CN105104712A - Microbiological feed additive and preparation method thereof - Google Patents
Microbiological feed additive and preparation method thereof Download PDFInfo
- Publication number
- CN105104712A CN105104712A CN201510649272.9A CN201510649272A CN105104712A CN 105104712 A CN105104712 A CN 105104712A CN 201510649272 A CN201510649272 A CN 201510649272A CN 105104712 A CN105104712 A CN 105104712A
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- Prior art keywords
- bacteriocin
- feed
- bacillus coagulans
- culture medium
- additive
- Prior art date
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Links
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a microbiological feed additive. The feed additive contains bacillus coagulans FM603 or a fermentative substance of the bacillus coagulans FM603; the fermentative substance of the bacillus coagulans FM603 contains bacteriocin and has antibacterial activity on gram-positive pathogenic bacteria such as Listeria monocytogenes, staphylococcus aureus, methicillin resistant staphylococcus aureus, clostridium perfringens, clostridium firmus and the like. The molecular weight of the bacteriocin is 4276.45 Da, a partial amino acid sequence is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro, and the bacteriocin is more stable under the processing of heat, acid, pepsin or trypsin, is likely to be degraded by pronase and loses activity. With the adoption of the feed additive, the egg laying rate of laying hens can be increased, the feed-egg ratio is reduced, and the egg quality is improved; the feed intake and the daily gain of piglets are increased, and the feed-meat ratio is decreased; the daily gain and the serum muramidase content of broilers are increased, and the feed-meat ratio and the death rate are decreased.
Description
Technical field
The present invention relates to microorganism and cultivation thereof and application technology, be specifically related to a kind of feed addictive containing bacillus coagulans FM603 and preparation method thereof.
Background technology
Antibiotic is the secondary metabolite that microorganism produces, and has antibacterial activity to other microorganism.The forties in 20th century, scientists finds that the antibiotic of feeding animals low dosage can improve the production performance of animal, increases economic efficiency.Within decades afterwards, antibiotic is widely used in livestock-raising industry, makes an addition in feed and is used as growth promoter, or is used as healing potion in cultivation site.Antibiotic use improves growth efficiency and the feed conversion rate of animal significantly, plays an important role to the development of livestock.But antibiotic use also brings problems, the medicament residue etc. in the propagation of such as drug-resistant bacteria, superinfection and animal product.What the appearance of drug-fast bacteria was serious threatens human health, and for anti-methicillinum staphylococcus aureus, the death rate infecting this drug resistance strain is three times of common bacterial strain, and medical expense and hospital stays also increase a lot.In the U.S. and Britain, about have the staphylococcus aureus of 40-60% to have Methicillin resistance, in China, the Escherichia coli of about 60-70% are insensitive to fluoroquinolone antibiotics.A large amount of use antibiotic, and violate withdrawal time and specify, easily cause the medicament residue in animal product to exceed standard, such animal product is difficult to enter international market, also can there is potential threat to the health of people.Report was had in the last few years during China's food security accident, fast-growing chicken event at the beginning of 2013 and the outburst of bird flu bring massive losses to poultry farming industry, the concern of consumers to livestock-raising industry also day by day increases, how to ensure the high productivity energy of animal, produce safe meat, egg, dairy products simultaneously, be related to the sustainable and stable development of livestock-raising industry.European Union completely forbade antibiotic in 2006 and is added in feed as growth promoter, and Korea S also have disabled antibiotics growth promoter in 2011.Nowadays whether China be also faced with and forbid adding antibiotic problem in feed, and this also impels feed and breeding enterprise, and the researcher of agriculture and animal husbandry universities and colleges is actively devoted to exploitation and the use of biologic product, to reduce or the application of substitute antibiotics.
Bacillus is Gram-positive, a sporiferous bacteroid, is present in occurring in nature widely.Bacillus can be converted into spore form under certain condition, and gemma to external world poor environment such as high temperature, high pressure, ultraviolet etc. has extremely strong resistance.Bacillus is industrially used as the production bacterial classification of amylase and protease, the also long-term making for the based food that ferments.Bacillus is of value to digestion and the intestinal health of food, can promote the production performance of animal, is therefore also used as food supplement and animal probio.A large amount of datas shows, bacillus can improve the production performance of animal, reduces pathogenic bacterial infection probability, and improve animal body immunity, reducing the ammonia concentration of breeding environment, is the favorable substitutes of antibiotics growth promoter.Bacillus is grown by stimulating animal intestinal villus, improves nospecific immunity, produces antibacterial substance and plays a role.Bacteriocin is one of antibacterial substance of bacillus generation, is the polypeptide class antibacterial material of microorganism by Ribosome biogenesis, comprises drug-fast bacteria have good antibacterial activity to a lot of pathogen.Bacillus can produce bacteriocin class antibacterial material, as subtilin, sublancin, subtilosin, lichenin etc.Bacteriocin is the important functional material of probio; the researcher of cock university finds; the lactic acid bacteria of bacteriocinogeny effectively can reduce the infection of listeria monocytogenes to mouse; but obtain the immunogene of this bacteriocin at listeria monocytogenes after, lactic acid bacteria loses the defencive function to mouse.There are some researches show, bacteriocin can reduce glucuroide in animal intestinal and glucuronidase activity, promotes broiler growth, and improve production performance, mechanism is similar to antibiotic.Therefore, compared with common bacillus, the production performance of bacillus to animal of bacteriocinogeny has more good facilitation, has larger application potential in actual production.
Summary of the invention
The object of the invention is to provide a kind of additive for microbe feedstuff, containing bacillus coagulans FM603 or its fermentation culture medium in described feed addictive.Containing bacteriocin in bacillus coagulans FM603 fermentation culture medium, to gram-positive bacterium, there is good antibacterial action.This bacteriocin molecular weight is 4276.45Da, and partial amino-acid series is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro.This bacteriocin can change gram-positive bacteria cell membrane permeability, causes efflux of K+ ions in cell.
Bacillus coagulans FM603 can adopt conventional medium under household condition fermented and cultured, but can obtain more high-biomass in improvement tryptone culture medium, improvement tryptone culture medium consists of: quality percent by volume, tryptone 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium chloride 0.1%, sodium dihydrogen phosphate 0.05%, distilled water 1000mL, pH7.0, condition of culture is 30 DEG C, and 150rpm cultivates 20h.
The invention provides a kind of preparation method of mentioned microorganism feed addictive, produce with bacillus coagulans FM603 strain fermentation, FM603 is comprised the steps: to line LB agar plate, 30 DEG C of incubated overnight, picking list colony inoculation is in 10mLLB culture medium, 150rpm, 30 DEG C of incubated overnight are as 1 grade of seed liquor, 1 grade of seed liquor is inoculated in 1000mL by 1% inoculum concentration and improves tryptone culture medium, 150rpm, 30 DEG C of incubated overnight obtain 2 grades of seed liquor, 2 grades of seed liquor are inoculated in 100L by 10% inoculum concentration and improve tryptone culture medium, 150rpm, 30 DEG C of fermentation 24h obtain zymotic fluid, zymotic fluid is mixed rear 30 DEG C of 36h that ferment with the palm kernel meal after sterilizing according to 1:1 ratio, dry to water content 10%, flour is to crossing 40 eye mesh screens and get final product.
Bacillus coagulans FM603 of the present invention, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 17th, 2014 and (is called for short CGMCC, address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is bacillus coagulans (Bacilluscoagulans), and preserving number is CGMCCNO.10221.
Bacterial strain FM603 of the present invention well-grown on normal temperature and conventional medium, bacterium colony is white on TA, irregular, rough surface.Gram-positive, produces gemma.By the 16SrRNA sequence of this bacterium in American National Biotechnology Information center (NCBI) database BLAST comparison, be 99% with the homology of bacillus coagulans, through API50CHB kit (bioMerieux, France) identify that the similarity of this bacterium and bacillus coagulans is 99.7%, therefore judge that this bacterium is as bacillus coagulans, name FM603.
The present invention also provides the method for the above-mentioned bacteriocin of a kind of fermenting and producing, comprises the steps:
1) fermented supernatant fluid preparation: bacillus coagulans FM603 is inoculated in 10mL LB liquid medium overnight incubation, 1mL nutrient solution is inoculated in 1000mL and improves tryptone culture medium, 200rpm, cultivate 20h for 30 DEG C, the centrifugal removing thalline of 10000rpm, obtains fermented supernatant fluid;
2) bacteriocin crude extract preparation: by ion column 50mM, the phosphate buffer balance of pH6.5, fermented supernatant fluid passes through ion column with the flow velocity of 50mL/min, supernatant by ion column is discarded, after rinsing ion column with phosphate buffer, with the phosphate buffer (50mM containing 1MNaCl, pH6.5) elution ionic post, flow velocity is 10mL/min, collect eluent 100mL, by eluent by C18 solid-phase extraction column, acetonitrile solid-phase extraction column with 100% also collects eluent, after eluent freeze drying, be dissolved in 50mM, in the phosphate buffer of pH6.5, bacterioid element crude extract is obtained after filtration,
3) purifying of bacteriocin: bacterioid element crude extract high-efficient liquid phase chromatogram purification, use C18 reversed-phase column (250mm × 10mm, 5 μ particle diameters), mobile phase is respectively A:100% acetonitrile, B: water, applied sample amount is 100 μ L, elution time is 40 minutes, flow velocity is 1mL/min, collects the solution of wash-out per minute, is repeatedly merged by the eluent of same time point after loading purifying, 50mM phosphate buffer is dissolved in after freeze drying, and detect the antibacterial activity of eluent, take Listeriainnocua as indicator bacteria, obtain the bacteriocin of purifying.
Compared with the bacillus bacteriocin reported, there is larger difference in the bacteriocin that FM603 produces in biological characteristics and antibacterial activity.
The bacteriocin that FM603 produces is stablized hot, acid, active in 60-70 DEG C of water-bath maintenance in 10 minutes more than 90%, active in 80-100 DEG C of water-bath maintenance in 10 minutes 80%; Within the scope of pH2-8, process 2h loss of activity be less than 10%, within the scope of pH9-10, process 2h activity retain 60%.This bacteriocin active Retention after pepsin, trypsase, papain, chymotrypsin, carboxypeptidase, amylase, lipase treatment 1h is greater than 90%, remains 20% activity (in detail in table 2) after pronase ferment treatment 1h.
This bacteriocin to Gram-positive pathogenic bacterium as staphylococcus aureus, listeria monocytogenes, bacillus cereus and the antibacterial activity (antibacterial circle diameter >20mm) as very strong in methicillin resistant Staphylococcus aureus has of drug-fast bacteria, the antibacterial activity (antibacterial circle diameter 17-18mm) as stronger in C.perfringens and strong clostridium have to anaerobism clostridium sporogene, to Gram-negative bacteria as Escherichia coli, salmonella, Yersinia, gram-positive bacteria is as bacillus subtilis, bacillus licheniformis, lactobacillus and saccharomycete are without antibacterial activity, to mould as Aspergillus flavus has certain antibacterial activity (in detail in table 3).
The bacteriocin of purifying of the present invention is through SDS-PAGE electrophoretic analysis, and there is a protein band at gel 4500Da place, and scaled off by gel and carry out antibacterial activity detection, result shows, this albumen has obvious antibacterial activity to Listeria.FM603 bacteriocin is through substance assistant laser desorpted flight time mass spectrum and Tandem Mass Spectrometry Analysis, and molecular weight is 4276.45Da, and partial amino-acid series is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro.This bacteriocin can change gram-positive bacteria cell film film potential and permeability, causes efflux of K+ ions in cell, thus kill bacteria.
Beneficial effect:
Animal test results shows, adds 500-1000g/T additive for microbe feedstuff provided by the invention in daily ration, can significantly improve laying rate of laying hen and Egg Quality, reduction feedstuff-egg ratio; Improve piglet daily gain and average daily feed intake, reduce feedstuff-meat ratio; Improve broiler chicken daily gain, reduce feedstuff-meat ratio and death rate.
Strain bacillus coagulans FM603 provided by the invention can be used as feed addictive and is applied to feed and aquaculture, the use of minimizing or substitute antibiotics, promotes breeding performonce fo animals, improves culture benefit.
Accompanying drawing explanation
The molecular weight of Fig. 1: FM603 bacteriocin;
The binding mode of Fig. 2: FM603 bacteriocin;
Fig. 3: FM603 bacteriocin is to the effect of film potential;
Fig. 4: FM603 bacteriocin is on the impact of potassium ion in cell;
Fig. 5: FM603 growth curve and bacteriocin produce rule;
Bacteriocin and protease is produced in Fig. 6: FM603 sweat; Wherein, Fig. 6 (A): after EDTA chelated metal ions, proteinase activity is suppressed, and bacteriocin activity is high; Fig. 6 (B): when existing without protease, bacteriocin is not degraded;
Fig. 7: FM603 gemma sprout time.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
The percentage sign " % " related in embodiment, if not specified, refers to mass percent, and the percentage of solution refers to the grams containing solute in 100ml, and the percentage between liquid, refers to the volume ratio of solution 25 DEG C time.
Embodiment 1: the Analysis of Antimicrobial Activity of bacillus coagulans FM603 bacteriocin
FM603 is lined LB agar plate, after 30 DEG C of incubated overnight, picking list colony inoculation is in 10mLLB culture medium, 150rpm, cultivate 24h for 30 DEG C, be inoculated in 1000mL by 0.2% inoculum concentration and improve tryptone culture medium, 150rpm, cultivates the centrifugal 15min of 24h, 10000rpm for 30 DEG C and obtains fermented supernatant fluid.By L.innocua, L.monocytogenes, S.aureus, B.cereus, M.luteus respectively in LB agar lining out 37 DEG C of overnight incubation, picking list colony inoculation is in LB culture medium, 37 DEG C of incubated overnight, draw 10ul nutrient solution and be inoculated in 10mLLB soft agar (the LB culture medium containing 0.75% agar), LB agar plate is laid in after mixing, the Oxford cup of sterilizing is placed after cooling, add the fermented supernatant fluid after 100 μ lFM603 fermented supernatant fluids or two times of gradient dilutions, 30 DEG C of overnight incubation, observe inhibition zone, calculate bacteriocin and tire.Tiring of bacteriocin adopts coubling dilution to measure, represent with AU/mL, specific as follows: fermented supernatant fluid or bacteriocin crude extract SPSS are carried out two times of gradient dilutions, each dilution gradient samples 100 μ l and joins in the cup of Oxford and detect antibacterial activity, occur that the most highly diluted multiple of inhibition zone is defined as an active unit, its inverse is multiplied by the antibacterial activity that extension rate is stoste tires (AU/mL).Result is as shown in table 1, and the activity of zymotic fluid to L.innocua, L.monocytogenes and M.luteus is tired as 2560AU/mL, tires as 1280AU/mL, tire as 5120AU/mL to the activity of B.cereus to the activity of S.aureus.
Table 1: bacteriocin antibacterial activity is tired
The preparation of embodiment 2:FM603 bacteriocin
1. fermented and cultured
Bacillus coagulans FM603 is cultivated at the flat lining out of LB, picking list colony inoculation is in 10mL LB liquid medium incubated overnight, 10mL incubated overnight seed liquor is inoculated in 1000mL and improves tryptone culture medium, its composition is: quality percent by volume, tryptone 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium chloride 0.1%, sodium dihydrogen phosphate 0.05%, distilled water 1000mL, pH7.0, condition of culture is 30 DEG C, and 150rpm cultivates 20 hours.
2. bacteriocin crude extract preparation
The centrifugal 15min of cultured FM603 zymotic fluid 10000 × g is got supernatant for subsequent use.By ion column 50mM, the phosphate buffer balance of pH6.5, fermented supernatant fluid passes through ion column with the flow velocity of 50mL/min, supernatant by ion column is discarded, after rinsing ion column with phosphate buffer, with the phosphate buffer (50mM containing 1MNaCl, pH6.5) elution ionic post, flow velocity is 10mL/min, collect eluent 100mL, by eluent by C18 solid-phase extraction column, acetonitrile solid-phase extraction column with 100% also collects eluent, after eluent freeze drying, be dissolved in 50mM, in the phosphate buffer of pH6.5, bacterioid element crude extract is obtained after filtration.
3. the purifying of bacteriocin
Bacteriocin crude extract high-efficient liquid phase chromatogram purification, use C18 reversed-phase column (250mm × 10mm, 5 μ particle diameters), mobile phase is respectively A:100% acetonitrile, B: water.Applied sample amount is 100 μ l, elution time is 40 minutes, flow velocity is 1mL/min, collect the solution of wash-out per minute, repeatedly after loading purifying, the eluent of same time point is merged, be dissolved in 50mM phosphate buffer after freeze drying, and detect the antibacterial activity of eluent, take L.innocua as indicator bacteria, obtain the bacteriocin of purifying.
The biological characteristics of embodiment 3:FM603 bacteriocin
1) to the tolerance of temperature: embodiment 2 fermented supernatant fluid is processed 10min respectively at 60,70,80,90,100 DEG C, with without heat treated sample for contrast, L.innocua is indicator bacteria, detect heat treatment to the impact of bacteriocin activity, represent antibacterial activity Retention with the antibacterial circle diameter of processed group and the ratio of control group antibacterial circle diameter, result is as shown in table 2, and 60-100 DEG C processes 10 minutes respectively, bacteriocin activity is substantially uninfluenced, and activity all remains on more than 90%.
2) to the sensitiveness of enzyme: Example 2 fermented supernatant fluid adds pepsin respectively, trypsase, pronase, lipase, amylase, the final concentration of enzyme is 1mg/mL, pepsin pH is 2.5, other ferment treatment pH is 6.5, after 37 DEG C of process 2h, take L.innocua as the Retention that indicator bacteria detects antibacterial activity, untreated fermented supernatant fluid is contrast, result is as shown in table 2, FM603 is to pepsin, trypsase, lipase and amylase insensitive, but antibacterial activity Retention is 20% after pronase ferment treatment 2h, illustrate that FM603 is a kind of protein, can be degraded by pronase.
3) to the tolerance of soda acid: get the bacteriocin crude extract of 1mL embodiment 2 respectively in test tube, with hydrochloric acid or the NaOH adjustment pH to 2,3,4,5,6,7,8,9,10 of 0.5M, after leaving standstill 2h, pH is adjusted to 7.0, take L.innocua as indicator bacteria, do not adjust the bacteriocin crude extract of pH for contrast, detect antibacterial activity.
Table 2: different temperatures, enzyme and pH are on the impact of bacteriocin antibacterial activity
As can be seen from the above table, the bacteriocin that bacillus coagulans FM603 produces has good tolerance to temperature, still the antibacterial activity of 80% has been retained after 100 DEG C of process, this bacteriocin can not by pepsin, trypsase, lipase and amylase degrades, but activity reduces to 20% after pronase ferment treatment, illustrate pronase responsive, also the protein attribute of this bacteriocin is demonstrated, but this protein has stronger resistance to pepsin and trypsase, is therefore not easy inactivation in animal intestinal.This bacteriocin has good tolerance to acid, and the process within the scope of pH2-5 does not almost affect activity, but after pH is greater than 8, the activity of this bacteriocin is obviously affected, and illustrates that this bacteriocin is suitable for applying under slant acidity condition.
The antimicrobial spectrum of embodiment 4:FM603 bacteriocin
Better bacteriocin can be applied in practice to the antibacterial activity of different microorganisms by detecting, the indicator bacteria detected in this test comprises: Escherichia coli CVCC195, CVCC249, staphylococcus aureus CVCC6538, methicillin resistant Staphylococcus aureus, listera innocua, listeria monocytogenes, VREF, bacillus subtilis CGMCC1.769, C.perfringens, strong clostridium etc., result is as shown in table 3:
The antimicrobial spectrum of table 3:FM603 bacteriocin
Bacillus coagulans FM603 to Gram-negative bacteria as Escherichia coli do not have antibacterial activity, but to Gram-positive pathogenic bacterium as staphylococcus aureus, listeria monocytogenes and methicillin-resistant Staphylococcus have good antibacterial activity, this bacteriocin has weak activity to the VREF that animal intestinal is separated, and does not have antibacterial activity to Lactobacillus plantarum and bacillus subtilis.
Embodiment 5: bacteriocin molecular weight and determined amino acid sequence
In order to verify the protein attribute of FM603 bacteriocin, the molecular weight of this bacteriocin of Simultaneously test, carry out SDS-PAGE test.SDS-PAGE resolving gel concentration 16%, using the bacteriocin of purifying as electrophoresis Sample, applied sample amount 10ul, 60V electrophoresis 1h, 100V electrophoresis 2h, adopt LMWP standard items in contrast, after electrophoresis terminates, glue is cut into two parts, comprise the part coomassie brilliant blue staining of protein standard substance and bacteriocin sample, another part only has bacteriocin sample, put into fixer (25% aqueous isopropanol) and fix 30 minutes, then with distilling washing 60 minutes.Washed glue is placed on LB agar plate, spreads the LB soft agar of inoculation L.innocua, flat board is placed on 37 DEG C of cultivation 16h in incubator after soft agar cooling, observes antibacterial activity.After dyeing, corresponding standard items 4500Da place, the runway of bacteriocin sample has a blue bands, illustrate that this bacteriocin is protein, molecular weight is about 4500Da, and another part of glue has obvious inhibition zone in identical position, confirms that this band is bacteriocin.
In order to obtain the accurate molecular weight of this bacteriocin, the bacteriocin sample of purifying is through substance assistant laser desorpted flying time mass spectrum analysis, and recording molecular weight is 4276.45Da, as shown in Figure 1.Sample cannot obtain amino acid sequence information through Edman degraded, illustrate that N end has modification group, the partial sequence of this bacteriocin is obtained: Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro through Tandem Mass Spectrometry Analysis, Dhb is two dehydrogenation Gamma Amino Butyric Acids, be that threonine is formed after modifying, illustrate that this bacteriocin may be a kind of Lantibiotics.
Embodiment 6:FM603 bacteriocin binding mode is studied
In two sterile tubes, add LB culture medium 9mL respectively, inoculate the L.innocua of 100 μ l incubated overnight, wherein a pipe adds bacteriocin crude extract 1mL, another pipe adds sterile distilled water 1mL, place them in 37 DEG C of cultivations, take out 1mL sample every 2h, detect the viable count of L.innocua.Result shows (shown in figure 2), along with the increase of incubation time, the viable bacteria content rapid development of control group, but the viable count of bacteriocin group tails off gradually, illustrate that L.innocua loses activity under the effect of bacteriocin, the binding mode of FM603 bacteriocin is sterilization.
Bacteriocin is to the effect of film potential: be inoculated in LB culture medium by after L.innocua incubated overnight by 1% inoculum concentration, 37 DEG C, and 200rpm cultivates 5h.Collected by centrifugation thalline, is suspended in 5mM dextrose buffer liquid, adds fluorescence probe DiSC3 to 0.5 μm of concentration, left at room temperature 15min after washing twice containing 5mMHEPES and glucose solution.Get 90 μ l and join NBS microwell plate, then add 10 μ lFM603 bacteriocins, detect change in fluorescence with fluophotometer.As shown in Figure 3, FM603 bacteriocin discharges fluorescence probe from cell membrane to result, illustrates that this bacteriocin can change cell membrane polarity.
Bacteriocin is on potassium ion impact in cell: be inoculated in LB culture medium by after L.innocua incubated overnight by 1% inoculum concentration, 37 DEG C, 200rpm cultivates 5h.Collected by centrifugation thalline, be suspended in 5mM dextrose buffer liquid after washing twice containing 5mMHEPES and glucose solution, PBFI (Potassiumsensitiveprobe) is added to 2 μm in cell suspension, get 90 μ l and join NBS microwell plate, add 10 μ lFM603 bacteriocins, detect change in fluorescence by fluophotometer.As shown in Figure 4, FM603 improves membrane passage to result, causes intracellular efflux of K+ ions.
Embodiment 7: the growth curve of bacillus coagulans FM603
FM603 is cultivated at the flat lining out of LB, picking list colony inoculation is in 10mLLB fluid nutrient medium, in 37 DEG C of overnight incubation, get 1mL nutrient solution and be inoculated in 500mL LB liquid medium, 37 DEG C, 200rpm cultivates 48h, detects zymotic fluid OD value and antibacterial activity, using L.innocua as indicator bacteria every 4h sampling.Result shows (shown in figure 5), and FM603 after inoculation 4h enters exponential phase, and 20h enters plateau, and bacteriocin activity is detected at 8h,
Embodiment 8: proteinase activity detects
FM603 is inoculated in 10mL LB liquid medium, cultivate 48h for 37 DEG C, 8000 × g gets supernatant after centrifugal 20 minutes, and with the filtering with microporous membrane of 0.22 μm, get 200 μ l filtrates add be placed on casein plate (25g/L skimmed milk power, 15g/L agar, 1000mL distilled water) Oxford cup in, cultivate 48h for 37 DEG C, detect the appearance of transparent circle.Result shows, occurs the transparent circle of about 25mm diameter around the cup of Oxford, illustrates that FM603 can produce protease.
Phase has a declining tendency the supernatant antibacterial activity of FM603 after incubation, may be because bacterial strain itself creates some protease have degraded effect to bacterium, in order to verify this hypothesis, bacterial strain is inoculated in respectively 3 500mL triangular flasks, each triangular flask has 200mLLB fluid nutrient medium, add protease inhibitors EDTA (0.1mM) or PMSF (0.1mM) after postvaccinal triangular flask is cultivated 18h at 30 DEG C, do not add the triangular flask of EDTA and PMSF in contrast.Cultivate after 48h, detect zymotic fluid in three triangular flasks respectively and detect antibacterial activity.Meanwhile, get the 18h fermentation broth sample not adding protease inhibitors, after filtration, be divided into three parts, add EDTA or PMSF (10mM) respectively, and detect antibacterial activity after cultivating 48h in 37 DEG C, do not add the filtrate of protease inhibitors in contrast.
As shown in Figure 6A, when EDTA to join in zymotic fluid and after cultivating 48h at late exponential stage, zymotic fluid still remains most active, and does not add the control group of protease inhibitors and add serpin PMSF group antibacterial activity after fermentation 48h and obviously reduce.No matter whether the filtrate of 18h fermentation broth sample, add protease inhibitors simultaneously, and active after cultivation 48h all do not have large change (Fig. 6 B).Phase is after fermentation described, FM603 creates a kind of metalloproteinases, its bacteriocin produced of can degrading, but after adding metal-chelator EDTA, metalloproteinases cannot normally play a role, and therefore bacteriocin activity is retained.
The amplification of embodiment 9:FM603 bacteriocin gene
Report about bacillus coagulans bacteriocin is less, the antibacterial material that FM603 produces shows obvious protein characteristic, in order to verify that whether this protein is consistent with the bacteriocin structure found, after extracting genomic DNA, adopt reported several to carry out pcr amplification to primer, primer is as shown in the table:
Table 4: increase the primer selected
Wherein Licfwd and Licrev designs according to lichenicidin structural gene, SB5 and SB6, LanBfw and LanBrev, LanCfwd and LanCrev are the degenerate primers designed according to the conserved sequence of bacteriocin modified protein, osboP1 and osboP2 is the amplimer of bacteriocin subtilosin.Adopt above primer to carry out pcr amplification respectively, amplified reaction terminates rear agarose electrophoresis test strip.Electrophoresis result shows, above primer does not all produce amplified production, illustrates that FM603 bacteriocin is inconsistent with the structure of above bacteriocin.
Embodiment 10:FM603 gemma sprout time
Gemma can be sprouted under optimum conditions for trophosome, and gemma works in animal intestinal also to be needed to sprout for trophosome, therefore can be understood sprout time and the rule of gemma by the quantity detecting gemma.By FM603 in LB agar lining out overnight incubation, picking list colony inoculation is in LB fluid nutrient medium, and 80 DEG C of water-bath 15min after cultivation 48h, after doing 10 times of gradient dilutions by the zymotic fluid after water-bath, choose suitable dilution factor and coat LB agar plate, calculate the quantity of gemma.Gemma liquid 1mL after water intaking bath is inoculated in 9mL LB liquid medium, cultivates 4h for 30 DEG C, respectively at 2h and 4h sampling, detects gemma and number of viable.
As shown in Figure 7, when 0h, the quantity of gemma is 1.0 × 10 to result
6cfu/mL, after 30 DEG C of cultivation 2h, trophosome quantity is 1.9 × 10
6cfu/mL, and Number of spores is 0, during to 4h, the quantity of trophosome is increased to 6 × 10
7cfu/mL, Number of spores is still 0.Illustrate under suitable conditions, gemma can be sprouted rapidly for trophosome, and the time is less than 2h.
The antibiotic susceptibility test of embodiment 11:FM603
FM603 is lined LB agar plate, 30 DEG C of incubated overnight, picking list colony inoculation is in LB liquid medium, and 30 DEG C, 150rpm incubated overnight, is diluted to 10 by nutrient solution
6cfu/mL, draw 10 μm and join in the LB soft agar medium of 10mL dissolving, after mixing, tiling is on LB agar plate, is positioned on agar plate, observes inhibition zone after 30 DEG C of overnight incubation after cooling by the susceptibility sheet containing different antibiotic concentration.Result is as shown in the table, when antibiotic concentration reaches 1000ppm, FM603 to the gram-positive bacteria antibiotic of test and broad-spectrum antibiotic comparatively responsive, and when antibiotic concentration is 50ppm, this bacterial strain is only responsive to Kitasamycin.
Table 5: bacterial strain FM603 is to the antibiotic sensitiveness * of difference
* +++ antibacterial circle diameter >20mm, ++ antibacterial circle diameter 15-20mm ,+antibacterial circle diameter <15mm ,-without inhibition zone.
Embodiment 12:FM603 fermentation palm kernel meal produces additive for microbe feedstuff
FM603 is lined LB agar plate, 30 DEG C of incubated overnight, picking list colony inoculation is in 10mL LB liquid medium, 150rpm, 30 DEG C of incubated overnight are as 1 grade of seed liquor, 1 grade of seed liquor is inoculated in 1000mL by 1% inoculum concentration and improves tryptone culture medium, 150rpm, 30 DEG C of incubated overnight obtain 2 grades of seed liquor, 2 grades of seed liquor are inoculated in 100L by 10% inoculum concentration and improve tryptone culture medium, 150rpm, 30 DEG C of fermentation 24h obtain zymotic fluid, zymotic fluid is mixed rear 30 DEG C of 36h that ferment with the palm kernel meal after sterilizing according to 1:1 ratio, dry to water content 10%, flour obtains FM603 fermentate to crossing 40 eye mesh screens.
Get fermentate and detect spore content, crude protein, crude fat, crude fibre, free amino acid, reduced sugar, result is as shown in table 6, the crude protein content of palm kernel meal is 17.45%, crude fat 11.8%, crude fibre 12.78%, after FM603 fermentation, crude protein improves 0.33 percentage point, crude fat declines 1.3 percentage points, and crude fibre improves 2.34 percentage points.This fermentate can be used as a kind of additive for microbe feedstuff containing FM603 gemma and uses.
Table 6:FM603 fermentate compares with palm kernel meal
Embodiment 13:FM603 is on the impact of performance in layers
In order to study the effect of FM603 as additive for microbe feedstuff, choosing the brown commercial generation laying hen of Luo Man in 25 week age 960, being divided into 3 process at random, each process 8 repetition, each repetition 40 chickens.Processed group 1 is blank group, basal diet of feeding; Processed group 2 and 3 is test group, adds the FM603 fermentate in 500g/T and 1000g/T embodiment 12 respectively in basal diet.Test period is 30 weeks, after off-test, calculates the indexs such as average feed intake, laying rate, feedstuff-egg ratio, picks up 30 pieces, egg at random respectively from each processed group, detects eggshell strength, shell thickness, Hough unit, yolk color and cholesterol level.Basal diet formula is as shown in table 7:
Table 7: Diet Formula and trophic level
Result is as shown in table 8, compared with control group, the raising that 500g/T and 1000g/TFM603 fermentative microorganism feed addictive finished product is conducive to Layer Production Performance and Egg Quality is added in basal diet, when adding 500g/TFM603 fermentative microorganism feed addictive, improve eggshell strength and yolk color significantly, when adding 1000g/TFM603 fermentative microorganism feed addictive finished product, significantly improving laying rate, eggshell strength and yolk color, reducing feedstuff-egg ratio.
Table 8:FM603 fermentate affects * to performance in layers
* different letter of going together represents significant difference.
Embodiment 14:FM603 is on the impact of weaned piglets
Choose the close male earner pig of 35 age in days body weight 90, be divided into 3 process at random, each process 6 repetition, each repetition 5 pigs.Process 1 is blank group, basal diet of feeding, and process 2 and 3 is test group, adds the FM603 fermentate in 500g/T and 1000g/T embodiment 12 respectively in basal diet.28 days experimental periods, free choice feeding and drinking-water, weigh after off-test, calculates average daily gain, feedstuff-meat ratio, average daily ingestion amount.Daily ration composition and nutritional labeling are in table 9.
Table 9: Diet Formula and trophic level
Result is as shown in table 10, and within experimental period, two test group (process 2,3) of adding FM603 fermentate are significantly higher than blank group (process 1) in feed intake and daily gain, and feedstuff-meat ratio is then remarkable in blank group.Illustrate that in daily ration, add FM603 fermentate is conducive to promoting feed intake, improves daily gain and efficiency of feed utilization, increases culture benefit.
Table 10:FM603 fermentate affects * to weaned piglets
* to go together different letter representation significant difference.
Embodiment 15:FM603 is on the impact of meat chicken production performance
Choose 1 age in days Broiler chicks 900, be divided into 3 process at random, each process 6 repetition, each repetition 50 chickens, free choice feeding is drunk water, process 1 is blank group, to feed basal diet, process 2 and 3 is test group, adds the FM603 fermentate in 500g/T and 1000g/T embodiment 12 respectively, 21 days experimental periods in basal diet, within 21st day, each processed group slaughters 12 chickens, weigh the weight of spleen, the bursa of farbricius and thymus gland, detect serum lysozyme concentration, record and calculate the daily gain of experimental period broiler chicken, feedstuff-meat ratio and the death rate.Day grain raw material and trophic component as shown in table 11.
Table 11: Diet Formula and trophic level
Result is as shown in table 12, add FM603 fermentate in daily ration after (process 2 and 3), improve broiler chicken daily gain significantly, reduce feedstuff-meat ratio, have effect to the reduction of the death rate, but difference is not remarkable compared with blank group (processing 1) yet.The content of two test group serum of broilers lysozymes is significantly higher than blank group, illustrate that the immunity of FM603 fermentate to broiler chicken has certain facilitation, but the spleen of blank group and test group, the bursa of farbricius and thymus gland relative weight do not have notable difference.
Table 12:FM603 fermentate affects * to meat chicken production performance
* to go together different letter representation significant difference.
Claims (3)
1. an additive for microbe feedstuff, is characterized in that, containing bacillus coagulans FM603 or its fermentation culture medium in described feed addictive, bacillus coagulans deposit number is CGMCCNO.10221.
2. additive for microbe feedstuff as claimed in claim 1, it is characterized in that, the preparation method of described additive for microbe feedstuff comprises the steps: bacillus coagulans FM603 to line LB agar plate, 30 DEG C of incubated overnight, picking list colony inoculation is in 10mlLB culture medium, 150rpm, 30 DEG C of incubated overnight are as 1 grade of seed liquor, 1 grade of seed liquor is inoculated in 1000mL by 1% inoculum concentration and improves tryptone culture medium, 150rpm, 30 DEG C of incubated overnight obtain 2 grades of seed liquor, 2 grades of seed liquor are inoculated in 100L by 10% inoculum concentration and improve tryptone culture medium, 150rpm, 30 DEG C of fermentation 24h obtain zymotic fluid, zymotic fluid is mixed rear 30 DEG C of 36h that ferment with the palm kernel meal after sterilizing according to 1:1 ratio, dry to water content 10%, flour is to crossing 40 eye mesh screens and get final product.
3. additive for microbe feedstuff as claimed in claim 2, it is characterized in that, described improvement tryptone culture medium consists of: quality percent by volume, tryptone 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium chloride 0.1%, sodium dihydrogen phosphate 0.05%, distilled water 1000mL, pH7.0.
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