CN103525714A - Method as well as special Bacillus thermoamylovorans for producing L-lactic acid - Google Patents
Method as well as special Bacillus thermoamylovorans for producing L-lactic acid Download PDFInfo
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Abstract
The invention discloses a method as well as special Bacillus thermoamylovorans for producing L-lactic acid. The Bacillus thermoamylovorans provided by the invention is specifically Bacillus thermoamylovorans CB3 with the preservation number in China General Microbiological Culture Collection Center (CGMCC) of CGMCC No.6153. According to the invention, L-lactic acid is produced by using Bacillus thermoamylovorans CB3 CGMCC No.6153 and taking 20g/L of cellobiose as a substrate through direct fermentation under a condition of 45-55 DEG C. The concentration of L-lactic acid is 12.2g/L, and the optical purity is greater than 98%. The Bacillus thermoamylovorans CB3 CGMCC No.6153 provided by the invention can be used for producing high-optically pure L-lactic acid by direct fermentation of cellobiose, so that reduction of cost of raw materials is facilitated. The method has an important actual industrial application value.
Description
Technical field
The present invention relates to a kind of method and special hot thereof of producing Pfansteihl and bite bacillus amyloliquefaciens.
Background technology
Lactic acid (Lactic Acid) has another name called one of the dihydroxypropionic acid ，Shi world three large organic acids.As a kind of traditional multi-usage fine chemicals, lactic acid can be used as acidic flavoring agent, perfume compound, sanitas, plant-growth regulator, biological degradable material, medicine and agricultural chemicals etc., is applied to food, pharmacy, brewages, in process hides, weaving, environmental protection and agricultural.The production method of lactic acid mainly contains three kinds of chemical synthesis, enzymic synthesis method and fermentation methods.Because synthesis method raw material used is acetaldehyde and violent in toxicity prussic acid, so synthesizing lactic acid is applied to food and can not be widely accepted, and its production cost is also higher in addition.Enzyme law catalysis can obtain optical purity lactic acid in specific manner, but its reaction process research difficulty is very large, is not yet applied to suitability for industrialized production.Production by Microorganism Fermentation lactic acid, can obtain by the selection of bacterial classification and culture condition D-ALPHA-Hydroxypropionic acid, Pfansteihl or both a certain proportion of mixtures, it is that fermenting raw materials is produced lactic acid that the renewable resourcess such as starch, Mierocrystalline cellulose of take decompose resulting glucose etc., and production cost is low, Product Safety is high, it is the main method of scale operation lactic acid.
The production cost of Pfansteihl is one of key factor of restriction poly(lactic acid) mass-producing application.In raising fermentative Production Pfansteihl process, in fermented liquid, the concentration of Pfansteihl is conducive to reduce costs.Fiber biological raw material has the advantage of non-grain resource, is conducive to reduce the raw materials cost of lactic acid-producing, is the effective way of controlling Pfansteihl production cost.Therefore, acquisition can DIRECT UTILIZATION OF CELLULOSE or the bacterial strain of the high optical pure L-lactic acid of fiber carbohydrate fermentative production there is important practical application in industry and be worth.
Summary of the invention
The object of this invention is to provide a strain heat and bite bacillus amyloliquefaciens and the application in producing Pfansteihl thereof.
Heat provided by the present invention is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) and is specially heat and bites in the active sludge of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3，Shi Cong Laiwu in Shandong province sewage work that screening obtains.This heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3 and on May 25th, 2012, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.6153.
Heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 somatic cells and is elongated rod-shaped, misaligned.Gramstaining is positive.At solid culture primary surface, thalline forms translucent oyster white bacterium colony, and bacterium colony is smooth, neat in edge.Atrichia, positive without gemma, growth temperature 30-65 ℃, catalase, its 16s rDNA sequence is as shown in sequence in sequence table 1.
A further object of the present invention is to provide a kind of microbial inoculum.
The activeconstituents of microbial inoculum provided by the present invention is that described heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153.
Another object of the present invention is to provide a kind of method of producing Pfansteihl.
The method of production Pfansteihl provided by the present invention is that the described heat of fermentation is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, or take its described microbial inoculum that is activeconstituents, obtains Pfansteihl.
In described method, described heat is bitten and in the fermention medium of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCCNo.6153, is contained carbon source, nitrogenous source and for the neutralizing agent of controlled fermentation liquid pH; Carbon source in described fermention medium can be at least one in cellobiose, glucose and fructose; The final concentration of described carbon source in described fermention medium can be 20-100 grams per liter (as 20-50 grams per liter).
In the present invention, the carbon source in described fermention medium can be cellobiose, and the final concentration of described cellobiose in described fermention medium specifically can be 20-50 grams per liter (as 20 grams per liters); Carbon source in described fermention medium also can be glucose, and the final concentration of described glucose in described fermention medium specifically can be 20-100 grams per liter (as 20-50 grams per liter, 20 grams per liters or 50 grams per liters).
In described method, the described nitrogenous source in described fermention medium is yeast extract (Angel Yeast Co.,Ltd), and described neutralizing agent is calcium carbonate; The final concentration of described yeast extract in described fermention medium can be 3-10 grams per liter (as 5 grams per liters), and the final concentration of described calcium carbonate in described fermention medium can be 10-40 grams per liter (as 12-30 grams per liter, 12 grams per liters or 30 grams per liters).
In described method, the pH of described fermention medium can be 5.5-7, concrete as 6.5.
In one embodiment of the invention, described fermention medium composed as follows: cellobiose or glucose 20-50 grams per liter, yeast extract 5 grams per liters, calcium carbonate 12-30 grams per liter, pH value is 6.5.
In described method, the condition of described fermentation can be 30-65 ℃ and cultivates 36-72 hour.Be specially in the present invention 45-55 ℃ (as 50 ℃) and cultivate 48-72 hour (as 48 hours or 72 hours).
In described method, the training method of described fermentation is to stir 1-2 time for every 12 hours, and each time of stirring is 30-45 minute, and mixing speed is 30-50 rev/min, and rotation radius is 30-40mm.According to practical situation, also can select standing cultivation.In one embodiment of the invention, the training method of described fermentation is specially every 12 hours and stirs 1 time, and each time of stirring is 30 minutes, and mixing speed is 30 revs/min, and rotation radius is 30-40mm.
In described method, also described heat can be bitten to bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 first accesses in following seed culture medium, at 45-55 ℃, cultivate 24-48 hour (as cultivated 48 hours at 50 ℃), then access described fermention medium; In every liter of described seed culture medium, contain: 20 grams of glucose, 5 grams of yeast powders, 12 grams, calcium carbonate, surplus is water; The pH of described seed culture medium is 6.5.
Another object of the present invention is to provide the preparation method of described microbial inoculum.
The method can comprise the steps: that described heat is bitten to bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, as activeconstituents, obtains described microbial inoculum.
The present invention utilizes heat to bite bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 and produces Pfansteihl, the 20 grams per liter cellobioses of take are substrate, under 45 ℃ of-55 ℃ of conditions, Pfansteihl is produced in direct fermentation, Pfansteihl concentration is 12.2 grams per liters, and optical purity is greater than 98%.Heat provided by the present invention is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 can directly utilize the high optical pure L-lactic acid of cellobiose fermentative production, be conducive to reduce raw materials cost, there is important practical application in industry and be worth.
Preservation explanation
Strain name: heat is bitten bacillus amyloliquefaciens
Latin name: (Bacillus thermoamylovorans)
Strain number: CB3
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on May 25th, 2012
The preservation center numbering of registering on the books: CGMCC No.6153
Accompanying drawing explanation
Fig. 1 is that heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 and utilized cellobiose direct fermentation to produce in Pfansteihl process, the Pfansteihl output at different fermentations temperature.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, the measuring method of related parameters is as follows:
The mensuration of Pfansteihl content: adopt Agilent1260 liquid chromatograph (Anjelen Sci. & Tech. Inc) to measure, organic acid post (U.S. Bio-Rad company, Aminex HPX-87H, W (5 μ m) 4.6ID * 250mm, organic acid is separated to be used), moving phase is 0.005mol/L sulfuric acid, flow velocity 0.6ml/min, sample size 5 μ L, UV-detector, detect wavelength 210nm, 65 ℃ of service temperatures.Pfansteihl standard substance are Sigma-Aldrich company product, and article No. is L1750.
The mensuration of Pfansteihl optical purity: adopt Agilent1260 liquid chromatograph, outfit chiral separation post (Mitsubishi chemical company, MCI GEL-CRS10W, 4.6mm ID * 50mm, optical isomer is separated to be used).Concrete operations condition is: the copper sulfate of 2mM is as moving phase, flow velocity 0.5ml/min, and sample size 10 μ l, UV-detector, detects wavelength 254nm, 25 ℃ of service temperatures.Utilize Pfansteihl and D-ALPHA-Hydroxypropionic acid standard substance to make typical curve, then according to typical curve, calculate the content of Pfansteihl and D-ALPHA-Hydroxypropionic acid in fermented liquid.Optical purity (optical purity) is to weigh in opticity sample the measuring of amount that an enantiomorph surpasses another enantiomorph, and its available enantiomeric excess value (enantiomeric excess) represents.In the present invention, the optical purity of Pfansteihl is calculated as follows: (Pfansteihl content-D-ALPHA-Hydroxypropionic acid content) ÷ (Pfansteihl content+D-ALPHA-Hydroxypropionic acid content) * 100%.
Glucose acid invert ratio is all defined as: the original bulk (grams per liter) * 100% of Pfansteihl output (grams per liter) ÷ cellobiose.
In following embodiment, yeast extract used is all purchased from Angel Yeast Co.,Ltd.
Embodiment 1, heat are bitten separation screening and the evaluation of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3
In the present embodiment, substratum used is composed as follows:
Solid slant culture base: cellobiose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, agar powder 15 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Liquid nutrient medium: cellobiose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
One, heat is bitten the separation screening of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3
From Chinese Laiwu in Shandong province sewage work, obtain active sludge sample, take 5 grams of mud samples in 250ml triangular flask, add 100ml physiological saline, after being fully mixed, with 10000 times of sterilized water dilutions, coat on the solid slant culture base that contains cellobiose, 50 ℃ of cultivations, after growing single bacterium colony, the single bacterium colony of part forms transparent circle owing to producing acid by the dissolution of calcium carbonate in periphery of bacterial colonies substratum.Single bacterium colony that picking transparent circle is larger, be inoculated in the liquid nutrient medium that 10ml contains cellobiose, 50 ℃ of standing cultivations 72 hours, measure Pfansteihl concentration in fermented liquid, the highest bacterial strain of picking one strain Pfansteihl output, be inoculated on the solid slant culture base that contains cellobiose, refrigerate standby.By this bacterial strain called after CB3.
Two, heat is bitten the evaluation of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3
Bacterial strain CB3 step 1 being obtained from form, physiological and biochemical property and the several aspects of 16s rDNA sequence homology identifies.Wherein, morphological specificity observation and physio-biochemical characteristics are identified the method for describing referring in " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) and " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying etc. write, Beijing: Science Press, 2001.2).
1, Morphological Identification
Through Morphological Identification, result shows that the bacterial strain CB3 that step 1 separation screening obtains is gram-positive microorganism, cell elongated rod shape, and long 2.5-3.5 μ m, wide 0.7-0.8 μ m, misaligned.At solid culture primary surface, thalline forms translucent oyster white bacterium colony, and bacterium colony is smooth, neat in edge.Atrichia, without gemma.
2, physiological and biochemical property is identified
Through physiological and biochemical property, identify, result shows that the bacterial strain CB3 that step 1 separation screening obtains can utilize trehalose, Sorbitol Powder, glucose, lactose, ribose, cellobiose, maltose, fructose, N.F,USP MANNITOL, melizitose, rhamnosyl, semi-lactosi, Vitamin C2, seminose; But can not utilize melibiose, amygdaloside, pectinose, inositol, raffinose.Oxidase negative.Hemolytic test is negative.Can be 30 ℃ of-65 ℃ of growths.
3,16s rDNA sequence homology analysis
The ethylenediamine tetraacetic acid (EDTA) (EDTA) of the Tutofusin tris of TE damping fluid: 10mmol/L (Tris), 1mmol/L, with hydrochloric acid, to adjust pH be 8.0.
CTAB/NaCl solution: the cetyltriethylammonium bromide that mass volume ratio is 10% (CTAB) is dissolved in the NaCl of 0.7mol/L.
The PCR reaction buffer of 10 times of concentration: the KCl of 500mmol/L, the dithiothreitol (DTT) of 30mmol/L (DTT), 100mmol/L adjust with hydrochloric acid Tris damping fluid, the bovine serum albumin of 1mg/ml, the 4 μ l25mmol/LMgCl that pH is 8.8
2, the mixture of 1 μ l4 kind dNTP.
The concrete operations of 16s rDNA sequence homology analysis are as follows:
The culture of the bacterial strain CB3 that the step 1 of cultivation 5ml obtains, to state of saturation, is got 1.5ml culture, centrifugal 2 minutes of 6000 turn/per minutes; Throw out adds the TE damping fluid of 565 μ l, with suction pipe, repeatedly blows and beats and makes it resuspended, adds the Proteinase K that sodium laurylsulfonate (SDS) that 30 μ l mass volume ratios are 10% and 5 μ l concentration are 20mg/mL, mixes, in 37 ℃ of incubations 1 hour; Add the NaCl that 100 μ l concentration are 5mol/L, fully mix, then add the CTAB/NaCl solution of 80 μ l, mix, in 65 ℃ of incubations 10 minutes; Add isopyknic chloroform/primary isoamyl alcohol (volume ratio=24:1 of chloroform and primary isoamyl alcohol), mix, centrifugal 5 minutes of 12000 turn/per minutes, proceed to supernatant liquor in a new pipe; Add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio=25:24:1 of phenol, chloroform and primary isoamyl alcohol), mix, centrifugal 5 minutes of 12000 turn/per minutes, proceed to supernatant in a new pipe; Add 0.6 times of volume Virahol, mix gently until DNA precipitates, with the centrifuge tube of a sealing, precipitation is transferred in 70% ethanol of 1ml and washs; Centrifugal 5 minutes of 12000 turn/per minutes, abandon supernatant, dry a little with Freeze Drying Equipment, are heavily dissolved in the TE damping fluid of 50 μ l, obtain the genomic dna of the bacterial strain CB3 that step 1 obtains as template.
The genomic dna extracting of take is template, utilizes eubacterium primer 2 7f and 1492r purchased from Shanghai Sheng Gong biotechnology company limited, carries out pcr amplification.Described 27f primer sequence is: 5 '-AGAGTTTGATCCTGGCTCAG-3 '; Described 1492r primer sequence is: 5 '-GGTTACCTTGTTACGACTT-3 '.In 50 μ l reaction systems, mix successively following reagent: 35 μ l H
2o, the PCR reaction buffer of 5 μ l10 times concentration, the 27f primer of 0.5 μ l, the 1492r primer of 0.5 μ l, 0.5 μ l template DNA is the genomic dna of the bacterial strain CB3 of step 1 acquisition, 0.5 μ l Taq archaeal dna polymerase, mixes rear centrifugal 5 seconds.Mixture is heated 5 minutes at 94 ℃.94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ are extended 2 minutes, 30 circulations altogether.After last circulation, at 72 ℃, be incubated 10 minutes, make reaction mixture amplification fully, obtain the pcr amplification product of the 16S rRNA gene of bacterial strain CB3, product is checked order.Sequence assembly and similarity analysis are used DNAStar software to complete, and sequence alignment completes online by American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov).
Sequencing result shows, the 16S rRNA gene order length of the bacterial strain CB3 that step 1 obtains is 1501bp, as shown in sequence in sequence table 1.
In view of above-mentioned form, analysis of physio biochemical characteristics and 16s rDNA sequence homology analysis result, the bacterial strain CB3 that step 1 separation screening is obtained is accredited as heat and bites bacillus amyloliquefaciens (Bacillus thermoamylovorans).This heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3 and on May 25th, 2012, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.6153.
In the present embodiment, substratum used is composed as follows:
Solid slant culture base: glucose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, agar powder 15 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Liquid nutrient medium: carbon source 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters.The initial pH of this substratum is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.Wherein carbon source is respectively: glucose, cellobiose, sucrose, fructose, wood sugar, pectinose and semi-lactosi, dosage is 20 grams per liters.
The method of fermentative Production Pfansteihl of the present invention comprises the following steps:
(1) slant culture: heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 bacterial classification and is inoculated on solid slant culture base, cultivates 36 hours for 50 ℃;
(2) seed culture: the bacterial strain that step (1) is cultivated, under aseptic condition, with inoculating articulating 2, encircle in the 100ml triangular flask of 30ml liquid nutrient medium is housed, 50 ℃ of standing cultivations 48 hours, make seed culture fluid;
(3) fermentation culture: access the seed nutrient solution of 3ml step (2) in the 100ml triangular flask of 30ml liquid nutrient medium is housed, is positioned over 50 ℃ of standing cultivations 48 hours, finish fermentation.
During fermentation ends, get fermented liquid, 10,000 revs/min centrifugal 5 minutes, get supernatant liquor, according to aforesaid method, detect the concentration of the Pfansteihl generating in fermented liquid.
Result shows, under 50 ℃ of culture temperature, utilizes glucose for carbon source, and producing Pfansteihl is 17 grams per liters; Utilize fructose for carbon source, producing Pfansteihl is 17 grams per liters; Utilize cellobiose for carbon source, producing Pfansteihl is 12 grams per liters; Utilize semi-lactosi for carbon source, producing Pfansteihl is 0.9 grams per liter; Utilize other carbohydrate not produce Pfansteihl for carbon source.Above result shows that it is hexose-glucose and fructose that heat is bitten the optimum carbon source of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, is secondly cellobiose.
Embodiment 3, heat are bitten the mensuration (triangular flask) that bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 utilizes glucose to produce the optimal reaction temperature of Pfansteihl for carbon source through fermentation
In the present embodiment, substratum used is composed as follows:
Solid slant culture base: glucose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, agar powder 15 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Liquid nutrient medium: glucose 50 grams per liters, yeast extract 5 grams per liters, calcium carbonate 30 grams per liters.The initial pH of this substratum is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
The method of fermentative Production Pfansteihl of the present invention comprises the following steps:
(1) slant culture: heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 bacterial classification and is inoculated on solid slant culture base, cultivates 36 hours for 50 ℃;
(2) seed culture: the bacterial strain that step (1) is cultivated, under aseptic condition, with inoculating articulating 2, encircle in the 100ml triangular flask of 30ml liquid nutrient medium is housed, 50 ℃ of standing cultivations 48 hours, make seed culture fluid;
(3) fermentation culture: access the seed nutrient solution of 3ml step (2) in the 100ml triangular flask of 30ml liquid nutrient medium is housed, is positioned over respectively 37 ℃, 42 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ of standing cultivations 48 hours, finish fermentation.
During fermentation ends, get fermented liquid, 10,000 revs/min centrifugal 5 minutes, get supernatant liquor, according to aforesaid method, detect the concentration of the Pfansteihl generating in fermented liquid.Test in triplicate results averaged.
Result is as table 1, and under 37 ℃ of culture temperature, Pfansteihl output is 20.8 grams per liters; Under 42 ℃ of culture temperature, Pfansteihl output is 26.2 for grams per liter; Under 50 ℃ of culture temperature, Pfansteihl output is 29.7 grams per liters; Under 55 ℃ of culture temperature, Pfansteihl output is 18.9 grams per liters; Under 60 ℃ of culture temperature, Pfansteihl output is 12.6 grams per liters; Under 65 ℃ of culture temperature, Pfansteihl output is 0.7 grams per liter.Above result shows heat bites bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 to utilize the optimum fermentation temp of glucose is 50 ℃.
Table 1 utilizes glucose at differing temps bottom fermentation Pfansteihl, to repeat experimental result 3 times as carbon source
| Temperature | Repeat 1 | |
Repeat 3 | Mean+ |
| 37℃ | 19.8 | 21.6 | 20.9 | 20.8±0.9 |
| 42℃ | 25.3 | 27.2 | 26.3 | 26.2±1.0 |
| 50℃ | 30.4 | 28.9 | 29.8 | 29.7±0.8 |
| 55℃ | 18.9 | 17.7 | 20.1 | 18.9±1.2 |
| 60℃ | 11.6 | 13.8 | 12.5 | 12.6±1.1 |
| 65℃ | 0.8 | 0.7 | 0.6 | 0.7±0.1 |
In the present embodiment, substratum used is composed as follows:
Solid slant culture base: glucose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, agar powder 15 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Liquid nutrient medium: cellobiose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters.The initial pH of this substratum is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
The method of fermentative Production Pfansteihl of the present invention comprises the following steps:
(1) slant culture: heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 bacterial classification and is inoculated on solid slant culture base, cultivates 36 hours for 50 ℃;
(2) seed culture: the bacterial strain that step (1) is cultivated, under aseptic condition, with inoculating articulating 2, encircle in the 100ml triangular flask of 30ml liquid nutrient medium is housed, 50 ℃ of standing cultivations 48 hours, make seed culture fluid;
(3) fermentation culture: access the seed nutrient solution of 3ml step (2) in the 100ml triangular flask of 30ml liquid nutrient medium is housed, is positioned over respectively 30 ℃, 37 ℃, 42 ℃, 50 ℃ and 60 ℃ of standing cultivations 48 hours, finish fermentation.
During fermentation ends, get fermented liquid, 10,000 revs/min centrifugal 5 minutes, get supernatant liquor, according to aforesaid method, detect the concentration of the Pfansteihl generating in fermented liquid.Test in triplicate results averaged.
Result is as shown in Fig. 1 and table 2, and under 30 ℃ of culture temperature, Pfansteihl output is 4.5 grams per liters; Under 37 ℃ of culture temperature, Pfansteihl output is 9.00 for grams per liter; Under 42 ℃ of culture temperature, Pfansteihl output is 10.8 grams per liters; Under 50 ℃ of culture temperature, Pfansteihl output is 12.4 grams per liters; Under 60 ℃ of culture temperature, Pfansteihl output is 0.1 grams per liter, and above result shows heat bites bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 to utilize the optimum fermentation temp of cellobiose be 50 ℃.
Table 2 utilizes cellobiose at differing temps bottom fermentation Pfansteihl, to repeat experimental result 3 times as carbon source
| Temperature | Repeat 1 | |
Repeat 3 | Mean+ |
| 30℃ | 3.9 | 4.5 | 5.0 | 4.5±0.6 |
| 37℃ | 8.2 | 9.5 | 9.2 | 9.00±0.7 |
| 42℃ | 10.9 | 10.4 | 11.2 | 10.8±0.4 |
| 50℃ | 13.4 | 12.1 | 11.7 | 12.4±0.9 |
| 60℃ | 0.1 | 0.1 | 0.1 | 0.1±0.0 |
Embodiment 5, heat are bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 and are directly utilized cellobiose fermentation production of L-lactic acid (fermentor tank)
In the present embodiment, substratum used is composed as follows:
Solid slant culture base: glucose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters, agar powder 15 grams per liters, pH value is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Seed culture medium: cellobiose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters.The initial pH of this substratum is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
Fermention medium: cellobiose 20 grams per liters, yeast extract 5 grams per liters, calcium carbonate 12 grams per liters.The initial pH of this substratum is 6.5.Under 115 ℃ of conditions, sterilizing is 20 minutes.
The method of fermentative Production Pfansteihl of the present invention comprises the following steps:
(1) slant culture: with embodiment 2;
(2) seed culture: the bacterial strain that step (1) is cultivated, under aseptic condition, with inoculating articulating 2, encircle in the 100ml triangular flask of 30ml seed culture medium is housed, cultivate altogether 7 bottles, 50 ℃ of standing cultivations 48 hours, make seed culture fluid;
(3) fermentation culture: Shanghai is protected emerging BIOTECH5 and risen in fermentor tank and add 1.8 liters of fermention mediums accesses the seed culture fluid that 210ml step (2) obtains in fermention medium, and 50 ℃ of standing cultivations 72 hours, finish fermentation.Wherein, every 12 hours, stir once.30 revs/min of mixing speed, rotation radius is 30-40mm, churning time 30 minutes.
During fermentation ends, get fermented liquid, 10,000 revs/min centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect Pfansteihl concentration in fermented liquid, glucose acid invert ratio, and the optical purity of Pfansteihl.Test in triplicate results averaged.
Result is as shown in table 3, and Pfansteihl concentration is 12.2 ± 0.7g/L, and optical purity is greater than 98%.This result shows, heat is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153 and can be utilized cellobiose to produce high optical pure L-lactic acid for sole carbon source direct fermentation, has certain prospects for commercial application.
Table 3 utilizes cellobiose direct fermentation Pfansteihl to repeat the result of experiment for 3 times
| Repeat | Pfansteihl output (g/L) | Glucose acid invert ratio (%) | Pfansteihl optical purity (%) |
| 1 | 12.2 | 60.8 | 98.2 |
| 2 | 11.5 | 57.6 | 97.9 |
| 3 | 13.0 | 65.0 | 98.6 |
| Mean+SD | 12.2±0.7 | 61.1±3.7 | 98.2±0.4 |
Claims (10)
1. to bite the deposit number at bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3，Ta China Committee for Culture Collection of Microorganisms common micro-organisms center be CGMCC No.6153 to heat.
2. a microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is that heat claimed in claim 1 is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153.
3. producing a method for Pfansteihl, is that fermentation heat claimed in claim 1 is bitten bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, or microbial inoculum claimed in claim 2, obtains Pfansteihl.
4. method according to claim 3, is characterized in that: described heat is bitten and in the fermention medium of bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, contained carbon source, nitrogenous source and for the neutralizing agent of controlled fermentation liquid pH; Carbon source in described fermention medium is at least one in cellobiose, glucose and fructose; The final concentration of described carbon source in described fermention medium is 20-100 grams per liter.
5. method according to claim 4, is characterized in that: the carbon source in described fermention medium is cellobiose, and the final concentration of described cellobiose in described fermention medium is 20-50 grams per liter; Or
Carbon source in described fermention medium is glucose, and the final concentration of described glucose in described fermention medium is 20-100 grams per liter.
6. according to the method described in claim 4 or 5, it is characterized in that: the described nitrogenous source in described fermention medium is yeast extract, described neutralizing agent is calcium carbonate; The final concentration of described yeast extract in described fermention medium is 3-10 grams per liter, and the final concentration of described calcium carbonate in described fermention medium is 10-40 grams per liter.
7. according to arbitrary described method in claim 4-6, it is characterized in that: the pH of described fermention medium is 5.5-7.
8. according to arbitrary described method in claim 3-7, it is characterized in that: the condition of described fermentation is 30-65 ℃ and cultivates 36-72 hour.
9. according to arbitrary described method in claim 3-8, it is characterized in that: the training method of described fermentation is to stir 1-2 time for every 12 hours, and each time of stirring is 30-45 minute, and mixing speed is 30-50 rev/min, and rotation radius is 30-40mm.
10. the preparation method of microbial inoculum described in claim 2, comprises the steps: that heat claimed in claim 1 is bitten to bacillus amyloliquefaciens (Bacillus thermoamylovorans) CB3CGMCC No.6153, as activeconstituents, obtains described microbial inoculum.
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| CN106222107A (en) * | 2016-08-05 | 2016-12-14 | 福建省农业科学院畜牧兽医研究所 | One strain is from the extreme thermophilic antibacterial of pig farm garbage |
| CN109402015A (en) * | 2018-11-20 | 2019-03-01 | 江南大学 | One plant of heat bites bacillus amyloliquefaciens and its application |
| CN111808775A (en) * | 2020-07-24 | 2020-10-23 | 南京高新工大生物技术研究院有限公司 | High-temperature-resistant thermophilic bacillus amylovorus and application thereof |
| CN115927086A (en) * | 2022-10-20 | 2023-04-07 | 中国科学院上海高等研究院 | Strain for enhancing methane production by organic solid waste anaerobic fermentation and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222107A (en) * | 2016-08-05 | 2016-12-14 | 福建省农业科学院畜牧兽医研究所 | One strain is from the extreme thermophilic antibacterial of pig farm garbage |
| CN106222107B (en) * | 2016-08-05 | 2019-07-26 | 福建省农业科学院畜牧兽医研究所 | One plant of extreme thermophilic bacterium from pig farm waste |
| CN109402015A (en) * | 2018-11-20 | 2019-03-01 | 江南大学 | One plant of heat bites bacillus amyloliquefaciens and its application |
| CN109402015B (en) * | 2018-11-20 | 2020-09-04 | 江南大学 | Bacillus amylovora and application thereof |
| CN111808775A (en) * | 2020-07-24 | 2020-10-23 | 南京高新工大生物技术研究院有限公司 | High-temperature-resistant thermophilic bacillus amylovorus and application thereof |
| CN115927086A (en) * | 2022-10-20 | 2023-04-07 | 中国科学院上海高等研究院 | Strain for enhancing methane production by organic solid waste anaerobic fermentation and application thereof |
| CN115927086B (en) * | 2022-10-20 | 2023-07-07 | 中国科学院上海高等研究院 | Strain for producing methane by reinforcing anaerobic fermentation of organic solid waste and application thereof |
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