CN109722392A - It can promote culture medium and its application of producing bacillus subtilis gemma - Google Patents
It can promote culture medium and its application of producing bacillus subtilis gemma Download PDFInfo
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Abstract
The present invention relates to field of microorganism engineering, the culture medium that can promote producing bacillus subtilis gemma and its application are disclosed.Culture medium provided by the invention contains the manganese sulfate of the yeast powder of 0.3-0.8 weight %, the corn pulp of 0.3-0.8 weight %, the molasses of 0.3-0.8 weight %, the corn flour of 1.5-2.5 weight %, the dregs of beans of 2-3 weight % and 0.008-0.02 weight %, pH7-7.5.The present invention also provides above-mentioned culture mediums to cultivate bacillus subtilis or prepare the purposes in animal feed.The present invention also provides the methods for preparing animal feed, including the culture medium is used to carry out Liquid Culture to bacillus subtilis, and the culture solution of acquisition is spray-dried, and obtained dried object is mixed with basal nutrient substance.The present invention can effectively facilitate producing bacillus subtilis gemma.
Description
Technical field
The present invention relates to field of microorganism engineering, and in particular to can promote producing bacillus subtilis gemma culture medium and
It is applied.
Background technique
With popularizing for fermented feed application range, the promotion of fermented feed yield is extremely urgent, and liquid seed liquor
Preparation is to restrict a key factor of yield；Liquid seed liquid preparing process is complicated, retention cycle is short；Therefore solid fungicide is answered
For fermented feed, it is a breakthrough progress, can solve the limitation in the directions such as equipment, technology, manpower, is to stablize and promoted
An important factor for product quality and raising yield.
Bacillus subtilis thallus itself can synthesize digestibility enzyme, such as protease, amylase, lipase；Full-time grain
It is fermenting with during storage, the enzyme system of generation can make small point that fiber, starch, protein degradation are easier to be absorbed and utilized by animal
Sub- substance improves the conversion ratio of feed.Bacillus subtilis plays a role in alimentary canal with endogenous enzymes jointly, improves feed and disappears
Rate.The growth and breeding in animal intestinal tract can generate a variety of nutriments such as vitamin, amino acid, organic acid, somatomedin
Deng participating in the metabolism of animal body, provide nutriment for animal body.
It is unfavorable for maintaining the work of bacillus subtilis with bacillus subtilis existing for trophosome however, adding into feed
Bacterium number, over time, the bacillus subtilis viable count of feed decline, can not effectively improve the digestibility of feed to
The mortality of raising animal is reduced, and the problem is not present then with spore form addition.Therefore, promote bacillus subtilis as far as possible
Bacterium, which produces gemma, has especially great Practical significance.
Summary of the invention
The purpose of the invention is to overcome the problems, such as that producing bacillus subtilis gemma number of the existing technology is lower, mention
For culture medium and its application of producing bacillus subtilis gemma can be promoted.
To achieve the goals above, one aspect of the present invention provides a kind of training that can promote producing bacillus subtilis gemma
Base is supported, which contains the yeast powder of 0.3-0.8 weight %, the corn pulp of 0.3-0.8 weight %, 0.3-0.8 weight %
Molasses, the corn flour of 1.5-2.5 weight %, the dregs of beans of 2-3 weight % and 0.008-0.02 weight % manganese sulfate, pH7-7.5.
Second aspect of the present invention provides above-mentioned culture medium and is cultivating bacillus subtilis or preparing the use in animal feed
On the way.
Third aspect present invention provides the method for culture bacillus subtilis, this method comprises: using above-mentioned culture medium
Liquid Culture is carried out to bacillus subtilis.
Fourth aspect present invention provides a kind of method for preparing animal feed, and this method includes according to the method described above to withered
Careless bacillus carries out Liquid Culture, and the culture solution of acquisition is spray-dried, and by obtained dried object and base nutrients
Matter mixing.
Through the above technical solutions, the present invention can effectively facilitate producing bacillus subtilis gemma, so that feed
Digestibility is significantly improved, and reduces the mortality of raising animal.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In the present invention, in the absence of explanation to the contrary, before the pH of culture medium refers both to initial pH value, namely sterilizing
PH value, other ingredients, dosage when being preparation (before sterilizing)；" CFU " used is Colony Forming Unit, can be passed through
The method of plate culture count measures；Number of spores is according to bacillus subtilis in GB/T 26428-2010 Application of Direct-fed Microbials
Detection method measures.
The culture medium provided by the invention that can promote producing bacillus subtilis gemma contains the yeast of 0.3-0.8 weight %
Powder, the corn pulp of 0.3-0.8 weight %, the molasses of 0.3-0.8 weight %, the corn flour of 1.5-2.5 weight %, 2-3 weight %
Dregs of beans and 0.008-0.02 weight % manganese sulfate, pH 7-7.5.
Preferred embodiment according to the present invention, the culture medium contain the yeast powder of 0.45-0.55 weight %, 0.45-
The corn pulp of 0.55 weight %, the molasses of 0.45-0.55 weight %, the corn flour of 1.9-2.1 weight %, 2.4-2.6 weight %
Dregs of beans and 0.009-0.012 weight % manganese sulfate, pH 7-7.4.
Culture medium of the invention typically contains water, and content can be selected as needed, for example, can be 92-96 weight
Measure %.Culture medium of the invention contains mentioned component or is made of the above ingredient and water.Wherein, yeast powder, corn pulp, molasses,
Corn flour and dregs of beans are the substance of the conventional culture medium for preparing microorganism, can be commercially available.
The present invention also provides above-mentioned culture mediums of the invention in culture bacillus subtilis or to prepare in animal feed
Purposes.
It is provided by the invention culture bacillus subtilis method include: using above-mentioned culture medium to bacillus subtilis into
Row Liquid Culture.
When culture, the inoculum concentration of bacillus subtilis is not required particularly, however, in preferred implementation side of the invention
In formula, relative to every milliliter of culture medium, the inoculum concentration of the bacillus subtilis is 1-2CFU.
In the present invention, the bacillus subtilis can be various bacillus subtilises commonly used in the art, can pass through
It is commercially available.
In the present invention, the condition of Liquid Culture is not required particularly, can be conventional culture bacillus subtilis
Condition.Under preferable case, the condition of Liquid Culture includes: that temperature is 28-32 DEG C, time 12-20h, and stirring rate is
110-130rpm。
It, can also be right before carrying out Liquid Culture to bacillus subtilis using culture medium of the invention in the present invention
Bacillus subtilis is activated, and activation can be carried out according to conventional activation method.For example, can be using containing peptone, ferment
The culture medium of female powder, sodium chloride and glucose is activated, and activation can also use second level by the way of level-one activation
The mode of activation.This will not be detailed here.
The method provided by the invention for preparing animal feed (such as bird feed, especially broiler fodder) includes according to above-mentioned
Method carries out Liquid Culture to bacillus subtilis, and the culture solution of acquisition is spray-dried, and by obtained dried object and base
The mixing of plinth nutriment.
According to the present invention, the spray drying carries out generally in spray dryer.The inlet temperature of spray dryer can
Think 150-200 DEG C, outlet temperature can be 100-120 DEG C.Being spray-dried duration can be 10-60s.Spray drying
Used carrier can be common various carriers, not influence the carrier of animal feed nutritive and mouthfeel generally, preferably
At least one of corn flour, maize peel, corncob and water soluble starch.Used carrier and object to be dried when spray drying
The weight ratio for expecting (culture solution) is (0.5-1.5): 1.
According to the preferred embodiment of the present invention, relative to every gram of basal nutrient substance, the dried object is with withered grass bud
The dosage of the Number of spores meter of spore bacillus is 1012-1015CFU。
According to the present invention, the basal nutrient substance can be the conventional nutriment for preparing animal fodder, packet
Include protein, lipid, carbohydrate, vitamin and mineral etc..The content of basal nutrient substance can be according to the battalion of animal
Feeding demand makes appropriate choice, and under preferable case, on the basis of the total weight of the animal feed, the content of protein is
20-55 weight %, lipid content be 15-20 weight %, the content of carbohydrate is 20-35 weight %, vitamin contains
Amount is 1-1.5 weight % and the content of minerals is 0.2-3 weight %.Basal nutrient substance can be by conventional for braking
The raw material of object feed provides, for example, the basal nutrient substance can be by animal fodder raw material (such as fish meal, liver powder, shrimp
Powder, shrimp shell meal, crab meat, crab shell powder, chicken meal, blood meal, silkworm chrysalis etc.), vegetality feedstuff (such as dregs of beans, peanut meal, flour,
Cotton dregs, rapeseed meal, single cell protein (yeast), the vinasse dregs of rice, corn protein powder etc.), glyceride stock (such as fish oil, phosphatide oil, soya-bean oil,
Cuttlefish cream, corn oil, palm oil, linseed oil etc.), vitamin (such as VCAnd VEDeng) etc. cooperations provide, for example, by " fish meal, big
The mixing of bean powder, peanut meal, yeast, soybean lecithin, shrimp shell meal, wheat flour, seed fat, calcium lactate and calcium dihydrogen phosphate "
Object provides, and carries out being mixed to get the mixture being that those skilled in the art can be easily determined using which kind of proportion,
This is repeated no more.
Generally, the animal feed contains a certain amount of water, for example, moisture content is generally 8-12 weight in animal feed
Measure %.In addition, additive can also be contained in the animal feed in order to further improve the meat of animal and promote to cultivate,
Such as phagostimulant, immunopotentiator, growth promoter, high temperature resistant enzyme preparation, antioxidant.The content of additive is usually 5-10
Weight %.
The present invention will be described in detail by way of examples below.In following embodiment, bacillus subtilis is obtained from peace
Emblem agriculture university Life Science College, number 951NA4；The daily ration of broiler chicken is purchased from China Agricultural University's animal experiment base (egg
The content of white matter is 41 weight %, the content of lipid is 20 weight %, the content of carbohydrate is 35 weight %, vitamin
Content is 1 weight % and the content of minerals is 3 weight %)；Bud ratio (%)=Number of spores/terminal clump count × 100%,
Wherein Number of spores is measured by the detection method of bacillus subtilis in GB/T 26428-2010 Application of Direct-fed Microbials.
Embodiment 1
(1) according to the culture medium and condition of table 1, using 250ml flask to bacillus subtilis carry out level-one shaking flask and
Second-level shake flask (activation) then spreads cultivation to bacillus subtilis using the tank that spreads cultivation that dischargeable capacity is 400L.
Table 1
(2) then the culture solution (characterization results such as bud ratio are shown in Table 1) in the tank that spreads cultivation is spray-dried, condition is such as
Under:
Spray carrier: corncob, the weight ratio with culture solution are 1:1
Inlet temperature: 150 DEG C
Outlet temperature: 100 DEG C
Duration: 10s
(3) dried object that step (2) obtain is added in the daily ration of broiler chicken, according to grain addition 5 × 10 of often setting a date14CFU's
The amount of bacterium is added, and feed 1 is obtained.
Embodiment 2
Feed is prepared according to the method for embodiment 1, unlike, according to grain addition 10 of often setting a date14The amount of the bacterium of CFU is added
Dried object obtains feed 2.
Embodiment 3
Feed is prepared according to the method for embodiment 1, unlike, the condition of spray drying is as follows:
Spray carrier: corn flour, the weight ratio with culture solution are 1:1
Inlet temperature: 200 DEG C
Outlet temperature: 120 DEG C
Duration: 10s
And according to grain addition 2 × 10 of often setting a date14The amount of the bacterium of CFU adds dried object, obtains feed 3.
Embodiment 4
Feed is prepared according to the method for embodiment 1, unlike, the condition of spray drying is as follows:
Spray carrier: corncob, the weight ratio with culture solution are 1:1
Inlet temperature: 150 DEG C
Outlet temperature: 100 DEG C
Duration: 60s
And according to grain addition 4 × 10 of often setting a date14The amount of the bacterium of CFU adds dried object, obtains feed 4.
Embodiment 5
Feed is prepared according to the method for embodiment 1, unlike, the content of each ingredient is such as in the culture medium in the tank that spreads cultivation
Shown in lower: the yeast powder of 0.45 weight %, the corn pulp of 0.55 weight %, the molasses of 0.45 weight %, 1.9 weight % corn
The manganese sulfate of powder, the dregs of beans of 2.6 weight % and 0.009 weight %, surplus are water, according to grain addition 10 of often setting a date15The bacterium of CFU
Amount addition dried object, obtains feed 5.Wherein, the characterization results such as the bud ratio of the culture solution to spread cultivation are shown in Table 2.
Embodiment 6
Feed is prepared according to the method for embodiment 1, unlike, the content of each ingredient is such as in the culture medium in the tank that spreads cultivation
Shown in lower: the yeast powder of 0.55 weight %, the corn pulp of 0.45 weight %, the molasses of 0.55 weight %, 2.1 weight % corn
The manganese sulfate of powder, the dregs of beans of 2.4 weight % and 0.012 weight %, surplus are water, according to grain addition 10 of often setting a date14The bacterium of CFU
Amount addition dried object, obtains feed 6.Wherein, the characterization results such as the bud ratio of the culture solution to spread cultivation are shown in Table 2.
Comparative example 1
Feed is prepared according to the method for embodiment 1, unlike, it is following that the culture medium in the tank that spreads cultivation is replaced by ingredient
Culture medium: 1.5 weight % of corn pulp, 0.5 weight % of molasses, 0.5 weight % of glucose, surplus is water, obtains feed D1.Its
In, the characterization results such as bud ratio of the culture solution to spread cultivation are shown in Table 2.
Comparative example 2
Feed is prepared according to the method for embodiment 1, unlike, in the used culture medium that spreads cultivation, with " zinc sulfate " generation
For " manganese sulfate ", feed D2 is obtained.Wherein, the characterization results such as the bud ratio of the culture solution to spread cultivation are shown in Table 2.
Comparative example 3
Feed is prepared according to the method for embodiment 1, unlike, the dosage of yeast powder is 2 weight %, the dosage of corn flour
For 0.5 weight %, feed D3 is obtained.Wherein, the characterization results such as the bud ratio of the culture solution to spread cultivation are shown in Table 2.
Table 2
| Terminal OD (100 times of dilution) | Terminal clump count (CFU/ml) | Bud ratio (%) | |
| Embodiment 5 | 0.731 | 1.3×1016 | 98 |
| Embodiment 6 | 0.742 | 1.5×1016 | 98.6 |
| Comparative example 1 | 0.450 | 5.5×1011 | 0 |
| Comparative example 2 | 0.526 | 7.0×1012 | 60.8 |
| Comparative example 3 | 0.620 | 2.3×1013 | 82.5 |
It can be seen that the reality that bacillus subtilis bacteria culture fluid is prepared using culture medium of the invention by the result of table 1 and 2
Applying example has the obvious better effect for improving bud ratio.
Test case 1
(1) withered grass is measured using the method for plate culture count after placing feed made from above-described embodiment and comparative example 30 days
The content of bacillus, the results are shown in Table 3.
(2) being raised table hens respectively using feed made from above-described embodiment and comparative example, (every group 100, chicken age is 5 to chick
It, average weight 100g), feed three times, weigh after feeding 42 days daily, and calculate feedstuff-meat ratio (feedstuff feeding weight and meat
Ratio between the weight of chicken) and mortality (percentage that the sum of death toll and diseased chicken number account for original chick quantity).Using not
The daily ration of dried object (bacillus subtilis) is added as control.The results are shown in Table 3.
Table 3
| Bacterium number (CFU/g) after saving 30 days | Average weight (kg) | Feedstuff-meat ratio | Mortality (%) | |
| Control group | — | 2.20 | 2.12 | 5.0 |
| Feed 1 | It is basically unchanged | 2.65 | 1.71 | 0.5 |
| Feed 2 | It is basically unchanged | 2.55 | 1.72 | 0.6 |
| Feed 3 | It is basically unchanged | 2.60 | 1.73 | 0.6 |
| Feed 4 | It is basically unchanged | 2.50 | 1.735 | 0.75 |
| Feed 5 | It is basically unchanged | 2.62 | 1.70 | 0.65 |
| Feed 6 | It is basically unchanged | 2.71 | 1.68 | 0.58 |
| Feed D1 | It reduces to 5.0 × 106 | 2.40 | 1.80 | 2.2 |
| Feed D2 | It reduces to 7.2 × 1010 | 2.45 | 1.78 | 2.0 |
| Feed D3 | It reduces to 6.5 × 1011 | 2.38 | 1.86 | 2.3 |
In table 3, it is basically unchanged and refers to that the order of magnitude under the decimal system remains unchanged.From the results shown in Table 3, this is used
The culture medium of invention, which prepares feed, can effectively improve the weight of broiler chicken, and reduce mortality.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
Claims (10)
1. the culture medium that one kind can promote producing bacillus subtilis gemma, which is characterized in that the culture medium contains 0.3-0.8 weight
Measure the yeast powder of %, the corn pulp of 0.3-0.8 weight %, the molasses of 0.3-0.8 weight %, 1.5-2.5 weight % corn flour,
The dregs of beans of 2-3 weight % and the manganese sulfate of 0.008-0.02 weight %, pH7-7.5.
2. culture medium according to claim 1, wherein the culture medium contain 0.45-0.55 weight % yeast powder,
The corn pulp of 0.45-0.55 weight %, the molasses of 0.45-0.55 weight %, the corn flour of 1.9-2.1 weight %, 2.4-2.6 weight
Measure the dregs of beans of % and the manganese sulfate of 0.009-0.012 weight %, pH7-7.4.
3. culture medium of any of claims 1 or 2 is cultivating bacillus subtilis or is preparing the purposes in animal feed.
4. a kind of method for cultivating bacillus subtilis, which is characterized in that this method comprises: using of any of claims 1 or 2
Culture medium carries out Liquid Culture to bacillus subtilis.
5. according to the method described in claim 4, wherein, relative to every milliliter of culture medium, the bacillus subtilis is connect
Kind amount is 1-2CFU.
6. method according to claim 4 or 5, wherein the condition of Liquid Culture includes: that temperature is 28-32 DEG C, and the time is
12-20h, stirring rate 110-130rpm.
7. a kind of method for preparing animal feed, which is characterized in that this method includes according to any one of claim 4-6 institute
The method stated carries out Liquid Culture, the dried object that the culture solution of acquisition is spray-dried, and will obtained to bacillus subtilis
It is mixed with basal nutrient substance.
8. according to the method described in claim 7, wherein, being spray-dried used carrier is corn flour, maize peel, corncob
At least one of with water soluble starch.
9. method according to claim 7 or 8, wherein relative to every gram of basal nutrient substance, the dried object is with withered
The dosage of the Number of spores meter of careless bacillus is 1012-1015CFU。
10. according to the method described in claim 7, wherein, the basal nutrient substance includes protein, lipid, carbon hydrate
Object, vitamin and mineral；On the basis of the total weight of the animal feed, the content of protein is 20-55 weight %, lipid
Content be 15-20 weight %, the content of carbohydrate is 20-35 weight %, the content of vitamin be 1-1.5 weight % and
The content of minerals is 0.2-3 weight %.
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| CN112175856A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | Solid culture medium for bacillus subtilis fermentation, bacillus subtilis solid microbial inoculum and preparation method and application thereof |
| CN115745700A (en) * | 2022-11-21 | 2023-03-07 | 山东农大肥业科技股份有限公司 | Development and application of plant source insect-repellent compound microbial fertilizer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112175856A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | Solid culture medium for bacillus subtilis fermentation, bacillus subtilis solid microbial inoculum and preparation method and application thereof |
| CN112175856B (en) * | 2019-07-05 | 2022-10-21 | 中粮生物科技股份有限公司 | Solid culture medium for bacillus subtilis fermentation, bacillus subtilis solid microbial inoculum and preparation method and application thereof |
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| CN115745700A (en) * | 2022-11-21 | 2023-03-07 | 山东农大肥业科技股份有限公司 | Development and application of plant source insect-repellent compound microbial fertilizer |
| CN115745700B (en) * | 2022-11-21 | 2024-06-25 | 山东农大肥业科技股份有限公司 | Development and application of plant-source insect-repellent compound microbial fertilizer |
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