CN103667107A - Enterococcus faecium strain capable of producing L-lactic acid - Google Patents
Enterococcus faecium strain capable of producing L-lactic acid Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and relates to an Enterococcus faecium strain capable of producing L-lactic acid. The Enterococcus faecium strain capable of producing L-lactic acid is named as Enterococcusfaeciums HY-U36. The Enterococcusfaeciums HY-U36 provided by the invention can ferment and convert glucose to directly generate and obtain the target product, namely L-lactic acid, the average conversion rate of glucose to L-lactic acid is 84%-91%, the conversion rate is high, the purity is as high as 98.4%, common byproducts, namely ethanol, malic acid, fumaric acid and the like are fewer, and the Enterococcusfaeciums HY-U36 is the L-lactic acid production strain with great research and development values.
Description
(1) technical field
The invention belongs to biological technical field, relate to a kind of manure enterococcin strain that produces Pfansteihl.
(2) background technology
Pfansteihl (L-lactic acid), formal name used at school L-2-hydroxy-propionic acid, molecular formula is C
3h
6o
3, by Sweden scientist C.W.Scheele, in milk, found the earliest, be one of three large organic acids of generally acknowledging in the world.Lactic acid has rotational isomerism, and Pfansteihl is its left-handed type, is the unique lactic acid type that can decompose utilization of human body
[1].
Pfansteihl is broadly divided into technical grade, food grade, plastics level and American Pharmacopeia level four classes by its purity in the world, is important biochemical product, in various fields such as food, medicine, agricultural, makeup and printing and dyeing, is widely used.The acid soft and stable in properties of Pfansteihl, directly katabolism and toxicological harmless effect in human body, by U.S. FDA, confirmed as safety (GRAS), a kind of good sanitas and pickling agent, in the food such as can, sauce salted vegetables, meat product, add Pfansteihl as sanitas, can substitute the Sodium Benzoate to the toxic effect of human body.In beer production, with Pfansteihl, substitute phosphoric acid and regulate pH value, can also play and promote saccharification and yeast to grow, suppress the effect that miscellaneous bacteria infects, improves beer flavor, extends beer shelf-life.At present, the U.S. has banned use of the inorganic acid for adjusting pH such as phosphoric acid, and all uses Pfansteihl instead
[2].At tobacco industry, add a certain amount of Pfansteihl, can eliminate sharp flavor, keep the humidity of tobacco, improve the quality of cigarette.In pharmaceutical industries, Pfansteihl or lactic acid salt can directly be mixed with drug use, because it is harmless, and have stronger sterilizing ability, and Pfansteihl ester is made the lubricant of compacting medicine body in pharmacy industry, can also be for the preparation of step-down class medicine
[3].The crude lactic acid ammonia solution being obtained by fermentation can be directly as ensilage additive application in agriculture feed, Pfansteihl also can be used as the agent of plant-growth vigor in addition, aquatic products is applied in agriculture fishery with bacteria-promoting agent etc.In addition, Pfansteihl can generate vertical PLLA chain or ring-type through polymerization, PLLA is a kind of nontoxic macromolecular compound, there is biocompatibility, in human body and occurring in nature can be broken down into Pfansteihl, can be made into operating sutures, fracture inside-fixture, the medical materials such as slow release capsule preparation
[3] [4].PLLA is for the production of biodegradable plastic, can replace PVC, PP class plastics for the production of the goods such as container film, fiber
[2], eliminate the ecocrisis that white pollution brings, there is good economic and social benefit.
The lactic acid of producing due to chemical synthesis is mainly DL-LACTIC ACID, and Pfansteihl is mainly by enzyme process and Production by Microorganism Fermentation at present.The synthetic Pfansteihl of enzyme process is so long as 1,2-chloropropionic acid enzymatic conversion method and pyruvic acid enzymatic conversion method method, but Production by Enzymes Pfansteihl is due to reasons such as cost is high, complex process, limited its suitability for industrialized production, and microbe fermentation method is because it has, raw material sources are extensive, production cost is low, optical purity of products high, become the topmost production method of current Pfansteihl.Production by Microorganism Fermentation Pfansteihl is that to take starch, glucose etc. be raw material, produces the method for Pfansteihl through microorganism fermentation, according to the kind of organism of fermentation, can be divided into root arrhizus fermentation and fermentation using bacteria.The industrial production of domestic Pfansteihl mainly be take Rhizopus oryzae as main, it can utilize the cheap raw material aerobic fermentations such as starch to produce Pfansteihl, but its sugared theoretical yield low (only having 75%), and the shortcoming such as fermentation has the easily conglomeration balling-up in fermented liquid of easy microbiological contamination, mycelia, power consumption is large, by product is many
[5], therefore limited it and further developed.In the last few years, fermentation using bacteria method was produced the focus that Pfansteihl becomes research
[6]this is mainly because compare with root arrhizus fermentation, bacterial fermentation process for preparing L-lactic acid has the high (theoretical yield 100% of transformation efficiency, in actual industrial production, can reach more than 90%), the feature such as fermentation byproduct is less, and downstream separation and purification technique are relatively simple, power consumption is few, production cost is low.
In decades, utilize the research of fermentation using bacteria method production Pfansteihl very active, content relates to bacterial screening, fermention medium optimization, fermentation, the separating-purifying of Pfansteihl, the many aspects such as the control of the metabolic pathway of synthesizing of Pfansteihl and bacterial classification genetic modification.The main production area of Pfansteihl is the U.S., West Europe and Japan etc. in the world at present, and nearly 80% manufacturer adopts fermentation method to produce.There are 2 Pfansteihl factories in the U.S., and they are the fermentative production ShangADM company of U.S.'s maximum and the joint venture company of Dutch PURAC and U.S. Cargill company, and yearly capacity is respectively 31000 tons and 34000 tons
[7].The existing lactic acid-producing ability of China is approximately 25000 tons/year, actual output is about 15000 tons/year, 90% product is DL-LACTIC ACID, and industrial scale, sour productive rate, product matter scape etc. are compared with world level and still had larger gap, and particularly the research and development of Pfansteihl are also not enough.Domestic for bacterial fermentation process for preparing L-lactic acid research Pseudomonas mainly take lactobacillus (Lactobacillus), streptococcus (Streptoccoccus), bacillus (Bacillus) as main, still with enterococcus spp strain fermentation, do not produce the research report of Pfansteihl, the strain enterococcus spp bacterial strain that this patent utilization screening obtains---faecium is that fermentation strain is produced Pfansteihl, having the features such as output is high, product purity is high, by product is less, is that a strain has the Pfansteihl industrial producing strain that research and development are worth.
(3) summary of the invention
The present invention, in order to make up the deficiencies in the prior art, provides a kind of manure enterococcin strain that produces Pfansteihl, take to realize to utilize saccharine material microbe fermentation method to obtain the hope that Pfansteihl is main purpose product.
The present invention is achieved through the following technical solutions:
Produce a manure enterococcin strain for Pfansteihl, its special character is: called after faecium Enterococcus faeciums HY-U36, and this bacterial strain 16S rDNA complete sequence is as follows:
TCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTT,Full-length 16S rDNA sequences above 800 nucleotides.
The faecium HY-U36 of above-mentioned production of high purity Pfansteihl, its biological property (9) is: on solid culture (isolation medium) flat board, cultivate and observe, bacterium colony can form the circular or oval bacterium colony that canescence, opaque, smooth surface, diameter are 0. 5～1mm, electron microscopic observation thalline is spherical, many paired or catenations, gramstaining result is positive.
The above-mentioned liquid nutrient medium of observing for thalli morphology forms (g/L): glucose 20, peptone 10, yeast extract paste 5, K
2hPO
42, sodium acetate 5, its pH value is 6.5.
The above-mentioned solid medium of observing for thalli morphology forms (g/L): glucose 20, peptone 10, yeast extract paste 5, K
2hPO
42, sodium acetate 5, agar 18, its pH value is 6.5.
Bacterial strain CGMCC N0.7274 physiological and biochemical property (10) is: bacterial strain HY-U36 optimum growth temperature is 30～37 ℃, can, 10 ℃ and 45 ℃ of growths, can account under 6.5%NaCl substratum and pH9.6 condition and grow at weight fraction; This bacterium can utilize glucose, fructose, maltose, sucrose, cellobiose, lactose to produce acid, and glucose fermentation is aerogenesis not, does not utilize Citrate trianion, and catalase is negative, reduction nitrate.
Bacterial strain CGMCC N0.7274 of the present invention is measured to the result demonstration of the portion gene sequence of 16S rRNA, this Pseudomonas is in enterococcus spp, and order-checking nucleotide sequence is as shown in SEQ ID NO.1.
By using (the National Center for Biotechnology Information of U.S. biotechnology information center, NCBI) blast program comparison, the gene order of finding the gene order of CGMCC N0.7274 bacterial strain 16S rRNA of the present invention and faecium (Enterococcus faeciums) the LMG 11423 16S rRNA of NCBI registration has high homology, and in conjunction with Physiology and biochemistry experiment, preliminary evaluation CGMCC N0.7274 bacterial strain is an Enterococcus faecalis (Enterococcus faeciums).
The basic skills of the faecium of production of high purity Pfansteihl of the present invention (Enterococcus faeciums) strain improvement is:
Use purpurum bromocresolis-calcium carbonate screening plate screening to obtain the manure enterococcin strain H-36 that a strain can produce Pfansteihl, take H-36 bacterial strain as starting strain, by ultraviolet ray, the physics such as nitrosoguanidine, chemical mutagen carries out mutagenesis to it, mutagenesis can be repeatedly, mutafacient system adopts ordinary method, after mutagenesis, coating screening is dull and stereotyped, picking list bacterium colony moves and connects inclined-plane, and then carry out shake flask fermentation, by measuring the wear rate of glucose and the yields screening object bacterial strain of Pfansteihl, choose consumption of glucose speed fast, the bacterial strain that Pfansteihl output is high, pass through again separating for several times purifying, obtain CGMCC N0.7274 bacterial strain.
The application of the faecium of production of high purity Pfansteihl of the present invention (Enterococcus faeciums) in preparing Pfansteihl.
Utilize the method for faecium (Enterococcus faeciums) fermentation production of L-lactic acid as follows:
Shake flask fermentation: get 35-40 ℃ of 37 ℃ of cultivation 10-16 hour of fresh inclined plane inoculating liquid seed culture medium that cultivate 1-2 days, inoculation fermentation substratum, 35-40 ℃ of shake flask fermentation 62-84 hour, in fermentation ends fermented liquid, can't detect glucose residual, conversion of glucose generate Pfansteihl transformation efficiency be 84-92%.
50L ferment tank: 37 ℃ of 35-40 ℃ of cultivation 10-16 hour of fresh inclined plane inoculating liquid seed culture medium that cultivate 1-2 days, by the inoculum size of 1-5%, be linked in preprepared fermention medium, 50L fermentor tank liquid amount 33L, carry out stir culture, by regulating fermentor tank rotating speed and ventilation ratio, control dissolved oxygen levels in the saturation ratio of 20-35%, fermented liquid initial pH value is 6.5-6.8, and the pH that maintains fermented liquid by Feeding ammonia water in fermenting process is in 6.2 left and right.After 24h, close breather valve and proceed to anaerobically fermenting, the glucose in fermenting process in period sampling measuring fermented liquid and the concentration of Pfansteihl, when Pfansteihl concentration no longer rises in fermented liquid, finish fermentation, and fermentation time is 62-84 hours.
Above-mentioned screening forms (g/L) with plate culture medium: glucose 20, peptone 10, yeast extract paste 5, corn steep liquor 5, dipotassium hydrogen phosphate 2, calcium lactate 60, purpurum bromocresolis 0.01, calcium carbonate 10, pH6.5-6.8;
Above-mentioned slant medium forms (g/L): glucose 20, peptone 10, yeast extract paste 5, potassium primary phosphate 2, agar 20, pH 6.5-6.8;
Aforesaid liquid seed culture medium forms (g/L): glucose 20, peptone 10, yeast extract paste 5, corn steep liquor 5, dipotassium hydrogen phosphate 2, pH6.5-6.8;
The composition of above-mentioned fermention medium (g/L): glucose 80-120, peptone 10-20, yeast extract paste 3-8, corn steep liquor 10-20, dipotassium hydrogen phosphate 1-3, diammonium hydrogen citrate 1-3, magnesium sulfate 0-1.5, manganous sulfate 0-0.2, tween 80 0.5-1.5mL/L, pH6.5-6.8;
The culture temperature of above-mentioned Pfansteihl fermentation is preferably 37 ℃.Above-mentioned on liquid seed culture medium incubation time be preferably 12-16 hour, fermention medium initial glucose concentration is preferably 100-120g/L, above-mentioned dissolved oxygen levels during the fermentation is preferably controlled at the saturation ratio of 2-5%.The above-mentioned 50L ferment tank cycle is generally 64-78 hour.The average conversion of conversion of glucose Pfansteihl is 84%-91%.
Above-mentioned glucose is measured as follows:
Use the SBA-4D type membrane bioreactor that Shandong Province academy sciences Biology Research Institute produces to measure.Measuring principle is to utilize the single-minded mensuration glucose content of fixation glucose dehydrogenation enzyme membrane.
Above-mentioned lactic acid is measured as follows:
High performance liquid chromatography (HPLC), chromatographic condition: chromatographic column: Prontosil 1202102C18H (10 μ m, 4.6mm i.d. * 250 mm); Flow velocity: 0.5mL/min; Sampling volume: 20 μ L; Detect wavelength: 210nm; Column temperature: 30 ℃; Moving phase: with redistilled water preparation 0.1mol/L KH2PO4, phosphorus acid for adjusting pH to 2.5.
Above-mentioned Pfansteihl is measured as follows:
Use the SBA-4D type membrane bioreactor that Shandong Province academy sciences Biology Research Institute produces to measure.Measuring principle is that immobilized enzyme can act on L (+) lactic acid specifically:
Lactic acid+O
2+ H
2o-> immobilization L (+) Lactate Oxidase-> pyruvic acid+H
2o
2
Doubly, the content of the Lactic Acid from Fermentation Broth of requirement dilution is within the scope of 0-50mg/dL for fermented liquid dilution 50-100.
Above-mentioned Pfansteihl purity testing is measured as follows:
Pfansteihl purity (%)=Pfansteihl concentration (g/L)/lactic acid concn (g/L)
The invention has the beneficial effects as follows: faecium of the present invention (Enterococcus faeciums) the HY-U36 transforming glucose that can ferment directly generates and obtain object product Pfansteihl, the average conversion that conversion of glucose generates Pfansteihl is 84%-91%, transformation efficiency is high, purity is up to 98.4%, the common by products such as ethanol, oxysuccinic acid and fumaric acid are less, are that a strain has the Pfansteihl production bacterial strain that research and development are worth.
(4) accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further illustrated.
Bacterial strain faecium provided by the invention (Enterococcus faeciums) HY-U36, on March 5th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100080, its deposit number is CGMCC N0.7274.
The 16S rDNA complete sequence figure of Fig. 1 faecium (Enterococcus faeciums) HY-U36;
Fig. 2 faecium (Enterococcus faeciums) HY-U36 violet staining picture;
Fig. 3 faecium (Enterococcus faeciums) HY-U36 gramstaining figure;
Fig. 4 faecium (Enterococcus faeciums) HY-U36 bacterial strain fermentation liquor high-efficient liquid phase chromatogram;
Fig. 5 faecium (Enterococcus faeciums) HY-U36 phylogenetic tree construction.
(5) embodiment
Embodiment 1: the screening of producing Pfansteihl manure enterococcin strain
Get according to a conventional method the samples such as animal excrement place soil, ox, sheep rumen content, under the condition of aseptic technique, get 2g sample, put into the 250mL triangular flask that 40mL sterilized water is housed, after concussion, getting 1mL is inoculated in the 250mL triangular flask that 40mL enrichment medium is housed in 37 ℃, 180r/min shaking table cultivation 24h, again that pregnant solution dilution spread is dull and stereotyped to the screening of purpurum bromocresolis-calcium carbonate, constant temperature culture in 37 ℃ of anaerobism bags, the bacterium colony puncture preservation that picking color changeable transparent circle is large after growing single bacterium colony.Then these preservation of bacteria strains are carried out to shake flask fermentation screening again, 40mL Medium of shaking flask fermentation is loaded in 250mL triangular flask, 1 connects 1 bottle graft and enters 1～2 ring slant strains, 37 ℃, 180r/min shaking culture are about 64 hours, then survey in fermented liquid extremely purity of Pfansteihl content, final screening obtains the bacterial strain of the pure Pfansteihl of a plant height, called after faecium (Enterococcus faeciums) H-36.
Embodiment 2: the seed selection of producing Pfansteihl faecium
By starting strain faecium H-36 inoculation liquid seed culture medium, 37 ℃ of cultivations are to logarithmic phase mid-term, centrifugal collection thalline, and stroke-physiological saline solution washed twice, then adds stroke-physiological saline solution and makes bacteria suspension, makes cell concn at 1 * 108-109/ml.Get above-mentioned bacteria suspension 10ml and put into plate, put under 30W ultraviolet lamp and irradiate, irradiation distance 15cm, irradiation time is 3 minutes, 5 minutes, 7 minutes, 10 minutes, interval sampling is suitably coated the purpurum bromocresolis-calcium carbonate screening flat board that contains weight fraction 6% calcium lactate after dilution, 37 ℃ of lucifuges are cultivated 1-2 days, picking upgrowth situation is good, single bacterium colony that color changeable transparent circle is large carries out preservation, cultivate 1-2 days for 37 ℃, inclined-plane maturation is inoculated the shaking flask that fermention medium is housed afterwards, 37 ℃ of shake flask fermentations 64 hours, get fermented liquid 4000r/min centrifugal 10 minutes, get supernatant liquor and measure residual glucose in fermented liquid, lactic acid and Pfansteihl content, choose concentration of residual glucose low, it is object bacterial strain that lactic acid produces acid amount bacterial strain high and that Pfansteihl purity is high.
Object bacterial strain is sieved with further separation screening again through shake flask fermentation again, and seed selection obtains the mutant strain H-U36 that the stable and Pfansteihl output of a strain sugar consumption rate and purity are all improved.
The fermention medium that H-U36 inoculation initial glucose concentration is 100g/L, 37 ℃ of shake-flask culture finish fermentation in 78 hours, can't detect glucose in fermented liquid, and lactic acid production is 75.7g/L, and Pfansteihl purity is 98.2%.
Embodiment 3: the seed selection of high-purity Pfansteihl bacterial strain faecium CGMCC N0.7274
In the mutant strain H-U36 inoculation liquid seed culture medium that embodiment 2 is screened, 37 ℃ of cultivations are to logarithmic phase mid-term, centrifugal collection thalline, stroke-physiological saline solution washed twice, then add stroke-physiological saline solution and make bacteria suspension, make cell concn at 1 * 108-109/ml, standby.
The preparation of nitrosoguanidine treatment solution: take nitrosoguanidine 20mg, be positioned in the aseptic triangular flask of 100ml, add acetone 2ml it is dissolved, then add 18ml Tris damping fluid (pH6.0,0.5mol/L) to mix, standby.
Get above-mentioned nitrosoguanidine treatment solution 10ml, add 10ml bacteria suspension, 30 ℃ of insulation vibration 50-60 minute, every sampling in 10 minutes once, after sampling, first dilute 1000 times of termination reactions, and then dilution 2-10 doubly, coating purpurum bromocresolis-calcium carbonate is dull and stereotyped, cultivate 1-2 days for 37 ℃, picking list bacterium colony moves and connects inclined-plane, inclined-plane maturation is inoculated the shaking flask that fermention medium is housed afterwards, 37 ℃ of shake flask fermentations 60 hours, centrifugal 10 minutes of fermented liquid 4000r/min, get supernatant liquor and measure glucose in fermented liquid, lactic acid and Pfansteihl content, choose concentration of residual glucose low, the bacterial strain that Pfansteihl output and purity are high is object bacterial strain.
Object bacterial strain is sieved again through shake flask fermentation again, through further separation screening obtains a strain stable hereditary property, Pfansteihl output is up to 81.2g/L, the mutant strain HY-U36 that purity is greater than 99.3%.
Above-mentioned bacterial strains faecium (Enterococcus faeciums) HY-U36, on March 5th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080, its deposit number is CGMCC N0.7274.
Above-mentioned enrichment medium forms (g/L): glucose 20, and peptone 10, yeast extract paste 5, dipotassium hydrogen phosphate 2L, pH is 6.5-6.8;
Above-mentioned plate isolation base forms (g/L): glucose 20, and peptone 10, yeast extract paste 5, dipotassium hydrogen phosphate 2, calcium lactate 60, purpurum bromocresolis 0.01, calcium carbonate 10, pH is 6.5-6.8;
Aforesaid liquid seed culture medium forms (g/L): glucose 20-30, and peptone 10, yeast extract paste 5, calcium carbonate 8, dipotassium hydrogen phosphate 2, pH is 6.5-6.8;
Above-mentioned Medium of shaking flask fermentation forms (g/L): glucose 80-120, peptone 5-15, yeast extract paste 2-8, dipotassium hydrogen phosphate 1-3, calcium carbonate 0-120, diammonium hydrogen citrate 1-3, magnesium sulfate 0-1.5, manganous sulfate 0-0.2, tween 80 0.5-1.5mL/L, pH is 6.5-6.8;
The faecium HY-U36 of the production of high purity Pfansteihl of the present embodiment, its biological property is: on solid culture (isolation medium) flat board, cultivate 36-24h observation for 28-37 ℃, bacterium colony can form the circular or oval bacterium colony that canescence, opaque, smooth surface, diameter are 0. 5～1mm, electron microscopic observation thalline is spherical, many in pairs or catenation as Fig. 2, gramstaining result is positive, as Fig. 3.
The above-mentioned liquid nutrient medium of observing for thalli morphology forms (g/L): glucose 20, peptone 10, yeast extract paste 5, K
2hPO
42, sodium acetate 5, its pH value is 6.5.
The above-mentioned solid medium of observing for thalli morphology forms (g/L): glucose 20, peptone 10, yeast extract paste 5, K
2hPO
42, sodium acetate 5, agar 18, its pH value is 6.5.
Bacterial strain CGMCC N0.7274 physiological and biochemical property is: bacterial strain HY-U36 optimum growth temperature is 30～37 ℃, can, 10 ℃ and 45 ℃ of growths, can account under 6.5%NaCl substratum and pH9.6 condition and grow at weight fraction; This bacterium can utilize glucose, fructose, maltose, sucrose, cellobiose, lactose to produce acid, and glucose fermentation is aerogenesis not, does not utilize Citrate trianion, and catalase is negative, reduction nitrate.
Bacterial strain CGMCC N0.7274 Physiology and biochemistry experimental result refers to table 1.
The part physiological and biochemical property (8) of table 1 bacterial strain CGMCC N0.7274
| Experimental project | Result |
| Utilization of carbon source | |
| Glucose | + |
| Maltose | + |
| Raffinose | - |
| Wood sugar | - |
| N.F,USP MANNITOL | + |
| Lactose | + |
| Fructose | + |
| Cellobiose | + |
| D-sorbose | - |
| Melibiose | + |
| L-arabinose | + |
| Citrate test | - |
| 0.04% selenite | - |
| Pyruvate fermentation | - |
| Starch Hydrolysis | - |
| Gelatine liquefication | - |
| |
- |
| Urease | - |
| V.P. test | + |
| Catalase | - |
| Anti-40% bile | + |
| Indole test | - |
| Mobility test | - |
| Pigment formation | - |
| 4%NaCl test | + |
| 6.5%NaCl test | + |
| Growth temperature (℃) | |
| 4 | - |
| 37 | + |
| 45 | + |
| 50 | + |
| 55 | - |
Note: "+" well-grown or be positive; "-" do not grow or is negative.
Embodiment 4: faecium (Enterococcus faeciums) CGMCC N0.7274 bacterium 16S rRNA gene sequencing
The Pfansteihl superior strain HY-U36 that embodiment 3 seed selections are obtained is that CGMCC N0.7274 bacterial strain entrusts Sangon Biotech (Shanghai) Co., Ltd. (Sangon Biotech (Shanghai) Co., Ltd) to carry out 16S rRNA gene sequencing.
Experimental technique is: picking slant culture is in 10 μ l aqua sterilisas, after 99 ℃ of sex change, centrifuging and taking supernatant liquor is as template, use TaKaRa 16S rDNA Bacterial Identification PCR Kit (Code No.D310), take Forward/Reverse primer2 as primer, amplification object fragment.Get 5 μ l and carry out agarose gel electrophoresis, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut glue and reclaim object segment, take Seq Forward, Seq Reverse Seq Internal carries out DNA sequencing as the above-mentioned recovery product of primer pair.
Sequencing result: faecium (Enterococcus faeciums) CGMCC N0.7274 16S rDNA order-checking nucleotide sequence is as shown in SEQ ID NO.1 and Fig. 1.
By using (the National Center for Biotechnology Information of U.S. biotechnology information center, NCBI) BLASTN program comparison, faecium (Enterococcus faeciums) the LMG 11423 16S rDNA sequences of finding CGMCC N0.7274 bacterial strain 16S rDNA sequence and NCBI registration have high homology, in conjunction with Physiology and biochemistry, test, illustrate that CGMCC N0.7274 bacterial strain is an Enterococcus faecalis (Enterococcus faeciums), as Fig. 5.
Bacterial strain 16S rDNA sequence table:
SEQ ID NO.1：
SEQUENCE LISTING
<110> Shandong Food Fermentative Industry Research and Design Inst.
<120> manure enterococcin strain that produces Pfansteihl
<130> 2012-11-15
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 800
<212> DNA
<213> faecium (Enterococcus faeciums)
<400> 1
tcgtacgctt ctttttccac cggagcttgc tccaccggaa aaagaggagt ggcgaacggg 60
tgagtaacac gtgggtaacc tgcccatcag aaggggataa cacttggaaa caggtgctaa 120
taccgtataa caatcgaaac cgcatggttt tgatttgaaa ggcgctttcg ggtgtcgctg 180
atggatggac ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggccacgat 240
gcatagccga cctgagaggg tgatcggcca cattgggact gagacacggc ccaaactcct 300
acgggaggca gcagtaggga atcttcggca atggacgaaa gtctgaccga gcaacgccgc 360
gtgagtgaag aaggttttcg gatcgtaaaa ctctgttgtt agagaagaac aaggatgaga 420
gtaactgttc atcccttgac ggtatctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agcgagcgca 540
ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600
tgggagactt gagtgcagaa gaggagagtg gaattccatg tgtagcggtg aaatgcgtag 660
atatatggag gaacaccagt ggcgaaggcg gctctctggt ctgtaactga cgctgaggct 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag 780
Embodiment 5: the application of faecium (Enterococcus faeciums) CGMCC N0.7274 bacterial strain
Faecium (Enterococcus faeciums) CGMCC N0.7274 bacterial strain is moved and connects slant activation;
Press the composition preparation seed culture medium of aforesaid liquid seed culture medium, initial glucose concentration actual measurement is 22-31 g/L, 250ml triangular flask liquid amount is 40ml, 115 ℃ of steam sterilizings 20 minutes, are cooled to room temperature, inoculation slant strains 2 rings, on to put rotation rotating speed and be 150-200r/min, rotation radius be 40mm shaking table, cultivate 12-16 hour for 37 ℃, obtain seed liquor, get 5 times of seed liquor dilutions and put 580nm to survey light absorption value be 0.92-0.97;
By the composition preparation fermention medium of above-mentioned fermention medium, initial glucose concentration actual measurement is 73-118g/L, 250ml triangular flask liquid amount is 40ml, 115 ℃ of steam sterilizings 20 minutes, be cooled to room temperature, inoculation seed liquor 1ml, putting rotation rotating speed is 150-200r/min, rotation radius is on 40mm shaking table, cultivate for 28-45 ℃ and within 78-96 hour, finish fermentation, fermented liquid centrifugal 10 minutes with 4000r/min, get supernatant liquor and measure glucose, lactic acid and Pfansteihl content are respectively 0g/L, 81.1g/L and 79.8g/L(are according to the corresponding increase and decrease of glucose initial content), the transformation efficiency that conversion of glucose generates Pfansteihl is 87.9%, Pfansteihl optical purity is 98.4%.
Or:
With 50L fermentor tank, press 30L liquid amount preparation fermention medium, adjust pH6.8,115 ℃ of real tank sterilizings 20 minutes, good seed liquor 600 ml of inoculation culture after circulating water to 37 ℃, after inoculation, sampling and measuring glucose concn is 72-117g/L immediately.
Initial fermentor tank parameter is set to: mixing speed is 200-350r/min, the ratio of ventilation ratio 1:0.05-0.1vvm(feed liquid volume and per minute air flow (measurement basis)), tank internal pressure 0.04-0.08MPa.In fermenting process, controlled fermentation temperature is 37 ± 0.2 ℃, by 24h before adjusting mixing speed and ventilation ratio control dissolved oxygen, is 20-35%, closes afterwards breather valve, pH6.2 ± 0.2.
Sampling in every 8 hours in fermenting process, measure content and the cell concentration of glucose, lactic acid and Pfansteihl in fermented liquid, when glucose content is during lower than 10 g/L, glucose, lactic acid and Pfansteihl content in every 2 hours sampling and measuring fermented liquids, when Pfansteihl content in fermented liquid no longer increases, finish fermentation, fermentation period is 78 hours.
After fermentation ends, get fermented liquid with 4000r/min centrifugal 10 minutes, getting supernatant liquor measures the content of glucose, lactic acid and Pfansteihl and is respectively 0g/L, 82.7g/L and 81.9g/L(according to the corresponding increase and decrease of glucose initial content), the transformation efficiency that conversion of glucose generates Pfansteihl is 89.8%, and Pfansteihl optical purity is 99.0%.
Above-mentioned glucose is measured as follows:
Use the SBA-4D type membrane bioreactor that Shandong Province academy sciences Biology Research Institute produces to measure.Measuring principle is to utilize the single-minded mensuration glucose content of fixation glucose dehydrogenation enzyme membrane.
Above-mentioned lactic acid is measured as follows:
High performance liquid chromatography (HPLC) is as Fig. 4, chromatographic condition:
Chromatographic column: Prontosil 1202102C18H (10 μ m, 4.6mm i.d. * 250 mm); Flow velocity: 0.5mL/min; Sampling volume: 20 μ L; Detect wavelength: 210 nm; Column temperature: 30 ℃; Moving phase: with redistilled water preparation 0.1mol/L KH2PO4, phosphorus acid for adjusting pH to 2.5.
Above-mentioned Pfansteihl is measured as follows:
Use the SBA-4D type membrane bioreactor that Shandong Province academy sciences Biology Research Institute produces to measure.Measuring principle is that immobilized enzyme can act on L (+) lactic acid specifically:
Lactic acid+O
2+ H
2o-> immobilization L (+) Lactate Oxidase-> pyruvic acid+H
2o
2
Doubly, the content of the Lactic Acid from Fermentation Broth of requirement dilution is within the scope of 0-50mg/dL for fermented liquid dilution 50-100.
Above-mentioned Pfansteihl purity testing is measured as follows:
Pfansteihl purity (%)=Pfansteihl concentration (g/L)/lactic acid concn (g/L).
Reference:
[1] Liu Weixiong. the latest developments of lactic acid and poly(lactic acid) [J]. food and fermentation industries, 2001.27 (3): 61～65
[2] DATTA R.Technological and economical potential of polylactic acid and lactic acid derivatives[J].FEMS Microbiology Reviews,1995,16:221-231.
[3] NIJU N, ROYCHOUDHURY P K, SRIVASTAVE A. L-(+)lactic acid fermentation and its product polymerization[J]. Electronic Journal of Biotechnology,2004,7(2):167-179.
[4] Young-Jung Wee,Jin-Nam Kim and Hwa-Won Ryu. Biotechnological Production of Lactic Acid and Its Recent Applications[J]. Food Technol Biotechnol ,2006,44(2):163–172.
[5] SOCCOL CR,STONOGA VI,REIMBAULT M.Production of L-lactic acid by Rhizopus species[J].Microbiology and Biotechnology,1994,10: 433–435.
[6] J.H. Litchfield.Microbiological production of lactic acid[J], Adv.Appl. Microbiol. 1996,42: 45–95.
[7] Datta R,Henry M.Lactic accid:recent advances in products processes and technologies-a review[J].Chem Technol Biotechnol,2006,81(7):1119-1129.
[8] eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, first version: 62-63.
[9] eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, first version: 353-363.
[10] eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, first version: 364-398.
SEQ ID NO.1
SEQUENCE LISTING
<110> Shandong Food Fermentative Industry Research and Design Inst.
<120> manure enterococcin strain that produces Pfansteihl
<130> 2012-11-15
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 800
<212> DNA
<213> faecium (Enterococcus faeciums)
<400> 1
tcgtacgctt ctttttccac cggagcttgc tccaccggaa aaagaggagt ggcgaacggg 60
tgagtaacac gtgggtaacc tgcccatcag aaggggataa cacttggaaa caggtgctaa 120
taccgtataa caatcgaaac cgcatggttt tgatttgaaa ggcgctttcg ggtgtcgctg 180
atggatggac ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggccacgat 240
gcatagccga cctgagaggg tgatcggcca cattgggact gagacacggc ccaaactcct 300
acgggaggca gcagtaggga atcttcggca atggacgaaa gtctgaccga gcaacgccgc 360
gtgagtgaag aaggttttcg gatcgtaaaa ctctgttgtt agagaagaac aaggatgaga 420
gtaactgttc atcccttgac ggtatctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agcgagcgca 540
ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600
tgggagactt gagtgcagaa gaggagagtg gaattccatg tgtagcggtg aaatgcgtag 660
atatatggag gaacaccagt ggcgaaggcg gctctctggt ctgtaactga cgctgaggct 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag 780
Claims (6)
1. a manure enterococcin strain that produces Pfansteihl, is characterized in that: called after faecium Enterococcus faeciums HY-U36, and the 16S rDNA complete sequence of this bacterial strain is as follows:
TCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTT,Full-length 16S rDNA sequences above 800 nucleotides.
2. the manure enterococcin strain of product Pfansteihl according to claim 1, it is characterized in that: adopt BLAST analytical method, by the Gene sequence comparison of the faecium LMG 11423 16S rRNA of the 16S rDNA complete sequence of faecium HY-U36 and NCBI registration, homology is 98.0%-99.0%.
3. the manure enterococcin strain of product Pfansteihl according to claim 1 and 2, it is characterized in that: on the flat board of solid culture or isolation medium, cultivate and observe, bacterium colony can form the circular or oval bacterium colony that canescence, opaque, smooth surface, diameter are 0. 5～1mm, electron microscopic observation thalline is spherical, many paired or catenations, gramstaining result is positive.
4. the manure enterococcin strain of product Pfansteihl according to claim 1 and 2, is characterized in that: strain growth temperature is 10-45 ℃, and optimum growth temperature is 30～37 ℃.
5. the manure enterococcin strain of product Pfansteihl according to claim 1 and 2, is characterized in that: this bacterial strain is to obtain Pfansteihl for saccharine material microbe fermentation method.
6. the manure enterococcin strain selection of product Pfansteihl according to claim 1 and 2, is characterized in that: comprise the following steps:
Use purpurum bromocresolis-calcium carbonate screening plate screening to obtain the manure enterococcin strain H-36 that a strain can produce Pfansteihl, take H-36 bacterial strain as starting strain, by ordinary method, it is carried out to mutagenesis, after mutagenesis, coating screening is dull and stereotyped, picking list bacterium colony moves and connects inclined-plane, and then carry out shake flask fermentation, by measuring the wear rate of glucose and the yields screening object bacterial strain of Pfansteihl, choose the bacterial strain that consumption of glucose speed is fast, Pfansteihl output is high, pass through again separating for several times purifying, obtain faecium HY-U36.
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| CN105524853A (en) * | 2014-09-30 | 2016-04-27 | 青岛康和食品有限公司 | PS agar medium and application thereof |
| CN110241043A (en) * | 2019-06-10 | 2019-09-17 | 大连理工大学 | The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid |
| CN111100802A (en) * | 2018-10-26 | 2020-05-05 | 中国石油化工股份有限公司 | Enterococcus faecalis and application thereof |
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| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
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| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
| CN103451244A (en) * | 2013-08-29 | 2013-12-18 | 山东省食品发酵工业研究设计院 | Application of enterococcus faecium in preparation of L-lactic acid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524853A (en) * | 2014-09-30 | 2016-04-27 | 青岛康和食品有限公司 | PS agar medium and application thereof |
| CN111100802A (en) * | 2018-10-26 | 2020-05-05 | 中国石油化工股份有限公司 | Enterococcus faecalis and application thereof |
| CN111100802B (en) * | 2018-10-26 | 2021-10-08 | 中国石油化工股份有限公司 | Enterococcus faecalis and application thereof |
| CN110241043A (en) * | 2019-06-10 | 2019-09-17 | 大连理工大学 | The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid |
| CN110241043B (en) * | 2019-06-10 | 2021-12-21 | 大连理工大学 | Strain for producing lactic acid through high-temperature fermentation and method for producing lactic acid |
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