CN107058194A - A kind of ensiling embedding microbial inoculum and its production method - Google Patents
A kind of ensiling embedding microbial inoculum and its production method Download PDFInfo
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Abstract
The present invention relates to a kind of ensiling embedding microbial inoculum and its production method, the embedding microbial inoculum is by the microbial inoculum A containing ester-producing yeast and Lactobacillus plantarum and bacillus subtilis microbial agent B according to weight ratio 2.5~3.5：1 composition.Ensilage prepared by the present invention exceedes commercially available microbial inoculum no matter in feed nutrition in itself, or on effective shelf-life, so the present invention improves the utilization rate of maize straw, improves the nutritive value of ensilage, has a good application prospect.
Description
【Technical field】
The invention belongs to agricultural zootechnical technical field.More particularly it relates to a kind of ensiling embedding microbial inoculum, also
It is related to the production method that the ensiling embeds microbial inoculum.
【Background technology】
China's stalk resource very abundant.Annual stalk yield accounts for the 20% of world's stalk total output up to 6,000 ten thousand tons or so
~30%.At present, all kinds of agricultural crop straws of China about 70% fall as domestic energy fuel consumption, not only waste of resource, and
Return environment to be negatively affected, so being efficiently treated through using the method for science to stalks such as maize straws, can both subtract
Little waste, environmental protection, the problem of can solving energy deficiency again.Therefore, relevant maize straw processing cost is low, simple to operate
And the feed Sativa Silage Technology of energy wide popularization and application is valued by people.The research of ensilage technology has up to a hundred with utilizing
Year history, maize straw quality by ensiling is fluffy, content of cellulose with sour fragrant mouthfeel and stalk declines, nutrition
Composition is substantially increased, and is the feed of domestic animal eating.Existing Sativa Silage Technology key is excellent ensiling microbes screening and culturing, straw ensiling
Pre-treatment, composite bacteria compounding and optimization of fermentation condition, domestic and international many scholars have put into these areas largely to grind
Study carefully work, but for that can solve how to suppress varied bacteria growing in fermentation process, reduction fermentation costs, degraded cellulose, carry
Many problems are still had in terms of high protein content.
CN 105331564A disclose a kind of side of immobilization composite bacteria agent direct fermentation sweet sorghum stalk Fodder making
Method, the key of the fabrication techniques sweet sorghum stalk feed is that microbial inoculum is handled using immobilization technology, so as to reach microbial inoculum
Can be with slow releasing function, but its microbial inoculum need to be used in mixed way with cellulase, and cost is too high, is unfavorable for large-scale promotion.CN
105379971A discloses a kind of preparation method of silage corn, and the key of the fabrication techniques silage corn is to utilize geotrichum candidum
Bacterium, production three kinds of bacterium of gastral cavity candidiasis and Lactobacillus plantarum handle corn simultaneously, so as to reach reduction corn stalk fiber element, carry
The effect of high feed nutrition, but because three kinds of bacterium act on simultaneously, its degraded cellulose improves the effect in terms of feed protein
It is unobvious.
These technological deficiencies existed for prior art, the present inventor passes through on the basis for summarizing prior art
Lot of experiments and analysis, complete the present invention finally.
【The content of the invention】
[technical problem to be solved]
It is an object of the invention to provide a kind of ensiling embedding microbial inoculum.
It is a further object to provide production method of the ensiling with embedding microbial inoculum.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of ensiling embedding microbial inoculum.The embedding microbial inoculum is by the bacterium containing ester-producing yeast and Lactobacillus plantarum
Agent A and bacillus subtilis microbial agent B is according to weight than 2.5~3.5：1 composition, the embedding microbial inoculum contains 2.5~3.5 × 108Individual/
G bacillus subtilises, 6.0~7.0 × 108Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
A preferred embodiment of the invention, the embedding microbial inoculum is by the bacterium containing ester-producing yeast and Lactobacillus plantarum
Agent A and bacillus subtilis microbial agent B is according to weight than 2.8~3.2:1 composition, the embedding microbial inoculum contains 2.8~3.2 × 108Individual/
G bacillus subtilises, 6.4~6.8 × 108Individual/g ester-producing yeasts and 2.2~2.4 × 109Individual/g Lactobacillus plantarums.
According to another preferred embodiment of the present invention, described bacillus subtilis is selected from Bacillus
Subtilis strain B48 (depositary institutions：Chinese industrial Microbiological Culture Collection administrative center CICC；Preserving number CICC
10089), Bacillus subtilis strain B49 (depositary institutions：Chinese industrial Microbiological Culture Collection administrative center
CICC；Preserving number CICC 10090) or Bacillus subtilis strain WL1021 (depositary institutions：The micro- life of Chinese industrial
Thing culture presevation administrative center CICC；Preserving number CICC10063) bacterial strain；Described ester-producing yeast is selected from Pichia anomala
(depositary institution China typical culture collection center CCTCC；Preserving number CICIM Y0297), Candida sp. (depositary institutions：
Chinese industrial Microbiological Culture Collection administrative center CICC；Preserving number CICC 1257) or Brettanomyces sp. (preservation lists
Position：China typical culture collection center CCTCC；Preserving number CCTCC AY 93142) bacterial strain；Described Lactobacillus plantarum is selected from
Lactobacillus plantarum strain F1 (depositary institutions：China typical culture collection center CCTCC；Preserving number
CCREM SDMCC 050029), Lactobacillus plantarum A15 (depositary institutions：Chinese industrial microorganism fungus kind protects
Hide administrative center CICC；Preserving number CICC 21814) or Lactobacillus plantarum Y15118 (depositary institutions：In
State Research for Industrial Microbial Germ preservation administrative center CICC；Preserving number CICC 20768) bacterial strain.
The present invention relates to production method of the above-mentioned ensiling with embedding microbial inoculum.
The step of ensiling is with embedding microbial inoculum production method is as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
By the ester-producing yeast bacterial strain selected from Pichia anomala, Candida sp. or Brettanomyces sp. according to
1% inoculum concentration is inoculated at 110~120 DEG C of temperature in 8~12min of sterilizing Shake flask medium, in temperature 30 in shaking flask
24~36h is cultivated under the conditions of DEG C, is obtained containing 2.0~2.5 × 108The shake flask culture of individual/g ester-producing yeasts；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 36~48h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 6.4~6.8 × 108Individual/
The fermentation culture of g ester-producing yeasts；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Will selected from Lactobacillus plantarum strain F1, Lactobacillus plantarum A15 or
Lactobacillus plantarum Y15118 lactobacillus plantarum strain according to 1% inoculum concentration be inoculated in temperature 110~
Sterilized at 120 DEG C in 8~12min Shake flask medium, cultivate 48~72h under the conditions of 30 DEG C of temperature in shaking flask, contained
Have 5.0~5.5 × 108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 60~72h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 2.2~2.4 × 109Individual/
The fermentation culture of g Lactobacillus plantarums；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Will selected from Bacillus subtilis strain B48, Bacillus subtilis strain B49 or
Bacillus subtilis strain WL1021 bacillus subtilis strain is inoculated in temperature 110 according to 1% inoculum concentration
Sterilized at~120 DEG C in 8~12min Shake flask medium, cultivate 24~36h under the conditions of 30 DEG C of temperature in shaking flask, obtain
Contain 7.5~8.0 × 107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 36~48h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 2.5~3.5 × 108Individual/
The fermentation culture of g bacillus subtilises；
IV, straw pretreatment
Straw is crushed, sieved, plant straw powder is collected, saturated vapor is reused and handles 1.0~1.5h, then
Room temperature is cooled to, sterilized plant powder of straw is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.0~1.5 are well mixed, according to the inoculum concentration of sterilized plant powder of straw dry weight meter 1.0~5.0%, well mixed
Fermentation culture be added in sterilized plant powder of straw, shake 8h in shaking table, eccentric cleaning obtains absorption ester-producing yeast with planting
The carrier of thing lactobacillus；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill pH6.0~6.8 contain with weight
Gauge 4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
By microbial inoculum A and microbial inoculum B according to weight than 2.5~3.5：1 is well mixed, and obtains described embedding microbial inoculum.
A preferred embodiment of the invention, described straw is maize straw, broomcorn straw, Wheat Straw
Stalk or rice straw.
According to another preferred embodiment of the present invention, the granularity of the plant straw powder is more than 80 mesh.
According to another preferred embodiment of the present invention, described embedding microbial inoculum is preserved at 4 DEG C of temperature.
According to another preferred embodiment of the present invention, described embedding microbial inoculum contains 2.5~3.5 × 108Individual/g is withered
Careless bacillus, 6.0~7.0 × 108Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
The present invention is described in more detail below.
The present invention relates to a kind of ensiling embedding microbial inoculum.
The straw ensiling microbial inoculum of China's market sale is mostly the Rapid Fermentation acid-producing bacteria agent based on lactic acid bacteria, and stalk is fine
Dimension element is substantially non-degradable, and protein lifting is also little, nor is adapted to the regional cold environment in Inner Mongol and applies.The present invention passes through
Bacillus subtilis, ester-producing yeast and the Lactobacillus plantarum for adopting to screen in sample from Inner Mongolia various regions, are inoculated with
When the stalks such as maize straw, quickly dissolved using microbial inoculum B, microorganism breeds rapidly, the cellulose in degrading straw is converted
For sugar, and slowly released using absorption-microbial inoculum A made from embedded-cross-linked technology after microbial inoculum B additions in 12h, utilize straw
The sugar of stalk itself and the sugar degraded by microbial inoculum B, can increase substantially the protein content of feed.
The embedding microbial inoculum is by the microbial inoculum A containing ester-producing yeast and Lactobacillus plantarum and bacillus subtilis microbial agent B according to weight
Measure ratio 2.5~3.5：1 composition, the embedding microbial inoculum contains 2.5~3.5 × 108Individual/g bacillus subtilises, 6.0~7.0 × 108
Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
Preferably, the embedding microbial inoculum is by the microbial inoculum A containing ester-producing yeast and Lactobacillus plantarum and bacillus subtilis microbial agent B
According to weight than 2.8~3.2:1 composition, the embedding microbial inoculum contains 2.8~3.2 × 108Individual/g bacillus subtilises, 6.4~6.8
×108Individual/g ester-producing yeasts and 2.2~2.4 × 109Individual/g Lactobacillus plantarums.
The bacillus subtilis that the present invention is used itself produces a kind of enzyme for being capable of decomposition of cellulose, therefore can be fully sharp
With the cellulose in stalk and the utilizable oligosaccharide of other bacteriums is degraded to, with enteron aisle can be made after cattle and sheep feeding
The advantages of other interior anaerobism probiotics amount reproductions, promotion digestion, raising immunity, be the improved seeds of straw ensiling strain.
According to the present invention, described bacillus subtilis be selected from Bacillus subtilis strain B48,
Bacillus subtilis strain B49 or Bacillus subtilis strain WL1021 bacterial strains；These bacterial strains are all
It is the bacterial strain that can be obtained from related microbial preservation mechanism.
The ester-producing yeast that the present invention is used can effectively lift the local flavor of ensilage, can utilize bacillus subtilis point
The oligosaccharide that solution cellulose is produced is bred.Ester-producing yeast protein content is very high, can effectively lift forage protein content, into
The single cell protein that can be eaten for cattle and sheep.
According to the present invention, described ester-producing yeast be selected from Pichia anomala, Candida sp. or
Brettanomyces sp. bacterial strains；These bacterial strains are all the bacterial strains that can be obtained from related microbial preservation mechanism.
The Lactobacillus plantarum that the present invention is used is amphimicrobe, as bacillus subtilis and ester-producing yeast are by original
The oxygen utilization in material gap occupies rapidly advantage and raised growth after finishing, this microbial inoculum can using absorption-embedded-cross-linked mode
So that Lactobacillus plantarum is slowly released, it is unlikely to cause because of the too fast growth of Lactobacillus plantarum in ensilage nutrition not
Foot, can cause saccharomycete, bacillus subtilis, Lactobacillus plantarum segmentation to turn into the dominant microflora in feed microenvironment, fermentation
Stalk can turn into that low cellulose, high protein, quality be fluffy, the sour fragrant high-quality cattle and sheep ensilage of smell.
According to the present invention, described Lactobacillus plantarum be selected from Lactobacillus plantarum strain F1,
Lactobacillus plantarum A15 or Lactobacillus plantarum Y15118 bacterial strains；These bacterial strains are all
The bacterial strain that can be obtained from related microbial preservation mechanism.
The present invention relates to production method of the ensiling with embedding microbial inoculum.
The step of ensiling is with embedding microbial inoculum production method is as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
Peptone, glucose and yeast extract are all the products sold in the market.
By the ester-producing yeast bacterial strain selected from Pichia anomala, Candida sp. or Brettanomyces sp. according to
1% inoculum concentration is inoculated at 110~120 DEG C of temperature in 8~12min of sterilizing Shake flask medium, in temperature 30 in shaking flask
24~36h is cultivated under the conditions of DEG C, is obtained containing 2.0~2.5 × 108The shake flask culture of individual/g ester-producing yeasts；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 36~48h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 6.4~6.8 × 108Individual/
The fermentation culture of g ester-producing yeasts；
Other raw materials that peptone, glucose, yeast extract and the follow-up preparation process that the step is used are used are all
It is the product sold in the market, is also the product sold in the market；What the step and subsequent step were used shakes
Bottle is equipment usually used in the art with fermentation tank, is also the product sold in the market, therefore, below will not
These technology contents are repeated again.
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Will selected from Lactobacillus plantarum strain F1, Lactobacillus plantarum A15 or
Lactobacillus plantarum Y15118 lactobacillus plantarum strain according to 1% inoculum concentration be inoculated in temperature 110~
Sterilized at 120 DEG C in 8~12min Shake flask medium, cultivate 48~72h under the conditions of 30 DEG C of temperature in shaking flask, contained
Have 5.0~5.5 × 108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 60~72h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 2.2~2.4 × 109Individual/
The fermentation culture of g Lactobacillus plantarums；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Will selected from Bacillus subtilis strain B48, Bacillus subtilis strain B49 or
Bacillus subtilis strain WL1021 bacillus subtilis strain is inoculated in temperature 110 according to 1% inoculum concentration
Sterilized at~120 DEG C in 8~12min Shake flask medium, cultivate 24~36h under the conditions of 30 DEG C of temperature in shaking flask, obtain
Contain 7.5~8.0 × 107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the hair for the 8~12min that sterilized at 110~120 DEG C of temperature according to 3% inoculum concentration
In ferment culture medium, 36~48h is cultivated under the conditions of 30~32 DEG C of temperature in fermentation tank, is obtained containing 2.5~3.5 × 108Individual/
The fermentation culture of g bacillus subtilises；
IV, straw pretreatment
Straw is crushed, sieved, plant straw powder is collected, saturated vapor is reused and handles 1.0~1.5h, then
Room temperature is cooled to, sterilized plant powder of straw is obtained；
The straw that the present invention is used is maize straw, broomcorn straw, wheat stalk or rice straw.Certainly, it is every
Can be decomposed by microbial inoculum of the present invention can't produce dysgenic other straws to its microbial inoculum, can be used for this hair
It is bright, also all within the scope of the present invention.
According to the present invention, the granularity of the plant straw powder is more than 80 mesh.
Crush, screening, equipment used in steam treatment are all equipment usually used in the art.
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.0~1.5 are well mixed, according to the inoculum concentration of sterilized plant powder of straw dry weight meter 1.0~5.0%, well mixed
Fermentation culture be added in sterilized plant powder of straw, shake 8h in shaking table, eccentric cleaning obtains absorption ester-producing yeast with planting
The carrier of thing lactobacillus；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill pH6.0~6.8 contain with weight
Gauge 4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
By microbial inoculum A and microbial inoculum B according to weight than 2.5~3.5：1 is well mixed, and obtains described embedding microbial inoculum.Described
Microbial inoculum is embedded to preserve at 4 DEG C of temperature.
Obtained embedding microbial inoculum prepared according to the methods of the invention contains 2.5~3.5 × 108Individual/g bacillus subtilises, 6.0
~7.0 × 108Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
Microbial inoculum is dissolved in 25~35 DEG C of water during inoculation, little sugar is added, then the maize straw after crushing through full
With steam treatment 1h, be cooled to 25 DEG C it is standby, liquid bacterial agent is accessed by siccative total amount 1%, after then mixing raw material and bacterium solution
Layering is inserted in pond, continues to add next layer of raw material after raw material is treaded after 20~30cm of addition, raw material is added and covered after finishing
Plastic sheeting, then tread after burying 10cm or so soil, spontaneous fermentation is then carried out, described ensilage is obtained.
[beneficial effect]
The beneficial effects of the invention are as follows：The commercially available straw ensiling microbial inoculum of China is mostly the Rapid Fermentation production based on lactic acid bacteria
Sour microbial inoculum, the cellulose that they contain to stalk in itself is substantially non-degradable, and protein lifting is also little, cold to Inner Mongol area
Environmental suitability is not also high.The present invention passes through the bacillus subtilis for adopting to be sieved in sample from Inner Mongol various regions, ester-producing yeast, plant
Thing lactobacillus, when being inoculated in maize straw, the cellulose degradation of stalk is converted into sugar by the rapidly dissolvable breedings of microbial inoculum B,
And microbial inoculum A is just slowly released after microbial inoculum B, obtained sugar of being degraded using stalk self sugar and by microbial inoculum B can
Forage protein content is increased substantially, for example, improves more than 50%.
The self-produced decomposition of cellulose enzyme of bacillus subtilis of the present invention, can make full use of the cellulose in stalk and be dropped
Solve as the utilizable oligosaccharide of other bacteriums, can make after cattle and sheep search for food other anaerobism probiotics amount reproductions in enteron aisle,
Promote digestion, raising immunity, therefore the present invention is the improved seeds of straw ensiling strain.
Ester-producing yeast of the present invention can effectively lift the local flavor of ensilage, and it can utilize bacillus subtilis
The oligosaccharide that bacterium decomposition of cellulose is produced is bred.The protein content of ester-producing yeast is very high, can effectively lift the egg of feed
Bai Hanliang, the single cell protein that can be eaten as cattle and sheep.
Lactobacillus plantarum of the present invention is amphimicrobe, as bacillus subtilis and ester-producing yeast are incited somebody to action
The oxygen utilization in raw material gap occupies rapidly advantage and raised growth after finishing, this microbial inoculum using absorption-embedded-cross-linked side
Formula can slowly release Lactobacillus plantarum, be unlikely to cause in ensilage because of the too fast growth of Lactobacillus plantarum
Subalimentation, can cause saccharomycete, bacillus subtilis, Lactobacillus plantarum segmentation turn into feed microenvironment in dominant bacteria
Group, stalk after fermentation can turn into that low cellulose, high protein, quality be fluffy, the sour fragrant high-quality cattle and sheep ensilage of smell.
Ensiling of the present invention is the bacterial strain that screens in the maize straw of Inner Mongol various regions and obtain with the strain in embedding microbial inoculum, it
Be very suitable for be directed to local cornstalk silage, the utilization rate of stalk can be effectively improved, while this microbial inoculum can be right
Stalk carries out quick ensiling, shortens feed maturation time.
【Embodiment】
The present invention is will be better understood that by following embodiments.
Embodiment 1：Production ensiling of the present invention embedding microbial inoculum
The implementation steps of the embodiment are as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
Pichia anomala ester-producing yeasts bacterial strains are inoculated at 110 DEG C of temperature the 12min that sterilizes according to 1% inoculum concentration
Shake flask medium in, cultivate 24h under the conditions of 30 DEG C of temperature in shaking flask, obtain containing 2.0 × 108Individual/g ester-producing yeasts
Shake flask culture；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 12min that sterilized at 110 DEG C of temperature according to 3% inoculum concentration
In, 44h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 6.4 × 108The fermented and cultured of individual/g ester-producing yeasts
Liquid；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Lactobacillus plantarum strainF1 lactobacillus plantarum strains are inoculated according to 1% inoculum concentration
Sterilized at 110 DEG C of temperature in 12min Shake flask medium, cultivate 48h under the conditions of 30 DEG C of temperature in shaking flask, contained
5.2×108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 12min that sterilized at 110 DEG C of temperature according to 3% inoculum concentration
In, 64h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 2.2 × 109The fermented and cultured of individual/g Lactobacillus plantarums
Liquid；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Bacillus subtilis strain B48 bacillus subtilis strains are inoculated in temperature according to 1% inoculum concentration
At 110 DEG C of degree in sterilizing 12min Shake flask medium, 28h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is obtained containing 7.6
×107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 12min that sterilized at 110 DEG C of temperature according to 3% inoculum concentration
In, 36h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 2.8 × 108The fermentation training of individual/g bacillus subtilises
Nutrient solution；
IV, straw pretreatment
By corn straw smashing, 80 mesh corn stalk powders are collected in screening, reuse saturated vapor processing 1.0h, then
Room temperature is cooled to, the corn stalk powder that sterilizes is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.0 are well mixed, according to sterilized plant powder of straw dry weight meter 3.01.05.04.0% inoculum concentrations, well mixed
Fermentation culture is added in sterilizing corn stalk powder, and 8h is shaken in shaking table, and eccentric cleaning obtains absorption ester-producing yeast and plant
The carrier of lactobacillus；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill containing by weight for pH6.0
4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
Microbial inoculum A and microbial inoculum B are compared 2.5 according to weight：1 is well mixed, and obtains described embedding microbial inoculum, and it contains 2.5 ×
108Individual/g bacillus subtilises, 6.2 × 108Individual/g ester-producing yeasts and 2.0 × 109Individual/g Lactobacillus plantarums.Described embedding bacterium
Agent is preserved at 4 DEG C of temperature.
Embodiment 2：Production ensiling of the present invention embedding microbial inoculum
The implementation steps of the embodiment are as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
Candida sp. ester-producing yeasts bacterial strains are inoculated in shaking at 120 DEG C of temperature sterilizing 8min according to 1% inoculum concentration
In bottle culture medium, 28h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is obtained containing 2.2 × 108The shaking flask of individual/g ester-producing yeasts
Nutrient solution；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 8min that sterilized at 120 DEG C of temperature according to 3% inoculum concentration
In, 36h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 6.8 × 108The fermented and cultured of individual/g ester-producing yeasts
Liquid；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Lactobacillus plantarum A15 lactobacillus plantarum strains are inoculated in temperature according to 1% inoculum concentration
Sterilized at 120 DEG C in 8min Shake flask medium, 72h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, obtain containing 5.0 ×
108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 8min that sterilized at 120 DEG C of temperature according to 3% inoculum concentration
In, 60h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 2.4 × 109The fermented and cultured of individual/g Lactobacillus plantarums
Liquid；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Bacillus subtilis strain B49 bacillus subtilis strains are inoculated in temperature according to 1% inoculum concentration
At 120 DEG C of degree in sterilizing 8min Shake flask medium, 24h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, obtain containing 7.5 ×
107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 8min that sterilized at 120 DEG C of temperature according to 3% inoculum concentration
In, 48h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 2.5 × 108The fermentation training of individual/g bacillus subtilises
Nutrient solution；
IV, straw pretreatment
Broomcorn straw is crushed, sieved, 100 height of eye fine strain of millet powder of straw are collected, saturated vapor processing 1.2h is reused, then
Room temperature is cooled to, the broomcorn straw powder that sterilizes is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.5 are well mixed, according to the sterilizing inoculum concentration of broomcorn straw dried bean noodles restatement 1.0%, well mixed fermented and cultured
Liquid is added in sterilized plant powder of straw, shakes 8h in shaking table, eccentric cleaning, obtains adsorbing ester-producing yeast and Lactobacillus plantarum
Carrier；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill containing by weight for pH6.4
4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
Microbial inoculum A and microbial inoculum B are compared 3.5 according to weight：1 is well mixed, and obtains described embedding microbial inoculum, and it contains 3.5 ×
108Individual/g bacillus subtilises, 6.0 × 108Individual/g ester-producing yeasts and 2.2 × 109Individual/g Lactobacillus plantarums.Described embedding bacterium
Agent is preserved at 4 DEG C of temperature.
Embodiment 3：Production ensiling of the present invention embedding microbial inoculum
The implementation steps of the embodiment are as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
Brettanomyces sp. ester-producing yeasts bacterial strains are inoculated according to 1% inoculum concentration and sterilized at 114 DEG C of temperature
In 9min Shake flask medium, 36h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is obtained containing 2.5 × 108Individual/g production ester ferment
Female shake flask culture；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 9min that sterilized at 114 DEG C of temperature according to 3% inoculum concentration
In, 48h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 6.4 × 108The fermented and cultured of individual/g ester-producing yeasts
Liquid；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Lactobacillus plantarum Y15118 lactobacillus plantarum strains are inoculated in temperature according to 1% inoculum concentration
At 114 DEG C of degree in sterilizing 9min Shake flask medium, 56h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, obtain containing 5.5 ×
108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 9min that sterilized at 114 DEG C of temperature according to 3% inoculum concentration
In, 72h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 2.3 × 109The fermented and cultured of individual/g Lactobacillus plantarums
Liquid；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Bacillus subtilis strain WL1021 bacillus subtilis strains are inoculated according to 1% inoculum concentration
At 114 DEG C of temperature in sterilizing 9min Shake flask medium, 36h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is contained
7.8×107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 9min that sterilized at 114 DEG C of temperature according to 3% inoculum concentration
In, 40h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 3.2 × 108The fermentation training of individual/g bacillus subtilises
Nutrient solution；
IV, straw pretreatment
Wheat stalk is crushed, sieved, 120 mesh wheat stalk powders are collected, saturated vapor processing is reused
1.01.21.51.4h, room temperature is then cooled to, the wheat stalk powder that sterilizes is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.4 are well mixed, according to the sterilizing inoculum concentration of wheat stalk powder dry weight meter 5.0%, well mixed fermented and cultured
Liquid is added in sterilized plant powder of straw, shakes 8h in shaking table, eccentric cleaning, obtains adsorbing ester-producing yeast and Lactobacillus plantarum
Carrier；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill containing by weight for pH 6.6
4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
Microbial inoculum A and microbial inoculum B are compared 2.8 according to weight：1 is well mixed, and obtains described embedding microbial inoculum, and it contains 3.2 ×
108Individual/g bacillus subtilises, 7.0 × 108Individual/g ester-producing yeasts and 2.4 × 109Individual/g Lactobacillus plantarums.Described embedding bacterium
Agent is preserved at 4 DEG C of temperature.
Embodiment 4：Production ensiling of the present invention embedding microbial inoculum
The implementation steps of the embodiment are as follows：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
Brettanomyces sp. ester-producing yeasts bacterial strains are inoculated according to 1% inoculum concentration and sterilized at 118 DEG C of temperature
In 10min Shake flask medium, 32h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is obtained containing 2.4 × 108Individual/g production esters
The shake flask culture of yeast；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 10min that sterilized at 118 DEG C of temperature according to 3% inoculum concentration
In, 40h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 6.6 × 108The fermented and cultured of individual/g ester-producing yeasts
Liquid；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L
Glucose, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate with
0.58g/L manganese sulfates；
Lactobacillus plantarum Y15118 lactobacillus plantarum strains are inoculated in temperature according to 1% inoculum concentration
At 118 DEG C of degree in sterilizing 10min Shake flask medium, 64h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is obtained containing 5.4
×108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose,
0.008g/L sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L sulfuric acid
Manganese；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 10min that sterilized at 118 DEG C of temperature according to 3% inoculum concentration
In, 68h is cultivated under the conditions of 30 DEG C of temperature in fermentation tank, is obtained containing 2.2 × 109The fermented and cultured of individual/g Lactobacillus plantarums
Liquid；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Bacillus subtilis strain WL1021 bacillus subtilis strains are inoculated according to 1% inoculum concentration
At 118 DEG C of temperature in sterilizing 10min Shake flask medium, 32h is cultivated under the conditions of 30 DEG C of temperature in shaking flask, is contained
8.0×107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen
Dipotassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Above-mentioned shake flask culture is inoculated in the fermentation medium for the 10min that sterilized at 118 DEG C of temperature according to 3% inoculum concentration
In, 44h is cultivated under the conditions of 32 DEG C of temperature in fermentation tank, is obtained containing 3.5 × 108The fermentation training of individual/g bacillus subtilises
Nutrient solution；
IV, straw pretreatment
Maize straw, broomcorn straw, wheat stalk or rice straw are crushed, 80100120100 mesh corns are collected in screening
Stalk, broomcorn straw, wheat stalk or rice straw powder, reuse saturated vapor processing 1.01.21.51.4h, then cool down
To room temperature, sterilized plant powder of straw is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that ester-producing yeast fermentation culture that step I is obtained is obtained with step II according to
Volume ratio 1：1.2 are well mixed, according to the inoculum concentration of sterilized plant powder of straw dry weight meter 4.0%, well mixed fermented and cultured
Liquid is added in sterilized plant powder of straw, shakes 8h in shaking table, eccentric cleaning, obtains adsorbing ester-producing yeast and Lactobacillus plantarum
Carrier；
6% polyvinyl alcohol, 0.3% sodium alginate it will gone in terms of mixed solution weight with 0.3% sodium carboxymethylcellulose
A night is soaked in ionized water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtains a kind of mixing
Solution；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill containing by weight for pH 6.8
4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight
Count 6% trehalose to be well mixed with 15% skimmed milk, then be placed in processing in freeze drying equipment and obtain described microbial inoculum B；
Microbial inoculum A and microbial inoculum B are compared 3.2 according to weight：1 is well mixed, and obtains described embedding microbial inoculum, and it contains 2.8 ×
108Individual/g bacillus subtilises, 6.8 × 108Individual/g ester-producing yeasts and 2.6 × 109Individual/g Lactobacillus plantarums.Described embedding bacterium
Agent is preserved at 4 DEG C of temperature.
Test example 1：Prepare ensilage
The implementation steps of the test example are as follows：
I, fermentation vat and raw material
Long 2m × wide 1.5m × depth 1.8m rectangle fermentation vats are selected to be tested.Fermentation vat surrounding laying plastics are thin
Film, selects fresh dark green maize straw, crushes, standby.
II, actication of culture
Prepared toward embodiment 1 in embedding microbial inoculum and add little sugar, then be placed in stirring and dissolving in the water of 25 DEG C~35 DEG C of temperature
1h, it is standby.
III, inoculation charging
By crushing maize stalk through saturated vapor handle 1h, be cooled to 25 DEG C it is standby, according to crushing maize stalk dry weight meter
The bacterium solution that 1% access step II is obtained, is then mixed, obtained raw material is inserted in fermentation vat, and filling thickness reaches 20~30cm
When tread, then continue to add raw material, carry out in the same fashion, until raw material is added, then plastic covering film, then fill about
10cm soil, is treaded, and about 7t raw materials are filled per pond.
It is prepared by IV, ensilage
Allow raw material to be fermented under field conditions (factors) in silage silo 15d, obtain described ensilage.
According to GB/T 6434-2006 standard method of analyses, crude fibre analysis is carried out to obtained ensilage；According to GB/
T 6432-1994 standard method of analyses, carry out crude protein analysis, these analysis results are listed in the table below 1 to obtained ensilage
In.Under conditions of 4 DEG C, effective holding time detection is carried out to obtained ensilage.
Test example 2：Prepare ensilage
The implementation steps of the test example are identical with test example 1, and simply the test example 2 uses embodiment 2
Embedding microbial inoculum, the fermentation time of preparation 20 days.The ensilage that the test example is obtained carries out crude fibre analysis, crude protein point
Analysis detects that these analysis results are also found in table 1 below with effective holding time.
Test example 3：Prepare ensilage
The implementation steps of the test example are identical with test example 1, and simply the test example 3 uses embodiment 3
The embedding microbial inoculum of preparation.The ensilage that the embodiment is obtained carries out crude fibre analysis, crude protein analysis and effective holding time
Detection, these analysis results are also found in table 1 below.
Test example 4：Prepare ensilage
The implementation steps of the test example are identical with test example 1, and simply the test example 3 uses embodiment 4
The embedding microbial inoculum of preparation.The ensilage that the test example is obtained carries out crude fibre analysis, crude protein analysis and effectively preservation
Time detecting, these analysis results are also found in table 1 below.
Test example 5：Prepare ensilage
The implementation steps of the test example are identical with test example 1, and simply the test example 5 does not add this hair
Bright embedding microbial inoculum.When the ensilage that the test example is obtained carries out crude fibre analysis, crude protein analysis with effectively preserving
Between detect, these analysis results are also found in table 1 below.
Test example 6：Prepare ensilage
The implementation steps of the test example are identical with test example 1, and simply the test example 6 adds presently commercially available
" agriculture Citroen zx " board stalk fermentation microbial inoculum.The ensilage that the test example is obtained carries out crude fibre analysis, crude protein analysis
Detected with effective holding time, these analysis results are also found in table 1 below.
Table 1：The present invention prepares the analysis result of ensilage
The result listed by table 1 can be clearly seen, by the ensilage obtained by the present invention no matter the battalion in feed in itself
On supporting, or on effective shelf-life, more than commercially available microbial inoculum, so, the present invention improves the utilization rate of maize straw, improves
The nutritive value of ensilage, has a good application prospect.
Claims (8)
1. a kind of ensiling embedding microbial inoculum, it is characterised in that the embedding microbial inoculum is by the microbial inoculum containing ester-producing yeast and Lactobacillus plantarum
A and bacillus subtilis microbial agent B is according to weight than 2.5~3.5：1 composition, the embedding microbial inoculum contains 2.5~3.5 × 108Individual/g
Bacillus subtilis, 6.0~7.0 × 108Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
2. embedding microbial inoculum according to claim 1, it is characterised in that the embedding microbial inoculum is by containing ester-producing yeast and plant breast
The microbial inoculum A and bacillus subtilis microbial agent B of bacillus are according to weight than 2.8~3.2:1 composition, the embedding microbial inoculum contains 2.8~3.2
×108Individual/g bacillus subtilises, 6.4~6.8 × 108Individual/g ester-producing yeasts and 2.2~2.4 × 109Individual/g Lactobacillus plantarums.
3. embedding microbial inoculum according to claim 1, it is characterised in that described bacillus subtilis is selected from Bacillus
Subtilis strainB48, CICC No.10089, Bacillus subtilis strain B49, CICCNo.10090 or
Bacillus subtilis strainWL1021, CICC No.10063；Described ester-producing yeast is selected from Pichia
Anomala, CCTCC No.CICIMY0297, Candida sp., CICC No.1257 or Brettanomyces sp., CCTCC
The bacterial strains of No.AY 93142；Described Lactobacillus plantarum is selected from Lactobacillus plantarum strain F1, CCTCC
No.CCREM SDMCC050029, Lactobacillus plantarum A15, CICC No.21814 or Lactobacillus
PlantarumY15118, CICCNo.20768 bacterial strain.
4. the production method of ensiling embedding microbial inoculum according to claim 1, it is characterised in that the step of the production method such as
Under：
It is prepared by I, ester-producing yeast nutrient solution：
Prepare the Shake flask medium with following compositions：20g/L peptones, 20g/L glucose and 10g/L yeast extracts；
By the ester-producing yeast bacterial strain selected from Pichia anomala, Candida sp. or Brettanomyces sp. according to 1%
Inoculum concentration is inoculated at 110~120 DEG C of temperature in 8~12min of sterilizing Shake flask medium, in 30 DEG C of bars of temperature in shaking flask
24~36h is cultivated under part, is obtained containing 2.0~2.5 × 108The shake flask culture of individual/g ester-producing yeasts；
Prepare the fermentation medium with following compositions：10g/L peptones, 10g/L glucose and 5g/L sodium chloride；
The fermentation that above-mentioned shake flask culture is inoculated in 8~12min of sterilizing at 110~120 DEG C of temperature is trained according to 3% inoculum concentration
Support in base, cultivate 36~48h under the conditions of 30~32 DEG C of temperature in fermentation tank, obtain containing 6.4~6.8 × 108Individual/g productions
The fermentation culture of ester yeast；
It is prepared by II, Lactobacillus plantarum nutrient solution：
Prepare the Shake flask medium with following compositions：10g/L beef extracts, 10g/L peptones, 10g/L yeast extracts, 5g/L grapes
Sugar, 20g/L sodium acetates, 5g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 2g/L Tween 80s, 1g/L magnesium sulfate and 0.58g/L sulphur
Sour manganese；
Will selected from Lactobacillus plantarum strainF1, Lactobacillus plantarumA15 or
Lactobacillus plantarumY15118 lactobacillus plantarum strain according to 1% inoculum concentration be inoculated in temperature 110~
Sterilized at 120 DEG C in 8~12min Shake flask medium, cultivate 48~72h under the conditions of 30 DEG C of temperature in shaking flask, contained
Have 5.0~5.5 × 108The shake flask culture of Lactobacillus plantarum；
Prepare the fermentation medium with following compositions：20g/L peptones, 10g/L yeast extracts, 5g/L glucose, 0.008g/L
Sodium chloride, 0.008g/L dipotassium hydrogen phosphates, 2g/L dibasic ammonium citrates, 0.008g/L magnesium sulfate and 0.008g/L manganese sulfates；
The fermentation that above-mentioned shake flask culture is inoculated in 8~12min of sterilizing at 110~120 DEG C of temperature is trained according to 3% inoculum concentration
Support in base, cultivate 60~72h under the conditions of 30~32 DEG C of temperature in fermentation tank, obtain containing 2.2~2.4 × 109Individual/g plants
The fermentation culture of thing lactobacillus；
It is prepared by III, bacillus subtilis bacteria culture fluid：
Prepare the Shake flask medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen two
Potassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
Bacillus subtilis strainB48, Bacillus subtilis strain B49 or Bacillus will be selected from
Subtilis strain WL1021 bacillus subtilis strain is inoculated at 110~120 DEG C of temperature according to 1% inoculum concentration
Sterilize in 8~12min Shake flask medium, cultivate 24~36h under the conditions of 30 DEG C of temperature in shaking flask, obtain containing 7.5~
8.0×107The shake flask culture of individual/g bacillus subtilises；
Prepare the fermentation medium with following compositions：5g/L sodium carboxymethylcelluloses, 2g/L ammonium sulfate, 1g/L phosphoric acid hydrogen two
Potassium, 0.5g/L sodium chloride and 0.5g/L magnesium sulfate；
The fermentation that above-mentioned shake flask culture is inoculated in 8~12min of sterilizing at 110~120 DEG C of temperature is trained according to 3% inoculum concentration
Support in base, cultivate 36~48h under the conditions of 30~32 DEG C of temperature in fermentation tank, obtain containing 2.5~3.5 × 108Individual/g is withered
The fermentation culture of careless bacillus；
IV, straw pretreatment
Straw is crushed, sieved, plant straw powder is collected, saturated vapor is reused and handles 1.0~1.5h, then cool down
To room temperature, sterilized plant powder of straw is obtained；
It is prepared by V, microbial inoculum
Microbial inoculum A preparation：
The Lactobacillus plantarum fermentation culture that the ester-producing yeast fermentation culture that step I is obtained is obtained with step II is according to volume
Than 1：1.0~1.5 are well mixed, according to the inoculum concentration of sterilized plant powder of straw dry weight meter 1.0~5.0%, well mixed hair
Ferment nutrient solution is added in sterilized plant powder of straw, and 8h is shaken in shaking table, and eccentric cleaning obtains absorption ester-producing yeast and plant breast
The carrier of bacillus；
Will in terms of mixed solution weight 6% polyvinyl alcohol, 0.3% sodium alginate and 0.3% sodium carboxymethylcellulose in deionization
A night is soaked in water, sterilize 20min under conditions of 121 DEG C of temperature, is then cooled to 40 DEG C of temperature, obtain a kind of mixing molten
Liquid；
The carrier and the mixed solution are compared 10 according to weight：1 mixing, then instill containing by weight for pH6.0~6.8
4%CaCl2Saturation boric acid solution in, stand 24h, obtain microbial inoculum A；
Microbial inoculum B preparation：
Fermentation of bacillus subtilis nutrient solution that step III is obtained, according to fermentation of bacillus subtilis nutrient solution weight meter 6%
Trehalose is well mixed with 15% skimmed milk, then is placed in freeze drying equipment processing and is obtained described microbial inoculum B；
By microbial inoculum A and microbial inoculum B according to weight than 2.5~3.5：1 is well mixed, and obtains described embedding microbial inoculum.
5. embedding microbial inoculum according to claim 4, it is characterised in that described straw is maize straw, sorghum stalks
Stalk, wheat stalk or rice straw.
6. embedding microbial inoculum according to claim 4, it is characterised in that the granularity of the plant straw powder is more than 80 mesh.
7. embedding microbial inoculum according to claim 4, it is characterised in that described embedding microbial inoculum is preserved at 4 DEG C of temperature.
8. embedding microbial inoculum according to claim 4, it is characterised in that described embedding microbial inoculum contains 2.5~3.5 × 108Individual/
G bacillus subtilises, 6.0~7.0 × 108Individual/g ester-producing yeasts and 2.0~2.6 × 109Individual/g Lactobacillus plantarums.
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