CN110241043B - Strain for producing lactic acid through high-temperature fermentation and method for producing lactic acid - Google Patents
Strain for producing lactic acid through high-temperature fermentation and method for producing lactic acid Download PDFInfo
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Abstract
The invention provides a strain for producing lactic acid by high-temperature fermentation and a method for producing the lactic acid, wherein the strain is named as: e.faecalis DUT1805, fermenting glucose at high temperature by using the strain in a gradient heating manner to produce lactic acid, wherein the gradient heating is 37-40-45-50 ℃ or 37-40-45-50 ℃ -55 ℃, the temperature is increased from 37 ℃ to 50 ℃ or 55 ℃, and the time required for the overall heating is 2h, 4h, 6h or 8 h. Faecalis DUT1805 has high production strength and lactic acid conversion; meanwhile, the E.faecalis DUT1805 can directly metabolize biomass raw materials such as rice flour or corn straw hydrolysate to produce lactic acid at a high temperature of 50 ℃ without gradient temperature rise.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a strain for producing lactic acid by high-temperature fermentation and a method for producing lactic acid by using the strain.
Background
Lactic acid, also known as α -hydroxypropionic acid, is one of the three major organic acids (carboxylic, hydroxyacid, ketoacid), an important fine chemical, and one of the 12 platform compounds of the petroleum replacement strategy identified by the U.S. department of energy (zhangli, bioprocessing, 2018, 16(4): 23-29). Lactic acid is widely used in the fields of food, chemical industry, medical treatment and the like, and among them, lactic acid is the most promising application for the synthesis of polylactic acid by using lactic acid as a monomer (Andrea Komesu et al, BioResources,2017,12(2): 4364-4383). With the exhaustion of non-renewable resources such as petroleum resources and the deterioration of environments such as white pollution and greenhouse effect, the establishment of a resource circulation type, environment-friendly and sustainable development mode has become a great trend. Among them, it is one of the important actions to promote the development and popularization of biodegradable polymer materials, and polylactic acid, as the first biodegradable material industrially produced at present, is called one of the most potential biomaterials (wangzhong, bio-industrial technology, 2017, 6: 17-22).
At present, most of the Lactic acid producing strains mainly comprise Lactic acid bacteria (Lactic acid bacteria), Bacillus (Bacillus strains), Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia coli), Rhizopus oryzae (Rhizopus oryzae) and the like, glucose is used as a carbon source, and the production cost is relatively high. The biomass raw material has the advantages of low price, easy obtaining and the like, thereby becoming a new raw material for producing lactic acid. It mainly comprises sugar-based raw materials (sugarcane, beet and the like), fiber raw materials (straws, rice straws, wood, rice hulls and the like) and starchiness raw materials (potatoes, grains and the like). There are many reports on the production of lactic acid from biomass materials, and Pediococcus acidilactici DQ2 can obtain 101.9g/L lactic acid from corn stalks at 48 ℃ (Kai Zhao et al, Bioresource Technology,2013,135: 481-489); enterococcus faecalis SI can obtain 59.8g/L lactic acid at 42 ℃ by using lignocellulose raw material, and the conversion rate can reach 0.95g/g (Shuo-Fu Yuan et al, Journal of Industrial Microbiology & Biotechnology,2018,45(11): 961-970); bacillus LA204 was obtained at 50 ℃ with 97.6g/L lactic acid from corn stover (Jinlong Hu et al, Bioresource Technology,2015,182: 251-; rhizopus oryzae DMKU 33 obtained 84.0g/L of lactic acid from cassava flour at 40 deg.C (Srisakul Trkarnpaibonon et al, Process Biochemistry,2017,63: 26-34). It has been reported that high-temperature fermentation is generally adopted for producing lactic acid from biomass raw materials, and the used strains mainly comprise heat-resistant lactic acid bacteria, bacillus and rhizopus oryzae.
The high-temperature fermentation is one of the important ways for strengthening synchronous saccharification of raw materials such as lignocellulose or rice, and the like, and the advantages are mainly shown in the following aspects:
(1) cellulase or amylase aspects: at present, the optimum action temperatures of cellulase and amylase are respectively 50 ℃ and 60 ℃. The conventional temperature synchronous fermentation reduces the activity of the cellulase or the amylase, thereby needing to increase the dosage of the cellulase or the amylase.
(2) Fermentation performance: the high temperature condition is beneficial to saccharification of raw materials such as lignocellulose or rice and the like, and better and faster mass transfer is realized. Meanwhile, the high-temperature microorganisms can tolerate the temperature distribution unevenness and fluctuation caused by a large-scale reactor, and the improvement of the fermentation performance of strains is facilitated.
(3) The production cost is as follows: high temperature fermentation can significantly reduce cooling costs, especially in hot seasons and tropical regions. Meanwhile, the risk of bacterial contamination is obviously reduced by high-temperature fermentation.
Based on the advantages of high-temperature fermentation, the temperature of the enterococcus faecalis fermented lactic acid reported at present can only reach 42 ℃ at most, so that a strain which is more resistant to high temperature and is used for producing lactic acid by fermentation is expected to be obtained.
Disclosure of Invention
In order to overcome the problem that the conventional enterococcus faecalis cannot ferment to produce lactic acid at the temperature of 50 ℃ or even higher than 50 ℃, the invention provides a strain for producing lactic acid by high-temperature fermentation and a method for producing lactic acid by fermenting the strain.
The strain is separated from fresh cow stomach contents, is named as Enterococcus faecalis DUT1805, and has been preserved in China microorganism culture collection (address: Beijing city rising district Beichen Xilu No.1, No. 3, postal code 100101) in 28 days 4 and 2019, and the preservation numbers are: CGMCC NO. 17640. The classification is named as: enterococcus faecalis DUT 1805.
The method for producing lactic acid by high-temperature fermentation adopts the strain, and produces lactic acid by fermenting glucose at the temperature of 50 ℃ or 55 ℃ by gradient temperature rise of 37-50 ℃ or 37-55 ℃.
In the method for producing lactic acid by high-temperature fermentation, the gradient temperature rise is as follows: the temperature is increased from 37 ℃ to 40 ℃ to 45 ℃ to 50 ℃ or from 37 ℃ to 40 ℃ to 45 ℃ to 50 ℃ to 55 ℃, and the time required for integral temperature increase is 2h, 4h, 6h or 8 h.
In the method for producing lactic acid by high-temperature fermentation, the fermentation process is not aerated.
The method for producing lactic acid by high-temperature fermentation adopts the strain to ferment rice flour or lignocellulose hydrolysate at the temperature of 37-50 ℃ to produce lactic acid.
The method for producing lactic acid by high-temperature fermentation does not sterilize the fermentation process.
In the method for producing lactic acid by high-temperature fermentation, the fermentation process is not aerated.
The application of the strain Enterococcus faecalis DUT1805 in the fermentation production of lactic acid.
The invention has the beneficial effects that:
(1) the invention provides a high-temperature-resistant strain-Enterococcus faecalis DUT1805 for preparing lactic acid by fermentation, which has the advantages of good fermentation stability, strong high-temperature tolerance, high production strength and capability of tolerating high temperature of more than 50 ℃.
(2) The invention provides a method for producing lactic acid by fermenting Enterococcus faecalis Faecalis DUT1805, and the temperature is controlled by gradient temperature rise in the fermentation process, thus improving the adaptability and tolerance of the strain to high temperature and providing a new method for strain acclimation.
(3) The invention takes rice or lignocellulose as raw materials, produces lactic acid by high-temperature fermentation, solves the defect of incongruity between enzymolysis temperature and fermentation temperature, can ferment enzyme at the optimum action temperature by using the strain of the invention to produce lactic acid, improves the utilization rate of the enzyme, realizes synchronous saccharification and conversion to produce lactic acid, and improves the efficiency of producing lactic acid.
(4) The high-temperature fermentation mode of the invention can reduce the cooling cost and the public engineering consumption, thereby reducing the production cost of the lactic acid.
Drawings
FIG. 1E. faecalis DUT1805 colony morphology;
figure 2 e. faecalis DUT1805 gram stain result;
figure 350 ℃ effect of glucose stress on e.faecalis DUT1805 fermentation performance;
figure 450 ℃ impact of different temperature control strategies on e.faecalis DUT1805 fermentation performance, a)40g/L glucose; B)80g/L glucose;
figure 550 ℃ impact of pH stress on e.faecalis DUT1805 fermentation performance;
fig. 6 effect of high temperature on e.faecalis DUT1805 fermentation performance;
figure 755 ℃, the impact of different temperature control strategies on the fermentation performance of e.faecalis DUT 1805;
FIG. 8E is a metabolic map of high temperature simultaneous saccharification of rice flour by faecalis DUT1805 to produce lactic acid, A) MRS medium, B) corn steep liquor dry powder medium;
fig. 9 e. faecalis DUT1805 metabolic profile for high temperature fermentation of lignocellulosic hydrolysate to produce lactic acid.
Detailed Description
The present invention will be further described with reference to the following detailed description. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and the reagents used were all available from chemical or biological reagents companies.
1. The media used in the following examples:
(1) seed medium (g/L): 30 parts of glucose, 10 parts of peptone, 10 parts of beef extract, 5 parts of yeast extract powder, (NH)4)2HC6H5O72，CH3COONa·3H2O 2，KH2PO4·3H2O 2，MgSO4·7H2O 0.2，MnSO4·H2O 0.05，MgCO3 12(pH＝6.3)。
(2) Enrichment medium (g/L): glucose 40, Yeast extract powder 5, C4H2Na2O45, monensin 0.1, K2HPO4·3H2O 3，NaCl 1，(NH4)2SO4 1，CaCl2 0.5，MgCl2 0.5。
(3) RCM Medium (g/L): 10 parts of peptone, 10 parts of beef extract, 3 parts of yeast extract powder, 5 parts of glucose, 1 part of soluble starch, 15 parts of NaCl and CH3COONa·3H2O3, L-cysteine hydrochloride 0.5.
(4) MRS medium (g/L): glucose 40, 80, 100, 120, 160 or rice flour 150 or corn straw hydrolysate, peptone 10, beef extract 10, yeast extract powder 5, (NH)4)3C6H5O7 2，CH3COONa 5，K2HPO4 2，MgSO4·7H2O 0.58，MnSO4·H2O0.25 (pH 6.5 or 6.0).
(5) Corn steep liquor dry powder medium (g/L): rice flour or corn stalk hydrolysate, corn steep liquor dry powder 16, (NH)4)3C6H5O7 2，CH3COONa 5，K2HPO4 2，MgSO4·7H2O 0.58，MnSO4·H2O 0.25(pH 6.5)。
2. Seed culture:
a250 mL penicillin bottle is adopted, and the liquid filling amount is 100 mL. After the culture medium is filled, nitrogen is continuously introduced into each bottle for 3min to remove oxygen, then the bottle is capped by a butyl rubber plug, and the bottle is sterilized for 20min at 121 ℃. In the experimental process, a disposable sterile needle tube is used for inoculation and sampling, the inoculation amount is 2% (v/v), the culture temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, and the culture time is 10-12 h.
3. Fermentation culture conditions:
A5L full-automatic fermentation tank is adopted, the liquid loading amount is 2L, the product of seed culture is inoculated into a fermentation medium according to the inoculation amount of 5% (v/v), glucose, rice flour and corn straw hydrolysate are respectively used as substrates, fermentation is carried out at the temperature of 37-50 or 37-55 ℃ and the rotating speed of 200r/min, and the pH value is controlled to be 6.5 or 6.0 by 8M NaOH in the fermentation process.
4. Temperature control strategy:
the temperature of the seed culture medium is 37 ℃, and the temperature of the fermentation liquid culture medium is 50 or 55 ℃. Considering that the strain has a certain adaptation period to the culture temperature, the temperature is increased to 50 ℃ according to 3 gradients of 37-40-45-50 ℃ or 4 gradients of 37-40-45-50-55 ℃, the reserved time for each temperature increase is about 20min, and the influence of different temperature increase times of 2h, 4h, 6h and 8h on the fermentation production of lactic acid by utilizing glucose by enterococcus faecalis considered. For biomass resources represented by rice flour or corn straw hydrolysate, direct fermentation at 50 ℃ is adopted, and gradient temperature rise is not needed. The gradient temperature rise operation is as follows:
heating to 50 ℃ for 2 h: the initial temperature is 37 ℃, the temperature is increased to 40 ℃ after 30min, the temperature is increased to 45 ℃ after 65min, and the temperature is increased to 50 ℃ after 105 min.
Heating to 50 ℃ for 4 h: the initial temperature is 37 ℃, the temperature is raised to 40 ℃ after 1.17h, the temperature is raised to 45 ℃ after 2.5h, and the temperature is raised to 50 ℃ after 3.5 h.
Heating to 50 ℃ for 6 h: the initial temperature is 37 ℃, the temperature is increased to 40 ℃ after 1.67h, the temperature is increased to 45 ℃ after 3.67h, and the temperature is increased to 50 ℃ after 5.67 h;
heating to 55 ℃ for 6 h: the initial temperature is 37 ℃, the temperature is raised to 40 ℃ after 1.50h, the temperature is raised to 45 ℃ after 2.83h, the temperature is raised to 50 ℃ after 4.2h, and the temperature is raised to 55 ℃ after 5.5 h.
Heating to 55 ℃ for 8 h: the initial temperature is 37 ℃, the temperature is increased to 40 ℃ after 1.67h, the temperature is increased to 45 ℃ after 3.67h, the temperature is increased to 50 ℃ after 5.67h, and the temperature is increased to 55 ℃ after 7.67 h.
Example 1
Screening preparation and identification of faecalis DUT 1805:
taking 0.1kg of fresh cow stomach contents in a triangular flask with 100mL of normal saline, carrying out vortex oscillation for 2min, inoculating the solid-liquid mixture in a penicillin bottle filled with 100mL of enrichment medium, and carrying out anaerobic enrichment culture. Continuously carrying out passage for 12 times to obtain the mixed bacteria with stable fermentation performance. The bacterial colony is inoculated in a 250mL penicillin bottle containing 100mL RCM culture medium, heat treatment is carried out for 20min at 90 ℃, then the bacterial colony is coated on an RCM plate, the plate is subjected to anaerobic culture at 37 ℃ for 24h, single bacterial colony is selected for streaking separation, and the bacterial colony is separated and purified for 3 generations and then is preserved. The isolated and purified single bacterium DNA is extracted, 16S rDNA bacteria universal primer is adopted for amplification (primer 27F: AGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT), and the single bacterium DNA is determined to be enterococcus faecalis through 16S rDNA comparison analysis and is named as E.faecalis DUT 1805. The single colony on the plate is round, opaque, neat in edge, milky white, and wet in surface protrusion, and the diameter of the colony is 0.5-1mm (figure 1). The cells were spherical, arranged in a double or chain form, and the size was 0.3 to 0.7 μm, and gram-positive bacteria were observed under an optical microscope (FIG. 2).
Example Effect of glucose stress on fermentation Performance of E.faecalis DUT1805 at 250 deg.C
Enterococcus faecalis E.faecalis DUT1805 passes through the seed culture medium, then is inoculated into a 5L fermentation tank filled with 2L MRS culture medium according to the volume ratio of 5 percent for culture, the temperature is increased from 37 ℃ to 50 ℃ in a gradient manner within 2h, the glucose concentration is gradually increased from 40g/L to 160g/L, the whole fermentation process is not aerated, and the fermentation result is shown in figure 3. The results show that: enterococcus faecalis DUT1805 can tolerate 160g/L glucose at high temperature of 50 ℃. Wherein the glucose concentration is 40g/L, the batch fermentation efficiency is highest, the final concentration of lactic acid is 37.92g/L, the conversion rate relative to glucose is 0.92g/g, the production intensity is 3.79g/(L.h), the metabolic byproducts comprise acetic acid, succinic acid and ethanol, and the concentrations are 4.57g/L, 1.30g/L and 1.55g/L respectively. At a glucose concentration of 80g/L, the final concentration of lactic acid was 60.65g/L, the conversion rate to glucose was 0.87g/g, and the production intensity was 1.26 g/(L.h); at a glucose concentration of 100g/L, the final concentration of lactic acid was 77.29g/L, the conversion rate to glucose was 0.89g/g, and the production intensity was 1.25 g/(L.h); the final concentration of lactic acid was 84.04g/L at a glucose concentration of 120g/L, the conversion rate to glucose was 0.92g/g, and the production intensity was 1.40 g/(L.h); at a glucose concentration of 160g/L, the final concentration of lactic acid was 79.35g/L, the conversion rate to glucose was 0.90g/g, and the production intensity was 1.10 g/(L.h).
Example effects of different temperature control strategies on the fermentation Performance of E.faecalis DUT1805 at 350 deg.C
Enterococcus faecalis DUT1805 is inoculated to 5L fermentation tank filled with 2L MRS culture medium for culture according to 5% volume ratio after passing through seed culture medium, the whole fermentation process is not ventilated, and the influence of different gradient temperature rise modes on the production of lactic acid by the E.faecalis DUT1805 through glucose fermentation is discussed. The fermentation results are shown in FIG. 4. When the substrate is glucose of 40g/L, the cell growth, the glucose consumption rate and the lactic acid generation rate of the experimental group which is heated from 37 ℃ to 50 ℃ in a gradient manner within 4h are better than those of the experimental group which is heated from 2h to 6h in a gradient manner to 50 ℃. The final concentration of lactic acid was 36.56g/L, the conversion rate relative to glucose was 0.92g/g, the production intensity was 4.57g/(L.h), and the concentrations of the metabolic byproducts acetic acid, succinic acid, and ethanol were 3.87g/L, 1.81g/L, and 2.44g/L, respectively. When the concentration of the substrate is lower (less than or equal to 40g/L), the optimal heating mode of raising the temperature to 50 ℃ in a gradient within 4h is selected. When the substrate is 80g/L glucose, the cell growth, the glucose consumption rate and the lactic acid generation rate of the experimental group which is heated from 37 ℃ to 50 ℃ in a gradient manner within 6h are better than those of the experimental group which is heated from 2h to 4h in a gradient manner to 50 ℃. The final concentration of the lactic acid is 65.41g/L, the yield of the lactic acid is 0.88g/g, the production intensity is 4.09g/(L.h), and the production intensity is much stronger than a 2h gradient heating strategy (1.26g/(L.h)) and a 4h gradient heating strategy (2.07 g/(L.h)); the concentrations of the metabolic byproducts acetic acid, succinic acid and ethanol were 4.98g/L, 1.89g/L and 5.38g/L, respectively. Therefore, when the concentration of the substrate is higher (more than or equal to 80g/L), the optimal heating mode of gradient heating to 50 ℃ within 6h is selected.
Example Effect of pH stress on fermentation Performance of E.faecalis DUT1805 at 450 deg.C
Enterococcus faecalis DUT1805 is inoculated to 5L fermentation tank filled with 2L MRS culture medium according to 5% volume ratio after passing through seed culture medium, initial sugar concentration is 40g/L, whole fermentation process is not aerated, influence of different fermentation pH on fermentation performance of the bacterium is discussed, and fermentation result is shown in FIG. 5. The comparison shows that the cell growth, the glucose consumption rate and the lactic acid generation rate are better than those of the experimental group with the pH of 6.0 at the pH of 6.5, the final concentration of the lactic acid is 37.92g/L, the conversion rate relative to the glucose is 0.92g/g, the production intensity is 3.79g/(L.h), and the concentrations of the metabolic byproducts, namely acetic acid, succinic acid and ethanol are respectively 4.57g/L, 1.30g/L and 1.55 g/L. pH 6.0 the experimental group had 3.57g/L residual sugar remaining after 36h fermentation, a final concentration of lactic acid of 33.60g/L, a conversion rate relative to glucose of 0.98g/g, a production intensity of 0.93g/(L.h), and concentrations of the metabolic byproducts acetic acid, succinic acid and ethanol of 4.23g/L, 0.55g/L and 2.53g/L, respectively. The research result shows that: the pH has a great influence on the fermentation performance of the E.faecalis DUT1805 at high temperature, the tolerance of the strain to low pH is poor, and the production intensity is sharply reduced along with the reduction of the pH.
Example 5 effect of high temperature on fermentation performance of e.faecalis DUT1805
Enterococcus faecalis DUT1805 is inoculated to 5L fermentation tank filled with 2L MRS culture medium according to 5% volume ratio after passing through seed culture medium, initial sugar concentration is 80g/L, gradient temperature rise is from 37 ℃ to 50 ℃ within 6h or from 37 ℃ to 55 ℃ within 6h, the whole fermentation process is not aerated, influence of high temperature on fermentation performance of the bacterium is discussed, and the fermentation result is shown in figure 6. The research result shows that the fermentation performance of the 50 ℃ experimental group is better than that of the 55 ℃ experimental group, the final concentration of the lactic acid is 65.41g/L, the conversion rate of the lactic acid relative to the glucose is 0.88g/g, the production intensity is 4.09g/(L.h), and the concentrations of the metabolic byproducts of acetic acid, succinic acid and ethanol are 4.98g/L, 1.89g/L and 5.38g/L respectively. The residual sugar of 13.92g/L is still remained after fermenting for 24 hours in the experiment group at the temperature of 55 ℃. Enterococcus faecalis DUT1805 can endure 55 ℃ high temperature, but the fermentation performance of 55 ℃ experimental group is general compared with that of 50 ℃. The glucose consumption rates of the two are not greatly different in the initial fermentation stage, and the glucose consumption rate and the production intensity are reduced due to the fact that the strain growth is inhibited in the later 55 ℃ experiment group.
Example effect of different temperature control strategies on fermentation performance of e.faecalis DUT1805 at 655 deg.c
Enterococcus faecalis DUT1805 is inoculated to 5L fermentation tank filled with 2L MRS culture medium for culture according to 5% volume ratio after passing through seed culture medium, initial sugar concentration is 80g/L, the whole fermentation process is not aerated, the influence of different gradient temperature rise modes on the fermentation performance of the bacterium is discussed, and the fermentation result is shown in FIG. 7. The temperature is respectively increased from 37 ℃ to 55 ℃ within 6h and 8h, and comparison shows that the gradient temperature rise time has larger influence on the biomass of the thalli and the consumption rate of the substrate, and the fermentation performance of the 8h temperature rise experimental group is superior to that of the 6h temperature rise experimental group. 13.92g/L of residual sugar still remains after the fermentation in the 6h heating test group, while the glucose is depleted in 24h in the 8h heating test group, the conversion rate of lactic acid relative to glucose is 0.82g/g, the production intensity is 2.49g/(L.h), and the concentrations of the metabolic byproducts of acetic acid, succinic acid and ethanol are 6.55g/L, 2.12g/L and 6.28g/L respectively. The research result shows that: the prolonged temperature rise time is beneficial to the adaptation of the thalli to a high-temperature environment, and the fermentation performance of the thalli is improved.
Example 7 E.Faecalis DUT1805 high temperature Simultaneous saccharification of Rice flour to produce lactic acid
(A) MRS medium
After the e.faecalis DUT1805 passes through the seed culture medium, it is inoculated into a 5L fermentation tank containing 2L MRS medium using rice flour as a carbon source at a volume ratio of 5% to culture, the concentration of the rice flour is 150g/L, the pH is 6.5, 0.075% (v/v) liquefying enzyme and saccharifying enzyme are added, respectively, the whole fermentation process is not aerated, the feasibility of producing lactic acid by high-temperature synchronous saccharification of the e.faecalis DUT1805 using rice flour is investigated, and the fermentation result is shown in fig. 8A. In the initial stage of synchronous saccharification, the glucose concentration is gradually increased, the product concentration is lower, and the substrate consumption rate and the product generation rate are higher after 12 hours, which fully indicates that in the rice flour culture, enterococcus faecalis DUT1805 can endure the high temperature of 50 ℃, but the seeds cultured at 37 ℃ are inoculated into a fermentation tank at 50 ℃ for culture, and the lag phase is 12 hours. The fermentation was terminated after 34h, no residual sugar was present in the tank, the final lactic acid concentration was 73.36g/L, the conversion rate relative to starch was 0.89g/g, the production intensity was 2.16g/(L.h), and the concentrations of the metabolic byproducts acetic acid and ethanol were 3.72g/L and 2.58g/L, respectively. The research result shows that: faecalis DUT1805 can simultaneously saccharify rice flour at a high temperature of 50 ℃ to produce lactic acid.
(B) Corn steep liquor dry powder culture medium
E.faecalis DUT1805 was inoculated into a 5L fermentor containing 2L of corn steep liquor dry powder medium containing rice flour as a carbon source at a volume ratio of 5% after passing through a seed medium, and cultured, the concentration of the rice flour was 150g/L, the pH was 6.5, 0.075% (v/v) of liquefying enzyme and saccharifying enzyme were added, respectively, the whole fermentation process was not aerated, the feasibility of e.faecalis DUT1805 for producing lactic acid by synchronously saccharifying rice flour at high temperature using a cheap medium was investigated, and the fermentation result is shown in fig. 8B. In the initial stage of synchronous saccharification, the glucose concentration is gradually increased, the product concentration is lower, and the substrate consumption rate and the product generation rate are higher after 8 hours, which fully indicates that in the rice flour culture, the E.faecalis DUT1805 can tolerate the high temperature of 50 ℃, but the seeds cultured at 37 ℃ are inoculated into a fermentation tank at 50 ℃ for culture, and the lag phase is 8 hours. The fermentation was terminated after 34h, no residual sugar was present in the tank, the final lactic acid concentration was 73.75g/L, the conversion rate relative to starch was 0.87g/g, the production intensity was 2.17g/(L.h), and the concentrations of the metabolic byproducts acetic acid and ethanol were 3.48g/L and 3.63g/L, respectively. The research result shows that: faecalis DUT1805 can produce lactic acid by simultaneous saccharification of rice flour at high temperature of 50 ℃ using corn steep liquor dry powder instead of conventional MRS medium.
Example 8 E.faecalis DUT1805 high temperature fermentation of lignocellulosic hydrolysate to produce lactic acid
Enterococcus faecalis DUT1805 is inoculated to 5L fermentation tank filled with 2L corn steep liquor dry powder culture medium according to 5% volume ratio after passing through seed culture medium and saccharifying corn stalk for 47h, the concentration of corn stalk is 100g/L, the whole fermentation process is not aerated, the feasibility of the bacterium for producing lactic acid by lignocellulose hydrolysate at high temperature is discussed, and the fermentation result is shown in figure 9. When 0.6% (v/v) cellulase was added to 100g/L of the acid-treated corn stover and saccharification was carried out for 47 hours, the concentrations of glucose and xylose were 17.9g/L and 2.4g/L, respectively. After 26h, the fermentation was terminated, and the final concentration of lactic acid was 21.59g/L, the conversion rate to glucose was 0.93g/g, the production intensity was 0.86g/(L.h), and the concentration of the metabolic by-product acetic acid was 0.77 g/L. The research result shows that: enterococcus faecalis DUT1805 can ferment lignocellulose hydrolysate at a high temperature of 50 ℃ to produce lactic acid.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
SEQUENCE LISTING
<110> university of Large Community
<120> strain for producing lactic acid by high-temperature fermentation and method for producing lactic acid
<130> 2019
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1468
<212> DNA
<213> E. faecalis DUT 180516S 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. A enterococcus faecalis (enterococcus faecium) DUT1805 for producing lactic acid by high-temperature fermentation has a preservation number of: CGMCC NO. 17640.
2. A method for producing lactic acid by high-temperature fermentation, which is characterized in that the method uses enterococcus faecalis (enterococcus faecis) DUT1805 according to claim 1 to produce lactic acid by fermenting glucose at 50 ℃ or 55 ℃ by gradient temperature rise of 37 ℃ to 50 ℃ or 37 ℃ to 55 ℃.
3. The method for producing lactic acid by high-temperature fermentation according to claim 2, wherein the gradient temperature rise is: the temperature is increased from 37 ℃ to 40 ℃ to 45 ℃ to 50 ℃ or from 37 ℃ to 40 ℃ to 45 ℃ to 50 ℃ to 55 ℃, and the time required for integral temperature increase is 2h, 4h, 6h or 8 h.
4. A method for producing lactic acid by high-temperature fermentation, characterized in that the method uses enterococcus faecalis (enterococcus faecium) DUT1805 according to claim 1 to ferment rice flour or lignocellulose hydrolysate at 37-50 ℃ to produce lactic acid.
5. The method for producing lactic acid by high-temperature fermentation according to claim 4, wherein the fermentation process is not sterilized.
6. The method for producing lactic acid by high temperature fermentation according to claim 2 or 4, wherein the fermentation process is not aerated.
7. Use of enterococcus faecalis (enterococcus faecium) DUT1805 for high temperature fermentation production of lactic acid according to claim 1 for fermentation production of lactic acid.
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| CN111206006A (en) * | 2020-03-25 | 2020-05-29 | 福建傲农生物科技集团股份有限公司 | Screening and domesticating method of high-temperature-resistant enterococcus faecalis |
| 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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