CN101215537A - Bacillus licheniformis and application thereof - Google Patents
Bacillus licheniformis and application thereof Download PDFInfo
- Publication number
- CN101215537A CN101215537A CNA2007103085374A CN200710308537A CN101215537A CN 101215537 A CN101215537 A CN 101215537A CN A2007103085374 A CNA2007103085374 A CN A2007103085374A CN 200710308537 A CN200710308537 A CN 200710308537A CN 101215537 A CN101215537 A CN 101215537A
- Authority
- CN
- China
- Prior art keywords
- bacillus licheniformis
- cgmcc
- application
- powder
- bacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a bacillus licheniformis and the application. The bacillus licheniformis is BL54 CGMCC No.2276. The bacillus licheniformis BL54 of the invention can utilize industrial or agricultural waste which contains cellulose as raw material such as plant straw, leaf, other industrial or agricultural waste tissue, grain stillage and the like and transmit the cellulose in these raw materials into reducing sugar, which improves the utility value of the waste greatly.
Description
Technical field
The present invention relates to a bacillus licheniformis and application thereof.
Background technology
Vegetable fibre class materials such as Mierocrystalline cellulose, hemicellulose and xylogen are prevalent in the middle of the plant, because their characteristic itself, if as feed, its ratio that is absorbed and used is very low, therefore often are taken as rubbish and meet with discarded.
Only taking annual gramineae Zea plant corn is example, and the vitality of this crop is very indomitable, except that extreme sandy soil and clay, all can cultivate.Investigation shows, the corn that every production is 100 kilograms can produce 17.5 kilograms corn ear (corn husk) and Stigma Maydis, the corn cob of 100 kilograms cornstalk and 41 kilograms.Calculate as can be known, the total amount of byproducts such as cornstalk and corn cob is 158.5% of principal yield, is a quite surprising numeral.But generally speaking, because the composition of cornstalk and corn cob is difficult to be utilized, so they often have been taken as waste, and its value only is 3.4% of principal product.
At present, because the price of cereal products such as corn and soya bean improves constantly, the someone proposes, and the plant processed side product of some rich cellulose is added in the middle of the animal-feed.But its effect is unsatisfactory.Because, in feed, directly add the plant processed side product of rich cellulose, though the cost of feed can be reduced, but because animal is lower to the digestive utilization ratio of these plant processed side products, causing feeding cost not obtain corresponding synchronous reduces, its consequence that directly causes is exactly that the youngling incubation rate descends, and the stage of fattening, the getting fat effect of fowl poultry was relatively poor, so usage quantity has been subjected to considerable restraint.Current only small part is used for mixing with clover, makes piece, and it is edible to supply with livestocks such as ox, sheep, horse.
The world energy sources shortage makes using yeast bacterium or other peculiar microorganism that high starch cereal is changed into alcohol and waits and provide the technical scheme of the energy to arise at the historic moment, and is corresponding, and how its byproduct that brings is used also becomes the very urgent problem that solves of needing.For the country that abounds with corn, Chinese sorghum, paddy rice, wheat, barley, cassava, the byproduct that is rich in vegetable fibre is very various, wood chip the like waste as rice straw, cornstalk, broomcorn straw, straw and forestry source mill, its raw materials cost is lower, and not influenced by staple food supply and price, have very large exploitation and be worth.
The utility value that how to improve high fiber cereal-maize straw the like waste will be following big business opportunity, and improve the digestibility of vegetable fibre, at first will convert it into oligosaccharides or monose.
Microorganism is the source that nature decomposes the enzyme maximum of plant cellulose, and industrial enzymes is produced used material generally all from microorganism at present.
Summary of the invention
The objective of the invention is a bacillus licheniformis and application thereof.
Bacillus licheniformis provided by the present invention is Bacillus licheniformis (Bacillus licheniformis) BL54CGMCC № .2276.
Described Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is for producing brood cell's Gram-positive bacillus.Bacterium colony size 6-8mm, colony shape is irregular, white.The condition of the most suitable growth: temperature is 45 ℃, pH6.5.This bacterial strain is an environmental microorganism.
Described Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276, be preserved in Chinese microorganism strain preservation board of trustee reason person on November 30th, 2007 and understand common micro-organisms center (abbreviation CGMCC, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .2276.
Bacillus licheniformis of the present invention (Bacillus licheniformis) BL54 can ferment and produce cellulolytic enzyme, proteolytic enzyme, the amylase of efficient, has collected good effect in small-sized fermentation test.The cellulolytic enzyme that this bacterial strain produces has stable properties at high temperature, meets present industrial working conditions.
Bacillus licheniformis of the present invention (Bacillus licheniformis) BL54 can utilize the agriculture and industry waste of cellulose to be raw material, as straw, blade, other agriculture waste tissue, vinasse etc., with these raw materials cellulose conversion wherein is reducing sugar, can improve the utility value of waste greatly.
Bacillus licheniformis of the present invention (Bacillus licheniformis) BL54 is owing to can produce cellulolytic enzyme, proteolytic enzyme, amylase, therefore also can utilize this strain fermentation production of cellulose lytic enzyme, proteolytic enzyme, amylase, on industrial production, have very high value.
Description of drawings
Fig. 1 is that each temperature of Bacillus licheniformis (Bacillus licheniformis) BL54 is cultivated the OD590 value after 18 hours
Fig. 2 is that each pH value of Bacillus licheniformis (Bacillus licheniformis) BL54 is cultivated the OD590 value after 18 hours
Fig. 3 is the carboxymethylcelluloenzyme enzyme activity of the different pH values of Bacillus licheniformis (Bacillus licheniformis) BL54 fermented liquid
Fig. 4 is the active bacterium colony detected result of the cellulolytic enzyme photo of Bacillus licheniformis (Bacillus licheniformis) BL54
Fig. 5 is the protease activity bacterium colony detection method photo of Bacillus licheniformis (Bacillus licheniformis) BL54
Fig. 6 is the substratum detected result photo of Bacillus licheniformis (Bacillus licheniformis) BL54 amylolytic enzyme activity bacterium colony
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The acquisition of embodiment 1, Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 and culture condition thereof detect
1, screening and the evaluation of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276
1) acquisition of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276
Respectively get a gram soil from Taichung County,Taiwan Province soil sample three place's different positionss, add 10mlTrypticase respectively
TM(every liter contains Tryptones (Tryptose) 20.0g to Soy Broth (TSB nutrient solution), glucose (Dextrose) 1.0g, Sodium phosphate dibasic (Disodium Phosphate) 2.0g, saltpetre (PotassiumNitrate) 1.0g) after concussion mixes in, (every liter contains peptone (Peptone) 1.0g to use the Mandels-Reese substratum respectively, carboxymethyl cellulose (CMC, Carboxymethyl cellulose) 10.0g, (NH
4)
2SO
41.4g, Urea0.3g, K
2HPO
42.0g, CaCl
2.H
2O0.4g, MgSO
4.7H
2O0.3g, FeSO
4.7H
2O5.0mg, MnSO
4.H
2O1.6mg, ZnSO
4.7H
2O1.4mg, CoCl
2.7H
2O2.0mg transfers to 7.0 with pH, and solid medium then adds 1.5%Agar) carry out 10 times of gradient dilutions to 10
-10Divide respectively in 4 dilution tubes of packing into, each gradient dilution liquid is incubated at 30 ℃, 40 ℃, 50 ℃ differing tempss respectively cultivated 24 hours, the bacterium that tentatively increases with the active bacterium of cellulase is screened.Get 100 μ l to TSA (Tryptic soy agar from each dilution tube again, Difco, Sparks U.S.A.) on the flat board, and smears evenly with L type glass rod, putting back 30 ℃, 40 ℃, 50 ℃ equally respectively cultivated after 24 hours, scratch again with on the new TSA of the single bacterium colony to of picked at random mode picking, and with three-step approach, repeat after the purifying subculture three times, on 40 ℃ of flat boards, obtain the bacterial strain that a strain has cellulase activity and protease activity, with its called after BL54.
2) bacterial classification biochemical identification
Observe the bacterium colony outward appearance kenel of BL54 purifying, and carry out gramstaining, confirm bacterium dyeing form.The result shows, BL54 bacterium colony outward appearance kenel, and gramstaining confirms that the result of bacterium dyeing form is as shown in table 1.
The BL54 bacterium that step 1) screening and purifying are obtained is inoculated in to be cultivated in the BUGM substratum 18～24 hours, the normal saline solution of adorning 18～20ml sterilization is to transparent glass tube, put into turbidometer (Turbidimeter, Biolog, Hayward, California, U.S.A.) in, the O.D. value is transferred to 100%, puts into turbidity standard pipe (turbiditystandards high and low limits, Biolog, Hayward, California, U.S.A.), the gram-positive microorganism scope is 35%～42%, and the bacterium amount is about 4.5 * 10
8/ ml.Then use disinfecting silk or cotton hedysarum scoparium fishing on substratum, be applied on the glass tube walls again, must make the O.D. value between the high value between the low value (35%～42%), pour into after finishing in the clean culture dish, utilize eight pipe extractor (8Channel Pipetman, Biolog, Hayward, California, U.S.A.) draw bacterium liquid, attention must be put back to culture dish with the uptake first time, and quantitatively (150 μ l) puts into BiologMicroPlate (Biolog, Hayward, California, U.S.A.) in, more than action must in 10 minutes, finish, the Biolog MicroPlate that will finish puts into incubator, effect 4,6, after 16～24 hours, see through 95 kinds of trioses, the metabolic of Amino acid or lipid acid is put into Biolog MicroStation machine automatic interpretation metabolism result, and its result is as shown in table 1.
The biochemical trait of table 1, BL54 and Bacillus licheniformis (Bacillus licheniformis) MTCC429 relatively
| BL54 | MTCC429 * | BL54 | MTCC429 * | ||
| Bacterium colony size (mm) | 6-8 | 5-7 | Metabolic substd | ||
| Colony shape | Irregular | Irregular | Melitose | + | - |
| Color | In vain | In vain | Sorbitol Powder | (+) | - |
| Gramstaining | + | + | Trehalose | + | - |
| Spread during cultivation | - | ND | Maltose | + | + |
| Nitrate reduction | + | ND | The black bearberry foline | + | ND |
| Catalase | + | + | Rough gentian second candy | + | ND |
| Growth | Glucose | + | + | ||
| 50℃ | + | - | Semi-lactosi | + | ND |
| 5％NaCl | + | + | Lactose | - | ND |
| Metabolic substd | Sucrose | + | ND | ||
| Dextrin | + | ND | Salicin | + | ND |
| Glycogen | + | ND | Wood sugar | + | ND |
| Acetamido glucose | + | + | Oxysuccinic acid | + | ND |
| Arabic glycocoll | - | - | Glycerine | + | ND |
| Cellobiose | + | + | Beta Alanine | + | - |
| Synanthrin | - | - | The silk amino acid | + | ND |
In the table 1 ,-: feminine gender; +: the positive; (+): the weak positive; ND: do not identify;
* data source: Shivaji S, Chaturvedi P, Suresh K, Reddy GS, Dutt CB, Wainwright M, Narlikar JV, Bhargava PM.Bacillus aerius sp.nov.Bacillus aerophilus sp.nov.Bacillusstratosphericus sp.nov.and Bacillus altitudinis sp.nov.isolated from cryogenic tubesused for collecting air samples from high altitudes.International Journal of Systematic andEvolutionary Microbiology (2006), 56,1465-1473.
3) four primers of 16S rDNA sequence retention zone design of the known bacterium of bacterial classification gene type assay evaluation-foundation, forward primer 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 530F:
5 '-GTGCCAGCAGCCGCGG-3 '; Reverse primer 907R:
5’-CCGTCAATTCCTTTGAGTTT-3’、1492R：
5’-TACGGCTACCTTGTTACGACTT-3’。And demarcate 700 and stain (the IRD of 800nm infrared ray absorption wavelength respectively
700Demarcate primer 2 7F, 530F, IRD
800Demarcate 907R, 1492R).Pcr amplification is with 27F and 1492R primer, and order-checking is with 530F and 907R primer, and SequiTherm Excel is adopted in reaction
TMII DNAsequencing Kit (Epicentre Technologies, Madison, Wisconsin, U.S.A.) and Perkin-ElmerkGeneAmp 9600 PCR system.The order-checking condition is 92 ℃ after 2 minutes earlier, 92 ℃ 30 seconds, 55 ℃ 15 seconds, 70 ℃ totally 30 circulations in 15 seconds then, last 4 ℃ of preservations; Reaction finishes the back and adds 3 μ l Stop solution respectively in each Eppendorf tube, place 92 ℃ of heating 3 minutes, the order-checking product is with 5.5%KB plus gel (LI-COR, Inc.Lincoln, Nebraska, U.S.A.) and LI-COR sequencing system (LI-COR, Inc.Lincoln, Nebraska U.S.A.) analyzes.Utilize Base ImageIR (LI-COR, Inc.Lincoln, Nebraska, U.S.A.) sequence of software acquisition electrophoresis, data are sent into GeneBase (Applied Maths, Belgium) arrange, construct and obtain BL54 bacterial strain 16S rDNA sequence, the nucleotide sequence that this 16S rDNA sequence has sequence 1 in the sequence table, BL54 bacterial strain complete 16S rDNA sequence and GenBank are carried out the bacterial classification comparison, comparison result and Bacillus licheniformis (Bacillus licheniformis) ATCC14580 (GENBANK number is CP000002.3) has 99.8% similarity, with the result of sequence alignment in conjunction with above-mentioned form and physiological and biochemical property, show that the BL54 bacterial strain belongs to Bacillus licheniformis (Bacillus licheniformis), i.e. Bacillus licheniformis (Bacillus licheniformis) BL54.
Bacillus licheniformis (Bacillus licheniformis) BL54, be preserved in Chinese microorganism strain preservation board of trustee reason person on November 30th, 2007 and understand common micro-organisms center (abbreviation CGMCC, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .2276.
2, the mensuration of the optimum growth temperature of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276
Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 with step 1 acquisition, concentration according to 106cfu/L is inoculated in the TSB nutrient solution, be divided into five parts, place respectively under 30 ℃, 40 ℃, 45 ℃, 50 ℃, the 60 ℃ conditions, shake training, cultivate after 18 hours under the culture temperature condition, get bacterium liquid and measure survey OD590 (is blank with TSB), the optimum growth temperature of screening this bacterial classification.
The OD590 value result of cultivation after 18 hours illustrates that Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is best at 45 ℃ of value-added effects as shown in Figure 1 under each culture temperature condition.
3, the mensuration of the suitableeest growth pH value of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276
With Bacillus licheniformis (Bacillus licheniformis) the BL54 CGMCC № .2276 of step 1 acquisition, according to 10
6The concentration of cfu/L is inoculated in the TSB nutrient solution, be divided into 5 groups, regulating the pH value respectively is 6,6.5,7,7.5,8, place 45 ℃ to shake training then, shake in the training process and to get bacterium liquid every 30 minutes and measure and survey OD590 (is blank with TSB), the OD590 value that respectively the cultivating under the pH value condition of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 cultivated after 18 hours as shown in Figure 2, the result shows that Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is that value-added effect is best under 6.5 conditions in the pH value.
The enzymic activity of the generation of embodiment 2, Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 detects
1, the carboxymethylcelluloenzyme enzyme activity identification of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276, and enzyme effect optimum pH is confirmed
1) preparation of the enzyme fluid samples of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 generation
With Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 according to 10
9The inoculum size of cfu/L is inoculated in the Mendels-Reese nutrient solution, in 45 ℃ cultivate 24 hours after, Bacillus licheniformis (Bacillus licheniformis) the BL54 CGMCC № .2276 zymocyte liquid that cultivation is obtained is in 4 ℃ 12, behind centrifugal 10 minutes of the 000rpm, is that 0.45 μ m filtering membrane filters with supernatant with the filter footpath, obtain enzyme liquid (fermented liquid supernatant liquid), be stored in-20 ℃.
2) carboxymethylcelluloenzyme enzyme of the enzyme liquid of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 generation is identified, and the affirmation of enzyme effect optimum pH
The 18mlMandels-Reese substratum is adjusted the pH value respectively to test conditions (pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0 or pH8.5), with 2ml above-mentioned steps 1) after Bacillus licheniformis (Bacilluslicheniformis) the BL57 enzyme liquid that obtains mixes, after 45 ℃ of enzyme optimum temperutures are cultivated 1 hour, measure sugared growing amount with DNS reducing sugar test method, and compare with glucose preparation standard curve.The cellulase relative reactivity calculates, and is 100% to carry out substitution ratio with the live vol of high reactivity pH6.5.
The result as shown in Figure 3, the result shows that the fermented liquid of Bacillus licheniformis (Bacillus licheniformis) BL54 all has good carboxymethylcelluloenzyme enzyme activity at pH6-8, but is best with pH6.5.
2, the cellulolytic enzyme of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is active detects
With Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 bacterium respectively with in the middle of the puncture needle inoculation TSA flat board, containing the quality percentage composition at media surface tiling 3.5ml respectively is that 1% carboxymethyl cellulose (CMC) and quality percentage composition are the substratum (being used to detect the endoglucanase activity) of 0.7% agarose, and culture dish is placed on 45 ℃ (about 18-24 hour) overnight cultivation.The mass percentage concentration that media surface after cultivation adds 10～15ml respectively is 0.1% Congo red solution.After acting on about 10～15 minutes, go excessive solution, with twice of the 1M NaCl of 10～15ml flushing.In red background, there is the periphery of bacterial colonies of endoglucanase activity (be in the substratum Mierocrystalline cellulose be degraded) can present white or pink.Above-mentioned experiment triplicate.
The result shows that to go into to contain the quality percentage composition be that 1% carboxymethyl cellulose and quality percentage composition are that the periphery of bacterial colonies of flat board of the substratum of 0.7% agarose presents rose pink (result as shown in Figure 4) containing the shop, the result shows that Bacillus licheniformis (Bacillus licheniformis) BL54 has hydrolytic action to carboxymethyl cellulose, promptly has the endo-type cellulolytic enzyme.
3, the hydrolase of proteolysis of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 detects
Hydrolase of proteolysis detects and adopts proteoclastic bacterium colony detection method, to contain the substratum of skimming milk (skin milk) matrix, detects bacterial classification proteolytic enzyme (proteinase) activity, and concrete grammar is as described below:
It is that 1.5% skimming milk (matrix) and quality percentage composition are the substratum of 1% agar powder that configuration contains the quality percentage composition, is poured in the culture dish, makes flat board.
20 μ l Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 bacterium liquid are seeded in the middle of above-mentioned preparation dull and stereotyped, and culture dish is placed on 45 ℃ does (about 18-24 hour) overnight cultivation.In dull and stereotyped oyster white background, there is the periphery of bacterial colonies of enzymic activity can present transparent hydrolysis circle, tranmittance is to the size of transparent hydrolysis circle, as can be known its activity difference.
The result as shown in Figure 5, the result shows, can find then that in above-mentioned bacterium colony detection method Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 protease activity is very strong, cultivation overnight is hydrolyzable contains skimming milk matrix more than 1/2 a substratum (Fig. 5).Among Fig. 5, transparent place is the enzymic hydrolysis circle.
4, the amylase activity of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 detects
Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is inoculated into to contain the quality percentage composition be that 1% starch and quality percentage composition are on the culture medium flat plate of 1% agar powder, cultivate overnight after, with mass percentage concentration is after 1% iodine solution dyes, starch is dyed brownish black, if bacterium colony produces amylase, the starch of periphery of bacterial colonies promptly is degraded, and can not be colored.
The result as shown in Figure 6, after the result shows that Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 is inoculated into the culture medium culturing that contains starch matrix (about 18-24 hour) overnight, after dyeing with iodine solution, in the undecomposed brownish black background of starch, as seen a yellowish transparent enzymic hydrolysis circle (yellow) shows that Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 has very strong amylase activity.
Embodiment 3, Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 contain application in the raw material production feed of vegetable fibre in utilization
Present embodiment is a raw material with Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 Receptaculum Helianthi powder, straw, green bean straw powder, millet bar powder, sunflower pole powder, corn cob meal, corn stalk or vinasse, carry out the fermentative production feed, concrete grammar is as described below:
With 500ml beaker dress raw material, scale 10g Receptaculum Helianthi powder, straw, green bean straw powder, millet bar powder, sunflower pole powder, corn cob meal, corn stalk or vinasse are raw material respectively, add 10 respectively in each beaker
6The Bacillus licheniformis of cfu/g raw material (Bacillus licheniformis) BL54 CGMCC № .2276, and add the 50ml deionized water, regulating the pH value is 6.5, after mixing, is statically placed in 45 ℃ of thermostat containers, ferments 24 hours.
Receptaculum Helianthi powder, straw, green bean straw powder, millet bar powder, sunflower pole powder, corn cob meal, corn stalk or vinasse after the fermentation are tiled on the Stainless Steel Disc, and thickness is thinner better, puts 60 ℃ of baking ovens blowing oven dry, to moisture content be below 8%.Pulverize the raw material that is feed with electric crusher, can be used for replacing in the pig chicken cattle and sheep complete diet pellet Semen Maydis powder (W-Gum) raw material.
Before and after the fermentation, measure fermenting raw materials front and back composition according to following method respectively:
(1) analysis of crude protein content
With smart 0.5 gram that claims of sample powder, put into and decompose pipe, and add the 15ml vitriol oil (8mol/L) and 1 catalyzer (Kjeltab catalyst), with crude protein determinator (B-435, B ü chi, Switzerland) hot digestion decomposed 2 hours, till the solution clarification.After the cooling, add 50ml distilled water, with crude protein water distilling apparatus (B-316, B ü chi, Switzerland) distillation Digestive system, and be that 4% boric acid solution adds 2 mixture indicators (methyl red: methyl blue=3: 2) as receiving liquid, distillation time is 5 minutes with the mass percentage concentration of 50ml, receive liquid with the titration of 0.1mol/L HCl solution again, when color is titration end point when rose pink by emerald green becoming.Record HCl drips of solution is quantitative, and calculates crude protein content (%) with following formula.Experimental repeatability: each sample is got two parallel samples and is measured, and is the result with its arithmetical av; When the crude protein mass content more than 25%, allowing relative deviation is 1%; When the crude protein mass content at 10-25%, allowing relative deviation is 2%; When the crude protein mass content below 10%, allowing relative deviation is 3%.
Among the formula I, a: with the used milliliter number of 0.1mol/LHCl drips of solution random sample product; B: with the blank used milliliter number of 0.1mol/LHCl titration; 6.25: nitrogenous coefficient; 0.0014:1ml0.1mol/LHCl solution is equivalent to the nitrogen amount of 0.0014g; HCl demarcates the power valency of concentration/0.1:0.1mol/LHCl solution.
(2) analysis of crude fat content
With smart 1 gram that claims of sample powder, parcel is gone into the filter paper of constant weight (Whatman No.1), put into the reflux condensing tube of Soxhlet device again, after 8 hours, take out filter paper with reflux extraction with anhydrous diethyl ether, place the cabinet of bleeding to make the ether volatilization, again with infra-red moisture meter (IR-200, Derner Instrument Company USA) is dried to filter paper constant weight and write down weight fast, calculates crude fat content (%) with following formula at last.
(3) determination of moisture
Weigh sample and place in the weighing bottle for about 5 g, in 105 ℃ to 110 ℃ loft drier, dry, weighing to the constant, its decrement is moisture.
Moisture (%)=(A/B) * 100
A=is dried decrement (g), B=sample weight (g)
Or with infra-red moisture meter (IR-200, Denver Instrument Company USA) measure its moisture content, and with 24 hours data average after, obtain the moisture content (%) of ight soil.
(4) crude fiber content is measured
Weigh sample 2g, extracting the back with ether puts in the flask together with asbestos 0.5g, adding 200ml sulfuric acid liquid (flask and water cooler are direct) heats immediately, must make solution boiling in the bottle in 1 minute, rotated flask once every 5 minutes, can thorough mixing in order to do solution in the bottle, and must note in the bottle can not being stained with sample on the liquid level.Be blown into air with foam-expelling, boil after 30 minutes and filter it with the Gooch crucible of putting the asbestos thin layer, with the boiling water flushing, to not containing till the acidity the inferior sodium hydroxide solution 200ml that uses, throw out and asbestos are washed in the bottle that flashes back, removed flask in 30 minutes with boiling after water cooler is connected, filter with original Gooch crucible again, with the boiling water flushing till do not contain acidity, use 95% alcohol 15ml again, wash it with ether 10ml then.Put Gooch crucible and in be dissolved in the baking oven with 110 ± 2 ℃ of oven dry of temperature, after cooling scale it.Move again on the ashing stove burn approximately to about 20 minutes braised in soy sauce removing carbon elimination, after the cooling again scale it, calculate the weight of being lost, be the robust fibre amount.
Robust fibre (%)=(A/B) * 100
A=ash content decrement (g), B=sample weight (g)
(5) coarse ash is measured
Weighing sample 5 to 10g is in porcelain crucible, and scorching hot to constant weight in 600 ℃ of temperature, residual amount is coarse ash.
Coarse ash (%)=(A/B) * 100
A=residue weight (g), B=sample weight (g)
(6) fermentation back carbohydrate generates and analyzes
Measuring method is with Miller, and the 1959 DNS reducing sugar test methods of being developed are measured, and compare with glucose preparation standard curve.The total reducing sugars amount that detects is the contained reducing sugar amount of 1 gram raw material, try to achieve the reducing sugar amount (discharging reducing sugar amount=experimental group (raw material after the fermentation) reducing sugar amount-control group (not fermentation raw material) reducing sugar amount) that the enzyme effect discharges through the formula conversion, and with the original reducing sugar amount of each raw material in contrast, to calculate its utilization ratio and effect effectiveness.
Receptaculum Helianthi powder, straw, green bean straw powder, millet bar powder, sunflower pole powder, corn cob meal, corn stalk or vinasse raw material major ingredient, the fiber content analysis result is as shown in table 2 below:
Composition before table 2. fermenting raw materials
| Kind | Crude protein % | Crude fat % | Coarse ash % | Robust fibre % |
| Corn stalk | 6.2 | 2.3 | 5.3 | 23.4 |
| Corn cob | 4.2 | 2.8 | 1.8 | 23.8 |
| Vinasse | 25.3 | 5.3 | 3.4 | 56.8 |
| The Receptaculum Helianthi powder | 7.45 | 3.32 | 8.66 | 15.43 |
| Straw | 1.67 | 1.04 | 5.94 | 38.80 |
| Green bean straw powder | 7.09 | 0.68 | 12.56 | 33.16 |
| Millet bar powder | 5.4 | 1.28 | 5.12 | 28.28 |
| The sunflower pole powder | 2.42 | 0.78 | 4.09 | 40.92 |
It is as shown in table 3 that the different material that utilizes step (6) to detect is handled back reducing sugar generation, and the reducing sugar Growth Rate Calculation formula in the table 3 is:
The reducing sugar generation of the feed that table 3. different material obtains after fermentative processing
| Different material | 1g raw material total reducing sugars amount (mg) before the fermentation | Total reducing sugars amount (mg) after the 1g fermenting raw materials | Reducing sugar rate of increase (%) | (%) renderd a service in effect * |
| The Receptaculum Helianthi powder | 80 | 100.6 | 25.8 | 81.8 |
| Straw | 42.5 | 69.2 | 62.8 | 56.2 |
| Green bean straw powder | 51.5 | 79.3 | 53.9 | 64.5 |
| Millet bar powder | 58.3 | 83.4 | 43.1 | 67.8 |
| The sunflower pole powder | 38.0 | 68.2 | 79.5 | 55.5 |
| Corn cob meal | 69.2 | 115.0 | 66.2 | 93.5. |
| Corn stalk | 46.5 | 91.0 | 95.7 | 75.6％ |
| Vinasse | 14.9 | 45.3 | 204.0 | 38.5％ |
* effect effectiveness is 100% with the original reducing sugar amount of Semen Maydis powder, with total reducing sugars amount after the fermenting raw materials effect divided by Semen Maydis powder reducing sugar amount (123mg) promptly.
Sequence table
<160>1
<210>1
<211>412
<212>DNA
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>1
gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 60
gcgcgcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 120
attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 180
aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 240
gctgaggcgc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 300
aacgatgagt gctaagtgtt agagggtttc cgccctttag tgctgcagca aacgcattaa 360
gcactccgcc tggggagtac ggcgcaagac tgaaactcaa aggaattgac gg 412
Claims (9)
1. Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276.
2. Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 contains application in the material of plant cellulose in degraded.
3. application according to claim 2 is characterized in that: the described material that contains plant cellulose is agriculture waste plant tissue and/or industrial produced wastes.
4. application according to claim 3, it is characterized in that: the method that described degraded contains the material of plant cellulose is to inoculate Bacillus licheniformis (Bacillus licheniformis) BL54CGMCC № .2276 in containing the material of plant cellulose, carries out fermentation culture.
5. application according to claim 4 is characterized in that: the temperature condition of described fermentation culture is 30-50 ℃; The pH value of described fermentation culture is 6-8.
6. application according to claim 5 is characterized in that: the temperature condition of described fermentation culture is 45 ℃; The pH value of described fermentation culture is 6.5.
7. the application of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 in the preparation cellulase.
8. the application of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 in preparation proteolytic enzyme.
9. the application of Bacillus licheniformis (Bacillus licheniformis) BL54 CGMCC № .2276 in preparation amylase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007103085374A CN101215537B (en) | 2007-12-29 | 2007-12-29 | Bacillus licheniformis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007103085374A CN101215537B (en) | 2007-12-29 | 2007-12-29 | Bacillus licheniformis and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101215537A true CN101215537A (en) | 2008-07-09 |
| CN101215537B CN101215537B (en) | 2010-06-16 |
Family
ID=39622047
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007103085374A Expired - Fee Related CN101215537B (en) | 2007-12-29 | 2007-12-29 | Bacillus licheniformis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101215537B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
| CN105524870A (en) * | 2016-01-20 | 2016-04-27 | 杭州富阳高博信息技术服务有限公司 | Environmentally-friendly preparation product capable of promoting straw degradation |
| CN105732132A (en) * | 2016-01-20 | 2016-07-06 | 杭州富阳高博信息技术服务有限公司 | Method for effectively decomposing agricultural waste by using composite microbial preparation |
| CN109482618A (en) * | 2015-12-01 | 2019-03-19 | 北京德瑞丰农业科技有限责任公司 | The purposes of bacillus M2 degradation agricultural wastes |
| CN111621432A (en) * | 2020-04-10 | 2020-09-04 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN111893079A (en) * | 2020-09-03 | 2020-11-06 | 广州希奕餐厨降解设备有限公司 | Compound microbial inoculum for vegetable market garbage compost as well as preparation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3046344B2 (en) * | 1990-10-24 | 2000-05-29 | 塩野義製薬株式会社 | New protease |
| AU4744900A (en) * | 1999-05-28 | 2000-12-18 | Novozymes A/S | Novel endo-beta-1,4-glucanases |
| CN1289659C (en) * | 2002-12-24 | 2006-12-13 | 新疆威仕达生物工程股份有限公司 | High temperature neutral protenase strains, high temperature neutral proleinase and process thereof |
| CN1769424A (en) * | 2005-09-20 | 2006-05-10 | 浙江大学 | Bacillus strain and its uses |
-
2007
- 2007-12-29 CN CN2007103085374A patent/CN101215537B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892182A (en) * | 2010-06-07 | 2010-11-24 | 中国农业大学 | Bacillus licheniformis and application thereof in promotion of cellulose degradation |
| CN109482618A (en) * | 2015-12-01 | 2019-03-19 | 北京德瑞丰农业科技有限责任公司 | The purposes of bacillus M2 degradation agricultural wastes |
| CN105524870A (en) * | 2016-01-20 | 2016-04-27 | 杭州富阳高博信息技术服务有限公司 | Environmentally-friendly preparation product capable of promoting straw degradation |
| CN105732132A (en) * | 2016-01-20 | 2016-07-06 | 杭州富阳高博信息技术服务有限公司 | Method for effectively decomposing agricultural waste by using composite microbial preparation |
| CN111621432A (en) * | 2020-04-10 | 2020-09-04 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN111621432B (en) * | 2020-04-10 | 2022-04-12 | 四川轻化工大学 | Bacillus licheniformis, screening method and application |
| CN111893079A (en) * | 2020-09-03 | 2020-11-06 | 广州希奕餐厨降解设备有限公司 | Compound microbial inoculum for vegetable market garbage compost as well as preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101215537B (en) | 2010-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102934736B (en) | Method for preparing sweet potato skin/ sweet potato powder dreg fermented feed | |
| CN102845592B (en) | Method for producing bio-protein feedstuff through solid-state fermentation of dreg silybum marianum dreg | |
| CN105961854A (en) | Preparation method of corn straw feed | |
| Ahmed et al. | Characterization and valuable applications of xylanase from endophytic fungus Aspergillus terreus KP900973 isolated from Corchorus olitorius | |
| CN101215537B (en) | Bacillus licheniformis and application thereof | |
| CN110218688B (en) | Bacillus marinus LY-23, microbial inoculum and application thereof and product using same | |
| CN106119171A (en) | The anti-microbial formulation of a kind of crop material feed fermentation and fermentation technology thereof | |
| CN105385616A (en) | Bacillus subtilis for effectively degrading zearalenone and application thereof | |
| CN105087444A (en) | Bacillus amyloliquefaciens capable of degrading ZEN (zearalenone) efficiently and application of bacillus amyloliquefaciens | |
| CN101215538B (en) | Bacillus licheniformis and application thereof | |
| CN105379947A (en) | Animal feed and preparation method thereof | |
| CN103667103B (en) | A kind of subtilis and application thereof of decomposing zearalenone and xylan | |
| CN102559508B (en) | Trichodermaviride used for producing cellulose degrading enzyme and its application in urban landscaping waste degradation | |
| CN113115858B (en) | Corn husk bacterial protein fermented feed and preparation method thereof | |
| Adetunji et al. | Production of phytotoxic metabolites with bioherbicidal activities from Lasiodiplodia pseudotheobromae produced on different agricultural wastes using solid-state fermentation | |
| CN1318582C (en) | Once aspergillus niger fermentation process of obtaining complex enzyme of several kinds of enzyme and with high enzyme activity | |
| CN109943502B (en) | Probiotics, leavening agent and application in Chinese herbal medicine preparation | |
| CN111134231A (en) | Zhangzhou bacillus and method for fermenting mulberry leaf powder by using Zhangzhou bacillus | |
| CN106804875A (en) | A kind of sweet potato residue fermented feed and preparation method and application | |
| CN101214004B (en) | Method for using rough material containing vegetable fibre to produce feed | |
| CN103642695B (en) | One Aspergillus oryzae and the application prepared at fermentable in fodder additives thereof | |
| CN109170364A (en) | A kind of fresh water fish feed and preparation method thereof | |
| Egbune et al. | Solid-state fermentation production of L-lysine by Corynebacterium glutamicum (ATCC 13032) using agricultural by-products as substrate | |
| Fadel et al. | Cellulases and animal feed production by solid-state fermentation by Aspergillus fumigatus NRCF-122 mutant | |
| CN104872379B (en) | A kind of fermentate and its fermentation process adding the molten slurry of fish in corn-soybean meal diet |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100616 Termination date: 20161229 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |