CN108707637A - A kind of preparation method of pigskin antibacterial polypeptide - Google Patents

A kind of preparation method of pigskin antibacterial polypeptide Download PDF

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Publication number
CN108707637A
CN108707637A CN201810356047.XA CN201810356047A CN108707637A CN 108707637 A CN108707637 A CN 108707637A CN 201810356047 A CN201810356047 A CN 201810356047A CN 108707637 A CN108707637 A CN 108707637A
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pigskin
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antibacterial peptide
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全盈园
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Jinhua Iron Knight Biotechnology Co Ltd
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    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract

The invention discloses a kind of preparation method of pigskin antibacterial polypeptide, the specific steps are：The aqueous solution of containing sodium carbonate and ethyl valerate will be added after fresh pig skin stripping and slicing, degreasing discards lye, is washed to after weakly acidic pH plus water is homogenized, boil, filter, dry that degreasing pigskin is rotten；Fermentation medium is prepared using degreasing pigskin gruel, inoculated aspergillus niger fermentation, centrifuged supernatant is zymotic fluid；Zymotic fluid ultrafiltration is obtained into antibacterial peptide crude extract；Antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, collects the higher component of antibacterial activity, be freeze-dried up to pigskin antibacterial peptide.It has the beneficial effect that：Extracting method simple possible of the present invention, extraction efficiency, yield and purity are high, and antibacterial peptide quality obtained is high, activity is high, the antibacterial characteristics that can quickly kill bacterium, have wide spectrum.

Description

A kind of preparation method of pigskin antibacterial polypeptide
Technical field
The present invention relates to technical field of biological extraction, more particularly to a kind of preparation method of pigskin antibacterial polypeptide.
Background technology
After antibiotic is found, since there is good fungistatic effect to be widely used in medical treatment, traditional beast for it In medicine and feed addictive, but also due to the excessive use of antibiotic, the problems such as drug resistance and medicament residue of pathogenic bacteria, gets over Come more serious, gradually cause the attention of people, find effective substitute of antibiotic, solves drug resistance and medicament residue is asked Topic is extremely urgent.And antibacterial peptide（Antimicrobial peptides, AMPs）It is a kind of prokaryotes and the equal energy of eucaryote Polypeptides matter generate, widely distributed and miscellaneous, with broad spectrum antibiotic activity, it is to gram-positive bacteria, leather Lan Shi negative bacteriums, fungi, protozoan, cancer cell, virus etc. have apparent inhibiting effect, are considered as before having greatly application The Substitutes For Antibiotic of scape becomes research hotspot both domestic and external.Antibacterial peptide is Sweden scientist Boman etc. earliest in a kind of name It cries and cherishs guppy giant silkworm（Hyalophora cecropia）Insect Immune inducing in vivo and isolate and purify, and be named as Cecropin（Cecropins）.Up to the present, people identify in various protokaryons and eucaryote and isolated one after another More than 1700 kinds of antibacterial peptide matters.
The type of antibacterial peptide is various, and distribution is also very extensive, as the research to antibacterial peptide is deep, the classification of antibacterial peptide It is more and more clear.Currently, antibacterial peptide is generally classified according to the following aspects：According to the route of synthesis of antibacterial peptide by antibacterial Peptide is divided into non-ribosomal synthetic antibacterial peptide（non-ribosomally synthesized peptides）It is anti-with Ribosome biogenesis Bacterium peptide（ribosomally synthesized peptides）Two classes.It is divided into according to the structure of antibacterial peptide：（1）Containing alpha-helix Linear antibacterial peptide；（2）Antibacterial peptide rich in certain amino acid residues；（3）Antibacterial peptide containing 1 disulfide bond；（4）With beta sheet The antibacterial peptide of structure；（5）The Tai &#91 with antibacterial activity derived from known peptide;31].It is divided into according to the source of antibacterial peptide：（1） Bacterium antibacterial peptide, such as polymyxins, bacitracin；（2）Insect antimicrobial peptide, such as cecropin, insect defensin；（3）Plant source Antibacterial peptide, such as plant alexin, balsamine element；（4）Frog antibacterial peptide, such as frog skin peptide；（5）Aquatic animal antibacterial peptide, Including fish, shellfish, crustacean class antibacterial peptide, the various complexity of type；（6）Mammalian Antimicrobial Peptides mainly have alexin （Defensins）, histatins（Histatins）With tri- kinds of Cathelicidins.At present both at home and abroad research in pig source antibacterial Antibacterial peptide in the relevant mainly chitterlings of peptide, and extracted in pigskin, purify antibacterial peptide and study grinding for its antibacterial activity Study carefully but seldom.
Invention content
The purpose of the present invention is to provide it is a kind of input it is low, easy to operate, protein degradation effect is good, antibacterial peptide yield and Purity is high, and antibacterial peptide quality obtained is high, activity is high, can quickly kill bacterium, a kind of pigskin for the antibacterial characteristics for having wide spectrum resists The preparation method of bacterium polypeptide.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
A kind of preparation method of pigskin antibacterial polypeptide, including pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, ultrafiltration, Purifying, the specific steps are：
Pretreatment of raw material：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:8-12（g/mL）Sodium carbonate containing 1-3% and 0.031- is added 0.038% ethyl valerate aqueous solution, degreasing 30- under conditions of temperature is 35-45 DEG C, ultrasonic frequency is 40-50kHz 60min discards lye, is washed to weakly acidic pH, then it is 1 to press solid-liquid ratio:8-12（g/mL）Water is added to be homogenized, heating is boiled, and is filtered, and is done Dry degreasing pigskin is rotten, which quickly can generate water-soluble soap salt and sweet under the booster action of ultrasonic wave by grease Oil and achieve the purpose that degreasing, degreasing rate is high, and ethyl valerate be added and on the one hand promote free aliphatic acid that can be combined with alkali Generate soap salt, improve degreasing efficiency and degreasing rate, and can by trimethylamine, (E, E) -2,4- heptadienals etc. on fishy smell influence compared with Big compound preferably removes, and improves the quality of antibacterial peptide, the H that on the other hand can be generated with ultrasonic cavitation effect2O2Hair Raw synergistic effect, can reduce the loss of protein, and can avoid excessive alkali destroy the protein in pigskin and other at Point, to the generation for the impurity that the bacterium peptide mouthfeel that avoids creating antagonism, smell and shelf-life influence, the final quality for improving antibacterial peptide And yield；
Fermentation medium is prepared：8-13 parts of extracting degreasing pigskin gruel, 18-22 parts of sucrose, 4-6 parts of sodium chloride, zinc chloride by weight 0.004-0.006 parts, 1.8-2.4 parts of potassium dihydrogen phosphate, 0.3-0.5 parts of magnesium sulfate, 0.005-0.015 parts of ferric sulfate, vitamin B10.001-0.002 parts, 900-1100 parts of sterile water are uniformly mixed to get fermentation medium, and the fermentation medium nutrition is non-lipid Comprehensively, it is able to maintain that the stability of pH, provides good growing environment for aspergillus niger, improve the yield of aspergillus niger metabolite；
Fermentation of Aspergillus niger：The pH for adjusting fermentation medium is 5.5-6.5, is 5-7% inoculated aspergillus nigers by inoculum concentration, is in temperature 25-30 DEG C, rotating speed ferment 2-3d under conditions of being 120-170r/min, it is that rhamnose is added in 7-9g/L then to press additive amount, after Supervention ferment 10-15h, centrifuged supernatant are zymotic fluid, and the addition of rhamnose can significantly reduce the surface tension of water and cell membrane, Change membrane passage, the acid protease in cell membrane is preferably secreted into extracellularly, increases acid in zymotic fluid The content of protease improves the utilization ratio of aspergillus niger protease, improves the degradation rate of pigskin gruel, additionally it is possible to inhibit microorganism Growth, reduces the utilization rate of product, the final yield for improving antibacterial peptide；It can be produced during Aspergillus Niger Growth breeding in the step Raw various metabolic enzymes, such as acid protease and neutral proteinase, effectively can be degraded to small molecule by macro-molecular protein Polypeptide, which has compared with using enzyme system degradation anti-freezing pigskin powder puts into low, easy to operate, protein degradation effect The advantage of good, antibacterial peptide yield；
Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 2-4kDa, obtains 2-4kDa peptide mixing below Object, then the NF membrane nanofiltration through 400-600Da obtain the antibacterial peptide crude extract of 400-600Da or more, spare, step separation Belong to physics mode, do not introduce chemical reagent, preferably saves the primary characteristic of small molecular protein peptide, and digest to pigskin Liquid, which has, preferably isolates and purifies effect；
Purifying：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted, is flowed with distilled water after loading Speed is 0.4-0.6mL/min, and automatic fraction collector timed collection detects often light absorption value A214 of the pipe collection liquid at 214nm, The antibacterial activity of each pipe collection liquid is measured, the higher component of antibacterial activity is collected, is freeze-dried up to pigskin antibacterial peptide, the antibacterial Peptide can quickly kill bacterium, and have the antibacterial characteristics of wide spectrum, to Escherichia coli, pseudomonas aeruginosa, Staphylococcus aureus Bacterium, saccharomycete all have apparent antibacterial action, and have the function of certain enhancing body's immunity, can be applied to food, Medicine, field of health care products.
Preferably, the weight ratio of L- rhamnoses and D- rhamnoses is 100 in rhamnose in fermentation of Aspergillus niger step: 2.57-2.63.The special proportioning of L- rhamnoses and D- rhamnoses can not only effectively reduce the acid of aspergillus niger metabolism in the rhamnose Property unproductive absorption of the protease to pigskin gruel, improve acid protease to the hydrophobic amino acids such as Phe, Trp, Tyr site Touch opportunity, and then carry out digestion, the vigor of acid protease is improved, but also memebrane protein can be dissolved, more preferably by thalline Interior protease is secreted into zymotic fluid, is increased the hydrolysis of pigskin gruel, is improved the yield of antibacterial polypeptide, in addition, the rhamnose The growth of aspergillus niger can also be inhibited by being added, the antibacterial polypeptide obtained using hydrolysis due to avoiding aspergillus niger from being needed because growing, to pig The increasing that skin gruel is converted into antibacterial polypeptide promotees significant effect, improves the yield of antibacterial polypeptide.
Compared with the prior art, the advantages of the present invention are as follows：1）Extracting method of the present invention have put into it is low, easy to operate, The advantage that protein degradation effect is good, antibacterial peptide yield and purity are high, is easy to industrial-scale production, has good using valence Value and market potential；2）Pretreatment of raw material of the present invention can make the degreasing efficiency of pigskin and degreasing rate high, can remove smelling removal substance, The loss of protein can be reduced simultaneously, and the generation for the impurity that bacterium peptide mouthfeel, smell and shelf-life influence of avoiding creating antagonism improves The quality of antibacterial peptide；3）The extracting method extracts antibacterial peptide using fermentation method, and Aspergillus Niger Growth will produce each during breeding Kind metabolic enzyme, such as acid protease and neutral proteinase, effectively can be degraded to micromolecule polypeptide by macro-molecular protein； 4）Antibacterial peptide made from the extracting method can quickly kill bacterium, and have the antibacterial characteristics of wide spectrum, have certain enhancing machine The effect of body immunity function can be applied to food, medicine, field of health care products.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of preparation method of pigskin antibacterial polypeptide, including pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, ultrafiltration, Purifying, the specific steps are：
1）Pretreatment of raw material：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:8（g/mL）The valeric acid containing 3% sodium carbonate and 0.031% is added Ethyl ester aqueous solution, degreasing 60min under conditions of temperature is 45 DEG C, ultrasonic frequency is 40kHz, discards lye, is washed to close Neutrality, then it is 1 to press solid-liquid ratio:8（g/mL）Water is added to be homogenized, heating is boiled, filtering, and dry that degreasing pigskin is rotten, the step is super Under the booster action of sound wave, can grease quickly be generated into water-soluble soap salt and glycerine and achieve the purpose that degreasing, degreasing rate Height, and ethyl valerate be added on the one hand promote free aliphatic acid can be combined with alkali generate soap salt, improve degreasing efficiency and take off Fat rate, and trimethylamine, (E, E) -2,4- heptadienals etc. can preferably remove the compound that fishy smell is affected, it improves The quality of antibacterial peptide, the H that on the other hand can be generated with ultrasonic cavitation effect2O2It acts synergistically, protein can be reduced Loss, and excessive alkali can be avoided to destroy protein and other compositions in pigskin, to the bacterium peptide mouth that avoids creating antagonism The generation for the impurity that sense, smell and shelf-life influence, the final quality and yield for improving antibacterial peptide；
2）Fermentation medium is prepared：13 parts of extracting degreasing pigskin gruel, 18 parts of sucrose, 6 parts of sodium chloride, zinc chloride 0.004 by weight Part, 2.4 parts of potassium dihydrogen phosphate, 0.3 part of magnesium sulfate, 0.015 part of ferric sulfate, vitamin B10.001 part, 1100 parts of sterile water mix It closes uniformly to get fermentation medium, the fermentation medium nutrition is non-lipid comprehensively, is able to maintain that the stability of pH, is carried for aspergillus niger For good growing environment, the yield of aspergillus niger metabolite is improved；
Fermentation of Aspergillus niger：The pH for adjusting fermentation medium is 5.5, is 7% inoculated aspergillus niger by inoculum concentration, is 25 DEG C, turns in temperature Speed is the 2d that ferments under conditions of 170r/min, and it is that rhamnose is added in 9g/L then to press additive amount, continues the 10h that ferments, centrifugation supernatant Liquid is zymotic fluid, and the addition of rhamnose can significantly reduce the surface tension of water and cell membrane, change membrane passage, will Acid protease in cell membrane is preferably secreted into extracellularly, is increased the content of acid protease in zymotic fluid, is improved black song The utilization ratio of fungi protease improves the degradation rate of pigskin gruel, additionally it is possible to which the growth for inhibiting microorganism reduces the utilization of product Rate, the final yield for improving antibacterial peptide；Various metabolic enzymes are will produce during Aspergillus Niger Growth breeding in the step, it is such as acid Macro-molecular protein effectively can be degraded to micromolecule polypeptide by protease and neutral proteinase etc., the fermentation step and profit It is compared with enzyme system degradation pigskin gruel with input is low, easy to operate, protein degradation effect is good, the advantage of antibacterial peptide yield；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 4kDa, obtains 4kDa peptide mixers below, NF membrane nanofiltration through 400Da again obtains the antibacterial peptide crude extract of 400Da or more, spare, and step separation belongs to physics side Formula does not introduce chemical reagent, preferably saves the primary characteristic of small molecular protein peptide, and has to pigskin enzymolysis liquid preferable Isolate and purify effect；
4）Purifying：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, Flow velocity is 0.6mL/min, automatic fraction collector timed collection, detects often light absorption value A214 of the pipe collection liquid at 214nm, is surveyed The antibacterial activity of fixed each pipe collection liquid, collects the higher component of antibacterial activity, is freeze-dried up to pigskin antibacterial peptide, the antibacterial peptide Bacterium can quickly be killed, and have the antibacterial characteristics of wide spectrum, to Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, Saccharomycete all has apparent antibacterial action, and has the function of certain enhancing body's immunity, can be applied to food, doctor Medicine, field of health care products.
The weight ratio of L- rhamnoses and D- rhamnoses is 100 in rhamnose in fermentation of Aspergillus niger step:2.57.The rhamnose The special proportioning of middle L- rhamnoses and D- rhamnoses can not only effectively reduce the acid protease of aspergillus niger metabolism to pigskin gruel Unproductive absorption improves touch opportunity of the acid protease to the hydrophobic amino acids such as Phe, Trp, Tyr site, and then carries out Digestion improves the vigor of acid protease, but also can dissolve memebrane protein, and endobacillary protease is more preferably secreted into fermentation In liquid, increases the hydrolysis of pigskin gruel, the yield of antibacterial polypeptide is improved, in addition, the addition of the rhamnose can also inhibit aspergillus niger Growth antibacterial polypeptide is converted into pigskin gruel using hydrolysis obtained antibacterial polypeptide due to avoiding aspergillus niger from being needed because growing Increase and promote significant effect, improves the yield of antibacterial polypeptide.
Embodiment 2：
A kind of preparation method of pigskin antibacterial polypeptide, including pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, ultrafiltration, Purifying, the specific steps are：
1）Pretreatment of raw material：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:10（g/mL）Penta containing 2% sodium carbonate and 0.035% is added Acetoacetic ester aqueous solution, degreasing 45min under conditions of temperature is 40 DEG C, ultrasonic frequency is 45kHz, discards lye, is washed to Weakly acidic pH, then it is 1 to press solid-liquid ratio:10（g/mL）Water is added to be homogenized, heating is boiled, filtering, dry that degreasing pigskin is rotten；
2）Fermentation medium is prepared：10 parts of extracting degreasing pigskin gruel, 20 parts of sucrose, 5 parts of sodium chloride, zinc chloride 0.005 by weight Part, 2.0 parts of potassium dihydrogen phosphate, 0.4 part of magnesium sulfate, 0.01 part of ferric sulfate, vitamin B10.0015 part, 1000 parts of sterile water mix It closes uniformly to get fermentation medium；
Fermentation of Aspergillus niger：The pH for adjusting fermentation medium is 6.0, is 6% inoculated aspergillus niger by inoculum concentration, is 27 DEG C, turns in temperature Speed is fermented 2.5d under conditions of being 150r/min, and it is that rhamnose is added in 8g/L then to press additive amount, continues the 12h that ferments, in centrifugation Clear liquid is zymotic fluid；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 3kDa, obtains 3kDa peptide mixers below, NF membrane nanofiltration through 500Da again obtains the antibacterial peptide crude extract of 500Da or more, spare；
4）Purifying：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, Flow velocity is 0.5mL/min, automatic fraction collector timed collection, detects often light absorption value A214 of the pipe collection liquid at 214nm, is surveyed The antibacterial activity of fixed each pipe collection liquid, collects the higher component of antibacterial activity, is freeze-dried up to pigskin antibacterial peptide.
The weight ratio of L- rhamnoses and D- rhamnoses is 100 in rhamnose in fermentation of Aspergillus niger step:2.6.
Embodiment 3：
A kind of preparation method of pigskin antibacterial polypeptide, including pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, ultrafiltration, Purifying, the specific steps are：
1）Pretreatment of raw material：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:10（g/mL）The aqueous solution containing 2% sodium carbonate is added, in temperature Degreasing 45min under conditions of degree is 40 DEG C, ultrasonic frequency is 45kHz, discards lye, is washed to weakly acidic pH, then press solid-liquid ratio It is 1:10（g/mL）Water is added to be homogenized, heating is boiled, filtering, dry that degreasing pigskin is rotten；
2）Fermentation medium is prepared：10 parts of extracting degreasing pigskin gruel, 20 parts of sucrose, 5 parts of sodium chloride, zinc chloride 0.005 by weight Part, 2.0 parts of potassium dihydrogen phosphate, 0.4 part of magnesium sulfate, 0.01 part of ferric sulfate, vitamin B10.0015 part, 1000 parts of sterile water mix It closes uniformly to get fermentation medium；
Fermentation of Aspergillus niger：The pH for adjusting fermentation medium is 6.0, is 6% inoculated aspergillus niger by inoculum concentration, is 27 DEG C, turns in temperature Speed is fermented 2.5d under conditions of being 150r/min, and it is that rhamnose is added in 8g/L then to press additive amount, continues the 12h that ferments, in centrifugation Clear liquid is zymotic fluid；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 3kDa, obtains 3kDa peptide mixers below, NF membrane nanofiltration through 500Da again obtains the antibacterial peptide crude extract of 500Da or more, spare；
4）Purifying：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, Flow velocity is 0.5mL/min, automatic fraction collector timed collection, detects often light absorption value A214 of the pipe collection liquid at 214nm, is surveyed The antibacterial activity of fixed each pipe collection liquid, collects the higher component of antibacterial activity, is freeze-dried up to pigskin antibacterial peptide.
The weight ratio of L- rhamnoses and D- rhamnoses is 100 in rhamnose in fermentation of Aspergillus niger step:2.6.
Embodiment 4：
A kind of preparation method of pigskin antibacterial polypeptide, including pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, ultrafiltration, Purifying, the specific steps are：
1）Pretreatment of raw material：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:10（g/mL）Penta containing 2% sodium carbonate and 0.035% is added Acetoacetic ester aqueous solution, degreasing 45min under conditions of temperature is 40 DEG C, ultrasonic frequency is 45kHz, discards lye, is washed to Weakly acidic pH, then it is 1 to press solid-liquid ratio:10（g/mL）Water is added to be homogenized, heating is boiled, filtering, dry that degreasing pigskin is rotten；
2）Fermentation medium is prepared：10 parts of extracting degreasing pigskin gruel, 20 parts of sucrose, 5 parts of sodium chloride, zinc chloride 0.005 by weight Part, 2.0 parts of potassium dihydrogen phosphate, 0.4 part of magnesium sulfate, 0.01 part of ferric sulfate, vitamin B10.0015 part, 1000 parts of sterile water mix It closes uniformly to get fermentation medium；
Fermentation of Aspergillus niger：The pH for adjusting fermentation medium is 6.0, is 6% inoculated aspergillus niger by inoculum concentration, is 27 DEG C, turns in temperature Speed is fermented 2.5d under conditions of being 150r/min, and it is that rhamnose is added in 8g/L then to press additive amount, continues the 12h that ferments, in centrifugation Clear liquid is zymotic fluid；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 3kDa, obtains 3kDa peptide mixers below, NF membrane nanofiltration through 500Da again obtains the antibacterial peptide crude extract of 500Da or more, spare；
4）Purifying：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, Flow velocity is 0.5mL/min, automatic fraction collector timed collection, detects often light absorption value A214 of the pipe collection liquid at 214nm, is surveyed The antibacterial activity of fixed each pipe collection liquid, collects the higher component of antibacterial activity, is freeze-dried up to pigskin antibacterial peptide.
Embodiment 5：
The measurement of pigskin gruel degradation rate
The zymotic fluid of selection example 2, embodiment 3, embodiment 4, respectively zymotic fluid filter to obtain supernatant through Buchner funnel, set It is soluble nitrogen sample, Kjeldahl nitrogen determination soluble nitrogen content that 6000r/min centrifugations 15min, which obtains supernatant,.It presses Formula calculates the degradation rate of protein in pigskin：Protein degradation rate（%）=soluble nitrogen sample (mgN/mL)/[In culture medium Nitrogen content (mgN/mL)s ]×100%.Result of calculation is as shown in table 1.
1 pigskin gruel degradation rate of table
As shown in Table 1, the degradation rate of protein is significantly larger than embodiment 3 and embodiment 4 in pigskin in embodiment 2, illustrates raw material Aqueous solution contains the loss that ethyl valerate can reduce protein in pre-treatment step, and then improves the degradation rate of protein, black The specific proportions of L- rhamnoses and D- rhamnoses can also improve pigskin gruel degradation rate in rhamnose in aspergillus fermentation step.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention, Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.

Claims (8)

1. a kind of preparation method of pigskin antibacterial polypeptide, including it is pretreatment of raw material, fermentation medium preparation, fermentation of Aspergillus niger, super Filter, purifying, it is characterised in that：The fermentation of Aspergillus niger step is：The pH of fermentation medium is adjusted, inoculated aspergillus niger is fermented, so After rhamnose is added, continue to ferment, centrifuged supernatant is zymotic fluid.
2. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：The inoculum concentration is 5- 7%, fermentation pH is 5.5-6.5, temperature is 25-30 DEG C, rotating speed 120-170r/min, time 2-3d.
3. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：The rhamnose adds Dosage is 7-9g/L, continues the 10-15h that ferments.
4. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：L- in the rhamnose The weight ratio of rhamnose and D- rhamnoses is 100:2.57-2.63.
5. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：The pretreatment of raw material Step is：It is 1 by solid-liquid ratio by fresh pig skin stripping and slicing:8-12（g/mL）It is added the penta of sodium carbonate containing 1-3% and 0.031-0.038% Acetoacetic ester aqueous solution, degreasing 30-60min, discards alkali under conditions of temperature is 35-45 DEG C, ultrasonic frequency is 40-50kHz Liquid is washed to weakly acidic pH, then it is 1 to press solid-liquid ratio:8-12（g/mL）Water is added to be homogenized, heating is boiled, filtering, dry degreasing pigskin It is rotten.
6. a kind of preparation method of pigskin antibacterial polypeptide according to claim 5, it is characterised in that：The fermentation medium Preparation steps are：8-13 parts of extracting degreasing pigskin gruel, 18-22 parts of sucrose, 4-6 parts of sodium chloride, zinc chloride 0.004- by weight 0.006 part, 1.8-2.4 parts of potassium dihydrogen phosphate, 0.3-0.5 parts of magnesium sulfate, 0.005-0.015 parts of ferric sulfate, vitamin B10.001-0.002 parts, 900-1100 parts of sterile water are uniformly mixed to get fermentation medium.
7. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：The ultrafiltration step For：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 2-4kDa, obtains 2-4kDa peptide mixers below, then pass through The NF membrane nanofiltration of 400-600Da obtains the antibacterial peptide crude extract of 400-600Da or more, spare.
8. a kind of preparation method of pigskin antibacterial polypeptide according to claim 1, it is characterised in that：The purification step For：Chromatographic column is balanced with distilled water, antibacterial peptide crude extract is subjected to gel chromatography, is eluted with distilled water after loading, flow velocity is 0.4-0.6mL/min, automatic fraction collector timed collection detect often light absorption value A214 of the pipe collection liquid at 214nm, measure The antibacterial activity of each pipe collection liquid collects the higher component of antibacterial activity, is freeze-dried up to pigskin antibacterial peptide.
CN201810356047.XA 2018-04-19 2018-04-19 A kind of preparation method of pigskin antibacterial polypeptide Withdrawn CN108707637A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110613094A (en) * 2019-11-12 2019-12-27 齐鲁工业大学 Processing method of low-fat steamed pork with rice stuffing

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110613094A (en) * 2019-11-12 2019-12-27 齐鲁工业大学 Processing method of low-fat steamed pork with rice stuffing

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