CN102120975A - Bacillus subtilis strain with stronger bacteriostatic action and application thereof - Google Patents
Bacillus subtilis strain with stronger bacteriostatic action and application thereof Download PDFInfo
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- CN102120975A CN102120975A CN2010105890745A CN201010589074A CN102120975A CN 102120975 A CN102120975 A CN 102120975A CN 2010105890745 A CN2010105890745 A CN 2010105890745A CN 201010589074 A CN201010589074 A CN 201010589074A CN 102120975 A CN102120975 A CN 102120975A
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Images
Abstract
The invention discloses a bacillus subtilis strain with stronger bacteriostatic action and application thereof. The strain is named as bacillus subtilis B7348 and preserved in a CCTCC (China Center for Type Culture Collection) on 12th October 2010 with a preservation number of CCTCC M2010260. Safety test and an efficiency test results indicate that the bacillus subtilis strain B7348 can be used as a feed additive. During the application, the bacillus subtilis strain B7348 is added in feed in a form of bacterium powder; and during the use, the bacillus subtilis strain B7348 is compounded with a plant bacterium lacticum LP-11 and metabolins thereof for reasonable collection, can meet demands of different animals at different cultivation stages, and is very suitable for the present state culture characteristics and culture mode.
Description
Technical field
The present invention relates to that a strain has the subtilis of strong bacteriostatic action and as the application of fodder additives.
Background technology
The animal products drug residue that causes because of abuse of antibiotics at present exceeds standard, the problem such as roll up of resistance pathogenic strains has caused people's great attention.From on January 1st, 2006, European Union completely forbade and uses the microbiotic feed additive for promoting growth in the feed.Last four kinds of permissions as the promotes growth purposes antibiotic feed additive---bambermycin, Validamycin, Salinomycin. and monensin are also stopped using.In the face of increasingly competitive world market, residue problems such as microbiotic are the bottlenecks of restriction China livestock product outlet always.So it is extremely urgent to seek the feeding antibiotic substitute.
Microbiotic is that certain micro-organisms changes primary metabolite into structural secondary metabolite by enzymatic reaction, by enzymatic reaction amino acid is changed into baroque compound such as linear gramicidins etc.Nisin bacteriocins such as (nisins) then needs to synthesize by rrna, from but real protein matter.Bacteriocin and antibiotic essential difference are: most of bacteriocin only has detrimental effect to the bacterium of nearly edge relation, and have nontoxic, have no side effect, noresidue, have no drug resistance, advantage such as simultaneously also free from environmental pollution.The use of therefore, the fodder additives of highly effective and safe---bacteriocin can reduce even replace antibiotic use under the part situation.
Use probiotics to solve the feeding antibiotic problem, obtain proof from mechanism: beneficial microorganism can produce multiple antibacterial substance its growth metabolism process, mainly comprises the antibacterial peptide of bacillus category generation, the bacteriocin that lactic acid bacteria class generates etc.Probiotics suppresses growth as intestinal bacteria, Salmonellas etc. on the one hand by these materials, and activation, the field planting for self provides favourable condition on the other hand.
At present, in the fodder additives, the genus bacillus of using has subtilis, Bacillus licheniformis, bacillus cereus, Japan genus bacillus etc., genus bacillus has the following advantages and characteristics: 1. have high temperature resistant, acid and alkali-resistance, characteristics such as withstand voltage, can tolerate the influence of granulated feed processing; 2. in storage, exist, do not consume the nutritive ingredient of feed, can guarantee quality of the fodder with spore form; 3. after entering enteron aisle, bring back to life rapidly at enteron aisle, the resurrection rate is near 100%; 4. genus bacillus can produce proteolytic enzyme, amylase, lipase and multiple amino acids; 5. genus bacillus can consume a large amount of oxygen, keeps the enteron aisle anaerobic environment, suppresses the growth of pathogenic bacterium, keeps the normal eubiosis of enteron aisle; 6. the effect that has balance and stable lactobacillus.
Because the subtilyne that subtilis produced has some gram-positive microorganisms of inhibition, do not produce resistance, noresidue, advantage such as free from environmental pollution, can infer, the subtilis meta-bolites substitutes the feeding antibiotic in the feed if can be used for fodder additives, will provide wide prospect for the feed microbe Application of Additives.Therefore, the subtilis meta-bolites is as antibiotic substitute, and its research and development is significant, and market outlook are wide.
At present, there is following defective in existing subtilis: the output of meta-bolites with bacteriostatic activity is not high, and bacteriostasis is general, is badly in need of the higher Bacillus subtilis strain of bacteriostatic activity.
Summary of the invention
At above-mentioned prior art, the present invention is intended to screen the high bacillus subtilis strain of bacteriostatic activity, and the highest technological condition for fermentation of its metabolite activity, antimicrobial spectrum studied, and test its effect on animal, for from now on industrialized developing lays the foundation.
The present invention is achieved by the following technical solutions:
One strain has the subtilis of strong bacteriostatic action, and this bacterial strain called after Bacillus subtilis B7348 is preserved in Chinese typical culture collection center on October 12nd, 2010, and its deposit number is CCTCC M 2010260.
It is as follows that the strain bio of described subtilis is learned characteristic: the cell size of this bacterial classification is for being generally (0.7 μ m～0.8 μ m) * (2.0 μ m～3.0 μ m), and is shaft-like, chaining seldom, and even dyeing, no pod membrane, the flagellum adnation can move; Gemma ovalize or column, middle life or wilfully, size is 0.8 μ m * (1.5 μ m～1.8 μ m); A little less than free spore surface is painted; Bacterium colony circle or irregular shape on the substratum, surface colour is dark, and thickening and opaque can be wrinkling, can be cream color or brown; Gram-positive, the V-P reaction can take place in the catalase positive; Can utilize glucose, pectinose, wood sugar and N.F,USP MANNITOL to produce acid, the energy hydrolyzed starch decomposes tryptophane and forms indoles; 45～55 ℃ of growth temperature maximums, 5～20 ℃ of Schwellenwerts, optimum temperuture is 37 ℃.
Described subtilis is identified by SDSS-PCR, and main antibacterial substance is the small-molecule substance of molecular weight at 7000～8000d in its meta-bolites.
Described subtilis is through safety testing and efficiency test, and the result shows subtilis Bacillus subtilisB7348, and CCTCC M 2010260 can be as fodder additives.
When described subtilis is used as fodder additives, add in the feed with the form of bacterium powder, described bacterium powder obtains by following steps:
(i) bacterial classification: select subtilis Bacillus subtilis B7348 for use, CCTCC M 2010260;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 20～28h at 32～40 ℃;
(iii) first order seed is cultivated: get cultured inclined-plane, be inoculated under aseptic condition in 50mL～100mL seed liquid nutrient medium, under 32～40 ℃ of conditions, leave standstill and cultivate 14～18h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 32～40 ℃ of conditions, leave standstill and cultivate 10～16h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 32～40 ℃ of conditions, leave standstill and cultivate 16～24h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 12000～15000cP, collects fermented liquid;
(vii) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (iii), (iv) described in seed liquid culture medium prescription be: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, during use, regulate the 20min that sterilizes under ℃ condition of pH to 6.5～7.5,115; Step (ii) described in the solid slant culture base be to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; (prescription of liquid fermentation medium v) is: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, sodium-chlor 5g/L during use, regulates pH to 6.5～7.5 to step.
That gram-positive microorganism is suppressed effect is better in view of subtilis B7348, and plant lactobacillus LP-11 (this bacterial strain called after Lactobacillus plantarum, be preserved in Chinese typical culture collection center on 06 21st, 2010, its deposit number is CCTCC M 2010150, be documented among the Chinese patent application CN201010240725.X) obvious to Gram-negative bacteria inhibition effect, simultaneously, both meta-bolitess have stronger bacteriostatic action, good complementary relationship is arranged between two bacterial classifications, and the associating result of use is better.Take all factors into consideration the different fungistatic effect of two strain bacterium, subtilis B7348 and plant lactobacillus LP-11 and meta-bolites thereof are carried out composite (wherein subtilis B7348 bacterium powder viable count is 2.0 * 10
8Cfu/g, milk-acid bacteria bacterium powder viable count is 1.0 * 10
8Cfu/g), carry out reasonably combinedly, form product peptide rhzomorph, to satisfy different animals, the demand in different breeding stage.
The using dosage of described peptide rhzomorph is as follows: to normal not ill stud bird, consumption is 0.2% of a feed total mass; To ill stud bird, consumption is 0.4% of a feed total mass; To pregnant sow, consumption is 0.1～0.2% of a feed total mass; To milking sow, consumption is 0.3～0.4% of a feed total mass; To suffering from the milk cow of mastitis, consumption is 80～120g/ head/sky, can use 120g/ head/sky earlier, two weeks, use 80g/ head/sky again, two weeks makes somatocyte (generally with threshold value that to contain 200,000 cells in every ml milk be subclinical mastitis, it is positive to surpass this value, and it is then normal to be lower than this value) be controlled at gradually below 200,000; To suffering from the milk cow of negative mastitis, consumption is 40g/ head/sky, and life-time service gets final product, and somatocyte is controlled in below 200,000 gradually after 1 month.
Description of drawings
Subtilis called after Bacillus subtilis B7348 with strong bacteriostatic action provided by the invention is preserved in Chinese typical culture collection center on October 12nd, 2010, and its deposit number is CCTCC M 2010260.
Fig. 1 is subtilis B7348 and the fungistatic effect figure of subtilis Nxc6 to different indicators, wherein, A is the fungistatic effect figure of B7348 to chicken colibacillosis O78, B is the fungistatic effect figure of B7348 to streptococcus aureus, C is the fungistatic effect figure of B7348 to swine escherichia coli 2116, and D is the fungistatic effect figure of Nxc6 to white dysentery Salmonellas C79-13.
Fig. 2 is that subtilis B7348 SDS-PAGE analyzes.
Fig. 3 is a safety testing rate of body weight gain data plot.
Fig. 4 respectively organizes the feed conversion rate of fryer for test.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
One, genus bacillus primary dcreening operation
Genus bacillus grows surely in animal body and can produce a large amount of amylase, proteolytic enzyme, cellulase and phytase etc.These enzymes help direct decomposition, digestion feed nutrient in animal intestinal, the non-starch polysaccharide in the degrading plant forage improves the physical property of digestive tube chyme or the antinutritional factor in the elimination daily ration, promotes growth and the production of animal.In addition, genus bacillus has produced a large amount of meta-bolitess, also comprises bacteriocin, for example subtilyne (subtilin) etc.Bacteriocin has direct bacteriostatic activity, by with actings in conjunction such as other meta-bolites such as acetate, propionic acid, butyric acid, can effectively suppress the pathogenic bacteria growth, improve the immune level of animal, finally improved the transformation efficiency of feed.
1. materials and methods
1.1 test strain subtilis N9, subtilis N9-1-35, Bacillus licheniformis S, cold water bacterium 1#, bacillus natto 1#, bacillus pumilus BT123, bacillus natto 4#, bacillus cereus etc.
1.11 fermention medium and culture condition glucose 1.5%, soybean cake powder 3.0%, peptone 0.2%, NaCl 0.1%, (NH
4)
2SO
40.5%, CaCO
30.6%, MgSO
40.6%, pH 7.2～7.5,37 ℃ of liquid 48h that cultivate.
1.1.2 the test strain pre-treatment is inoculated into slant strains in the 500mL triangular flask that the 100mL fermention medium is housed, 37 ℃ of constant temperature vibrations (180rpm) are cultured to the gemma rate more than 10%, 5000rpm centrifugation thalline and supernatant liquor, and it is standby to get supernatant liquor.
1.2 indicator
Swine escherichia coli 1565, Sarcina lutea, streptococcus aureus.
Substratum and culture condition: glucose 0.2%, peptone 1.0%, NaCl 0.5%, extractum carnis 0.5%, 7.0,37 ℃ of liquid 24h that cultivate of pH.
All dilute 100 times before more than cultivating the test of gained indicator, between the OD value 0.2～0.3.
1.3 extracorporeal bacteria inhibitor test-cup-plate method
The preparation of two dish: the culture dish of cut-off footpath 90mm, inject nutrient agar medium (1.5%) 15mL of sterilization, horizontal positioned makes it to solidify, as bottom, other gets indicator substratum (concentration is 0.8%, is chilled to about 50 ℃) and an amount of mixing of indicator bacterium liquid, gets 6mL and is layered on the bottom substratum, horizontal positioned makes it to solidify, as the bacterium layer.
Add sample: with the sterilized Oxford of aseptic nipper gripping cup, open the ware lid, be placed on the substratum.In the cup of Oxford, fill it up with the antimicrobial fluid (300 μ L) of same amount, three repetitions of each sample.The culture dish that adds sample is carefully put into 37 ℃ of thermostat containers, take out behind the cultivation 18h and measure antibacterial circle diameter.
1.4 key instrument equipment
2. result and analysis
Concrete test-results is as shown in table 1:
Table 1 genus bacillus primary dcreening operation result
Test-results shows that subtilis N9-1-35, bacillus natto 1# do not have the obvious suppression effect to three kinds of pathogenic bacterium listed above; Subtilis N9, Bacillus licheniformis S, cold water bacterium 1#, bacillus natto 4#, bacillus pumilus BT123 have than the obvious suppression effect two kinds of pathogenic bacterium at least, so tentatively selected subtilis N9, cold water bacterium 1#, Bacillus licheniformis S, bacillus natto 4#, bacillus pumilus BT123 are as next step test strain, in order to obtain to have the genus bacillus of higher bacteriostasis property, several bacillus in next step test, have been screened again simultaneously.
Two, genus bacillus is sieved again
1 materials and methods
1.1 material
Fresh cow dung, pig manure;
Indicator: intestinal bacteria O78, swine escherichia coli 2116, fowl typhoid Salmonellas C79-20, white dysentery Salmonellas C79-13, chicken colibacillosis 249, streptococcus aureus.
1.2 sample collecting and processing
Take by weighing fresh cow dung, chicken manure 10g respectively, place the triangular flask that 100mL physiological saline is housed, in 85 ℃ water-bath, handle 15min, centrifugal (3000rpm) 10min.It is standby to get supernatant liquor.
1.3 strains separation
Get quantity of sample supernatant liquor and intestinal bacteria O78 fermented liquid mixing spread plate, place 37 ℃ of constant temperature culture 24h, choosing periphery of bacterial colonies has the bacterium colony of inhibition zone to carry out separation and purification, and carries out extracorporeal bacteria inhibitor test.
1.4 the pre-treatment of test strain and the cultivation of indicator: concrete grammar is with the genus bacillus primary dcreening operation.
1.5 extracorporeal bacteria inhibitor test-cup-plate method (concrete grammar is with the genus bacillus primary dcreening operation).
1.6 bacterial strain and the primary dcreening operation bacterial strain that newly filters out compared test, adopts cup-plate method to carry out extracorporeal bacteria inhibitor test.
1.7pH value is to the influence of genus bacillus fungistatic effect
1.7.1 test strain pre-treatment
37 ℃ of constant temperature vibrations (180rpm) are cultivated, and observe the gemma production rate, reach at 10% o'clock and promptly can be used for test.By aseptic technique fermented liquid pH is transferred to 3.0,4.0,5.0,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0,11.0, the centrifugal 10min of 5000rpm, it is stand-by to get supernatant.
1.7.2 control group is handled
Get the blank keynote pH to 3.0,4.0,5.0,6.0,6.5,7.0,7.5,8.0,8.5,9.0,10.0,11.0 that cultivates, the centrifugal 10min of 5000rpm, it is stand-by to get supernatant.
1.8 produce the neutral protease enzyme work-employing folin's methods in the enzyme performance detection assay fermentation of bacillus liquid.
1.9 drug sensitive test adopts ordinary method to measure the bacteriostatic action of microbiotic to genus bacillus.
2 results and analysis
2.1 newly screen bacterial strain: strain bacillus test isolates 40 altogether from cow dung, pig manure surplus, gramstaining is positive, and gemma is arranged, and preliminary evaluation is a genus bacillus.To this surplus 40 the strain bacterium carry out the bacteriostasis property test, wherein the performance with subtilis B7348, Nxc6 bacterial strain is the most outstanding.Bacteriostatic test the results are shown in Figure 1 and table 2.
Table 2 bacteriostatic test result (unit: mm)
2.2 newly sieve the comparison test of bacterial strain and primary dcreening operation bacterial strain, the results are shown in Table 3.
Bacteriostatic test effect (the unit: mm) of table 38 bacillus
Annotate :+expression antibacterial circle diameter is 9～13mm, ++ the expression antibacterial circle diameter is＞13mm;-expression inhibition zone is not obvious.
By test-results as can be seen, Bacillus licheniformis S only has good inhibitory effect to gram-positive microorganism, subtilis N9 only has good inhibitory effect to Gram-negative bacteria, B7348, Nxc6 show stronger restraining effect to gram-positive microorganism, simultaneously Gram-negative bacteria also there is certain restraining effect, bacillus pumilus BT123 does not have the obvious suppression effect to several indicators, cold water bacterium 1# has certain restraining effect to Gram-negative bacteria, and bacillus natto 4# only has restraining effect to gram-positive microorganism.
By multiple test-results of sieving as can be seen, subtilis N9, B7348, Nxc6, Bacillus licheniformis S and cold water bacterium 1# have certain restraining effect to testing used a few strain indicators, further screened by extracorporeal bacteria inhibitor test again, be the results are shown in Table 4.
Fungistatic effect (the unit: mm) of the multiple sieve of table 4 back residue bacterial strain
Test-results shows that subtilis B7348 and Nxc6 have more satisfactory fungistatic effect to used indicator, and especially to Gram-positive pathogenic bacteria-streptococcus aureus, it is fairly obvious to suppress effect, and concrete fungistatic effect figure sees shown in Figure 1.
2.3pH value is to the influence of genus bacillus fungistatic effect
2.3.1pH value the results are shown in Table 5 to the influence (because Bacillus licheniformis S only has the obvious suppression effect to gram-positive microorganism, so indicator is streptococcus aureus and Sarcina lutea in this test) of Bacillus licheniformis S fungistatic effect.
Table 5pH value is to the influence (unit: mm) of Bacillus licheniformis s fungistatic effect
Test-results shows, the meta-bolites of Bacillus licheniformis S can produce the material with obvious inhibition gram-positive microorganism, and its bacteriostatic activity reached the highest at 7.0～8.0 o'clock, and in the scope of pH value 3.0-11.0, its bacteriostatic activity presents the characteristic that raises and afterwards reduce earlier.
2.3.2pH value the results are shown in Table 6 to the influence of subtilis B7348 fungistatic effect.
Table 6pH value is to the influence (unit: mm) of subtilis B7348 fungistatic effect
Test-results shows, subtilis B7348 can produce the meta-bolites with obvious inhibition gram-positive microorganism, and its bacteriostatic activity is no significant difference in the scope of pH6.5～8.5, and in the scope of pH value 3.0-11.0, its bacteriostatic activity presents the characteristic that raises and afterwards reduce earlier.
2.3.3pH value the results are shown in Table 7 to the influence of subtilis Nxc6 fungistatic effect.
Table 7pH value is to the influence (unit: mm) of subtilis Nxc6 fungistatic effect
Test-results shows, subtilis Nxc6 can produce the meta-bolites with obvious inhibition gram-positive microorganism, and its bacteriostatic activity is no significant difference in the scope of pH6.5～8.5, and in the scope of pH value 3.0-11.0, its bacteriostatic activity presents the characteristic that raises and afterwards reduce earlier.
2.4 the product enzyme performance of genus bacillus the results are shown in Table 8.
Table 8 neutral protease measurement result
By test-results as can be seen, subtilis B7348 neutral protease vigor screens bacterial strain apparently higher than other.
2.5 the result is as shown in table 9 in the drug sensitive test drug sensitive test.
Table 9 microbiotic is to subtilis B7348, and the bacteriostatic test of Nxc6 is antibacterial circle diameter (mm) as a result
Test-results shows that B7348, Nxc6 belong to extremely sensitive to Ofloxacine USP 23, and B7348 belongs to extremely sensitive to penicillin, and Nxc6 belongs to medium sensitivity to penicillin, B7348, Nxc6 to Streptomyces in extremely sensitive.
3. conclusion
3.1pH value influences not obvious to the pH bacteriostatic activity to sporeformer in 6.5～8.5 scopes that influences of genus bacillus fungistatic effect; Genus bacillus is cultivated and finishes the neutral alkali partially of back pH generally speaking, and promptly pH is 7.0～8.5.
3.2 each bacterial strain fungistatic effect relatively draws by test, many bacillus of test have notable difference to the fungistatic effect of different pathogenic bacterium, on the whole the gram-positive microorganism effect are better than Gram-negative bacteria.Wherein the most stable with B7348 and Nxc6, antimicrobial spectrum is the widest, fungistatic effect the best.Bacillus licheniformis S is only obvious to the effect of gram-positive microorganisms such as streptococcus aureus.
3.3 screening strain enzyme-producing characteristic is produced neutral protease vigor by analyzing several bacillus, it is best that discovery subtilis B7348 produces the enzyme characteristic, and neutral protease vigor can reach 140U/mL.
3.4 the bacteriostasis of definite comprehensive bacterial strain of test strain, produce enzyme performance and, finally choose the alternative bacterial strain of subtilis B7348 as follow-up test to many-sided factors such as antibiotic susceptibility.
The preliminary study of embodiment 2 subtilis B7348 meta-bolites physico-chemical properties
The purpose and meaning of 1 research
Subtilis is very extensive in distributed in nature, studies have shown that in a large number it can produce the antibacterial substance of multiple inhibition phytopathogen, the antimicrobial substance of most genus bacillus has has a broad antifungal spectrum, less demanding to potential of hydrogen, advantages such as Heat stability is good, these all provide good condition for development of new natural antiseptic agent and biological prevention and control agent.At present, utilize research report that the character of producing bacillus subtilis antibacterial substance is applied to animal microecological formulation but seldom, the antimicrobial substance of subtilis has the function of promotes growth, health care and treatment disease to livestock and poultry, belong to have no side effect, a class environment-friendly type preparation that noresidue, nothing cause bacterial drug resistance, be a new researchdevelopment direction on the present feed as a kind of novel fodder additive.
A large amount of experimental studies show, the subtilis meta-bolites is to intestinal bacteria (Escherichia coli), Salmonellas various pathogens such as (Salmonella Lignieres) all has stronger restraining effect, but the physico-chemical property of the antibacterial substance that produces and the composition of antibacterial substance are still waiting to continue experimental study, this research is intended the physico-chemical property of subtilis meta-bolites antibacterial substance is carried out preliminary study, and it is carried out the initial gross separation purification experiment, in the hope of providing foundation in the further development and application aspect the animal microecological formulation for the subtilis meta-bolites.
2 test materialss
2.1 strains tested
Subtilis N9,7348, Nxc6.
2.2 indicator
Chicken colibacillosis O
1(Escherichia coli O
1), streptococcus aureus (Staphy loccocus aureus).
2.3 substratum
Genus bacillus slant medium: peptone 10g, extractum carnis 5g, NaCl 5g, agar 15g, distilled water 1000mL, pH value 7.0.
Genus bacillus liquid seed culture medium: glucose 2g, yeast extract paste 5g, peptone 10g, NaCl 5g, distilled water 1000mL, pH value 7.0.
Fermentation of bacillus substratum: glucose 5g, peptone 10g, extractum carnis 3g, distilled water 1000mL, pH value 7.0.
Indicator fermention medium: with the subtilis liquid seed culture medium.
2.4 instrument and equipment
Constant incubator, autoclaving pot, Bechtop, microscope, acidometer, constant-temperature shaking culture case, liquid-transfering gun, Oxford cup, potlery tile lid etc.
3 test methods
3.1 extracorporeal bacteria inhibitor test-cup-plate method
The preparation of two dish: the culture dish of cut-off footpath 90mm, inject nutrient agar medium (1.5%) 15mL of sterilization, horizontal positioned makes it to solidify, as bottom, other gets indicator substratum (concentration is 0.8%, is chilled to about 50 ℃) and an amount of mixing of indicator bacterium liquid, gets 6mL and is layered on the bottom substratum, horizontal positioned makes it to solidify, as the bacterium layer.
Add sample: with the sterilized Oxford of aseptic nipper gripping cup, open the ware lid, be placed on the substratum.In the cup of Oxford, fill it up with the antimicrobial fluid (300 μ L) of same amount, three repetitions of each sample.The culture dish that adds sample is carefully put into 37 ℃ of thermostat containers, take out behind the cultivation 18h and measure antibacterial circle diameter.
3.2 the preparation of meta-bolites
The preparation of subtilis meta-bolites: with bacterial classification inoculation in slant medium, cultivate rejuvenation for 37 ℃, go down to posterity behind the 24h in the 250mL triangular flask that the 50mL liquid seed culture medium is housed, 37 ℃ of constant-temperature shaking culture (180rpm) 24h, be inoculated into then in the 500mL triangular flask that the 100mL fermention medium is housed, 37 ℃ of constant temperature vibrations (180rpm), be cultured to the gemma rate more than 10%, 5000rpm centrifugation thalline and supernatant liquor, get supernatant liquor and filter (the filter membrane diameter is 0.22 μ m), collect the meta-bolites that filtrate is subtilis through biofilter; With cup-plate method meta-bolites being carried out bacteriostatic activity then measures.
3.3H
2O
2Bacteriostatic action is got rid of test.
Prepare the H of 20mg/mL with the phosphate buffer solution of 50mmol/L
2O
2Enzyme solution adds 0.4mL and cultivates meta-bolites in the 1mL enzyme solution, the final concentration that makes enzyme is 4mg/mL, and other gets 0.1mL phosphate buffer solution adding equivalent culture supernatant and compares.Behind 37 ℃ of water-bath 2h with handle before the fermented supernatant fluid size of bacteriostatic activity relatively, to get rid of the H in the meta-bolites
2O
2Bacteriostatic action.
3.4 milk-acid bacteria acid product bacteriostatic action is got rid of test
The milk-acid bacteria meta-bolites is transferred pH to 4.0, compare, measure the inhibition zone size, determine to get rid of in the meta-bolites lactic acid antibacterial substance in addition with cup-plate method with the lactic acid of pH value 4.0 and the MRS substratum of pH value 4.0.
3.5 meta-bolites antibacterial substance specificity analysis
3.5.1 temperature stability
Respectively the meta-bolites of each strains tested is handled 10min, 30min, 60min respectively under 60 ℃, 80 ℃, 100 ℃, do bacteriostatic test after the cooling, measure bacteriostatic activity, control group is set simultaneously.
3.5.2pH value is to the influence of antibacterial substance bacteriostatic activity
It is 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 that meta-bolites is transferred pH with NaOH and HCl respectively, at room temperature places 24h (control group places 4 ℃ of refrigerator 24h), measures its external fungistatic effect with the Oxford agar diffusion method then.
3.5.3 proteolytic enzyme is to the active influence of antibacterial substance
Subtilis B7348 meta-bolites transfers to the optimum pH (being respectively 2.0,7.5,7.5 and 6.5) of stomach en-, trypsinase, Proteinase K and papoid effect, add above-mentioned each enzyme liquid respectively by whole mass concentration 1.0mg/mL, behind the water bath with thermostatic control enzymolysis 3h that (is respectively 40 ℃, 37 ℃, 58 ℃ and 60 ℃) under the optimum temperuture of above-mentioned each proteolytic enzyme, the pH value of enzymolysis solution is recalled to the pH value of original fermented solution, with streptococcus aureus and intestinal bacteria O
1Being indicator, is contrast with the meta-bolites of enzymolysis processing not, and cup-plate method is carried out extracorporeal bacteria inhibitor test, do three parallel.
3.6 the preliminary extraction of antibacterial substance
Adopt ammonium sulfate precipitation method, ammonium sulfate extraction effect optimization testing program to bacillus subtilis B7348 design different concns, find out best ammonium sulfate extraction concentration, and antibacterial substance in each test group bacillus subtilis strain meta-bolites has been carried out preliminary extraction.
The selection of best ammonium sulfate concentrations: cultured fermented liquid in the centrifugal 20min of 4000rpm, is removed thalline.Supernatant liquor is on average poured in 8 Erlenmeyer flasks, slowly added anhydrous slufuric acid ammonium powder while stirring, make the final quality mark of solution reach 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% respectively through grinding.Be placed in 4 ℃ of refrigerators and leave standstill 8～12h, albumen precipitation is come out.In the centrifugal 10min of 5000rpm, abandon supernatant liquor then, will precipitate redissolution, dialyse in the dialysis tubing of falling people (spending the night) with deionized water.Measure the anti-microbial activity of 8 concentration dialyzates and supernatant liquor respectively, determine the best working concentration of ammonium sulfate precipitation according to the size of inhibition zone.
4 results and discussion
4.1H
2O
2Bacteriostatic action is got rid of test
Table 10 subtilis H
2O
2Enzymolysis test-results antibacterial circle diameter: (mm)
The result is as shown in table 10, and test-results shows that to the aureus with inhibition decrease to some degree, illustrate in the subtilis B7348 meta-bolites has certain H to subtilis B7348 behind hydrogen peroxide enzyme enzymolysis
2O
2, but also have a large amount of other materials to have bacteriostatic activity equally.
4.2 milk-acid bacteria acid product bacteriostatic action is got rid of test
Table 11 milk-acid bacteria acid product bacteriostatic action is got rid of the test-results antibacterial circle diameter: (mm)
Annotate: indicator is a streptococcus aureus.
The result is as shown in table 11, test-results shows, the milk-acid bacteria LP meta-bolites and the MRS substratum (transferring pH with lactic acid) that are pH value 4.0 equally all have tangible fungistatic effect, and the antibacterial circle diameter of meta-bolites is slightly larger than the substratum antibacterial circle diameter, but the lactic acid of simple pH value 4.0 does not have fungistatic effect, meta-bolites or certain antibacterial substance in the MRS substratum of this explanation milk-acid bacteria LP have certain bacteriostatic activity under acidic conditions, and simple lactic acid does not have bacteriostatic action.
4.3 temperature stability test
Table 12 subtilis B7348 treatment of different temperature test antibacterial circle diameter: (mm)
The result is as shown in table 12, and test-results shows that the antibacterial substance in the subtilis B7348 meta-bolites is through 60 ℃, 80 ℃, 100 ℃ processing 10min, 30min, 60min, to chicken colibacillosis O
1Slightly reduce with the rising of handling temperature and the prolongation of time with aureus with inhibition, but all in all, subtilis B7348 has better thermostability.
4.4pH value is to the influence of antibacterial substance bacteriostatic activity
Table 13 subtilis B7348pH value stabilization test antibacterial circle diameter: (mm)
The result is as shown in table 13, test-results shows, antibacterial substance in the meta-bolites of subtilis B7348 is at room temperature placed its bacteriostatic activity of 24h and is slightly reduced, activity reaches the highest when pH value 8.0, in the scope of pH value 4.0-11.0, its bacteriostatic activity presents the characteristic that raises and afterwards reduce earlier.
4.5 the stability of proteolytic enzyme
Table 14 subtilis B7348 protease hydrolyzed test antibacterial circle diameter: (mm)
The result is as shown in table 14, test-results shows that subtilis B7348 meta-bolites behind each protease hydrolyzed, is had only the part bacteriostatic activity in addition of trypsinase and pepsin, and after Proteinase K and papoid processing, lost bacteriostatic activity fully.The result confirms to contain the Partial Protein constituents in the antibacterial substance of subtilis B7348 meta-bolites.
4.6 the preliminary extraction of antibacterial substance
4.6.1 the selection of best ammonium sulfate concentrations
With subtilis B7348 is test strain, and it is as shown in Table 15 to carry out best ammonium sulfate concentrations test-results.
The different ammonium sulfate concentrations of table 15 subtilis B7348 meta-bolites are carried the test-results antibacterial circle diameter mutually: (mm)
The result is as shown in Table 15, and by test-results as can be seen, ammonium sulfate concentrations is extracted the back to intestinal bacteria O 10%～70%
1The unrestraint effect; Ammonium sulfate concentrations is 30%, 40% and 50% o'clock, and extracting the back has restraining effect to streptococcus aureus, and extracted at 30% o'clock more thorough, the antibacterial circle diameter of supernatant liquor is minimum, so ammonium sulfate precipitation concentration is good with 30%.
5 conclusions
5.1 mainly produce one of bacterial classification as probiotics, research and the application of bacillus subtilis formulation in animal produces is the focus that livestock industry research and production personnel pay close attention to always, but it is fewer that the characteristic of antibacterial substance that subtilis produces is applied in animal diseases control aspect report, this test is carried out preliminary study to the physico-chemical property of subtilis B7348 meta-bolites antibacterial substance, and it is tentatively extracted test, in the hope of providing foundation in the further development and application aspect the animal diseases control for subtilis.
5.2 in this test strain, subtilis antibacterial substance that B7348 produces has certain thermostability, 60 ℃, 80 ℃, 100 ℃ after handling 30min active do not change or change very little, there was no significant difference between each is handled.The thermostability of antibacterial substance shows that it can stand pyroprocessing in suitability for industrialized production with in food-processing, can reduce the condition restriction of its suitability for industrialized production, thereby reduce the cost of its production and application, for its large-scale industrial production and application provide may.
Effectively antipathogenic composition analysis in the embodiment 3 subtilis B7348 meta-bolitess
1. materials and methods
Adopt the SDS-PAGE method to analyze.
Gum concentration is 15%, sample applied sample amount 20 μ L, Maker applied sample amount 10 μ L.Two ends are Maker.
Sample process different treatment: subtilis thalline, bacillus subtilis bacteria culture fluid, the centrifugal 5min of bacillus subtilis bacteria culture fluid 4000rpm, get supernatant.
Three kinds of processing add to 4,5,6 swimming lanes respectively, analyze.
2. test-results
Subtilis B7348 SDS-PAGE result as shown in Figure 2.
3. analyze and discuss
Subtilis B7348 three dense bands all occurred about 8kd in three swimming lanes of 4,5,6, analyze from molecular weight, and this three band is the small-molecule peptide material.Wherein all this band can occur in nutrient solution supernatant and the precipitation, illustrate that subtilis B7348 can secrete this active small peptide.The author carries out bacteriostatic test with this material again, verifies that it has stronger fungistatic effect, thereby confirms that subtilis B7348 has the effect of stronger inhibition cause of disease indicator.
The preparation technology of embodiment 4 bacterium powder
(i) bacterial classification: select subtilis (Bacillus subtilis) B7348 for use, CCTCC M 2010260;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 24h at 37 ℃;
(iii) first order seed is cultivated: with cultured inclined-plane, encircle in 100mL seed liquid nutrient medium with inoculation articulating 2 under aseptic condition, under 37 ℃ of conditions, leave standstill and cultivate 16h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in the 1000mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 12h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 37 ℃ of conditions, leave standstill and cultivate 20h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 14000cP, collects fermented liquid;
(vii) after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately.
Above-mentioned steps is (iii), (iv) described seed liquid culture medium prescription is: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, regulate pH7.0, and 20min sterilizes under 115 ℃ of conditions; The (ii) described solid slant culture base of step is to add 1.5% agar powder in the above-mentioned seed liquid nutrient medium; (prescription of liquid fermentation medium v) is: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, sodium-chlor 5g/L, pH 7.0 for step.
Because it is better that subtilis B7348 suppresses effect to gram-positive microorganism, and plant lactobacillus LP-11 is obvious to Gram-negative bacteria inhibition effect, both have good complementary relationship, and the associating result of use is better.Take all factors into consideration the different fungistatic effect of two strain bacterium, subtilis B7348 and plant lactobacillus LP-11 and meta-bolites thereof are carried out composite (wherein subtilis B7348 bacterium powder viable count is 2.0 * 10
8Cfu/g, milk-acid bacteria bacterium powder viable count is 1.0 * 10
8Cfu/g.), carry out reasonably combinedly, form product peptide rhzomorph, satisfy different animals, the demand in different breeding stage.
The peptide rhzomorph is normal 0.2% at the recommendation consumption of stud bird, when ill 0.4%; The peptide rhzomorph by 0.1～0.2% consumption, is pressed 0.3～0.4% consumption milking sow pregnant sow; For clinical mastitis, peptide rhzomorph consumption is 80～120g/ head/sky, can be earlier with 120 gram two weeks, again with 80 gram two weeks, (generally with the threshold value that to contain 200,000 cells in every ml milk be subclinical mastitis, it is positive to surpass this value, and it is then normal to be lower than this value for somatocyte.) be controlled at gradually below 200,000; For negative mastitis, peptide rhzomorph consumption is 40g/ head/sky, and life-time service gets final product, and somatocyte was controlled at below 200,000 gradually in 1 month.
1 materials and methods
1.1 material
1.1.1 test strain: subtilis B7348 and Nxc6 are obtained by previous embodiment 1 screening and separating purifying.
1.1.2 experimental animal: the blue brown young cock in sea, livestock corporation buys by Eastern Mountain, Tai.
1.1.3 the basal diet prescription: composition sees Table 16.
Table 16 basal diet prescription
Annotate: do not add feeding antibiotic in the Preblend
1.2 test site: ecological pasture, Tai'an Ba Fubafu agricultural science and technology company limited Culai Mountain.
1.3 testing program
1.3.1 test grouping and feeding and management: 450 the 1 blue brown young cocks in age in days sea, chicken size, healthy state are basic identical, are basal diet not contain antibiotic complete diet pellet.Be divided into 11 groups at random, promptly 1 control group and 10 test group are established 2 repetitions for every group, each repeats 20 chickens, add the B7348 and the Nxc6 spray powder of different concns respectively and test on the basis of basal diet, wherein 1～7d is for raising the phase in advance, and the official test phase is 14d; According to routine hen house is carried out disinfection, adopt the mode of raising in cages, free choice feeding, drinking-water.Carry out immunization by the chicken house normal procedure; Test design sees Table 17, and (wherein the viable count of subtilis B7348 and Nxc6 spray powder is 2 * 10
8Cfu/g).
Table 17 test design
1.3.2 test rating
(1) observation of appearance character
(2) the pathology situation of observation internal organs
(3) mensuration of growth indexes
2 results and discussion
2.1 after two weeks fed, chick growth was grown normal, outward appearance and the ight soil of observing chick there is no unusually.
After 2.2 experimental animal is dissected, the no abnormality seen pathology.
2.3 influence to growth performance
After growth test finished, every group of chick gross weight of weighing was to calculate indexs such as last counterpoise, rate of body weight gain, growth ratio, and the result is shown in table 18.
Rate of body weight gain=(W
t-W
0)/W
0
Growth ratio (%)=(ln W
t-ln W
0)/T
Wt-tests last chick counterpoise (g), W
0-test is chick counterpoise (g) just, and T-tests fate.
Table 18 subtilis is to the influence of growth performance of chicks
To rate of body weight gain and two groups of data analyses of growth ratio, as shown in Figure 3.
Test-results shows, except that 7 (2) and N (3), rate of body weight gain and growth ratios of all the other each groups all are higher than control group, especially 7 (3), 7 (5) group rates of body weight gain improve 10.0%, 11.1% than control group, growth ratio improves 5.4%, 6.7% than control group.
Test-results shows that further subtilis B7348 is better than subtilis Nxc6 to the gaining effect of chick.
The efficiency test of embodiment 7 subtilis B7348
1 test objective:
The test objective of this test is to detect subtilis B7348 and whether the compound lactobacillus meta-bolites has certain immune effect to the animal of curing the disease, and investigates it and whether have comparability with the microbiotic contrast aspect production performance that improves fryer.
2 test materialss
2.1 the white plumage fryer of experimental animal: AA+ is available from fryer field, the Tai province village.
2.2 test strain: subtilis B7348 (be preserved in Chinese typical culture collection center on October 12nd, 2010,
Its deposit number is CCTCC M 2010260), plant lactobacillus LP (this bacterial strain called after Lactobacillusplantarum, be preserved in Chinese typical culture collection center on 06 21st, 2010, its deposit number is CCTCCM 2010150, is documented among the Chinese patent application CN201010240725.X).
2.3 test site: ecological pasture, Tai'an Ba Fubafu agricultural science and technology company limited Culai Mountain.
2.4 basal diet prescription: see Table 19.
Table 19 basal diet prescription
3 test methods
3.1 test grouping and feeding and management: 240 the 1 white plumage fryer of age in days AA+, chicken size, healthy state are basic identical, are basal diet not contain antibiotic complete diet pellet.Be divided into 4 groups at random, promptly 1 control group and 3 test group are established 2 repetitions for every group, and each repeats 30 chickens.Test design sees Table 20.Chick goes out shell beginning in second day, attacks poison with white dysentery Salmonellas 24h culture, only attacks poison amount 0.5mL/.Treat to observe about chick seven ages in days the variation of chick ight soil, indexs such as the statistics sickness rate and the course of disease.According to routine hen house is carried out disinfection, adopt the mode of raising in cages, free choice feeding, drinking-water.21 days trial periods.
Table 20 test design scheme
* annotate: subtilis B7348 bacterium powder viable count is 2 * 10
8Cfu/g, milk-acid bacteria bacterium powder viable count is 1 * 10
8Cfu/g, Ofloxacine USP 23 purity is 2%.
3.2 detection index
3.2.1 respectively organize the mensuration of the chicken sickness rate and the course of disease
3.2.2 growth traits analysis: weightening finish and the feed conversion rate of measuring 7,14,21 ages in days respectively.
4 results and analysis
4.1 efficiency analysis: the result is shown in table 21.
The incidence behind the merit poison is respectively organized in table 21 test
Wherein the control group sickness rate is more than 90%, and still has 10% not recovery from illness when 21 ages in days, and the sickness rate and the course of disease are apparently higher than other each group.Secondly, III group sickness rate is just fully recovered in back three days in morbidity about 60%, and the sickness rate and the course of disease are starkly lower than other each group.In addition, I group and II group sickness rate are about 70%, and the I group has also been fully recovered in the back four day time of morbidity, and the II group course of disease is a little longer, and these the two groups of sickness rate and the course of disease are between negative control and positive control.
Test-results shows that interpolation subtilis B7348 and plant lactobacillus LP can obviously be alleviated the fryer diarrhea symptom that causes because of Salmonella infection in the daily ration of fryer.
4.2 production traits analysis: the result is shown in table 22, and makes comparison diagram according to table 22, as shown in Figure 4.
The growth performance situation of fryer is respectively organized in table 22 test
The test-results of table 22 and Fig. 4 shows that from average feed conversion rate, III organizes a little higher than other test group of feed conversion rate.In first week, the microbiotic test group has obvious superiority than other test group.Mainly be that it can play the effect of protection intestinal microflora because test initial stage microbiotic is obvious to the inhibition effect of assorted bacterium.Along with the prolongation of test period, animal body and other assorted bacterium have all produced certain resistance to microbiotic, cause the downtrending of microbiotic group feed conversion rate obvious, have just lost superiority to testing the later stage.On the contrary, viable bacteria field planting in animal body has some cycles, more early takes in probiotic bacteriums such as subtilis, plant lactobacillus and can help it to form floras such as advantage milk-acid bacteria as early as possible, helps animal to improve resistibility, avoids the infection of external unwanted bacteria.Therefore it is with the obvious advantage than the microbiotic group that I and II organize the test later stage.
Test-results shows that interpolation subtilis B7348 and milk-acid bacteria LP-11 can obviously be alleviated the fryer diarrhea symptom that causes because of Salmonella infection in the daily ration of fryer.Compare with feeding antibiotic, use two probiotics and meta-bolites thereof,, reduce aspects such as sickness rate, reached and surpassed the effect of feeding antibiotic improving Abwehrkraft des Koepers.Especially obvious to animal later stage recovery effects.
In a word, can draw by test-results, animal is taken in the probiotics that contains subtilis B7348, plant lactobacillus LP and LP fermented product, the effects such as resistance against diseases of improving food conversion ratio, improve animal body can be played, the effect that substitutes feeding antibiotic can be played to a certain extent.
Embodiment 8 peptide rhzomorphs are to the action effect of the meat kind chicken of raising in cages
The peptide rhzomorph contains the multiple viable bacteria composition useful to chicken, their can be in the enteron aisle of chicken specific position breedings form specific flora advantage, suppress the growth of pernicious bacteria by producing a series of meta-bolitess, thereby kept intestinal health, reduced the generation of intestinal tract disease.Probiotics can produce some digestive ferments and vitamin B group, promote the absorption of the nutritive substance in the enteron aisle, improve feeding effect, can also reduce in the enteron aisle and excrete the formation of ammonia of ight soil and the quantity of harmful bacterium, very beneficial to improving Air quality and environment of chicken house.
Probiotics is generally acknowledged the beneficial effect of chicken, but the problem that economic benefit is the user to be concerned about the most, and selected several major techniques of this test and economic target are come analyzing evaluation the feed effect of peptide rhzomorph of meat kind chicken of raising in cages.
1 materials and methods
1.1 peptide rhzomorph: be the mixing of subtilis B7348 bacterium powder and plant lactobacillus LP-11 bacterium powder, wherein, subtilis B7348 bacterium powder viable count is 2.0 * 10
8Cfu/g, plant lactobacillus LP-11 bacterium powder viable count is 1.0 * 10
8Cfu/g; Subtilis B7348 bacterium powder is pressed method preparation among the embodiment 4.
1.2 test chicken AA father and mother are for meat kind chicken
1.3 test period
On January 17th, 1 20010 on November 17th, 2009, test chicken week age: 0-61 age in week
1.4 test chicken grouping
The finishing period 1.4.1 brood (0-22 age in week)
When going into to give up, will be divided into two groups immediately with batch 1 Japanese instar chickling (mother), 11088 of test group, 11039 of control groups, every component is supported in 5 are brooded house.
1.4.3 laying period (23--61 age in week)
Change the finishing period test chicken over to 6 identical hen houses of the same field condition of laying eggs, each 3 of test group control groups, 5856 every group.
1.4.3 feeding and management
The extra 0.1% peptide rhzomorph that adds no longer adds any medicine in the test group daily ration, and control group is by raising according to a conventional method and managing, and whole process is raised in cages.
1.5 examination statistical item
The finishing period 1.5.1 brood: surviving rate, breed qualification rate, feed consumption rate, expenses for medicine cost, chicken group routine inspection;
1.5.2 laying period: death rate, go into to give up the chicken egg number, go into to give up chicken and produce qualified egg number, plant egg rate, scrambled egg rate, expenses for medicine cost, chicken group routine inspection.
2 results and analysis
The finishing period 2.1 brood
Table 23 brood time laying hen statistic data unit: (only, %, kg, unit)
* the chicken number of moving out is maternal cock number, when being calculated to be motility rate this number is shootd off from go into to give up the chicken number.
The result is shown in table 23, and the result shows that the test group comparison is high 0.14 percentage point according to forming motility rate, and it is high 0.2 percentage point to breed qualification rate, and every chicken feed consumption reduces 0.41g; The ight soil of test chicken is obviously than control group drying, and form is normal, and the ammonia flavor in the hen house alleviates (sense organ judgement).
2.2 laying period
Table 24 laying period laying hen statistic data unit: (only, %, piece/only, kg)
The result is shown in table 24, and the result shows, test group than control group go into to give up chicken count egg number Duo 6.56 pieces/only, many 8.17 pieces of qualified egg/only.These difference mainly are to be caused by death rate and the difference of planting the egg rate, low 6.2 percentage points of the death rate of test group, and kind egg rate is high 1.34 percentage points.
2.3 economic benefit
The finishing period 2.3.1 brood
Expenses for medicine cost test group than control group low 0.383 yuan/only, save expenses for medicine 57.4% (other project is disregarded).
2.3.1 laying period
Qualified kind of egg overcharged into 11.03 yuan/for 8.17 pieces by 1.35 yuan/piece;
Test group is compared according to forming many 363 of live chickens, and many 6.8 yuan an of price is on average overcharged into 0.4215 yuan/;
Test group is only average with peptide rhzomorph 41.01g, by 12 yuan/kg, average 0.4921 yuan/only;
Many feed consumptions of test group 1.62kg/, by 1.35 yuan/kg, many 2.187 yuan/only;
Average 0.7644 yuan/of control group expenses for medicine.
2.3.3 adding up to, increases income and economizes on spending the full phase
Output: 11.03+0.383-0.22+0.4215-2.187-0.4921=8.9354 unit/only
Drop into: 0.284+0.984+0.4921+2.187=3.9471 unit/only
Input-output ratio=3.9471: 8.9354=1: 2.26
3 conclusions
3.1 add 0.1% peptide rhzomorph in the meat kind daily grain of chicken under this test conditions, no longer add any medicine, can guarantee chicken group health, it is normal to grow, and the production performance performance is good.
3.2 test group than control group, is brooded the finishing period surviving rate, to breed qualification rate slightly high, the feed consumption number is slightly high, saves expenses for medicine 57.4%, and the ight soil form is significantly better than control group, hen house ammonia flavor alleviates; Laying period, can obviously reduce death rate, improves laying rate and plant the egg rate, to going into to give up 8.17 pieces of qualified kind of eggs of hen fecund every of 61 week.
3.3 add peptide rhzomorph remarkable in economical benefits in the meat kind chicken feed, input-output ratio is more than 1: 2.26.
Comprehensive above experiment conclusion, the subtilis B7348 that the present invention's screening obtains has good fungistatic effect, can be used as fodder additives uses, for common being seen subtilis not available, now be preserved in Chinese typical culture collection center on October 12nd, 2010, its deposit number is CCTCC M 2010260.
Claims (7)
1. a strain has the subtilis of strong bacteriostatic action, it is characterized in that: this bacterial strain called after Bacillus subtilisB7348, be preserved in Chinese typical culture collection center on October 12nd, 2010, and its deposit number is CCTCCM 2010260.
2. the subtilis with strong bacteriostatic action according to claim 1, it is characterized in that: it is as follows that the strain bio of described subtilis is learned characteristic: the cell size of this bacterial classification is for being generally (0.7 μ m～0.8 μ m) * (2.0 μ m～3.0 μ m), shaft-like, chaining seldom, even dyeing, no pod membrane, the flagellum adnation can move; Gemma ovalize or column, middle life or wilfully, size is 0.8 μ m * (1.5 μ m～1.8 μ m); A little less than free spore surface is painted; Bacterium colony circle or irregular shape on the substratum, surface colour is dark, and thickening and opaque can be wrinkling, can be cream color or brown; Gram-positive, the V-P reaction can take place in the catalase positive; Can utilize glucose, pectinose, wood sugar and N.F,USP MANNITOL to produce acid, the energy hydrolyzed starch decomposes tryptophane and forms indoles; 45～55 ℃ of growth temperature maximums, 5～20 ℃ of Schwellenwerts, optimum temperuture is 37 ℃.
3. the described application of claim 1 with subtilis of strong bacteriostatic action as fodder additives.
4. application according to claim 3 is characterized in that: during application, add in the feed with the form of bacterium powder, described bacterium powder obtains by following steps:
(i) bacterial classification: select subtilis Bacillus subtilis B7348 for use, CCTCC M 2010260;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 20～28h at 32～40 ℃;
(iii) first order seed is cultivated: get cultured inclined-plane, be inoculated under aseptic condition in 50mL～100mL seed liquid nutrient medium, under 32～40 ℃ of conditions, leave standstill and cultivate 14～18h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 32～40 ℃ of conditions, leave standstill and cultivate 10～16h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 32～40 ℃ of conditions, leave standstill and cultivate 16～24h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 12000～15000cP, collects fermented liquid;
(vii) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (iii), (iv) described in seed liquid culture medium prescription be: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L during use, regulates the 20min that sterilizes under ℃ condition of pH to 6.5～7.5,115; Step (ii) described in the solid slant culture base be to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; Step (prescription of liquid fermentation medium v) is: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, sodium-chlor 5g/L, during use, regulate pH to 6.5～7.5.
5. application according to claim 3 is characterized in that: during application, add plant lactobacillus LP-11, form product: the peptide rhzomorph joins in the feed as fodder additives.
6. application according to claim 5 is characterized in that: in the described peptide rhzomorph, the viable count of subtilis B7348 bacterium powder is 2.0 * 10
8Cfu/g, the viable count of plant lactobacillus LP-11 bacterium powder is 1.0 * 10
8Cfu/g.
7. application according to claim 6 is characterized in that: described peptide rhzomorph is as follows as the consumption of fodder additives:
To normal not ill stud bird, consumption is 0.2% of a feed total mass;
To ill stud bird, consumption is 0.4% of a feed total mass;
To pregnant sow, consumption is 0.1～0.2% of a feed total mass;
To milking sow, consumption is 0.3～0.4% of a feed total mass;
To suffering from the milk cow of mastitis, consumption is 80～120g/ head/sky;
To suffering from the milk cow of negative mastitis, consumption is 40g/ head/sky.
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