CN109423466A

CN109423466A - A kind of composite fermentation microbial inoculum and its application

Info

Publication number: CN109423466A
Application number: CN201710786611.7A
Authority: CN
Inventors: 吴培均; 李富伟; 骆骅; 李兆勇
Original assignee: Beijing Crvab Bio Tech Co ltd
Current assignee: Beijing Crvab Bio Tech Co ltd
Priority date: 2017-09-04
Filing date: 2017-09-04
Publication date: 2019-03-05
Anticipated expiration: 2037-09-04
Also published as: CN109423466B

Abstract

The invention discloses a kind of composite fermentation microbial inoculum and its applications, and using bacillus subtilis, bacillus licheniformis and enterococcus faecium as active constituent, three is cooperated according to bacterium number than 1~5:1~5:0.5~5 ratio, and total bacteria count is hundred million CFU/g of 20-200.The present invention carries out the production of fermented feed, realizes antibacterial, acidification, predigestion and reduces multiple functions such as mycotoxin, has a extensive future.

Description

A kind of composite fermentation microbial inoculum and its application

Technical field

The present invention relates to field of biotechnology more particularly to a kind of high-performance solid composite fermentation microbial inoculum and its applications.

Background technique

Fermented feed is by the way that multiple beneficial microorganism by science compounding, is inoculated into feedstuff with certain proportion In, and ferment under suitable conditions, a kind of organic biological feed of the bioactive functions ultimately produced.Fermentation is raised Material can not only reduce the anti-nutritional factors in feedstuff (such as cotton dregs, dregs of beans), improve its utility value；Also it can use Unconventional water resources resource (such as potato residues, vinasse, apple pomace) improves nutritional ingredient and palatability.Therefore, fermented feed is not Can only alleviate feed resource shortage problem can also economize on resources even waste discharge.

China, using biotechnology as the emerging development strategy industry of country, will push biotechnology to lead in December, 2012 The development in an all-round way in domain.Currently, China's animal husbandry is in the critical period that the mode of accelerating development changes, Demonstration And Extension green, ring It protects, safe and efficient probiotics preparation, to ensureing livestock products effective supply, promotion increasing peasant income, preserving the ecological environment, promotes The good and fast development of animal husbandry is of great significance.Ministry of Agriculture's rule in " catalogue of feed additive varieties (2013) " newly revised The China Fan Liao allow using Tiny ecosystem type and application, expand allow the probiotic used in feed type and use Object encourages the research and development and application of green safe micro-ecological feed, has pushed probiotics from the perspective of policy in animal husbandry It promotes the use of.Therefore, it is supported according to national policy and developing direction, the efficient micro-ecological feed of Low-cost has the epoch special Sign, meets national industry developing direction, meets current animal husbandry development demand, has future development and applies future and potentiality.

Summary of the invention

The purpose of the present invention is to provide a kind of solid composite fermentation microbial inoculum, the fermented feed which produces can subtract Few antibiotic dosage reduces aflatoxin content and has predigestion effect to feed during the fermentation.

In order to achieve the above objectives, present invention employs the following technical solutions:

A kind of composite fermentation microbial inoculum, using bacillus subtilis, bacillus licheniformis and enterococcus faecium as active constituent, by three Person cooperates according to bacterium number than 1~5:1~5:0.5~5 ratio, and total bacteria count is 20~20,000,000,000 CFU/g.

The enterococcus faecium Enterococcus Faecium high-yield lactic acid saves number CGMCC No.9666；Describedly Clothing bacillus Baclicuslincheniformis high yield neutral proteinase and amylase, saving number is CGMCC No.14441；The bacillus subtilis Bacillus subtilis can reduce aflatoxin in feed in solid fermentation Content, saving number is CGMCC No.14442.

Bacillus subtilis (Bacillus subtilis) of the invention and bacillus licheniformis (Bacillus Licheniformis it) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on July 19th, 2017 The heart (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, 100101), deposit number is respectively CGMCC NO.14442,CGMCC NO.14441.Enterococcus faecium (Enterococcus Faecium) is on September 16th, 2014 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.9666.

The invention mainly relates to the enterococcus faecium of a plant height lactic acid producing, a plant height produces the lichens of neutral proteinase and amylase Bacillus；One plant of bacillus subtilis that can reduce aflatoxin content in feed in solid fermentation.

The production method of bacillus subtilis or bacillus licheniformis of the present invention includes the following steps:

Primary inclined plane culture: bacillus is inoculated on primary inclined plane culture medium, and 24~36 are cultivated at 24~37 DEG C Hour, obtain level-one Bacillus colonies；Culture medium composition: 2.5~5.0g of beef extract, 5.0~15.0g of soy peptone, 1.0~5.0g of yeast extract, 1.0~5.0g of sodium chloride, 5.0~10.0g of glucose, 5.0~20g of agar, distilled water 1000ml, pH It is 6.5~7.5；

Secondary liquid culture: the level-one Bacillus colonies of culture are inoculated in second level culture solution, liquid amount is 150ml/500ml cultivates 15~20 hours under conditions of 30~37 DEG C, 110~220 revs/min, obtains second level bacillus kind Sub- liquid；Second level culture solution configuration proportion: 5.0~15g of peptone, 1.0~5.0g of beef extract, 1.0~5.0g of sodium chloride, distilled water 1000mL, pH are 6.5~7.5；

Three-level expands culture: second level bacillus seed liquor is inoculated in fermentor with 0.1%~0.5% weight ratio, It is cultivated 24~36 hours under conditions of on culture medium in 30~37 DEG C, 110~220 revs/min using conventional method, obtains three-level Bacillus seed liquor；Culture medium composition: corn flour 0.1~0.5%, glucose 0.1~1.0%, beancake powder 0.1~5.0%, Fish meal 0.1~0.5%, calcium carbonate 1.0~5.0%, ammonium sulfate 0.01~0.05%, dipotassium hydrogen phosphate 0.01~0.05%, sulphur Sour magnesium 0.01~0.05% and manganese sulfate 0.01~0.05%；

Liquid fermentation: fermentor leads to steam in 100~150 DEG C of insulated sterilizings 40~after sixty minutes, and fermentation substrate is crushed It being put into fermentor to 80 mesh, adds water, the weight and fermentation substrate weight ratio of water be added are 80~120:1~10, Then lead to high steam and be warming up to 100~150 DEG C, and keep the temperature 40~natural cooling 40~60 minutes after sixty minutes, lead to cooling water 20~40 DEG C are cooled to, three-level bacillus subtilis seed liquor is accessed under aseptic condition, with weight ratio meter, inoculum concentration is 0.1~ 1.0%, tank pressure is 0.05MPa, and speed of agitator is 120~220 revs/min, and with volume basis, ventilating ratio is 0.5~1:0.5~1, Fermentation time is 24~48 hours；L.35 × 10 detecting zymotic fluid viable count is¹⁰CFU/ml；With weight ratio meter, the fermentation Substrate includes: soybean meal hydrolysate 1%~5%, glucose 0.1~1.5%, the cornstarch 0.1 of yeast powder 1%~5%, 3% ~1.5%, calcium carbonate 0.1~1.0%, pH 7.0-7.5；The fermentation liquid is that content reaches 10 by filtering¹⁰CFU/ml's Liquid bacillus.

The present invention provides a kind of enterococcus faecium fermentation process, and steps are as follows:

It is seed culture medium with MRS culture medium, enterococcus faecium 18~24 is cultivated in the case where 35~45 DEG C aerobic or facultative conditions Hour, become first order seed；

Primary seed solution is seeded in seeding tank and is expanded culture, uses MRS culture medium for seed culture medium, 35 It is cultivated 18~24 hours under~45 DEG C aerobic or facultative conditions, becomes secondary seed；

The secondary seed solution inoculation of acquisition is subjected to fermented and cultured in the fermenter, uses the MRS culture medium of improvement for hair Ferment culture medium, in the case where 30~45 DEG C aerobic or facultative conditions, using timing or continuous feeding batch fermentation manner culture 18~36 Hour；

Preferably, the MRS culture medium for enterococcus faecium of fermenting be in every liter of MRS culture medium added with soy peptone 5~ 30g, 1~10g of glucose, 1~10g of yeast powder, 1~10g of sodium acetate twist lemon acid diamine 0.1~8g, Tween800.1~5g, 0.1~5g of dipotassium hydrogen phosphate, 0.05~lg of magnesium sulfate, 0.001~0.2g of manganese sulfate, 1~30g of calcium carbonate, are adjusted by 1 liter of pure water Save pH value 5.5~7.5.

The present invention also provides application of the above-mentioned composite fermentation microbial inoculum in fermentation solid culture medium.

Preferably, the inoculum concentration of the composite fermentation microbial inoculum 5-15% in mass ratio is added in solid fermentation material and is used for admittedly Body fermentation.

Further, include the following steps: for the specific production method of solid fermentation

(1) it takes fermenting microbe and carbon source to be added in 30-40 DEG C of water, is sufficiently stirred, place 5-12 hours；

(2) step (1) obtains in solution addition solid fermentation material, and water supplement makes the water content of solid fermentation material exist Between 35%-40%, ferment 12 hours to 36 hours.

An optimal technical scheme according to the present invention carries out solid fermentation, step using fermenting microbe provided by the invention It is rapid as follows:

Prepare the 100-300 kilograms of plastic barrel that can be filled with water, takes fermenting microbe and 1000 grams of brown sugar to be put into plastic barrel, so Afterwards bucket topped up with water, water temperature range is best at 30-40 DEG C, is sufficiently stirred, and places 5-12 hours.No-sundries and pollution are used as far as possible Clean water source, extraneous contact is also reduced in placement process.

Calculating first needs water weight to be added, adds the water content of material after water between 35%-40%, with one ton For feed, aqueous 14% in chow diet, then feed per ton needs volume external adding water weight between 400 kilograms~430 kilograms, Amount of water is reduced when temperature is high, and amount of water is improved when temperature is low.The water weight of the water weight for needing additionally to add=activated+ Water weight to be added is also needed, after also needing water to be added to mix with activated water, then is uniformly mixed with feed.The water temperature used As far as possible close to 30 DEG C, be conducive to microorganism Rapid Fermentation in this way.

By feed stacking on clean impermeable cement flooring, prevents from seeping ground under moisture, be piled into 1 meter or so as far as possible Thickness is then covered with one layer of plastic paper above feed.Under the conditions of clean as far as possible at feed stacking, warm, calm, rain-proof, winter is most Amount heat preservation, summer keep away direct sunlight.Fermentation time is generally close 12 hours to 36 hours and specific environment temperature relation, the summer Its time shortens, and the time in winter extends.Deepen with attenuation degree, feed internal temperature can rise.(winter 24 is small within 12 hours for fermentation When) after, a feed can be stirred every 10 hours promotes uniform fermentation.

The bacillus subtilis that the present invention uses, which is one plant, can reduce aflatoxin content in feed in solid fermentation Bacillus, save number be CGMCC No.14442.The present invention provides one kind with this enterococcus faecium combination withered grass gemma The method that bacillus and bacillus licheniformis carry out production high-performance solid fermentation.The fermented feed that the strain is produced can be reduced Antibiotic dosage reduces aflatoxin content and has predigestion effect to feed during the fermentation.Make in the present invention The bacillus of high yield neutral proteinase and amylase while using, the bacillus can promote lactic acid bacteria to be proliferated.The present invention The enterococcus faecium screened can be proliferated rapidly, generate organic acid, quickly reduced the pH value in fermentation process, inhibited varied bacteria growing. The bacillus licheniformis used in the present invention can reduce aflatoxin content in feed in solid fermentation.Preferably, originally The high-performance solid fermentation special bacteria that invention provides makes two bacillus and enterococcus faecium according to bacterium number ratio 2:2:1 cooperation With carrying out the production of fermented feed, realize antibacterial, acidification, predigestion and to reduce multiple functions, the application prospects such as mycotoxin wide It is wealthy.

Detailed description of the invention

Fig. 1 is the selection result of bacteria produced proteinase strain；

Fig. 2 is the selection result of alpha-amylase producing bacterial strain；

Fig. 3 is enterococcus faecium colonial morphology of the present invention.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The screening of embodiment one protease and Alpha-amylase high yielding strain

1 test method

The screening of 1.1 bacteria produced proteinase strains

Primary dcreening operation: transparent circle method is hydrolyzed using casein plate, the bacterial strain dibbling after isolating and purifying is in casein medium Plate, in cultivating 48h in insulating box, picking generates the bacterium colony (as shown in Figure 1) of hydrolysis transparent circle, measures L value (L value=transparent Loop diameter/colony diameter) after, 4 DEG C save backup.

Secondary screening: obtaining generating the bacterial strain of transparent circle through primary dcreening operation, draws 1mL bacteria suspension (10⁸CFU/mL) it is inoculated in fermentation training It supports in base, stirs evenly, natural ph, cultivated in 37 DEG C of incubator and measure prolease activity afterwards for 24 hours.Enzyme activity determination: Take enzyme solution to be measured.40 DEG C of water bath with thermostatic control 2min, are added casein 2mL (shaking up), then trichlorine is added in 40 DEG C of water bath with thermostatic control 10min Acetic acid 4mL (shakes up), centrifuging and taking supernatant, surveys OD275nm value.Make blank control simultaneously.

The screening of 1.2 alpha-amylase producing bacterial strains

Primary dcreening operation: in strain inoculated to sterilized starch agar medium, 37 DEG C of stationary cultures for 24 hours after, transfer into dress Have in the liquid triangular flask of the 25mL culture medium, with 37 DEG C, 220r/min shaking table culture for 24 hours.Take 1mL culture solution sterile water terraced Degree dilution respectively takes 0.15mL bacteria suspension to be respectively coated on plate isolation base, and 37 DEG C of cultures for 24 hours, spray iodine solution on plate, The single colonie for having transparent circle is chosen into (as shown in Figure 2), further after scribing line separation, is chosen to slant preservation culture medium, 37 DEG C Culture for 24 hours, saves at 4 DEG C.

Secondary screening: take 1 ring primary dcreening operation strain access seed culture medium, in 37 DEG C, 220r/min shaking table culture for 24 hours.1mL is taken to access Solid-state culture medium measures alpha-amylase enzyme activity after 30 DEG C of constant temperature incubation 72h, and the optimal bacterium of activity is saved at 4 DEG C Kind.

The measuring method of enzyme activity: taking substrate 5mL to preheat 10min in 40 DEG C of water-baths, and 0.5mL enzyme solution is added, accurate to keep the temperature 5min takes 0.5mL mixed liquor that the H of 5mL 0.1mol/L is added₂SO₄Reaction is terminated, therefrom takes 0.5mL that the dilute iodine solution of 5mL is added aobvious Color measures light absorption value at 660nm.With 0.5mL buffer instead of the enzyme solution of 0.5mL to compare, using distilled water as colorimetric Blank.

2 experimental results

2.1 primary dcreening operations primary dcreening operation from 30 plants of bacteriums obtains generating totally 15 plants of casein hydrolysis circle bacterial strain, accounts for all test bacterium The 50% of strain.Its L value (being shown in Table 1) of Preliminary Determination, data are shown, wherein the L value of bacterial strain Y27 is maximum, are 3.01.

2.2 secondary screenings and enzyme activity determination will be singled out the biggish 5 plants of bacterium of L value, measure its proteinase activity after fermented and cultured (being shown in Table 2), in 5 plants of bacterium, L value and all biggish bacterial strain of enzyme activity are Y27, and L value is 3.01, and protease activity is 324.72U/mL.

The screening of 2.3 alpha-amylase producing bacterial strains is produced amylase experiment discovery, is shared 9 plants of bacterium α-amylase Producers, is accounted for The 30% of test strain.Further secondary screening discovery, Y8, Y20 and Y27 α-amylase Producer vigor are preferable, wherein Y27 strain enzyme-producing compared with Height, enzyme activity reach 225.76U/g, and it is Y30 bacterial strain that wherein enzyme activity is lower, and enzyme activity 67.9U/g the results are shown in Table 3.It is comprehensively compared The enzymatic productivity of strains tested, then protease and alpha-amylase activity it is highest be bacterial strain Y27.

1 bacteria produced proteinase strain primary dcreening operation of table

The secondary screening of 2 bacterial strain of table

3 alpha-amylase activity measurement result of table

The identification of 2.4 bacterial strains

It is identified by gyrB gene order, the gene order (SEQ ID NO.1) of Y27 bacterial strain and bacillus licheniformis The similitude of (Bacillus licheniformis) is 99%, is determined as bacillus licheniformis (Bacillus licheniformis).Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on July 19th, 2017 Object center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal It compiles 100101), deposit number is CGMCC NO.14441.

The screening of 2 high acid lactic acid bacteria of embodiment

1 test method

1.1 acid-producing bacteria strains screening

It is separated to 5 plants of acid-producing bacteria strains from healthy chicken intestinal in conjunction with enrichment culture method and the test of molten calcium circle, bacterial strain will be purified Be seeded to 5mL sterilizing MRS broth bouillon in, 37 DEG C of anaerobic fermentations for 24 hours afterwards detection fermentation liquid centrifugation supernatant (4 DEG C, 5000g, PH and lactic acid content 10min).

When measuring lactic acid content, prior to measuring lactate standard liquid (10,20,30,40 and 50mg/L) under 560nm ultraviolet light Absorbance value, then using lactate standard liquid concentration as abscissa, absorbance value is that ordinate draws curve.When sample liquid processing, 4g active carbon is added first in 40mL centrifuged supernatant, filter paper filters after 65 DEG C of stirring in water bath decolourize 40min, then repeatedly Handle filtrate for several times, until filtrate, which becomes, clarifies, this clear solution is organic acid crude extract referring finally to exploitations such as beams Lactic acid method pre-processes MRS meat soup fermentation supernatant, then does blank control with distilled water, in 560nm measurement sample fermentation liquid Absorbance value.

The pH and its Lactic Acid from Fermentation Broth content (mg/L) of 4 streptococcus acidi lactici fermented solution of table

The molecular biology identification of 1.2 high-yield lactic acid bacterial strains

The analysis of 16SDNA sequence: the extraction of above-mentioned YB-5 bacteria total DNA uses bacterial genomes DNA extraction kit (day Root biochemistry (Beijing) Science and Technology Ltd., Tiangen DP302-02) it extracts.16S rDNA amplimer draws using bacterium is general Object, primer sequence are as follows: forward primer is 27F (corresponding to Escherichia coil 8-27 bit base): 5'- AGAGTTTGATCCTGGCTC AG-3'；Reverse primer is that 1495R (corresponds to 1495-1515 alkali of Escherichia coil Base): 5'-CTACGGCTACCTTGTTACGA-3'.Reaction system (50 μ L) is as shown in table 2:

5 PCR amplification system of table

PCR amplification program are as follows: 95 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min；58 DEG C of annealing 1min；72 DEG C of extension 2min, Carry out 30 circulations, last 72 DEG C of extensions 10min.PCR product is detected using 1% agarose gel electrophoresis, and fragment length is about Biotechnology (Beijing) Co., Ltd, the positive products of 1500bp purified Hou Songrui Boxing section carries out sequencing, YB-5 bacterium The nucleotides sequence of encoding gene of 16S rRNA be classified as the sequence in sequence table.

Obtained gene order (SEQ ID NO.2) carried out in GenBank database BLAST (http: // Blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis.Qualification result finds YB-5 and enterococcus faecium The homology of (Enterococcus faecium) is 99%, is determined as Enterococcus faecium.Bacterial strain was in 2014 9 The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Beijing on the 16th The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC No.9666, classification naming are enterococcus faecium (Enterococcus Faecium), and colonial morphology is as shown in Figure 3.

The combination and compatibility of 3 high-performance solid fermenting microbe of embodiment is studied

(1) combination matching in bacillus subtilis and bacillus licheniformis solid fermentation material is tested

Using corn-soybean meal as culture medium, it is inoculated with the different proportion combination of bacillus subtilis and bacillus licheniformis, is passed through The variation of bacterium number determines the most suitable adding proportion between two strains after measurement solid fermentation.Add 100g total according to 1 ton of fermentation substrate The bacterium powder of bacterium number 20,000,000,000 is matched, and packet design see the table below 6.7 parts of 100g corn-soybean meal (7:3) is accurately weighed, with newspaper packet Good, 120 DEG C, sterilize 30min.Bacillus subtilis and bacillus licheniformis are added to the corn-soybean meal of sterilizing by corresponding proportion In, it stirs, and water is added to make the water content 40% of final corn-soybean meal culture medium.It is put into 37 DEG C of incubators, Cultivate 36h.The total bacteria count of each test group is calculated using a mixing flat board counting method.

6 packet design of table

It can be seen that by table 6 and 7, total bacteria count highest, this result illustrate withered in the 4th group after the 4th group of fermentation in seven combinations The ratio of careless bacillus and bacillus licheniformis be most suitable for, can mutually compatibility improve solid fermentation strain proliferative capacity.

Viable count after table 7 ferments

(2) the combination matching experiment of enterococcus faecium and bacillus in solid fermentation material

This bacillus subtilis to screen enterococcus faecium the combination high yield neutral proteinase and amylase of high-yield lactic acid Bacterium and bacillus licheniformis combine three kinds of bacterium in laboratory in varing proportions, ferment to feed, by fermentation after pH, The indexs such as total bacteria count and feed resolution ratio evaluate different strain ratio to the fermentation discomposing effect of feed, determine fermented feed type The most proper combination compatibility of compound micro-ecological preparation.The bacterium powder that 100g bacterium number is 20,000,000,000 CFU/g is added to carry out according to 1 ton of fermentation substrate Proportion, packet design see the table below 8.

8 packet design of table

Blank control group: the sterile water with test group equivalent is added in feed sample, water and material are stirred.

Test group: the bacterium solution sufficiently dissolved by the feed sample of sterilizing and in proportion is poured into beaker, then is rinsed with 50ml water Triangular flask rinses bacterium solution well, is fully transferred in beaker, and water and material are stirred.It is put into 37 DEG C of incubators, Cultivate 36h.

Bacterium number and pH value variation after the fermentation of 9 each group of table

10 feed resolution ratio of table

Feed soluble matter/%=itself (feed substance weight-blank dry matter weight itself)/feed dry matter weight itself × 100%；Resolution ratio/%=(blank dry matter weight-sample dry matter the weight)/feed dry matter weight of microorganism to feed itself × 100%；

The ratio for showing lactic acid bacteria and bacillus from the combination and compatibility result of study of table 3-10 compound micro-ecological preparation is Synergistic effect can be played when 2:8 to the greatest extent.

Application of the embodiment 4 using the feed of high-performance solid fermenting microbe fermentation on piglet

This test selects parity, weight close, Du × length × Dasanyuan hybridization for the 28 ages in days wean being in a good state of health Pig 100,2 groups are randomly divided into, every group is divided into 5 repetitions, each repetition 10.Control group: feeding basal diet, test Group: the solid fermentation material that feeding is obtained using high-efficiency fermenting strain fermentation.

Test is begun from 28 age in days of weanling pig, and experimental period 21 days.It is sufficiently rinsed and strict sterilization pigsty before test.Experimental period Between feeding 5 times daily, using dry mixing material, with the slightly surplus material of crib for principle.Test pig free choice feeding, free water.Colony house is natural Ventilation is periodically swept, and traditional vaccination is immune.

Influence of the solid fermentation material to piglet growth performance and diarrhea rate the results are shown in Table 11.

Influence of the 11 solid fermentation material of table to piglet growth performance and diarrhea rate

Note: indicating different letter persons after colleague's data indicates significant difference (P < 0.05), and same letter indicates that difference is not shown It writes (p > 0.05).As a result it is indicated with average ± standard deviation, similarly hereinafter.

As shown in table 11, the average daily gain peace of piglet is remarkably improved using the solid fermentation material that fermenting microbe ferments Equal daily ingestion amount, while reducing diarrhea rate.

Influence of the solid fermentation material to piglet dry matter digestibility the results are shown in Table 12

12 pig dry matter digestibility test result of table compares

By table 12 as it can be seen that the dry matter digestibility of the test group of feeding solid fermentation material is improved compared with control group.

Feeding can significantly improve the feed intake of piglet using the feed that high-performance solid fermenting microbe ferments and averagely increase day by day Weight, feedstuff-meat ratio have the tendency that reduction, reduce grice diarrhoea rate, improve dry matter digestion, promote the absorption and utilization of nutriment, It improves immunity of organisms and then promotes pig farm culture efficiency, reduce environmental pollution.

It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Although ginseng It is described the invention in detail according to embodiment, it will be apparent to an ordinarily skilled person in the art that technical side of the invention Case is modified or replaced equivalently, and without departure from the spirit and scope of technical solution of the present invention, should all be covered in the present invention Scope of the claims in.

Sequence table

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catcattcag ttgggcactc tagcaagact gccggtgaca aaccggagga aggtggggat 1140

gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tgggaagtac 1200

aacgagttgc gaagtcgcga ggctaagcta atctcttaaa gcttctctca gttcggattg 1260

caggctgcaa ctcgcctgca tgaagccgga atcgctagta atcgcggatc agcacgccgc 1320

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380

ccgaagtcgg tgaggtaacc tttttggagc cagccgccta aggtgggata gatgattggg 1440

gtgaagtcgt aacaaggtaa cca 1463

Claims

1. a kind of composite fermentation microbial inoculum, which is characterized in that with bacillus subtilis, bacillus licheniformis and enterococcus faecium be activity Ingredient cooperates three according to bacterium number than 1~5:1~5:0.5~5 ratio, and total bacteria count is hundred million CFU/g of 20-200.

2. composite fermentation microbial inoculum according to claim 1, which is characterized in that the enterococcus faecium Enterococcus Faecium high-yield lactic acid saves number CGMCC No.9666；The bacillus licheniformis high yield neutral proteinase and amylase, Saving number is CGMCC No.14441；The bacillus subtilis can reduce aflatoxin in feed in solid fermentation and contain Amount, saving number is CGMCC No.14442.

3. composite fermentation microbial inoculum according to claim 1, which is characterized in that the bacillus subtilis or lichens gemma bar The production method of bacterium includes the following steps:

1) bacillus subtilis or bacillus licheniformis are inoculated on primary inclined plane culture medium, at 24~37 DEG C cultivate 24~ 36 hours, obtain level-one Bacillus colonies；Culture medium composition: 2.5~5.0g of beef extract, soy peptone 5.0~ 15.0g, 1.0~5.0g of yeast extract, 1.0~5.0g of sodium chloride, 5.0~10.0g of glucose, 5.0~20g of agar, distilled water 1000ml, pH are 6.5~7.5；

2) the level-one Bacillus colonies of culture are inoculated in second level culture solution, liquid amount 150ml/500ml, 30~37 DEG C, cultivate 15~20 hours under conditions of 110~220 revs/min, obtain second level bacillus seed liquor；Second level culture solution is prepared Ratio: 5.0~15g of peptone, 1.0~5.0g of beef extract, 1.0~5.0g of sodium chloride, distilled water 1000mL, pH is 6.5~ 7.5；

3) second level bacillus seed liquor is inoculated in fermentor with 0.1%~0.5% weight ratio, is being trained using conventional method It supports and is cultivated 24~36 hours under conditions of 30~37 DEG C, 110~220 revs/min on base, obtain three-level bacillus seed liquor； Culture medium composition: corn flour 0.1~0.5%, glucose 0.1~1.0%, beancake powder 0.1~5.0%, fish meal 0.1~0.5%, Calcium carbonate 1.0~5.0%, ammonium sulfate 0.01~0.05%, dipotassium hydrogen phosphate 0.01~0.05%, magnesium sulfate 0.01~0.05% With manganese sulfate 0.01~0.05%；

4) fermentor leads to steam in 100~150 DEG C of insulated sterilizings 40~after sixty minutes, and fermentation substrate is crushed to 80 mesh and is put into hair In fermentation tank, water is added, the weight and fermentation substrate weight ratio of water be added are 80~120:1~10, then lead to high pressure and steam Vapour is warming up to 100~150 DEG C, and keep the temperature 40~natural cooling 40~60 minutes, logical cooling are water-cooled to 20~40 after sixty minutes DEG C, three-level bacillus subtilis seed liquor is accessed under aseptic condition, with weight ratio meter, inoculum concentration is 0.1~1.0%, and tank pressure is 0.05MPa, speed of agitator are 120~220 revs/min, and with volume basis, ventilating ratio is 0.5~1:0.5~1, fermentation time 24 ~48 hours；L.35 × 10 detecting zymotic fluid viable count is¹⁰CFU/ml；With weight ratio meter, the fermentation substrate includes: yeast Soybean meal hydrolysate 1%~5%, glucose 0.1~1.5%, the cornstarch 0.1~1.5%, calcium carbonate of powder 1%~5%, 3% 0.1~1.0%, pH 7.0-7.5；The fermentation liquid is that content reaches 10 by filtering¹⁰The liquid bacillus of CFU/ml.

4. composite fermentation microbial inoculum according to claim 1, which is characterized in that the production method of the enterococcus faecium includes such as Lower step:

It 1) is seed culture medium with MRS culture medium, in the case where 35~45 DEG C aerobic or facultative conditions, culture enterococcus faecium 18~24 is small When, become first order seed；

2) step 1) primary seed solution obtained is seeded in seeding tank and is expanded culture, use MRS culture medium for kind Sub- culture medium is cultivated 18~24 hours in the case where 35~45 DEG C aerobic or facultative conditions, becomes secondary seed；

3) step 2) secondary seed solution inoculation obtained is subjected to fermented and cultured in the fermenter, is cultivated using the MRS of improvement Base is fermentation medium, in the case where 30~45 DEG C aerobic or facultative conditions, using timing or continuous feeding batch fermentation manner culture 18~36 hours.

5. composite fermentation microbial inoculum according to claim 4, which is characterized in that the modified MRS culture medium in the step 3) It is in every liter of MRS culture medium added with 5~30g of soy peptone, 1~10g of glucose, 1~10g of yeast powder, sodium acetate 1~ 10g twists 0.1~5g of lemon acid diamine 0.1~8g, Tween80,0.1~5g of dipotassium hydrogen phosphate, 0.05~lg of magnesium sulfate, manganese sulfate 0.001~0.2g, 1~30g of calcium carbonate, adjust pH value 5.5~7.5 by 1 liter of pure water.

6. application of the described in any item composite fermentation microbial inoculums of claim 1-5 in fermentation solid culture medium.

7. application of the composite fermentation microbial inoculum according to claim 6 in fermentation solid culture medium, which is characterized in that by institute The inoculum concentration for stating composite fermentation microbial inoculum 5-15% in mass ratio is added in solid fermentation material for solid fermentation.

8. application of the composite fermentation microbial inoculum according to claim 7 in fermentation solid culture medium, which is characterized in that be used for The specific production method of solid fermentation includes the following steps:

1) it takes fermenting microbe and carbon source to be added in 30~40 DEG C of water, is sufficiently stirred, place 5-12 hours；

2) step 1) obtains in solution addition solid fermentation material, and water supplement makes the water content of solid fermentation material 35%~40% Between, it ferments 12 hours to 36 hours.