CN103468604B - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN103468604B CN103468604B CN201310327938.XA CN201310327938A CN103468604B CN 103468604 B CN103468604 B CN 103468604B CN 201310327938 A CN201310327938 A CN 201310327938A CN 103468604 B CN103468604 B CN 103468604B
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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 at raising efficiency of feed utilization, increases production performance, regulating intestinal canal microecological balance, increases the aspects such as immunizing power and has significant promoter action.On feed and animal productiong, more microorganism is used to have milk-acid bacteria, yeast, genus bacillus and photosynthetic bacterium at present, wherein genus bacillus has the biological nature of the uniqueness such as strong stress resistance, high temperature high voltage resistant, acid and alkali-resistance cholate, easily storage, evaluate from animal productiong and fodder industry angle, it can meet the requirement that feed microorganism modern industry is produced better.
Subtilis can produce a large amount of proteolytic enzyme, amylase, lipase and other beneficial agents in metabolic process kind, enter after in organism and can surely grow fast, good micro-ecological environment is formed in enteron aisle, have and strengthen animal digestion ability, degraded antinutritional factor, increase efficiency of feed utilization, strengthen the effects such as animal immunizing power.What the Ministry of Agriculture of China announced just can include subtilis in the feed level microbe additive bacterial classification of Direct-fed animal, and it, as safe probiotic bacterium, is now widely used in field of fodder.
Dregs of beans is the most important vegetable protein sources of livestock and poultry, and its crude protein content up to 43% ~ 48%, and is made up of the amino acid comparatively balanced, and especially containing the Methionin that other plant feed easily lacks, its content is up to 2. 5% ~ 2. 8%.But also there is multiple antinutritional factor in dregs of beans, greatly have impact on the feeding value of dregs of beans.Trypsin inhibitor is antinutritional factor main in dregs of beans, and the content in dregs of beans is about 2%.Trypsin inhibitor can hinder the effect of proteolytic ferment in enteron aisle and protein digestibility is declined, pancreas itself can also be acted on, the pancreas enzyme of stimulating pancreas supersecretion, protein endogenous metabolism is caused to lose, cause animal to occur digestion and absorption function imbalance or disorderly, suppress the growth phenomenon of body.Utilize microorganism solid fermentation can improve this situation.Subtilis has very strong proteolytic enzyme, lipase, amylase isoreactivity.
But, now report that the microorganism strains of production exists problems, as long in 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 (acid-soluble albumen) content is not high after fermentation.
Summary of the invention
For prior art Problems existing, the object of the invention is to design the technical scheme that a kind of subtilis and application thereof are provided.
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 bioactive peptide protein fodder in fermentative production.
Described 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 cultivated through plate culture medium activation, seed flask, 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 carries out drying, pulverizing, namely obtains being rich in bioactive peptide protein fodder.
A kind of described method utilizing bacillus subtilis strain KF-2 fermentative production to be rich in 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 utilizing bacillus subtilis strain KF-2 fermentative production to be rich in bioactive peptide protein fodder, it is characterized in that described step 2) in solid state fermentation conditions be: fully stir during inoculation, monitor temperature change in fermenting process, when temperature is higher than 50 DEG C, carry out stirring process, 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 order.
Mutagenesis of the present invention obtains a kind of bacillus subtilis strain KF-2, and this bacterial strain can secrete the enzyme systems such as abundant proteolytic enzyme, has the ability of significant fermented bean dregs.Utilize this bacterial strain and corresponding zymotechnique to carry out solid state fermentation process to dregs of beans, the protein of macromolecule in degradable bean cake powder, make it change into and there is bioactive little peptide protein, facilitate the absorption of animal.Meanwhile, after bean pulp fermentation, antinutritional factor wherein and antigen protein obtain and effectively degrade, and 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.
The nutritive value of dregs of beans is significantly improved after using this strain fermentation dregs of beans, measuring some index results is: the crude protein content of fermented bean dregs reaches more than 51%, acid-soluble protein content reaches more than 8%, viable count reaches 4,000,000,000/gram of 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, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention, NM specific experiment method in the following example, experimental technique carries out routinely usually.
Embodiment 1: the separation of bacillus subtilis strain KF-2, purifying, mutagenesis and qualification
From natural condition, screening obtains subtilis, after after many wheels selection by mutation of our company, bacterial strain obtains genetic improvement, its degraded and transform the ability of dregs of beans and be significantly improved, and the production performance of bacterial strain improves greatly.
Prepare beef-protein medium flat board, separation and Culture from the organic matters such as beans, obtain subtilis, the bacterial strain of acquisition is observed through bacterium colony, morphology microscopical identification is subtilis.
Utilize fermentation of bacillus subtilis dregs of beans, repeated screening and purification and rejuvenation (step: separation and Culture subtilis from fermentation good soybean products, obtain purifying bacterial strain to continue to cultivate, then further fermented bean dregs, its fermentation capacity is improved constantly), the performance of strain fermentation dregs of beans is improved, after showing fermented bean dregs, viable count strengthens, (dregs of beans protein content can reach more than 5% in the aspects such as acid-soluble protein content raising, the little peptide content such as acid-soluble albumen improves more than 10%, lactic acid content improves more than 2%), this Strain Designation is bacillus subtilis strain KF-2.
Bacillus subtilis strain KF-2 cultivates and propagation method:
Bacillus subtilis strain KF-2 is good at beef-protein medium grow on plates, and this substratum can be used for the Growth and reproduction of bacillus subtilis strain KF-2.Culture temperature 32 DEG C.Beef-protein medium content is extractum carnis 5g, peptone 10g, sodium-chlor 5g, agar 15 ~ 20g, distilled water 1000mL, 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 basis of microscopic observation, somatic cells size is 0.68 ~ 0.85 × 2.0 ~ 3.1 microns, thalline uniform coloring, after cultivating certain hour, can form gemma in somatic cells, gemma size is 0.6 ~ 0.9 × 1.0 ~ 1.5 microns.Gemma ellipse is to column, and be positioned at thalline central authorities or slightly inclined position, 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 DEG C, cultivate after 3 days, bacterium colony presents surface irregularity, opaque, shallow white or micro-yellow.Cultivate bacterium colony and show there is gauffer, bacterium colony shows slightly dry, more smooth, not easily provokes.
After subtilis KF-2 inoculates, after beef-protein medium grow on plates 15h, a large amount of gemma is just had to produce.
Physiological property and metabolic characteristic: subtilis KF-2 easily cultivates, can utilize the gas chromatography matter such as protein, multiple sugar and starch.Be aerophil, the free oxygen in environment can be consumed rapidly, can cultivate as animal intestinal bacterium, cause enteron aisle hypoxemia, promote that useful anerobe grows.
Subtilis KF-2 fermented organic can produce the organic acid such as lactic acid, propionic acid.
The biological enzyme kind that subtilis KF-2 produces is more, as proteolytic enzyme, amylase, cellulase etc.The substratum such as soybean protein isolate, casein has and is significantly hydrolyzed circle, demonstrate comparatively high protein enzymic activity.On starch flat board, observe through iodine staining and have transparent circle, demonstrate Starch Hydrolysis enzymic activity.
The concrete purposes of subtilis KF-2 and effect: subtilis (
bacillus subtilis) bacterial strain KF-2 can be used for fermented bean dregs, produces the high protein feed being rich in active small peptide.
Dregs of beans protein content after fermentation can reach more than 5%, and the little peptide content such as acid-soluble albumen improves obviously, can improve more than 10%, and lactic acid content improves more than 2%.In tunning, be rich in the enzyme system that proteolytic enzyme, amylase etc. are abundant, the objectionable impuritiess such as the antinutritional factor can degraded in dregs of beans.Meanwhile, containing the bacillus subtilis spore that a large amount of resistance is stronger in fermented bean dregs, be convenient to storage and transport.After animal edible, subtilis breeds rapidly in enteron aisle, significantly suppresses the growth of harmful bacteria, improves animal intestinal health, improves the utilization ratio of feed, improves Animal nutrition level, produce 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 cultivated through plate culture medium activation, seed flask, one-level shake-flask culture or fermentor tank amplification culture step by step, obtain seed liquor, above-mentioned plate culture medium, Shake flask medium are PDA substratum; Plate culture medium activation is that the bacterial strain preserved from inclined-plane is transferred to flat board, 32 DEG C of cultivations, then from dull and stereotyped picking 3 single bacterium colonies to seed flask, seed flask 32 DEG C, under 150 r/min shaking table conditions, after cultivating 15 h, seed culture fluid is all 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 flask substratum is: above-mentioned plate culture medium does not add agar, fermention medium: same to seed culture medium.
2) strain liquid in the fermentor tank obtained through step 1) is seeded in dregs of beans by 8 ~ 12% of dregs of beans weight and carries out solid state fermentation, fully stir during inoculation, guarantee that bacterial classification and dregs of beans mix, the time of fermentation is 48 h, monitor temperature change in fermenting process, when temperature is higher than 50 DEG C, carry out stirring process, fully ventilative cooling, when temperature is lower, proper extension fermentation time, in addition, also need in fermenting process to observe fermentation whether normal, smell whether have in the fragrant and yeast wine flavour of beans, fermented product whether turn black, go mouldy, caking etc.
3) by step 2) tunning that obtains dries to moisture≤12%, is crushed to fineness 40 ~ 60 order, namely obtains being rich in peptide protein feed alive.
Through qualification, what embodiment 2 obtained be rich in crude protein content in peptide protein feed alive reaches more than 51%, acid-soluble protein content reaches more than 6%, viable count reaches 2,000,000,000/gram of 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 acid-soluble albumen improves more than 4%, and lactic acid content improves more than 1%.
Claims (2)
1. subtilis (
bacillus subtilis) bacterial strain KF-2, deposit number is CGMCC No.7690.
2. utilize bacillus subtilis strain KF-2 fermentative production as claimed in claim 1 to be rich in a method for bioactive peptide protein fodder, it is characterized in that comprising following processing step:
1) described bacillus subtilis strain KF-2 is cultivated through plate culture medium activation, seed flask, 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, and inoculum size is 8 ~ 12% of dregs of beans weight, fully stirs during inoculation, monitor temperature change in fermenting process, when temperature is higher than 50 DEG C, carry out stirring process, fermentation time is 48 h;
3) by step 2) tunning that obtains dries to moisture≤12%, and smashing fineness 40 ~ 60 order, namely obtains being rich in bioactive peptide protein fodder.
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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 |
| 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 |
| 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 |
| 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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Denomination of invention: A Bacillus subtilis and its application Effective date of registration: 20221114 Granted publication date: 20150715 Pledgee: Hangzhou United Rural Commercial Bank Co.,Ltd. Haining sub branch Pledgor: ZHEJIANG COFINE BIOTECH. Inc.,Ltd. Registration number: Y2022980021846 |
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Date of cancellation: 20230823 Granted publication date: 20150715 Pledgee: Hangzhou United Rural Commercial Bank Co.,Ltd. Haining sub branch Pledgor: ZHEJIANG COFINE BIOTECH. Inc.,Ltd. Registration number: Y2022980021846 |
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Denomination of invention: A Bacillus subtilis and its application Effective date of registration: 20230901 Granted publication date: 20150715 Pledgee: Hangzhou United Rural Commercial Bank Co.,Ltd. Haining sub branch Pledgor: ZHEJIANG COFINE BIOTECH. Inc.,Ltd. Registration number: Y2023980054897 |