CN1831111A - Lactobacillus brucei preparation, its preparation method and its use - Google Patents
Lactobacillus brucei preparation, its preparation method and its use Download PDFInfo
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- 241000186660 Lactobacillus Species 0.000 title claims abstract description 49
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 48
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- 240000008042 Zea mays Species 0.000 claims abstract description 24
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 24
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- 235000005822 corn Nutrition 0.000 claims abstract description 22
- 230000001332 colony forming effect Effects 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims description 64
- 241000186679 Lactobacillus buchneri Species 0.000 claims description 55
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种布氏乳酸杆菌制剂，其每克含1×109～1×1011个布氏乳酸杆菌菌落形成单位。本发明还公开了所述布氏乳酸杆菌制剂的制备方法及其用途，所述布氏乳酸杆菌制剂可改善青贮饲料开窖后稳定性，并能有效提高玉米青贮饲料的品质，化解有害微生物大量增殖带来的饲料安全风险。The invention discloses a preparation of Lactobacillus brucei, which contains 1×10 9 -1×10 11 colony-forming units of Lactobacillus brunii per gram. The invention also discloses the preparation method and application of the Lactobacillus bransonii preparation, the Lactobacillus brauchneri preparation can improve the stability of the silage after opening the cellar, can effectively improve the quality of the corn silage, and dissolve a large amount of harmful microorganisms Feed safety risks from multiplication.
Description
Technical field
The invention belongs to field of fodder, relate to a kind of function bacterial classification-Bu Shi lactobacillus (L.buchneri) preparation, its preparation method, and adopt the Bu Shi lactobacillus preparation to improve the purposes that the whole corn silage feed is opened the cellar for storing things rear stability.
Background technology
Characteristics such as silage is of a great variety, has the source extensively, and cost is low, and it is convenient to gather, and good palatability and nutrient are more comprehensive are the main feeds of relevant a vegetarian animal, such as the desirable feed that is ox, sheep etc.General gramineous crop, leguminous crop, piece root, stem tuber and aquatic feed and leaf etc. all can be used to ensiling.At present with the most use is the corn (band grain ear) that special plantation is used for ensiling, secondly is corn stalk, sorghum stalks and the sweet potato vine of plucking behind the fringe.
The principle of ensiling is under suitable condition, by anaerobically fermenting, produces sour environment, suppresses the harmful microbe procreation, thereby reaches the purpose of preserving feed.Silage is the microorganisms symbiosis system of a complexity, mainly comprises milk-acid bacteria, yeast, clostridium and genus bacillus, acetic acid bacteria, mould, wherein the value volume and range of product with milk-acid bacteria is maximum, also is the microorganism that plays a major role in silage fermatation.Milk-acid bacteria produce acid and acidproof ability all very strong, they can utilize the carbohydrate in the raw material to carry out lactic fermentation, form lactic acid and small amount of acetic acid, can make that lactic acid content reaches 1.5%～2% in the silage, make the pH reduction.Owing to added natural fast fermentation agent in the silage, so at earlier fermentation, can reduce environment pH value fast, the rapid decline of pH value, not only suppress the breeding of butyric bacteria, also can stop other bad bacterium,, thereby help improving the fermentation quality of silage as the growth of filamentous fungus, yeast and mould.
When making, whole strain stalk (comprising bar, stem, leaf) all can be used for ensiling, and the green of stalk and blade can be preserved.So, can preserve the nutrient more than 85% in the crop material.And owing to silage soft and succulency, the sour-sweet fragrance of smell, good palatability very is suitable for the beef cattle of feeding, and beef cattle also is delithted with and searches for food; And the secretion of energy promoting digestion gland, for the digestibility that improves feed good action is arranged.
Maize silage is the requisite common feedstuffs of cattle-raising, a head of cattle is searched for food about 20 kilograms average every day, therefore silage can overcome winter-spring season owing to lack the disadvantageous effect that the fine green and rough feeds brings cattle-raising because can annual equilibrium supply.Maize silage is the main feed of China's cattle-raising, and whole corn silage worldwide has been widely adopted, and along with the fast development of milk industry, generally is accepted and adopts in China's cattle-raising in recent years.
Whole-plant corn is a kind of main raw material for preparing silage, and its preparation is simple and easy, and is historical remote.Yet, the maize silage of traditional method preparation is owing to adopt lactic fermentation, its acetate after ensiling is finished, the propionic acid isoconcentration is on the low side, open the back, cellar for storing things because anaerobic environment is destroyed, the consumption aerobic microorganism grows again in a large number, causes that its stability sharply descends, and principal character is that yeast, mould grow in a large number, glucose oxidase speeds up, CO
2Generation increases fast, not only causes the maize silage quality to descend, and brings very big risk owing to mould grows the mycotoxin that is produced in a large number to feed safety.As untimely utilization or utilize speed slow excessively and since its stability sharply decline can cause a large amount of losses.
Summary of the invention
The purpose of this invention is to provide a kind of silage that improves opens the cellar for storing things rear stability and improves whole corn silage quality Bu Shi lactobacillus (L.buchneri) preparation.
Another object of the present invention provides a kind of preparation method of Bu Shi lactobacillus preparation.
A further object of the present invention provides a kind of Bu Shi lactobacillus preparation and opens the purposes of cellar for storing things aspect the rear stability improving silage, can effectively improve the quality of maize silage, dissolves the feed safety risks that a large amount of propagation of harmful microorganism are brought.
In order to realize the object of the invention, technical scheme of the present invention is:
A kind of Bu Shi lactobacillus preparation, every gram contains 1 * 10
9～1 * 10
11Individual Bu Shi lactobacillus (L.buchneri) colony-forming unit (cfu/g).
Be preferably, every gram contains 1 * 10
9～1 * 10
10Individual L.buchneri colony-forming unit (cfu/g).
Described Bu Shi lactobacillus (L.buchneri) is available from Chinese microorganism strain preservation center (CICC), and CICC is numbered 6007.
The biological characteristics of Bu Shi lactobacillus:
(1) bacillus, short usually and straight, 0.7～1.0 * 2.0～4.0 microns, two terminal circle, single or one-tenth short chain.
(2) bacterium colony is general coarse or be osculant, and is flat, may be close to transparent.
(3) heterofermentation, it is sour gentle to produce from autoclaved dextrose culture-medium, is aerobic katabolism to the glucose of sterilizing respectively.Tunning is DL-lactic acid (total carbon of 50% glucose), and other more important products are acetate, ethanol and CO
2
(4) temperature of generation maximum acid is 23～30 ℃, 30 ℃ of growth optimum temperutures.
Use and minimizing viable bacteria freeze-drying loss for the ease of transportation, prolong agent of lactic acid bacteria product shelf-lives, agent of lactic acid bacteria can be made lyophilized powder.When described Bu Shi lactobacillus preparation is freeze-dried formulation, also contain lyophilized vaccine in total formulation weight amount 30～40%.
Described lyophilized vaccine is glycerine and skimmed milk, the volume ratio of glycerine and skimmed milk 1: 1～1: 2.
Described Bu Shi lactobacillus preparation adopts that Bu Shi lactobacillus (L.buchneri) is activated, inoculation culture, centrifugal, lyophilize form.
For realizing another object of the present invention, the preparation method of Bu Shi lactobacillus preparation of the present invention comprises the steps:
1) with Lactobacillus buchneri kind 28～32 ℃ of following activation culture 15～20h in activation medium; Adopt plate streak in 28～32 ℃ of following purifying substratum, to carry out purifying then and cultivated 1～2 day, get purifying bacterium liquid;
2) purifying bacterium liquid is inoculated in the fermentor tank with 3～7% inoculum size, the pH value is controlled at 5.0～7.0 again, and 28～32 ℃ of following mixed culture 30～40h get fermented liquid in fermention medium;
3) described then fermented liquid is under 20～26 ℃, and rotating speed carried out centrifugal treating 30～40 minutes for 3000～4000 rev/mins, gets zymophyte mud;
4) in zymophyte mud, behind the described lyophilized vaccine of adding, carry out lyophilize and get the lactic acid bacteria freeze drying powder at last.
Wherein, described activation medium is the MRS liquid nutrient medium, and its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3～6.5.
Described purifying substratum is the MRS nutrient agar that adds lime carbonate, and MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3～6.5.In above-mentioned 1000ml liquid nutrient medium, add 15～20g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 15～20mim are standby.
Described fermention medium also can adopt the MRS liquid nutrient medium, and its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3～6.5.
The plate streak that purifying of the present invention is adopted when cultivating is this area method commonly used.
Fermentor tank of the present invention is this area fermentor tank commonly used, can be specifications such as 5L, 10L or 50L.
The control of pH value can be adopted the lactic acid regulation and control in the described fermenting process.
Described lyophilized vaccine is glycerine and skimmed milk, and the concentration of glycerine solution is 8～15%, and skimmed milk solution is 8～15%, mixes in 1: 1～1: 2 with volumetric ratio.
Described bacterium mud (cell mashed prod) and protective material volume ratio 1: 3～1: 4.
Used freeze drying process adopts this area refrigerating apparatus and processing step commonly used to get final product.
Such as, at-30～-40 ℃ of pre-freeze 5～7h, it evenly is frozen in around the bottle on the inwall, use ALPHA-2 vacuum-freeze-dry machine then, when equitemperature is reduced to-40～-50 ℃, open the vacuum pump suction to 0.1mbar, the drying bottle of packing into, dry 12～24h.The dry powder goods are deposited in 4 ℃ of refrigerators.
Specifically, the preparation method of described Bu Shi lactobacillus preparation is:
1) activation culture of Lactobacillus buchneri
Under aseptic situation, Lactobacillus buchneri bacterium powder is inoculated in the MRS liquid nutrient medium, cultivate 15～20h at 28～32 ℃ of following shaking tables.
The MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3～6.5.
2) the separation pure culture (plate streak) of Lactobacillus buchneri
Pour the MRS nutrient agar that adds lime carbonate into plate, treat culture medium solidifying after, dip in the disinfection inoculation ring and to get the bacterium liquid that has shaken and rule, each bacterial classification is made three plates, after drawing well, seal plate, be upside down in 28～32 ℃ interior the cultivation 1～2 day of biochemical incubator with sealing compound.Choose individual uniform single bacterium colony purifying and cultivate, test standby.Plate can be preserved at 4 ℃ of refrigerators.
The MRS nutrient agar that adds lime carbonate, MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3～6.5.In above-mentioned 1000ml liquid nutrient medium, add 15～20g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 15～20mim are standby.
3) expansion of purifying bacterium liquid is numerous, cultivates
With the inoculum size of purifying bacterium liquid with Lactobacillus buchneri 3～7%, be inoculated in the Erlenmeyer flask of 1000ml, the volume of substratum is 475ml in the Erlenmeyer flask, then inoculating the bacterium liquid measure accordingly is 25ml; 28～32 ℃ of following pH values is 5.0～7.0, cultivates 30～40h;
4) centrifugal
Under 20～26 ℃, centrifugal 30～40 minutes of 3000～4000 rev/mins of rotating speeds are abandoned supernatant liquor, bacterium mud;
5) lyophilize
Earlier with 8～15% glycerine solutions and 8～15% skim-milk solution, with volumetric ratio be mixing in 1: 1～1: 2 as protective material, sterilising treatment again with bacterium mud (cell mashed prod), is to mix in 1: 3～1: 4 with bacterium mud and protective material volume ratio;-40～-50 ℃ of lyophilizes 12～24 hours, make the bacterium powder at last.
Bu Shi lactobacillus preparation of the present invention can be used for improving silage and opens the cellar for storing things rear stability, can effectively improve the quality of maize silage, dissolves the feed safety risk that a large amount of propagation of harmful microorganism are brought.
The Bu Shi lactobacillus preparation is used to prepare the method for silage, comprises that feed cutting, every layer of feed add microbe additive, and sealing and fermenting after the compacting connects the bacterium amount and contains 10 for every gram feed during ensiling
6～10
8Individual L.buchneri colony-forming unit (cfu).
Described feed is fresh corn stalk or whole-plant corn.
Described microbe additive adopts the Bu Shi lactobacillus preparation to add deionized water and is made into suspension, and concentration is controlled between 1%～5%, and this suspension is sprayed on ensiling limit, limit, gets final product according to the preparation process of conventional silage.
That is to say that silage preparation method of the present invention is to be raw material with maize straw or whole-plant corn, at normal temperatures the stable maize silage of producing through operations such as chopping, filling, inoculation, compacting, sealing, fermentation, storages of high-quality and safety.
Concrete steps are as follows:
The whole-plant corn cutting that to gather in the crops dough stage earlier is into about 2～3cm; Corn after will cutting then is filled in the horizental silo; In filling the process of horizental silo, the limit ensiling, the special microorganism additive is added on the limit, and every kg corn material adds the aaerosol solutions of 25 grams with the microbe additive of deionized water mixing, makes to connect the bacterium amount and reach every gram feed and contain 10
6～10
8Individual L.buchneri colony-forming unit (cfu); Compacting material in the process of fill material is eliminated air in the horizental silo as far as possible; After filling finishes, the material of sealing institute ensiling; Ferment and to open the cellar for storing things utilization after 3 months.
The present invention adopts Bu Shi lactobacillus (L.buchneri) to be used for the preparation of maize silage, the maize silage that has solved the traditional method preparation is being opened the problem that the cellar for storing things rear stability sharply descends, and can effectively improve the quality of maize silage, dissolve the feed safety risks that a large amount of propagation of harmful microorganism are brought.
The prepared maize silage high-quality and safety of the present invention is stablized, good palatability, and its pH value is 3.8～4.31, dry-matter 26～30%, ethanol 0.6～1.4%, acetate 5.0～7.2%, lactic acid 2.0～13.5%, propionic acid 0.5～0.9%, propyl alcohol 1.1～2.0%.In the centre of horizental silo, yeast and mould all do not have and detect in the maize silage; And the logarithmic value of top layer yeast colony number is less than 3.0, and no mould detects.Comparing with traditional Silaging method, is that unit calculates with weight, and milk-acid bacteria bacterium colony total amount improves 10～100 times in the maize silage, and yeast quantity reduces to 1/8～1/10.Stability improves (1 ℃ of required time of temperature rising is more than 260 hours) more than 1 times at normal temperatures.
Description of drawings
Fig. 1 is the silage lay up period dry-matter loss of different treatment.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The Bu Shi lactobacillus preparation adopts Bu Shi lactobacillus (L.buchneri) fermentation culture, lyophilize to form; Every gram contains 9.5 * 10
10Individual L.buchneri colony-forming unit (cfu/g).
Wherein, the Bu Shi lactobacillus is available from Chinese microorganism strain preservation center (CICC), and CICC is numbered 6007.
Contain 33% lyophilized vaccine in the Bu Shi lactobacillus preparation, lyophilized vaccine is glycerine and skimmed milk, and volume ratio is 1: 1.
Concrete preparation process is as follows:
1. the activation culture of Lactobacillus buchneri
In Bechtop, in the place's calcination of spirit lamp internal flame, inoculation Lactobacillus buchneri bacterium powder is in the MRS liquid nutrient medium with transfering loop, and calcination test tube mouth and plug have been filled in test tube.The shaking table that Lactobacillus buchneri is put into 30 ℃ is cultivated 18h.
The MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3.
2, the separation pure culture (plate streak) of Lactobacillus buchneri
Pour the MRS nutrient agar that adds lime carbonate into plate, treat culture medium solidifying after, dip in the disinfection inoculation ring and to get the bacterium liquid that has shaken and rule, each bacterial classification is made three plates, after drawing well, seal plate, be upside down in 30 ℃ interior the cultivation 2 days of biochemical incubator with sealing compound.Choose individual uniform single bacterium colony purifying and cultivate, test standby.Plate can be preserved at 4 ℃ of refrigerators.
Wherein, add the MRS nutrient agar of lime carbonate, MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.5.In above-mentioned 1000ml liquid nutrient medium, add 20g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 15mim are standby.
3, the expansion of purifying bacterium liquid is numerous, cultivates
With the inoculum size of purifying bacterium liquid with Lactobacillus buchneri 5%, be inoculated in the Erlenmeyer flask of 1000ml, the volume of substratum is 475ml in the Erlenmeyer flask, then inoculating the bacterium liquid measure accordingly is 25ml; At 30 ℃ of following pH values is 6.0, cultivates 36h;
4, centrifugal
At 26 ℃, 4000 rev/mins were descended centrifugal 30 minutes, abandoned supernatant liquor, got bacterium mud;
5, lyophilize
Earlier with 10% glycerine solution and 10% skim-milk solution, with volumetric ratio be mixing in 1: 1 as protective material, sterilising treatment again with bacterium mud (cell mashed prod), is to mix at 3: 1 with protective material and bacterium mud volume ratio;-50 ℃ of lyophilizes 24 hours, make the bacterium powder at last.
Embodiment 2
The Bu Shi lactobacillus preparation adopts Bu Shi lactobacillus (L.buchneri) fermentation culture, lyophilize to form; Every gram contains 1.2 * 10
10Individual L.buchneri colony-forming unit (cfu/g).
Wherein contain 40% lyophilized vaccine, lyophilized vaccine is glycerine and skimmed milk, and volume ratio is 1: 2.
Concrete preparation process is as follows:
1. the activation culture of Lactobacillus buchneri
In Bechtop, in the place's calcination of spirit lamp internal flame, inoculation Lactobacillus buchneri bacterium powder is in the MRS liquid nutrient medium with transfering loop, and calcination test tube mouth and plug have been filled in test tube.The shaking table that Lactobacillus buchneri is put into 32 ℃ is cultivated 15h.
The MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.5.
2, the separation pure culture (plate streak) of Lactobacillus buchneri
Pour the MRS nutrient agar that adds lime carbonate into plate, treat culture medium solidifying after, dip in the disinfection inoculation ring and to get the bacterium liquid that has shaken and rule, each bacterial classification is made three plates, after drawing well, seal plate, be upside down in 30 ℃ interior the cultivation 1 day of biochemical incubator with sealing compound.Choose individual uniform single bacterium colony purifying and cultivate, test standby.Plate can be preserved at 4 ℃ of refrigerators.
Wherein, add the MRS nutrient agar of lime carbonate, MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.3.In above-mentioned 1000ml liquid nutrient medium, add 15g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 18mim are standby;
3, the expansion of purifying bacterium liquid is numerous, cultivates
With the inoculum size of purifying bacterium liquid with Lactobacillus buchneri 3%, be inoculated in the Erlenmeyer flask of 1000ml, the volume of substratum is 475ml in the Erlenmeyer flask, then inoculating the bacterium liquid measure accordingly is 25ml; At 30 ℃ of following pH values is 5.0, cultivates 30h;
4, centrifugal
At 26 ℃, 5000 rev/mins were descended centrifugal 30 minutes, abandoned supernatant liquor, got bacterium mud;
5, lyophilize
Earlier with 8% glycerine solution and 12% skim-milk solution, with volumetric ratio be mixing in 1: 2 as protective material, sterilising treatment again with bacterium mud (cell mashed prod), is to mix at 4: 1 with protective material and bacterium mud volume ratio;-50 ℃ of lyophilizes 24 hours, make the bacterium powder at last.
Embodiment 3
The Bu Shi lactobacillus preparation adopts Bu Shi lactobacillus (L.buchneri) fermentation culture, lyophilize to form; Every gram contains 1.0 * 10
11Individual L.buchneri colony-forming unit (cfu/g).
Wherein contain 30% lyophilized vaccine, lyophilized vaccine is glycerine and skimmed milk, and volume ratio is 1: 1.
Concrete preparation process is as follows:
1. the activation culture of Lactobacillus buchneri
In super quiet worktable, in the place's calcination of spirit lamp internal flame, inoculation Lactobacillus buchneri bacterium powder is in the MRS liquid nutrient medium with transfering loop, and calcination test tube mouth and plug have been filled in test tube.The shaking table that Lactobacillus buchneri is put into 28 ℃ is cultivated 20h.
The MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.5.
2, the separation pure culture (plate streak) of Lactobacillus buchneri
Pour the MRS nutrient agar that adds lime carbonate into plate, treat culture medium solidifying after, dip in the disinfection inoculation ring and to get the bacterium liquid that has shaken and rule, each bacterial classification is made three plates, after drawing well, seal plate, be upside down in 30 ℃ interior the cultivation 2 days of biochemical incubator with sealing compound.Choose individual uniform single bacterium colony purifying and cultivate, test standby.Plate can be preserved at 4 ℃ of refrigerators.
Wherein, add the MRS nutrient agar of lime carbonate, MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first (MgSO
47H
2O 11.5g+MnSO
44H
2O 2.8g+ distilled water 100ml) 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, and distilled water adds to 1000ml, and proofreading and correct the pH value is 6.5.In above-mentioned 1000ml liquid nutrient medium, add 18g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 20mim are standby.
3, the expansion of purifying bacterium liquid is numerous, cultivates
With the inoculum size of purifying bacterium liquid with Lactobacillus buchneri 7%, be inoculated in the Erlenmeyer flask of 1000ml, the volume of substratum is 475ml in the Erlenmeyer flask, then inoculating the bacterium liquid measure accordingly is 25ml; At 28 ℃ of following pH values is 7.0, cultivates 40h;
4, centrifugal
At 26 ℃, 3000 rev/mins were descended centrifugal 40 minutes, abandoned supernatant liquor, got bacterium mud;
5, lyophilize
Earlier with 8% glycerine solution and 15% skim-milk solution, with volumetric ratio be mixing in 1: 1 as protective material, sterilising treatment again with bacterium mud (cell mashed prod), is to mix at 3: 1 with protective material and bacterium mud volume ratio;-50 ℃ of lyophilizes 24 hours, make the bacterium powder at last.
Embodiment 4
The preparation method is with embodiment 1, and different is that the every gram of described Bu Shi lactobacillus preparation contains 1.0 * 10
9Individual L.buchneri colony-forming unit (cfu).Wherein contain 35% lyophilized vaccine, lyophilized vaccine is glycerine and skimmed milk, and volume ratio is 1: 1.
Embodiment 5
The preparation method is with embodiment 1, and different is that the every gram of described Bu Shi lactobacillus preparation contains 1.3 * 10
11Individual L.buchneri colony-forming unit (cfu).Wherein contain 40% lyophilized vaccine, lyophilized vaccine is glycerine and skimmed milk, and volume ratio is 1: 2.
Experimental example 1
The Bu Shi lactobacillus preparation that adopts embodiment 1 to obtain prepares silage, and its preparation method is as follows:
The whole-plant corn that to gather in the crops dough stage cuts into 2cm, corn after the cutting is filled in the horizental silo, in filling process, add the aaerosol solution of 25 grams according to every kg corn material with the Bu Shi lactobacillus preparation of deionized water mixing, every gram corn material provides 1 * 10
6Individual L.buchneri colony-forming unit (cfu); With forklift compacting material, note not staying dead angle, purpose is to eliminate air in the horizental silo as far as possible; After filling finishes, cover the material of institute's ensiling completely with the sealed polyethylene plastic of black, with soil or other material compacting plastics film, purpose is to prevent in the fermenting process owing to produce gas and cause leaking gas the destruction of causing the ensiling anaerobic environment around the horizental silo; Ferment and to open the cellar for storing things utilization after 3 months.
Prepared maize silage high-quality and safety is stable, good palatability, and its pH value is 4.3, dry-matter 26%, ethanol 0.8%, acetate 6.2%, lactic acid 2.0%, propionic acid 0.9%, propyl alcohol 2.0%.In the centre of horizental silo, yeast and mould all do not have and detect in the maize silage; And the logarithmic value of top layer yeast colony number is less than 3.0, and no mould detects.
Experimental example 2
This experimental example purpose is to study maize silage of the present invention and opens cellar for storing things rear stability (Aerobicstability).
1, definition of stability: for temperature raises 1 ℃ of needed time, unit: hour (h).
2, measuring method: get in the Plastic Bottle that the 0.3kg maize silage is loaded on opening, be placed under 20 ℃ the room temperature, measured the temperature variation of silage central position in continuous 260 hours.
3, weigh the index of opening the cellar for storing things rear stability
(1), leading indicator
Stability (Aerobic stability): use the additive rear stability by more than 260 hours of raising in 135 hours, biology statistical test difference is (p＜0.01) extremely significantly.
(2), reference index: open back, cellar for storing things microbial numbers and change unit: log cfu g-1
The A probiotics
The logarithmic value of milk-acid bacteria total amount (LAB) quantity is increased to 9.07 by 8.31, and biology statistical test difference is (p＜0.01) extremely significantly;
B is harmful to bacterium
The logarithmic value of yeast (Yeast) quantity reduces to 2.46 by 3.23, and biology statistical test difference is remarkable (p＞0.01) not;
Mould (Mould): do not detect
Three, other discoveries: acetate, propionic acid content obviously increase (p＜0.01) in the tunning, and these two products all have restraining effect to harmful bacterium.
Four, the addition of milk-acid bacteria in the feed: for every kg corn material adds the aaerosol solution of 25 grams with the special microorganism additive of deionized water mixing, for every gram corn material provides 10
6～10
8Individual L.buchneri colony-forming unit (cfu).It is about 1～2 kilogram per ton with bacterial classification.
Five, test processing spec:
Have 6 tests during ensiling and handle, each handles 3 bags.
The Con=control group, every kg ensilage adds the 25g deionized water;
1 group of g1=dextrose treatment, every kg ensilage adds 37.5g glucose solution (wherein containing glucose 12.5g);
2 groups of g2=dextrose treatment, every kg ensilage adds 75g glucose solution (wherein containing glucose 25g);
L.b=Lactobacillus buchneri (L.buchneri) treatment group, every kg ensilage are added 25g L.buchneri aaerosol solution and (be can be every g ensilage and provide 10
6Cfu L.buchneri);
1 group of L.b+g1=Lactobacillus buchneri (L.buchneri)+dextrose treatment, every kg ensilage add 25g L.buchneri aaerosol solution and (can be every g ensilage and provide 10
6Cfu L.buchneri)+every kg ensilage interpolation 37.5g glucose solution (wherein containing glucose 12.5g)
2 groups of L.b+g2=Lactobacillus buchneri (L.buchneri)+dextrose treatment, every kg ensilage add 25g L.buchneri aaerosol solution and (can be every g ensilage and provide 10
6Cfu L.buchneri)+every kg ensilage interpolation 75g glucose solution (wherein containing glucose 25g)
Table 1 is formed for the composition measurement and the microorganism of examination whole-plant corn
| Index | Con | g 1 | g 2 | L.b | L.b+g 1 | L.b+g 2 |
| Dry-matter, g kg -1 | 290 | 299.5 | 299.8 | 291.7 | 303.3 | 302.2 |
| Soluble-carbohydrate, g kg -1 DM | 85.7 | 116.1 | 138.8 | 88 | 113.2 | 132.9 |
| Total nitrogen, g kg -1 DM | 10.3 | - | - | 10.1 | - | - |
| Neutral detergent fiber (NDF), g kg -1 DM | 462.9 | - | - | 456.5 | - | - |
| Acid detergent fiber (ADF), g kg -1 DM | 222.1 | - | - | 243.3 | - | - |
| Milk-acid bacteria total amount (LAB), log 10 cfu g -1 | 5.99 | - | - | 6.2 | - | - |
| Lactobacillus (Lactobacilli), log 10 cfu g -1 | 5.64 | - | - | 6.2 | - | - |
| Yeast (Yeast), log 10 cfu g -1 | 4.7 | - | - | 4.3 | - | - |
| Mould (Mould), log 10 cfu g -1 | 3.7 | - | - | 3.6 | - | - |
Chemical constitution when table 2 maize silage is stored 91 days, tunning concentration, microorganism are formed (the ensiling device is a polyethylene double-layer plastic bag, and every bag has the 2-kg whole-plant corn)
| Index | Control group (con) | Add glucose group 1 (g 1 | Add glucose group 2 (g 2 | L.b | L.b+g 1 | L.b+g 2 | SEM |
| pH | 3.82 c-C | 3.73 d-D | 3.71 d-D | 4.31 a-A | 4.03 b-B | 4.03 b-B | 0.02 |
| Dry-matter, g kg -1 | 268.3 abc-A | 268.7 abc-A | 274.6 ab-A | 259.2 bc-A | 264.5 abc-A | 256.9 c-A | 7.32 |
| Soluble sugar, g kg -1 DM | 4.46 b-A | 4.56 ab-A | 4.84 a-A | 4.56 ab-A | 4.58 ab-A | 4.79 a-A | 0.16 |
| The dry-matter loss, g kg -1 DM | 27.37 f-F | 37.99 e-E | 51.55 b-B | 42.35 d-D | 46.7 c-C | 60.51 a-A | 1.19 |
| Ethanol, g kg -1 DM | 7.58 d-D | 23.42 b-B | 38.22 a-A | 11.78 c-C | 22.11 b-B | 37.09 a-A | 0.87 |
| Acetate, g kg -1 DM | 43.63 c-CD | 38.43 d-D | 37.83 d-D | 68.17 a-A | 54.72 b-B | 56.94 b-B | 2.57 |
| Lactic acid, g kg -1 DM | 74.4 b-B | 88.48 a-A | 86.21 a-A | 16.69 e-D | 48.26 c-C | 39.51 d-C | 3.85 |
| Propionic acid, g kg -1 DM | 1.73 c-C | 0 d-D | 0 d-D | 8.04 a-A | 4.34 b-B | 4.3 b-B | 0.17 |
| Butyric acid, g kg -1 DM | 0 c-C | 0 c-C | 0 c-C | 0.79 a-A | 0.78 a-A | 0.7 b-B | 0.02 |
| Lactic acid/acetate | 1.71 b-B | 2.31 a-A | 2.31 a-A | 0.25 d-D | 0.88 c-C | 0.70 c-C | 0.15 |
| Milk-acid bacteria total amount (LAB), log cfu g -1 | 8.31 b-B | 7.92 c-C | 7.97 c-C | 9.07 a-A | 8.84 a-A | 8.83 a-A | 0.11 |
| Yeast (Yeast), log cfu g -1 | 3.23 a | 3.16 a | 3.22 a | 2.46 a | 2.59 a | 2.56 a | 0.57 |
| Mould (Mould), log cfu g -1 | ＜2 a | ＜2 a | ＜2 a | ＜2 a | ＜2 a | ＜2 a | 0.04 |
| Open cellar for storing things rear stability (Aerobic stability), h | 135 cd-BC | 107 d-C | 92 d-C | ＞260 a-A | 209 ab-AB | 196 bc-AB | 29.6 |
A, b, c, d, e, fThe different lowercase persons of colleague's shoulder motes represent significant difference (p＜0.05)
A, B, C, D, E, FThe different capitalization persons of colleague's shoulder motes represent difference extremely significantly (p＜0.01)
Conclusion: Fig. 1 is the silage lay up period dry-matter loss of different treatment, adopt Lactobacillus buchneri to lack than the loss of control group dry-matter, and adopt Lactobacillus buchneri to improve 10～100 times in the maize silage than control group milk-acid bacteria bacterium colony total amount, and yeast quantity reduces to 1/8～1/10.Stability improves (1 ℃ of required time of temperature rising is more than 260 hours) more than 1 times at normal temperatures.
Claims (10)
1, a kind of Bu Shi lactobacillus preparation, it is characterized in that: every gram contains 1 * 10
9～1 * 10
11Individual Bu Shi lactobacillus (CICC is numbered 6007) colony-forming unit.
2, Bu Shi lactobacillus preparation as claimed in claim 1, it is characterized in that: every gram contains 1 * 10
9～1 * 10
10Individual L.buchneri colony-forming unit.
3, Bu Shi lactobacillus preparation according to claim 1 and 2 is characterized in that: contain the lyophilized vaccine in total formulation weight amount 30～40%.
4, Bu Shi lactobacillus preparation according to claim 3 is characterized in that: described lyophilized vaccine is glycerine and skimmed milk, the volume ratio of glycerine and skimmed milk 1: 1～1: 2.
5, a kind of method for preparing the described Bu Shi lactobacillus preparation of arbitrary claim in the claim 1～4 is characterized in that, comprises the steps:
1) with Lactobacillus buchneri kind 28～32 ℃ of following activation culture 15～20h in activation medium; Adopt plate streak in 28～32 ℃ of following purifying substratum, to carry out purifying then and cultivated 1～2 day, get purifying bacterium liquid;
2) purifying bacterium liquid is inoculated in the fermentor tank with 3～7% inoculum size, the pH value is controlled at 5.0～7.0 again, and 28～32 ℃ of following mixed culture 30～40h get fermented liquid in fermention medium;
3) described then fermented liquid is under 20～26 ℃, and rotating speed carried out centrifugal treating 30～40 minutes for 3000～4000 rev/mins, gets zymophyte mud;
4) in zymophyte mud, behind the described lyophilized vaccine of adding, carry out lyophilize and get the lactic acid bacteria freeze drying powder at last.
According to the preparation method of the described Bu Shi lactobacillus preparation of claim 5, it is characterized in that 6, described activation medium is the MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, distilled water adds to 1000ml, and the pH value is 6.3～6.5.
According to the preparation method of the described Bu Shi lactobacillus preparation of claim 5, it is characterized in that 7, described purifying substratum is the MRS nutrient agar that adds lime carbonate, MRS nutrient agar composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, distilled water adds to 1000ml, and the pH value is 6.3～6.5; In above-mentioned 1000mi liquid nutrient medium, add 15～20g agar, 7.5g lime carbonate, 115 ℃ of autoclaving 15～20mim are standby.
According to the preparation method of the described Bu Shi lactobacillus preparation of claim 5, it is characterized in that 8, described fermention medium is the MRS liquid nutrient medium, its composition is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, tween-80 1ml, ammonium citrate 2g, sodium-acetate 5g, dipotassium hydrogen phosphate 2g, hydrochloric acid first 5ml, each composition heated to be dissolved in the distilled water, filters, and adds glucose 20g, distilled water adds to 1000ml, and the pH value is 6.3～6.5.
9, according to the preparation method of the described Bu Shi lactobacillus preparation of claim 5; it is characterized in that; described lyophilized vaccine is glycerine and skimmed milk; the concentration of glycerine solution is 8～15%; skimmed milk solution is 8～15%; mixed described bacterium mud and protective material volume ratio 1: 3～1: 4 in 1: 1～1: 2 with volumetric ratio.
10, according to the described Bu Shi lactobacillus preparation of claim 1 in the application that is used for preparing maize straw or whole corn silage feed.
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| CN1958786B (en) * | 2005-12-28 | 2011-07-27 | 东北农业大学 | Agent of lactic acid bacteria in use for silage of maize, and preparation method |
| CN102987435A (en) * | 2012-11-13 | 2013-03-27 | 西华大学 | Preparation method for multifunctional complex-fermented powder for tenderization and fermentation and method for preparing fermented yak meat particles |
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| CN1958786B (en) * | 2005-12-28 | 2011-07-27 | 东北农业大学 | Agent of lactic acid bacteria in use for silage of maize, and preparation method |
| CN102987435A (en) * | 2012-11-13 | 2013-03-27 | 西华大学 | Preparation method for multifunctional complex-fermented powder for tenderization and fermentation and method for preparing fermented yak meat particles |
| CN104738356A (en) * | 2013-12-27 | 2015-07-01 | 先锋国际良种公司 | Phenotype and genotype variant of lactobacillus buchneri |
| CN106615609A (en) * | 2016-03-30 | 2017-05-10 | 中国农业科学院特产研究所 | Lactobacillus buchneri capable of improving feed ensiling qualities and application thereof |
| CN105941872A (en) * | 2016-05-16 | 2016-09-21 | 中国农业大学 | Lactobacillus buchneri inoculant as well as preparation method and application thereof |
| CN106244509A (en) * | 2016-10-12 | 2016-12-21 | 汉臣氏（沈阳）儿童制品有限公司 | Lactobacillus rhamnosus culture medium and cultural method |
| CN107981034A (en) * | 2017-11-14 | 2018-05-04 | 北京好实沃生物技术有限公司 | One plant of lactobacillus buchneri HEW-A666 and its application |
| CN107981034B (en) * | 2017-11-14 | 2021-03-02 | 北京好实沃生物技术有限公司 | Lactobacillus buchneri HEW-A666 and application thereof |
| CN114698767A (en) * | 2022-06-07 | 2022-07-05 | 山东健源生物科技有限公司 | Biological agent for removing mycotoxin in feed as well as preparation method and application of biological agent |
| CN114698767B (en) * | 2022-06-07 | 2022-08-23 | 山东健源生物科技有限公司 | Biological agent for removing mycotoxin in feed as well as preparation method and application of biological agent |
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