % of substratum, 2 %, 4 %(g/g), take basic medium as control group, fruit bat is placed in temperature (25 ± 1) ℃, cultivate in the water isolation type constant incubator of humidity 55 %～65 %.Every 24 h observed and recordeds are organized the fruit bat mortality, and record survival number of days, until the whole natural deaths of fruit bat.The mean lifetime of 5 fruit bats of every group of last death, as its Mean longest life.Adopt SPSS10.0 to carry out data analysis.
The results are shown in Table 1, Fig. 3, Fig. 4:
The lengthen the life effect of table 1 fly maggot peptide to fruit bat
a?:?
P?<?0.05，b:?
P?<?0.01
Can find out from table 1, Fig. 3, Fig. 4, aspergillus oryzae Aspergillus oryzae 3042 transforms the fly maggot peptide that fly maggot protein 96h forms, and the middle and high concentration group in this experiment, have the effect of utmost point significant prolongation to life span of drosophila melanogaster.Illustrate that the fly maggot peptide has good anti-aging effects.
Claims (9)
1. the preparation method of a fly maggot peptide, it comprises the following steps:
A, the microbial strains that can produce proteolytic enzyme are inoculated in aseptic culture medium, and aerated culture, obtain microbial spore or thalline;
B, microbial spore or thalline are inoculated in substratum take fly maggot protein as only nitrogen source, fermentation culture 12 ~ 100 hours, remove mycelium pellet or thalline, obtains the fly maggot peptide solution;
C, with the fly maggot peptide solution, cryodrying, obtain the fly maggot peptide.
2. the preparation method of fly maggot peptide according to claim 1 is characterized in that: the described safe microorganisms bacterial classification that produces proteolytic enzyme be in aspergillus oryzae, aspergillus niger, subtilis, Bacillus licheniformis, actinomycetes any one.
3. the preparation method of described fly maggot peptide according to claim 1 and 2 is characterized in that: the described substratum of a step be in czapek's solution, PDA substratum, wheat bran dregs of beans substratum, beef-protein medium, Gause I substratum any one.
4. the Preparation Method of fly maggot peptide according to claim 3, is characterized in that described wheat bran dregs of beans substratum, and its formula is by percentage to the quality, dregs of beans 30-40%, and wheat bran 10-_15_%, fermented bean dregs extractive substance 0.1-1.0%, all the other are water.
5. the preparation method of described fly maggot peptide according to claim 1 and 2, it is characterized in that: the described aerated culture of a step, its temperature are 20-40 ℃, the time is 2-7 days.
6. the preparation method of described fly maggot peptide according to claim 1 and 2, it is characterized in that: the described substratum take fly maggot protein as only nitrogen source of b step, its preparation method is to the phosphate buffered saline buffer that adds physiological saline or 0.1-0.2mol/L pH6.5-8.0 after fresh fly maggot or dry fly maggot are fully soaked, under 4-10 ℃, homogenate or grinding 10-20h, centrifugal or filter, remove solid insoluble, collect liquid, being adjusted to protein concn is 20-30mg/mL, add wherein carbohydrate, the mass concentration that makes carbohydrate is 2-8%.
7. the preparation method of fly maggot peptide according to claim 6, it is levied and is: described carbohydrate be in sucrose, glucose, molasses, starch, Nulomoline any one.
8. the preparation method of fly maggot peptide according to claim 6, it is levied and is: described phosphate buffered saline buffer be in Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic-potassium phosphate buffer any one.
9. the preparation method of described fly maggot peptide according to claim 1 and 2, it is levied and is: the described fermentation culture of b step, its condition are 20-40 ℃, the concussion of 10-400r/min shaking flask, aerated culture 12-100h.
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Inventor after: Wu Jinxia Inventor after: Zhang Heying Inventor after: Zhang Ruiying Inventor after: Liu Qing Inventor after: Dong Linxuan Inventor before: Wu Jinxia Inventor before: Zhang Heying |
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