CN102559560A - Bacillus licheniformis for producing lactic acid at high yield after fermentation, and preparation and application of Bacillus licheniformis - Google Patents
Bacillus licheniformis for producing lactic acid at high yield after fermentation, and preparation and application of Bacillus licheniformis Download PDFInfo
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- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 171
- 238000002360 preparation method Methods 0.000 title claims abstract description 93
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 238000000855 fermentation Methods 0.000 title claims abstract description 49
- 230000004151 fermentation Effects 0.000 title claims abstract description 49
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 28
- 239000004310 lactic acid Substances 0.000 title claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 112
- 239000000843 powder Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of microorganism application, in particular to Bacillus licheniformis for producing lactic acid at high yield after fermentation, and preparations and application of the Bacillus licheniformis. The invention provides Bacillus licheniformis CRVAB001 for producing lactic acid at high yield after fermentation in the collection number of CGMCC No.4850. The invention also provides a microecological liquid preparation comprising the Bacillus licheniformis, a preparation method for the microecological liquid preparation, microecological powder comprising the Bacillus licheniformis, and a preparation method for the microecological powder. The Bacillus licheniformis can be efficiently and quickly fermented to produce the lactic acid, and has great industrial application prospect. The viable content of the liquid preparation is more than 1*10<10> colony-forming units (CFU)/mL, the viable content of the powder is more than 1*10<11>CFU/g, and the effectiveness of the Bacillus licheniformis preparations is fully guaranteed.
Description
Technical field
The present invention relates to the mikrobe Application Areas, particularly, relate to a kind of Bacillus licheniformis and preparation and application of the high lactic acid producing in back of fermenting.
Background technology
Bacillus licheniformis (Bacillus licheniformis) is one of feed grade bacterial strain of China Ministry of Agriculture 2008 No. 1126 bulletin approvals use.Bacillus licheniformis belongs to the amphimicrobe that can produce gemma.The thalline reproduction speed is fast, strong stress resistance, and ability extracellular proteinase, glycase, lypase and multiple amino acids.Compare with bifidus bacillus etc. with milk-acid bacteria, the advantage of genus bacillus be it to external world poor environment very strong resistibility is arranged, compare with the subtilis that is all genus bacillus, the Bacillus licheniformis growth temperature is high 5～7 ℃; Secondly, its growth velocity is moderate, makes the unsuitable folding albumen of just striding film fully folding easily; In addition, the ability in Bacillus licheniformis secretory protein to the substratum approximately is 2 times of subtilis, and the heterologous protein expression level has been reported and reached 20～25mg/mL, is expression level soprano in all genus bacillus of finding report.Bacillus licheniformis treatment diarrhea is fine with the diarrhoea effect, is the desirable strain of making probiotics.In China, Shenyang first pharmacy is in " whole intestines are given birth to " of list marketing in 1992, and its staple is a Bacillus licheniformis just, and the diarrhea effect is remarkable, and has no side effect.
The production technique of genus bacillus mainly contains two kinds at present: solid fermentation method and solution fermentation.Solid fermentation method yields poorly, and labour intensity is big, is prone to pollute, but invests less.This growth pattern of at present domestic many small-scale many employings of genus bacillus manufacturer, production level is uneven, and the product viable count does not wait to hundreds of hundred million from every gram tens.The advantage that liquid fermentation process has that output is big, mechanization degree is high, be easy to monitor, the submerged fermentation transformation efficiency is high, but facility investment is high, and power consumption is bigger.This mode of production constant product quality, through the centrifugal genus bacillus product that obtains that concentrates, its viable count is the highest can to reach every gram more than 1,000,000,000,000.
CN101986865A discloses the preparation method of a kind of Bacillus licheniformis, adopts liquid submerged fermentation of three grade, the continuous feeding batch fermentation, and the gemma rate can reach more than 90%.Liquid fermenting is easy to control, reduce and pollute, but this patent does not disclose the viable count of the Bacillus licheniformis that utilizes this technology fermentation.Patent CN1202522A discloses a bacillus licheniformis and probiotics thereof; This method is utilized solid fermentation method or solution fermentation fermentation Bacillus licheniformis, and is simple to operate, is easy to realize; But this owned by France in the laboratory development, be unfavorable for big popularization of producing.Patent CN102021131A discloses a kind of preparation method of Bacillus licheniformis liquid preparation, liquid submerged fermentation, viable count 2 * 10
9During CFU/mL, obtain needed fermented liquid, this method preparation belongs to typical liquid fermenting, and fermented liquid can be used to prevent and treat watermelon blight; Solution fermentation is difficult for microbiological contamination, but the viable count of this prepared is lower.Patent CN102120972A discloses the preparation method of a kind of Bacillus licheniformis, and this method adopts three thermogrades that Bacillus licheniformis freeze-dried vaccine powder is recovered, and the Bacillus licheniformis after the recovery prepares fermented liquid behind inclined-plane, triangular flask, fermentor tank; Fermented liquid is through 60 ± 2 ℃ of hot air circulation oven dry; Through the Bacillus licheniformis bacterium powder for preparing after three grades of recoveries, viable count is high, and this fermentation method post-processing operation is simple; But the heat oven dry is consuming time oversize, and the viable count loss is bigger.Wang Xinlei etc. (2010) have studied the fermentation condition of Bacillus licheniformis converted starch production lactic acid, and it is high to optimize back lactic acid producing amount, and rate of producing acid is fast, but does not disclose viable count and gemma rate of formation.
Summary of the invention
In order to address the above problem, contriver of the present invention proposes and has accomplished the present invention.
The Bacillus licheniformis CRVAB001 that the purpose of this invention is to provide a kind of high lactic acid producing in back of fermenting.
Another object of the present invention provides a kind of little ecological liquid preparation.
Another object of the present invention provides the preparation method of above-mentioned little ecological liquid preparation.
Another object of the present invention provides a kind of little ecological pulvis.
Another object of the present invention provides the preparation method of above-mentioned little ecological pulvis.
The present invention separates a kind of Bacillus licheniformis of lactic acid producing, called after Bacillus licheniformis CRVAB001 (Bacillus licheniformis).This bacterial strain is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, 100101) on May 17th, 2011, and its deposit number is: CGMCC No.4850.
The invention provides a kind of little ecological liquid preparation, comprise above-mentioned Bacillus licheniformis CRVAB001.
The present invention also provides the preparation method of above-mentioned little ecological liquid preparation, may further comprise the steps:
1) the thalline primary inclined plane is cultivated: Bacillus licheniformis CRVAB001 is inoculated in slant medium cultivates, cultivate down in 30～45 ℃ and obtained the thalline primary inclined plane in 18～36 hours;
2) thalline secondary liquid culture: step 1) gained thalline primary inclined plane is inoculated in liquid nutrient medium cultivates, under 30～45 ℃, the condition of 150～250r/min, cultivate and obtain Bacillus licheniformis liquid, bacterium number>=1.0 * 10
10Stop during CFU/mL cultivating, obtain bacterium liquid;
3) three grades of seed liquor of thalline are cultivated: with step 2) gained bacterium liquid is inoculated in liquid nutrient medium with 1%～5% inoculum size (w/w), and under 30～45 ℃, the condition of 150～250r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain seed liquor;
4) liquid fermenting: earlier with steam to the tank body sky 20～40min that disappears, then fermention medium is added in the fermentor tank, logical HP steam is warming up to 110～130 ℃, and is incubated 20～40min; Logical afterwards water quench to 30～45 ℃ is inoculated in step 3) gained seed liquor in the fermention medium under the aseptic condition, and inoculum size is 2%～5% (w/w), tank pressure 0.03～0.10MPa; 30～45 ℃ of leavening temperatures, rotating speed 150～250r/min adopts aerobic-anaerobism conversion fermentation; Ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, and ventilation in 7～14 hours is than being 1: 1, and (reasonably ventilation is than guaranteeing that oxygen dissolving value is not less than 20% to carry out aerobic fermentation; Help the formation of thalline fermentation and gemma), aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation, collects fermented liquid, obtains little ecological liquid preparation.
Wherein, said slant culture based formulas is: Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, and sodium-chlor 5g/L, agar 20g/L, water 1000mL, pH 7.0～7.5, and is aseptic.
Preparing method according to little ecological liquid preparation of the present invention; Wherein, Said liquid culture based formulas is: count by weight percentage Semen Maydis powder 0.5%～4.0%, dregs of beans 0.5%～2.0%, fish meal 0.1%～0.5%, glucose 0.1%～1.0%, sal epsom 0.01%～0.1%, manganous sulfate 0.01%～0.05%, water surplus; PH 7.0～7.5, and is aseptic.
According to the preparation method of little ecological liquid preparation of the present invention, wherein, said liquid culture based formulas is: count by weight percentage glucose 5%～10%, steeping water 0.03%～0.07%, ammonium sulfate 0.05%～0.20%, MgSO
40.01%～0.03%, FeSO
40.001～0.003%, NaCl 0.1%～0.5%, CaCO
31%～5%, KH
2PO
40.06%～0.16%, water surplus is aseptic.
The present invention also provides a kind of little ecological pulvis, comprises above-mentioned Bacillus licheniformis CRVAB001.
The present invention also provides a kind of preparation method of little ecological pulvis, may further comprise the steps:
1) the thalline primary inclined plane is cultivated: Bacillus licheniformis CRVAB001 is inoculated in slant medium cultivates, cultivate down in 30～45 ℃ and obtained the thalline primary inclined plane in 18～36 hours;
2) thalline secondary liquid culture: step 1) gained thalline primary inclined plane is inoculated in liquid nutrient medium cultivates, under 30～45 ℃, the condition of 150～250r/min, cultivate and obtain Bacillus licheniformis liquid, bacterium number>=1.0 * 10
10Stop during CFU/mL cultivating, obtain bacterium liquid;
3) three grades of seed liquor of thalline are cultivated: with step 2) gained bacterium liquid is inoculated in liquid nutrient medium with 1%～5% inoculum size (w/w), and under 30～45 ℃, the condition of 150～250r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain seed liquor;
4) liquid fermenting: earlier with steam to the tank body sky 20～40min that disappears, then fermention medium is added in the fermentor tank, logical HP steam is warming up to 110～130 ℃, and is incubated 20～40min; Logical afterwards water quench to 30～45 ℃ is inoculated in step 3) gained seed liquor in the fermention medium under the aseptic condition, and inoculum size is 2%～5% (w/w); Tank pressure 0.03～0.10MPa, 30～45 ℃ of leavening temperatures, rotating speed 150～250r/min; Adopt aerobic-anaerobism conversion fermentation, ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate in 7～14 hours than being 1: 1; Carry out aerobic fermentation, aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation, collects fermented liquid.
5) saltout: in step 4) gained fermented liquid, add the aqueous solution of 1%～5% (g/mL) Sodium phosphate, dibasic, 1%～5% (g/mL) calcium chloride, 1%～5% (g/mL) sodium hydroxide, stir 30～60min;
6) filter: in the solution of step 1), add flocculating aids, stir 10～20min, leave standstill 10～90min, get into flame filter press and filter, filtration temperature is 30～50 ℃, and filter pressure is 3～5kg/cm
2, the material moisture of Plate Filtration remains on 30%～50% (weight ratio), obtains filter cake,
Wherein, said flocculating aids is to count by weight percentage one or more in 1%～6% light calcium carbonate, 1%～5% perlite and 1%～5% zeyssatite;
The gained filter cake is directly carried out drying, preferably get into flash dryer, EAT is 120～180 ℃; Input speed is 80～200r/min; The material outlet temperature is 60～90 ℃, and final material moisture is 0.5%～10% (weight ratio), obtains little ecological pulvis.
The present invention also provides above-mentioned little ecological liquid preparation as fodder additives and in the application that improves aspect the aquaculture water environment.
The present invention also provides above-mentioned little ecological pulvis as fodder additives and in the application that improves aspect the aquaculture water environment.
Of the present inventionly contain the little ecological liquid preparation of Bacillus licheniformis or the method for use of pulvis is:
1) Bacillus licheniformis liquid preparation or pulvis are mixed or spice with feed; Adding proportion in the complete diet pellet of pig is 0.1 ‰～2 ‰ (w/w); Adding proportion in fryer and meat duck complete diet pellet is 0.1 ‰～2 ‰ (w/w); Adding proportion in laying hen and egg duck material is 0.1 ‰～2 ‰ (w/w); Adding proportion in ruminating animal fine fodders such as ox and sheep is 0.2 ‰～3 ‰ (w/w); Adding proportion in aquatic animal feed is 500～1000g/ (a mu rice);
2) also can be directly oral or add in the entry and drink.
Two kinds of Bacillus licheniformis probioticses provided by the invention, the quality standard below they have reached respectively:
Liquid preparation is pale brown look liquid, denseer sour fragrance; Viable count>=1 * 10
10CFU/mL, gemma rate of formation>=90%, lactic acid content>=30g/L; Plumbous (in Pb)≤40mg/kg, inorganic arsenic (in As)≤10mg/kg, fluorine (in F)≤1000mg/kg, Salmonellas does not see and detects, and meets national forage health standard.
Pulvis is the yellowish white powder; Viable count>=1 * 10
11CFU/g; Dry pulvis moisture content is 0.5%～10%; Plumbous (in Pb)≤40mg/kg, inorganic arsenic (in As)≤10mg/kg, fluorine (in F)≤1000mg/kg, Salmonellas does not see and detects, and meets national forage health standard.
The little ecological liquid preparation that contains Bacillus licheniformis of the present invention or pulvis and preparation method thereof compared with prior art have following significant advantage:
1) Bacillus licheniformis CRVAB001 of the present invention can decompose multiple sugar and produce acid, produces proteolytic enzyme and glycase, and is high temperature resistant, acidproof, under high salt concn can grow;
2) the present invention adopts the liquid submerged fermentation method; Make preparation technology's efficient be greatly enhanced; The composition and the technological condition for fermentation of the substratum that adopts through each step of further optimization; Make that the viable bacteria content of the Bacillus licheniformis in the finished product is increased substantially, wherein the viable bacteria content in the liquid preparation can reach 1 * 10
10More than the CFU/mL, the viable bacteria content in the pulvis can reach 1 * 10
11More than the CFU/g, guaranteed the validity of Bacillus licheniformis preparation fully;
3) the used Bacillus licheniformis of the present invention can efficiently utilize sugared source fermentation lactic acid producing such as glucose fast, has stronger prospects for commercial application;
4) the used liquid submerged fermentation method of the present invention adopts aerobic-anaerobism conversion fermentation mode, is beneficial to the accumulation of Bacillus licheniformis lactic acid producing;
5) acidproof, the bile tolerance of Bacillus licheniformis preparation of the present invention's preparation keeps efficient stable, for extensively promoting the use of of Bacillus licheniformis preparation laid a good foundation in intestinal environment;
6) the gemma rate of formation of Bacillus licheniformis is high in the Bacillus licheniformis preparation of the present invention, becomes spore rate >=90%, and is more high temperature resistant, is convenient to processing and preservation;
7) Bacillus licheniformis preparation of the present invention have no side effect, have no drug resistance, noresidue, cost is low, effect is remarkable;
8) Bacillus licheniformis preparation of the present invention can improve breeding performonce fo animals as in the fodder additives; Be used in the aquaculture water, can improve breeding environment.
Description of drawings
Bacillus licheniformis CRVAB001 (Bacillus licheniformis); Be stored in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on May 17th, 2011; Institute of Microorganism, Academia Sinica; 100101), its deposit number is: CGMCC No.4850.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, the embodiment that provides has been merely and has illustrated the present invention, rather than in order to limit scope of the present invention.
Strains separation, the purifying of embodiment 1 Bacillus licheniformis CRVAB001 (Bacillus licheniformis)
Respectively from the gi tract collected specimens of broiler chicken, laying hen, piglet, sow; Carry out mikrobe separation, purifying in the laboratory; Therefrom be separated to 114 bacillus; Select 12 bacillus through resistance testing sieves such as acidproof, bile tolerance, resistant protease and heatproofs, select 1 plant height lactic acid producing bacterial strain through the lactic acid producing testing sieve again, can reach 20 μ g/mL through its lactic acid content of gas Chromatographic Determination.This strain number is CRVAB001, is accredited as Bacillus licheniformis (Bacillus licheniformis) through 16S rRNA gene order.This bacterial strain is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, 100101) on May 17th, 2011, and its deposit number is: CGMCC No.4850.
The biological property of embodiment 2 Bacillus licheniformis CRVAB001 (Bacillus licheniformis)
" uncle Jie Shi Bacteria Identification handbook is carried out for ne ar and physicochemical characteristics appraisal basis.This Bacillus licheniformis CRVAB001 microscopic morphology and physicochemical characteristics test-results are as shown in table 1 below.
Table 1 Bacillus licheniformis ne ar and physics and chemistry test-results
| Test subject | The result | Test subject | The result |
| Gramstaining | Positive | Glucide produces acid | |
| The bacterium shape | Shaft-like | Glucose | + |
| Cell dia＞1 μ m | - | Pectinose | + |
| Form gemma | + | Wood sugar | + |
| Gemma expands | - | N.F,USP MANNITOL | + |
| Gemma is circular | - | Lactose | - |
| Catalase | + | The glucose fermentation aerogenesis | - |
| Oxydase | + | The starch hydrolysis | + |
| Anaerobic growth | + | The casein hydrolysis | + |
| The VP test | + | Utilize Citrate trianion | + |
| VP bacterium liquid pH＞7 | - | 50 ℃ of growths | + |
| VP bacterium liquid pH＜6 | + | The 7%NaCl growth | + |
| The MR test | + | PH 5.7 growths | + |
| Nitrate reduction test | + |
The novel Bacillus licheniformis liquor-Y1 of embodiment 3 preparations
1) the Bacillus licheniformis primary inclined plane is cultivated: adopt ordinary method that Bacillus licheniformis CRVAB001 is inoculated in slant medium, cultivate down in 30 ℃ and obtained the Bacillus licheniformis primary inclined plane in 36 hours; Described substratum is the mixture (g/L) in following ratio preparation: Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, and sodium-chlor 5g/L, agar 20g/L, water 1000mL, pH 7.0～7.5;
2) Bacillus licheniformis secondary liquid culture: the Bacillus licheniformis primary inclined plane that step 1) is obtained is inoculated in liquid nutrient medium, under 30 ℃, the condition of 250r/min, cultivates and obtains Bacillus licheniformis liquid, viable count>=1 * 10
10Stop during CFU/mL cultivating; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 4.0%, dregs of beans 2.0%, fish meal 0.5%, glucose 1.0%, sal epsom 0.1%, manganous sulfate 0.05%, and pH 7.0～7.5;
3) the Bacillus licheniformis liquid that three grades of seed liquor cultivations of Bacillus licheniformis: with step 2) obtains is inoculated in seed culture medium with 1% inoculum size (w/w); Under 30 ℃, the condition of 250r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1 * 10
10CFU/mL; Count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 4.0%, dregs of beans 2.0%, fish meal 0.5%, glucose 1.0%, sal epsom 0.1%, manganous sulfate 0.05%, and pH 7.0～7.5;
4) Bacillus licheniformis liquid fermenting: earlier with steam to the tank body sky 40min that disappears, fermention medium is put into fermentor tank, logical then HP steam is warming up to 110 ℃, and insulation 40min; Logical afterwards water quench to 30 ℃, aseptic condition inoculation is down gone into the described Bacillus licheniformis seed liquor of step 3), and inoculum size is 2% (w/w), tank pressure 0.03MPa; 30 ℃ of leavening temperatures, rotating speed 250r/min, adopt aerobic-anaerobism conversion fermentation: ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate in 7～14 hours than being 1: 1; Carry out aerobic fermentation, aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): glucose 10%, steeping water 0.07%, ammonium sulfate 0.20%, MgSO
40.03%, FeSO
40.003%, NaCl 0.5%, CaCO
35%, KH
2PO
40.16%.This lichen bacillus ferments liquid can be directly as the Bacillus licheniformis liquid preparation.
Embodiment 4 preparation Bacillus licheniformis liquid preparation-Y2
1) the Bacillus licheniformis primary inclined plane is cultivated: adopt ordinary method that Bacillus licheniformis CRVAB001 is inoculated in slant medium, cultivate down in 45 ℃ and obtained the Bacillus licheniformis primary inclined plane in 18 hours; Described substratum is the mixture (g/L) in following ratio preparation: Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, and sodium-chlor 5g/L, agar 20g/L, water 1000mL, pH 7.0～7.5;
2) Bacillus licheniformis secondary liquid culture: the Bacillus licheniformis primary inclined plane that step 1) is obtained is inoculated in liquid nutrient medium, under 45 ℃, the condition of 150r/min, cultivates and obtains Bacillus licheniformis liquid, viable count>=1 * 10
10Stop during CFU/mL cultivating; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 0.5%, dregs of beans 0.5%, fish meal 0.1%, glucose 0.1%, sal epsom 0.01%, manganous sulfate 0.01%, and pH 7.0～7.5;
3) the Bacillus licheniformis liquid that three grades of seed liquor cultivations of Bacillus licheniformis: with step 2) obtains is inoculated in seed culture medium with 5% inoculum size (w/w); Under 45 ℃, the condition of 150r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1 * 10
10CFU/mL; Count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 0.5%, dregs of beans 0.5%, fish meal 0.1%, glucose 0.1%, sal epsom 0.01%, manganous sulfate 0.01%, and pH 7.0～7.5;
4) Bacillus licheniformis liquid fermenting: earlier with steam to the tank body sky 20min that disappears, fermention medium is put into fermentor tank, logical then HP steam is warming up to 130 ℃, and insulation 20min; Logical afterwards water quench to 45 ℃, aseptic condition inoculation is down gone into the described Bacillus licheniformis seed liquor of step 3), and inoculum size is 5% (w/w), tank pressure 0.10MPa; 45 ℃ of leavening temperatures, rotating speed 150r/min, adopt aerobic-anaerobism conversion fermentation: ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate in 7～14 hours than being 1: 1; Carry out aerobic fermentation, aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): glucose 5%, steeping water 0.03%, ammonium sulfate 0.05%, MgSO
40.01%, FeSO
40.001%, NaCl 0.1%, CaCO
31%, KH
2PO
40.06%.This lichen bacillus ferments liquid can be directly as the Bacillus licheniformis liquid preparation.
Embodiment 5 preparation Bacillus licheniformis liquid preparation-Y3
1) the Bacillus licheniformis primary inclined plane is cultivated: adopt ordinary method that Bacillus licheniformis CRVAB001 is inoculated in slant medium, cultivate down in 35 ℃ and obtained the Bacillus licheniformis primary inclined plane in 20 hours; Described substratum is the mixture (g/L) in following ratio preparation: Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, and sodium-chlor 5g/L, agar 20g/L, water 1000mL, pH 7.0～7.5;
2) Bacillus licheniformis secondary liquid culture: the Bacillus licheniformis primary inclined plane that step 1) is obtained is inoculated in liquid nutrient medium, under 35 ℃, the condition of 200r/min, cultivates and obtains Bacillus licheniformis liquid, viable count>=1 * 10
10Stop during CFU/mL cultivating; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 3.0%, dregs of beans 1.0%, fish meal 0.3%, glucose 0.5%, sal epsom 0.05%, manganous sulfate 0.03%, and pH 7.0～7.5;
3) the Bacillus licheniformis liquid that three grades of seed liquor cultivations of Bacillus licheniformis: with step 2) obtains is inoculated in seed culture medium with 3% inoculum size (w/w); Under 35 ℃, the condition of 200r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1 * 10
10CFU/mL; Count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 3.0%, dregs of beans 1.0%, fish meal 0.3%, glucose 0.5%, sal epsom 0.05%, manganous sulfate 0.03%, and pH 7.0～7.5;
4) Bacillus licheniformis liquid fermenting: earlier with steam to the tank body sky 30min that disappears, fermention medium is put into fermentor tank, logical then HP steam is warming up to 126 ℃, and insulation 30min; Logical afterwards water quench to 35 ℃, aseptic condition inoculation is down gone into the described Bacillus licheniformis seed liquor of step 3), and inoculum size is 3% (w/w), tank pressure 0.06MPa; 35 ℃ of leavening temperatures, rotating speed 200r/min, adopt aerobic-anaerobism conversion fermentation: ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate in 7～14 hours than being 1: 1; Carry out aerobic fermentation, aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): glucose 7%, steeping water 0.05%, ammonium sulfate 0.15%, MgSO
40.02%, FeSO
40.002%, NaCl 0.3%, CaCO
33%, KH
2PO
40.11%.This lichen bacillus ferments liquid can be directly as the Bacillus licheniformis liquid preparation.
Embodiment 6 preparation Bacillus licheniformis liquid preparation-Y4
1) the Bacillus licheniformis primary inclined plane is cultivated: adopt ordinary method that Bacillus licheniformis CRVAB001 is inoculated in slant medium, cultivate down in 37 ℃ and obtained the Bacillus licheniformis primary inclined plane in 24 hours; Described substratum is the mixture (g/L) in following ratio preparation: Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, and sodium-chlor 5g/L, agar 20g/L, water 1000mL, pH 7.0～7.5;
2) Bacillus licheniformis secondary liquid culture: the Bacillus licheniformis primary inclined plane that step 1) is obtained is inoculated in liquid nutrient medium, under 37 ℃, the condition of 210r/min, cultivates and obtains Bacillus licheniformis liquid, viable count>=1 * 10
10Stop during CFU/mL cultivating; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 3.2%, dregs of beans 1.5%, fish meal 0.3%, glucose 0.5%, sal epsom 0.015%, manganous sulfate 0.03%, and pH 7.0～7.5;
3) the Bacillus licheniformis liquid that three grades of seed liquor cultivations of Bacillus licheniformis: with step 2) obtains is inoculated in seed culture medium with 2% inoculum size (w/w); Under 37 ℃, the condition of 210r/min, cultivate and obtain Bacillus licheniformis seed liquor, viable count>=1 * 10
10CFU/mL; Count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): Semen Maydis powder 3.0%, dregs of beans 1.5%, fish meal 0.3%, glucose 0.5%, sal epsom 0.02%, manganous sulfate 0.03%, and pH 7.0～7.5;
4) Bacillus licheniformis liquid fermenting: earlier with steam to the tank body sky 30min that disappears, fermention medium is put into fermentor tank, logical then HP steam is warming up to 121 ℃, and insulation 30min; Logical afterwards water quench to 37 ℃, aseptic condition inoculation is down gone into the described Bacillus licheniformis seed liquor of step 3), and inoculum size is 2% (w/w), tank pressure 0.05MPa; 37 ℃ of leavening temperatures, rotating speed 210r/min, adopt aerobic-anaerobism conversion fermentation: ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate in 7～14 hours than being 1: 1; Carry out aerobic fermentation, aerobic fermentation stage, 0～8 hour; Dissolved oxygen 20%, 9～14 hours, dissolved oxygen 35%; Stop oxygen supply after 14 hours, intermittently stir, carry out anaerobically fermenting, viable count>=1 * 10
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation; Wherein, count by weight percentage, described fermention medium is in following ratio liquid mixture prepared (%): glucose 8%, steeping water 0.05%, ammonium sulfate 0.10%, MgSO
40.02%, FeSO
40.001%, NaCl 0.2%, CaCO
33%, KH
2PO
40.132%.This lichen bacillus ferments liquid can be directly as the Bacillus licheniformis liquid preparation.
Embodiment 7 preparation Bacillus licheniformis pulvis-F1
After in the lichen bacillus ferments liquid of embodiment 1 preparation, adding the aqueous solution of 1% (g/mL) Sodium phosphate, dibasic, 1% (g/mL) calcium chloride, 1% (g/mL) sodium hydroxide; Stir 30min, add 1% light calcium carbonate, 1% perlite again, stir 10min; Leave standstill 10min; Get into flame filter press then and filter, filtration temperature is 30 ℃, and filter pressure is 3kg/cm
2, the material moisture of Plate Filtration remains on 50% (weight ratio); Filter cake behind the Plate Filtration directly gets into flash dryer and carries out drying, and EAT is 180 ℃, and input speed is 200r/min, and the material outlet temperature is 90 ℃, and final material moisture is 10% (weight ratio), the viable count of the Bacillus licheniformis that obtains>=1 * 10
11CFU/g.
Embodiment 8 preparation Bacillus licheniformis pulvis-F2
After in the lichen bacillus ferments liquid of embodiment 2 preparations, adding the aqueous solution of 5% (g/mL) Sodium phosphate, dibasic, 5% (g/mL) calcium chloride, 5% (g/mL) sodium hydroxide; Stir 60min, add 6% light calcium carbonate, 1% zeyssatite again, stir 20min; Leave standstill 90min; Get into flame filter press then and filter, filtration temperature is 50 ℃, and filter pressure is 5kg/cm
2, the material moisture of Plate Filtration remains on 30% (weight ratio); Filter cake behind the Plate Filtration directly gets into flash dryer and carries out drying, and EAT is 120 ℃, and input speed is 80r/min, and the material outlet temperature is 60 ℃, and final material moisture is 0.5% (weight ratio), the viable count of the Bacillus licheniformis that obtains>=1 * 10
11CFU/g.
Embodiment 9 preparation Bacillus licheniformis pulvis-F3
After in the lichen bacillus ferments liquid of embodiment 3 preparations, adding the aqueous solution of 3% (g/mL) Sodium phosphate, dibasic, 3% (g/mL) calcium chloride, 3% (g/mL) sodium hydroxide; Stir 30min, add 1% perlite, 5% zeyssatite again, stir 20min; Leave standstill 40min; Get into flame filter press then and filter, filtration temperature is 40 ℃, and filter pressure is 4kg/cm
2, the material moisture of Plate Filtration remains on 38% weight ratio); Filter cake behind the Plate Filtration directly gets into flash dryer and carries out drying, and EAT is 160 ℃, and input speed is 150r/min, and the material outlet temperature is 80 ℃, and final material moisture is 2% (weight ratio), the viable count of the Bacillus licheniformis that obtains>=1 * 10
11CFU/g.
Embodiment 10 preparation Bacillus licheniformis pulvis-F4
After in the lichen bacillus ferments liquid of embodiment 4 preparations, adding the aqueous solution of 5% (g/mL) Sodium phosphate, dibasic, 5% (g/mL) calcium chloride, 5% (g/mL) sodium hydroxide; Stir 30min, add 2% light calcium carbonate, 5% perlite again, stir 20min; Leave standstill 20min; Get into flame filter press then and filter, filtration temperature is 42 ℃, and filter pressure is 4kg/cm
2, the material moisture of Plate Filtration remains on 35% (weight ratio); Filter cake behind the Plate Filtration directly gets into flash dryer and carries out drying, and EAT is 160 ℃, and input speed is 200r/min, and the material outlet temperature is 85 ℃, and final material moisture is 1% (weight ratio), the viable count of the Bacillus licheniformis that obtains>=1 * 10
11CFU/g.
The safety research of embodiment 11 Bacillus licheniformis pulvis
With being inoculated in liquid nutrient medium after the Bacillus licheniformis CRVAB001 activation, cultivate 24h for 37 ℃, get 100 μ L bacterium drops and be added in the blood agar, observing behind 37 ℃ of cultivation 48h has not transparent circle.Select 180 of the local thin and small hen chick in 21 age in days Beijing, be divided into 2 groups (test group and control groups) at random, every group of 3 repetitions, each repeats 30 chickens, every intramuscular injection 1mL of test group chicken Bacillus licheniformis CRVAB001 (1.5 * 10
9CFU/mL), every every day oral 1.5 * 10 in addition
9CFU/mL Bacillus licheniformis CRVAB001 bacterium liquid 1mL; Every chicken of control group injection 1mL zero(ppm) water and every day oral 1mL Bacillus licheniformis CRVAB001 substratum, dissect after 14 days.The result finds that blood agar is cultivated and do not seen haemolysis; The test group chicken health and the mental status are good, search for food, drinking-water, ight soil is all normal, and animal does not have death, does not see toxicity symptom in 14 days; Test group chicken when dissected is not also found chicken bladder, common hepatic duct calculus; Test group chicken routine blood test each item index all (is seen table 2) in range of normal value, and there are no significant between control group difference; Viewing test group liver,spleen,kidney, the fabricius bursa, duodenum etc. are not all seen pathological change under the mirror.Explain that Bacillus licheniformis CRVAB001 to animal safety, has no side effect, can relieved use in fodder additives.
Table 2 Bacillus licheniformis safety testing result
The acid research of the product of embodiment 12 Bacillus licheniformis liquid preparations
The bacillus licheniformis liquid preparation of embodiment 4 preparations is handled through removing albumen, carried out chromatography of ions after the centrifuging and detect, the content of measuring lactic acid is 32.8g/L.This result shows that Bacillus licheniformis CRVAB001 has good lactic acid producing ability.
The acid resistance research of embodiment 13 Bacillus licheniformis pulvis
PH is lower in the animal stomach, presents acidity, and the pig stomach pH that grows up is generally 1～2; The pH variation of weanling pig stomach is bigger, is generally 2.60～5.83, and food is 2～6h through the emptying time of stomach; Therefore, Bacillus licheniformis must can tolerate lower pH, could play a role at animal digestive system.The Bacillus licheniformis pulvis of embodiment 5 preparations was handled 2 hours under 37 ℃ in manual simulation's gastric juice (pH is 2.0,3.0,4.0), and the bacterial strain survival rate reaches 90.8%, 92.1% and 96.3% respectively; In manual simulation's gastric juice (pH is 2.0,3.0,4.0), handled 6 hours down for 37 ℃, the survival rate of bacterial strain reaches 89.5%, 90.5% and 95.8% (seeing table 3) respectively; Explain that this Bacillus licheniformis preparation can arrive enteron aisle through also keeping quite high activity behind the gastric juice.
Table 3 Bacillus licheniformis pulvis is 37 ℃ of survival results (* 10 that handle down after 2 or 6 hours in manual simulation's gastric juice
11CFU/g)
The bile tolerance research of embodiment 14 Bacillus licheniformis pulvis
Excretory bile is another obstacle that Bacillus licheniformis plays a role in the digestive tube; Thalline only has higher cholate tolerance; Could keep enough viable counts, thus the performance prebiotic effect, and the bile salt levels fluctuation range is 0.03%～0.3% in the animal small intestine.The viable bacteria survival rate is respectively 99.8%, 98.4%, 96.8% and 91.5% (seeing table 4) the Bacillus licheniformis pulvis of embodiment 8 preparation handled 6h in the pig cholate solution of 0.03%, 0.1%, 0.2% and 0.3% different concns after.Explain that this Bacillus licheniformis preparation has very strong tolerance to cholate.
The survival result (* 10 of table 4 Bacillus licheniformis pulvis after different pig cholate are handled
11CFU/g)
The high temperature resistant research of embodiment 15 Bacillus licheniformis preparations
The Bacillus licheniformis pulvis of embodiment 7 preparation is added into 0.1 ‰ ratio carries out pelletization treatment in the complete diet pellet, and detect the survival rate of bacterial strain.80,90 and 100 ℃ of following pelletization treatment 10 minutes, the Bacillus licheniformis survival rate was respectively at 97.2%, 92.1% and 82.4% (seeing table 5).Explain that this Bacillus licheniformis can the withstand high temperatures pelletization treatment and keep good activity.
The survival rate (* 10 of table 5 Bacillus licheniformis preparation after handling 10 minutes under the different pelleting temperatures
11CFU/g)
The stable storing Journal of Sex Research of embodiment 16 Bacillus licheniformis preparations
Weight ratio with 4% was stored the Bacillus licheniformis pulvis of embodiment 7 preparations 3 months, 6 months and 12 months in Preblend; Its bacterial strain survival rate is respectively 97.8%, 90.1% and 79.8% (seeing table 6); The storage characteristics that this Bacillus licheniformis preparation is described is good; Can in certain period, keep higher number of viable, guarantee the activity of preparation.
Table 6 Bacillus licheniformis pulvis preserves 3,6 and the bacterial strain survival rate (* 10 after 12 months in Preblend
11CFU/g)
The application of embodiment 17 Bacillus licheniformis preparations in animal produces
160 AA broiler chicken are divided into control group and Bacillus licheniformis group at random; Every group of 4 repetitions; 20 chickens of every repetition, control group fed conventional corn-dregs of beans type daily ration are as basal diet, and test group is added the Bacillus licheniformis pulvis of embodiment 8 preparations in basal diet; Add by 0.3% addition, trial period is 21 days.The result shows (seeing table 7), compares with control group, and in the day weight gain raising of 7,14,21 age in days Bacillus licheniformis preparation groups, daily ingestion amount and feedstuff-meat ratio, wherein, the Bacillus licheniformis preparation extremely significantly reduces the average food consumption (P＜0.01) of 7 age in days broiler chicken.
Table 7 Bacillus licheniformis is to the influence of 1～21 age in days broiler chicken production performance
Indicate significant difference (P＜0.05) with indicating different lowercases behind the line data; Indicate not difference extremely significantly (P＜0.01) of different capitalization primary and secondary tables.
Embodiment 18 application of Bacillus licheniformis preparation in aquaculture water
Hazardous and noxious substances such as the ammonia nitrogen in the breeding environment, nitrite and hydrogen sulfide worsen aquaculture water, and havoc is played in benign ecological cycles to water surrounding, and the research report uses probiotics that aquaculture water is had good improvement effect.Back 3 days of cultivating pool water inlet sterilization, control group did not use Bacillus licheniformis, and test group applies the Bacillus licheniformis preparation (viable bacteria content 1.24 * 10 of embodiment 8 preparations by the consumption of 1.5mg/L
11CFU/g), test duration 5 months is put behind the seedling and to be used 1 Bacillus licheniformis in per 10 days, and consumption is 0.75mg/L.Put ammonia nitrogen and the nitrous acid nitrogen content of measuring 1 cultivating pool water body behind the seedling in per 20 days.The result shows that the ammonia nitrogen of control group and nitrous acid nitrogen concentration are passed constantly with culturing time and raise; Compare with control group, test group is tangible downtrending, cultures certainly that its ammonia nitrogen and nitrous acid nitrogen concentration remain at lower level after 35 days, and its MV is respectively 0.12 and 0.015mg/L, significantly is lower than control group (P＜0.05).Explain that Bacillus licheniformis has good effect for the improvement of aquaculture water.
Claims (9)
1. the Bacillus licheniformis CRVAB001 (Bacillus licheniformis) of the high lactic acid producing in back that ferments is characterized in that deposit number is: CGMCC No.4850.
2. a little ecological liquid preparation is characterized in that, comprises the described Bacillus licheniformis CRVAB001 of claim 1.
3. the preparation method of a little ecological liquid preparation is characterized in that, may further comprise the steps:
1) the thalline primary inclined plane is cultivated: with deposit number is that the Bacillus licheniformis CRVAB001 of CGMCC No.4850 is inoculated in slant medium and cultivates, and obtains the thalline primary inclined plane;
2) thalline secondary liquid culture: step 1) gained thalline primary inclined plane is inoculated in liquid nutrient medium cultivates viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain bacterium liquid;
3) three grades of seed liquor cultivations of thalline: with step 2) gained bacterium liquid is inoculated in liquid nutrient medium and cultivates, viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain seed liquor;
4) liquid fermenting: step 3) gained seed liquor is inoculated in the fermention medium, adopts aerobic-anaerobism conversion fermentation, ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate than being 1: 1 viable count>=1 * 10 in 7～14 hours
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation, collects fermented liquid, obtains little ecological liquid preparation.
4. according to the preparation method of the said little ecological liquid preparation of claim 3; It is characterized in that; Said liquid culture based formulas is: count by weight percentage; Semen Maydis powder 0.5%～4.0%, dregs of beans 0.5%～2.0%, fish meal 0.1%～0.5%, glucose 0.1%～1.0%, sal epsom 0.01%～0.1%, manganous sulfate 0.01%～0.05%, water surplus, pH 7.0～7.5.
5. according to the preparation method of the said little ecological liquid preparation of claim 3, it is characterized in that said liquid culture based formulas is: count by weight percentage glucose 5%～10%, steeping water 0.03%～0.07%, ammonium sulfate 0.05%～0.20%, MgSO
40.01%～0.03%, FeSO
40.001～0.003%, NaCl 0.1%～0.5%, CaCO
31%～5%, KH
2PO
40.06%～0.16%, water surplus.
6. a little ecological pulvis is characterized in that, comprises the described Bacillus licheniformis CRVAB001 of claim 1.
7. the preparation method of a little ecological pulvis is characterized in that, may further comprise the steps:
1) the thalline primary inclined plane is cultivated: with deposit number is that the Bacillus licheniformis CRVAB001 of CGMCC No.4850 is inoculated in slant medium and cultivates, and obtains the thalline primary inclined plane;
2) thalline secondary liquid culture: step 1) gained thalline primary inclined plane is inoculated in liquid nutrient medium cultivates viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain bacterium liquid;
3) three grades of seed liquor cultivations of thalline: with step 2) gained bacterium liquid is inoculated in liquid nutrient medium and cultivates, viable count>=1.0 * 10
10Stop during CFU/mL cultivating, obtain seed liquor;
4) liquid fermenting: step 3) gained seed liquor is inoculated in the fermention medium, adopts aerobic-anaerobism conversion fermentation, ventilations in preceding 6 hours of fermentation beginning are than being 1: 0.8, ventilate than being 1: 1 viable count>=1 * 10 in 7～14 hours
10CFU/mL, the gemma rate of formation reaches more than 90%, and lactic acid content>=30g/L stops fermentation, collects fermented liquid;
5) saltout: in step 4) gained fermented liquid, add the aqueous solution of 1%～5% (g/mL) Sodium phosphate, dibasic, 1%～5% (g/mL) calcium chloride, 1%～5% (g/mL) sodium hydroxide, stir;
6) in the solution of step 1), add flocculating aids, stir, leave standstill, filter, obtain filter cake, obtain little ecological pulvis through drying,
Wherein, said flocculating aids is to count by weight percentage one or more in 1%～6% light calcium carbonate, 1%～5% perlite and 1%～5% zeyssatite.
8. the said little ecological liquid preparation of claim 2 is as fodder additives and in the application that improves aspect the aquaculture water environment.
9. the said little ecological pulvis of claim 7 is as fodder additives and in the application that improves aspect the aquaculture water environment.
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| CN103805267B (en) * | 2012-11-07 | 2016-02-10 | 中国石油化工股份有限公司 | A kind of hydrofinishing process of inferior patrol |
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