CN103667107B - A kind of manure enterococcin strain producing Pfansteihl - Google Patents
A kind of manure enterococcin strain producing Pfansteihl Download PDFInfo
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- CN103667107B CN103667107B CN201310474859.1A CN201310474859A CN103667107B CN 103667107 B CN103667107 B CN 103667107B CN 201310474859 A CN201310474859 A CN 201310474859A CN 103667107 B CN103667107 B CN 103667107B
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- Prior art keywords
- pfansteihl
- lactic acid
- fermentation
- vref
- glucose
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to biological technical field, relate to a kind of manure enterococcin strain producing L lactic acid.The manure enterococcin strain of this product L lactic acid, named VREF Enterococcus faeciums HY U36, VREF of the present invention (Enterococcus faeciums) HY U36 can directly generate and obtain purpose product L lactic acid by microbe conversion glucose, it is 84% 91% that glucose is converted into the average conversion of L lactic acid, conversion ratio is high, high purity 98.4%, the common by-products such as ethanol, malic acid and fumaric acid are less, are the L production of lactic acid bacterial strains of a strain great research and development value.
Description
(1) technical field
The invention belongs to biological technical field, relate to a kind of manure enterococcin strain producing Pfansteihl.
(2) background technology
Pfansteihl (L-lactic acid), formal name used at school L-2-hydracrylic acid, molecular formula is C3H6O3, the earliest by Sweden scientist
C.W.Scheele finds in milk, is one of big organic acid of three generally acknowledged in the world.Lactic acid has optical isomerism, L-
Lactic acid is its left-handed type, is the human body lactic acid type that uniquely can decompose utilization[1]。
Pfansteihl is broadly divided into technical grade, food-grade, plastics level and American Pharmacopeia level four class by its purity in the world, is
Important biochemical product, is widely used in various fields such as food, medicine, agricultural, cosmetics and printing and dyeing.Pfansteihl acid
Soft and the stable in properties of property, can be confirmed as safety with Direct Resolution metabolism and nonhazardous effect by U.S. FDA in human body
(GRAS), it is a kind of excellent preservative and pickling agent, in the food such as can, sauce salted vegetables, meat products, adds Pfansteihl conduct
Preservative, can substitute the Sodium Benzoate of effect toxic to human body.In Beer Brewage, substitute phosphoric acid with Pfansteihl and regulate
PH value, moreover it is possible to play and promote saccharification and yeast development, suppression miscellaneous bacteria to infect, improve beer flavor, extend beer shelf-life
Effect.At present, the U.S. has prohibitted the use of the inorganic acid for adjusting pH such as phosphoric acid, and all uses Pfansteihl instead[2].At tobacco business, add
Add a certain amount of Pfansteihl, acid can be eliminated, keep the humidity of tobacco, improve the quality of cigarette.At pharmaceuticals industry, L-breast
Acid or lactate can be formulated directly into drug use, owing to it is harmless, and have stronger sterilizing ability,
Pfansteihl ester makees to suppress the lubricant of medicine body in pharmacy industry, it is also possible to be used for preparing step-down class medicine[3].Obtained by fermentation
Crude lactic acid ammonia solution can be directly as ensilage additive application in agricultural feed, and additionally Pfansteihl is alternatively arranged as plant growth
Vigor agent, aquatic products bacteria-promoting agent etc. is applied in agriculture fishery.Additionally, Pfansteihl can generate vertical chain or ring-type L-through polymerization
PLA, PLLA is a kind of nontoxic macromolecular compound, has biocompatibility, can quilt in human body and in nature
Resolve into Pfansteihl, can be made into the medical materials such as operation suture thread, fracture inside-fixture, slow release capsule preparation[3][4].The poly-breast of L-
Acid is used for producing biodegradable plastic, can substitute for the life for the goods such as container film, fiber of PVC, PP class plastics
Produce[2], eliminate the environmental crisis that white pollution is brought, there is good economic and social benefit.
The lactic acid produced due to chemical synthesis is mainly DL-LACTIC ACID, and current Pfansteihl is mainly by enzyme process and microorganism
Fermentation method produces.As long as enzymatic clarification Pfansteihl 1,2-chloropropionic acid enzymatic conversion method and pyruvic acid enzymatic conversion method method, but enzyme process
Production Pfansteihl, due to reasons such as cost height, complex process, limits its industrialized production, and microbe fermentation method has because of it
Raw material sources are extensive, production cost is low, optical purity of products high, it has also become the topmost producer of Pfansteihl at present
Method.Production by Microorganism Fermentation Pfansteihl is with starch, glucose etc. as raw material, produces the side of Pfansteihl through fermentable
Method, can be divided into root arrhizus fermentation and bacterial fermentation according to the kind of fermentative microorganism.The industrial production of domestic Pfansteihl is mainly with rice
Head mold is main, and it can utilize the cheap raw material aerobic fermentations such as starch to produce Pfansteihl, but its sugar theoretical yield is low (only
75%), and fermentation there is the shortcomings such as easy microbiological contamination, mycelia easy conglomeration balling-up in zymotic fluid, power consumption is big, accessory substance is many[5], because of
This limits it and further develops.In the last few years, bacterial fermentation process produced Pfansteihl becomes the focus of research[6], this is mainly
Because compared with root arrhizus fermentation, bacterial fermentation process for preparing L-lactic acid has conversion ratio height, and (theoretical yield 100%, in actual industrial life
Product can reach more than 90%), fermentation byproduct less, downstream separation and purification technique is relatively easy, power consumption is few,
The features such as production cost is low.
In decades, the research utilizing bacterial fermentation process production Pfansteihl is the most active, and content relates to bacterial screening, fermentation
Medium optimization, fermentation, the separating-purifying of Pfansteihl, the metabolic pathway of synthesizing of Pfansteihl control and bacterial classification gene
The many aspects such as transformation.The main production area of Pfansteihl is the U.S., West Europe and Japan etc., the production of nearly 80%
Producer uses fermentation method to produce.There are 2 Pfansteihl factories in the U.S., and they are the fermenting and producing business ADM public affairs that the U.S. is maximum
Department and Holland PURAC and the joint venture company of Cargill company of the U.S., yearly productive capacity is respectively 31000 tons and 34000 tons[7]。
The existing lactic acid production capacity of China is about 25000 tons/year, and actual production is about 15000 tons/year, and the product of 90% is DL-breast
Acid, production scale, acid productivity, product matter scape etc. still have bigger gap compared with world level, particularly the research of Pfansteihl with
Develop the most not enough.Domestic for bacterial fermentation process for preparing L-lactic acid research Pseudomonas mainly with lactobacillus
(Lactobacillus), streptococcus (Streptoccoccus), bacillus (Bacillus) be main, still not with intestines
Coccus strain fermentation produces the research report of Pfansteihl, and this patent utilizes the strain Enterococcus strain dung that screening obtains
Enterococcus is that fermentation strain produces Pfansteihl, has the features such as yield is high, product purity is high, accessory substance is less, is that a strain is great
The Pfansteihl industrial producing strain that research and development are worth.
(3) summary of the invention
The present invention is in order to make up the deficiencies in the prior art, it is provided that a kind of manure enterococcin strain producing Pfansteihl, to realize
Saccharine material microbe fermentation method is utilized to obtain the hope that Pfansteihl is main purpose product.
The present invention is achieved through the following technical solutions:
A kind of manure enterococcin strain producing Pfansteihl, it is characterized in that named VREF Enterococcus
Faeciums HY-U36, this bacterial strain 16S rDNA complete sequence is as follows:
TCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTA
ACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGAT
TTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAA
GGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGG
CAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCG
TAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACG
GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGC
AGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGC
AGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCT
CTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGT
AAACGATGAGTGCTAAGTGTTGGAGGGTTT, above-mentioned 800 nucleotides of 16S rDNA sequence.
The VREF HY-U36 of above-mentioned production of high purity Pfansteihl, its biological property (9) is: (separate in solid culture
Culture medium) to cultivate on flat board and observe, bacterium colony can form canescence, opaque, smooth surface, a diameter of 0. 5～the circle of 1mm
Or ellipse bacterium colony, electron microscopic observation thalline is spherical, how paired or catenation, and Gram's staining result is positive.
Above-mentioned fluid nutrient medium composition (g/L) observed for thalli morphology: glucose 20, peptone 10, yeast extract 5,
K2HPO42, sodium acetate 5, its pH value is 6.5.
Above-mentioned solid medium composition (g/L) observed for thalli morphology: glucose 20, peptone 10, yeast extract 5,
K2HPO42, 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 DEG C,
Can grow under the conditions of weight fraction accounts for 6.5%NaCl culture medium and pH9.6 10 DEG C and 45 DEG C growths；This bacterium may utilize Portugal
Grape sugar, fructose, maltose, sucrose, cellobiose, lactose produce acid, glucose fermentation not aerogenesis, do not utilize citrate, contact
Enzyme is negative, reduction nitrate.
The result of the partial gene sequence that the bacterial strain CGMCC N0.7274 of the present invention measures 16S rRNA is shown, this bacterium
Belonging to enterococcus spp, order-checking nucleotide sequence is as shown in SEQ ID NO.1.
By using U.S.'s Biotechnology Information center (National Center for Biotechnology
Information, NCBI) blast program comparison, find the gene of the CGMCC N0.7274 bacterial strain 16S rRNA of the present invention
Sequence has with the gene order of VREF (Enterococcus faeciums) the LMG 11423 16S rRNA of NCBI registration
Having high homology, and combine bio-chemical characteristics, Preliminary Identification CGMCC N0.7274 bacterial strain is an Enterococcus faecalis
(Enterococcus faeciums).
VREF (Enterococcus faeciums) strain improvement of production of high purity Pfansteihl of the present invention
Basic skills is:
Bromocresol purple-calcium carbonate screening plate screening is used to obtain a strain and can produce the manure enterococcin strain H-of Pfansteihl
36, with H-36 bacterial strain as starting strain, by the physics such as ultraviolet, nitrosoguanidine, chemical mutagen, it is carried out mutagenesis, mutagenesis
Can be repeated several times, method of mutagenesis uses conventional method, coating screening flat board after mutagenesis, picking list bacterium colony transposing inclined-plane, the most again
Carry out shake flask fermentation, by measuring wear rate and the yields screening purpose bacterial strain of Pfansteihl of glucose, choose consumption grape
The bacterial strain that sugar speed is fast, Pfansteihl yield is high, then purify through separating for several times, it is thus achieved that CGMCC N0.7274 bacterial strain.
The VREF (Enterococcus faeciums) of production of high purity Pfansteihl of the present invention is at preparation L-breast
Application in acid.
The method utilizing VREF (Enterococcus faeciums) fermentation production of L-lactic acid is as follows:
Shake flask fermentation: take the 35-40 DEG C of fresh inclined plane inoculating liquid seed culture medium 37 DEG C cultivated 1-2 days and cultivate 10-16
Hour, inoculation fermentation culture medium, 35-40 DEG C of shake flask fermentation 62-84 hour, fermentation ends zymotic fluid can't detect glucose residual
Stay, glucose be converted into Pfansteihl conversion ratio be 84-92%.
50L ferment tank: 37 DEG C of 35-40 DEG C of cultivations of fresh inclined plane inoculating liquid seed culture medium cultivating 1-2 days
10-16 hour, it is linked in preprepared fermentation medium by the inoculum concentration of 1-5%, 50L fermentation tank liquid amount 33L, enters
Row stir culture, is controlled at the beginning of the dissolved oxygen levels saturation degree at 20-35%, zymotic fluid by regulation fermentation tank rotating speed and ventilation ratio
Beginning pH value is 6.5-6.8, maintains the pH of zymotic fluid about 6.2 by Feeding ammonia water in sweat.After 24h, close logical
Air valve proceeds to anaerobic fermentation, and in sweat, the glucose in period sampling measuring zymotic fluid and the concentration of Pfansteihl, work as fermentation
When Pfansteihl concentration no longer rises in liquid, terminating fermentation, fermentation time is 62-84 hours.
Above-mentioned screening plating medium composition (g/L): glucose 20, peptone 10, yeast extract 5, corn steep liquor 5, phosphoric acid
Hydrogen dipotassium 2, calcium lactate 60, bromocresol purple 0.01, calcium carbonate 10, pH6.5-6.8；
Above-mentioned slant medium composition (g/L): glucose 20, peptone 10, yeast extract 5, potassium dihydrogen phosphate 2, agar 20,
pH 6.5-6.8；
Aforesaid liquid seed culture medium composition (g/L): glucose 20, peptone 10, yeast extract 5, corn steep liquor 5, phosphoric acid hydrogen
Dipotassium 2, pH6.5-6.8；
The composition (g/L) of above-mentioned fermentation medium: glucose 80-120, peptone 10-20, yeast extract 3-8, corn steep liquor
10-20, dipotassium hydrogen phosphate 1-3, diammonium hydrogen citrate 1-3, magnesium sulfate 0-1.5, manganese sulfate 0-0.2, Tween 80 0.5-
1.5mL/L, pH6.5-6.8；
The cultivation temperature of above-mentioned Pfansteihl fermentation is preferably 37 DEG C.Above-mentioned on liquid seed culture medium incubation time preferred
For 12-16 hour, fermentation medium initial glucose concentration was preferably 100-120g/L, above-mentioned dissolved oxygen water during the fermentation
The flat saturation degree being preferably controlled in 2-5%.The above-mentioned 50L ferment tank cycle is generally 64-78 hour.Glucose converts L-breast
The average conversion of acid is 84%-91%.
Above-mentioned glucose measures as follows:
The SBA-4D type membrane bioreactor using Shandong Province academy sciences Biology Research Institute to produce measures.Measuring principle is profit
With fixation glucose dehydrogenation enzyme membrane single-minded mensuration glucose content.
Above-mentioned lactic acid measures as follows:
High performance liquid chromatography (HPLC), chromatographic condition: chromatographic column: Prontosil 1202102C18H (10 μm,
4.6mm i.d.×250 mm)；Flow velocity: 0.5mL/min；Sampling volume: 20 μ L；Detection wavelength: 210nm；Column temperature: 30 DEG C；Stream
Dynamic phase: prepare 0.1mol/L KH2PO4, phosphorus acid for adjusting pH to 2.5 with redistilled water.
Above-mentioned Pfansteihl measures as follows:
The SBA-4D type membrane bioreactor using Shandong Province academy sciences Biology Research Institute to produce measures.Measuring principle is solid
Surely change enzyme can specifically act on L (+) lactic acid:
Lactic acid+O2+H2O-> immobilization L (+) LO-> pyruvic acid+H2O2
Zymotic fluid dilutes 50-100 times, it is desirable to the content of the Lactic Acid from Fermentation Broth of dilution is in the range of 0-50mg/dL.
Above-mentioned Pfansteihl purity testing measures as follows:
Pfansteihl purity (%)=Pfansteihl concentration (g/L)/lactic acid concn (g/L)
The invention has the beneficial effects as follows: VREF of the present invention (Enterococcus faeciums) HY-U36
Can directly generate and obtain purpose product Pfansteihl by microbe conversion glucose, glucose is converted into the average conversion of Pfansteihl
For 84%-91%, conversion ratio is high, high purity 98.4%, and the common by-products such as ethanol, malic acid and fumaric acid is less, is a strain pole
The Pfansteihl that tool research and development are worth produces bacterial strain.
(4) accompanying drawing explanation
The present invention is further illustrated below in conjunction with the accompanying drawings.
Bacterial strain VREF (Enterococcus faeciums) HY-U36 that the present invention provides, March 5 in 2013
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: the Chaoyang District, Beijing City North Star
West Road 1 institute, postcode: 100080, its deposit number is CGMCC N0.7274.
The 16S rDNA complete sequence figure of Fig. 1 VREF (Enterococcus faeciums) HY-U36；
Fig. 2 VREF (Enterococcus faeciums) HY-U36 violet staining picture；
Fig. 3 VREF (Enterococcus faeciums) HY-U36 Gram's staining figure；
Fig. 4 VREF (Enterococcus faeciums) HY-U36 bacterial strain fermentation liquor high-efficient liquid phase chromatogram；
Fig. 5 VREF (Enterococcus faeciums) HY-U36 phylogenetic tree construction.
(5) detailed description of the invention
Embodiment 1: produce the screening of Pfansteihl manure enterococcin strain
Take the samples such as soil at animal wastes, ox, sheep rumen content according to a conventional method, take under conditions of sterile working
2g sample, puts into equipped with in the 250mL triangular flask of 40mL sterilized water, takes 1mL and be inoculated into equipped with 40mL enriched medium after concussion
250mL triangular flask in 37 DEG C, 180r/min shaking table cultivate 24h, then by pregnant solution dilution spread to bromocresol purple-calcium carbonate
Screening flat board, incubated in 37 DEG C of anaerobism bags, the bacterium colony that picking color changeable transparent circle is big after growing single bacterium colony punctures preservation.
Then these preservation of bacteria strains carry out shake flask fermentation screening again, and 40mL Medium of shaking flask fermentation is loaded in 250mL triangular flask, 1
Connect 1 bottle graft and enter 1～2 ring slant strains, 37 DEG C, 180r/min shaken cultivation about 64 hours, then survey Pfansteihl in zymotic fluid
Content extremely purity, final screening obtains the bacterial strain of a pure Pfansteihl of plant height, named VREF (Enterococcus
Faeciums) H-36.
Embodiment 2: produce the seed selection of Pfansteihl VREF
Starting strain VREF H-36 is inoculated liquid seed culture medium, cultivates exponential phase mid-term for 37 DEG C, from
The heart collects thalline, and SPSS washes twice, and then adds SPSS and makes bacteria suspension, make cell concentration 1 ×
108-109/ml.Take above-mentioned bacteria suspension 10ml and put in plate, put and irradiate under 30W uviol lamp, irradiation distance 15cm, irradiates
Time is 3 minutes, 5 minutes, 7 minutes, 10 minutes, coats containing weight fraction 6% calcium lactate after interval sampling suitably dilution
Bromocresol purple-calcium carbonate screens flat board, and 37 DEG C of lucifuges are cultivated 1-2 days, single bacterium that picking upgrowth situation is good, color changeable transparent circle is big
Dropping into row preservation, cultivate 1-2 days, inoculate the shaking flask equipped with fermentation medium after inclined-plane maturation for 37 DEG C, 37 DEG C of shake flask fermentations 64 are little
Time, take zymotic fluid 4000r/min and be centrifuged 10 minutes, take residual glucose, lactic acid and Pfansteihl in supernatant mensuration zymotic fluid and contain
Amount, chooses bacterial strain for the purpose of the bacterial strain that concentration of residual glucose is low, lactic acid product acid amount is high and Pfansteihl purity is high.
Purpose bacterial strain is sieved with further separation screening through shake flask fermentation more again, seed selection obtain a strain sugar consumption rate stable and
The mutant strain H-U36 that Pfansteihl yield and purity are all improved.
H-U36 inoculation initial glucose concentration is the fermentation medium of 100g/L, 78 hours knots of 37 DEG C of Shaking culture
Bundle fermentation, can't detect glucose in zymotic fluid, lactic acid production is 75.7g/L, and Pfansteihl purity is 98.2%.
Embodiment 3: the seed selection of high-purity Pfansteihl bacterial strain VREF CGMCC N0.7274
In mutant strain H-U36 inoculation liquid seed culture medium embodiment 2 screened, cultivate logarithm for 37 DEG C
In mid-term in growth period, centrifugal collection thalline, SPSS washes twice, and then adds SPSS and makes bacteria suspension, makes
Cell concentration is at 1 × 108-109/ml, standby.
Nitrosoguanidine treatment fluid is prepared: weighs nitrosoguanidine 20mg, is positioned in the aseptic triangular flask of 100ml, adds acetone 2ml
Make it dissolve, add 18ml Tris buffer solution (pH6.0,0.5mol/L) mixing, standby.
Taking above-mentioned nitrosoguanidine treatment fluid 10ml, add 10ml bacteria suspension, 30 DEG C of insulations are vibrated 50-60 minute, every 10
Minute sampling once, first dilute after sampling 1000 times terminate reaction, dilute 2-10 times the most again, coating bromocresol purple-carbonic acid
Calcium flat board, cultivates 1-2 days for 37 DEG C, picking list bacterium colony transposing inclined-plane, inoculates the shaking flask equipped with fermentation medium after inclined-plane maturation, and 37
DEG C shake flask fermentation 60 hours, zymotic fluid 4000r/min is centrifuged 10 minutes, take supernatant measure glucose in zymotic fluid, lactic acid and
L-lactic acid, chooses that concentration of residual glucose is low, bacterial strain for the purpose of Pfansteihl yield and the high bacterial strain of purity.
Purpose bacterial strain is sieved again through shake flask fermentation again, obtains a strain stable hereditary property, L-breast through further separation screening
Acid yield is up to 81.2g/L, the purity mutant strain HY-U36 more than 99.3%.
Above-mentioned bacterial strains VREF (Enterococcus faeciums) HY-U36, is preserved on March 5th, 2013
China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: BeiYiTiao, ZhongGuanCun, Haidian District, BeiJing City
No. 13, postcode: 100080, its deposit number is CGMCC N0.7274.
Above-mentioned enriched medium composition (g/L): glucose 20, peptone 10, yeast extract 5, dipotassium hydrogen phosphate 2L, pH are
6.5-6.8；
Above-mentioned plate isolation base composition (g/L): glucose 20, peptone 10, yeast extract 5, dipotassium hydrogen phosphate 2, breast
Acid calcium 60, bromocresol purple 0.01, calcium carbonate 10, pH is 6.5-6.8；
Aforesaid liquid seed culture medium composition (g/L): glucose 20-30, peptone 10, yeast extract 5, calcium carbonate 8, phosphoric acid
Hydrogen dipotassium 2, pH is 6.5-6.8；
Above-mentioned Medium of shaking flask fermentation composition (g/L): glucose 80-120, peptone 5-15, yeast extract 2-8, phosphoric acid hydrogen
Dipotassium 1-3, calcium carbonate 0-120, diammonium hydrogen citrate 1-3, magnesium sulfate 0-1.5, manganese sulfate 0-0.2, Tween 80 0.5-
1.5mL/L, pH are 6.5-6.8；
The VREF HY-U36 of the production of high purity Pfansteihl of the present embodiment, its biological property is: (divide in solid culture
From culture medium) on flat board 28-37 DEG C cultivate 36-24h and observe, bacterium colony can form canescence, opaque, smooth surface, a diameter of
The circle of 0. 5～1mm or oval bacterium colony, electron microscopic observation thalline is spherical, and how paired or catenation such as Fig. 2, gram contaminates
Look result is positive, such as Fig. 3.
Above-mentioned fluid nutrient medium composition (g/L) observed for thalli morphology: glucose 20, peptone 10, yeast extract 5,
K2HPO42, sodium acetate 5, its pH value is 6.5.
Above-mentioned solid medium composition (g/L) observed for thalli morphology: glucose 20, peptone 10, yeast extract 5,
K2HPO42, 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 DEG C, can be
10 DEG C and 45 DEG C growths, can grow under the conditions of weight fraction accounts for 6.5%NaCl culture medium and pH9.6；This bacterium may utilize grape
Sugar, fructose, maltose, sucrose, cellobiose, lactose produce acid, glucose fermentation not aerogenesis, do not utilize citrate, catalase
Feminine gender, reduction nitrate.
Bacterial strain CGMCC N0.7274 bio-chemical characteristics is the results detailed in 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 | + |
| Gossypose | - |
| Wood sugar | - |
| Mannitol | + |
| Lactose | + |
| Fructose | + |
| Cellobiose | + |
| D-sorbose | - |
| Melibiose | + |
| Arabinose | + |
| Citrate test | - |
| 0.04% selenite | - |
| Pyruvate fermentation | - |
| Starch Hydrolysis | - |
| Gelatin liquefaction | - |
| H202Enzyme test | - |
| Urease | - |
| V.P. test | + |
| Catalase | - |
| Anti-40% bile | + |
| Indole test | - |
| Mobility test | - |
| Pigment produces | - |
| 4%NaCl tests | + |
| 6.5%NaCl tests | + |
| Growth temperature (DEG C) | |
| 4 | - |
| 37 | + |
| 45 | + |
| 50 | + |
| 55 | - |
Note: "+" well-grown or be positive；"-" does not grows or is negative.
Embodiment 4: VREF (Enterococcus faeciums) CGMCC N0.7274 bacterium 16S rRNA gene is surveyed
Sequence
Pfansteihl superior strain HY-U36 i.e. CGMCC N0.7274 bacterial strain student on commission's work embodiment 3 seed selection obtained is raw
Thing engineering (Shanghai) Co., Ltd. (Sangon Biotech (Shanghai) Co., Ltd) carries out 16S rRNA gene sequencing.
Experimental technique is: picking slant culture is in 10 μ l aqua sterilisas, and after 99 DEG C of sex change, centrifuging and taking supernatant is as mould
Plate, uses TaKaRa 16S rDNA Bacterial Identification PCR Kit (Code No.D310), with
Forward/Reverse primer2 is primer, expands purpose fragment.Take 5 μ l and carry out agarose gel electrophoresis, use TaKaRa
Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) cuts glue and reclaims purpose segment, with
Seq Forward, Seq Reverse Seq Internal are that primer carries out DNA sequencing to above-mentioned recovery product.
Sequencing result: VREF (Enterococcus faeciums) CGMCC N0.7274 16S rDNA checks order core
Nucleotide sequence is as shown in SEQ ID NO.1 and Fig. 1.
By using U.S.'s Biotechnology Information center (National Center for Biotechnology
Information, NCBI) BLASTN program comparison, find that CGMCC N0.7274 bacterial strain 16S rDNA sequence is noted with NCBI
VREF (Enterococcus faeciums) the LMG 11423 16S rDNA sequence of volume has high homology, in conjunction with
Bio-chemical characteristics, illustrates that CGMCC N0.7274 bacterial strain is an Enterococcus faecalis (Enterococcus faeciums), such as figure
5。
Bacterial strain 16S rDNA sequence table:
SEQ ID NO.1:
SEQUENCE LISTING
<110>Shandong Food Fermentative Industry Research and Design Inst.
<120>a kind of manure enterococcin strain producing Pfansteihl
<130> 2012-11-15
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 800
<212> DNA
<213>VREF (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
tgctaagtgt tggagggttt 800
Embodiment 5: the application of VREF (Enterococcus faeciums) CGMCC N0.7274 bacterial strain
By VREF (Enterococcus faeciums) CGMCC N0.7274 bacterial strain transposing slant activation；
Preparing seed culture medium by the composition of aforesaid liquid seed culture medium, initial glucose concentration actual measurement is 22-31 g/
L, 250ml triangular flask liquid amount is 40ml, 115 DEG C of steam sterilizings 20 minutes, is cooled to room temperature, inoculates slant strains 2 ring, puts rotation
Speed of walking around is on the shaking table of 40mm for 150-200r/min, radius of turn, cultivates 12-16 hour, obtains seed liquor, take seed for 37 DEG C
Liquid dilutes 5 times and put 580nm to survey light absorption value is 0.92-0.97；
Preparing fermentation medium by the composition of above-mentioned fermentation medium, initial glucose concentration actual measurement is 73-118g/L,
250ml triangular flask liquid amount is 40ml, 115 DEG C of steam sterilizings 20 minutes, is cooled to room temperature, inoculates seed liquor 1ml, puts rotation and turns
Speed is on 40mm shaking table for 150-200r/min, radius of turn, cultivates 78-96 hour for 28-45 DEG C and terminates fermentation, zymotic fluid with
4000r/min is centrifuged 10 minutes, take that supernatant measures glucose, lactic acid and L-lactic acid are respectively 0g/L, 81.1g/L and
79.8g/L(increases and decreases accordingly according to glucose initial content), it is 87.9% that glucose is converted into the conversion ratio of Pfansteihl, L-breast
Acid optical purity is 98.4%.
Or:
Press 30L liquid amount preparation fermentation medium with 50L fermentation tank, adjust pH6.8,115 DEG C of real tank sterilizings 20 minutes, follow
Ring inoculates cultured seed liquor 600 ml after being water-cooled to 37 DEG C, after inoculation, sampling and measuring concentration of glucose is 72-immediately
117g/L。
Preliminary fermentation tank parameter is set to: speed of agitator is 200-350r/min, ventilation ratio 1:0.05-0.1vvm(feed liquid
Volume and the ratio of throughput per minute (measurement basis)), pressure inside the tank 0.04-0.08MPa.Sweat controls fermentation temperature
Spend 37 ± 0.2 DEG C, be 20-35% by 24h before regulation speed of agitator and ventilation ratio control dissolved oxygen, close breather valve afterwards,
pH6.2±0.2。
Sweat samples for every eight hours, measures glucose, lactic acid and the content of Pfansteihl and thalline in zymotic fluid dense
Degree, when glucose content is less than 10 g/L, glucose, lactic acid and L-lactic acid in every 2 hours sampling and measuring zymotic fluids, extremely
In zymotic fluid, L-lactic acid terminates fermentation when not being further added by, and fermentation period is 78 hours.
Take zymotic fluid after fermentation ends to be centrifuged 10 minutes with 4000r/min, take supernatant and measure glucose, lactic acid and L-breast
The content of acid is respectively 0g/L, 82.7g/L and 81.9g/L(and increases and decreases accordingly according to glucose initial content), glucose converts raw
The conversion ratio becoming Pfansteihl is 89.8%, and Pfansteihl optical purity is 99.0%.
Above-mentioned glucose measures as follows:
The SBA-4D type membrane bioreactor using Shandong Province academy sciences Biology Research Institute to produce measures.Measuring principle is profit
With fixation glucose dehydrogenation enzyme membrane single-minded mensuration glucose content.
Above-mentioned lactic acid measures as follows:
High performance liquid chromatography (HPLC) such as Fig. 4, chromatographic condition:
Chromatographic column: Prontosil 1202102C18H (10 μm, 4.6mm i.d. × 250 mm)；Flow velocity: 0.5mL/
min；Sampling volume: 20 μ L；Detection wavelength: 210 nm；Column temperature: 30 DEG C；Flowing phase: prepare 0.1mol/L with redistilled water
KH2PO4, phosphorus acid for adjusting pH to 2.5.
Above-mentioned Pfansteihl measures as follows:
The SBA-4D type membrane bioreactor using Shandong Province academy sciences Biology Research Institute to produce measures.Measuring principle is solid
Surely change enzyme can specifically act on L (+) lactic acid:
Lactic acid+O2+H2O-> immobilization L (+) LO-> pyruvic acid+H2O2
Zymotic fluid dilutes 50-100 times, it is desirable to the content of the Lactic Acid from Fermentation Broth of dilution is in the range of 0-50mg/dL.
Above-mentioned Pfansteihl purity testing measures as follows:
Pfansteihl purity (%)=Pfansteihl concentration (g/L)/lactic acid concn (g/L).
Bibliography:
[1] Liu Weixiong. the latest developments [J] of lactic acid and PLA. 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] the elegant pearl in east, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, the first edition: 62-
63。
[9] the elegant pearl in east, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, the first edition: 353-
363。
[10] the elegant pearl in east, Cai Miaoying. common bacteria system identification handbook [M]. Science Press, 2001, the first edition: 364-
398。
SEQ ID NO.1
SEQUENCE LISTING
<110>Shandong Food Fermentative Industry Research and Design Inst.
<120>a kind of manure enterococcin strain producing Pfansteihl
<130> 2012-11-15
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 800
<212> DNA
<213>VREF (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
tgctaagtgt tggagggttt 800
Claims (1)
1. the manure enterococcin strain producing Pfansteihl, it is characterised in that: named VREF Enterococcus
Faeciums HY-U36, deposit number is CGMCC N0.7274, and the 16S rDNA complete sequence of this bacterial strain is as follows:
TCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTG
GGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTT
TGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCA
CCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGG
GAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGG
ATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGC
CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA
GCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGA
GTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGC
GGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTT, above-mentioned 800 nucleotides of 16S rDNA sequence,
Use BLAST analytic approach, by the VREF LMG of the 16S rDNA complete sequence of VREF HY-U36 with NCBI registration
The Gene sequence comparison of 11423 16S rRNA, homology is 98.0%-99.0%,
Cultivating on the flat board of solid culture or isolation medium and observe, bacterium colony forms canescence, opaque, smooth surface, straight
Footpath is the circle of 0. 5～1mm or oval bacterium colony, and electron microscopic observation thalline is spherical, and how paired or catenation, gram contaminates
Look result is positive,
Strain growth temperature is 10-45 DEG C, and optimum growth temperature is 30～37 DEG C,
This bacterial strain is to obtain Pfansteihl for saccharine material microbe fermentation method.
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| 7株鸡源肠球菌的分离、鉴定及益生性能比较;惠明等;《中国粮油学报》;20120725;77-80 * |
| Direct l-lactic acid fermentation with sago starch by a novel amylolytic lactic acid bacterium, Enterococcus faecium;Keisuke Shibata等;《Enzyme and Microbial Technology》;20061228;149-155 * |
| 新疆传统发酵酸乳中屎肠球菌Enterococcus faecom的高密度培养;胡敏等;《中国乳品工业》;20121231;12-17 * |
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