CN105018397A - Bacillus subtilis culture medium and preparation method thereof - Google Patents

Bacillus subtilis culture medium and preparation method thereof Download PDF

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CN105018397A
CN105018397A CN201510537657.6A CN201510537657A CN105018397A CN 105018397 A CN105018397 A CN 105018397A CN 201510537657 A CN201510537657 A CN 201510537657A CN 105018397 A CN105018397 A CN 105018397A
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culture medium
bacillus subtilis
sugarcane
mao
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CN105018397B (en
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马艳辉
张磊
周爱凤
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Guangdong Zhongshan Maohui Biotechnology Co., Ltd.
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Jinan Shunhao Biotechnology Co Ltd
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Abstract

The invention discloses a Bacillus subtilis culture medium and a preparation method thereof. The culture medium is prepared from components in parts by weight as follows: 4-8 parts of sweet potatoes, 7-13 parts of peptone, 0.05-0.3 parts of ammonium nitrate, 0.4-0.8 parts of potassium hydroxide, 0.5-1 part of calcium hydrophosphate, 0.3-0.6 parts of magnesium bicarbonate, 8-12 parts of Chinese wolfberries, 6-13 parts of sugarcane, 7-10 parts of seguin chestnuts, 5-8 parts of snakegourd roots, 0.05-0.2 parts of sodium salicylate, 0.01-0.3 parts of 8-hydroxyquinoline and 70-90 parts of deionized water. The Bacillus subtilis culture medium and the preparation method thereof have the benefits as follows: the culture medium is applied to culture of Bacillus subtilis, the spore yield is high, the cost is low, and the obtained Bacillus subtilis has the higher anti-bacterial activity and the good stability.

Description

A kind of bacillus subtilis bacterium culture medium and preparation method thereof
Technical field
The present invention relates to substratum field, particularly a kind of bacillus subtilis bacterium culture medium and preparation method thereof.
Background technology
Bacillus subtillis is the one of Bacillus.Individual cells 0.7 ~ 0.8 × 2 ~ 3 microns, uniform coloring.Without pod membrane, peritrichous, can move.Gram-positive microorganism, gemma 0.6 ~ 0.9 × 1.0 ~ 1.5 microns, oval to column, be positioned at thalline central authorities or slightly inclined, after sporulation, thalline does not expand.Bacterium colony surface irregularity is opaque, dirty white or micro-yellow, when growing in liquid medium within, and normal formation wrinkle mould.Aerophil.Available protein, multiple sugar and starch, decompose tryptophane and form indoles.Be widely used in genetics research, to the route of synthesis of the purine nucleotides of this bacterium and its regulation mechanism research clearer.Extensively be distributed in the organism of soil and corruption, easily breed in withered grass leaching juice, therefore named.
The mechanism of action: the 1. subtilyne produced in subtilis thalli growth process, polymyxin, nystatin, linear gramicidins isoreactivity material, the conditioned pathogen of these active substances to pathogenic bacterium or autogenous infection has obvious restraining effect.2. subtilis consumes rapidly the free oxygen in environment, causes enteron aisle hypoxemia, promotes that useful anerobe grows, and produces the organic acids such as lactic acid, reduces enteron aisle pH value, indirectly suppresses the growth of other pathogenic bacterium.3. the growing of stimulating animal immune organ, activates T, bone-marrow-derived lymphocyte, improves immunoglobulin (Ig) and antibody horizontal, strengthens cellular immunization and humoral immune function, improves herd immunity.4. subtilis thalline self synthesizes the enzymes such as α-amylase, proteolytic enzyme, lipase, cellulase, together plays a role in digestive tube with the digestive enzymes in animal body.5. the multiple vitamin B group such as energy synthesise vitamins B1, B2, B6, nicotinic acid, improves the activity of animal body internal interference element and scavenger cell.
Subtilis, as useful microorganism, achieves good effect at present, more and more gets more and more people's extensive concerning in recent years in purifying aquatic product cultivating environment, peaceful control of crop disease, additive for farm animal feed etc.
At present, the substratum of subtilis, exists that gemma productive rate is low, cost is high, and the subtilis activity generated is low, and stability is not high.These problems are problems that the present invention needs to solve.
Summary of the invention
The object of the present invention is to provide a kind of bacillus subtilis bacterium culture medium and preparation method thereof, thus solution gemma productive rate is low, cost is high, subtilis activity is low, the problem that stability is not high.
In order to achieve the above object, the invention provides a kind of bacillus subtilis bacterium culture medium, wherein, obtained by the raw material of following parts by weight: sweet potato 4-8 part, peptone 7-13 part, ammonium nitrate 0.05-0.3 part, potassium hydroxide 0.4-0.8 part, secondary calcium phosphate 0.5-1 part, Magnesium hydrogen carbonate 0.3-0.6 part, matrimony vine 8-12 part, sugarcane 6-13 part, Mao Li 7-10 part, Snakegourd Root 5-8 part, cynomorium songaricum 7-10 part, salicylic acid sodium 0.05-0.2 part, oxine 0.01-0.3 part and deionized water 150-200 part.
Wherein, the raw material of following parts by weight is had to obtain: sweet potato 5-7 part, peptone 8-12 part, ammonium nitrate 0.1-0.2 part, potassium hydroxide 0.4-0.8 part, secondary calcium phosphate 0.5-1 part, Magnesium hydrogen carbonate 0.3-0.6 part, matrimony vine 9-12 part, sugarcane 6-10 part, Mao Li 7-10 part, Snakegourd Root 5-8 part, cynomorium songaricum 8-10 part, salicylic acid sodium 0.1-0.2 part, oxine 0.1-0.3 part and deionized water 150-180 part.
Wherein, the raw material of following parts by weight is had to obtain: sweet potato 6 parts, peptone 10 parts, 0.15 part, ammonium nitrate, 0.5 part, potassium hydroxide, secondary calcium phosphate 0.6 part, Magnesium hydrogen carbonate 0.5 part, matrimony vine 10 parts, sugarcane 8 parts, Mao Li 8 parts, Snakegourd Root 7 parts, cynomorium songaricum 8 parts, 0.1 part, salicylic acid sodium, oxine 0.2 part and deionized water 160 parts.
In order to achieve the above object, present invention also offers a kind of preparation method of described bacillus subtilis bacterium culture medium, wherein,
The first step, gets sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds deionized water, soaks 1-2 hour, then boils and continue 20-30 minute, filtration, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.2-0.22um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 80-90 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 30-40 minute, working concentration is that the NaOH solution of 60-80% carries out PH adjustment, be adjusted to 7.2-7.3, then at 100-110 DEG C, sterilizing 10-12 minute under 100-120kPa condition, obtain bacillus subtilis bacterium culture medium.
Matrimony vine: [source] this product is the dry mature fruit of plant of Solanaceae lycium barbarum.Gather when autumn, fruit took on a red color, hot-air seasoning, removing carpopodium.[nature and flavor] are sweet, flat.Containing carotene 3.39 milligrams of %, VitB1 0.23 milligram of %, riboflavin 0.33 milligram of %, nicotinic acid 1.7 milligrams of %, xitix 3 milligrams of % in matrimony vine.Still isolate β-sitosterol, linolic acid.[function cures mainly] nourshing kidney, moistening lung, tonifying liver, improving eyesight.
Sugarcane: the stem stalk that [source] is grass sugarcane.Gather after autumn, cut and get over-ground part.Top tip leaf of pruning is tied up, and air-dryly dries out.[habitats distribution] southern each province.[Chemical Composition] is containing sugarcane candy 13 ~ 27%, caproic acid, oxyacetic acid (gallcollic acid), glycine, equisetic acid etc.[habitats distribution] southern each province.[Chemical Composition] is containing sugarcane candy 13 ~ 27%, caproic acid, oxyacetic acid (gallcollic acid), glycine, equisetic acid etc.
Mao Li: involucre, bark, root that [source] is Fagaceae Mao Li.Each province on the south [habitats distribution] Henan, Shaanxi and the Yangtze valley.[Chemical Composition] bark contains tannin.[function cures mainly] has a sleepless night, digestion-promoting gas, pulmonary tuberculosis, pneumonia.
Snakegourd Root: [source] this product is the dry root of cucurbitaceous plant snakegourd or trichosanthes rosthornii Harms.Autumn, season in winter two excavate, and clean, removing crust, and segment is dry.[Chemical Composition] isolates Trichosanthin.Also obtain multiple amino acids: α hydroxymethylserine, aspartic acid, citrulline, Serine, L-glutamic acid, Threonine, glycine, α-amino-isovaleric acid, tyrosine, phenylalanine, Histidine, Methionin, arginine, ornithine and peptide class ribose, wood sugar, pectinose, glucose, semi-lactosi etc.[nature and flavor] taste is sweet; Micro-hardship; Cold nature.[return through] returns lung; Stomach warp.[function cures mainly] clearing heat and promoting fluid; Moistening the lung and resolving the phlegm; Row is dense in detumescence.
Cynomorium songaricum: [source] this product is the dry meat stem of Cynomoriaceae plant cynomorium songaricum.Spring excavates, and removing inflorescence, segment, dries.[Chemical Composition] is containing anthocyanin, triterpenoid saponin and tannin.[nature and flavor] are sweet, temperature.1. amplification on Canon of Materia Medica: " sweet." 2. " detailed outline ": " is sweet, temperature, nontoxic." 3. " book on Chinese herbal medicine is looked for the truth ": " is sweet salty, warm in nature." 4. " Ningxia herbal medicine handbook ": " is sweet puckery, prevents." [return through] enters liver, kidney channel.[function cures mainly] kidney tonifying ease constipation.
The beneficial effect that technical scheme of the present invention is brought is: substratum of the present invention is applied to the cultivation of subtilis, and gemma productive rate is high, and cost is low, and the subtilis obtained has higher bacteriostasis property, good stability.
Embodiment
In order to solve above-mentioned purpose, the present invention enumerates following examples and is described.
Embodiment 1
Formula: sweet potato 4g, peptone 7g, ammonium nitrate 0.05g, potassium hydroxide 0.4g, secondary calcium phosphate 0.5g, Magnesium hydrogen carbonate 0.3g, matrimony vine 8g, sugarcane 6g, Mao Li 7g, Snakegourd Root 5g, cynomorium songaricum 7g, salicylic acid sodium 0.05g, oxine 0.01g and deionized water 150g.
Preparation process:
The first step, gets sweet potato, matrimony vine and cynomorium songaricum, adds deionized water, soaks 1 hour, then boils and continue 20 minutes, filters, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.2um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 80 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 30 minutes, working concentration be 60% NaOH solution carry out PH adjustment, be adjusted to 7.2, then 100 DEG C, sterilizing 10 minutes under 100kPa condition, obtain bacillus subtilis bacterium culture medium.
Embodiment 2
Formula: sweet potato 8g, peptone 13g, ammonium nitrate 0.3g, potassium hydroxide 0.8g, secondary calcium phosphate 1g, Magnesium hydrogen carbonate 0.6g, matrimony vine 12g, sugarcane 13g, Mao Li 10g, Snakegourd Root 8g, cynomorium songaricum 10g, salicylic acid sodium 0.2g, oxine 0.3g and deionized water 200g.
Preparation process:
The first step, gets sweet potato, matrimony vine and cynomorium songaricum, adds deionized water, soaks 2 hours, then boils and continue 30 minutes, filters, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.22um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 90 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 40 minutes, working concentration is that the NaOH solution of 60-80% carries out PH adjustment, be adjusted to 7.3, then 110 DEG C, sterilizing 12 minutes under 120kPa condition, obtain bacillus subtilis bacterium culture medium.
Embodiment 3
Formula: sweet potato 5g, peptone 8g, ammonium nitrate 0.1g, potassium hydroxide 0.4g, secondary calcium phosphate 0.5g, Magnesium hydrogen carbonate 0.3g, matrimony vine 9g, sugarcane 6g, Mao Li 7g, Snakegourd Root 5g, cynomorium songaricum 8g, salicylic acid sodium 0.1g, oxine 0.1g and deionized water 150g.
Preparation process:
The first step, gets sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds deionized water, soaks 2 hours, then boils and continue 30 minutes, filters, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.21um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 85 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 35 minutes, working concentration be 70% NaOH solution carry out PH adjustment, be adjusted to 7.25, then 110 DEG C, sterilizing 12 minutes under 110kPa condition, obtain bacillus subtilis bacterium culture medium.
Embodiment 4
Formula: sweet potato 7g, peptone 12g, ammonium nitrate 0.2g, potassium hydroxide 0.8g, secondary calcium phosphate 1g, Magnesium hydrogen carbonate 0.6g, matrimony vine 12g, sugarcane 10g, Mao Li 10g, Snakegourd Root 8g, cynomorium songaricum 10g, salicylic acid sodium 0.2g, oxine 0.3g and deionized water 180g.
Preparation process:
The first step, gets sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds deionized water, soaks 1 hour, then boils and continue 30 minutes, filters, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.22um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 90 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 40 minutes, working concentration be 80% NaOH solution carry out PH adjustment, be adjusted to 7.3, then 110 DEG C, sterilizing 12 minutes under 120kPa condition, obtain bacillus subtilis bacterium culture medium.
Embodiment 5
Formula: sweet potato 6g, peptone 10g, ammonium nitrate 0.15g, potassium hydroxide 0.5g, secondary calcium phosphate 0.6g, Magnesium hydrogen carbonate 0.5g, matrimony vine 10g, sugarcane 8g, Mao Li 8g, Snakegourd Root 7g, cynomorium songaricum 8g, salicylic acid sodium 0.1g, oxine 0.2g and deionized water 160g.
Preparation process:
The first step, gets sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds deionized water, soaks 2 hours, then boils and continue 30 minutes, filters, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.22um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 90 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 40 minutes, working concentration be 80% NaOH solution carry out PH adjustment, be adjusted to 7.3, then 110 DEG C, sterilizing 12 minutes under 120kPa condition, obtain bacillus subtilis bacterium culture medium.
Experimental subjects
The fermentation of bacillus subtilis substratum of Example 1-5 is cultivated subtilis, and get traditional bacillus subtilis bacterium culture medium as a comparison group carry out cultivation contrast.
The nutrient media components of comparative group: glucose 4.50 parts, peptone 1 part, yeast extract paste 0.50 part, potassium primary phosphate 0.8 part and 0.5 part, calcium carbonate.
Gemma productive rate contrasts
Incubation time: 21 hours.
Culture temperature: 36 DEG C.
Bacterial classification uses: subtilis is buied by Beijing Bioisystech Co., Ltd of Bo Yihui section, and production code member is CMCC (B) 63501.
Comparison of test results
Table 1 is the gemma productive rate contrast of each group
Group Incubation time Culture temperature Gemma productive rate
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%
Comparative group 21 36 68%
Can be learnt by table 1, the substratum of embodiments of the invention 1-5, the gemma productive rate obtaining subtilis is higher.
Bacteriostasis property contrasts
Above five groups of bacillus subtilis strains obtained as a comparison experimental subjects carry out bacteriostasis property contrast.
Former bacterium mycelia: julienne potatoes pyrenomycetes, root-rot rhizoctonia, botrytis cinerea.
Mycelial growth suppresses method: be transferred on PDA flat board by the former bacterium mycelia that 4 DEG C are preserved and activate, 4-6d is cultivated in 28 DEG C of thermostat containers, bacterium dish of the same size is broken at pathogenic bacteria thalline marginal position respectively with the aseptic punch tool that diameter is 5mm, access in PDA flat board respectively with inoculating needle picking bacterium dish, utilize some connection access subtilis at distance bacterium dish 3mm place, be placed in the inhibition zone radius measuring five groups of embodiments and control group subtilis after 3-5d cultivated by 28 DEG C of thermostat containers respectively.
Comparing result is as follows:
Table 2 is inhibition zone radius (cm) contrast of each group
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 subtilis of five embodiments is compared with control group subtilis, and the successful of bacteriostatic action is higher than control group bacterial strain.
To sum up, substratum of the present invention is applied to the cultivation of subtilis, and gemma productive rate is high, and cost is low, and the subtilis obtained has higher bacteriostasis property, good stability.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (4)

1. a bacillus subtilis bacterium culture medium, it is characterized in that, obtained by the raw material of following parts by weight: sweet potato 4-8 part, peptone 7-13 part, ammonium nitrate 0.05-0.3 part, potassium hydroxide 0.4-0.8 part, secondary calcium phosphate 0.5-1 part, Magnesium hydrogen carbonate 0.3-0.6 part, matrimony vine 8-12 part, sugarcane 6-13 part, Mao Li 7-10 part, Snakegourd Root 5-8 part, cynomorium songaricum 7-10 part, salicylic acid sodium 0.05-0.2 part, oxine 0.01-0.3 part and deionized water 150-200 part.
2. bacillus subtilis bacterium culture medium according to claim 1, it is characterized in that having the raw material of following parts by weight to obtain: sweet potato 5-7 part, peptone 8-12 part, ammonium nitrate 0.1-0.2 part, potassium hydroxide 0.4-0.8 part, secondary calcium phosphate 0.5-1 part, Magnesium hydrogen carbonate 0.3-0.6 part, matrimony vine 9-12 part, sugarcane 6-10 part, Mao Li 7-10 part, Snakegourd Root 5-8 part, cynomorium songaricum 8-10 part, salicylic acid sodium 0.1-0.2 part, oxine 0.1-0.3 part and deionized water 150-180 part.
3. bacillus subtilis bacterium culture medium according to claim 1 and 2, it is characterized in that having the raw material of following parts by weight to obtain: sweet potato 6 parts, peptone 10 parts, 0.15 part, ammonium nitrate, 0.5 part, potassium hydroxide, secondary calcium phosphate 0.6 part, Magnesium hydrogen carbonate 0.5 part, matrimony vine 10 parts, sugarcane 8 parts, Mao Li 8 parts, Snakegourd Root 7 parts, cynomorium songaricum 8 parts, 0.1 part, salicylic acid sodium, oxine 0.2 part and deionized water 160 parts.
4., according to a preparation method for the arbitrary described bacillus subtilis bacterium culture medium of claim 1-3, it is characterized in that,
The first step, gets sweet potato, matrimony vine and cynomorium songaricum according to proportioning, adds deionized water, soaks 1-2 hour, then boils and continue 20-30 minute, filtration, and it is for subsequent use to get filtrate;
Second step, pulverizes sugarcane, Snakegourd Root and Mao Li mixed grinding, and crosses filtration 0.2-0.22um, and filter and obtain fine powder, fine powder filterability reaches 100%;
3rd step, the filtrate that the cooling the first step obtains is 80-90 DEG C to temperature, the fine powder obtained by second step and surplus stock described in other mix according to proportioning, by mixture uniform stirring 30-40 minute, working concentration is that the NaOH solution of 60-80% carries out PH adjustment, be adjusted to 7.2-7.3, then at 100-110 DEG C, sterilizing 10-12 minute under 100-120kPa condition, obtain bacillus subtilis bacterium culture medium.
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