CN105341425A - Pig feed containing bacteriostatic microbial ecological agent and application thereof - Google Patents

Abstract

The invention belongs to the technical field of feed, and in particular relates to pig feed containing a bacteriostatic microbial ecological agent capable of replacing antibiotic and application thereof. The bacteriostatic microbial ecological agent provided by the invention is prepared by compounding high acid-produced enterococcus faecium and bacteriocin generated by bacillus subtilis. Through feeding with the feed, the antibiotic is replaced with the used efficient bacteriostatic microbial ecological agent, so that intestinal microecological balance of a pig is effectively maintained, and growth performance and immunity are improved.

Description

A kind of pig feed containing antibacterial probiotics and application thereof

Technical field

The present invention relates to field of feed, particularly, the present invention relates to a kind of pig feed containing antibacterial probiotics and application thereof.

Background technology

Antibiotic is as feed addictive Be very effective in livestock and poultry animal diseases prevention, disease-resistant and growth promotion etc., and major contribution has been made in the development for intensive animal husbandry.But, it is found that antibiotic is while bringing great economic benefit, also result in very large harm, long-term abuse of antibiotics causes pathogen to produce the immunologic function etc. of microecological balance and reduction animal in drug resistance, suprainfection, destruction animal body, these all bring serious threat to animal, aquaculture and feed industry, also injure the health of the mankind.

Along with people's living standard improves constantly; to the quality of animal products and the requirement of security also more and more higher; in addition the attention degree of environmental protection is strengthened year by year; many countries are to the kind of feeding antibiotic; using method, the aspect such as dosage and compatibility has carried out strictly limiting or forbidding, various countries start to research and develop green, safe animal feed additive successively simultaneously; carry out substitute antibiotics, guarantee interests and the food security of culturist.In recent years, the kind of feeding antibiotic substitute is more, mainly contains probiotics, enzyme preparation, oligosaccharides, plant extracts, saccharicter-penin, acidulant etc.Wherein probiotics with its noresidue, have no drug resistance, have no side effect and Be very effective, low cost and other advantages and day by day coming into one's own.For reducing or antibiotic use in alternative Production of Livestock and Poultry process, it is simple, convenient to the invention provides, and can maintain the intestinal microecology balance of pig, improve the pig feed of the growth performance of pig and a kind of Efficient antibacterial probiotics substitute antibiotics of immunity.

Summary of the invention

It is simple, convenient to the object of this invention is to provide, and can maintain the intestinal microecology balance of pig, improve the pig feed of the growth performance of pig and a kind of Efficient antibacterial probiotics substitute antibiotics of immunity.

Another object of the present invention is to provide the application of above-mentioned pig feed on swine rearing.

To achieve these goals, technical scheme provided by the invention is as follows:

Weanling pig feed containing antibacterial probiotics of the present invention, comprise weanling pig daily ration and antibacterial probiotics, described antibacterial probiotics weight accounts for 0.50 ‰ ~ 1.00 ‰ of weanling pig daily ration weight; Described antibacterial probiotics is the mixture of intestines dung coccus and producing bacillus subtilis bacteriocin, and wherein, the ratio of described VREF and bacteriocin is 2 × 10 ⁸~ 5 × 10 ⁸the VREF of CFU: 1g bacteriocin.

According to weanling pig feed of the present invention, wherein, described weanling pig Diet Formula raw materials by weight comprises: corn 55.0% ~ 65.0%, wheat bran 2.0% ~ 5.0%, dregs of beans 20.0% ~ 25.0%, fish meal 1.0% ~ 5.0%, whey powder 3% ~ 8.00%, soya-bean oil 1.0% ~ 4.0%, calcium monohydrogen phosphate 0.1% ~ 1.0%, stone flour 0.5% ~ 1.5%, lysine 0.1% ~ 0.5%, salt 0.1% ~ 0.3%, premix 1.0%.Preferably, described weanling pig Diet Formula raw materials by weight comprises: corn 60.0%, wheat bran 3.0%, dregs of beans 23.0%, fish meal 4.0%, whey powder 5.00%, soya-bean oil 2.0%, calcium monohydrogen phosphate 0.7%, stone flour 0.8%, lysine 0.2%, salt 0.3%, premix 1.0%

Growing and fattening pig feed containing antibacterial probiotics of the present invention, wherein, described growing and fattening pig feed comprises growing-finishing pigs diets and antibacterial probiotics, and described antibacterial probiotics weight accounts for 0.50 ‰ ~ 1.00 ‰ of growing-finishing pigs diets weight; Described antibacterial probiotics is the mixture of intestines dung coccus and producing bacillus subtilis bacteriocin, and wherein, the ratio of described VREF and bacteriocin is 2 × 10 ⁸~ 5 × 10 ⁸the VREF of CFU: 1g bacteriocin.

According to growing and fattening pig feed of the present invention, it is characterized in that, described growing-finishing pigs diets formula material comprises by weight percentage: corn 60% ~ 70%, dregs of beans 25% ~ 30%, soya-bean oil 0.5% ~ 1.5%, calcium monohydrogen phosphate 0.5% ~ 2.0%, salt 0.1% ~ 0.5%, stone flour 0.5% ~ 1.5%, premix 1.0%.Preferably, described growing-finishing pigs diets formula material comprises by weight percentage: corn 67.8%, dregs of beans 28.0%, soya-bean oil 1.3%, calcium monohydrogen phosphate 1.0%, salt 0.4%, stone flour 0.5%, premix 1.0%.

According to the above-mentioned arbitrary described pig feed (weanling pig feed or growing and fattening pig feed) of the present invention, wherein, described VREF is VREF CE001 (Enterococcusfaecium), and its culture presevation is numbered: CGMCCNO.9666.This VREF, has good product acid activity.

Bacillus subtilis used in the present invention can be any bacterial strain of existing bacteriocin, especially preferred, and bacillus subtilis of the present invention is selected purchased from Chinese agriculture Microbiological Culture Collection administrative center, and its preserving number is the bacterial strain of ACCC10619.

According to pig feed of the present invention, wherein, the preparation method of described antibacterial probiotics, comprises the following steps:

1) bacillus subtilis of preparation institute bacteriocinogeny is joined in proportion in the VREF zymotic fluid in fermentation later stage, be uniformly mixed;

2) mixed zymotic fluid Separation of Solid and Liquid is obtained bacterium mud; drying protectant is added in bacterium mud; the mass volume ratio (g/L) of described bacterium mud and drying protectant is 5% ~ 25%, adopts granulation, freeze-drying or spraying dry to make antibacterial probiotics.

Further, preparation in accordance with the present invention, the preparation of described bacteriocin comprises the following steps:

1) bacillus subtilis strain is inoculated in fermentation nutrient solution, under 37 ~ 39 DEG C of conditions, cultivates 48 ~ 50h, obtain zymotic fluid;

2) supernatant is got by centrifugal for fermentation liquor 5000 ~ 12000r/m 5-30min, in supernatant, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation degree reaches 20 ~ 80%, then under 4 ~ 5 DEG C of conditions, 4 ~ 12h is left standstill, again under 2 ~ 10 DEG C of conditions, with the centrifugal 10 ~ 30min of 5000 ~ 12000r/m rotating speed, abandoning supernatant obtains bacteriocin.

Bacillus subtilis strain fermentation nutrient solution of the present invention can use well known in the art arbitrarily for the nutrient solution of bacillus subtilis, preferably, nutrient solution used comprises (weight ratio): corn flour 0.5% ~ 3.5%, beancake powder 1.0% ~ 5.0%, fish meal 0.01% ~ 0.5%, glucose 0.2% ~ 2.0%, calcium carbonate 0.5% ~ 5.0%, ammonium sulfate 0.001% ~ 0.2%, dipotassium hydrogen phosphate 0.001% ~ 0.2%, magnesium sulfate 0.001% ~ 0.2%, manganese sulfate 0.001% ~ 0.2%, pure water 1 liter, pH is 7.0 ~ 7.5.

The unit of activity of described bacteriocin can reach 17361.68U/mg, can suppress multiple Gram-negative and Gram-positive pathogens bacterium.

Preparation in accordance with the present invention, the preparation method of described VREF zymotic fluid comprises the following steps:

1) in seed culture medium, cultivate VREF 18 ~ 24 hours under 35 ~ 45 DEG C of aerobics or facultative conditions, become first order seed;

2) primary seed solution is seeded in seeding tank carry out expansion cultivate, under 35 ~ 45 DEG C of aerobics or facultative conditions cultivate 18 ~ 24 hours, become secondary seed;

3) secondary seed solution of acquisition is seeded in fermentation tank carries out fermented and cultured, under 30 ~ 45 DEG C of aerobics or facultative conditions, adopt timing or continuous feeding batch fermentation manner to cultivate 18 ~ 36 hours;

As preferably, step 1) and 2) use MRS culture medium to be seed culture medium.

As preferably, step 3) fermentation adopts the MRS culture medium of improvement, and the MRS culture medium of described improvement is added with soy peptone 5 ~ 30g in often liter of MRS culture medium, glucose 1 ~ 10g, dusty yeast 1 ~ 10g, sodium acetate 1 ~ 10g, citric acid diamines 0.1 ~ 8g, Tween800.1 ~ 5g, dipotassium hydrogen phosphate 0.1 ~ 5g, magnesium sulfate 0.05 ~ lg, manganese sulfate 0.001 ~ 0.2g, calcium carbonate 1 ~ 30g, pure water 1 liter, adjust ph to 5.5 ~ 7.5.

Present invention also offers the application of described weanling pig feed in weaning pig feeding, and the application of described growing and fattening pig feed in growing and fattening swine rearing.

Beneficial effect of the present invention is that proportioning is simple, easy to use, can maintain the intestinal microecology balance of pig, improve growth performance and immunity, be specially:

By the pig feed adding Efficient antibacterial probiotics of feeding, the gastrointestinol microorganism flora of pig can be improved, strengthen immunity of organisms, reduce diarrhea rate, significantly improve growth performance.Meanwhile, in feeding process, decrease the use of antibiotic medicine, guarantee is provided to the livestock products producing green safety.

Accompanying drawing explanation

Fig. 1 is the bacterium colony that VREF of the present invention (Enterococcusfaecium) bacterial strain is formed on MRS agar medium.

Fig. 2 is the dull and stereotyped schematic diagram of Oxford cup that bacteriocin bacteriostatic activity of the present invention detects, and wherein SH represents high dose sample solution, and SL represents low-dosage sample solution, and BH represents high dose standard liquid, and BL represents Low-dose Standard solution.

Fig. 3 is that the bacteriostatic activity of bacteriocin of the present invention under different pH condition compares (embodiment 3 table 6 group 1).

Fig. 4 is that probiotics of the present invention compares with the bacteriostatic activity of compound micro-ecological preparation under same concentrations of VREF with single bacteriocin, single bacillus subtilis and bacillus subtilis.

VREF CE001 (Enterococcusfaecium) of the present invention is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2014, Institute of Microorganism, Academia Sinica, 100101), deposit number is CGMCCNO.9666.

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Culture medium used in following embodiment is filled a prescription as follows if no special instructions:

MRS culture medium:

Peptone 10g, powdered beef 5g, glucose 20g, Tween 80 ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, citric acid tri-amonia 2g, epsom salt 0.2g, manganese sulfate monohydrate 0.05g, dusty yeast 4g, is settled to 1L with distilled water.

MRS agar medium:

Agar 15g is added in 1LMRS culture medium.

The Isolation and Identification of embodiment 1 VREF (Enterococcusfaecium)

(1) isolation and purification of VREF (Enterococcusfaecium)

The separation of 1.1 bacterial strains is cultivated

Healthy chicken is lethal, dissect after, get small intestinal mucosa and each 1g of intestinal contents with aseptic procedure respectively, add in the bottle that 9mL sterile saline is housed, carry out doubling dilution after mixing, get 10 ^-4, 10 ^-5, 10 ^-6dilution suspension 100mL, is added drop-wise on 3 MRS solid mediums respectively, smears evenly with glass bar, then in 37 DEG C of anaerobism and aerobic cultivation 48h, observes colonial morphology and whether has molten calcium ring.The lactic acid bacteria colony inoculation selecting molten calcium ring, in MRS fluid nutrient medium, carries out line separation and purification.Observe meat soup and whether become muddy, have muddy placement 4 DEG C of refrigerator storages for subsequent use.

1.2 Gram's staining

Draw a small amount of MRS broth culture with asepsis injector, drop on slide, alcolhol burner flame is dried gently fixing.Drip violet staining liquid, dye 30s, washing; Drip Gram's iodine solution mordant dyeing, effect 1min, washing; Decolour 30s to drip acetone ethanol mixed liquor (acetone: 95% ethanol=3:7), washing; Drip husky yellow dyeing liquor and redye 1min, washing, wait to do, observe under ordinary optical microscope, thalline takes on a red color as feminine gender, purple be the positive.In the coccus that Gram-positive form is consistent, carry out catalase experiment further.

1.3 catalase experiments

Do MRS slant medium, get culture 0.2mL, inject and MRS agar medium inclined-plane is housed, 5%CO ₂incubator, cultivates 24h, after growing bacterium colony, is added drop-wise on bacterium colony by 3% hydrogenperoxide steam generator for 37 DEG C, if it is negative for not having bubble to produce explanation, if it is positive for having bubble to produce explanation.Genus lactubacillus (Lactobacillus) tentatively can be thought through Gram-positive and catalase experiment feminine gender by the culture of MRS culture medium Anaerobic culturel.

Animal intestinal sample, through separation and purification, isolates 5 strain Lactococcus called after YB-1, YB-2, YB-3, YB-4, YB-5 respectively altogether in conjunction with Gram's staining and catalase experiment.

(2) acid-producing bacteria strain screening study

5 strain test strains of above-mentioned screening are inoculated in 5mLMRS fluid nutrient medium by the inoculum concentration of 2% (v/v), after 37 DEG C of anaerobic fermentation 24h, detect pH value and the lactic acid content of the centrifugal supernatant of zymotic fluid.The results are shown in Table 1.The product acid amount of YB-5 lactic bacteria strain, higher than all the other bacterial strains, shows that this strains of lactic acid bacteria has good product acid activity.

The pH of table 1 streptococcus acidi lactici fermented solution and Lactic Acid from Fermentation Broth content (mg/L) thereof

Strain number	PH value	Lactic acid content
			YB-1	4.4	3637.05
YB-2	4.8	3152.51
			YB-3	4.2	4082.13
YB-4	4.2	4141.62
			YB-5	3.6	5092.51

(3) extracting genome DNA and 16SrRNA order-checking

16SrRNA gene sequencing: the extraction of above-mentioned YB-5 bacteria total DNA adopts bacterial genomes DNA extraction kit (sky root biochemistry (Beijing) Science and Technology Ltd., TiangenDP302-02) to extract.16SrRNA gene magnification primer adopts bacterial universal primers, and its primer sequence is: forward primer is 27F (corresponding to Escherichiacoil8-27 bit base): 5'-AGAGTTTGATCCTGGCTCAG-3'; Reverse primer is 1495R (corresponding to Escherichiacoil1495-1515 bit base): 5'-CTACGGCTACCTTGTTACGA-3'.Reaction system (50 μ L) is as shown in table 2:

Table 2PCR amplification system

Pcr amplification program is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min; 58 DEG C of annealing 1min; 72 DEG C extend 2min, carry out 30 circulations, and last 72 DEG C extend 10min.PCR primer utilizes 1% agarose gel electrophoresis to detect, biotechnology (Beijing) Co., Ltd of positive products purified Hou Songrui Boxing section that fragment length is about 1500bp carries out sequencing, and the nucleotide sequence of the encoding gene of the 16SrRNA of YB-5 bacterium is as shown in SEQIDNo.1.

The gene order obtained carries out BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis in GenBank database.Qualification result finds that the homology of YB-5 and VREF (Enterococcusfaecium) is 99%, is defined as Enterococcusfaecium.Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.9666, and its Classification And Nomenclature is VREF CE001 (Enterococcusfaecium).

The cell of VREF CE001 (Enterococcusfaecium) (CGMCCNo.9666) is spherical, and Gram-positive, other biological characteristics is as shown in table 3.This VREF (Enterococcusfaecium) is the one of enterococcus spp (Enterococcussp.).

The biological characteristics of table 3 VREF

The preparation of embodiment 2 producing bacillus subtilis bacteriocin

(1) with the inoculum concentration of 0.5%, Bacillus subtilis strain is inoculated in fermentation tank, adopts conventional method under the condition of 37 DEG C, 150 revs/min, to cultivate 50h on culture medium, obtain zymotic fluid.Described culture medium comprises (weight ratio): corn flour 0.5%, beancake powder 3%, fish meal 0.1%, glucose 1.0%, calcium carbonate 1.59%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.01%, manganese sulfate 0.01%, pure water 1 liter, pH is 7.0 ~ 7.5;

(2) supernatant is got after the centrifugal 20min of fermentation liquor 10000r/m, in supernatant, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation degree reaches 80%, then under 4 DEG C of conditions, 12h is left standstill, again under 5 DEG C of conditions, 12000r/m, centrifugal 30min, abandoning supernatant obtains bacteriocin.

The Performance Detection that embodiment 3 bacteriocin is antibacterial

(1) antimicrobial spectrum of bacteriocin

The bacteriocin precipitation of getting 1g obtained adds 5 respectively, 10, in 20mL nutrient broth, make 3 nutrient broths (20% containing different bacterium element concentration, 10%, 5%), with the nutrient broth not containing bacteriocin in contrast, inoculate salmonella typhi (Salmonellatyphi respectively, CVCC2212, National Veterinary Culture Collection), staphylococcus aureus (Staphylococcusaureus, CVCC1882, National Veterinary Culture Collection), E.coli K88 (EscherichiaColi, CMCC44742, Chinese medicine bacterium preservation administrative center) and colon bacillus 0157 (EscherichiaColi, purchased from China Veterinary Drugs Supervisory Inst.), inoculum concentration is 5% of nutrient solution, put 37 DEG C, 5%CO ₂24h cultivated by incubator.

Detect the clump count often organizing culture, the results are shown in Table 4.

Table 4 micro-organisms result of the test

As can be seen from the table, the bacteriocin that producing bacillus subtilis is raw under variable concentrations all has good inhibition to salmonella typhi, staphylococcus aureus and two strain Escherichia coli, can make its viable count decline 4-5 order of magnitude.

(2) bacteriostatic activity of bacteriocin detects

Get the sodium acetate buffer (pH6.5) that 1g bacteriocin precipitation is dissolved in 20mL0.02mol/L.Odontothrips loti is adopted to detect its bacteriostatic activity.Bacteriostatic activity analytical method:

(1) colistine sulfate standard items 0.0360g is accurately taken, be settled to 10mL with phosphate buffer, 2000rpm, dilute 10 times after centrifugal 10min again as high dose concentration, and high dose concentration is diluted 1 times again, make it to be made into high low dosage two concentration gradient solution;

(2) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;

(3) get and be diluted to about 10 ⁷the indicator bacteria of the fresh cultured of cfu/mL--Escherichia coli (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the flatness of agar gelling wild Oryza species plane;

(4) Oxford cup aseptic nipper is positioned on flat board gently, after 200 μ L bacteriocin solution are added Oxford cup, 12h is left standstill under 4 ~ 5 DEG C of conditions, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background, by the size of each inhibition zone of vernier caliper measurement, get its mean value

Calculate by formula (1) and tire.

$\log \frac{c_{SH}}{c_{BH}}=\frac{(x_{SH}+x_{SL})-(x_{BH}+x_{BL})}{(x_{SH}+x_{BH})-(x_{SL}+x_{BL})}\×\log k---\left(1\right)$

In formula:

C _sH---tiring of sample solution, unit is U/mg;

C _bH---tiring of standard liquid, unit is U/mg;

X _sH---the antibacterial circle diameter caused by high dose sample solution, unit is millimeter (mm);

X _sL---the antibacterial circle diameter caused by low-dosage sample solution, unit is millimeter (mm);

X _bH---the antibacterial circle diameter caused by high dose standard liquid, unit is millimeter (mm);

X _bL---the antibacterial circle diameter caused by Low-dose Standard solution, unit is millimeter (mm);

K---the ratio of high dose multiple and low dosage multiple.

C in the present embodiment _bHfor 22782U/mg, experimental result is as table 5, and the bacteriocin that producing bacillus subtilis is raw is after testing tired as 17361.68U/mg.

Table 5 inhibition zone measurement result (mm)

(3) bacteriostatic activity of bacteriocin is with pH value situation of change

Get the sodium acetate buffer that 1g bacteriocin precipitation is dissolved in 20mL0.02mol/L, pH value of solution is adjusted to 4.0,5.0,6.0,7.0 respectively.Getting 1g bacteriocin precipitation is again dissolved in 20mL VREF zymotic fluid (pH4.0), adopts Odontothrips loti to detect its bacteriostasis property.Bacteriostasis property analytical method:

(1) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;

(2) get and be diluted to about 10 ⁷the indicator bacteria of the fresh cultured of cfu/mL--Escherichia coli (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the flatness of agar gelling wild Oryza species plane;

(3) Oxford cup aseptic nipper is positioned on flat board gently, after each solution of 200 μ L is added Oxford cup, 12h is left standstill under 4-5 DEG C of condition, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background, by the size of each inhibition zone of vernier caliper measurement.Setup Experiments three groups is parallel.Result is as shown in table 6.

Table 6 inhibition zone measurement result (mm)

The result of table 6 shows that the biocidal property of bacteriocin can promote along with the reduction of pH value, and in the VREF zymotic fluid of pH4.0, the biocidal property of bacteriocin significantly improves.

The preparation of the probiotics of embodiment 4 Efficient antibacterial

Probiotics 1:

(1) be seed culture medium with MRS culture medium, under 37 DEG C of aerobics or facultative conditions, cultivate VREF 18 hours, become first order seed;

(2) by primary seed solution by 0.5% inoculum concentration (volume ratio) be seeded in seeding tank and carry out expansion and cultivate, adopt MRS culture medium to be seed culture medium, cultivate 18 hours under 37 DEG C of aerobics or facultative conditions, become secondary seed;

(3) secondary seed solution that step (2) obtains is seeded in fermentation tank carries out fermented and cultured, the MRS culture medium adopting improvement is fermentation medium, under 37 DEG C of aerobics or facultative conditions, timing or continuous feeding batch fermentation manner is adopted to cultivate 36 hours; The MRS culture medium of described improvement is added with soy peptone 10g in often liter of MRS culture medium, glucose 10g, dusty yeast 5g, sodium acetate 1g, citric acid diamines 0.8g, Tween801g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.08g, manganese sulfate 0.1g, calcium carbonate 20g, pure water 1 liter, adjust ph 7.0.

(4) bacteriocin prepared is joined VREF zymotic fluid according to 1g bacteriocin than the ratio of 500,000,000 VREF viable counts, be uniformly mixed.Under 1000rpm condition, zymocyte liquid is carried out collected by centrifugation thalline.

(5) after centrifugal complete bacterium mud is weighed; bacterium mud is mixed according to mass volume ratio (g/L) 1:5 with protective agent, in-70 DEG C of pre-freeze 2 ~ 3h, in advance open cold lyophilizer; temperature to be frozen is down to-40 DEG C, rapidly the thalline that pre-freeze is good is put into freezing 8h.Wherein said protective agent formula is: skimmed milk power 14%, sucrose 10%, FOS 16% and sodium glutamate 10%, sterilized water 1L.

Probiotics 2:

Except the bacteriocin prepared joins except VREF zymotic fluid according to 1g bacteriocin than the ratio of 200,000,000 VREF viable counts by step (4), other preparation method is with sample 1.

The fungistatic effect research of embodiment 5 probiotics

Odontothrips loti is adopted to be contrasted by the bacteriostasis property of prepare 1% (g/mL) concentration probiotics and the single bacteriocin of 1% (g/mL) concentration, 1% (g/mL) single bacillus subtilis solution and the bacillus subtilis of collecting from market and VREF (ratio of bacillus subtilis and VREF is 8:2) compound micro-ecological preparation 1% (g/mL) solution.Bacteriostasis property analytical method:

(1) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;

(2) get and be diluted to about 10 ⁷the indicator bacteria of the fresh cultured of cfu/mL--Escherichia coli (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the flatness of agar gelling wild Oryza species plane;

(3) Oxford cup aseptic nipper is positioned on flat board gently, after each solution of 200 μ L is added Oxford cup, under 4-5 DEG C of condition, leave standstill 12h, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background.Result as shown in Figure 4.

Under result shows same concentrations, the biocidal property of probiotics of the present invention is significantly higher than single bacteriocin.Bacillus subtilis formulation and bacillus subtilis and VREF compound formulation produce without obvious inhibition zone.

Embodiment 6 Efficient antibacterial probiotics is on the impact of piglet growth performance and gut flora

This test selects parity, body weight close, and Du × length × Dasanyuan hybridized pig 100 of the 28 age in days wean be in a good state of health, be divided into 2 groups at random, every component is 5 repetitions, each repetition 10.Piglet diet formula: corn 60.0%, wheat bran 3.0%, dregs of beans 23.0%, fish meal 4.0%, whey powder 5.00%, soya-bean oil 2.0%, calcium monohydrogen phosphate 0.7%, stone flour 0.8%, lysine 0.2%, salt 0.3%, premix 1.0%.Control group: 1000kg Basic drawing adds+40g gentamicin and 20g colistine sulfate, test group: 1000kg Basic drawing+1.0kg Efficient antibacterial probiotics.

Test and begin from weanling pig 28 age in days, 21 days experimental periods.Fully rinse and strict sterilization pigsty before test.Duration of test feeding every day 5 times, adopts dry mixing material, with the slightly surplus material of crib for principle.Test pig free choice feeding, freely to drink water.Colony house gravity-flow ventilation, regularly sweeps, traditional vaccination immunity.

The impact of Efficient antibacterial probiotics on piglet growth performance and diarrhea rate the results are shown in Table 7.

Table 7 Efficient antibacterial probiotics is on the impact of piglet growth performance and diarrhea rate

Note: indicate different alphabetical person after colleague's data and represent significant difference (P<0.05), same letter represents that difference is not remarkable.

As shown in table 1, Efficient antibacterial probiotics can significantly improve the average daily gain of piglet and average daily ingestion amount, reduces diarrhea rate simultaneously.

The impact of Efficient antibacterial probiotics on piglet gastrointestinal bacterial flora the results are shown in Table 8.

Table 8 Efficient antibacterial probiotics is on the impact (lgCFU/g) of intestine of young pigs flora

Note: indicate different alphabetical person after colleague's data and represent significant difference (P<0.05), same letter represents that difference is not remarkable.

As shown in table 8, Efficient antibacterial probiotics significantly can reduce the quantity of beneficial bacterium lactic acid bacteria in intestines and stomach, reduces the colibacillary quantity of harmful microorganism, is conducive to intestine of young pigs Tiny ecosystem healthy.

Embodiment 7 Efficient antibacterial probiotics is on the impact of microorganism species in grower pigs growth performance and ight soil

This test selects 100 ages in days, parity close, and body weight is at Du × length × Dasanyuan hybridized pig of about 20kg, and male and female respectively accounts for 1/2, is divided into two groups at random, often organizes 5 repetitions, each repetition 10 pigs.Grower pigs Diet Formula: corn 67.8%, dregs of beans 28.0%, soya-bean oil 1.3%, calcium monohydrogen phosphate 1.0%, salt 0.4%, stone flour 0.5%, premix 1.0%.Control group: 1000kg basal diet+150g aureomycin; Test group: 1000kg basal diet+0.5kg Efficient antibacterial probiotics.

Swinery house is in the same size, and towards identical, duration of test feeding every day 2 times, adopt siccative to mix humidogene and feed, have enough and do not remain, water fountain supplies water.Colony house gravity-flow ventilation, regularly sweeps, traditional vaccination immunity.

The impact of Efficient antibacterial probiotics on grower pigs pig growth performance the results are shown in Table 9.

Table 9 Efficient antibacterial probiotics is on the impact of grower pigs growth performance

Note: indicate different alphabetical person after colleague's data and represent significant difference (P<0.05), same letter represents that difference is not remarkable.

As shown in table 9, Efficient antibacterial probiotics can significantly improve the average daily gain of grower pigs and average daily ingestion amount, reduces feed-weight ratio.

Efficient antibacterial probiotics the results are shown in Table 10 to the impact of microorganism species in grower pigs ight soil.

Table 10 Efficient antibacterial probiotics is on the impact (lgCFU/g) of microorganism species in grower pigs ight soil

Note: indicate different alphabetical person after colleague's data and represent significant difference (P<0.05), same letter represents that difference is not remarkable.

As shown in table 10, Efficient antibacterial probiotics can significantly improve content of lactic acid bacteria in grower pigs ight soil, reduces Escherichia coli and salmonella content, illustrates can improve grower pigs intestinal microecology from another angle, is conducive to keeping intestinal health.

Certainly; the present invention can also have various embodiments; when not deviating from the present invention's spirit and essence thereof; those of ordinary skill in the art can openly make various corresponding change and modification according to of the present invention, but these change accordingly and are out of shape the protection domain that all should belong to the claim appended by the present invention.

Claims (8)

1., containing a weanling pig feed for antibacterial probiotics, it is characterized in that, described weanling pig feed comprises weanling pig daily ration and antibacterial probiotics, and described antibacterial probiotics weight accounts for 0.50 ‰ ~ 1.00 ‰ of weanling pig daily ration weight; Described antibacterial probiotics is the mixture of intestines dung coccus and producing bacillus subtilis bacteriocin, and wherein, the ratio of described VREF and bacteriocin is 2 × 10 ⁸~ 5 × 10 ⁸the VREF of CFU: 1g bacteriocin.

2. weanling pig feed according to claim 1, it is characterized in that, described weanling pig Diet Formula raw materials by weight comprises: corn 55.0% ~ 65.0%, wheat bran 2.0% ~ 5.0%, dregs of beans 20.0% ~ 25.0%, fish meal 1.0% ~ 5.0%, whey powder 3% ~ 8.00%, soya-bean oil 1.0% ~ 4.0%, calcium monohydrogen phosphate 0.1% ~ 1.0%, stone flour 0.5% ~ 1.5%, lysine 0.1% ~ 0.5%, salt 0.1% ~ 0.3%, premix 1.0%.

3. the growing and fattening pig feed containing antibacterial probiotics, it is characterized in that, described growing and fattening pig feed comprises growing-finishing pigs diets and antibacterial probiotics, and described antibacterial probiotics weight accounts for 0.50 ‰ ~ 1.00 ‰ of growing-finishing pigs diets weight; Described antibacterial probiotics is the mixture of intestines dung coccus and producing bacillus subtilis bacteriocin, and wherein, the ratio of described VREF and bacteriocin is 2 × 10 ⁸~ 5 × 10 ⁸the VREF of CFU: 1g bacteriocin.

4. growing and fattening pig feed according to claim 3, it is characterized in that, described growing-finishing pigs diets formula material comprises by weight percentage: corn 60% ~ 70%, dregs of beans 25% ~ 30%, soya-bean oil 0.5% ~ 1.5%, calcium monohydrogen phosphate 0.5% ~ 2.0%, salt 0.1% ~ 0.5%, stone flour 0.5% ~ 1.5%, premix 1.0%.

5., according to the arbitrary described pig feed of claim 1-4, it is characterized in that, the culture presevation of described VREF is numbered: CGMCCNO.9666.

6., according to the arbitrary described pig feed of claim 1-4, it is characterized in that, the preparation method of described antibacterial probiotics, comprises the following steps:

1) bacillus subtilis of preparation institute bacteriocinogeny is joined in proportion in the VREF zymotic fluid in fermentation later stage, be uniformly mixed;

2) mixed zymotic fluid Separation of Solid and Liquid is obtained bacterium mud; drying protectant is added in bacterium mud; the mass volume ratio (g/L) of described bacterium mud and drying protectant is 5% ~ 25%, adopts granulation, freeze-drying or spraying dry to make antibacterial probiotics.

7. the application of weanling pig feed in weaning pig feeding described in claim 1 or 2.

8. the application of growing and fattening pig feed in growing and fattening swine rearing described in claim 3 or 4.