CN110241043A - The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid - Google Patents
The bacterial strain of one plant height temperature fermenting lactic acid and the method for producing lactic acid Download PDFInfo
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Abstract
The present invention provides the bacterial strain of a plant height temperature fermenting lactic acid and the methods for producing lactic acid, Strain Designation are as follows: E.faecalis DUT1805, hot fermentation glucose production lactic acid under in such a way that the bacterial strain is in gradient increased temperature, gradient increased temperature is 37-40-45-50 DEG C of gradient or 37-40-45-50 DEG C of -55 DEG C of gradients, it is warming up to 50 DEG C or 55 DEG C from 37 DEG C, is 2h, 4h, 6h or 8h the time required to whole heating.E.faecalis DUT1805 has high production intensity and rotational rate of lactic acid；Meanwhile E.faecalis DUT1805 direct metabolism biological matter raw material such as paddy meal or corn stalk hydrolysis conversion can produce lactic acid under 50 DEG C of high temperature without gradient increased temperature.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to the bacterial strain of a plant height temperature fermenting lactic acid and utilization should
The method of bacterial strain production lactic acid.
Background technique
Lactic acid, also referred to as α-hydracrylate are one of three big organic acids (carboxylic acid, carboxylic acid, ketone acid), are important fine
Chemicals, be one of strategic 12 platform chemicals of oil replacement that U.S. Department of Energy determines (Zhang Li, biological processing,
2018,16 (4): 23-29).Lactic acid is widely used in the fields such as food, chemical industry, medical treatment, wherein synthesizes using lactic acid as monomer poly-
Lactic acid is application (Andrea Komesu et al., BioResources, 2017,12 (2): 4364- of lactic acid most prospect
4383).With the exhaustion of the non-renewable resources such as petroleum resources, the deterioration of the environment such as white pollution, greenhouse effects constructs resource
Circular form, environmentally friendly, sustainable development development model have become the general trend of events.Wherein, Biodegradable high-molecular material is pushed
The exploitation and popularization of material are one of important measures, biodegradable material of the polylactic acid as current first industrialized production,
One of referred to as most potential biomaterial (Wang Yue, biological industry technology, 2017,6:17-22).
It mainly includes lactic acid bacteria (Lactic acid bacteria), gemma that the production strain of lactic acid, which is studied more, at present
Bacillus (Bacillus strains), enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia
Coli), Rhizopus oryzae (Rhizopus oryzae) etc., using glucose as carbon source, production cost is relatively high.Biomass is former
Material has that cheap, the advantages such as be easy to get, thus becomes the new raw material of production of lactic acid.It mainly includes glycosyl raw material (sugarcane, beet
Deng), fiber-like raw material (stalk, straw, wood and rice husk etc.) and starchy material (potato, cereal etc.).Related biomass is former
The report of material production lactic acid has very much, and Pediococcus acidilactici DQ2 can get the lactic acid of 101.9g/L by corn stover at 48 DEG C
(Kai Zhao et al.,Bioresource Technology,2013,135:481-489)；Enterococcus faecalis SI at 42 DEG C,
It can get the lactic acid of 59.8g/L by lignocellulosic material, conversion ratio can reach 0.95g/g (Shuo-Fu Yuan et
al.,Journal of Industrial Microbiology&Biotechnology,2018,45(11):961-970)；Bud
Spore bacillus LA204 at 50 DEG C, using by corn stover obtain 97.6g/L lactic acid (Jinlong Hu et al.,
Bioresource Technology,2015,182:251-257)；Rhizopus oryzae DMKU 33 is obtained at 40 DEG C by tapioca starch
Lactic acid (Srisakul Trakarnpaiboon et al., the Process Biochemistry, 2017,63:26- of 84.0g/L
34).Existing report generally uses hot fermentation it is found that producing lactic acid by biomass material, and the bacterial strain used mainly has resistance to
This three categories of hot lactic acid bacteria, bacillus and Rhizopus oryzae.
Hot fermentation is one of the important channel for strengthening the raw materials synchronous saccharification such as lignocellulosic or paddy, this advantage master
It shows in the following areas:
(1) cellulase or amylase aspect: cellulase and amylase optimum temperature are respectively all at 50 DEG C at present
With 60 DEG C.Ordinary temperature synchronous fermentation, reduces the activity of cellulase or amylase, to need to increase cellulase or shallow lake
Powder enzyme dosage.
(2) in terms of fermenting property: hot conditions are conducive to the saccharification of the raw materials such as lignocellulosic or paddy, realize more preferably faster
Mass transfer.Meanwhile high temperature microbe is resistant to temperature distributing disproportionation caused by large-scale reactor and fluctuation, is conducive to strain and sends out
The promotion of ferment performance.
(3) in terms of production cost: hot fermentation can be substantially reduced cooling cost, especially in high temperature season and torrid zone ground
Area.Meanwhile hot fermentation significantly reduces the risk of microbiological contamination.
The temperature of advantage based on hot fermentation, the Enterococcus faecalis fermentation lactic acid reported at present only up to reach 42 DEG C,
It is therefore desirable for obtaining the bacterial strain of one plant of fermenting lactic acid more resistant to high temperature.
Summary of the invention
In order to overcome the problems, such as that existing enterococcus faecalis cannot be even higher than 50 DEG C of at a temperature of fermenting lactic acid at 50 DEG C,
The present invention provides the bacterial strain of a plant height temperature fermenting lactic acid and the method using strain fermentation production lactic acid.
For strain isolation of the invention from the fresh content of ox stomach, Strain Designation is Enterococcus faecalis
DUT1805, the bacterial strain is on April 28th, 2019 in Chinese microorganism strain collection preservation (address are as follows: Beijing's southern exposure
The institute 3 of area North Star West Road 1, postcode 100101), deposit number are as follows: CGMCC NO.17640.Classification naming are as follows: enterococcus faecalis
Enterococcus faecalis DUT1805。
The method of hot fermentation production lactic acid of the present invention, the method pass through 37~50 DEG C of gradient increased temperature with above-mentioned bacterial strains
Or 37~55 DEG C, glucose fermentation produces lactic acid at a temperature of 50 DEG C or 55 DEG C.
In the method for above-mentioned hot fermentation production lactic acid, gradient increased temperature are as follows: 37 DEG C of -40 DEG C of -45 DEG C of -50 DEG C of gradients or 37
DEG C -40 DEG C of -45 DEG C of -50 DEG C of -55 DEG C of gradients are warming up to 50 DEG C or 55 DEG C from 37 DEG C, are 2h, 4h, 6h the time required to whole heating
Or 8h.
In the method for above-mentioned hot fermentation production lactic acid, fermentation process stuffiness.
The method of hot fermentation production lactic acid of the present invention, the method are fermented at a temperature of 37~50 DEG C with above-mentioned bacterial strains
Paddy meal or ligno-cellulose hydrolysate produce lactic acid.
Fermentation process is unsterilised in the method for above-mentioned hot fermentation production lactic acid.
In the method for above-mentioned hot fermentation production lactic acid, fermentation process stuffiness.
Application of the bacterial strain Enterococcus faecalis DUT1805 of the present invention in fermenting lactic acid.
Beneficial effects of the present invention:
(1) the present invention provides high-temperature resistant strain-enterococcus faecalis Enterococcus that a kind of fermentation prepares lactic acid
Faecalis DUT1805, fermenting stability is good, and high temperature resistance is strong, can tolerate 50 DEG C or more high temperature, and production intensity is high.
(2) the present invention provides utilize enterococcus faecalis Enterococcus faecalis DUT1805 fermenting lactic acid
Method, take the mode of gradient increased temperature to carry out temperature control in fermentation process, improve adaptability, tolerance of the strain to high temperature,
New method is provided for strain domestication.
(3) for the present invention using paddy or lignocellulosic as raw material, hot fermentation produces lactic acid, solves hydrolysis temperature and hair
The uncoordinated disadvantage of ferment temperature can make enzyme fermenting lactic acid under the conditions of optimum temperature using bacterial strain of the present invention, improve
The utilization rate of enzyme realizes synchronous saccharification conversion production lactic acid, improves the efficiency of production lactic acid.
(4) mode of hot fermentation of the invention can reduce cooling cost, public work consumption be reduced, to reduce cream
Sour production cost.
Detailed description of the invention
Fig. 1 E.faecalis DUT1805 colonial morphology figure；
Fig. 2 E.faecalis DUT1805 Gram's staining result；
At 50 DEG C of Fig. 3, influence of the grape carbohydrate stress to E.faecalis DUT1805 fermenting property；
At 50 DEG C of Fig. 4, influence of the different temperature control strategies to E.faecalis DUT1805 fermenting property, A) 40g/L grape
Sugar；B) 80g/L glucose；
At 50 DEG C of Fig. 5, pH coerces the influence of E.faecalis DUT1805 fermenting property；
Influence of Fig. 6 high temperature to E.faecalis DUT1805 fermenting property；
At 55 DEG C of Fig. 7, influence of the different temperature control strategies to E.faecalis DUT1805 fermenting property；
Fig. 8 E.faecalis DUT1805 high temperature synchronous saccharification paddy meal produces the metabolic map of lactic acid, A) MRS culture medium,
B) Dried Corn Steep Liquor Powder culture medium；
The metabolic map of Fig. 9 E.faecalis DUT1805 hot fermentation ligno-cellulose hydrolysate production lactic acid.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.In following embodiments, unless otherwise specified, institute
The experimental method used is conventional method, and reagent used etc. can chemically or biological reagent company is bought.
1. the culture medium that following embodiments use:
(1) seed culture medium (g/L): glucose 30, peptone 10, beef extract 10, yeast extract 5, (NH4)2HC6H5O7
2, CH3COONa·3H2O 2, KH2PO4·3H2O 2, MgSO4·7H2O 0.2, MnSO4·H2O 0.05, MgCO312 (pH=
6.3)。
(2) enriched medium (g/L): glucose 40, yeast extract 5, C4H2Na2O45, coban 0.1, K2HPO4·
3H2O 3, NaCl 1, (NH4)2SO41, CaCl20.5, MgCl2 0.5。
(3) RCM culture medium (g/L): peptone 10, beef extract 10, yeast extract 3, glucose 5, soluble starch 1,
NaCl 15, CH3COONa·3H2O 3, L-cysteine hydrochloride 0.5.
(4) MRS culture medium (g/L): glucose 40,80,100,120,160 or paddy meal 150 or corn stalk hydrolysis,
Peptone 10, beef extract 10, yeast extract 5, (NH4)3C6H5O72, CH3COONa 5, K2HPO42, MgSO4·7H2O 0.58,
MnSO4·H2O 0.25 (pH=6.5 or 6.0).
(5) Dried Corn Steep Liquor Powder culture medium (g/L): paddy meal or corn stalk hydrolysis, Dried Corn Steep Liquor Powder 16, (NH4)3C6H5O72, CH3COONa 5, K2HPO42, MgSO4·7H2O 0.58, MnSO4·H2O 0.25(pH 6.5)。
2. seed culture:
Using 250mL cillin bottle, liquid amount 100mL.It is housed to after culture medium and persistently leads to the deoxygenation of 3min nitrogen for every bottle, later
It is bound with butyl rubber plug, 121 DEG C of sterilizing 20min.It is inoculated with and is sampled, inoculum concentration 2% with disposable sterilized needle tubing in experimentation
(v/v), 37 DEG C of cultivation temperature, shaking speed 200r/min, incubation time 10-12h.
3. fermentation culture conditions:
Using 5L automatic fermenter, the product of seed culture is seeded to hair with inoculum concentration 5% (v/v) by liquid amount 2L
In ferment culture medium, respectively using glucose, paddy meal, corn stalk hydrolysis as substrate, in fermentation jar temperature 37-50 or 37-55
DEG C, ferment under revolving speed 200r/min, it is 6.5 or 6.0 that fermentation process 8M NaOH, which controls pH,.
4. temperature control strategy:
Seed culture medium temperature is 37 DEG C, and fermentation liquid culture medium temperature is 50 or 55 DEG C.In view of strain is to cultivation temperature
Have certain laundering period, thus select by 37-40-45-50 DEG C of this 3 gradient increased temperatures to 50 DEG C or 37-40-45-50-55 DEG C this
4 gradient increased temperatures are to 55 DEG C, and heating is set aside some time as 20min or so every time, and have investigated 2h, 4h, 6h, 8h different heating-up times
The influence of glucose fermentation production lactic acid is utilized to enterococcus faecalis.For using paddy meal or corn stalk hydrolysis as the life of representative
Physical resources are fermented using direct 50 DEG C, are not necessarily to gradient increased temperature.Gradient increased temperature concrete operations are as follows:
2h is warming up to 50 DEG C: 37 DEG C of initial temperature, 40 DEG C are warming up to after 30min, 45 DEG C are warming up to after 65min, 105min
After be warming up to 50 DEG C.
4h is warming up to 50 DEG C: 37 DEG C of initial temperature, 40 DEG C are warming up to after 1.17h, 45 DEG C are warming up to after 2.5h, is risen after 3.5h
Temperature is to 50 DEG C.
6h is warming up to 50 DEG C: 37 DEG C of initial temperature, 40 DEG C are warming up to after 1.67h, 45 DEG C, after 5.67h are warming up to after 3.67h
It is warming up to 50 DEG C；
6h is warming up to 55 DEG C: 37 DEG C of initial temperature, 40 DEG C are warming up to after 1.50h, 45 DEG C, after 4.2h are warming up to after 2.83h
50 DEG C are warming up to, 55 DEG C are warming up to after 5.5h.
8h is warming up to 55 DEG C: 37 DEG C of initial temperature, 40 DEG C are warming up to after 1.67h, 45 DEG C, after 5.67h are warming up to after 3.67h
50 DEG C are warming up to, 55 DEG C are warming up to after 7.67h.
Embodiment 1
The screening of E.faecalis DUT1805 prepares and identification:
Take the fresh content of 0.1kg ox stomach in the triangular flask of 100mL physiological saline, vortex oscillation 2min, solid-liquid mixing
Object is inoculated in the cillin bottle equipped with 100mL enriched medium, carries out anaerobism enrichment culture.Continuous passage 12 times, obtain Fermented
The stable mixed bacterium of energy.The flora is inoculated in the cillin bottle that 250mL contains 100mL RCM culture medium, 90 DEG C of heat treatment 20min, so
After be coated on RCM plate, plate 37 DEG C of Anaerobic culturels for 24 hours after, picking single colonie carries out scribing line separation, isolates and purifies for 3 generations
Conservation afterwards.The single bacterium DNA of extraction separation and purification, expanded using 16S rDNA bacterial universal primers (primer 2 7F:
AGTTTGATCMTGGCTCAG；1492R:GGTTACCTTGTTACGACTT), it is compared and is analyzed by 16S rDNA, determine that it is excrement
Enterococcus is named as E.faecalis DUT1805.Single colonie on plate is rounded, opaque, neat in edge, and bacterium colony is in cream
White, surface bulge is wet, colony diameter 0.5-1mm (Fig. 1).Observation optical microphotograph knows thallus under the microscope under the microscope
To be spherical, in pairs or catenation, 0.3-0.7 μm of size, bacterial Gram stained positive (Fig. 2).
At 2 50 DEG C of embodiment, influence of the grape carbohydrate stress to E.faecalis DUT1805 fermenting property
Enterococcus faecalis E.faecalis DUT1805 is seeded to by 5% volume ratio equipped with 2L after seed culture medium
It is cultivated in the 5L fermentor of MRS culture medium, from 37 DEG C of gradient increased temperatures to 50 DEG C in 2h, concentration of glucose is gradually mentioned from 40g/L
It is raised to 160g/L, entire fermentation process stuffiness, fermentation results are as shown in Figure 3.The result shows that: enterococcus faecalis DUT1805 high temperature
It can tolerate the glucose of 160g/L at 50 DEG C.Wherein, batch fermentation efficiency highest when concentration of glucose is 40g/L, lactic acid is most
Final concentration of 37.92g/L, the conversion ratio relative to glucose are 0.92g/g, and production intensity is 3.79g/ (L.h), Metabolic byproducts
Object has acetic acid, succinic acid and ethyl alcohol, and concentration is respectively 4.57g/L, 1.30g/L and 1.55g/L.When concentration of glucose is 80g/L,
The final concentration 60.65g/L of lactic acid, the conversion ratio relative to glucose are 0.87g/g, and production intensity is 1.26g/ (L.h)；Grape
When sugared concentration is 100g/L, the ultimate density of lactic acid is 77.29g/L, and the conversion ratio relative to glucose is 0.89g/g, production
Intensity is 1.25g/ (L.h)；When concentration of glucose is 120g/L, the final concentration of 84.04g/L of lactic acid, relative to glucose
Conversion ratio is 0.92g/g, and production intensity is 1.40g/ (L.h)；Concentration of glucose be 160g/L when, lactic acid it is final concentration of
79.35g/L, the conversion ratio relative to glucose are 0.90g/g, and production intensity is 1.10g/ (L.h).
At 3 50 DEG C of embodiment, influence of the different temperature control strategies to E.faecalis DUT1805 fermenting property
Enterococcus faecalis DUT1805 is seeded to the 5L equipped with 2L MRS culture medium after seed culture medium, by 5% volume ratio
It is cultivated in fermentor, entire fermentation process stuffiness, inquires into different gradient increased temperature modes and E.faecalis DUT1805 is utilized
The influence of glucose fermentation production lactic acid.Fermentation results are as shown in Figure 4.When substrate is the glucose of 40g/L, from 37 DEG C in 4h
The generating rate of the experimental group of gradient increased temperature to 50 DEG C, cell growth, glucose consumption rate and lactic acid is better than 2h, 6h inside gradient
It is warming up to 50 DEG C of experimental groups.The ultimate density of lactic acid is 36.56g/L, and the conversion ratio relative to glucose is 0.92g/g, production
Intensity be 4.57g/ (L.h), the concentration of metabolic byproducts acetic acid, succinic acid and ethyl alcohol be respectively 3.87g/L, 1.81g/L and
2.44g/L.When concentration of substrate is lower (≤40g/L), 4h inside gradient is selected to be warming up to 50 DEG C as optimum temperature rise mode.Work as substrate
For 80g/L glucose when, from 37 DEG C of gradient increased temperatures to 50 DEG C of experimental group in 6h, cell growth, glucose consumption rate and
The generating rate of lactic acid is warming up to 50 DEG C of experimental groups better than 2h, 4h inside gradient.The ultimate density of lactic acid is 65.41g/L, and lactic acid produces
Rate is 0.88g/g, and production intensity is 4.09g/ (L.h), production intensity much stronger than 2h gradient increased temperature strategy (1.26g/ (L.h)) and
4h gradient increased temperature strategy (2.07g/ (L.h))；The concentration of metabolic byproducts acetic acid, succinic acid and ethyl alcohol be respectively 4.98g/L,
1.89g/L and 5.38g/L.Therefore it when concentration of substrate is higher (>=80g/L), selects 6h inside gradient to be warming up to 50 DEG C and is risen to be best
Warm mode.
At 4 50 DEG C of embodiment, pH coerces the influence to E.faecalis DUT1805 fermenting property
Enterococcus faecalis DUT1805 is seeded to the 5L equipped with 2L MRS culture medium after seed culture medium, by 5% volume ratio
It is cultivated in fermentor, Initial sugar concentration 40g/L, entire fermentation process stuffiness, inquires into different fermentations pH to the bacterium Fermented
The influence of energy, fermentation results are as shown in Figure 5.Comparison discovery, cell growth, glucose consumption rate and lactic acid generate when pH 6.5
Rate is better than 6.0 experimental group of pH, and the ultimate density 37.92g/L of lactic acid, the conversion ratio relative to glucose is 0.92g/g, raw
Production intensity be 3.79g/ (L.h), the concentration of metabolic byproducts acetic acid, succinic acid and ethyl alcohol be respectively 4.57g/L, 1.30g/L and
1.55g/L.PH=6.0 experimental group fermentation 36h still has the residual sugar of 3.57g/L, the final concentration of 33.60g/L of lactic acid, relative to Portugal
The conversion ratio of grape sugar is 0.98g/g, and production intensity is 0.93g/ (L.h), the concentration of metabolic byproducts acetic acid, succinic acid and ethyl alcohol
Respectively 4.23g/L, 0.55g/L and 2.53g/L.Result of study shows: under high temperature, pH ferments to E.faecalis DUT1805
Performance is affected, and the bacterium is poor to the tolerance of low pH, with the decline of pH, produces intensity and sharply declines.
Influence of 5 high temperature of embodiment to E.faecalis DUT1805 fermenting property
Enterococcus faecalis DUT1805 is seeded to the 5L equipped with 2L MRS culture medium after seed culture medium, by 5% volume ratio
It is cultivated in fermentor, Initial sugar concentration 80g/L, from 37 DEG C of gradient increased temperatures in from 37 DEG C of gradient increased temperatures to 50 DEG C or 6h in 6h
To 55 DEG C, influence of the high temperature to the bacterium fermenting property is inquired into entire fermentation process stuffiness, and fermentation results are as shown in Figure 6.Research
The result shows that 50 DEG C of experimental group fermenting properties are better than 55 DEG C of experimental groups, the ultimate density of lactic acid is 65.41g/L, lactic acid relative to
The conversion ratio of glucose is 0.88g/g, and production intensity is 4.09g/ (L.h), metabolic byproducts acetic acid, succinic acid and ethyl alcohol it is dense
Degree is respectively 4.98g/L, 1.89g/L and 5.38g/L.55 DEG C of experimental groups still have the residual sugar of 13.92g/L after fermentation for 24 hours.Excrement intestines
Tolerable 55 DEG C of the high temperature of coccus DUT1805, but for 50 DEG C of experimental groups, 55 DEG C of experimental group fermenting properties are general.Two
Person's glucose consumption rate is not much different at fermentation initial stage, and 55 DEG C of experimental groups of later period lead to Portugal since growth is suppressed
Grape sugar consumption rate and production strength reduction.
At 6 55 DEG C of embodiment, influence of the different temperature control strategies to E.faecalis DUT1805 fermenting property
Enterococcus faecalis DUT1805 is seeded to the 5L equipped with 2L MRS culture medium after seed culture medium, by 5% volume ratio
It is cultivated in fermentor, Initial sugar concentration 80g/L, entire fermentation process stuffiness inquires into different gradient increased temperature modes to the bacterium
The influence of fermenting property, fermentation results are as shown in Figure 7.Temperature is risen to 55 DEG C from 37 DEG C in 6h, 8h respectively, comparison is found,
The gradient increased temperature time is affected to Fungal biodiversity and base consumption rate, and the fermenting property of 8h heating experimental group is better than 6h liter
Warm experimental group.6h heating experimental group fermentation ends still have a residual sugar of 13.92g/L, and 8h heats up experimental group, glucose for 24 hours it
It is inside consumed totally, lactic acid is 0.82g/g relative to the conversion ratio of glucose, and production intensity is 2.49g/ (L.h), Metabolic byproducts
The concentration of object acetic acid, succinic acid and ethyl alcohol is respectively 6.55g/L, 2.12g/L and 6.28g/L.Result of study shows: extending heating
Time facilitates thallus and adapts to hot environment, improves the fermenting property of thallus.
7 E.faecalis DUT1805 high temperature synchronous saccharification paddy meal of embodiment produces lactic acid
(A) MRS culture medium
E.faecalis DUT1805 is seeded to by 5% volume ratio after seed culture medium and is with paddy meal equipped with 2L
It is cultivated in the 5L fermentor of the MRS culture medium of carbon source, paddy meal concentration 150g/L, pH=6.5, adds 0.075% (v/v) respectively
α-amylase and carbohydrase, entire fermentation process stuffiness inquire into E.faecalis DUT1805 using paddy meal high temperature and synchronize sugar
The feasibility of metaplasia lactic acid producing, fermentation results are as shown in Figure 8 A.At synchronous saccharification initial stage, concentration of glucose is gradually increased, product
Concentration is lower, and base consumption rate and product formation rate are very fast after 12h, absolutely proves, in paddy meal culture, enterococcus faecalis
DUT1805 can be resistant to 50 DEG C of high temperature, but the seed of 37 DEG C of cultures is linked into 50 DEG C of fermentor and cultivates, and has prolonging for 12h
Demurrage.Terminate fermentation after 34h, without residual sugar in tank, lactic acid final concentration 73.36g/L, the conversion ratio relative to starch is
0.89g/g, production intensity are 2.16g/ (L.h), and the concentration of metabolic byproducts acetic acid and ethyl alcohol is respectively 3.72g/L and 2.58g/
L.Result of study shows: E.faecalis DUT1805 can produce lactic acid by synchronous saccharification paddy meal at a high temperature of 50 DEG C.
(B) Dried Corn Steep Liquor Powder culture medium
E.faecalis DUT1805 is seeded to by 5% volume ratio after seed culture medium and is with paddy meal equipped with 2L
It is cultivated in the 5L fermentor of the Dried Corn Steep Liquor Powder culture medium of carbon source, paddy meal concentration 150g/L, pH=6.5 are added respectively
0.075% (v/v) α-amylase and carbohydrase, entire fermentation process stuffiness inquire into E.faecalis DUT1805 using inexpensively
Culture medium high temperature synchronous saccharification paddy meal produces the feasibility of lactic acid, and fermentation results are as shown in Figure 8 B.At synchronous saccharification initial stage, Portugal
Grape sugar concentration gradually increases, and production concentration is lower, and base consumption rate and product formation rate are very fast after 8h, absolutely proves,
In paddy meal culture, E.faecalis DUT1805 can be resistant to 50 DEG C of high temperature, but the seed of 37 DEG C of cultures is linked into 50 DEG C
Fermentor in cultivate, have the lag phase of 8h.Terminate fermentation after 34h, without residual sugar in tank, lactic acid final concentration 73.75g/L,
Conversion ratio relative to starch is 0.87g/g, and production intensity is 2.17g/ (L.h), the concentration of metabolic byproducts acetic acid and ethyl alcohol
Respectively 3.48g/L and 3.63g/L.Result of study shows: E.faecalis DUT1805 can use Dried Corn Steep Liquor Powder substitution
Traditional MRS culture medium synchronous saccharification paddy meal at a high temperature of 50 DEG C produces lactic acid.
8 E.faecalis DUT1805 hot fermentation ligno-cellulose hydrolysate of embodiment produces lactic acid
Enterococcus faecalis DUT1805 after corn stover is saccharified 47h, is seeded to after seed culture medium by 5% volume ratio
It is cultivated in 5L fermentor equipped with 2L Dried Corn Steep Liquor Powder culture medium, corn stover concentration is 100g/L, and entire fermentation process is obstructed
Gas, inquires into the feasibility that the bacterium utilizes ligno-cellulose hydrolysate high temperature production lactic acid, and fermentation results are as shown in Figure 9.100g/L
The corn stover of acid processing adds 0.6% (v/v) cellulase, and when saccharification carries out 47h, glucose and xylose concentration is respectively
17.9g/L,2.4g/L.Terminate fermentation, the ultimate density 21.59g/L of lactic acid after 26h, the conversion ratio relative to glucose is
0.93g/g, production intensity are 0.86g/ (L.h), and metabolic byproducts acetic acid concentration is 0.77g/L.Result of study shows: excrement intestines ball
Bacterium DUT1805 can produce lactic acid by fermenting lignocellulose hydrolyzate at a high temperature of 50 DEG C.
For any person skilled in the art, without departing from the scope of the technical proposal of the invention, all
Many possible changes and modifications are made to technical solution of the present invention using the technology contents of the disclosure above, or are revised as equivalent
The equivalent embodiment of variation.Therefore, anything that does not depart from the technical scheme of the invention, according to the technical essence of the invention to
Any simple modifications, equivalents, and modifications that upper embodiment is done should all still fall within the range of technical solution of the present invention protection
It is interior.
SEQUENCE LISTING
<110>Dalian University of Technology
The bacterial strain of<120>one plant height temperature fermenting lactic acids and the method for producing lactic acid
<130> 2019
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1468
<212> DNA
<213>E. faecalis DUT1805 16S rDNA sequence
<400> 1
gggcgatggg gggcggctat acatgcaagt cgaacgcttc tttcctcccg agtgcttgca 60
ctcaattgga aagaggagtg gcggacgggt gagtaacacg tgggtaacct acccatcaga 120
gggggataac acttggaaac aggtgctaat accgcataac agtttatgcc gcatggcata 180
agagtgaaag gcgctttcgg gtgtcgctga tggatggacc cgcggtgcat tagctagttg 240
gtgaggtaac ggctcaccaa ggccacgatg catagccgac ctgagagggt gatcggccac 300
actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttcggcaa 360
tggacgaaag tctgaccgag caacgccgcg tgagtgaaga aggttttcgg atcgtaaaac 420
tctgttgtta gagaagaaca aggacgttag taactgaacg tcccctgacg gtatctaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540
ccggatttat tgggcgtaaa gcgagcgcag gcggtttctt aagtctgatg tgaaagcccc 600
cggctcaacc ggggagggtc attggaaact gggagacttg agtgcagaag aggagagtgg 660
aattccatgt gtagcggtga aatgcgtaga tatatggagg aacaccagtg gcgaaggcgg 720
ctctctggtc tgtaactgac gctgaggctc gaaagcgtgg ggagcaaaca ggattagata 780
ccctggtagt ccacgccgta aacgatgagt gctaagtgtt ggagggtttc cgcccttcag 840
tgctgcagca aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggtcttg acatcctttg accactctag agatagagct ttcccttcgg 1020
ggacaaagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttattgtta gttgccatca tttagttggg cactctagcg 1140
agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggga agtacaacga gtcgctagac cgcgaggtca 1260
tgcaaatctc ttaaagcttc tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag 1320
ccggaatcgc tagtaatcgc ggatcagcac gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taaccttttg 1440
gagccagccg cctaaggtga ttaaaggg 1468
Claims (7)
1. the bacterial strain Enterococcus faecalis DUT1805 of a plant height temperature fermenting lactic acid, deposit number are as follows:
CGMCC NO.17640。
2. a kind of method of hot fermentation production lactic acid, which is characterized in that the method is passed through with bacterial strain described in claim 1
37~50 DEG C or 37~55 DEG C of gradient increased temperature, glucose fermentation produces lactic acid at a temperature of 50 DEG C or 55 DEG C.
3. the method for hot fermentation production lactic acid according to claim 2, which is characterized in that the gradient increased temperature are as follows: 37
DEG C -40 DEG C of -45 DEG C of -50 DEG C of gradients or 37 DEG C of -40 DEG C of -45 DEG C of -50 DEG C of -55 DEG C of gradients, are warming up to 50 DEG C or 55 DEG C from 37 DEG C, whole
It is 2h, 4h, 6h or 8h the time required to body heating.
4. a kind of method of hot fermentation production lactic acid, which is characterized in that the method is with bacterial strain described in claim 1,37
Ferment paddy meal or ligno-cellulose hydrolysate production lactic acid at a temperature of~50 DEG C.
5. the method for hot fermentation production lactic acid according to claim 4, which is characterized in that fermentation process in the method
It is unsterilised.
6. the method for producing lactic acid according to hot fermentation described in claim 2 and 4, which is characterized in that ferment in the method
Process stuffiness.
7. hot fermentation described in claim 1 produces application of the bacterial strain of lactic acid in fermenting lactic acid.
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| CN113174343B (en) * | 2021-04-13 | 2022-12-20 | 大连理工大学 | Microbial flora for producing lactic acid by utilizing lignocellulose and fermentation method |
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