CN105018397B - A kind of bacillus subtilis bacterium culture medium and preparation method thereof - Google Patents
A kind of bacillus subtilis bacterium culture medium and preparation method thereof Download PDFInfo
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- CN105018397B CN105018397B CN201510537657.6A CN201510537657A CN105018397B CN 105018397 B CN105018397 B CN 105018397B CN 201510537657 A CN201510537657 A CN 201510537657A CN 105018397 B CN105018397 B CN 105018397B
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of bacillus subtilis bacterium culture medium and preparation method thereof, wherein, the ingredient that culture medium has following parts by weight is made：70 90 parts of 48 parts of sweet potato, 7 13 parts of peptone, 0.05 0.3 parts of ammonium nitrate, 0.4 0.8 parts of potassium hydroxide, 0.5 1 parts of calcium monohydrogen phosphate, 0.3 0.6 parts of magnesium bicarbonate, 8 12 parts of matrimony vine, 6 13 parts of sugarcane, 7 10 parts of Mao Li, 58 parts of root of Chinese trichosanthes, 0.05 0.2 parts of septichen sodium, 0.01 0.3 parts of 8 oxyquinoline and deionized water.And disclose preparation method.The beneficial effects of the invention are as follows：The culture medium of the present invention is applied to the culture of bacillus subtilis, and gemma yield is high, at low cost, and obtained bacillus subtilis has higher bacteriostasis property, and stability is good.
Description
Technical field
The present invention relates to culture medium field, more particularly to a kind of bacillus subtilis bacterium culture medium and preparation method thereof.
Background technology
Bacillus subtillis is one kind of Bacillus.0.7 ~ 0.8 × 2 ~ 3 microns of individual cells, uniform coloring.Nothing
Pod membrane, peritrichous can move.Gram-positive bacteria, 0.6 ~ 0.9 × 1.0 ~ 1.5 microns of gemma, ellipse to column, positioned at bacterium
Body is central or slightly inclined, and thalline does not expand after sporulation.Bacterium colony rough surface is opaque, and dirty white or yellowish is trained in liquid
When being grown in foster base, wrinkle mould is commonly formed.Aerobic bacteria.Available protein, a variety of sugar and starch, decompose tryptophan and form indoles.
It is widely used in genetics research, it is clearer to the route of synthesis and its regulation mechanism research of the purine nucleotides of this bacterium.Extensively
It is general to be distributed in the organic matter of soil and corruption, easily bred in withered grass soaks juice, it is therefore named.
The mechanism of action：1. the subtilin generated during bacillus subtilis thalli growth, polymyxins, mildew making
Element, gramicidins isoreactivity substance, these active materials have apparent suppression to the conditioned pathogen of pathogenic bacteria or autogenous infection
It makes and uses.2. bacillus subtilis consumes rapidly the free oxygen in environment, enteron aisle hypoxemia is caused, promotes to grow beneficial to anaerobic bacteria,
And the organic acids such as lactic acid are generated, enteron aisle pH value is reduced, other pathogenic bacteria is inhibited to grow indirectly.3. stimulate animal immune organ
Growth and development, activation T, bone-marrow-derived lymphocyte, improves immunoglobulin and antibody level, enhances cellular immunity and humoral immune function,
Improve herd immunity.4. the enzymes such as bacillus subtilis thalline itself synthesis alpha-amylase, protease, lipase, cellulase
Class plays a role in alimentary canal together with the digestive enzymes in animal body.5. it is more to synthesize vitamin B1, B2, B6, niacin etc.
Kind B family vitamin, improves the activity of interferon and macrophage in animal body.
Bacillus subtilis is as beneficial microorganism, at present in purifying aquatic product cultivating environment, peaceful control of crop disease, poultry
Feed additive for fowls etc. achieves good application effect, increasingly gets more and more people's extensive concerning in recent years.
At present, the culture medium of bacillus subtilis, there are gemma low yield, bacillus subtilis of high cost, and generating
Bacterium activity is low, and stability is not high.These problems are problems to be solved of the present invention.
Invention content
The purpose of the present invention is to provide a kind of bacillus subtilis bacterium culture medium and preparation method thereof, so as to solve gemma production
The problem of rate is low, of high cost, and bacillus subtilis activity is low, and stability is not high.
In order to achieve the above object, the present invention provides a kind of bacillus subtilis bacterium culture medium, wherein, by following parts by weight
Several raw materials are made：4-8 parts of sweet potato, 7-13 parts of peptone, 0.05-0.3 parts of ammonium nitrate, 0.4-0.8 parts of potassium hydroxide, phosphoric acid hydrogen
0.5-1 parts of calcium, 0.3-0.6 parts of magnesium bicarbonate, 8-12 parts of matrimony vine, 6-13 parts of sugarcane, 7-10 parts of Mao Li, 5-8 parts of root of Chinese trichosanthes, cynomorium songaricum
150-200 parts of 7-10 parts, 0.05-0.2 parts of septichen sodium, 0.01-0.3 parts of 8-hydroxyquinoline and deionized water.
Wherein, the raw material for having following parts by weight is made：5-7 parts of sweet potato, 8-12 parts of peptone, 0.1-0.2 parts of ammonium nitrate,
0.4-0.8 parts of potassium hydroxide, 0.5-1 parts of calcium monohydrogen phosphate, 0.3-0.6 parts of magnesium bicarbonate, 9-12 parts of matrimony vine, 6-10 parts of sugarcane, thatch
7-10 parts of chestnut, 5-8 parts of root of Chinese trichosanthes, 8-10 parts of cynomorium songaricum, 0.1-0.2 parts of septichen sodium, 0.1-0.3 parts of 8-hydroxyquinoline and
150-180 parts of deionized water.
Wherein, the raw material for having following parts by weight is made：6 parts of sweet potato, 10 parts of peptone, 0.15 part of ammonium nitrate, hydroxide
0.5 part of potassium, 0.6 part of calcium monohydrogen phosphate, 0.5 part of magnesium bicarbonate, 10 parts of matrimony vine, 8 parts of sugarcane, 8 parts of Mao Li, 7 parts of root of Chinese trichosanthes, cynomorium songaricum 8
Part, 0.1 part of septichen sodium, 0.2 part of 8-hydroxyquinoline and 160 parts of deionized water.
In order to achieve the above object, the present invention also provides a kind of preparation sides of the bacillus subtilis bacterium culture medium
Method, wherein,
The first step takes sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds in deionized water, impregnates 1-2 hours, then boil simultaneously
Continue 20-30 minutes, filtering takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.2-0.22um, is obtained by filtration thin
Powder, fine powder filterability reach 100%；
Third walks, and the filtrate that the cooling first step obtains to temperature is 80-90 DEG C, the fine powder that second step is obtained and other institutes
Surplus stock is stated to mix according to proportioning, by mixture uniform stirring 30-40 minutes, using a concentration of 60-80% NaOH solution into
Row PH is adjusted, and is adjusted to 7.2-7.3, is then sterilized 10-12 minutes under the conditions of 100-110 DEG C, 100-120kPa, obtain withered grass
Bacillus culture medium.
Matrimony vine：【Source】This product is the dry mature fruit of plant of Solanaceae lycium barbarum.Autumn fruit harvests when taking on a red color,
Hot-air seasoning removes carpopodium.【Nature and flavor】Sweet and neutral.3.39 milligrams of % containing carrotene in matrimony vine, 0.23 milligram of % of thiamine, core yellow
Element 0.33 milligram of %, 1.7 milligrams of % of niacin, 3 milligrams of % of ascorbic acid.Still isolate cupreol, linoleic acid.【Major function】
Nourshing kidney, moistening lung, tonifying liver, improving eyesight.
Sugarcane：【Source】Stalk for grass sugarcane.It harvests after autumn, cuts and take aerial part.Prune top tip leaf
It ties up, air-dries and dry out.【Habitats distribution】Southern each province.【Chemical analysis】Candy containing sugarcane 13~27%, caproic acid, glycolic
（gallcollic acid）, glycine, aconitic acid etc..【Habitats distribution】Southern each province.【Chemical analysis】Candy containing sugarcane 13~
27%th, caproic acid, glycolic（gallcollic acid）, glycine, aconitic acid etc..
Mao Li：【Source】Involucre, bark, root for Fagaceae Mao Li.【Habitats distribution】Henan, Shaanxi and the Changjiang river stream
Each province on the south domain.【Chemical analysis】Bark contains tannin.【Major function】Insomnia, digestion-promoting gas, pulmonary tuberculosis, pneumonia.
Root of Chinese trichosanthes：【Source】Dry root of this product for cucurbitaceous plant snakegourd or trichosanthes rosthornii Harms.Autumn, the excavation of two season of winter, are washed
Only, crust is removed, segment is dry.【Chemical analysis】Isolate trichosanthin.Also obtain a variety of amino acid：α methylol silk ammonia
Acid, aspartic acid, citrulling, serine, glutamic acid, threonine, glycine, valine, tyrosine, phenylalanine, histidine,
Lysine, arginine, ornithine and peptides ribose, xylose, arabinose, glucose, galactolipin etc..【Nature and flavor】It is sweet in flavor；It is micro-
It is bitter；Cold nature.【Channel tropism】Return lung；Stomach.【Major function】Clearing heat and promoting fluid；Moistening lung for removing phlegm；Detumescence row is dense.
Cynomorium songaricum：【Source】This product is the dry meat stem of Cynomoriaceae plant cynomorium songaricum.Spring excavates, and removes inflorescence, and segment is shone
It is dry.【Chemical analysis】Containing anthocyanin, triterpenoid saponin and tannin.【Nature and flavor】Sweet, warm.①《Amplification on Materia Medica》：It is " sweet."②《Detailed outline》："
Sweet, warm, it is nontoxic."③《Book on Chinese herbal medicine is looked for the truth》：" it is sweet salty, it is warm-natured."④《Ningxia Chinese herbal medicine handbook》：" it is sweet puckery, it prevents."【Channel tropism】Enter liver,
Kidney channel.【Major function】Kidney tonifying ease constipation.
The advantageous effect that technical scheme of the present invention is brought is：The culture medium of the present invention is applied to the training of bacillus subtilis
It supports, gemma yield is high, at low cost, and obtained bacillus subtilis has higher bacteriostasis property, and stability is good.
Specific embodiment
In order to solve above-mentioned purpose, the present invention enumerates following embodiment and illustrates.
Embodiment 1
Formula：Sweet potato 4g, peptone 7g, ammonium nitrate 0.05g, potassium hydroxide 0.4g, calcium monohydrogen phosphate 0.5g, magnesium bicarbonate
0.3g, matrimony vine 8g, sugarcane 6g, Mao Li 7g, root of Chinese trichosanthes 5g, cynomorium songaricum 7g, septichen sodium 0.05g, 8-hydroxyquinoline 0.01g
With deionized water 150g.
Preparation process：
The first step takes sweet potato, matrimony vine and cynomorium songaricum, adds in deionized water, impregnates 1 hour, then boils and continue 20 minutes,
Filtering, takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.2um, fine powder is obtained by filtration, carefully
Powder filterability reaches 100%；
Third walks, and the obtained filtrate of the cooling first step to temperature is 80 DEG C, the fine powder that second step is obtained with described in other
Surplus stock is mixed according to proportioning, by mixture uniform stirring 30 minutes, a concentration of 60% NaOH solution is used to carry out PH tune
Section, adjusts to 7.2, then sterilizes 10 minutes under the conditions of 100 DEG C, 100kPa, obtain bacillus subtilis bacterium culture medium.
Embodiment 2
Formula：Sweet potato 8g, peptone 13g, ammonium nitrate 0.3g, potassium hydroxide 0.8g, calcium monohydrogen phosphate 1g, magnesium bicarbonate
0.6g, matrimony vine 12g, sugarcane 13g, Mao Li 10g, root of Chinese trichosanthes 8g, cynomorium songaricum 10g, septichen sodium 0.2g, 8-hydroxyquinoline
0.3g and deionized water 200g.
Preparation process：
The first step takes sweet potato, matrimony vine and cynomorium songaricum, adds in deionized water, impregnates 2 hours, then boils and continue 30 minutes,
Filtering, takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.22um, fine powder is obtained by filtration, carefully
Powder filterability reaches 100%；
Third walks, and the obtained filtrate of the cooling first step to temperature is 90 DEG C, the fine powder that second step is obtained with described in other
Surplus stock is mixed according to proportioning, by mixture uniform stirring 40 minutes, the NaOH solution of a concentration of 60-80% is used to carry out PH
It adjusts, adjusts to 7.3, then sterilize 12 minutes under the conditions of 110 DEG C, 120kPa, obtain bacillus subtilis bacterium culture medium.
Embodiment 3
Formula：Sweet potato 5g, peptone 8g, ammonium nitrate 0.1g, potassium hydroxide 0.4g, calcium monohydrogen phosphate 0.5g, magnesium bicarbonate
0.3g, matrimony vine 9g, sugarcane 6g, Mao Li 7g, root of Chinese trichosanthes 5g, cynomorium songaricum 8g, septichen sodium 0.1g, 8-hydroxyquinoline 0.1g and
Deionized water 150g.
Preparation process：
The first step takes sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds in deionized water, impregnates 2 hours, then boil and hold
30 minutes continuous, filtering takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.21um, fine powder is obtained by filtration, carefully
Powder filterability reaches 100%；
Third walks, and the obtained filtrate of the cooling first step to temperature is 85 DEG C, the fine powder that second step is obtained with described in other
Surplus stock is mixed according to proportioning, by mixture uniform stirring 35 minutes, a concentration of 70% NaOH solution is used to carry out PH tune
Section, adjusts to 7.25, then sterilizes 12 minutes under the conditions of 110 DEG C, 110kPa, obtain bacillus subtilis bacterium culture medium.
Embodiment 4
Formula：Sweet potato 7g, peptone 12g, ammonium nitrate 0.2g, potassium hydroxide 0.8g, calcium monohydrogen phosphate 1g, magnesium bicarbonate
0.6g, matrimony vine 12g, sugarcane 10g, Mao Li 10g, root of Chinese trichosanthes 8g, cynomorium songaricum 10g, septichen sodium 0.2g, 8-hydroxyquinoline
0.3g and deionized water 180g.
Preparation process：
The first step takes sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds in deionized water, impregnates 1 hour, then boil and hold
30 minutes continuous, filtering takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.22um, fine powder is obtained by filtration, carefully
Powder filterability reaches 100%；
Third walks, and the obtained filtrate of the cooling first step to temperature is 90 DEG C, the fine powder that second step is obtained with described in other
Surplus stock is mixed according to proportioning, by mixture uniform stirring 40 minutes, a concentration of 80% NaOH solution is used to carry out PH tune
Section, adjusts to 7.3, then sterilizes 12 minutes under the conditions of 110 DEG C, 120kPa, obtain bacillus subtilis bacterium culture medium.
Embodiment 5
Formula：Sweet potato 6g, peptone 10g, ammonium nitrate 0.15g, potassium hydroxide 0.5g, calcium monohydrogen phosphate 0.6g, magnesium bicarbonate
0.5g, matrimony vine 10g, sugarcane 8g, Mao Li 8g, root of Chinese trichosanthes 7g, cynomorium songaricum 8g, septichen sodium 0.1g, 8-hydroxyquinoline 0.2g
With deionized water 160g.
Preparation process：
The first step takes sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds in deionized water, impregnates 2 hours, then boil and hold
30 minutes continuous, filtering takes filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses filtering 0.22um, fine powder is obtained by filtration, carefully
Powder filterability reaches 100%；
Third walks, and the obtained filtrate of the cooling first step to temperature is 90 DEG C, the fine powder that second step is obtained with described in other
Surplus stock is mixed according to proportioning, by mixture uniform stirring 40 minutes, a concentration of 80% NaOH solution is used to carry out PH tune
Section, adjusts to 7.3, then sterilizes 12 minutes under the conditions of 110 DEG C, 120kPa, obtain bacillus subtilis bacterium culture medium.
Experimental subjects
The fermentation of bacillus subtilis culture medium of Example 1-5 cultivates bacillus subtilis, and takes traditional
Group carries out culture comparison to bacillus subtilis bacterium culture medium as a comparison.
The nutrient media components of contrast groups：4.50 parts of glucose, 1 part of peptone, 0.50 part of yeast extract, potassium dihydrogen phosphate 0.8
0.5 part of part and calcium carbonate.
Gemma yield compares
Incubation time：21 hours.
Cultivation temperature：36℃.
Strain uses：Bacillus subtilis is bought by Beijing Bioisystech Co., Ltd of Bo Yihui sections, and product identification is
CMCC(B)63501。
Comparison of test results
Table 1 is compared for the gemma yield of each group
| Group | Incubation time | Cultivation temperature | Gemma yield |
| Embodiment 1 | 21 | 36 | 97.9% |
| Embodiment 2 | 21 | 36 | 98.1% |
| Embodiment 3 | 21 | 36 | 98.2% |
| Embodiment 4 | 21 | 36 | 97.7% |
| Embodiment 5 | 21 | 36 | 98% |
| Contrast groups | 21 | 36 | 68% |
By table 1 it is known that the culture medium of the embodiment of the present invention 1-5, obtain the gemma yield of bacillus subtilis compared with
It is high.
Bacteriostasis property compares
Experimental subjects carries out bacteriostasis property comparison to five groups of obtained bacillus subtilis strains as a comparison above.
Opportunistic pathogen mycelia：Julienne potatoes pyrenomycetes, root-rot rhizoctonia, botrytis cinerea.
Mycelia growth inhibition assay：The opportunistic pathogen mycelia of 4 DEG C of preservations is transferred on PDA tablets and is activated, at 28 DEG C
Culture 4-6d, size one is broken into the aseptic card punch of a diameter of 5mm in pathogen thalline marginal position respectively in insulating box
The bacterium dish of cause is respectively connected to transfer needle picking bacterium dish in PDA tablets, is being accessed at bacterium dish 3mm using point connection
Bacillus subtilis is placed in 28 DEG C of insulating boxs after cultivating 3-5d and measures five groups of embodiments and control group withered grass bud respectively
The inhibition zone radius of spore bacillus.
Comparing result is as follows：
Table 2 is the inhibition zone radius of each group（cm）Comparison
| Group | Julienne potatoes pyrenomycetes | Root-rot rhizoctonia | Botrytis cinerea |
| Embodiment 1 | 0.53 | 1.41 | 1.32 |
| Embodiment 2 | 0.54 | 1.42 | 1.33 |
| Embodiment 3 | 0.56 | 1.43 | 1.34 |
| Embodiment 4 | 0.53 | 1.42 | 1.33 |
| Embodiment 5 | 0.53 | 1.42 | 1.32 |
| Control group | 0.3 | 0.68 | 0.81 |
As can be seen from Table 2, the bacillus subtilis of five embodiments is compared with control group bacillus subtilis, antibacterial work
It is with obvious effects to be higher than control group bacterial strain.
To sum up, culture medium of the invention is applied to the culture of bacillus subtilis, and gemma yield is high, at low cost, and
The bacillus subtilis arrived has higher bacteriostasis property, and stability is good.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of bacillus subtilis bacterium culture medium, which is characterized in that be made by the raw material of following parts by weight：4-8 parts of sweet potato, egg
White 7-13 parts of peptone, 0.05-0.3 parts of ammonium nitrate, 0.4-0.8 parts of potassium hydroxide, 0.5-1 parts of calcium monohydrogen phosphate, magnesium bicarbonate 0.3-0.6
Part, 8-12 parts of matrimony vine, 6-13 parts of sugarcane, 7-10 parts of Mao Li, 5-8 parts of root of Chinese trichosanthes, 7-10 parts of cynomorium songaricum, septichen sodium
150-200 parts of 0.05-0.2 parts, 0.01-0.3 parts of 8-hydroxyquinoline and deionized water；
The preparation method of the bacillus subtilis bacterium culture medium is：
The first step takes sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds in deionized water, impregnates 1-2 hours, then boils and continues
20-30 minutes, filtering took filtrate spare；
Second step crushes sugarcane, root of Chinese trichosanthes and Mao Li mixed grindings, and crosses the filter membrane of 0.2-0.22um, and fine powder is obtained by filtration,
Fine powder filterability reaches 100%；
Third walks, and the filtrate that the cooling first step obtains to temperature is 80-90 DEG C, and the fine powder that second step is obtained is described surplus with other
Remaining raw material is mixed according to proportioning, by mixture uniform stirring 30-40 minute, uses the NaOH solution progress PH of a concentration of 60-80%
It adjusts, adjusts to 7.2-7.3, then sterilize 10-12 minutes under the conditions of 100-110 DEG C, 100-120kPa, obtain withered grass gemma
Baccilus medium.
2. bacillus subtilis bacterium culture medium according to claim 1, which is characterized in that by the raw material system of following parts by weight
：5-7 parts of sweet potato, 8-12 parts of peptone, 0.1-0.2 parts of ammonium nitrate, 0.4-0.8 parts of potassium hydroxide, 0.5-1 parts of calcium monohydrogen phosphate,
0.3-0.6 parts of magnesium bicarbonate, 9-12 parts of matrimony vine, 6-10 parts of sugarcane, 7-10 parts of Mao Li, 5-8 parts of root of Chinese trichosanthes, 8-10 parts of cynomorium songaricum, neighbour
150-180 parts of 0.1-0.2 parts of hydroxybenzoic acid sodium, 0.1-0.3 parts of 8-hydroxyquinoline and deionized water.
3. bacillus subtilis bacterium culture medium according to claim 1 or 2, which is characterized in that by the original of following parts by weight
Material is made：6 parts of sweet potato, 10 parts of peptone, 0.15 part of ammonium nitrate, 0.5 part of potassium hydroxide, 0.6 part of calcium monohydrogen phosphate, magnesium bicarbonate
0.5 part, 10 parts of matrimony vine, 8 parts of sugarcane, 8 parts of Mao Li, 7 parts of root of Chinese trichosanthes, 8 parts of cynomorium songaricum, 0.1 part of septichen sodium, 8- hydroxyl quinolines
160 parts of 0.2 part of quinoline and deionized water.
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| CN101595893B (en) * | 2009-06-05 | 2011-11-02 | 云南省烟草农业科学研究院 | Bacillus subtilis microbial agent for preventing and curing alternaria alternate and preparation method thereof |
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