CN105175518A - Bacteriocin generated by bacillus coagulans FM603 and preparing method thereof - Google Patents

Abstract

The invention provides bacteriocin and a preparing method thereof. The bacteriocin is generated by bacillus coagulans FM603 with the collection number of CGMCC NO.10221, and has antimicrobial activity on Gram-positive pathogenic bacteria such as Listeria monocytogenes, staphylococcus aureus, methicillin resistant staphylococcus aureus, clostridium perfringens and clostridium firmus. The molecular weight of the bacteriocin is 4276.45 Da, a part of the amino acid sequence is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro, and the bacteriocin is stable under treatment of heat, acid, pepsin or trypsin and can be easily degraded by pronase to be inactivated. The bacteriocin can be used as microorganism feed additives, the laying rate of laying hens can be increased, the feed-egg ratio can be reduced, and egg quality can be improved; the feed intake and the daily gain of piglets can be increased, and the feed-meat ratio can be reduced; the daily gain of broilers and the content of lysozyme can be increased, and the feed-meat ratio and the death rate can be reduced.

Description

Bacteriocin that Bacillus coagulans FM603 produces and preparation method thereof

Technical field

The present invention relates to microorganism and cultivation thereof and utilisation technology, be specifically related to the preparation method of a kind of bacteriocin of being produced by Bacillus coagulans FM603 and this bacteriocin.

Background technology

Microbiotic is the secondary metabolite that microorganism produces, and has anti-microbial activity to other microorganism.The forties in 20th century, scientists finds that the microbiotic of feeding animals low dosage can improve the production performance of animal, increases economic efficiency.Within decades afterwards, microbiotic is widely used in livestock-raising industry, makes an addition in feed and is used as growth stimulant, 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 drug residue etc. in the propagation of such as drug-resistant bacteria, superinfection and animal product.What the appearance of resistant organism was serious threatens human health, and for anti-methicillinum streptococcus aureus, the mortality ratio 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 streptococcus aureus of 40-60% to have Methicillin resistance, in China, the intestinal bacteria of about 60-70% are insensitive to fluoroquinolone antibiotics.A large amount of use microbiotic, and violate withdrawal time and specify, easily cause the drug residue in animal product to exceed standard, such animal product is difficult to enter world market, also can there is potential threat to the health of people.Report was had in the last few years during China's food safety 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 human 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 microbiotic in 2006 and is added in feed as growth stimulant, and Korea S also have disabled antibiotics growth stimulant 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 researchist of agriculture and animal husbandry universities and colleges is actively devoted to exploitation and the use of biotechnological formulation, to reduce or the application of substitute antibiotics.

Genus bacillus is Gram-positive, a sporiferous genera bacillus, is present in occurring in nature widely.Genus 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 resistibility.Genus bacillus is industrially used as the production bacterial classification of amylase and proteolytic enzyme, the also long-term making for the based food that ferments.Genus 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 probiotic bacterium.A large amount of datas shows, genus 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 stimulant.Genus bacillus is grown by stimulating animal intestinal villus, improves non-specific immunity, produces antibacterial substance and plays a role.Bacteriocin is one of antibacterial substance of genus bacillus generation, is the polypeptide class antimicrobial substance of microorganism by Ribosome biogenesis, comprises resistant organism have good anti-microbial activity to a lot of pathogenic bacteria.Genus bacillus can produce bacteriocin class antimicrobial substance, as subtilin, sublancin, subtilosin, lichenin etc.Bacteriocin is the important functional material of probiotic bacterium; the researchist of cock university finds; the milk-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, milk-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 microbiotic.Therefore, compared with common genus bacillus, the production performance of genus bacillus to animal of bacteriocinogeny has more good promoter action, has larger application potential in actual production.

Summary of the invention

One object of the present invention is to provide a kind of bacteriocin produced by Bacillus coagulans FM603.Bacillus coagulans FM603 fermentation culture produces bacteriocin, has good anti-microbial effect to gram positive bacterium.This bacteriocin molecular weight is 4276.45Da, and partial amino-acid series is Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro.

Another object of the present invention is to provide a kind of preparation method of bacteriocin, 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 Tryptones substratum, 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 buffered saline buffer balance of pH6.5, fermented supernatant fluid passes through ion column with the flow velocity of 50mL/min, supernatant liquor by ion column is discarded, after rinsing ion column with phosphate buffered saline buffer, with the phosphate buffered saline buffer (50mM containing 1MNaCl, pH6.5) elution ionic post, flow velocity is 10mL/min, collect elutriant 100mL, by elutriant by C18 solid-phase extraction column, acetonitrile solid-phase extraction column with 100% also collects elutriant, after elutriant lyophilize, be dissolved in 50mM, in the phosphate buffered saline 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), moving 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 per minute wash-out, is repeatedly merged by the elutriant of same time point after loading purifying, 50mM phosphate buffered saline buffer is dissolved in after lyophilize, and detect the anti-microbial activity of elutriant, take Listeriainnocua as indicator, obtain the bacteriocin of purifying.

Compared with the genus bacillus bacteriocin reported, there is larger difference in the bacteriocin that FM603 produces in biological characteristics and anti-microbial 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 stomach en-, trypsinase, papoid, 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 streptococcus aureus, listeria monocytogenes, bacillus cereus and the anti-microbial activity (antibacterial circle diameter >20mm) as very strong in methicillin resistant Staphylococcus aureus has of resistant organism, the anti-microbial activity (antibacterial circle diameter 17-18mm) as stronger in clostridium perfringens and strong clostridium have to anaerobism clostridium sporogene, to Gram-negative bacteria as intestinal bacteria, Salmonellas, Yersinia, gram-positive microorganism is as subtilis, Bacillus licheniformis, Bacterium lacticum and yeast are without anti-microbial activity, to mould as Aspergillus flavus has certain anti-microbial 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 anti-microbial activity detection, result shows, this albumen has obvious anti-microbial activity to listeria bacteria.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 membrane potential and permeability, causes efflux of K+ ions in cell, thus kill bacteria.

Bacteriocin provided by the invention can directly apply to after extraction purification prepares fodder additives.Also the producing strains Bacillus coagulans FM603 of bacteriocin of the present invention or its fermenting culture can be applied to and prepare feed interpolation.

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, surface irregularity.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 test 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.

FM603 can adopt conventional medium under household condition to cultivate, but can obtain more high-biomass in improvement Tryptones substratum, improvement Tryptones substratum consists of: quality volume percent, Tryptones 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium-chlor 0.1%, SODIUM PHOSPHATE, MONOBASIC 0.05%, distilled water 1000mL, pH7.0, culture condition is 30 DEG C, and 150rpm cultivates 20h.

The present invention also provides a kind of microorganism feed addictive, by Bacillus coagulans FM603 fermentative production, its production method is as follows: FM603 is lined LB agar plate, 30 DEG C of incubated overnight, picking list colony inoculation is in 10mlLB substratum, 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 size and improves Tryptones substratum, 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 size and improve Tryptones substratum, 150rpm, 30 DEG C of fermentation 24h obtain fermented liquid, fermented liquid 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.

Beneficial effect:

Animal test results shows, adds the microorganism feed addictive that 500-1000g/T is produced by aforesaid method in daily ration, can significantly improve laying rate of laying hen and Egg Quality, reduction feedstuff-egg ratio; Improve piglet day weight gain and average daily food consumption, reduce feedstuff-meat ratio; Improve broiler chicken day weight gain, reduce feedstuff-meat ratio and death rate.

Strain bacillus coagulans FM603 provided by the invention can be used as fodder additives 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 membrane 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 proteolytic enzyme is produced in Fig. 6: FM603 fermenting process; Wherein, Fig. 6 (A): after EDTA chelated metal ions, protease activity is suppressed, and bacteriocin activity is high; Fig. 6 (B): when existing without proteolytic enzyme, bacteriocin is not degraded;

Fig. 7: FM603 gemma sprout time.

Embodiment

The present invention is described below by specific embodiment.Unless stated otherwise, technique 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 per-cent of solution refers to the grams containing solute in 100ml, and the per-cent 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 substratum, 150rpm, cultivate 24h for 30 DEG C, be inoculated in 1000mL by 0.2% inoculum size and improve Tryptones substratum, 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 substratum, 37 DEG C of incubated overnight, draw 10ul nutrient solution and be inoculated in 10mLLB soft agar (the LB substratum 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 stroke-physiological saline solution are carried out two times of gradient dilutions, each dilution gradient samples 100 μ l and joins in the cup of Oxford and detect anti-microbial activity, occur that the most highly diluted multiple of inhibition zone is defined as an activity unit, its inverse is multiplied by the anti-microbial activity that extension rate is stoste tires (AU/mL).Result is as shown in table 1, and the activity of fermented liquid 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 anti-microbial activity is tired

The preparation of embodiment 2:FM603 bacteriocin

1. fermentation culture

Bacillus coagulans FM603 is streak culture on LB flat board, picking list colony inoculation is in 10mL LB liquid medium incubated overnight, 10mL incubated overnight seed liquor is inoculated in 1000mL and improves Tryptones substratum, its composition is: quality volume percent, Tryptones 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium-chlor 0.1%, SODIUM PHOSPHATE, MONOBASIC 0.05%, distilled water 1000mL, pH7.0, culture condition is 30 DEG C, and 150rpm cultivates 20 hours.

2. bacteriocin crude extract preparation

The centrifugal 15min of cultured FM603 fermented liquid 10000 × g is got supernatant liquor for subsequent use.By ion column 50mM, the phosphate buffered saline buffer balance of pH6.5, fermented supernatant fluid passes through ion column with the flow velocity of 50mL/min, supernatant liquor by ion column is discarded, after rinsing ion column with phosphate buffered saline buffer, with the phosphate buffered saline buffer (50mM containing 1MNaCl, pH6.5) elution ionic post, flow velocity is 10mL/min, collect elutriant 100mL, by elutriant by C18 solid-phase extraction column, acetonitrile solid-phase extraction column with 100% also collects elutriant, after elutriant lyophilize, be dissolved in 50mM, in the phosphate buffered saline 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), moving 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 per minute wash-out, repeatedly after loading purifying, the elutriant of same time point is merged, be dissolved in 50mM phosphate buffered saline buffer after lyophilize, and detect the anti-microbial activity of elutriant, take L.innocua as indicator, 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, detect thermal treatment to the impact of bacteriocin activity, represent anti-microbial activity Retention with the antibacterial circle diameter for the treatment of 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 susceptibility of enzyme: Example 2 fermented supernatant fluid adds stomach en-respectively, trypsinase, PRONASE A, 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 detects anti-microbial activity, untreated fermented supernatant fluid is contrast, result is as shown in table 2, FM603 is to stomach en-, trypsinase, lipase and amylase insensitive, but anti-microbial activity Retention is 20% after pronase ferment treatment 2h, illustrate that FM603 is a kind of protein, can by pronase enzyme liberating.

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 sodium hydroxide 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, do not adjust the bacteriocin crude extract of pH for contrast, detect anti-microbial activity.

Table 2: differing temps, enzyme and pH are on the impact of bacteriocin anti-microbial activity

As can be seen from the above table, the bacteriocin that Bacillus coagulans FM603 produces has good tolerance to temperature, still the anti-microbial activity of 80% has been retained after 100 DEG C of process, this bacteriocin can not by stomach en-, trypsinase, lipase and amylase degrades, but activity reduces to 20% after pronase ferment treatment, illustrate PRONASE A responsive, also the protein attribute of this bacteriocin is demonstrated, but this protein has stronger resistance to stomach en-and trypsinase, 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 anti-microbial activity of different microorganisms by detecting, the indicator detected in this test comprises: intestinal bacteria CVCC195, CVCC249, streptococcus aureus CVCC6538, methicillin resistant Staphylococcus aureus, listera innocua, listeria monocytogenes, faecium, subtilis CGMCC1.769, clostridium 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 intestinal bacteria do not have anti-microbial activity, but to Gram-positive pathogenic bacterium as streptococcus aureus, listeria monocytogenes and methicillin-resistant Staphylococcus have good anti-microbial activity, this bacteriocin has weak activity to the faecium that animal intestinal is separated, and does not have anti-microbial activity to plant lactobacillus and 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 low molecular weight protein (LMWP) standard substance in contrast, after electrophoresis terminates, glue is cut into two portions, comprise the part coomassie brilliant blue staining of protein standard substance and bacteriocin sample, another part only has bacteriocin sample, put into stationary liquid (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 anti-microbial activity.After dyeing, corresponding standard substance 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-terminal 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 substratum 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 membrane potential: be inoculated in LB substratum by after L.innocua incubated overnight by 1% inoculum size, 37 DEG C, and 200rpm cultivates 5h.Collected by centrifugation thalline, is suspended in 5mM dextrose buffer liquid, adds fluorescent 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 fluorophotometer.As shown in Figure 3, FM603 bacteriocin discharges fluorescent probe from cytolemma to result, illustrates that this bacteriocin can change cytolemma polarity.

Bacteriocin is on potassium ion impact in cell: be inoculated in LB substratum by after L.innocua incubated overnight by 1% inoculum size, 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 fluorophotometer.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 streak culture on LB flat board, picking list colony inoculation is in 10mLLB liquid 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 fermented liquid OD value and anti-microbial activity, using L.innocua as indicator every 4h sampling.Result shows (shown in figure 5), and FM603 after inoculation 4h enters logarithmic phase, and 20h enters plateau, and bacteriocin activity is detected at 8h,

Embodiment 8: protease activity detects

FM603 is inoculated in 10mL LB liquid medium, cultivate 48h for 37 DEG C, 8000 × g gets supernatant liquor 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 skim-milk, 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 proteolytic enzyme.

Phase has a declining tendency the supernatant liquor anti-microbial activity of FM603 after incubation, may be because bacterial strain itself creates some proteolytic enzyme 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 liquid nutrient medium, add proteinase inhibitor 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 fermented liquid in three triangular flasks respectively and detect anti-microbial activity.Meanwhile, get the 18h fermentation broth sample not adding proteinase inhibitor, after filtration, be divided into three parts, add EDTA or PMSF (10mM) respectively, and detect anti-microbial activity after cultivating 48h in 37 DEG C, do not add the filtrate of proteinase inhibitor in contrast.

As shown in Figure 6A, when EDTA to join in fermented liquid and after cultivating 48h at late exponential stage, fermented liquid still remains most active, and does not add the control group of proteinase inhibitor and add serpin PMSF group anti-microbial activity after fermentation 48h and obviously reduce.No matter whether the filtrate of 18h fermentation broth sample, add proteinase inhibitor 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 metalloprotease, its bacteriocin produced of can degrading, but after adding metal chelator EDTA, metalloprotease 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 antimicrobial substance 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 structure gene, SB5 and SB6, LanBfw and LanBrev, LanCfwd and LanCrev are the degenerated 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 nourishing body, and gemma works in animal intestinal also to be needed to sprout for nourishing body, therefore can be understood sprout time and the rule of gemma by the quantity detecting gemma.Spend the night streak culture on LB agar plate for FM603, picking list colony inoculation is in LB liquid nutrient medium, and 80 DEG C of water-bath 15min after cultivation 48h, after doing 10 times of gradient dilutions by the fermented liquid after water-bath, choose suitable extent of dilution 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, nourishing body quantity is 1.9 × 10 ⁶cfu/mL, and Number of spores is 0, during to 4h, the quantity of nourishing body is increased to 6 × 10 ⁷cfu/mL, Number of spores is still 0.Illustrate under suitable conditions, gemma can be sprouted rapidly for nourishing body, 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 microorganism microbiotic of test and Broad spectrum antibiotics 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 susceptibility * 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 microorganism feed addictive

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 size and improves Tryptones substratum, 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 size and improve Tryptones substratum, 150rpm, 30 DEG C of fermentation 24h obtain fermented liquid, fermented liquid 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 fermented product to crossing 40 eye mesh screens.

Get fermented product and detect spore content, crude protein, crude fat, robust fibre, total free aminoacids, reducing sugar, result is as shown in table 6, the crude protein content of palm kernel meal is 17.45%, crude fat 11.8%, robust fibre 12.78%, after FM603 fermentation, crude protein improves 0.33 percentage point, crude fat declines 1.3 percentage points, and robust fibre improves 2.34 percentage points.This fermented product can be used as a kind of microorganism feed addictive containing FM603 gemma and uses.

Table 6:FM603 fermented product 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 microorganism feed addictive, 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.Treatment group 1 is blank group, basal diet of feeding; Treatment group 2 and 3 is test group, adds the FM603 fermented product 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 food consumption, laying rate, feedstuff-egg ratio, picks up 30 pieces, egg at random respectively from each treatment 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 organism of fermentation fodder additives finished product is conducive to Layer Production Performance and Egg Quality is added in basal diet, when adding 500g/TFM603 organism of fermentation fodder additives, improve eggshell strength and yolk color significantly, when adding 1000g/TFM603 organism of fermentation fodder additives finished product, significantly improving laying rate, eggshell strength and yolk color, reducing feedstuff-egg ratio.

Table 8:FM603 fermented product 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 fermented product in 500g/T and 1000g/T embodiment 12 respectively in basal diet.28 days trial 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 nutritive ingredient are in table 9.

Table 9: Diet Formula and trophic level

Result is as shown in table 10, and within trial period, two test group (process 2,3) of adding FM603 fermented product are significantly higher than blank group (process 1) in food consumption and day weight gain, and feedstuff-meat ratio is then remarkable in blank group.Illustrate that in daily ration, add FM603 fermented product is conducive to promoting feed intake, improves day weight gain and efficiency of feed utilization, increases culture benefit.

Table 10:FM603 fermented product 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 chicken 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 fermented product in 500g/T and 1000g/T embodiment 12 respectively, 21 days trial periods in basal diet, within 21st day, each treatment group slaughters 12 chickens, weigh the weight of spleen, the fabricius bursa and thymus gland, detect serum lysozyme concentration, record and calculate the day weight gain of trial period broiler chicken, feedstuff-meat ratio and mortality ratio.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 fermented product in daily ration after (process 2 and 3), improve broiler chicken day weight gain significantly, reduce feedstuff-meat ratio, have effect to the reduction of mortality ratio, but difference is not remarkable compared with blank group (processing 1) yet.The content of two test group serum of broilers N,O-Diacetylmuramidases is significantly higher than blank group, illustrate that the immunizing power of FM603 fermented product to broiler chicken has certain promoter action, but the spleen of blank group and test group, the fabricius bursa and thymus gland relative weight do not have notable difference.

Table 12:FM603 fermented product affects * to meat chicken production performance

* to go together different letter representation significant difference

Claims (4)

1. the bacteriocin produced by Bacillus coagulans, is characterized in that, the protein antibacterial substance of described bacteriocin to be molecular weight be 4276.45Da, the deposit number of described Bacillus coagulans is CGMCCNO.10221.

2. prepare a method for bacteriocin described in claim 1, comprise the steps:

(1) fermented supernatant fluid preparation: Bacillus coagulans CGMCC NO.10221 is inoculated in 10mL LB liquid medium overnight incubation, 1mL nutrient solution is inoculated in 1000mL and improves Tryptones substratum, 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 buffered saline buffer balance of pH6.5, fermented supernatant fluid passes through ion column with the flow velocity of 50mL/min, supernatant liquor by ion column is discarded, after rinsing ion column with phosphate buffered saline buffer, with 50mM, pH6.5 is containing the phosphate buffered saline buffer elution ionic post of 1MNaCl, flow velocity is 10mL/min, collect elutriant 100mL, by elutriant by C18 solid-phase extraction column, acetonitrile solid-phase extraction column with 100% also collects elutriant, after elutriant lyophilize, be dissolved in 50mM, in the phosphate buffered saline 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, moving phase is respectively A:100% acetonitrile, B: water, applied sample amount is 100 μ L, and elution time is 40 minutes, and flow velocity is 1mL/min, collect the solution of per minute wash-out, repeatedly after loading purifying, the elutriant of same time point is merged, be dissolved in 50mM phosphate buffered saline buffer after lyophilize, and detect the anti-microbial activity of elutriant, take Listeriainnocua as indicator, obtain the bacteriocin of purifying.

3. the preparation method of bacteriocin as claimed in claim 2, it is characterized in that, described improvement Tryptones substratum consists of: mass volume ratio, Tryptones 1.5%, glucose 1.5%, yeast leaching powder 0.5%, Dried Corn Steep Liquor Powder 0.2%, ammonium sulfate 0.05%, sodium-chlor 0.1%, SODIUM PHOSPHATE, MONOBASIC 0.05%, distilled water 1000mL, pH7.0.

4. the application of bacteriocin according to claim 1 in fodder additives.