CN104839448A - A micro ecological feed additive for regulating rumens and a preparation method thereof - Google Patents
A micro ecological feed additive for regulating rumens and a preparation method thereof Download PDFInfo
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Abstract
The present invention belongs to the field of microbial fermentation technology, and particularly to a micro ecological feed additive for regulating rumens and a preparation method thereof. The additive consists of metabolites and intracellular substances of yeast, and metabolites of aspergillus oryzae with a mass ratio of 15: 1, 20: 1 or 25: 1. Single strain industrial fermentation technology is used to produce a specific anti-stress metabolite composite, the method is stable in process, and effectively solves the defects that viable organism and enzyme preparations are easily inactivated and unstable, which greatly expands the application range of the additive, and is conducive to achieve the targeted product development of different animals and different physiological stages of the animals. The additive is not a viable organism preparation, but is the specific anti-stress metabolite composite produced by viable organisms after high density fermentation, and the additive is easy to store so that active ingredient activity cannot be decreased with prolonged storage time, which ensures active ingredient contents of the additive.
Description
Technical field
The invention belongs to technical field of microbial fermentation, particularly a kind of regulating rumen microbial forage additive and preparation method thereof.
Background technology
Probiotics, refers under the guidance of microecology theory, and the imbalance of adjustment microecological balance, keeps microecological balance, improves the normal flora of host health level and the substance preparation general name of metabolite and the growth of selective promotion host normal flora thereof.The Main Function of microbial forage additive is: regulate animal intestinal micro-ecology balance, improve growth of animal rate, improve efficiency of feed utilization, promotes that animal is to the absorption of calcium, magnesium, iron; Suppress the breeding of harmful intestinal tract microorganism, improve animal health level, strengthen animal immunizing power; Have no side effect, have no drug resistance, not pathogenic, be regarded as one of green feed additive.
The advantage of microbial forage additive is: the negative effect that (1) avoids long-term abuse of antibiotics to bring is as the insecurity etc. of medicament residue, body generation antibody-resistant bacterium, animal body immunity degradation, environmental pollution, food.(2) avoid active bacteria formulation not easily preservation for a long time, non-refractory, active to reduce gradually, in animal intestinal field planting difficulty, not strong by inactivation capacity anti-during intestines and stomach along with the storage time.In use there is single, the drawback such as sphere of action is narrow, the easy inactivation of functional component of effect in enzyme preparation.
Yeast is the unicellular lower eukaryotes of a class, and namely it have similar prokaryotes and easily cultivate, breed soon, be convenient to the characteristics such as genetic manipulation, has again molecule and the characteristics of cell biology of classical eukaryotic.Saccharomyces cerevisiae is human use microorganism the earliest, now the field such as extensive use and food, feed, medicine, brewery industry.Yeast metabolism thing be under specific process conditions controls by preferred saccharomyces cerevisiae in suitable fluid nutrient medium through a large amount of breedings, after reaching the required order of magnitude, by in different process conditions access liquid anaerobic fermentation medium, under complete anaerobic condition, it is made to produce a large amount of metabolites increment of cud beneficial bacterium being had to facilitation by regulating and controlling different fermentation conditions.It is different from common yeast product, it is complex metabolism thing group cud beneficial bacterium being had to Pasitive Regulation Effect of Genseng produced under special process regulation by specific yeast strain, it is rich in B family vitamin, mineral matter, digestive ferment, more complete amino acid, little peptide and UGF, it is the feed functional additive integrating nutrition and health care, its action effect does not rely on viable yeast bacterium, long shelf-life, properties of product are stablized.
The production technology mainly solid anaerobic digestion method that yeast metabolism thing is general.Solid state fermentation technique is simple, although eliminate liquid fermentation to need dehydration, collection, dry supervisor, but bacterial classification infects through multistage cultivation and the open type fermented miscellaneous bacteria that is subject to, production time is long, and floor space is large, and labour intensity is large, the bad control of various condition, effective metabolite content is low, and product quality is difficult to reach standard, and wherein living contaminants is ubiquitous problem during solid fermentation is produced.And liquid anaerobic fermentation, process control is more flexible, accurate, metabolin can be produced to specific direction by controlling zymotechnique, liquid anaerobic fermentation is in a complete airtight environment in addition, not easily be bacterial contamination, production time is short, production efficiency is high, is convenient to suitability for industrialized production.
Aspergillus oryzae is the main bacteria seed that soy sauce is produced, and aspergillus oryzae, natural widely distributed, has again the enzyme system that various active is high, and cellulase-producing ability is strong again, the safe bacterial classification approved in the world especially.The fungi preparation that Aspergillus Oryzae extract is through more fermentation and obtains, ruminant can be mainly used in by Direct-fed to animal, aspergillus oryzae culture is added on the growth of ruminating and effectively can stimulate Ruminal Fibre decomposer in feed, effectively can stimulate cellulose-decomposing bacteria eccrine fiber element enzyme, the digestion peak of rumen microorganism to fibroid roughage is impelled to arrive in advance, slow down the Rapid Fermentation of soluble-carbohydrate, serve a kind of competitive protection effect, after avoiding feed intake in cud feed Rapid Fermentation and the pH value short time is declined phenomenon fast, thus stability maintenance ruminal pH value, the generation of high yield cow acid poisoning phenomenon in effective reduction pasture.To improve in cud fiber and half Cellulolytic bacteria to the digestion of fiber in feed, and then also improve the total amount of acetic acid and propionic acid volatile fatty acid in their metabolite, regulate Rumen physiology function, improve metabolism, reduce the generation of metabolic disease, and then improve the production performance of animal.
Rumen Microbial Ecosystem decides the nutrient distribution process of cow feeding dry, and then directly affects milk cow integral production performance, and therefore rumen Microbial Ecosystem plays an important role in dairy cow nutrition.Microflora in ruminant tumor gastric, its main purpose is exactly by the balance of microflora in regulation and control cud thus the production performance of raising ruminant.Aspergillus oryzae and saccharomyces cerevisiae not only can improve Rumen Internal Environment as the adjusting control agent of lumen fermentation, and can not produce similar antibiotic medicament residue problem, therefore more and more receive the concern of people, have been widely used in husbandry sector at present.The research of single product is more, but composite use therebetween, and how composite, have no report.
Summary of the invention
For solving above-mentioned prior art Problems existing, the object of the present invention is to provide a kind of regulating rumen microbial forage additive and preparation method thereof.
For achieving the above object, the technical solution used in the present invention is: a kind of regulating rumen microbial forage additive, be made up of thing, aspergillus oryzae metabolin in yeast metabolism thing and born of the same parents, in described yeast metabolism thing and born of the same parents, the mass ratio of thing, aspergillus oryzae metabolin is 15:1,20:1 or 25:1.
In described yeast metabolism thing and born of the same parents, thing preferably adopts thing in the yeast metabolism thing of spray-dried process and born of the same parents.
Described aspergillus oryzae metabolin be preferably through low temperature drying pulverization process aspergillus oryzae metabolin.
The preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: saccharomyces cerevisiae bacterial classification is inoculated in YPD slant medium by (1), 28 ~ 30 DEG C of static gas wave refrigerator 48 hours, obtain the saccharomyces cerevisiae bacterial classification brought back to life; (2) the saccharomyces cerevisiae bacterial classification of resurrection is seeded in liquid YPD medium, cultivates to obtain primary seed solution; (3) by primary seed solution with 4 ~ 10% amount be seeded to aerobic fermentation culture medium, 28 ~ 30 DEG C of aerobic fermentation 24 ~ 48h, obtain secondary seed solution; (4) liquid anaerobic fermentation: by secondary seed solution with 10 ~ 20% inoculum concentration be seeded in liquid anaerobic fermentation medium, 15 ~ 20 DEG C of anaerobic fermentation 24 ~ 48h, obtain anaerobic fermentation product; (5) aqtocytolysis: papain is added in anaerobic fermentation product by the amount of saccharomyces cerevisiae dry cell weight 1-2%, 50 ~ 55 DEG C of self-dissolvings 36 ~ 48 hours, obtain yeast cells autolysate, spray-dried process, obtain thing in yeast metabolism thing and born of the same parents;
The preparation of step 2, aspergillus oryzae metabolin: aspergillus oryzae is inoculated in PDA slant medium by (1), 30 DEG C of static gas wave refrigerator 3-5 days, obtain the aspergillus oryzae bacterial classification brought back to life; (2) be seeded in PDA plating medium by the aspergillus oryzae bacterial classification of resurrection, 30 DEG C of static gas wave refrigerator 3-5 days, obtain first order seed; (3) primary seed solution is used physiological saline wash-out, put into the triangular flask that glass agitation beads is housed, the vibration 20min of 200r/min, filter, get filtrate, adjustment spore concentration is 1x10
8individual/ml, obtains spore suspension; (4) by spore suspension with 2 ~ 4% amount be seeded in fluid nutrient medium, 30 DEG C cultivate 48h, obtain aspergillus oryzae liquid; (5) aspergillus oryzae liquid is inoculated in solid fermentation culture medium by 14% inoculum concentration, cultivates 5 days, through 40 DEG C of crushed after being dried, obtain aspergillus oryzae metabolin for 30 DEG C;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 15:1,20:1 or 25:1 mix, obtain regulating rumen microbial forage additive.
Anaerobic fermentation culture medium described in step one is grouped into by the following one-tenth by mass fraction: molasses 12 ~ 20%, 0.05 ~ 0.15% sodium chloride, 0.05 ~ 0.15% calcium chloride, 0.05 ~ 0.15% magnesium sulfate, 0.05 ~ 0.2% peptone, 0.05 ~ 0.1% potassium chloride, 0.05 ~ 0.1% dipotassium hydrogen phosphate, 0.05 ~ 0.1% ammonium sulfate, surplus is water, and regulates pH to be 6.5 ~ 7.5.
Solid fermentation culture medium described in step 2 is grouped into by the following one-tenth by mass fraction: wheat bran 10g, urea 0.6%, dipotassium hydrogen phosphate 0.20%, magnesium sulfate 0.05%, ferrous sulfate 0.05%, sodium chloride 0.05%, and material-water ratio is 5:6, temperature 30 DEG C.
The stressed condition being unsuitable for Yeast Growth is adopted in liquid anaerobic sweat in step one, but saccharomycetic activity to be guaranteed simultaneously, make it under stressed condition, produce specific resisting stress metabolite, the pH of culture medium controls 6.5 ~ 7.5, temperature controls at 15 ~ 20 DEG C, inoculum concentration controls 10 ~ 20%, and incubation time controls at 24 ~ 48h.
The external batch cultivation experimental result of simulation rumen is adopted to be index in step 2 in solid fermentation process, solid fermentation culture medium and fermentation condition are screened, make aspergillus oryzae can screen in fermentation system produce be conducive to regulate and control rumen microorganism balance and improve crude fiber digestibility extra-metabolite.
Preferably, the preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: saccharomyces cerevisiae bacterial classification is inoculated in YPD slant medium by (1), 28 ~ 30 DEG C of static gas wave refrigerator 48 hours, obtain the saccharomyces cerevisiae bacterial classification brought back to life; (2) the saccharomyces cerevisiae bacterial classification of resurrection is seeded in liquid YPD medium, cultivates to obtain primary seed solution; (3) by primary seed solution with 4% amount be seeded to aerobic fermentation culture medium, 30 DEG C of aerobic fermentation 24h, obtain secondary seed solution; (4) liquid anaerobic fermentation: by secondary seed solution with 20% inoculum concentration be seeded in liquid anaerobic fermentation medium, 15 DEG C of anaerobic fermentation 24h, obtain anaerobic fermentation product; (5) aqtocytolysis: papain is added in anaerobic fermentation product by the amount of saccharomyces cerevisiae dry cell weight 1%, 55 DEG C of self-dissolvings 36 hours, obtain yeast cells autolysate, spray-dried process, obtain thing in yeast metabolism thing and born of the same parents;
The preparation of step 2, aspergillus oryzae metabolin: aspergillus oryzae is inoculated in PDA slant medium by (1), 30 DEG C of static gas wave refrigerator 3-5 days, obtain the aspergillus oryzae bacterial classification brought back to life; (2) be seeded in PDA plating medium by the aspergillus oryzae bacterial classification of resurrection, 30 DEG C of static gas wave refrigerator 3-5 days, obtain first order seed; (3) primary seed solution is used physiological saline wash-out, put into the triangular flask that glass agitation beads is housed, the vibration 20min of 200r/min, filter, get filtrate, adjustment spore concentration is 1x10
8individual/ml, obtains spore suspension; (4) by spore suspension with 2 ~ 4% amount be seeded in fluid nutrient medium, 30 DEG C cultivate 48h, obtain aspergillus oryzae liquid; (5) aspergillus oryzae liquid is inoculated in solid fermentation culture medium by 14% inoculum concentration, cultivates 5 days, through 40 DEG C of crushed after being dried, obtain aspergillus oryzae metabolin for 30 DEG C;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 20:1 mix, obtain regulating rumen microbial forage additive.
Anaerobic fermentation liquid culture medium described in step one is grouped into by the following one-tenth by mass fraction: 20% molasses, 0.05% sodium chloride, 0.05% calcium chloride, 0.05% magnesium sulfate, 0.2% peptone, 0.05% potassium chloride, 0.1% dipotassium hydrogen phosphate, 0.1% ammonium sulfate, surplus is water, pH7.5.
Relative to prior art, beneficial effect of the present invention is:
(1) the present invention adopts single culture industrial fermentation technology to produce specific resisting stress metabolome, and this preparation method stablizes, and efficiently solves active bacteria formulation and the easy inactivation of enzyme preparation, unstable defect, has greatly widened the scope of application of product; Adopt two single strain fermentation metabolome compounded technologies, widen the functional of product further, and be conducive to realizing the exploitation of the specific aim product in different animals and physiology (children age, enclose product etc.) stage.
(2) the regulating rumen microbial forage additive prepared is best in quality, through Animal experiment, good stability proves that it is by force both effectiveness, as feed addictive balanced in nutrition, can improve breeding performonce fo animals and feed conversion rate.Regulating rumen microbial forage additive of the present invention is not active bacteria formulation, but the anti-specificity produced by viable bacteria after high density fermentation stress metabolite, easy storage, can not reduce with the prolongation activity of holding time, ensure that the content of product active ingredient.
(3) preparation technology of the present invention is simple, and fermentation period is short, greatly reduces living contaminants degree, improves the content of effective metabolism.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail:
Embodiment 1
The preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: saccharomyces cerevisiae bacterial classification is inoculated in YPD slant medium by (1), 28 DEG C of static gas wave refrigerator 48 hours, obtain the saccharomyces cerevisiae bacterial classification brought back to life; (2) the saccharomyces cerevisiae bacterial classification of resurrection is seeded in liquid YPD medium, cultivates to obtain primary seed solution; (3) by primary seed solution with 4% amount be seeded to aerobic fermentation culture medium, 30 DEG C of aerobic fermentation 24h, obtain secondary seed solution; (4) liquid anaerobic fermentation: by secondary seed solution with 20% inoculum concentration be seeded in liquid anaerobic fermentation medium, 15 DEG C of anaerobic fermentation 24h, obtain anaerobic fermentation product; (5) aqtocytolysis: papain is added in anaerobic fermentation product by the amount of saccharomyces cerevisiae dry cell weight 1%, 55 DEG C of self-dissolvings 36 hours, obtain yeast cells autolysate, spray-dried process, obtain thing in yeast metabolism thing and born of the same parents;
The preparation of step 2, aspergillus oryzae metabolin: aspergillus oryzae is inoculated in PDA slant medium by (1), 30 DEG C of static gas wave refrigerator 3 days, obtain the aspergillus oryzae bacterial classification brought back to life; (2) be seeded in PDA plating medium by the aspergillus oryzae bacterial classification of resurrection, 30 DEG C of static gas wave refrigerator 3 days, obtain first order seed; (3) primary seed solution is used physiological saline wash-out, put into the triangular flask that glass agitation beads is housed, the vibration 20min of 200r/min, filter, get filtrate, adjustment spore concentration is 1x10
8individual/ml, obtains spore suspension; (4) by spore suspension with 2% amount be seeded in fluid nutrient medium, 30 DEG C cultivate 48h, obtain aspergillus oryzae liquid; (5) aspergillus oryzae liquid is inoculated in solid fermentation culture medium by 14% inoculum concentration, cultivates 5 days, through 40 DEG C of crushed after being dried, obtain aspergillus oryzae metabolin for 30 DEG C;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 20:1 mix, obtain regulating rumen microbial forage additive.
Anaerobic fermentation liquid culture medium described in step one is grouped into by the following one-tenth by mass fraction: 20% molasses, 0.05% sodium chloride, 0.05% calcium chloride, 0.05% magnesium sulfate, 0.2% peptone, 0.05% potassium chloride, 0.1% dipotassium hydrogen phosphate, 0.1% ammonium sulfate, surplus is water, pH 7.5.
Solid fermentation culture medium described in step 2 is grouped into by the following one-tenth by mass fraction: wheat bran 10g, urea 0.6%, dipotassium hydrogen phosphate 0.20%, magnesium sulfate 0.05%, ferrous sulfate 0.05%, sodium chloride 0.05%, condition of culture is inoculum concentration 14%, incubation time 5 days, material-water ratio is 5:6, temperature 30 DEG C.
Embodiment 2
The preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: with the step one of embodiment 1;
The preparation of step 2, aspergillus oryzae metabolin: with the step 2 of embodiment 1;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 15:1 mix, obtain regulating rumen microbial forage additive.
Embodiment 3
The preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: with the step one of embodiment 1;
The preparation of step 2, aspergillus oryzae metabolin: with the step 2 of embodiment 1;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 25:1 mix, obtain regulating rumen microbial forage additive.
Product efficacy is verified
1, experimental animal
Choose the sheep (18 monthly age) that 4 body conditions are good, permanence lymphoma stomach fistulization pipe is installed, for the collection of rumen fluid.Essence is thick than being set as 40:60, green hay (sheep's hay) free choice feeding; Secondary feed every day respectively at morning 8:00,17:00, and concentrate supplement is adding respectively sooner or later; Ensure sufficient, clean drinking-water, and keep the clean of drinking trough.
2, daily ration and experimental design is tested
Test setting essence is thick than being 5:5 and 0.15g fine fodder+0.15g sheep's hay, totally 6 processed group, each process 4 repetition
3, the outer batch cultivation method of rumen fluid
3.1, external batch cultivation device
Batch cultivation device designed, designed, main body water bath with thermostatic control shaking table, bath temperature and frequency of oscillation adjustable; Cover bottle (volume is 250ml) by indigo plant and make blake bottle.
3.2, buffer
Buffer reagent and macroelement solution----A liquid: take K
2hPO
4382.5mg, KH
2pO
4292mg, (NH
4)
2sO
4480mg, NaCl 200mg, MgSO
47H
2o 100mg and Na
2cO
34000mg.Add distilled water after mentioned reagent being mixed and be settled to 1000ml.The preparation in one day before use of this solution is stand-by.
PFNNINGS trace element solution----B liquid: accurately take EDTA 500mg, FeSO
47H
2o200mg, MnCl
24H
2o 200mg, ZnSO
47H
2o 10mg, H
3bO
330mg, CoCl
26H
2o20mg, CuCl
22H
2o1mg, NiCl
26H
2o 2mg and NaMoO
43mg.Adding distil water after mentioned reagent mixing is settled to 1000ml.Continue to pass into CO
2after 18 hours, cover tightly bottleneck, be placed in refrigerator for subsequent use.
Reductant solution----C liquid: take 25g Na
2s9H
2o is placed in after 100ml volumetric flask adds the dissolving of 80ml distilled water and is settled to 100ml, continues to be filled with CO
2after 20min, cover tightly bottleneck, be placed in refrigerator for subsequent use.
Prepared by buffer solution: before cultivation, 1h accurately measures 988ml A liquid, 10ml B liquid, and 2ml C liquid, fully mixes, logical CO
2then be sub-packed in (each blake bottle adds 50ml) in blake bottle to colourless, continue to pass into CO
2gas 10min, covers tightly bottle cap, and be placed in water bath with thermostatic control be preheated to 39 DEG C stand-by.
3.3, the collection of rumen fluid and the preparation of nutrient solution
Morning raises (08:00) first 1 hour by sheep rumen up and down different loci hard PVC pipe gather enough rumen fluids, pour into and reach 39 DEG C through preheating and be connected with CO
2vacuum flask in, cover tightly bottleneck immediately after filling, return laboratory rapidly, through four layers of filtered through gauze (temperature should be remained on about 39 DEG C and the least possible contacting with air by period), continue afterwards to pass into CO
2gas 5min, then rapidly packing to above-mentioned preheated and be connected with CO
2blake bottle in (each blake bottle adds 20ml rumen fluid, buffer solution 40ml), start cultivate.
3.4, sample collection
Within 24th hour, gather whole processed group rumen fluid in what cultivate, measure pH immediately, get 1.5ml nutrient solution after 10000r/min, 10min are centrifugal, get 1ml supernatant, add 25% metaphosphoric acid solution 0.2mL and mix, preserve VFA to be measured for-20 DEG C; Separately get 1.5ml nutrient solution-20 DEG C preservation, NH to be measured
3-N.Gather nutrient solution four layers of filtered through gauze, it is to be measured to get filtrate, collection residue.
3.5 sample determination
3.5.1pH
Measure and adopt direct measuring method;
3.5.2VFA
Measure and adopt Agilent Technologies 7890A gas chromatograph to carry out measuring (Cao Qingyun 2006);
3.5.3NH
3-N
Measure and adopt sodium nitroprusside colorimetric method for determining (Feng ancestor kind 1993).
3.5.4DM, NDF and ADF content assaying method is with reference to " forage analysis and determination of feeds quality technology "
4, data statistic analysis
Test data Excel software arranges, and adopts SPSS17.0 software to compare analysis.Experimental result is in table 1.
Table 1 different metabolic thing is on the impact of cud nutrient metabolism
| Group | pH | Nitrogen mg/100ml | Total VFAmmol/L | NDF digestibility % | ADF digestibility % | DM digestibility % |
| Control group | 6.55 | 19.14 | 65.04 | 39.52 | 20.42 | 41.47 |
| Yeast metabolism thing | 6.52 | 18.46 | 76.85 | 40.12 | 20.29 | 42.03 |
| Aspergillus oryzae metabolin | 6.50 | 18.96 | 72.20 | 46.43 | 23.41 | 46.52 |
| Embodiment 1 | 6.50 | 18.04 | 77.13 | 47.79 | 24.78 | 47.12 |
| Embodiment 2 | 6.51 | 17.36 | 80.21 | 49.33 | 25.09 | 48.56 |
| Embodiment 3 | 6.50 | 18.26 | 78.56 | 47.25 | 24.89 | 48.32 |
From table 1, for cud PH, add different microbial forage additive groups, all have a declining tendency compared with control group, but between each additive group, difference is not remarkable, different microbial forage additives can play the effect of stable ruminal pH value; All have for each additive in this test ammoniacal nitrogen the trend reducing ammonia nitrogen concentration, wherein the additive package of embodiment 2 is to NH
3the amplitude that-N concentration reduces is the most remarkable; Concerning total volatile fatty acid, each additive improves the content of general volatile aliphatic acid all in varying degrees, wherein the increase rate of embodiment 2 pairs of general volatile content of fatty acid is maximum, is significantly higher than other each group (p<0.05); In dry matter degradability, add separately yeast culture to dry, neutral detergent fiber and acid detergent fiber DeGrain (p>0.05); Independent interpolation aspergillus oryzae metabolin, has been significantly increased to the degradation rate (p<0.05) of dry, neutral detergent fiber and acid detergent fiber compared with control group.
After adding by optimum additive capacity after two kinds of metabolins being mixed in the ratio in embodiment 2, comparatively control group general volatile aliphatic acid of comparing improves 23.34%, DM degradation rate improves 17.10%, neutral detergent fiber degradation rate improves 24.82%, acid detergent fiber degradation rate improves 22.87%, to adjustment and the dry matter degradability of function of rumen, the better effects if when degradation rate of neutral detergent fiber and acid detergent fiber adds more separately, there is the synergistic effect of effect, but only under suitable mixing ratio row, just can produce synergistic effect, this proportioning awaits further optimum organization in work from now on, thus explore the potentiality of this product to a greater extent.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (8)
1. a regulating rumen microbial forage additive, it is characterized in that, described regulating rumen microbial forage additive is made up of thing, aspergillus oryzae metabolin in yeast metabolism thing and born of the same parents, and in described yeast metabolism thing and born of the same parents, the mass ratio of thing, aspergillus oryzae metabolin is 15:1,20:1 or 25:1.
2. regulating rumen microbial forage additive according to claim 1, is characterized in that, in described yeast metabolism thing and born of the same parents, thing is thing in the yeast metabolism thing of spray-dried process and born of the same parents.
3. regulating rumen microbial forage additive according to claim 1, is characterized in that, described aspergillus oryzae metabolin be through low temperature drying pulverization process aspergillus oryzae metabolin.
4. the preparation method of the regulating rumen microbial forage additive described in any one of claim 1-3, is characterized in that, comprise the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: saccharomyces cerevisiae bacterial classification is inoculated in YPD slant medium by (1), 28 ~ 30 DEG C of static gas wave refrigerator 48 hours, obtain the saccharomyces cerevisiae bacterial classification brought back to life; (2) the saccharomyces cerevisiae bacterial classification of resurrection is seeded in liquid YPD medium, cultivates to obtain primary seed solution; (3) by primary seed solution with 4 ~ 10% amount be seeded to aerobic fermentation culture medium, 28 ~ 30 DEG C of aerobic fermentation 24 ~ 48h, obtain secondary seed solution; (4) liquid anaerobic fermentation: by secondary seed solution with 10 ~ 20% inoculum concentration be seeded in liquid anaerobic fermentation medium, 15 ~ 20 DEG C of anaerobic fermentation 24 ~ 48h, obtain anaerobic fermentation product; (5) aqtocytolysis: papain is added in anaerobic fermentation product by the amount of saccharomyces cerevisiae dry cell weight 1-2%, 50 ~ 55 DEG C of self-dissolvings 36 ~ 48 hours, obtain yeast cells autolysate, spray-dried process, obtain thing in yeast metabolism thing and born of the same parents;
The preparation of step 2, aspergillus oryzae metabolin: aspergillus oryzae is inoculated in PDA slant medium by (1), 30 DEG C of static gas wave refrigerator 3-5 days, obtain the aspergillus oryzae bacterial classification brought back to life; (2) be seeded in PDA plating medium by the aspergillus oryzae bacterial classification of resurrection, 30 DEG C of static gas wave refrigerator 3-5 days, obtain first order seed; (3) primary seed solution is used physiological saline wash-out, put into the triangular flask that glass agitation beads is housed, the vibration 20min of 200r/min, filter, get filtrate, adjustment spore concentration is 1x10
8individual/ml, obtains spore suspension; (4) by spore suspension with 2 ~ 4% amount be seeded in fluid nutrient medium, 30 DEG C cultivate 48h, obtain aspergillus oryzae liquid; (5) aspergillus oryzae liquid is inoculated in solid fermentation culture medium by 14% inoculum concentration, cultivates 5 days, through 40 DEG C of crushed after being dried, obtain aspergillus oryzae metabolin for 30 DEG C;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 15:1,20:1 or 25:1 mix, obtain regulating rumen microbial forage additive.
5. the preparation method of regulating rumen microbial forage additive according to claim 4, it is characterized in that, anaerobic fermentation culture medium described in step one is grouped into by the following one-tenth by mass fraction: molasses 12 ~ 20%, 0.05 ~ 0.15% sodium chloride, 0.05 ~ 0.15% calcium chloride, 0.05 ~ 0.15% magnesium sulfate, 0.05 ~ 0.2% peptone, 0.05 ~ 0.1% potassium chloride, 0.05 ~ 0.1% dipotassium hydrogen phosphate, 0.05 ~ 0.1% ammonium sulfate, surplus is water, and pH is 6.5 ~ 7.5.
6. the preparation method of regulating rumen microbial forage additive according to claim 4, it is characterized in that, solid fermentation culture medium described in step 2 is grouped into by the following one-tenth by mass fraction: wheat bran 10g, urea 0.6%, dipotassium hydrogen phosphate 0.20%, magnesium sulfate 0.05%, ferrous sulfate 0.05%, sodium chloride 0.05%, pH is 6.5 ~ 7.5.
7. the preparation method of regulating rumen microbial forage additive according to claim 4, is characterized in that, the preparation method of described regulating rumen microbial forage additive, comprises the steps:
The preparation of thing in step one, yeast metabolism thing and born of the same parents: saccharomyces cerevisiae bacterial classification is inoculated in YPD slant medium by (1), 28 ~ 30 DEG C of static gas wave refrigerator 48 hours, obtain the saccharomyces cerevisiae bacterial classification brought back to life; (2) the saccharomyces cerevisiae bacterial classification of resurrection is seeded in liquid YPD medium, cultivates to obtain primary seed solution; (3) by primary seed solution with 4% amount be seeded to aerobic fermentation culture medium, 30 DEG C of aerobic fermentation 24h, obtain secondary seed solution; (4) liquid anaerobic fermentation: by secondary seed solution with 20% inoculum concentration be seeded in liquid anaerobic fermentation medium, 15 DEG C of anaerobic fermentation 24h, obtain anaerobic fermentation product; (5) aqtocytolysis: papain is added in anaerobic fermentation product by the amount of saccharomyces cerevisiae dry cell weight 1%, 55 DEG C of self-dissolvings 36 hours, obtain yeast cells autolysate, spray-dried process, obtain thing in yeast metabolism thing and born of the same parents;
The preparation of step 2, aspergillus oryzae metabolin: aspergillus oryzae is inoculated in PDA slant medium by (1), 30 DEG C of static gas wave refrigerator 3-5 days, obtain the aspergillus oryzae bacterial classification brought back to life; (2) be seeded in PDA plating medium by the aspergillus oryzae bacterial classification of resurrection, 30 DEG C of static gas wave refrigerator 3-5 days, obtain first order seed; (3) primary seed solution is used physiological saline wash-out, put into the triangular flask that glass agitation beads is housed, the vibration 20min of 200r/min, filter, get filtrate, adjustment spore concentration is 1x10
8individual/ml, obtains spore suspension; (4) by spore suspension with 2 ~ 4% amount be seeded in fluid nutrient medium, 30 DEG C cultivate 48h, obtain aspergillus oryzae liquid; (5) aspergillus oryzae liquid is inoculated in solid fermentation culture medium by 14% inoculum concentration, cultivates 5 days, through 40 DEG C of crushed after being dried, obtain aspergillus oryzae metabolin for 30 DEG C;
The preparation of step 3, regulating rumen microbial forage additive: by the aspergillus oryzae metabolin in thing, step 2 in the yeast metabolism thing in step one and born of the same parents in mass ratio 20:1 mix, obtain regulating rumen microbial forage additive.
8. the preparation method of regulating rumen microbial forage additive according to claim 7, it is characterized in that, anaerobic fermentation culture medium described in step one is grouped into by the following one-tenth by mass fraction: 20% molasses, 0.05% sodium chloride, 0.05% calcium chloride, 0.05% magnesium sulfate, 0.2% peptone, 0.05% potassium chloride, 0.1% dipotassium hydrogen phosphate, 0.1% ammonium sulfate, surplus is water, pH 7.5.
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