CN102558294B - Method for rapidly extracting forage antibacterial peptides from fermentation liquid - Google Patents
Method for rapidly extracting forage antibacterial peptides from fermentation liquid Download PDFInfo
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- CN102558294B CN102558294B CN201210019952.9A CN201210019952A CN102558294B CN 102558294 B CN102558294 B CN 102558294B CN 201210019952 A CN201210019952 A CN 201210019952A CN 102558294 B CN102558294 B CN 102558294B
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Abstract
The invention provides a method for rapidly extracting forage antibacterial peptides from a fermentation liquid. The method which comprises steps of sterilizing, releasing the antibacterial peptides, adsorbing by wheat bran, filtering, drying and packaging is adopted to rapidly extract the antibacterial peptides from the fermentation liquid of microbes, so a forage-grade antibacterial peptide biological antiseptic can be obtained. The method adopts the cheap wheat bran as an adsorbent or a carrier, has the advantages of rapid extraction, high recovery rate, simultaneous realization of high antibacterial activity and long preservation period, simple technology, low cost, and rapidness and time saving, and allows the safe and stable forage antibacterial peptide biological antiseptic to be prepared.
Description
Technical field
The present invention relates to the sharp separation preparation method of microbial antibacterial peptide, especially relate to a kind of method of utilizing wheat bran to extract antibacterial peptide as sorbent material and feeding carrier from the fermented liquid of microorganism, belong to biological technical field.
Background technology
Antibacterial peptide (Antimicrobial peptide, AMP) is that biogenic one has bioactive micromolecule polypeptide, and that majority has is safe, has a broad antifungal spectrum, feature that thermostability is high.In recent years, microbe-derived antibacterial peptide receives much attention, the antibacterial peptide that particularly milk-acid bacteria, genus bacillus and Brevibacillus laterosporus produce has efficient anti-microbial activity to pathogenic bacterium and the spoilage organism of food source property, can be by human body alimentary canal proteases for decomposing, be the natural biological antiseptic agent nontoxic to human body, be widely used in food and feed industry.
Extensive extracting method is the key that realizes antibacterial peptide suitability for industrialized production cheaply, is also the principal element that restricts at present the antibacterial peptide market competitiveness.The traditional method of extracting antibacterial peptide from fermented liquid has the means such as ammonium sulfate precipitation, organic solvent deposit and vacuum concentration, as the first step that bacteriocin extracts is generally used ammonium sulfate precipitation, but these traditional methods exist conventionally, and the rate of recovery is low, the time long, pollute the shortcomings such as large, be difficult to realize extensive preparation.Therefore,, find lactic acid chain bacterium peptide (Nisin) in the fermented liquid of nineteen forty-four at Lactococcus lactis since, people strive to find the feasible method that extracts antibacterial peptide from microbial fermentation solution always.
In recent years, investigators both domestic and external try to explore to be undertaken by the method for sorbent material adsorbing and extracting antibacterial peptide the extensive preparation of antibacterial peptide.1992, the people such as the U.S. Yang of university of Wyoming reported a kind of easy bacteriocin separation purification method, and the method is first adjusted to 5.5 by fermented liquid pH, and part antibacterial peptide is adsorbed onto on somatic cells, then reduces pH antibacterial peptide is dissociated from somatic cells.But investigator only reports successively and carry out sorption and desorption with thalline itself, sorption and desorption efficiency is all very low, as nisin total recovery only has 10%.1996, the people such as Coventry use Calucium Silicate powder antisticking agent adsorption antibacterial peptide from the fermented liquid of milk-acid bacteria of trade(brand)name " Micro-Cel ", but antibacterial peptide-adsorption complex desorption that the method obtains is very difficult, even all antibacterial peptide can not be disintegrated down in the scope of pH1.0-12.0, only have 1% the sodium laurylsulfonate (SDS) can desorption.But SDS is a kind of washing agent, the antibacterial peptide standby by this legal system should not be used in food.1998, the people such as Janes extract and concentrated antibacterial peptide as sorbent material by silicic acid and rice hull ash from the fermented liquid of milk-acid bacteria, equally, the adsorption complex desorption of using silicic acid to obtain as sorbent material is also very difficult, the actual technological line that they adopt is that concentrated this step reaches by lyophilize.And be the low-temperature and high-speed bactofugation that has adopted 22100g before lyophilize step, its desorption process must be spent the night 4 ℃ of following stirrings again, and the major defect of the method is: technical requirements harshness, cost is higher, is not suitable for for large-scale production.
In the domestic rear extraction research work about antibacterial peptide, also people's patent report in 2003 such as Lian Dong first will adsorb with sorbent materials such as diatomite after fermentation of nisin liquid high-temperature inactivation thalline, then with polyoxyethylene glycol and sodium chloride aqueous solution, carry out desorb, but this patent is not reported method how to remove surfactant polyethylene after desorption.The people such as Zhuan Xuliang patent report in 2006 a kind of desorb reclaim the novel method of nisin, it combines tensio-active agent desorb and nanofiltration method, first regulate the pH, dress desorption column, tensio-active agent desorption of absorption mixed solution, the method that then adopts nanofiltration that the surfactant SDS in solution is removed, when removing SDS, the solution that contains Nisin is concentrated, concentrated solution adopts spray drying process to make dry powder again, finally reclaims biological preservative nisin.
On the other hand, the strategy of employing is that the antibacterial peptide in fermented liquid is adsorbed on to food-grade adsorbent surface, does not carry out desorption and directly applies to food preservation.As within 1996, once reported, antibacterial peptide was adsorbed on to food grade surface; Within 2010, once there is report that bacteriocin is adsorbed on the sorbent material of two kinds of food grade.But food-grade adsorbent price is comparatively expensive, is difficult to large-scale production, does not up to the present also report both at home and abroad and adopt sorbent material that antibacterial peptide is adsorbed on feed.
In sum, the attached preparation method of absorption-desorption of current use does not relate to antibacterial peptide is adsorbed on feeding carrier directly to performance anti-microbial effect and extends preservation term, the method of adsorbing and extracting antibacterial peptide is comparatively loaded down with trivial details, cost is higher, or in product, contain the chemical substance that is difficult for removal, thereby be difficult to large-scale promotion application.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing wheat bran to extract antibacterial peptide as carrier sharp separation from microbial fermentation solution.
For the defect existing in above-mentioned prior art, the inventor, through long-term research and experiment, creates technical scheme of the present invention.Contriver searches out the feeding carrier with adsorptive power, has studied adsorption and the anti-microbial activity of antibacterial peptide and feeding carrier.
Technical scheme of the present invention is: the technological line that employing sterilization release antibacterial peptide, wheat bran adsorb, filter, are dried and pack, thereby realization Quick taking-up antibacterial peptide from microbial fermentation solution.
Provided by the invention a kind of from microbial fermentation solution the method for the feeding antibacterial peptide of rapid extraction, comprise following steps:
1) fermented liquid is processed, sterilization discharges antibacterial peptide;
2) add wheat bran, take wheat bran as sorbent material and carrier carry out wheat bran absorption;
3) filter, collect filtrate and obtain feeding antibacterial peptide.
Also comprise filtrate is dried, vacuum packaging.
Wherein step 1) described fermentation liquor treatment is that fermented liquid acid is adjusted to pH to 3-6,121 ℃ of heating 15-30min, kill and produce bacterium and antibacterial peptide is discharged into fermented liquid from thalline completely.
Wherein step 2) described in add the 4%-10% that the amount of wheat bran is fermentating liquid volume, adsorption temp 30-50 ℃, 100-200r/min stirs 15-60min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran.
Step 3) described being filtered into gauze or strainer filtering, discard liquid, collect antibacterial peptide-wheat bran mixture.
Described filter screen is 50 orders.
Described dry be after the tiling of antibacterial peptide-wheat bran mixture, to be dried, adopt nature to dry or 40-70 ℃ of warm air drying.
Provided by the invention a kind of from microbial fermentation solution the method for the feeding antibacterial peptide of rapid extraction by following steps, formed:
(1) fermented liquid is processed, discharged antibacterial peptide: pH to 3-6 is adjusted in the fermented liquid that microorganism fermentation is produced acid, 121 ℃ of heating 15min, kill and produce bacterium and antibacterial peptide is discharged into fermented liquid from thalline completely;
(2) wheat bran absorption: add the wheat bran of fermentating liquid volume 4%-10% to adsorb, adsorption temp 30-50 ℃, 100-200r/min stirs 15-60min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran;
(3) filter: with gauze or strainer filtering, discard liquid, collect antibacterial peptide-wheat bran mixture;
(4) dry: will after the tiling of antibacterial peptide-wheat bran mixture, to be dried, to adopt nature to dry or 40-70 ℃ of warm air drying;
(5) vacuum packaging: dried wheat bran mixture is carried out to vacuum packaging, obtain the feed grade antibacterial peptide product of ADSORPTION STATE.
Sorbent material of the present invention is wheat bran, and type has sheet or strip.
Utilize the method for extraction antibacterial peptide of the present invention from side genus bacillus (Brevibacillus laterosporu) fermented liquid, to extract the antibacterial peptide of feed grade.
The antibacterial peptide that method provided by the invention obtains can be used in prepares biological preservative.
The antibacterial peptide that method provided by the invention obtains can be used in prepares feed.
The antibacterial peptide that method provided by the invention obtains can be used in prepares fodder additives.
Provided by the invention from fermented liquid the method for rapid extraction antibacterial peptide overcome the drawback of prior art, use sorbent material wheat bran cheaply, antibacterial peptide is directly adsorbed on wheat bran, its adsorption rate is up to 80%-90%, reach the object of utilizing cheap sorbent material to prepare antibacterial peptide from microbial fermentation solution, when obtaining antibacterial peptide high-recovery, realize high antibacterial activity and extended preservation term.The inventive method extraction process is simple, environmental protection, with low cost, shortening time, the method is prepared safe, stable solid-state antibacterial peptide product, can be widely used in fodder industry, for the extensive preparation of antibacterial peptide provides a kind of novel method, there is more extensive and practical economical effectiveness.
Accompanying drawing explanation
Fig. 1 is process flow diagram of the present invention;
Fig. 2 is the anti-microbial activity figure of product of the present invention, and in figure, 1 is wheat bran, and 2 is antibacterial peptide-wheat bran mixture; Left figure is the preservation antibacterial activity test of 7 days under 4 ℃ of conditions, and right figure is the preservation antibacterial activity test of 42 days under 4 ℃ of conditions.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, in embodiment, the each raw material adding, except special instruction, is commercially available.
Embodiment 1
From the fermented liquid of Brevibacillus laterosporus AS1.2738, extract antibacterial peptide, obtain feed grade antibacterial peptide product, step is as follows:
(1) sterilization discharges antibacterial peptide: the fermented liquid that microorganism fermentation is produced is adjusted pH to 3 with acid, and 121 ℃ of heating 15min, kill and produce bacterium, inactivated proteases, precipitation foreign protein and antibacterial peptide is discharged into fermented liquid from thalline completely.
(2) wheat bran absorption: add the wheat bran of fermentating liquid volume 4% to adsorb, 30 ℃ of adsorption temps, 100r/min stirs 15min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran.
(3) filter: 50 mesh filter screens filter, and discard liquid, collect antibacterial peptide-wheat bran mixture.
(4) dry: by being dried after the tiling of antibacterial peptide-wheat bran mixture, can to adopt nature to dry or 40 ℃ of warm air dryings.
(5) vacuum packaging: dried wheat bran mixture is carried out to vacuum packaging, obtain the feed grade antibacterial peptide product of ADSORPTION STATE.
In method of the present invention, the adsorption rate of wheat bran adsorption antibacterial peptide in embodiment 1 flow process is measured, result shows that adsorption rate is 80%.
From the fermented liquid of the full bacillus brevis AS1.2739 of side, extract antibacterial peptide, obtain feed grade antibacterial peptide product, concrete steps are as follows:
(1) sterilization discharges antibacterial peptide: the fermented liquid that microorganism fermentation is produced is adjusted pH to 5 with acid, and 121 ℃ of heating 30min, kill and produce bacterium, inactivated proteases, precipitation foreign protein and antibacterial peptide is discharged into fermented liquid from thalline completely.
(2) wheat bran absorption: add the wheat bran of fermentating liquid volume 7% to adsorb, 40 ℃ of adsorption temps, 150r/min stirs 30min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran.
(3) filter: with gauze or filter-cloth filtering, discard liquid, collect antibacterial peptide-wheat bran mixture.
(4) dry: by being dried after the tiling of antibacterial peptide-wheat bran mixture, can to adopt nature to dry or 55 ℃ of warm air dryings.
(5) vacuum packaging: dried wheat bran mixture is carried out to vacuum packaging, obtain the feed grade antibacterial peptide product of ADSORPTION STATE.
In method of the present invention, the adsorption rate of wheat bran adsorption antibacterial peptide in embodiment 1 flow process is measured, result shows that adsorption rate is 90%.
Embodiment 3
From the fermented liquid of Brevibacillus laterosporus AS1.864, extract antibacterial peptide, obtain feed grade antibacterial peptide product, step is as follows:
(1) sterilization discharges antibacterial peptide: the fermented liquid that microorganism fermentation is produced is adjusted pH to 6 with acid, and 121 ℃ of heating 20min, kill and produce bacterium, inactivated proteases, precipitation foreign protein and antibacterial peptide is discharged into fermented liquid from thalline completely.
(2) wheat bran absorption: add the wheat bran of fermentating liquid volume 10% to adsorb, 50 ℃ of adsorption temps, 200r/min stirs 60min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran.
(3) filter: with gauze or filter-cloth filtering, discard liquid, collect antibacterial peptide-wheat bran mixture.
(4) dry: by being dried after the tiling of antibacterial peptide-wheat bran mixture, can to adopt nature to dry or 70 ℃ of warm air dryings.
(5) vacuum packaging: dried wheat bran mixture is carried out to vacuum packaging, obtain the feed grade antibacterial peptide product of ADSORPTION STATE.
In method of the present invention, the adsorption rate of wheat bran adsorption antibacterial peptide in embodiment 1 flow process is measured, result shows that adsorption rate is for approaching 97%.
Embodiment 4
Definite employing internationally recognized " proteolytic enzyme By Sensitivity Method " of antibacterial peptide, the fermented liquid that embodiment 1-3 is obtained is handled as follows respectively: 1, the centrifugal 15min of 10000rpm removes thalline and obtains supernatant liquor, then through 0.22 μ m membrane filtration, removes and guarantees to remove thalline; 2, supernatant liquor pH is adjusted to 6.5 to get rid of organic acid effect; 3, get equivalent sample liquid, respectively with proteolytic enzyme (stomach en-, proteolytic enzyme E, trypsinase, Proteinase K) 1: 1 volume mixture of solution of 10mg/mL, in 37 ℃ of thermostat water baths, react 2h, reaction finishes to boil in rear 100 ℃ of boiling water baths the 5min enzyme that goes out; 4, get respectively the sample before and after protease treatment, measure antibacterial circle diameter with agar diffusion method, and compare.Result shows: fermented liquid is to proteolytic enzyme E and stomach en-sensitivity, and antibacterial circle diameter is decreased to respectively 8.0 and 14.0mm, and particularly after proteolytic enzyme E processes, bacteriostatic activity loses completely, and this shows that this material that embodiment 1-3 makes is antibacterial peptide.
Embodiment 5 preservation term experiments
The feeding antibacterial peptide that embodiment 1-3 is made preservation 6 weeks under 4 ℃ of conditions respectively, in preservation, within the 7th day and the 42nd day, take out respectively 0.05g wheat bran state product, adopt agar diffusion method to detect its bacteriostatic activity, in 37 ℃ of standing incubators of constant temperature, cultivate 12h, observe inhibition zone changing conditions., the results are shown in shown in accompanying drawing 2, the preservation product of 42 days is compared with preservation on the 7th day, and antibacterial circle diameter is basic identical, and the antibacterial effect of visible wheat bran state antibacterial peptide has no obvious decline.Therefore, the feeding antibacterial peptide product that the present invention obtains not only has higher anti-microbial activity, and can keep the preservation term of growing.
Embodiment 6 fungistatic effect experiments
The feed grade antibacterial peptide that embodiment 3 is made carries out bacteriostatic experiment, adopt agar diffusion method to measure the antimicrobial spectrum of antibacterial peptide product, found that feeding antibacterial peptide product provided by the invention---wheat bran ADSORPTION STATE antibacterial peptide has higher anti-microbial activity, can not only suppress gram-positive microorganism, as streptococcus aureus, bacillus cereus, Listeria monocytogenes, micrococcus luteus, Lactococcus lactis etc., and as Salmonellas, intestinal bacteria and Pseudomonas aeruginosa, it all has good fungistatic effect to Gram-negative bacteria.The feeding antibacterial peptide product that the results show the present invention obtains has higher anti-microbial activity, and multiple food source property spoilage organism and pathogenic bacterium are had to significant fungistatic effect.
The antimicrobial spectrum of table 1 wheat bran ADSORPTION STATE antibacterial peptide
Note: " ++++" represent that antibacterial circle diameter is greater than 20mm; " +++ " represents that antibacterial circle diameter is greater than 15mm and is less than 20mm.
Embodiment 7 feeding effect experiments
The feeding antibacterial peptide that embodiment 1-3 is made is for the raising of broiler chicken, addition means is shown in Table 2, result shows that broiler feeding is after 30 days, adds the low dose group of antibacterial peptide product and compares with the control group that does not add antibacterial peptide with high dose group, and survival rate improves respectively 5%, 10%; Body weight generally increases, and high dose group be greater than low dose group (compare with contrast, low dose group average weight gain 69g, rate of body weight gain is 5.24%; High dose group average weight gain 210g, rate of body weight gain is 15.95%).This shows that feeding antibacterial peptide is safe, and broiler chicken is had to somatotrophic effect.
The feeding antibacterial peptide of table 2 is for the addition means of broiler fodder
Claims (5)
1. a method for the feeding antibacterial peptide of rapid extraction from side genus bacillus (Brevibacillus laterosporu) fermented liquid, is characterized in that comprising following steps:
1) fermented liquid is processed, antibacterial peptide is discharged from thalline; Described fermentation liquor treatment is that fermented liquid acid is adjusted to pH to 5-6, and 121 ℃ of heating 15-30min, kill and produce bacterium and antibacterial peptide is discharged into fermented liquid from thalline completely;
2) add wheat bran, take wheat bran as sorbent material and carrier carry out wheat bran absorption; The 7%-10% that the described amount that adds wheat bran is fermentating liquid volume, adsorption temp 30-50 ℃, 100-200r/min stirs 15-60min, makes antibacterial peptide fully be adsorbed on wheat bran surface, makes the mixture of antibacterial peptide-wheat bran; Wherein, step 2) in, adsorption rate reaches 80%-90%;
3) use strainer filtering, described filter screen is 50 orders, discards liquid, collects filtrate and obtains feeding antibacterial peptide;
4) filtrate is dry, vacuum packaging, described dry be after the tiling of antibacterial peptide-wheat bran mixture, to be dried, adopt nature to dry or 40-70 ℃ of warm air drying.
2. the antibacterial peptide obtaining according to the method for claim 1.
3. antibacterial peptide claimed in claim 2 is in the application of preparing in biological preservative.
4. antibacterial peptide claimed in claim 2 is in the application of preparing in feed.
5. wheat bran application in method described in claim 1 as sorbent material.
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| CN107936087B (en) * | 2017-11-20 | 2021-02-02 | 河北科技大学 | Green and efficient extraction and preparation method of microbial source antibacterial peptide additive |
| CN111118091A (en) * | 2018-10-31 | 2020-05-08 | 宁夏乙征生物工程有限公司 | Production method of antibacterial peptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293201A (en) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | Process for separating and extracting nisin from fermented liquid |
| CN1623412A (en) * | 2003-12-03 | 2005-06-08 | 孙振钧 | Earthworm active peptide antibacerial feed material and its production method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293201A (en) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | Process for separating and extracting nisin from fermented liquid |
| CN1623412A (en) * | 2003-12-03 | 2005-06-08 | 孙振钧 | Earthworm active peptide antibacerial feed material and its production method |
Non-Patent Citations (2)
| Title |
|---|
| 侧孢短芽孢杆菌S62-9产抗菌物质的分离纯化及部分特性的研究;杨倩 等;《河北农业大学学报》;20100331;第33卷(第2期);74-78 * |
| 杨倩 等.侧孢短芽孢杆菌S62-9产抗菌物质的分离纯化及部分特性的研究.《河北农业大学学报》.2010,第33卷(第2期),74-78. |
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