CN107821739A

CN107821739A - The application of composite feed additive, the preparation method of antivirotic and antivirotic

Info

Publication number: CN107821739A
Application number: CN201711171746.9A
Authority: CN
Inventors: 潘韵; 陈泽田; 王逢久; 仵桂仓; 郑文官
Original assignee: Henan Yihong Shancheng Biotechnology Co ltd
Current assignee: Henan Yihong Shancheng Biotechnology Co ltd
Priority date: 2017-11-22
Filing date: 2017-11-22
Publication date: 2018-03-23

Abstract

The invention discloses composite feed additive prepared by a kind of preparation method of composite feed additive and the preparation method; the method and antivirotic that antivirotic is prepared using the composite feed additive are also disclosed, while also discloses that the application that application and protection zooblast confrontation vesicular stomatitis virus of the antivirotic in vesicular stomatitis virus is suppressed are infectd.The beneficial effects of the invention are as follows：1st, for composite feed additive using the double bacterium mixed fermentations of saccharomyces cerevisiae and Methylotrophic bacillus, nutritional ingredient and viable bacteria are more abundant, greatly enhance the premunition of animal, its feeding with it is significant in the application on feed industry；2nd, antivirotic is composite feed additive leachate, there is obvious activity and Proliferation Ability to VSV；3rd, antivirotic effectively protects zooblast to avoid being infected by VSV.

Description

The application of composite feed additive, the preparation method of antivirotic and antivirotic

Technical field

The present invention relates to mixed feed addition prepared by a kind of preparation method of composite feed additive and the preparation method Agent, the application of antivirotic and the antivirotic prepared using composite feed additive is further related to, belongs to biological technical field.

Background technology

Today's society aquaculture development is rapid, but a large amount of of antibiotic to make with there is also problems, antibiotic simultaneously Pathogen is easily set to produce drug resistance with overlong time.Moreover, if antibiotic easily remains in body, easily pass through food chain The immune effect of humans and animals is affected, hidden some dangers for for control epidemic disease.Meanwhile antibiotic can be destroyed in enteron aisle Microecological balance, at this moment micro-organisms in vitro be possible to cause cross-infection in invasion body.And probiotics can adjust animal intestine Road balances, and can suppress many pathogenic bacteria, and contributes to animal intestinal tract to wriggle, and promotes digestion, therefore, how in animal husbandry Widely using the feed addictive of probiotics progress fermenting and producing turns into a new problem.

Methylotrophic bacillus, there is antibacterium virus and hemolytic activity, in addition, also have and promote Biofilm formation, anti-oxidant, the removing various active such as ultra-oxygen anion free radical and anti-lipid peroxidation.Meanwhile methyl is sought Gemma, environment strong stress resistance, energy high temperature resistant, resist drying, anti-hydrochloric acid in gastric juice can be formed by supporting type bacillus, and be easy to store.Mesh Before, Methylotrophic bacillus is mainly used in the middle of bio-feritlizer, and the application among feed addictive is rarely reported.

Vesicular stomatitis virus (VSV）Importance with animal doctor above and economically, because they can make ox and pig lethal. Infected animal shows as generating heat, drowsiness, loss of appetite, occurs blister venereal disease stove and leg between oral cavity, nipple, toe and on coronet The can-like annulus in portion.Bubble is easily rupturable, exposes granulation tissue, and take on a red color erosion, the epithelium that surrounding scratches again, often in 7 ~ 10 days It is completely recovered.Dead due to vesicular stomatitis is less, still, local secondary bacterial and fungal infection can be caused, so as to lead Cause limping, Body weight loss, go out milk decline and mastitis, bring heavy economic losses.

Oxen and horses pig metainfective incubation period is generally 1 ~ 7 day, Early manifestation be heating, blunt, anorexia, salivate it is more.Then White to bois de rose bubble, the inside for 0.5 to several centimeters size occur is full of yellow liquid, generally assembled in groups.Bubble is common In tongue, gum, nose and lip, bubble can also occur in the mouth of pig and the ear of horse.Contain substantial amounts of viral (average out in bubble Every milliliter of 10-10 infectious unit), there are viremia virusemia and general infection, the visible lymphatic vessel hyperplasia of Histopathologic changes, infection 4 After it, cerebral nerve spongiocyte and the monocyte infiltration of brain and cardiac muscle.The easy rehabilitation of this sickly look, even if the state of an illness is very heavy, 7 ~ Also can fully recover within 10 days.

It is known in VSV category to there are 5 strains of Indiana, NJ, Alagoas, Piry and Chandipura to make one pathogenic. 20 ~ 30 h start to break out after people's infection, may start from conjunctiva, influenza-like symptom then occur:Ague, Nausea and vomiting, flesh Bitterly, pharyngitis, conjunctivitis, lymphnoditis.Child's infection can cause encephalitis.The course of disease continues 3 ~ 6 days, no complication and lethal.

Due to VSV being widely current property, hyperinfection, variability, the particularity of antibody protection, a kind of peace is there is no at present The anti-system of complete effective vaccine.Once this disease occurs, it is necessary to take the measures such as urgent isolation, block, sterilization immediately.Therefore, VSV Preventing and treating turns into a great problem of current livestock cultivation.

The content of the invention

In order to overcome drawbacks described above, the invention provides disclose a kind of preparation method of composite feed additive.

The invention also discloses a kind of composite feed additive.

The invention also discloses a kind of preparation method of antivirotic.

The invention also discloses a kind of antivirotic.

The invention also discloses a kind of application of antivirotic in vesicular stomatitis virus is suppressed.

The invention also discloses a kind of application of antivirotic on protection vesicular stomatitis virus infection cell.

To achieve these goals, the technical scheme is that：A kind of preparation method of composite feed additive, including Following steps：

Step 1）, weigh solid matrix and crush, mix；

Step 2）, nutritional ingredient, fermentation by saccharomyces cerevisiae liquid and Methylotrophic fermentation of bacillus liquid be added in blending tank, and Add water stirring and dissolving；

Step 3）, by step 2）Mixed liquor in blending tank is transported to continuous mixer, is well mixed with solid matrix, and tiles In carrying out solid fermentation in fermentation bed；

Step 4）, solid fermentation product dried, obtain composite feed additive；

Saccharomyces cerevisiae is the saccharomyces cerevisiae for being preserved in Guangdong Province's Culture Collection（Saccharomyces cerevisiae）5-1, preserving number are GDMCC No：60243, the preservation time is September in 2017 13, preservation address：It is Chinese wide 5 building, the building of compound the 59th of Dong Sheng Xianlie Middle Road, Guangzhou Cities 100；

Methylotrophic bacillus is the Methylotrophic bacillus for being preserved in Guangdong Province's Culture Collection （Bacillus methylotrophilus）2-3, preserving number are GDMCC No：60242, the preservation time is September 13 in 2017 Day, preservation address：5 building, the building of compound the 59th of Xianlie Mid Road, Guangzhou City, Guangdong Province, China 100.

Preferably, the step 1）The composition of middle solid matrix includes：By weight percentage, the % of corn 15 ~ 25, straw The % of stalk 10 ~ 30, the % of wheat bran 10 ~ 20, the % of dregs of beans 10 ~ 25, the % of vinasse 15 ~ 30；Solid matrix water content is measured afterwards, Calculate the weight of dry；

Step 3）Middle solid fermentation condition is：In terms of percent by weight of dry matter, the % of fermentation bed stone moisture 40 ~ 60, connect 10 ~ 20 % of kind amount；Fermentation bed fermentation temperature is 25 ~ 45 °C, and stirring is controlled during fermentation 2 ~ 3 Secondary, fermentation time is controlled in 36 ~ 60 h.

Preferably, the nutritional ingredient is based on percent by weight of dry matter：The % of glucose 2 ~ 10, magnesium sulfate 0.1 ~ 0.5 %, the % of potassium dihydrogen phosphate 0.1 ~ 1.0, the % of calcium chloride 0.1 ~ 1.0, the % of cobalt chloride 0.01 ~ 0.1, zinc chloride 0.01 ~ 0.1 %, the % of selenium chloride 0.01 ~ 0.1.

Preferably, the step 2）Middle fermentation by saccharomyces cerevisiae liquid and Methylotrophic fermentation of bacillus liquid press dry respectively 2 ~ 10 % of matter weight are added in blending tank.

A kind of composite feed additive.

A kind of preparation method of antivirotic, after composite feed additive adds tri-distilled water, water-bath, concussion, in centrifuging and taking Clearly, composite feed additive leachate is made after aseptic filtration, the composite feed additive leachate is antivirotic.

A kind of antivirotic.

A kind of application of antivirotic in vesicular stomatitis virus is suppressed.

The application that a kind of antivirotic is infectd in protection zooblast confrontation vesicular stomatitis virus.

The beneficial effects of the invention are as follows：1st, composite feed additive is double using saccharomyces cerevisiae and Methylotrophic bacillus Bacterium mixed fermentation, nutritional ingredient and viable bacteria are more abundant, the premunition of animal are greatly enhanced, in its feeding and feed industry On application on it is significant；

2nd, antivirotic is composite feed additive leachate, there is obvious activity and Proliferation Ability to VSV.

3rd, antivirotic effectively protects zooblast to avoid being infected by VSV.

Brief description of the drawings

Fig. 1 is the fluorogram for adding the h restrovirus control groups of VSV virus-4s 8.

Fig. 2 is the fluorogram of C1 groups after the h of addition VSV virus-4s 8.

Fig. 3 is the Vero cells for adding the h restrovirus control groups of VSV virus-4s 8.

Fig. 4 is the Vero cells of C1 groups after the h of addition VSV virus-4s 8.

Embodiment

Technical scheme is clearly and completely described below in conjunction with accompanying drawing, it is clear that described implementation Example is part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill The every other embodiment that personnel are obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.

Embodiment 1

First, the preparation of fermentation by saccharomyces cerevisiae liquid

1）It is prepared by S. cervisiae seed liquor：S. cervisiae is seeded in S. cervisiae liquid seed culture medium 30 DEG C, The r/min of rotating speed 180 cultivates 20 h, obtains S. cervisiae seed liquor.

2）It is prepared by fermentation by saccharomyces cerevisiae liquid：Wine S. cervisiae seed liquor being seeded to by 5 % inoculum concentrations in fermentation tank In brewer yeast bacteria liquid fermentation medium, amplification culture, the m3/h of air mass flow 30, the r/min of speed of agitator 150,30 ° of temperature C, the h of incubation time 18, obtains fermentation by saccharomyces cerevisiae liquid.Now, yeast count >=3.0 × 10⁸ CFU/mL。

Wherein,

S. cervisiae liquid seed culture medium composition is：The g/L of tryptone 20, the g/L of DEXTROSE ANHYDROUS 20, yeast extract 5 G/L, pH 7.0；

S. cervisiae liquid fermentation medium composition is：The g/L of peptone 65, the g/L of sodium chloride 10, the g/L of epsom salt 2, The g/L of calcium chloride 0.3, the g/L of potassium dihydrogen phosphate 0.2, glucose 40 g/L, pH 7.0.

2nd, the preparation of Methylotrophic fermentation of bacillus liquid

1）It is prepared by Methylotrophic bacillus seed liquor：Methylotrophic bacillus is seeded to Methylotrophic gemma bar In bacteria liquid seed culture medium, the r/min of rotating speed 180,36 °C of temperature, the h of incubation time 24, Methylotrophic gemma is obtained Bacillus seed liquor.

2）It is prepared by Methylotrophic fermentation of bacillus liquid：Methylotrophic bacillus seed liquor is pressed into 5 % inoculum concentrations It is seeded in the Methylotrophic bacillus liquid fermentation medium in fermentation tank, amplification culture, the m3/h of air mass flow 25, The r/min of speed of agitator 150,36 °C of temperature, the h of incubation time 36, obtain Methylotrophic fermentation of bacillus liquid.This When, Methylotrophic bacillus number >=1.0 × 10¹⁰ CFU/mL。

Wherein,

Methylotrophic bacillus liquid seed culture medium composition is：The g/L of beef extract 5, peptone 10 g/L, NaCl 5 g/ L, pH 7.2 ± 0.2；

Methylotrophic bacillus liquid fermentation medium components are：The g/L of corn flour 50, the g/L of beancake powder 25, starch 15 G/L, yeast extract 20 g/L, NaCl 80 g/L, MgSO₄·7H₂O 1 g/L, pH 7.2 ± 0.2.

3rd, added using S. cervisiae and the combined solid fermented technique productions mixed feed of Methylotrophic bacillus Agent

1）Solid fermentation culture medium is prepared by following mass percent, weighs the % of corn 20, the % of stalk 20, the % of wheat bran 15, dregs of beans Crushed after 20 %, the % of vinasse 25, measure its water content, calculate the weight of dry, then mix, obtain matrix.

2）In 2 tons of blending tanks plus water, amount of water is 60 % of dry matter weight, by every gram of dry containing saccharomycete and Methylotrophic bacillus is >=5.0 × 10⁸, by following mass percent（The percentage of dry matter weight）Weigh grape 5 % of sugar, the % of magnesium sulfate 0.3, the % of potassium dihydrogen phosphate 0.5, the % of calcium chloride 0.8, the % of cobalt chloride 0.03, the % of zinc chloride 0.03, chlorination The % of selenium 0.03, is added in blending tank, by two kinds of strain fermentating liquids being prepared by the above method respectively by dry matter weight 5 % be driven into by bacterium solution pump in 2 tons of blending tanks, with agitator by DDGS it is well mixed after company got to by bacterium solution pump In continuous mixer.Bacterium solution pump discharge meter flow control ensures solid-liquid mix moisture in 40 % or so in 35 ~ 37 L/min.

3）The matrix for connecting strain is fermented 48 h under the conditions of 30 ~ 40 °C, then by solid fermentation thing at 50 °C Lower dries pulverizing, that is, obtain finished product composite feed additive.

High, identified yeast total viable count >=1.0 × 10 by above-mentioned steps production composite feed additive viable bacteria content⁹ Cfu/g, Methylotrophic bacillus living sum >=2.0 × 10¹⁰cfu/g.In the composite feed additive production process, Fermentation by saccharomyces cerevisiae matrix produces the nutriments such as substantial amounts of amino acid, small peptide, manna oligosacchride, and Methylotrophic bacillus makees Have for the characteristic bacterial strain of this product to vesicular stomatitis virus（VSV）There is significant inhibitory action.

Embodiment 2

First, the preparation of antivirotic

The composite feed additive in 1 g embodiments 1 is weighed, 2 ml tri-distilled waters is added, shakes 30 in 37 °C of water-baths 1000 rpm are centrifuged 10 minutes after min, draw supernatant, and PH to 7.0,0.22 is adjusted with 0.5 M NaOHμThe sterile filters of m Device filters, and prepares stoste, note stoste is C1, with 4 times of doubling dilutions, respectively C2, C3, C4.

2nd, the green ape and monkey kidney in Africa（Vero）The culture of cell and VSV viruses

Recovery Vero cells, by Vero cells with after 0.25 % pancreatin digestion, piping and druming disperses cell, is trained completely with DMEM Nutrient solution（Containing 2 % hyclones, 10⁵U/L benzyl penicillins sylvite and 100 g/L streptomycin sulphates, pH 7.2）It is incubated at In T25 bottles, 37 °C are put, 5 % CO₂Quiescent culture in incubator.Most cells are adherent after cultivating 24 h, discard bottle Middle nutrient solutions, the fresh DMEM complete culture solutions of 5 ml, 37 °C of preheatings are added, Tissue Culture Flask is placed in 37 °C, 5% CO₂Continue the individual layer that culture to cell grows up to densification in incubator.

After Vero passages 3 times, the Vero cells for having grown up to fine and close individual layer are taken, discard nutrient solution, used PBS cleans cell surface 3 times, every time 2 ml.Exhaust remaining PBS, is inoculated with 0.3 ml VSV virus liquids, 37 °C of suctions Attached 1.5 h, rocked 1 time every 20 min.Unadsorbed virus liquid is abandoned in suction, 5 ml DMEM maintaining liquids is added, in 37 °C、5 % CO₂Cultivated in incubator and observe cytopathic effect（cytopathogenic effeet, CPE）, after 13 h, when When there is lesion in 80 % cell, harvest virus.By cell culture under the conditions of -20 °C/37 °C multigelation 3 Secondary, then in 4 °C, 1800 rpm centrifuge 10 min, take supernatant（Virus liquid）Packing is stored in -80 °C of refrigerators.

3rd, the measure of VSV virus titers

By the PBS buffer solution for cleaning cell surface 3 times of well-grown Vero cells in blake bottle, 2 ml every time, 0.25 % trypsin solution vitellophags, 1000 rpm centrifuge 5 min, cell count, and adjustment cell density is 5 × 10³ Individual/hole（0.1 ml/ holes）96 porocyte culture plates are seeded to, in 37 DEG C, 5 % CO₂24 h are cultivated in incubator.Will be to be measured Vesicular stomatitis virus carries out 10 times of serial dilutions with the DMEM nutrient solutions containing 2 % hyclones（10^-1~10^-9）, Each ml of dilution factor 1.24 h of culture Vero cells are taken out, discard nutrient solution, 0.2 ml PBS bufferings are added per hole Liquid cleaning cell 1 time, discards PBS, and the VSV virus liquids of 0.1 ml dilutions are inoculated with per hole, and each dilution factor 8 is multiple Hole, while the negative control for not being inoculated with VSV viruses is set up, 37 DEG C are put, 5 % CO₂After 1 h is adsorbed in incubator, Taking-up discards nutrient solution, continues to put 37 DEG C after adding 0.2 ml DMEM cell maintenance mediums, 5 % CO₂Cultivated in incubator, often 12 h are observed and are recorded the lesion situation of each hole cell.TCID is calculated by Reed-Muench Liang Shi methods₅₀, obtain VSV's TCID₅₀It is 10^-8.125/0.1 ml。

4th, antivirotic is to VSV virus In-vitro Inhibitory Effects

After 12 orifice plate Cultivation of Vero, 24 h, cell length to about 90 % discards nutrient solution in hole, and with D-Hanks liquid Wash twice, per hole by the amount access VSV viruses of MOI=10, every 20 min is rocked once, and after adsorbing 2 h, liquid in hole is abandoned in suction Body, then washed 2 times with D-Hanks liquid, it is separately added into antivirotic C1, C2, C3 and C4 of 1 ml various concentrations.It is each dense Degree 1 repetition of setting, while virus control and negative control (control group respectively adds 1 ml DMEM nutrient solutions) are provided with, culture is seen Examine.

After 12 36 h of orifice plate cell infection VSV viruses, low concentration C₄There are the % lesions of a hole 20, other hole cells are almost Normally；After 48h, latter two concentration（C₃、C₄）Antivirotic has 90 % cytopathy.C₁And C₂Normally, virus control is entirely sick Become, negative control is normal.Harvest viral supernatants.Illustrate antivirotic concentration in C₁ - C₂In the range of have inhibitory activity to VSV. The Sample supernatants of collection are subjected to TCID₅₀Measure.

The virus titer of above-mentioned sample is surveyed by the methods described of embodiment 6（TCID₅₀）.According to observing and recording for 72 h, profit Calculated with Reed-Muench Liang Shi methods.4 concentration Cs of antivirotic₁、C₂、C₃、C₄TCID₅₀It is 10 respectively^-2.125、10^-5.625、10^-6.125、10^-8.5, the TCID50 of virus control is 10^-8.7。

Define antivirotic TCID₅₀Reduce≤10⁶And> 10³When be 1 active unit, take 10^3.075For 1 work Property unit：

The derivation LogTCID of the different groups of table 1₅₀C/V

As shown in table 1, with the raising of antivirotic concentration, viral TCID₅₀That reduces is more, illustrates that antivirotic suppresses VSV It is proliferated into dose-dependence.

5th, fluorescent test

After 12 orifice plate Cultivation of Vero, 24h, cell length to individual layer, washed 2 times with D-Hanks liquid, by the amount of MOI=10 VSV viruses are accessed, every 20 min is rocked once, and after adsorbing 2 h, liquid in hole is abandoned in suction, then is washed 2 times with D-Hanks liquid, point The antivirotic C of 1 ml various concentrations is not added₁、C₂、C₃And C₄.1 repetition of each concentration, culture observation.After 12 h, examination Test group and virus control group cell is all normal；After 24 h, virus control group cell whole lesion, negative control and antivirotic C₁Concentrations of cells is normal, and other concentration different degrees of cell degeneration occur and come off.Counted according to fluorescence, antivirotic 4 Concentration C₁、C₂、C₃、C₄Fluorescence volume be 3.5 × 10 respectively³Individual/mL, 1.4 × 10⁴Individual/mL, 3.5 × 10⁵Individual/mL, 5.6 × 10⁵ Individual/mL, virus control are 7 × 10⁵；

Press down light quantity=virus group fluorescence volume-antivirotic group fluorescence volume.Define antivirotic suppression light quantity≤6 × 10⁵And>2× 10⁵When be 1 active unit, take 3.5 × 10⁵For 1 active unit.

Fluorescence volume, suppression light quantity, Log suppression light quantities and the derivation Log values of the different groups of table 2

As shown in table 2, with the raising of antivirotic concentration, viral TCID₅₀That reduces is more, illustrates that antivirotic suppresses VSV It is proliferated into dose-dependence.

Two groups of Vero cells are separately added into the VSV viruses of isodose, fluorescence it can be seen from Fig. 1 and Fig. 2 fluorescence picture It has been shown that, a large amount of propagation of control group virus, full of whole visual field.And C₁Experimental group viral growth is suppressed, it is seen that FLuorescent It is less.

Fluorescent test shows that antivirotic has the function that significantly to suppress VSV viruses.

6th, cytopathic effect is tested（Cytopathic effect, CPE）

After 96 orifice plate culture cells, 24 h, cell length to individual layer, D-Hanks liquid is washed 2 times, is accessed by the amount of MOI=10 VSV viruses, every 20 min are rocked once, and after adsorbing 2 h, liquid in hole is abandoned in suction, and D-Hanks liquid is washed 2 times, adds various concentrations Antivirotic C₁、C₂、C₃And C₄Each 100 mL, i.e. final concentration are respectively 62.5μg/mL（The 4 of stoste^-2Times）、15.625μ g/mL（The 4 of stoste^-3Times）、7.8125μg/mL（The 4 of stoste^-4Times）、3.9063μg/mL（The 4 of stoste^-5Times）, each concentration 7 Individual repetition, while virus control and negative control are set, culture observation.When virus control has half cytopathy, add MTT 10μAfter L, 4 h, liquid in hole is abandoned in suction, adds 200 per holeμL DMSO, 10 min are vibrated, OD values are surveyed in ELIASA.Calculate thin Born of the same parents' survival rate.

After 24 h, it has been observed that C₁Concentrations of cells is almost normal, C₂Concentrations of cells deforms, C₃、C₄Concentration whole lesion.MTT Method surveys OD values.According to formula：OD_C-VValue=（Experimental group group OD values-virus control group OD values）, such as following table：

OD values, ODc-v, Log suppression light quantity and the derivation Log values of the different groups of table 3

As table 3 shows, with the raising of antivirotic concentration, its inhibitory action to VSV viruses also strengthens, closed in dose-dependant System.

By two groups of Vero cells it can be seen from Fig. 3 and Fig. 4 be separately added into the h of VSV virus-4s 8 of isodose after observe, compare The mortality of group cell, come off to form plaque.And C₁Experimental group cell growth is vigorous, has no obvious lesion.

Cytopathic effect experiment shows that antivirotic has the function that significantly to protect zooblast.

Technical scheme is not limited to the limitation of above-mentioned specific embodiment, and every technique according to the invention scheme is done The technology deformation gone out, each falls within protection scope of the present invention.

Claims

A kind of 1. preparation method of composite feed additive, it is characterised in that：Comprise the following steps：

Step 1）, weigh solid matrix and crush, mix；

Step 2）, nutritional ingredient, fermentation by saccharomyces cerevisiae liquid and Methylotrophic fermentation of bacillus liquid be added in blending tank, and Add water stirring and dissolving；

Step 3）, by step 2）Mixed liquor in blending tank is transported to continuous mixer, is well mixed with solid matrix, and tiles In carrying out solid fermentation in fermentation bed；

Step 4）, solid fermentation product dried, obtain composite feed additive；

Saccharomyces cerevisiae is the saccharomyces cerevisiae for being preserved in Guangdong Province's Culture Collection（Saccharomyces cerevisiae）5-1, preserving number are GDMCC No：60243；

Methylotrophic bacillus is the Methylotrophic bacillus for being preserved in Guangdong Province's Culture Collection （Bacillus methylotrophilus）2-3, preserving number are GDMCC No：60242.
2. the preparation method of composite feed additive according to claim 1, it is characterised in that：The step 1）Middle solid The composition of matrix includes：By weight percentage, the % of corn 15 ~ 25, the % of stalk 10 ~ 30, the % of wheat bran 10 ~ 20, dregs of beans 10 ~ 25 %, the % of vinasse 15 ~ 30；Solid matrix water content is measured afterwards, calculates the weight of dry；

Step 3）Middle solid fermentation condition is：In terms of percent by weight of dry matter, the % of fermentation bed stone moisture 40 ~ 60, connect 10 ~ 20 % of kind amount；Fermentation bed fermentation temperature is 25 ~ 45 °C, and stirring is controlled during fermentation 2 ~ 3 Secondary, fermentation time is controlled in 36 ~ 60 h.
3. the preparation method of composite feed additive according to claim 2, it is characterised in that：The nutritional ingredient is by dry Substance weight percentages：The % of glucose 2 ~ 10, the % of magnesium sulfate 0.1 ~ 0.5, the % of potassium dihydrogen phosphate 0.1 ~ 1.0, chlorination The % of calcium 0.1 ~ 1.0, the % of cobalt chloride 0.01 ~ 0.1, the % of zinc chloride 0.01 ~ 0.1, the % of selenium chloride 0.01 ~ 0.1.
4. the preparation method of the composite feed additive according to Claims 2 or 3, it is characterised in that：The step 2）In Fermentation by saccharomyces cerevisiae liquid and Methylotrophic fermentation of bacillus liquid are added to mixing by 2 ~ 10 % of dry matter weight respectively In tank.
A kind of 5. composite feed additive as prepared by claim any one of 1-4.
A kind of 6. preparation method of antivirotic, it is characterised in that：Composite feed additive as claimed in claim 5 adds three After steaming water, water-bath, concussion, centrifuging and taking supernatant, composite feed additive leachate, the mixed feed are made after aseptic filtration Additive leachate is antivirotic.
A kind of 7. antivirotic as prepared by claim 6.
A kind of 8. application of the antivirotic in vesicular stomatitis virus is suppressed as claimed in claim 7.
9. a kind of antivirotic as claimed in claim 7 is answered what protection zooblast confrontation vesicular stomatitis virus was infectd With.