CN102845614B - Method for producing lactic acid-producing bacillus subtilis symbiotic feed additive bacteria solution - Google Patents
Method for producing lactic acid-producing bacillus subtilis symbiotic feed additive bacteria solution Download PDFInfo
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- CN102845614B CN102845614B CN201210334412.XA CN201210334412A CN102845614B CN 102845614 B CN102845614 B CN 102845614B CN 201210334412 A CN201210334412 A CN 201210334412A CN 102845614 B CN102845614 B CN 102845614B
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Abstract
The invention belongs to the field of biology, and especially relates to a fermentation method and a fermentation formula of lactic acid-producing bacillus subtilis. The formula comprises the components of, by weight: 1-5 parts of peptone, 1-3 parts of beef extract paste, 1-10 parts of a yeast extract, 5-15 parts of sodium chloride, and 10-100 parts of fructooligosaccharide. The fermentation method comprises the steps that: a seed liquid producing the lactic acid-producing bacillus subtilis is inoculated into fermentation tank loaded with the culture medium. The fermentation condition is that: the materials are continuously cultured under 30-37 DEG C for 24-72h, and is subjected to standing and is cultured for 96-120h, such that the lactic acid-producing bacillus subtilis symbiotic feed additive bacteria solution is obtained. The culturing method of the culture medium can be applied in large-scale productions of the lactic acid-producing bacillus subtilis symbiotic feed additive bacteria solution. The process is stable, probiotic content is high, and production cost is low.
Description
Technical field
The invention belongs to biological field, particularly the cultural method of lactic acid-producing bacillus subtilis.
Background technology
Along with the fast development of national economy and the raising day by day of living standards of the people, people have proposed requirement higher, that upgrade to breeding environment and safety, healthy animal product.And in the last few years, the animal foodstuff security incident of exposure again and again, as " clenbuterol hydrochloride event ", " melamine milk event " etc., none has not caused animal farming industry and has hit weightily, has caused serious trust crisis.China is as the first in the world animal-breeding big country, animal product outlet is very important in national economy, but food-safety problem be take as barrier in some countries such as European, Japanese in recent years, and the part of refusing Chinese animal product enters, cause unnecessary loss to China's economy.This series of problem is all due to " animal production safety " problem, has become the bottleneck of the healthy and sustainable development of the Chinese aquaculture science of restriction.
Generally believe at present in the world, probiotics will have huge application prospect in following animal-breeding, to solving animal production safety, will play larger effect.
Although China is more late than American-European starting aspect the research and development of probiotics, develops quite rapid over nearly 20 years.The producer that produces probiotics was by tens 1600 of developing into 2004 of 2000.According to incompletely statistics, by 2004 only, the output of the various probioticses of China has reached 150,000 tons.What industrialization level was high, larger has Sichuan how bright clearly by 8501,8701,8801,8901,901 series of bacillus development, is respectively used to piglet, growing and fattening pigs, fish, fowl, broiler chicken." cereobiogen " of Dalian Medical College's triturate, " the short Kang Sheng " that Nanjing agricultural university grinds, the DM423 bacterium powder that Songjiang pharmaceutical factory produces, " the dysentery health pulvis " that Heilungkiang veterinary institute is produced, " the increasing rhzomorph " that Beijing nutrient research is produced, lactic acid bacteria complexing agent that Hangzhou Commercial College produces etc.On market, apply more, effect and be mostly preferably that to take bacillus and Bacillus acidi lactici be main compound probiotic oral formulations.Lactic acid-producing bacillus subtilis is carried out to fermenting and producing, and the advantage of comprehensive bacillus subtilis and Bacillus acidi lactici, can make probiotics performance obtain qualitative leap, reaches the object of update.The raw lactic acid-producing bacillus subtilis bacterium of large scale fermentation liquid is the basis and one of key that novel probiotics is produced, but does not have the zymotechnique of the raw lactic acid-producing bacillus subtilis bacterium of batch production large scale fermentation liquid at present.
Summary of the invention
The object of the present invention is to provide the culture medium of a kind of culture medium prescription, culture medium preparation method and acquisition thereof, this formula and culture medium cost are low, draw materials conveniently, are applicable to the cultivation of lactic acid-producing bacillus subtilis; The preparation method of this culture medium is simple, is applicable to extensive preparation.
For achieving the above object, technical scheme of the present invention is:
The culture medium prescription of lactic acid-producing bacillus subtilis, specifically composed of the following components by weight: peptone 1-5 part, beef extract 1-3 part, yeast extract 1-10 part, sodium chloride 5-15 part, FOS 10-100 part.
Further, the culture medium prescription of lactic acid-producing bacillus subtilis, specifically composed of the following components by weight: 3 parts of peptones, 2 parts of beef extracts, 7 parts of yeast extracts, 11 parts, sodium chloride, 75 parts of FOSs.FOS, is called again levulan, FOS, FOS, and molecular formula is that (wherein G is glucose to G-F-Fn, and F is fructose, n=13).
Described culture medium prescription is prepared the method for culture medium, gets peptone, beef extract, yeast extract and the sodium chloride of formula ratio, and adding appropriate water to make the quality volume fraction of peptone is 1-5g/L, and each component is fully dissolved, and obtains mixing material; By described mixing material sterilizing, add the FOS of formula ratio to mix, obtain culture medium.
Culture medium prepared by described method.This culture medium is the special culture media of lactic acid-producing bacillus subtilis, under specific fermentation condition, can obtain good ferment effect.
Two of object of the present invention is to provide produces the method for bacterium liquid for lactic acid-producing bacillus subtilis symphysis unit feed addictive, and the method is simple to operate, and stability is high, and productive rate is high.
For achieving the above object, technical scheme of the present invention is:
Described culture medium is produced the method for bacterium liquid for lactic acid-producing bacillus subtilis symphysis unit feed addictive, specifically comprise the following steps: by producing lactic acid-producing bacillus subtilis seed liquor, be inoculated into and in the fermentation tank that described culture medium is housed, carry out dynamic fermentation, dynamic fermentation condition is: Continuous Cultivation 24-72 hour under 30-37 ℃ of condition, throughput is 0.1-0.5V/Vmin, standing cultivation 96-120 hour, obtains lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid.
Further, described dynamic fermentation condition is dynamic agitation fermentation under 2-20 rev/min of condition.
As preferably, the volume ratio of described seed liquor and culture medium is 1-4: 10, and the liquid amount of described fermentation tank is 10-20ml/100mL, the concentration of described seed liquor is 3-15 * 10
8cfu/mL, the liquid amount of fermentation tank is preferably 20mL/100mL.
Wherein, described seed liquor is secondary seed solution.
Wherein, the preparation method of described secondary seed solution specifically comprises the following steps:
The acquisition of A primary seed solution
By lactic acid-producing bacillus subtilis bacterial classification access nutrient broth, cultivate 24 hours for 37 ℃; Then streak inoculation is on nutrient agar panel, in biochemical cultivation case, through 37 ℃, 24-48 hour, selects some, colonies typical inoculation nutrient agar inclined-plane after cultivating, and puts in 37 ℃, biochemical cultivation case and cultivates 24 hours, as first order seed; During lactic acid-producing bacillus subtilis bacterial classification access nutrient broth, the preferred 1ml/100mL of inoculum concentration;
The acquisition of B secondary seed solution
Get first order seed and be inoculated in nutrient broth, put in 37 ℃ of biochemical cultivation cases and cultivate 24 hours, obtain secondary seed, first order seed is inoculated in the preferred 2ml/100mL of nutrient broth inoculum concentration;
The preparation method of nutrient broth is: peptone 10g, and beef extract 3g, sodium chloride 5g, distilled water, to 1000mL, regulates pH7.4; Press mentioned component and mix, dissolve post-equalization pH, packing flask, every bottle of 225mL, 121 ℃ of autoclaving 15min, obtain nutrient broth.
Beneficial effect of the present invention is: the cultural method based on described culture medium, can be used for large-scale production lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid, and its process stabilizing, probio content is high, and production cost is low.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
In embodiment, lactic acid-producing bacillus subtilis CSY1-3-10-1 strain is as bacterial classification.
The preparation of embodiment 1 culture medium
Formula one: the culture medium prescription of lactic acid-producing bacillus subtilis, specifically composed of the following components by weight: 1 part of peptone, 1 part of beef extract, 1 part of yeast extract, 15 parts, sodium chloride, 100 parts of FOSs.
Formula two: the culture medium prescription of lactic acid-producing bacillus subtilis, specifically composed of the following components by weight: 5 parts of peptones, 3 parts of beef extracts, 10 parts of yeast extracts, 5 parts, sodium chloride, 10 parts of FOSs.
Formula three: the culture medium prescription of lactic acid-producing bacillus subtilis, specifically composed of the following components by weight: 3 parts of peptones, 2 parts of beef extracts, 7 parts of yeast extracts, 11 parts, sodium chloride, 75 parts of FOSs.
Above-mentioned three kinds of formulas are prepared according to the following steps: peptone, beef extract, yeast extract and the sodium chloride of getting formula ratio, adding appropriate water to make the quality volume fraction of peptone is 3g/L (1-5g/L all can), and each component is fully dissolved, obtain mixing material; By described mixing material sterilizing, add the FOS of formula ratio to mix, formula one, two and three obtains culture medium one, culture medium two and culture medium three successively.
The application of embodiment 2 culture mediums
One
Described culture medium is produced the method for bacterium liquid for lactic acid-producing bacillus subtilis symphysis unit feed addictive, specifically comprises the following steps: (concentration is 3 * 10 will to produce lactic acid-producing bacillus subtilis seed liquor
8cfu/mL) be inoculated in the fermentation tank that described culture medium (formula three) is housed and carry out 10 revs/min of dynamic fermentations, fermentation condition is: 30 ℃ of Continuous Cultivation are after 24 hours, standing cultivation is 96 hours again, obtains lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid.Through counting, lactic acid-producing bacillus subtilis symphysis unit feed addictive is greater than 1.80 * 10 with the bacterium number that contains of lactic acid-producing bacillus subtilis in bacterium liquid
10cFU/mL.
Two
Lactic acid-producing bacillus subtilis bacterial classification is accessed to described culture medium (formula two), cultivate 24 hours for 37 ± 1 ℃; Then streak inoculation, on nutrient agar panel, in biochemical cultivation case, is cultivated 24-48 hour under 37 ± 1 ℃ of conditions, selects colonies typical inoculation nutrient agar inclined-plane, puts in 37 ± 1 ℃, biochemical cultivation case and cultivates 24 hours, as first order seed.
Three
Get first order seed and be inoculated in described culture medium (formula one), put in 37 ± 1 ℃ of biochemical cultivation cases and cultivate 24 hours, obtain secondary seed.
Embodiment 3 produces the method for bacterium liquid for lactic acid-producing bacillus subtilis symphysis unit feed addictive
The preparation method of 1 secondary seed solution
Specifically comprise the following steps:
A) acquisition of primary seed solution
By lactic acid-producing bacillus subtilis bacterial classification access nutrient broth (inoculum concentration 1ml/100mL), cultivate 24 hours for 37 ± 1 ℃; Then streak inoculation is on nutrient agar panel, in biochemical cultivation case, selects some, colonies typical inoculation nutrient agar inclined-plane after 37 ℃, 48 hours cultivate, and puts in 37 ℃, biochemical cultivation case and cultivates 24 hours, and as primary seed solution, (concentration is 3 * 10
8cfu/mL);
B) acquisition of secondary seed solution
Get first order seed and be inoculated in nutrient broth (inoculum concentration 2ml/100mL), put in 37 ± 1 ℃ of biochemical cultivation cases and cultivate 24 hours, (concentration is 15 * 10 to obtain secondary seed solution
8cfu/mL).
The method of bacterium liquid for 2 lactic acid-producing bacillus subtilis symphysis unit feed addictives
Specifically comprise the following steps: the described secondary seed of producing lactic acid-producing bacillus subtilis is inoculated in the fermentation tank that described culture medium (the described culture medium three in embodiment 1) is housed and carries out dynamic fermentation, fermentation condition is: 37 ℃ of Continuous Cultivation (2-20 rev/min of dynamic agitation fermentations, throughput is 0.1-0.5V/Vmin) after 72 hours, standing cultivation is 120 hours again, obtains lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid.The volume ratio of described seed liquor and culture medium is 4: 10, and the liquid amount of described fermentation tank is 20ml/100mL.Through counting, lactic acid-producing bacillus subtilis symphysis unit feed addictive is greater than 2.20 * 10 with the bacterium number that contains of lactic acid-producing bacillus subtilis in bacterium liquid
10cFU/mL.
Embodiment 4
With the method preparation of embodiment 3, produce lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid, spray-dried etc. (parameter: drying capacity: 0.1-2.0Kg water/hour; Drying time: 1.0-1.5 second; Maximum air flow amount: 35m
3/ h; Compressed air require: 1000-3000L/h; Temperature: 90-220 ℃; Heated for controlling temperature precision: ± 2 ℃) finally make the first feed addictive of the micro-ecological symphysis of lactic acid-producing bacillus subtilis.
5 batches of first feed addictives of the micro-ecological symphysis of double centner laboratory system have altogether been produced in laboratory trial-production, and lot number is respectively 2011001,2011002,2011003,2011004,2011005.In Additive Production process, strictly by semi-finished product and product inspection regulation, test, semi-finished product are produced and survey report the results are shown in Table 1, and end product quality check situation is in Table 2 (the original survey report of 5 batches of laboratory trial product finished products refers to rear).
The micro-ecological symphysis unit's feed addictive semi-finished product production of table 1 lactic acid bacillus subtilis and assay
Table 2 2011001-2011005 criticizes the first feed addictive laboratory product inspection of the micro-ecological symphysis of lactic acid bacillus subtilis result
The first feed addictive (20110011,20110021,20110031,20110041,20110051) of the micro-ecological symphysis of above-mentioned 5 batches of laboratories lactic acid bacillus subtilis processed.Safe in whole production process, smooth and easy.Each Interventions Requested of semi-finished product and finished product all reach on the indices of < < microbial forage additive (enterprise) quality standard > >.Fully proved this Additive Production process stabilizing; the additive steady quality of formulating according to < < microbial forage additive production standard > > (draft), safe and effective, can carry out large-scale production.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (6)
1. produce the method for bacterium liquid for lactic acid-producing bacillus subtilis symphysis unit feed addictive, it is characterized in that, specifically comprise the following steps: lactic acid-producing bacillus subtilis seed liquor is inoculated into and in the fermentation tank that culture medium is housed, carries out dynamic fermentation, fermentation condition is: Continuous Cultivation 24-72 hour under 30-37 ℃ of condition, throughput is 0.1-0.5V/Vmin, standing cultivation 96-120 hour, obtains lactic acid-producing bacillus subtilis symphysis unit feed addictive bacterium liquid again; The culture medium prescription of described lactic acid-producing bacillus subtilis is specifically composed of the following components by weight: peptone 1-5 part, beef extract 1-3 part, yeast extract 1-10 part, sodium chloride 5-15 part, FOS 10-100 part; Get peptone, beef extract, yeast extract and the sodium chloride of formula ratio, adding appropriate water to make the quality volume parts of peptone is 1-5g/L, and each component is fully dissolved, and obtains mixing material; By described mixing material sterilizing, add the FOS of formula ratio to mix, obtain culture medium.
2. the method for bacterium liquid for production lactic acid-producing bacillus subtilis symphysis according to claim 1 unit feed addictive, it is characterized in that, the culture medium prescription of described lactic acid-producing bacillus subtilis is specifically composed of the following components by weight: 3 parts of peptones, 2 parts of beef extracts, 7 parts of yeast extracts, 11 parts, sodium chloride, 75 parts of FOSs.
3. method according to claim 1, is characterized in that, described dynamic fermentation condition is dynamic agitation fermentation under 2-20 rev/min of condition.
4. method according to claim 1, is characterized in that, the volume ratio of described seed liquor and described culture medium is 1-4:10, and the liquid amount of described fermentation tank is 10-20mL/100mL, and the concentration of described seed liquor is 3-15 * 10
8cfu/mL.
5. method according to claim 1, is characterized in that, described seed liquor is secondary seed solution.
6. method according to claim 1, is characterized in that, the preparation method of described secondary seed solution specifically comprises the following steps:
The acquisition of A first order seed
By lactic acid-producing bacillus subtilis bacterial classification access nutrient broth, cultivate 24 hours for 37 ± 1 ℃; Then streak inoculation, on nutrient agar panel, in biochemical cultivation case, is cultivated 24-48 hour under 37 ± 1 ℃ of conditions, selects colonies typical inoculation nutrient agar inclined-plane, puts in the biochemical cultivation case of 37 ± 1 ℃ and cultivates 24 hours, as first order seed;
The acquisition of B secondary seed solution
Get first order seed and be inoculated in nutrient broth, put in 37 ± 1 ℃ of biochemical cultivation cases and cultivate 24 hours, obtain secondary seed solution.
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