CN104762328A - Fermentation method of increasing anti-bacterial capability of fermentation liquid of marine bacillus subtilis - Google Patents
Fermentation method of increasing anti-bacterial capability of fermentation liquid of marine bacillus subtilis Download PDFInfo
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Abstract
The invention relates to the technical field of bio-fermentation and particularly provides a fermentation method of significantly increasing anti-bacterial capability of fermentation liquid of marine bacillus subtilis with nano aluminum oxide for increasing the anti-bacterial capability of the fermentation liquid. The fermentation method is characterized in that cultivation of the marine bacillus subtilis is divided into two fermentation steps, wherein the first step fermentation promotes the thallus to growth rapidly and in the second fermentation step, the nano aluminum oxide is added to the fermentation liquid obtained in the first step for induce the marine bacillus subtilis to increase the anti-bacterial capability of the fermentation liquid. Compared with a method in the prior art, the fermentation method can significantly increase the anti-bacterial capability of the fermentation liquid of the marine bacillus subtilis. The fermentation method is safe and reliable, is simple, is low in cost, is easy to industrially perform, and further can lay the foundation for industrially economically producing anti-bacterial substances.
Description
Technical field
The present invention relates to technical field of biological fermentation, specifically a kind of fermentation process of the raising sea bacillus subtilis fermented liquid antibacterial ability utilizing nano aluminium oxide that fermented liquid antibacterial ability is significantly improved.
Background technology
Subtilis (
bacillus subtilis) be the one of bacillus.Individual cells 0.7 ~ 0.8*2 ~ 3 micron, uniform coloring.Without pod membrane, peritrichous, can move.Gram-positive microorganism, gemma 0.6-0.9*1.0-1.5 micron, 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 microscler one-tenth 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 modulation scheme research clearer, be extensively distributed in the organism of soil and corruption, easily breed in withered grass leaching juice, therefore named.
The 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.
Subtilis has very strong restraining effect to harmful microorganisms such as the vibrios in aquatic products, intestinal bacteria and baculoviruss, and effectively prevention aquatic products animal intestine is scorching, the diseases such as gill rot.Secrete the function of a large amount of chitinase, the cell walls of chitinase decomposable asymmetric choice net pathogenic fungi and Antifungi disease, decompose the hazardous and noxious substances in culturing pool, purify water; In decomposing pool, residual bait, ight soil, organism etc., have the short grained effect of rubbish in very strong cleaning water;
Current research shows, the composition of substratum, culture temperature, potential of hydrogen can both affect the synthesis of subtilis antimicrobial substance.These work provides good material and phenotype for studying the biosynthetic regulatory mechanism of genus bacillus antimicrobial substance.But the raising of these methods to antibacterial ability has limitation, be difficult to obtain more high antibacterial activity by simply cultivating.And nano material was widely used in the fields such as engineering manufacture, medical science in recent years owing to having some properties, and can nano material cause Physiological Effect also seldom to report to microorganism.
Summary of the invention
The object of the present invention is to provide the fermentation process of the raising sea bacillus subtilis fermented liquid antibacterial ability utilizing nano aluminium oxide that fermented liquid antibacterial ability is significantly improved.
The present invention can be achieved through the following technical solutions:
A kind of method improving sea bacillus subtilis fermented liquid antibacterial ability, it is characterized in that this sea bacillus subtilis is cultivated and be divided into two stage fermentations: first stage fermentation promotes thalline ramp, subordinate phase is fermented and is added nano aluminium oxide in the fermented liquid that obtains by fermenting to the first stage and induce sea bacillus subtilis, its fermented liquid antibacterial ability is improved, and above-mentioned method comprises the following steps:
(1) to Landy inoculation of medium bacillus marinus liquid spawn, temperature 28 ~ 30 DEG C, under rotating speed 120 ~ 150 r/min condition, carry out first stage fermentation, fermentation time is 6-24 hour;
(2) after first stage fermentation ends, in fermented liquid, add nano aluminium oxide, proceed subordinate phase fermentation, fermentation time is 48-66 hour.
The interpolation concentration of described nano aluminium oxide is 0.1mmol/L ~ 10mmol/L.
The inoculum size of described liquid spawn is the 1%-4% of Landy culture volume.
The formula of 1 liter of described Landy substratum is: glucose 20 grams, Pidolidone 5g, yeast extract 1 gram, potassium primary phosphate 1 gram, magnesium sulfate heptahydrate 0.5 gram, 0.5 gram, Repone K, manganous sulfate 5 milligrams, cupric sulfate pentahydrate 0.16 milligram, 0.15 milligram, ferric sulfate, be settled to 1 liter with water, regulate pH to 7.0,115 DEG C of sterilizings 25 ~ 40 minutes.
The method of raising sea bacillus subtilis fermented liquid antibacterial ability of the present invention, adds nano aluminium oxide and induces the raw more antimicrobial substance of producing bacillus subtilis when sea bacillus subtilis liquid fermenting.The nano aluminium oxide safety used, amount is few, the antibacterial ability of fermented liquid can be significantly improved, compared with prior art, sea bacillus subtilis fermented liquid antibacterial ability can be significantly improved, and safe and reliable, method is simple, with low cost, adopt the inventive method that the antibacterial ability of fermented liquid can be made to improve 2-3 doubly compared with conventional Landy cultural method.
accompanying drawing illustrates:
Accompanying drawing 1 is first stage incubation time and 2 mM Al in the embodiment of the present invention 1
2o
3the fermented supernatant fluid antibacterial effect of different induction time.
Accompanying drawing 2 is subordinate phase different concns Al in the embodiment of the present invention 2
2o
3fermented supernatant fluid antibacterial effect.
Accompanying drawing 3 is different nano aluminium oxide induction fermentation liquid antibacterial effect schematic diagram adding concentration in the embodiment of the present invention 2.
embodiment:
Below in conjunction with example, the present invention is described in further details:
embodiment one
1. configure Landy substratum, filling a prescription is: glucose 20 grams, Pidolidone 5g, yeast extract 1 gram, potassium primary phosphate 1 gram, magnesium sulfate heptahydrate 0.5 gram, 0.5 gram, Repone K, manganous sulfate 5 milligrams, cupric sulfate pentahydrate 0.16 milligram, 0.15 milligram, ferric sulfate.Be settled to 1 liter with water, regulate pH to 7.0.115 DEG C of sterilizings 25 ~ 40 minutes.
2. in the Landy substratum configured, access Bacillus subtilis strain with the inoculum size of 2 %, temperature 28 DEG C, under rotating speed 150 r/min condition, carry out the fermentation of first stage, fermentation time can be 6 or 12 or 18 or 24 hours.
3. respectively after first stage fermentation ends, aseptically respectively to adding the nano aluminium oxide that ultimate density is 2mmol/L in fermented liquid, carrying out subordinate phase fermentation, adding up to fermentation 72 hours with the first stage.
4., after subordinate phase fermentation ends, the centrifugal thalline of 1200 rpm, collects supernatant liquor, fermented supernatant fluid is detected to the fungistatic effect of Acinetobacter bauamnnii with Odontothrips loti, add 200 μ L supernatant liquors in the cup of Oxford, culture dish is cultivated 24 hours as in 28 DEG C of incubators, statistics antibacterial circle diameter.Detected result is shown in Fig. 1.Antibacterial ability increases obviously compared with comparative example, increases per-cent and sees Fig. 1.
embodiment two
1. configure Landy substratum, filling a prescription is: glucose 20 grams, Pidolidone 5g, yeast extract 1 gram, potassium primary phosphate 1 gram, magnesium sulfate heptahydrate 0.5 gram, 0.5 gram, Repone K, manganous sulfate 5 milligrams, cupric sulfate pentahydrate 0.16 milligram, 0.15 milligram, ferric sulfate.Be settled to 1 liter with water, regulate pH to 7.0.115 DEG C of sterilizings 25 ~ 40 minutes.
2. in the Landy substratum configured, access Bacillus subtilis strain with the inoculum size of 2 %, temperature 28 DEG C, under rotating speed 150 r/min condition, carry out the fermentation of first stage, fermentation time is 12 hours.
3. when first stage fermentation ends, aseptically respectively to the nano aluminium oxide adding different concns in fermented liquid, carry out subordinate phase fermentation, fermentation time is 60 hours, the ultimate density of adding nano aluminium oxide in secondary fermentation liquid is respectively 0.1mmol/L, 0.5mmol/L, 1mmol/L, 5mmol/L, 10mmol/L.
4., after subordinate phase fermentation ends, the centrifugal thalline of 1200 rpm, collects supernatant liquor, fermented supernatant fluid is detected to the fungistatic effect of Acinetobacter bauamnnii with Odontothrips loti, add 200 μ L supernatant liquors in the cup of Oxford, culture dish is cultivated 24 hours as in 28 DEG C of incubators, statistics antibacterial circle diameter.Detected result is shown in Fig. 2.Antibacterial ability increases obviously compared with comparative example, increases per-cent and sees Fig. 2 and accompanying drawing 3.
comparative example:
1. according to the method preparation Landy substratum in embodiment 1, if three repetitions.115 DEG C of sterilizings 30 minutes.With 2 % inoculum size access Bacillus subtilis strains, 28 DEG C, 150 r/min cultivate 72 hours.
2., after fermentation ends, the centrifugal thalline of 1200 rpm, collects supernatant liquor, fermented supernatant fluid is detected to the fungistatic effect of Acinetobacter bauamnnii with Odontothrips loti, add 200 μ L supernatant liquors in the cup of Oxford, culture dish is cultivated 24 hours as in 28 DEG C of incubators, statistics antibacterial circle diameter.
The method of raising sea bacillus subtilis fermented liquid antibacterial ability of the present invention, adds nano aluminium oxide and induces the raw more antimicrobial substance of producing bacillus subtilis when sea bacillus subtilis liquid fermenting.The nano aluminium oxide safety used, amount is few, the antibacterial ability of fermented liquid can be significantly improved, compared with prior art, sea bacillus subtilis fermented liquid antibacterial ability can be significantly improved, and safe and reliable, method is simple, with low cost, adopt the inventive method that the antibacterial ability of fermented liquid can be made to improve 2-3 doubly compared with conventional Landy cultural method.
Claims (7)
1. one kind is improved the fermentation process of sea bacillus subtilis antibacterial ability, it is characterized in that this sea bacillus subtilis is cultivated and be divided into two stage fermentations: first stage fermentation promotes thalline ramp, subordinate phase is fermented and is added nano aluminium oxide in the fermented liquid that obtains by fermenting to the first stage and induce sea bacillus subtilis, and its fermented liquid antibacterial ability is improved.
2. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 1, is characterized in that comprising the following steps:
Step 1: to Landy inoculation of medium bacillus marinus liquid spawn, temperature 28 ~ 30 DEG C, under rotating speed 120 ~ 150 r/min condition, carry out first stage fermentation, fermentation time is 6-24 hour;
Step 2: after first stage fermentation ends, adds nano aluminium oxide in fermented liquid, proceeds subordinate phase fermentation, altogether ferments 72 hours.
3. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 2, is characterized in that the interpolation concentration of described nano aluminium oxide is 0.1 mmol/L ~ 10 mmol/L.
4. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 2, is characterized in that the inoculum size of described liquid spawn is the 1%-4% of Landy culture volume.
5. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 2, is characterized in that the formula of 1 liter of described Landy substratum is: glucose 20 grams, Pidolidone 5g, yeast extract 1 gram, potassium primary phosphate 1 gram, magnesium sulfate heptahydrate 0.5 gram, 0.5 gram, Repone K, manganous sulfate 5 milligrams, cupric sulfate pentahydrate 0.16 milligram, 0.15 milligram, ferric sulfate, is settled to 1 liter with water, regulate pH to 7.0,115 DEG C of sterilizings 25 ~ 40 minutes.
6. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 2, is characterized in that the interpolation concentration of described nano aluminium oxide is 0.1mmol/L or 0.5mmol/L or 1mmol/L or 2mmol/L or 5mmol/L or 10mmol/L.
7. a kind of fermentation process improving sea bacillus subtilis antibacterial ability according to claim 2, is characterized in that first stage fermentation time is 6 or 12 or 18 or 24 hours.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107913607A (en) * | 2017-11-21 | 2018-04-17 | 常州达奥新材料科技有限公司 | A kind of preparation method of ecological bacteriostatic type PES ultrafiltration membrane materials |
| CN110982734A (en) * | 2019-11-21 | 2020-04-10 | 枣庄市杰诺生物酶有限公司 | Marine-derived bacillus subtilis 2713, antibacterial substance and preparation method and application thereof |
| CN111733119A (en) * | 2020-08-25 | 2020-10-02 | 烟台水禾土生物科技有限公司 | High-density fermentation method of bacillus subtilis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107913607A (en) * | 2017-11-21 | 2018-04-17 | 常州达奥新材料科技有限公司 | A kind of preparation method of ecological bacteriostatic type PES ultrafiltration membrane materials |
| CN110982734A (en) * | 2019-11-21 | 2020-04-10 | 枣庄市杰诺生物酶有限公司 | Marine-derived bacillus subtilis 2713, antibacterial substance and preparation method and application thereof |
| CN110982734B (en) * | 2019-11-21 | 2022-01-14 | 枣庄市杰诺生物酶有限公司 | Marine-derived bacillus subtilis 2713, antibacterial substance and preparation method and application thereof |
| CN111733119A (en) * | 2020-08-25 | 2020-10-02 | 烟台水禾土生物科技有限公司 | High-density fermentation method of bacillus subtilis |
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