CN103468604A - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN103468604A CN103468604A CN201310327938XA CN201310327938A CN103468604A CN 103468604 A CN103468604 A CN 103468604A CN 201310327938X A CN201310327938X A CN 201310327938XA CN 201310327938 A CN201310327938 A CN 201310327938A CN 103468604 A CN103468604 A CN 103468604A
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Abstract
The invention relates to bacillus subtilis and an application thereof, and belongs to the technical field of biological engineering. The invention provides a bacillus subtilis strain KF-2 with a preservation number of CGMCC No. 7690. Besides, the invention provides a method for applying the bacillus subtilis in fermentation production of active peptide-rich protein feed. A plurality of indexes are determined after soybean meal is fermented by the strain. Crude protein content reaches over 51%; acid soluble protein content reaches over 6%; viable counts reach 2 billion per gram of the fermented soybean meal; and volatile salt-based nitrogen is lower than 50 mg/100 g.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of subtilis and application thereof.
Background technology
Feed level microbe additive is the class novel fodder additive that development in recent years is got up, and improving efficiency of feed utilization, increases production performance, the regulating intestinal canal microecological balance, and the aspects such as increase immunizing power have significant promoter action.In feed and animal production, use more microorganism that milk-acid bacteria, yeast, genus bacillus and photosynthetic bacterium are arranged at present, wherein genus bacillus has unique biological natures such as strong stress resistance, high temperature high voltage resistant, acid and alkali-resistance cholate, easy storage, from animal, produce and the evaluation of fodder industry angle, it can meet the requirement that feed is produced with the microorganism modern industry better.
Subtilis can produce a large amount of proteolytic enzyme, amylase, lipase and other useful factors in the metabolic process kind, after entering in organism, can surely grow fast, form good micro-ecological environment in enteron aisle, there is the animal digestion of reinforcement ability, the degraded antinutritional factor, increase efficiency of feed utilization, strengthen the effects such as animal immunizing power.What China Ministry of Agriculture announced can just comprise subtilis in the feed level microbe additive bacterial classification of Direct-fed animal, and it,, as safe probiotic bacterium, now is widely used in field of fodder.
Dregs of beans is the most important vegetable protein of livestock and poultry source, and its crude protein content is up to 43%～48%, and is comprised of the amino acid than balance, especially contains easily scarce Methionin of other plant feed, and its content is up to 2. 5%～2. 8%.But also there is multiple antinutritional factor in dregs of beans, greatly affected the feeding value of dregs of beans.The trypsinase statin is antinutritional factor main in dregs of beans, and the content in dregs of beans is 2% left and right.The trypsinase statin can hinder the effect of proteolytic ferment in enteron aisle and protein digestibility is descended, can also act on pancreas itself, the pancreas enzyme of stimulating pancreas supersecretion, cause the loss of protein endogenous metabolism, cause animal digestion and absorption function imbalance or disorderly to occur, suppress the growth phenomenon of body.Utilize microorganism solid fermentation can improve this situation.Subtilis has very strong proteolytic enzyme, lipase, amylase isoreactivity.
But, there are problems in the microorganism strains that now report is produced, as long as fermentation time (therefore easily cause and go mouldy), fermenting process moisture are too high, the degraded of antinutritional factor not thoroughly, crude protein and the problem such as little peptide (sour molten albumen) content is not high after fermentation.
Summary of the invention
The problem existed for prior art, the object of the invention is to the technical scheme that design provides a kind of subtilis and application thereof.
Subtilis in the present invention (
bacillus subtilis) bacterial strain KF-2, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation day: on 06 09th, 2013, preserving number: CGMCCNo.7690.
Described bacillus subtilis strain KF-2 is rich in the application in the bioactive peptide protein fodder in fermentative production.
Describedly a kind ofly utilize bacillus subtilis strain KF-2 fermentative production as claimed in claim 1 to be rich in the method for bioactive peptide protein fodder, it is characterized in that comprising following processing step:
1) described bacillus subtilis strain KF-2 is passed through to plate culture medium activation, seed shake-flask culture, one-level shake-flask culture or fermentor tank amplification culture step by step, obtain seed liquor;
2) seed liquor obtained through step 1) is seeded in dregs of beans carries out solid state fermentation;
3) by step 2) tunning that obtains dried, pulverized, and obtains being rich in the bioactive peptide protein fodder.
A kind of described method of utilizing bacillus subtilis strain KF-2 fermentative production to be rich in the bioactive peptide protein fodder, is characterized in that described step 2) in inoculum size be 8～12% of dregs of beans weight.
A kind of described method of utilizing bacillus subtilis strain KF-2 fermentative production to be rich in the bioactive peptide protein fodder, it is characterized in that described step 2) in solid state fermentation conditions be: during inoculation, fully stir, in fermenting process, monitor temperature changes, temperature is during higher than 50 ℃, carry out the stirring processing, fermentation time is 48 h.
Described a kind of bacillus subtilis strain KF-2 fermentative production is rich in the method for bioactive peptide protein fodder, it is characterized in that in described step 3), tunning is dried to moisture≤12%, smashing fineness 40～60 orders.
Mutagenesis of the present invention obtains a kind of bacillus subtilis strain KF-2, and this bacterial strain can be secreted the abundant enzymes such as proteolytic enzyme system, has the ability of significant fermented bean dregs.Utilize this bacterial strain and corresponding zymotechnique to carry out the solid state fermentation processing to dregs of beans, the protein of macromolecule in the degradable bean cake powder, change into it and have bioactive little peptide protein, promoted the absorption of animal.Simultaneously, after bean pulp fermentation, antinutritional factor wherein and antigen protein have obtained degraded effectively, significantly improve the nutritional quality of dregs of beans, improve animal to the utilising efficiency of dregs of beans and improve animal intestinal and promotion health.
Use after this strain fermentation dregs of beans the nutritive value that has significantly improved dregs of beans, measuring some index results is: the crude protein content of fermented bean dregs reaches more than 51%, the molten protein content of acid reaches more than 8%, viable count reaches 4,000,000,000/gram fermented bean dregs, total volatile basic nitrogen, lower than 50 mg/100g, contains higher protease activity simultaneously.
Embodiment
In order to make the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described, NM concrete experimental technique in the following example, experimental technique carries out routinely usually.
Embodiment 1: the separation of bacillus subtilis strain KF-2, purifying, mutagenesis and evaluation
From natural condition, screening obtains subtilis, after after many wheels selection by mutation of our company, bacterial strain has obtained genetic improvement, its degraded and the ability that transforms dregs of beans are significantly improved, the production performance of bacterial strain improves greatly.
Prepare the beef-protein medium flat board, separation and Culture from the organic matters such as beans, obtain subtilis, and the bacterial strain of acquisition is subtilis through bacterium colony observation, morphology microscopical identification.
Utilize the fermentation of bacillus subtilis dregs of beans, repeated screening and purification and rejuvenation (step: separation and Culture subtilis from the soybean products that ferments preferably, obtaining the purifying bacterial strain continues to cultivate, then further fermented bean dregs, its fermentation capacity is improved constantly), make the performance of strain fermentation dregs of beans be improved, after showing fermented bean dregs, viable count strengthens, (the dregs of beans protein content can reach more than 5% in the aspects such as the molten protein content raising of acid, the little peptide contents such as the molten albumen of acid improve more than 10%, lactic acid content improves more than 2%), this bacterial strain called after bacillus subtilis strain KF-2.
Bacillus subtilis strain KF-2 cultivates and propagation method:
Bacillus subtilis strain KF-2 is well-grown on the beef-protein medium flat board, and this substratum can be used for the Growth and reproduction of bacillus subtilis strain KF-2.32 ℃ of culture temperature.Beef-protein medium content is extractum carnis 5g, peptone 10g, sodium-chlor 5g, agar 15～20g, distilled water 1000mL, and pH 7.0～7.2.
The morphological feature of bacillus subtilis strain KF-2:
Subtilis KF-2 belongs to bacillus (Bacillus), for gram-positive microorganism, the thalline of the visible rod-short of micro-Microscopic observation, the somatic cells size is 0.68～0.85 * 2.0～3.1 microns, the thalline uniform coloring, after cultivating certain hour, in somatic cells, can form gemma, the gemma size is 0.6～0.9 * 1.0～1.5 microns.Gemma is oval to column, is positioned at thalline central authorities or slightly inclined to one side position, and after sporulation, thalline does not expand.Subtilis KF-2 is without pod membrane, and peritrichous, can move.
Cultural characters: after subtilis KF-2 bacterial strain list colony inoculation, on beef-protein medium 32 ℃, cultivate 3 days after, bacterium colony presents surface irregularity, opaque, shallow white or micro-yellow.Cultivate bacterium colony and show that gauffer is arranged, it is dry, more smooth that bacterium colony shows slightly, and is difficult for provoking.
After subtilis KF-2 inoculation, on the beef-protein medium flat board, after growth 15h, just there is a large amount of gemma to produce.
Physiological property and metabolic characteristic: subtilis KF-2 easily cultivates, and can utilize the gas chromatography matter such as protein, multiple sugar and starch.Be aerophil, can consume rapidly the free oxygen in environment, can be used as the animal intestinal bacterium and cultivate, cause the enteron aisle hypoxemia, promote useful anerobe growth.
Subtilis KF-2 fermented organic can produce the organic acids such as lactic acid, propionic acid.
The biological enzyme kind that subtilis KF-2 produces is more, as proteolytic enzyme, amylase, cellulase etc.Obvious hydrolysis circle is arranged on the substratum such as soybean protein isolate, casein, demonstrate than the high protein enzymic activity.On the starch flat board, observe transparent circle is arranged through iodine staining, demonstrate the Starch Hydrolysis enzymic activity.
Concrete purposes and the effect of subtilis KF-2: subtilis (
bacillus subtilis) bacterial strain KF-2 can be used for fermented bean dregs, produces the high protein feed that is rich in active small peptide.
Dregs of beans protein content after fermentation can reach more than 5%, and the little peptide content such as sour molten albumen improves obviously, can improve more than 10%, and lactic acid content improves more than 2%.In tunning, be rich in the abundant enzyme system such as proteolytic enzyme, amylase, the objectionable impuritiess such as antinutritional factor in the dregs of beans of can degrading.Simultaneously, contain the bacillus subtilis spore that a large amount of resistance is stronger in fermented bean dregs, be convenient to store and transportation.After animal edible, subtilis is breeding rapidly in enteron aisle, significantly suppresses the growth of harmful bacteria, improves animal intestinal health, improves the utilization ratio of feed, improves the Animal nutrition level, produces good economic benefit and ecological benefits.
Embodiment 2: utilize subtilis KF-2 fermentative production to be rich in the method for bioactive peptide protein fodder
1) described subtilis KF-2 is passed through to plate culture medium activation, seed shake-flask culture, one-level shake-flask culture or fermentor tank amplification culture step by step, obtain seed liquor, above-mentioned plate culture medium, shake-flask culture base are the PDA substratum; The plate culture medium activation is that the bacterial strain of preserving from inclined-plane is transferred to flat board, and 32 ℃ of cultivations, then from 3 single bacterium colonies of dull and stereotyped picking to the seed shaking flask, the seed shaking flask is under 32 ℃, 150 r/min shaking table conditions, after cultivating 15 h, seed culture fluid all is inoculated into fermentor tank, fermentor tank aerated culture 24h.
Above-mentioned plate culture medium is: extractum carnis, protein culture medium (extractum carnis 5 g, peptone 10 g, sodium-chlor 5 g, agar 15～20 g, distilled water 1000 mL, pH 7.0～7.2), seed shake-flask culture base is: above-mentioned plate culture medium does not add agar, fermention medium: with the seed substratum.
2) strain liquid in the fermentor tank obtained through step 1) is seeded in dregs of beans and carries out solid state fermentation by 8～12% of dregs of beans weight, during inoculation, fully stir, guarantee that bacterial classification and dregs of beans mix, the time of fermentation is 48 h, in fermenting process, monitor temperature changes, temperature is during higher than 50 ℃, carry out the stirring processing, fully ventilative cooling, when temperature is low, the proper extension fermentation time, in addition, in fermenting process, also need to observe whether fermentation normal, whether smell has beans perfume (or spice) and yeast wine flavour, fermented product in whether turn black, go mouldy, caking etc.
3) by step 2) tunning that obtains dries to moisture≤12%, is crushed to fineness 40～60 orders, obtains being rich in peptide protein feed alive.
Through identifying, the crude protein content in peptide protein feed alive that is rich in that embodiment 2 obtains reaches more than 51%, the molten protein content of acid reaches more than 6%, viable count reaches 2,000,000,000/gram fermented bean dregs, total volatile basic nitrogen is lower than 50mg/100g, protein content can improve more than 5% than initial dregs of beans raw material, and the little peptide content such as sour molten albumen improves more than 4%, and lactic acid content improves more than 1%.
Claims (6)
- Subtilis ( bacillus subtilis) bacterial strain KF-2, deposit number is CGMCCNo.7690.
- 2. bacillus subtilis strain KF-2 as claimed in claim 1 is rich in the application in the bioactive peptide protein fodder in fermentative production.
- 3. one kind is utilized bacillus subtilis strain KF-2 fermentative production as claimed in claim 1 to be rich in the method for bioactive peptide protein fodder, it is characterized in that comprising following processing step:1) described bacillus subtilis strain KF-2 is passed through to plate culture medium activation, seed shake-flask culture, one-level shake-flask culture or fermentor tank amplification culture step by step, obtain seed liquor;2) seed liquor obtained through step 1) is seeded in dregs of beans carries out solid state fermentation;3) by step 2) tunning that obtains dried, pulverized, and obtains being rich in the bioactive peptide protein fodder.
- 4. a kind of method of utilizing bacillus subtilis strain KF-2 fermentative production to be rich in the bioactive peptide protein fodder as claimed in claim 3, is characterized in that described step 2) in inoculum size be 8～12% of dregs of beans weight.
- 5. a kind of method of utilizing bacillus subtilis strain KF-2 fermentative production to be rich in the bioactive peptide protein fodder as claimed in claim 3, it is characterized in that described step 2) in solid state fermentation conditions be: during inoculation, fully stir, in fermenting process, monitor temperature changes, temperature is during higher than 50 ℃, carry out the stirring processing, fermentation time is 48 h.
- 6. a kind of bacillus subtilis strain KF-2 fermentative production as claimed in claim 3 is rich in the method for bioactive peptide protein fodder, it is characterized in that in described step 3), tunning is dried to moisture≤12%, smashing fineness 40～60 orders.
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Cited By (9)
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|---|---|---|---|---|
| CN103766586A (en) * | 2014-02-07 | 2014-05-07 | 范红玉 | Method for promoting formation of bacillus subtilis spores by using metal ions |
| CN105603031A (en) * | 2016-03-09 | 2016-05-25 | 广东海洋大学 | Aquatic protein active peptide preparing method without adopting high-temperature treatment |
| CN107535671A (en) * | 2017-09-27 | 2018-01-05 | 浙江大学 | Improve the microbial fermentation yellow wine lees feed and preparation method of ruminal protein utilization rate |
| CN107821844A (en) * | 2017-11-30 | 2018-03-23 | 山东和美集团有限公司 | Improve feed of duck's egg quality and preparation method thereof |
| CN108497174A (en) * | 2017-02-23 | 2018-09-07 | 丽水学院 | A method of solid state fermentation being carried out to wood fibre hydrolysis raw material using bacillus subtilis and produces fermented feed |
| CN109321619A (en) * | 2018-09-17 | 2019-02-12 | 江苏大学 | The method that pulsed magnetic field sterilization combines high temperature bacterium solid state fermentation grouts to prepare oligopeptide |
| CN109965082A (en) * | 2019-04-15 | 2019-07-05 | 广东大广生物科技有限公司 | A kind of child care pig fermented feed and preparation method thereof |
| CN113801825A (en) * | 2021-10-21 | 2021-12-17 | 厦门海嘉成生物科技有限公司 | Bacillus subtilis for producing active peptide and application thereof |
| CN114231445A (en) * | 2021-11-30 | 2022-03-25 | 唐山仟客莱生物科技有限公司 | Mixed fermentation medium of composite probiotics and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766586A (en) * | 2014-02-07 | 2014-05-07 | 范红玉 | Method for promoting formation of bacillus subtilis spores by using metal ions |
| CN105603031A (en) * | 2016-03-09 | 2016-05-25 | 广东海洋大学 | Aquatic protein active peptide preparing method without adopting high-temperature treatment |
| CN108497174A (en) * | 2017-02-23 | 2018-09-07 | 丽水学院 | A method of solid state fermentation being carried out to wood fibre hydrolysis raw material using bacillus subtilis and produces fermented feed |
| CN107535671A (en) * | 2017-09-27 | 2018-01-05 | 浙江大学 | Improve the microbial fermentation yellow wine lees feed and preparation method of ruminal protein utilization rate |
| CN107535671B (en) * | 2017-09-27 | 2020-09-11 | 浙江大学 | Microbial fermentation yellow wine lees feed for improving rumen protein utilization rate and preparation method thereof |
| CN107821844A (en) * | 2017-11-30 | 2018-03-23 | 山东和美集团有限公司 | Improve feed of duck's egg quality and preparation method thereof |
| CN109321619A (en) * | 2018-09-17 | 2019-02-12 | 江苏大学 | The method that pulsed magnetic field sterilization combines high temperature bacterium solid state fermentation grouts to prepare oligopeptide |
| CN109965082A (en) * | 2019-04-15 | 2019-07-05 | 广东大广生物科技有限公司 | A kind of child care pig fermented feed and preparation method thereof |
| CN113801825A (en) * | 2021-10-21 | 2021-12-17 | 厦门海嘉成生物科技有限公司 | Bacillus subtilis for producing active peptide and application thereof |
| CN114231445A (en) * | 2021-11-30 | 2022-03-25 | 唐山仟客莱生物科技有限公司 | Mixed fermentation medium of composite probiotics and application thereof |
| CN114231445B (en) * | 2021-11-30 | 2024-03-26 | 唐山仟客莱生物科技有限公司 | Mixed fermentation medium of composite probiotics and application thereof |
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