CN102352315A - Microbial inoculum, preparation method thereof, and application of microbial inoculum to preparation of silage - Google Patents
Microbial inoculum, preparation method thereof, and application of microbial inoculum to preparation of silage Download PDFInfo
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Abstract
The invention relates to a microbial inoculum, a preparation method thereof, and application of the microbial inoculum to preparation of silage. The microbial inoculum provided by the invention consists of trichoderma viride, aspergillus niger and yeast mutagenic bacteria. The invention also provides the preparation method for the microbial inoculum and application of the microbial inoculum to preparation of the silage. The silage prepared by fermentation of the microbial inoculum has short fermentation time, wherein on the tenth day of fermentation, cellulose content and protein content are changed obviously; on the twenty-fifth day of fermentation, the cellulose content and the protein content are not changed basically, the protein content reaches over 9.3 percent and the cellulose content is degraded by 10 to 19 percent; the prepared silage has strong fragrance, and the utilization of raw materials of the silage is increased and the nutritional value is improved; a pilot plant test is performed, so large-scale production can be directly performed; and when the silage is used for feeding animals, the silage is easy to digest after domestic animals eat the silage, so the ingestion time of the animals is shortened, the ingestion quantity is increased, and the weight-increasing speed of the domestic animals is high.
Description
Technical field
The present invention relates to a kind of microbiobacterial agent, the invention still further relates to the preparation method and its application in the preparation silage of this microbiobacterial agent.
Background technology
Silage is a domestic animal in winter, spring VITAMIN, mineral substance, proteinic important source, and it can improve breeding potential and the lactation power of domestic animal, and promotes growing of cub, is the indispensable basal feed of feeding stock animals.Very long in the winter time northern China, cold weather, seasonal strong, vegetative period is short, and green grass or young crops is cut fodder production and is restricted, and silage provides the optimal mode of succulent fodder in winter, also is that the ideal when famine year, feed was short is stocked feed.
Be the silage of ensiling raw material with the agricultural crop straw at present; The production of its mass-producing mainly is to add yeast to carry out the fermentation of ensiling raw material; This method fermentation time is long; Usually need 2-3 months; The silage protein content that obtains is low; Its quality percentage composition only is about 4%, Mierocrystalline cellulose does not have degraded basically, fragrance is lighter, and the edible back of domestic animal digestibility is low, and the ensiling utilization ratio of raw materials is low.
Summary of the invention
The purpose of this invention is to provide a kind of microbiobacterial agent; With the agricultural crop straw is the ensiling raw material; Utilize the silage fermatation time of this microbiobacterial agent fermentative preparation short, protein content is high, the cellulose degradation amplitude is big, aromatic flavour; The edible back of domestic animal readily digested, and the ensiling utilization ratio of raw materials is high; For this reason, the present invention also provides a kind of preparation method and its application in the preparation silage of this microbiobacterial agent.
The present invention provides a kind of microbiobacterial agent to achieve these goals, and said microbiobacterial agent is made up of viride, aspergillus niger, yeast mutagenesis bacterium.
Preferably, described yeast mutagenesis bacterium be yeast via
12C
+ 6Radioinduction obtains.
Preferably, said viride: aspergillus niger: the volume parts ratio of yeast mutagenesis bacterium is 1-3:1-2:1-3.
More preferably, said viride: aspergillus niger: the volume parts ratio of yeast mutagenesis bacterium is 1:1:1,2:1:2 or 3:2:3.
Again preferably, said microbiobacterial agent also comprises nutritive salt, and the component of said nutritive salt is counted by weight: 10 parts in urea, 3 parts in sal epsom, 20 parts of potassium primary phosphates.
Again preferably, said microbiobacterial agent also comprises milk-acid bacteria.The adding milk-acid bacteria can increase the mouthfeel of silage, i.e. palatability.
The present invention also provides the preparation method of microbiobacterial agent, and said preparing method's step is:
A, yeast is carried out mutagenesis obtain yeast mutagenesis bacterium;
B, yeast mutagenesis bacterium, viride, aspergillus niger are carried out slant culture with the PDA substratum;
C, the viride through slant culture, the aspergillus niger that obtains among the step b carried out enlarged culturing respectively in mould seed flask culture base;
D, the yeast mutagenesis bacterium through slant culture that obtains among the step b is carried out enlarged culturing in the sub-flask culture base of yeast mutagenesis bacterial classification;
E, the yeast mutagenesis bacterium after the enlarged culturing is mixed with viride, aspergillus niger, blending ratio according to volume parts than viride: aspergillus niger: yeast mutagenesis bacterium is 1-3:1-2:1-3, makes microbiobacterial agent.
The present invention also provides the application of this microbiobacterial agent in the preparation silage.
Preferably, the fermentation time of said silage is 25 days.
More preferably, the ensiling raw material of said silage during the fermentation the pH value be 6.0.
Under this pH value, can also can suppress the growth of other assorted bacterium simultaneously for mould provides good growing environment,
The silage fermatation time of using microbiobacterial agent fermentative preparation of the present invention is short; Fermenting, Mierocrystalline cellulose and protein content have just had obvious variation 10 days the time; Fermenting, content of cellulose and protein content basically no longer change after 25 days; Protein content has reached more than 9.3%; The content of cellulose 10%-19% that degraded; The silage aromatic flavour of processing has improved the ensiling utilization ratio of raw materials, has improved the nutritive value of silage; Carry out pilot plant test, can directly carry out scale operation; With this silage nutrition purposes, especially for feeding animals, the edible back of domestic animal readily digested has shortened animal and has searched for food the time, has improved food consumption, and the domestic animal speed of weight increment is fast.
Embodiment
Experimental technique among the following embodiment and used reagent and equipment if no special instructions, are ordinary method, conventional reagent and equipment.
Embodiment 1
1.1 bacterial classification
Viride bacterial strain: available from Research for Industrial Microbial Germ preservation center, Gansu Province, deposit number: GSICC 62010;
Yeast strain: available from Research for Industrial Microbial Germ preservation center, Gansu Province, deposit number: GSICC is revised as 51951;
Aspergillus niger strain: available from Research for Industrial Microbial Germ preservation center, Gansu Province, deposit number: GSICC 60108;
Milk-acid bacteria: available from Chinese agriculture microbial strains preservation administrative center, deposit number: ACCC 11026.
1.2 instrument used in this experiment is following
Greenhouse solid fermentation incubator: available from: the Shanghai letter is electronics technology company limited model all: DPN-9272B;
The robust fibre determinator: available from: Jinan Sheng Tai Instr Ltd. model: ST-06;
The crude protein determinator: available from: Jinan Sheng Tai Instr Ltd. model: ST-04C;
High-pressure steam sterilizing pan.
1.3 substratum.
(1) preparation of slant medium:
Milk-acid bacteria slant culture based formulas: extractum carnis: 0.15g; Peptone: 0.5g; NaCl:0.25g; Agar: 1g; Water: 50ml.
Potato sugar nutrient agar (PDA) prescription (being used for viride, yeast mutagenesis bacterium, aspergillus niger): potato (be cut into small pieces after the peeling, boil half an hour): 20g; Water: 100ml; Agar: 2g; Sugar (use sucrose when cultivating mould, use glucose during culturing yeast): 2g.
(2) preparation of seed flask culture base:
Milk-acid bacteria seed flask culture based formulas:
Tryptones: 2g; Yeast extract paste: 0.5g; Peptone: 0.5g; Glucose: 0.5g; Tween 80: 0.005g; Tomato juice 25g, water 1000 ml.
Mould seed flask culture based formulas: (being used for aspergillus niger and viride)
Peptone: 3g; Ammonium sulfate: 5g; Sal epsom 0.4g, sucrose 30g, potassium primary phosphate 2g, water 1000ml.
The sub-flask culture based formulas of yeast mutagenesis bacterial classification:
Glucose: 10g; Peptone: 10g; Yeast extract powder: 5g; Potassium primary phosphate: 3g; Water: 1000ml.
1.4 the preparation method of bacterial classification
1.4.1 the cultivation of viride bacterial classification
1.4.1.1 the switching of the inclined-plane of viride bacterial classification spreads cultivation foster:
Get one of the viride bacterial classification of purchase, be connected on the PDA inclined-plane for preparing with the line mode with inoculating needle picking spore in the Bechtop, put into 35 ℃ ± 0.5 ℃ hot-house culture case and cultivate 3d.
1.4.1.2 trichoderma viride kind seed flask culture:
Take out among the 1.4.1.1 the viride bacterial classification of growth and maturity and observe, transfer with the inoculation shovel good spore that takes a certain amount of length and carry out enlarged culturing, with 220r/min rotation bottle swingging machine, at 35 ℃ ± 0.5 ℃ temperature bottom fermentation 3d in seed culture medium.
1.4.2 the cultivation of Aspergillus niger strain
1.4.2.1 the switching of the inclined-plane of aspergillus niger strain spreads cultivation foster
Get one of the aspergillus niger strain of purchase, be connected on the PDA inclined-plane for preparing with the line mode with inoculating needle picking spore in the Bechtop, put into 28 ℃ ± 0.5 ℃ hot-house culture case and cultivate 3d.
1.4.2.2 aspergillus niger strain seed flask culture
Take out among the 1.4.2.1 the aspergillus niger strain of growth and maturity and observe, transfer with the inoculation shovel good spore that takes a certain amount of length and carry out enlarged culturing, with 220r/min rotation bottle swingging machine, at 28 ℃ ± 0.5 ℃ temperature bottom fermentation 3d in seed culture medium.
1.4.3 the cultivation of lactic bacterium strains
1.4.3.1 the switching of the inclined-plane of lactic acid bacteria culturers spreads cultivation foster:
Get one of the lactic acid bacteria culturers of purchase, be connected on the milk-acid bacteria inclined-plane for preparing with the line mode with inoculating needle picking spore in the Bechtop, put into 37 ℃ ± 0.5 ℃ hot-house culture case and cultivate 20h.
1.4.3.2 lactic acid bacteria culturers seed flask culture
Take out among the 1.4.3.2 the lactic acid bacteria culturers of growth and maturity and observe, transfer with the inoculation shovel good bacterium that takes a certain amount of length and carry out enlarged culturing, put into 37 ℃ ± 0.5 ℃ hot-house culture case and cultivate 20h in seed culture medium.
1.4.4 the heavy ion beam irradiation mutagenesis of barms:
1.4.4.1 the activation of bacterial classification:
Get known tiring and one of barms that production performance is good, be placed on (28 ℃ ± 0.5 ℃) activation 30min between the constant temperature buffering.
1.4.4.2
12C
+ 6Ionic fluid penetrates mutagenesis:
To the yeast different irradiation doses of design multipoint in batches, provide with heavy ion accelerator
12C
+ 6Ionic fluid carries out irradiation mutagenesis to two samples of barms spore suspension, and irradiation dose is respectively 0 Gy, 20 Gy; 40 Gy; 60 Gy, 80 Gy, 100 Gy; 120Gy; The spore suspension gradient dilution that to handle is then accurately drawn the spore suspension 0.2ml/ plate of a sample, is poured on the separating plate substratum that makes; Separate single bacterium colony, to pursue best mutagenesis dosage.Simultaneously, separate contrast with method with the common separation plate culture medium, so that carry out the colonial morphology contrast with another sample spore suspension of bacterial classification that sets out.Treat the separating plate substratum 28 ℃ ± 0.5 ℃ cultivation 6-7d growth and maturity, 2-4 ℃ of preservation is subsequent use in refrigerator.The research heavy ion beam
12C
+ 6To the dosage of the induction mutation of bacterium that sets out select with mutagenesis after filter out the ratio of superior strain.
1.4.4.3 the screening of the high bacterial strain of tiring:
(1) selecting of single bacterium colony:
On the flat board of separator well, carefully choosing the plentiful single bacterium colony of spore on the Bechtop; And colonial morphology has single bacterium colony of definitive variation; After having write down detailed form of bacterium colony and size, transfer on the PDA slant medium; In thermostat container, cultivate; Culture temperature is that (28 ℃ ± 0.6 ℃), incubation time are 6-7d, cultivates the back and refrigerates subsequent use.
(2) shake a bottle primary dcreening operation
Taking out the bacterial classification of switching on the PDA inclined-plane of growth and maturity observes; The good spore of getting a certain amount of length is transferred and in the sub-flask culture base of yeast mutagenesis bacterial classification, is carried out enlarged culturing; With 200r/min rotation bottle swingging machine, carry out the fermentation of seed bottle under constant temperature, the constant humidity condition.
Get step cultured seed liquid and transfer with certain inoculum size and carry out second order fermentation in yeast mutagenesis bacteria fermentation culture medium (60ml/500ml triangular flask), the prescription of yeast mutagenesis bacteria fermentation culture medium is identical with the sub-flask culture based formulas of yeast mutagenesis bacterial classification.Speed setting is 200r/min, cultivates 48h under constant temperature, the constant humidity condition.
1.4.4.4 mensuration result:
The fermentation of sweet sorghum slag is measured
10g sweet sorghum slag and 20g water are put into Erlenmeyer flask,, each irradiation dose is inoculated in the ratio of 1% (100g inoculates 1ml).Inoculation is placed on 27 ℃ of cultivation 7d in the thermostat container.Every day observation experiment phenomenon and carry out record, after 7 days with the content of the crude protein in the kjeldahl apparatus test sample article.The result sees the following form 1.
Table 1
Can find out by table 1; When irradiation dose is 60 Gy; Rising has appearred in crude protein content; When irradiation dose is 80 Gy; Crude protein content can rise to 8.9%, has improved 16% than control group, and ascending amount is comparatively remarkable; Therefore when the preparation complex micro organism fungicide, can select yeast mutagenic strain for use through seed selection behind the 80Gy irradiation.
Embodiment 2
1, the experiment of microbiobacterial agent fermenting sweet sorghum slag
1.1, the laboratory experiment of microbiobacterial agent fermenting sweet sorghum slag
1.1.1, the proportioning of microbiobacterial agent is to the influence experiment of fermenting sweet sorghum slag
Every group of bacterial classification mixed by following volume ratio:
Group one: viride and yeast mutagenesis bacterium 1:1;
Group two: aspergillus niger and yeast mutagenesis bacterium 1:1;
Group three: viride: aspergillus niger: yeast mutagenesis bacterium is 1:1:1;
Group four: blank (do not add bacterial classification and only add nutritive salt);
Every component is established three parallel controls.
50g sweet sorghum slag and 100g water are put into Erlenmeyer flask, add nutritive salt, the prescription of nutritive salt is following: urea: 0.1g, and sal epsom: 0.03g, potassium primary phosphate: 0.2g forms sweet sorghum slag fermentation raw material.In high-pressure steam sterilizing pan, sterilized 30 minutes for 121 ℃, respectively four groups of bacterial classifications are inoculated in following ratio: inoculate 5ml in per 100 gram sweet sorghum slags, vaccination ways ferments in the Erlenmeyer flask for spraying, and 28 ° of C of leavening temperature, fermentation time are 10 days.Observe color, sense organ and the texture of product, do preliminary blending ratio and judge the further design of the chamber experimental program that experimentizes.The result is following:
Group one: color is a deep green, and colony growth is vigorous, can smell light fragrance;
Group two: color is an aterrimus, and colony growth is vigorous, can smell light fragrance;
Group three: color is blackish green, and colony growth is very vigorous, can smell dense fragrance;
Blank control group: yellow stalk, British plain spirits, the aseptic length of being born.
Can find out according to experiment: group three color, colony growth situation and smell are best, so, prepare microbiobacterial agent with the mixture of viride, aspergillus niger and yeast mutagenesis bacterium.
1.1.2, nutritive salt are tested the influence of microbiobacterial agent fermenting sweet sorghum slag; Wherein the prescription of nutritive salt is following: urea: 0.1g, sal epsom, 0.03g, potassium primary phosphate, 0.2g.
Experiment is established four groups:
First group is added nutritive salt, viride: aspergillus niger: yeast mutagenesis bacterium is pressed the volume ratio of 1:1:1,2:1:2,3:2:3 respectively and mixes;
Second group is not added nutritive salt, viride: aspergillus niger: yeast mutagenesis bacterium is pressed the volume ratio of 1:1:1,2:1:2,3:2:3 respectively and mixes;
Two groups of components of contrast: the 3rd group is the interpolation nutritive salt group of blank, the 4th group be blank do not add the nutritive salt group;
Every component is established three parallel controls.
100g water is put into Erlenmeyer flask; First group with each different proportionings of the 3rd group in add 5g nutritive salt respectively; The ratio of quality and the number of copies of each component is in the nutritive salt: 10 parts in urea, 3 parts in sal epsom, 20 parts of potassium primary phosphates; Four groups of solution that mix are poured into respectively in the 50g sweet sorghum slag; In pressure kettle, sterilized 30 minutes for 121 ℃; Respectively four groups of bacterial classifications are inoculated in following ratio: inoculate 5ml in per 100 gram sweet sorghum slags; Vaccination ways is for spraying; Ferment in the Erlenmeyer flask; 28 ° of C of leavening temperature, fermentation time is 10 days.Sample quartering uses robust fibre determinator and crude protein determinator to survey its Mierocrystalline cellulose and proteic content.Measure the result and see table 2.
Table 2 nutritive salt is to the influence of microbiobacterial agent fermentation capacity
﹡ P＜0.001 is with remarkable to photograph group ratio heteropole; ° P＜0.001, to compare difference extremely remarkable with adding the nutritive salt group.
Can know from table 2: first group and second group is compared, and first group is higher than second group on protein content, and the cellulose degradation ability is better than second group, therefore, adds the growth that nutritive salt can promote bacterial classification, strengthens the effect of bacterial classification.Second group and the 4th group is compared, and content of cellulose significantly reduces, and protein content significantly improves, and therefore, does not add nutritive salt and also can reach comparatively ideal effect.
From table 2, it can also be seen that: the 1:1:1 group is comparatively outstanding in proteic raising; Nutritious salt group is respectively 9.03% and 7.39% with no nutritive salt group; And the 3:2:3 group is comparatively outstanding on the ability of cellulose degradation, and nutritious salt group is respectively 9.07% and 10.08% with no nutritive salt group.
1.1.3, sterilization is to the influence of microbiobacterial agent fermenting sweet sorghum slag experiment
Every group of bacterial classification mixed by following volume ratio:
First group is the sterilization group: viride: aspergillus niger: yeast mutagenesis bacterium is pressed the volume ratio of 1:1:1,2:1:2,3:2:3 respectively and mixes;
Second group is unsterilised group, viride: aspergillus niger: yeast mutagenesis bacterium is pressed the volume ratio of 1:1:1,2:1:2,3:2:3 respectively and mixes;
Two groups of contrast components: the 3rd group is the sterilization group of blank, and the 4th group is unsterilised group of blank;
Every component is established three parallel controls.
100g water is put into Erlenmeyer flask; Add 5g nutritive salt respectively in the different proportionings of each of every group; The weight proportion of nutritive salt is: 10 parts in urea; 3 parts in sal epsom; 20 parts of potassium primary phosphates; Four groups of solution that mix are poured into respectively in the 50g sweet sorghum slag; All using the HCl adjusting pH of 0.25ml/mol with four groups is 6.0; With first group and the 3rd group of 121 ℃ of sterilization 30min in high-pressure steam sterilizing pan; Respectively four groups of bacterial classifications are inoculated in following ratio: inoculate 5ml in per 100 gram sweet sorghum slags; Vaccination ways is for spraying; Ferment in the Erlenmeyer flask; 28 ° of C of temperature; Fermentation time is 10d; Sample quartering uses robust fibre determinator and crude protein determinator to measure every group Mierocrystalline cellulose and proteic content.Experimental result is seen table 3.
Whether table 3 sterilizes to the influence of microbiobacterial agent fermentation capacity
﹡P＜0.001, extremely remarkable with control group difference, ° P＜0.001, extremely remarkable with sterilization group difference,
&P＜0.01, with sterilization group significant difference, ^P＜0.05 is not remarkable with sterilization group difference.
Can find out that from table 3 sterilization group and unsterilised group difference on the ability of cellulose degradation is extremely significant; But difference is little in the raising of protein content: the sterilization group of 1:1:1 and unsterilised group differ 0.64%; The sterilization group of 2:1:2 and unsterilised group differ 0.26%; The sterilization group of 3:2:3 and unsterilised group of difference have only 0.06%; So; Whether sterilize to not significantly influence of proteic raising, unsterilisedly also can reach ferment effect preferably.
It can also be seen that from table 3 1:1:1 group is comparatively outstanding in proteic raising, sterilization group and unsterilised group are respectively 8.03% and 7.39%; And 3:2:3 group is comparatively outstanding on the ability of cellulose degradation, and sterilization group and unsterilised group are respectively 9.09% and 12.31%.
1.2, the pilot experiment of microbiobacterial agent fermenting sweet sorghum slag
According to laboratory experiment result and data, carry out small-scale and simulate suitability for industrialized production, under the temperature that bacterium and natural condition are arranged, viride, aspergillus niger, yeast mutagenesis bacterium are respectively by the following volume ratio 1:1:1 that experimentizes, 2:1:2,3:2:3.Experiment is divided into two aspects and carries out: test under the spontaneous fermentation condition nutritive salt on the one hand to the influence of microbiobacterial agent fermenting sweet sorghum slag; On the other hand microbiobacterial agent of the present invention and like product " little storage king " stalk decomposition agent are compared; Temperature is a room temperature; And, use robust fibre determinator and crude protein determinator to survey its Mierocrystalline cellulose and proteic content through fermentation time being carried out quartering, interim sampling acquisition experimental data.Concrete experimental procedure and experimental result are following:
Nutritive salt is to the influence of microbiobacterial agent fermenting sweet sorghum slag under
1.2.1, the spontaneous fermentation condition.Wherein the composition of nutritive salt is by weight: 10 parts in urea, 3 parts in sal epsom, 20 parts of potassium primary phosphates.
Experiment is established four groups:
First group is added nutritive salt, the volume ratio 1:1:1 of viride, aspergillus niger, yeast mutagenesis bacterium, 2:1:2,3:2:3;
Second group is not added nutritive salt, and the volume ratio of viride, aspergillus niger, yeast mutagenesis bacterium is respectively 1:1:1,2:1:2,3:2:3;
Two groups of components of contrast: the 3rd group is the interpolation nutritive salt group of blank, the 4th group be blank do not add the nutritive salt group.
Every group different proportionings are got 3kg water; First group with each different proportionings of the 3rd group in add 150g nutritive salt; The ratio of quality and the number of copies of each component is in the nutritive salt: 10 parts in urea, 3 parts in sal epsom, 20 parts of potassium primary phosphates; Using the HCl of 0.25ml/mol to regulate pH each group is 6.0, and the different proportionings interpolations of each of every group are counted 2% milk-acid bacteria with sweet sorghum slag amount.The above-mentioned solution that mixes is poured into respectively in the sweet sorghum slag that 3kg just squeezed the juice; Respectively the bacterial classification of each different proportionings of four groups is inoculated in following ratio: per 100 gram sweet sorghum slags inoculation 5ml; Vaccination ways is for spraying; Fermentation at room temperature; By stages, quartering is taken a sample use robust fibre determinator and crude protein determinator its Mierocrystalline cellulose of survey and proteic content.Experimental result is seen table 4 and table 5.
Table 4 nutritive salt and different fermentations time are to the influence of Mierocrystalline cellulose (%) ability in the microbiobacterial agent degraded sweet sorghum slag
﹡P＜0.001, extremely remarkable with control group difference, ° P＜0.01 and interpolation nutritive medium group significant difference, ^P＜0.05, nutritive medium group difference is not remarkable with adding.
As shown in table 4: relatively reaching variance analysis and can draw through the cellulose degradation amount: difference is extremely remarkable between two groups of second group and blank; Second group is not so good as first group on cellulose degradation, can know that adding nutritive salt can better degrade the Mierocrystalline cellulose in the sweet sorghum; But in order to satisfy the production needs, reduce production costs during actual production, it also is feasible selecting not add nutritive salt.
In interim sampling of fermentation fate and mensuration process, find: before 25 days, the cellulose degradation of each assembly ratio is very rapidly, and after 25 days, cellulose degradation is slow, and it is fixed to keep steady to 70 Tian Bao at 55 days, basically no longer changes.Therefore, when actual production, preferably fermenting prepared silage over 25 days.
Three kinds of different proportionings to bacterial classification compare discovery: the 3:2:3 group is comparatively outstanding on the ability of cellulose degradation; Nutritious salt group is respectively 10.11% and 10.51% with the content of cellulose of no nutritive salt group; Be lower than 1:1:1 group and 2:1:2 group; Kept consistent with the result that the laboratory draws, promptly the cellulose degradation ability of 3:2:3 group is best.
Table 5 different fermentations time and nutritive salt improve the influence of albumen (%) ability to microbiobacterial agent
﹡P＜0.001, extremely remarkable with control group difference, ° P＜0.01, with interpolation nutritive medium group significant difference,
^P＜0.05, nutritive medium group difference is not remarkable with adding.
Can know from table 5: the data analysis and the variance analysis that improve through protein content can draw: difference is extremely remarkable between two groups of second group and blank; Second group is not so good as first group on protein content, can know that adding nutritive salt can make the better of sweet sorghum slag fermentation; But in order to satisfy the production needs, reduce production costs during actual production, it also is feasible selecting not add nutritive salt.
In interim sampling of fermentation fate and mensuration process, find: before 25 days, it is very rapidly that the albumen of each assembly ratio improves, and after 25 days, albumen improves slowly, and it is fixed to keep steady to 70 Tian Bao at 55 days, basically no longer changes.Therefore, when actual production, preferably fermenting prepared silage over 25 days.
Three kinds of different proportionings to bacterial classification compare discovery: the 1:1:1 group is comparatively outstanding on the ability that albumen improves; Add the nutritive salt group and do not add nutritive salt histone content and be respectively 10.60% and 9.94%; Be higher than 2:1:2 group and 3:2:3 group; The result who draws with the laboratory is consistent, and promptly 1:1:1 group effect in proteic raising is best.
1.2.2,
With " little storage king " stalk decomposition agent (available from the neat starfish agricultural cience and farming techniques application service station of oolong wood) comparative experiments
Experiment is established three groups: and " little storage king " stalk decomposition agent group, blank group, microbiobacterial agent group (volume ratio of viride, aspergillus niger, Candida utilis mutagenesis bacterium is respectively 1:1:1,2:1:2,3:2:3).Each experimental group is established three parallel controls.
" little storage king " stalk decomposition agent group experimentizes according to the operation instruction of " little storage king " stalk decomposition agent; With " little storage king " stalk fermentation dried bacterium one bag alive; Pour in the 200ml clear water; Stir; Bacterial classification activates and to be blended into the 200ml mass percent behind the 1-2h is 1% salt solution; It is inserted 3kg sweet sorghum slag with the mode of spraying, ferment under the natural condition, by stages, quartering takes a sample.
The blank group: 3kg sweet sorghum slag and 3kg water are mixed, and using the HCl adjusting pH of 0.25ml/mol is 6.0, it is inserted sweet sorghum slag 3Kg, the state of nature fermentation.
The microbiobacterial agent group: the volume ratio of viride, aspergillus niger, Candida utilis mutagenesis bacterium is respectively 1:1:1,2:1:2,3:2:3.Get 3kg water for every group, using the HCl adjusting pH of 0.25ml/mol is 6.0, and every assembly is counted 2% milk-acid bacteria than adding with sweet sorghum slag amount.The above-mentioned solution that mixes is poured into respectively in the sweet sorghum slag that 3kg just squeezed the juice, respectively three kinds of different proportioning bacterial classifications are inoculated in following ratio: per 100 gram sweet sorghum slags inoculation 5ml, vaccination ways is for spraying the natural condition bottom fermentation.By stages, quartering is taken a sample use robust fibre determinator and crude protein determinator its Mierocrystalline cellulose of survey and proteic content.Experimental result is seen table 6.
Table 6 different fermentations asynchronism(-nization) microbiobacterial agent fermenting sweet sorghum slag effect table
﹡P＜0.001, extremely remarkable with control group difference, ° P＜0.001, extremely remarkable with biological decomposition agent group difference
^P＜0.05, not remarkable with contrast difference.
Can find out that from table 6 " little storage king " stalk decomposition agent and blank group compare, the content of cellulose significant difference, albumen improves not remarkable." little storage king " stalk decomposition agent and microbiobacterial agent are relatively; Difference is extremely remarkable: use " little storage king " stalk decomposition agent fermenting sweet sorghum slag cellulosic quality percentage composition 16.34% after 70 days; Proteic quality percentage composition 4.73%; And the 1:1:1 of microbiobacterial agent; 2:1:2; The Mierocrystalline cellulose quality percentage composition of three different proportionings of 3:2:3 is respectively 10.77%, 10.60%.10.51%, proteic quality percentage composition is respectively 9.94%, 9.43%, 9.33%, so the effect of " little storage king " stalk decomposition agent does not have the effective of microbiobacterial agent of the present invention.This shows; The present invention is through adding milk-acid bacteria for three kinds of flora mixed fermentation sweet sorghum slags and cooperation; Aspect degraded cellulose, the raising albumen effect is preferably being arranged; Thereby and because added milk-acid bacteria and increased palatability; Simulate large-scale sweet sorghum slag fermentation through pilot plant test; Verified that experiment is used for scale operation, realizes the feasibility of industrialization.
1.3, conclusion
Experimental data according to table 4-table 6 can be found out; In changing in time; Protein content is improving; Behind the different time; The protein content significant difference; Improve preferably with the protein content of 1:1:1 group, the effect of measuring with the laboratory is consistent, and more excellent group of can be used as in the microbial bacteria agent prescription of this group produced.Cellulose degradation also has significant difference over time, and is wherein best with the cellulose degradation effect of 3:2:3 group, and also the result who measures with the laboratory is consistent.After one month, silage fragrance is dense, and fragrant and sweet smell is arranged after the oven dry in the fermentation of low temperature and room temperature, and color is deep yellow, and protein content increases substantially, and the roughage improvement is concentrated feed, effectively raises the feeding value of sweet sorghum slag.Can find out obviously that in experimental data during the fermentation initial stage arrived fermentation 55d, content of cellulose was in the decline state always, the rising that protein content is then steady is being fermented behind 55d, and Mierocrystalline cellulose and protein content no longer include obvious variation basically.Add in every group of different proportioning behind the milk-acid bacteria of quality percentage composition 2% through the effect of hybrid bacterial strain, silage shows: color yellow, dense fragrance is arranged, and sensory effects is good, is fit to feeding.
The 1:1:1 combination has good effect in the lifting of protein content; Effect is preferably also arranged in cellulosic degraded, but make up the effective of degraded cellulose,, can play good albumen and promote effect so it is not that very high agricultural crop straw ferments that this combination can be used for content of cellulose not as good as 3:2:3.The 2:1:2 combination, degraded cellulose compares with the proteic ability of lifting and other two proportionings, and is comparatively placed in the middle.The cellulose degradation ability of 3:2:3 combination is best, and it is better that albumen promotes effect.So this group can be used for the fermentation of the high agricultural crop straw of content of cellulose, can play good cellulose degradation effect, improve proteic content simultaneously.Through three kinds of different proportionings, can ferment to different agricultural crop straws, to reach required silage.
The present invention has verified breadboard effect through laboratory experiment and pilot plant test are compared, and can effectively instruct real attenuation production.With the pH regulator to 6.0 of microbiobacterial agent, the flora growing environment is adjusted, reach good ferment effect.After in microbiobacterial agent, adding milk-acid bacteria, effectively raise the palatability of silage, strongly fragrant; And do not influence the fermentation of other bacterial classifications.
2, the experiment of microbiobacterial agent fermented maize stalk
Experiment is divided into four groups: viride: aspergillus niger: the volume ratio of yeast mutagenesis bacterium is respectively 1:1:1,2:1:2,3:2:3, one group of blank.Establish three parallel controls respectively for every group.Get 3kg water for every group, with the HCl adjusting pH to 6.0 of 0.25ml/mol, every group of interpolation counted 2% milk-acid bacteria with the maize straw quality.The above-mentioned solution that mixes is poured into respectively in the 3kg maize straw; Respectively four groups of bacterial classifications are inoculated in following ratio: per 100 gram maize straw inoculation 5ml; Vaccination ways is for spraying; The natural condition bottom fermentation; Adopt by stages, sample quartering experimentizes;, use robust fibre determinator and crude protein determinator to survey its Mierocrystalline cellulose and proteic content.Experimental result is seen table 7.
Table 7 different fermentations time microbiobacterial agent fermented maize stalk effect table
﹡P＜0.001 is extremely remarkable with control group difference, and the content of cellulose significant difference is compared with the 3:2:3 group in ° P＜0.01, and relatively protein content difference is not remarkable with the 1:1:1 group in ^P＜0.05.
The content of cellulose and the protein content of each assembly ratio are compared with control group simultaneously, can find out with microbiobacterial agent fermented maize stalk to obtain effect preferably.
Because of the 3:2:3 group is one group best on the cellulose degradation effect, so, compare as content of cellulose control group and other groups with the 3:2:3 group; From table 9, can find out; Fine on the 3:2:3 group cellulose degradation ability, be 10.22%, content of cellulose is lower than 1:1:1 group and 2:1:2 group.Because of the 1:1:1 group is to improve one group best on the effect at protein content; So; Compare as protein content control group and other groups with the 1:1:1 group; From table 9, can find out; The protein content difference that itself and 2:1:2 group, 3:2:3 organize is not remarkable; Take all factors into consideration above each group situation, can select the biological decomposition agent fermentation proportioning of 3:2:3 group for use as maize straw.
Can know by table 7: but ruminant domestic animal passes through the Digestive tract decomposition of cellulose of self, the silage that can select for use the fermentation of 1:1:1 group to obtain is fed, and but the cellulose degradation effect of this group is not preferably albumen raising amount maximum; And the good decomposition of cellulose of the Digestive tract of other domestic animals self, the silage that can select for use the fermentation of 3:2:3 group to obtain is fed, and the cellulose degradation effect of this group is best.
3, the experiment of microbiobacterial agent fermentation broomcorn straw
Broomcorn straw is as the main feed resource of China, and its content of cellulose is very high, and its quality percentage composition is approximately 31%, and the ruminant domestic animals such as cattle and sheep that are used for feeding at present mix with high protein feed and feed usually.In order broomcorn straw to be can be used in feed all domestic animals, reduce its content of cellulose, increase percent protein, it is changed into the high-quality concentrated feed can improve its palatability and nutritive value is highly significant.
Experiment is divided into four groups: viride, aspergillus niger, yeast mutagenesis bacterium are respectively 1:1:1 by volume, 2:1:2, and 3:2:3, one group of blank is established three parallel controls respectively for every group.Get 3kg water for every group, using the HCl adjusting pH of 0.25ml/mol is 6.0, and every assembly is counted 2% milk-acid bacteria than adding with the broomcorn straw quality.The above-mentioned solution that mixes is poured into respectively in the 3kg broomcorn straw; Respectively four groups are inoculated in following ratio: per 100 gram broomcorn straws inoculation 5ml; Vaccination ways is for spraying; The natural condition bottom fermentation; Adopt by stages four, the point-score sampling experimentizes;, use robust fibre determinator and crude protein determinator to survey its Mierocrystalline cellulose and proteic content.Experimental result is seen table 8.
Mierocrystalline cellulose and protein content (%) table behind the different fermentations time microbiobacterial agent fermentation broomcorn straw in table 8 pilot plant test
﹡P＜0.001, extremely remarkable with control group difference, the content of cellulose significant difference is compared with the 3:2:3 group in ° P＜0.01, and relatively protein content difference is not remarkable with the 1:1:1 group in ^P＜0.05.
From table 8, can obtain: each assembly is compared with control group than content of cellulose under identical fermentation time and protein content simultaneously, can find out with microbiobacterial agent fermentation broomcorn straw to obtain effect preferably.
From table 8, can also obtain: 2:1:2 organizes on protein content, and is not remarkable with the good 1:1:1 group of albumen raising effect difference; In cellulosic degraded, well 3:2:3 group difference is not remarkable with the cellulose degradation ability.So; Decomposition experiment for broomcorn straw; We are when producing the albumen high-quality feed; Can select viride mutagenesis bacterium, aspergillus niger mutagenesis bacterium, Candida utilis mutagenesis bacterium is that the proportioning of 2:1:2 is fermented, and not only can effectively reduce content of cellulose and also can well increase percent protein.
Embodiment 3
The experiment that the maize straw of microbiobacterial agent fermentation is fed sheep
1. experiment material and method
1.1 laboratory animal and grouping
From the flock of sheep of Yongdeng County, Lanzhou one Yang Chang, select 20 of local sheep of close, healthy 6 anosis monthly ages of body weight, be divided into experimental group and control group (seeing table 9) at random, two groups of sheep body weight difference not remarkable (P>0.05).The feed maize straw of microbiobacterial agent of the present invention fermentation of experimental group, the common dried maize straw of control group fed.
The experiment grouping information slip that the maize straw of table 9 microbiobacterial agent fermentation is fed sheep:
1.2 feed is handled
Mixed concentrate is grouped into by the one-tenth of following quality percentage composition: corn 45%, and cottonseed cake 41%, wheat bran 12%, bone meal 2% is formed, and additive has salt (adding by standard), yeast powder (pressing 0. 5% of daily ration total amount adds) and trace element and multivitamin.
1.3 feeding and management
Experimental group and control group are raised in identical adjacent two hurdles house of conditionally complete.Give before the experiment that two groups of sheep are weighed, expelling parasite, feeding coal is controlled at 120% of random foraging amount, throws something and feeds for three times every day, calculates and respectively organizes the average daily feed intake of sheep; 0. 5 kilograms of two groups of mixed concentrates that sheep every day, supplementary feeding was identical simultaneously/only, whole day is supplied with clean competent drinking-water.Be 50 days experimental period.
2. testing index
2.1 food consumption
Food consumption=feeding coal-surplus material amount
2.2 grazing rate
Grazing rate=feeding coal-surplus material amount/feeding coal * 100%
2.3 gain in weight
Gain in weight=whole opisthosoma weighs-initial body weight
3. experimental result
3.1 food consumption and grazing rate
Search for food average every day 0.51 kilogram of common dried maize straw/only of search for food average every day 0. 59 kilograms of the maize straws of microbiobacterial agent fermentation/only of experimental period, the sheep of experimental group, the sheep of control group.The maize straw of observing the microbiobacterial agent fermentation is to the search for food influence of time of sheep, and average every meal time of searching for food of experimental group is 45.2 minutes, the grazing rate 87.36% of the maize straw of microbiobacterial agent fermentation; Control group is 55.4 minutes, and the grazing rate of common dried maize straw is 71.69%.Because of the maize straw quality of microbiobacterial agent fermentation soft; Good palatability; Thereby the average every meal of the experimental group sheep maize straw time ratio control group that microbiobacterial agent fermented of searching for food is short, but average every sheep daily ingestion amount is increased to 0. 59 kilograms by 0.51 kilogram, has increased by 15.7%; Grazing rate is increased to 87.36% by 71.69%, has improved 21.86%, and food consumption and grazing rate all are enhanced.
3.2 the effect of gain in weight is seen table 10.
The experiment gaining effect contrast table that the maize straw of table 10 microbiobacterial agent fermentation is fed sheep:
Can find out through table 10: the experimental group gaining effect is comparatively obvious, and total augment weight manys 25Kg than control group, and average daily gain has improved 26.46% than control group, and gaining effect is obvious.
4. Economic and Efficiency Analysis
Experimental group sheep is Duoed 25 kilograms than the absolute weightening finish of control group, and per kilogram is increased income 700 yuan in 28 yuan/kilogram of existing market price live-weights.The deduction maize straw increases by 630 yuan of net return, remarkable in economical benefits through 70 yuan of microbiobacterial agent fermentation costs.
5. discuss
Maize straw quality through the microbiobacterial agent fermentation is soft, palatability strengthens; Compare with common dried maize straw, can improve the nutritive value of common dried maize straw, thereby shorten searching for food the time of sheep, improve food consumption, digestibility and feed conversion rate, gaining effect is remarkable.The maize straw fabrication techniques cost of microbiobacterial agent fermentation is low, easy and simple to handle, and the technology easy master is a practical technique that comprehensive benefit is arranged, be worth promoting.
Embodiment 4
The experiment of the sweet sorghum slag feeding cow of microbiobacterial agent fermentation
1. experiment material and method
1.1 laboratory animal
The German He Sitan lactating cows in 3 years old age that the experiment milk cow selects for use one diary farm, Yongdeng County, Lanzhou to raise; Select the age; Body weight; Parity; The lactation moon; Milk fat content; 12 of milk production and physiological property basically identical or close lactating cowss; Be divided into two groups at random; Every group 6; One group of the sweet sorghum slag that the microbiobacterial agent of feeding fermented is experimental group; Feed another group of undressed sweet sorghum slag for control group; Carry out the 7d preliminary trial period after the grouping earlier; In this phase, number; Weigh; Expelling parasite; Survey milk fat content; Adjust two groups of body weight and milk production; Handle two groups of differences in back not remarkable (p>0.05) through biometric data, change trial period then over to.
1.2 feeding and management
All take drylot feeding to fasten raising for two groups; Feed by the same poultry raiser; Feed every day; Milk; Drinking-water each 3 times; Two groups of mixed concentrates are identical with green and rough feeds; Mixed concentrate every 9kg that limits the quantity of every day; Two groups of green and rough feedss are principle cannot not eat up surplusly grass then; Measure quantity-unlimiting; Supply with the outer motion of competent drinking-water and house every day; Other control measures all are consistent for two groups, do milk production in the experimental period; Relevant datas such as the feed consumption rate record and the situation of searching for food are observed, after waiting to test end; Calculate the milk production of two groups of lactating cowss respectively, milk is expected economic benefit situation analysis when.Be 30 days experimental period.
2. testing index
2.1 food consumption
Food consumption=feeding coal-surplus material amount
2.2 average daily milk production
Average daily milk production=total milk production/experiment fate
2.3 milk material ratio
Milk material ratio=total milk yield/total feeding coal
3. experimental result and analysis
3.1 food consumption
The feed experimental group of the sweet sorghum slag that fermented with microbiobacterial agent; Because the sweet sorghum slag that fermented through microbiobacterial agent is comparatively loose, and good palatability, lactating cows shows milk cow happiness food in experimental period, food consumption increases; Ight soil is normal, and no abnormal situation takes place.
3.2 average daily milk production is expected than seeing the following form 11 with milk
Average daily milk production and milk material compared information slip during the sweet sorghum slag that table 11 microbiobacterial agent fermented was fed and tested
Can find out the experimental group of the sweet sorghum slag that the microorganism fodder bacteria fermentation is crossed by table 11, every average daily milk production of lactating cows is 26.39 kg in the experimental period, and control group is 24.12 kg, and experimental group is enhanced than control group; The experimental group milk material of the sweet sorghum slag that the microorganism fodder bacteria fermentation is crossed is than being 2.36:1; And control group is 2.07:1, promptly every consumption lkg mixed concentrate, the comparable control group fecund of experimental group 0.29kg milk; The milk material is than having improved 14%, and the milk material compare group of experimental group also is enhanced.
4. Economic and Efficiency Analysis
Comparison shows than situation analysis result with the milk material by the average daily milk production of two groups of lactating cowss of table 11: the feed test group of the sweet sorghum slag that microbiobacterial agent fermented of lactating cows; The milk production of experimental group milk cow is 158.34kg; And the milk production of control group is 144.72kg, and every day, milk production increased 13.62kg; Experimental group consumes feed 67.08kg altogether, and control group consumes feed 69.9kg altogether.By 3.20 yuan/kilogram of existing fresh milk market value; Experimental group day is taken in 506.69 yuan; Control group day is taken in 463.1 yuan; Deduction feed cost and handling cost separately; Experimental group day gets a profit 372.53 yuan; Control group day gets a profit 323.3 yuan, 49.23 yuan of day increase net return, and a day level of profitability has improved 15.23%.
Confirm through test; The feed experimental group of the sweet sorghum slag that microbiobacterial agent fermented of milk cow; The palatability and the nutritive ingredient of sweet sorghum slag have not only been improved; And herbage intake, efficiency of feed utilization, lactation amount and the economic benefit etc. of milk cow all had good result; Therefore, the method for handling the sweet sorghum slag with microbiobacterial agent is feasible, and this method is safe and reliable; Simple, be convenient to operation and promote.
Claims (10)
1. microbiobacterial agent, it is characterized in that: said microbiobacterial agent is made up of viride, aspergillus niger, yeast mutagenesis bacterium.
2. microbiobacterial agent according to claim 1 is characterized in that: described yeast mutagenesis bacterium be yeast via
12C
+ 6Radioinduction obtains.
3. microbiobacterial agent according to claim 2 is characterized in that: said viride: aspergillus niger: the volume parts of yeast mutagenesis bacterium compares 1-3:1-2:1-3.
4. microbiobacterial agent according to claim 3 is characterized in that: said viride: aspergillus niger: the volume parts of yeast mutagenesis bacterium is than 1:1:1,2:1:2 or 3:2:3.
5. according to the described microbiobacterial agent of the arbitrary claim of claim 1-4, it is characterized in that: said microbiobacterial agent also comprises nutritive salt, and the component of said nutritive salt is counted by weight: 10 parts in urea, 3 parts in sal epsom, 20 parts of potassium primary phosphates.
6. according to the described microbiobacterial agent of the arbitrary claim of claim 1-4, it is characterized in that: said microbiobacterial agent also comprises milk-acid bacteria.
7. the preparation method of the described microbiobacterial agent of claim 1, it is characterized in that: said preparing method's step is:
A, yeast is carried out mutagenesis obtain yeast mutagenesis bacterium;
B, yeast mutagenesis bacterium, viride, aspergillus niger are carried out slant culture with the PDA substratum;
C, the viride through slant culture, the aspergillus niger that obtains among the step b carried out enlarged culturing respectively in mould seed flask culture base;
D, the yeast mutagenesis bacterium through slant culture that obtains among the step b is carried out enlarged culturing in the sub-flask culture base of yeast mutagenesis bacterial classification;
E, the yeast mutagenesis bacterium after the enlarged culturing is mixed with viride, aspergillus niger, blending ratio according to volume parts than viride: aspergillus niger: yeast mutagenesis bacterium is 1-3:1-2:1-3, makes microbiobacterial agent.
8. the application of the described microbiobacterial agent of the arbitrary claim of claim 1-6 in the preparation silage.
9. application according to claim 8 is characterized in that: the fermentation time of said silage is 25 days.
10. application according to claim 8 is characterized in that: the ensiling raw material of said silage pH value during the fermentation is 6.0.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102613392A (en) * | 2012-04-01 | 2012-08-01 | 贵州省生物研究所 | Preparation method of straw feed |
| CN104017739A (en) * | 2014-05-07 | 2014-09-03 | 中国科学院近代物理研究所 | Composite microbial inoculant and preparation method thereof, and application of composite microbial inoculant in producing high-protein potato residue feed |
| CN104232493A (en) * | 2014-05-07 | 2014-12-24 | 中国科学院近代物理研究所 | Compound microorganism bacterium agent and silage prepared from bacterium agent |
| CN107232396A (en) * | 2017-06-15 | 2017-10-10 | 李广新 | A kind of utilize compounds the method that microbial inoculum prepares alfalfa silage |
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2011
- 2011-09-14 CN CN2011102714271A patent/CN102352315A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102613392A (en) * | 2012-04-01 | 2012-08-01 | 贵州省生物研究所 | Preparation method of straw feed |
| CN104017739A (en) * | 2014-05-07 | 2014-09-03 | 中国科学院近代物理研究所 | Composite microbial inoculant and preparation method thereof, and application of composite microbial inoculant in producing high-protein potato residue feed |
| CN104232493A (en) * | 2014-05-07 | 2014-12-24 | 中国科学院近代物理研究所 | Compound microorganism bacterium agent and silage prepared from bacterium agent |
| CN104017739B (en) * | 2014-05-07 | 2017-12-22 | 中国科学院近代物理研究所 | Complex micro organism fungicide, its preparation method and its application in high protein potato dreg fodder is produced |
| CN107232396A (en) * | 2017-06-15 | 2017-10-10 | 李广新 | A kind of utilize compounds the method that microbial inoculum prepares alfalfa silage |
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