CN114958667A - Lactococcus capable of highly producing L-lactic acid and application thereof - Google Patents

Lactococcus capable of highly producing L-lactic acid and application thereof Download PDF

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CN114958667A
CN114958667A CN202210585002.6A CN202210585002A CN114958667A CN 114958667 A CN114958667 A CN 114958667A CN 202210585002 A CN202210585002 A CN 202210585002A CN 114958667 A CN114958667 A CN 114958667A
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lactococcus
lactic acid
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耿阿蕾
李娜娜
孙建中
谢蓉蓉
涂茁炜
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Zhu Mi
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Jiangsu University
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Abstract

The invention provides lactococcus for high yield of L-lactic acid and application thereof, belonging to the technical field of biological engineering; in the invention, a Lactococcus which is screened from the intestinal tract of the Laurencia formosanus and can efficiently produce lactic acid in the same type is marked as Lactococcus X1(Lactococcus sp.X1), wherein the Lactococcus X1 can produce L-lactic acid by utilizing carbon sources such as glucose, sucrose, cellobiose, xylose and the like, and is Lactococcus which has low requirement on nutrient factors, can ferment various carbon sources, can produce water-soluble vitamins and can efficiently produce lactic acid in the same type.

Description

Lactococcus capable of highly producing L-lactic acid and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to lactococcus capable of highly producing L-lactic acid and application thereof.
Background
Lactic acid, also known as 2-hydroxypropionic acid, is an important multipurpose natural organic acid and is currently applied in the fields of chemical industry, food, cosmetics, medicine, light industry, environmental protection and the like. Lactic acid is classified into three types, D-type, L-type and DL-type, according to its configuration and optical activity, wherein L-lactic acid has its unique application value. For example: only L-lactate dehydrogenase exists in human body, so that only L-type lactate can be metabolized, if DL-type or D-type lactate is excessively taken, metabolic disturbance of human body can be caused, even acidosis and other adverse reactions are caused, the world health organization clearly defines that DL-type or D-type lactate cannot be added into food drunk by infants, and D-type lactate taken by adults every day is strictly controlled to be less than 100mg/Kg of body weight. In addition, the biodegradable environment-friendly polymer material poly L-lactic acid can be produced by polymerization by using L-lactic acid as a monomer, and has important significance for better solving the serious problems of white pollution and the like faced by human at present.
At present, the method mainly adopts a microbial fermentation method to synthesize the L-lactic acid in industry, and has the advantages of relatively high optical purity of products, simple production process, few byproducts, low energy consumption and the like. But still has some disadvantages, firstly, the acid production amount of lactic acid is not high, the conversion rate is low; secondly, the product contains more D-lactic acid, and the optical purity of the target product L-lactic acid is lower, so that the separation and purification of the L-lactic acid are difficult; thirdly, the producing strain has high requirements on nutrient substrates, and can obtain higher lactic acid yield by using expensive yeast powder or yeast extract as a nitrogen source, so that the fermentation cost is increased; fourthly, the carbon source range available for the strains is narrow, so that the carbon source is wasted. These drawbacks greatly limit the large-scale industrial production of L-lactic acid. The fermentation cost of the L-lactic acid is about 30-40%, so that the screening of high-yield strains of the L-lactic acid and the adoption of cheap culture medium raw materials to reduce the fermentation cost have important significance for promoting and expanding the industrial production and application of the L-lactic acid.
Disclosure of Invention
Aiming at some defects in the prior art, the invention provides lactococcus capable of highly producing L-lactic acid and application thereof. In the invention, a Lactococcus which is high in efficiency and can homologously produce lactic acid and is screened from the intestinal tract of the Lactococcus formosanus is marked as Lactococcus X1(Lactococcus sp.X1), wherein Lactococcus X1 can produce L-lactic acid by utilizing carbon sources such as glucose, sucrose, cellobiose, xylose and the like, and is a Lactococcus which can ferment various carbon sources, has low requirements on nutritional factors and can efficiently homologously produce lactic acid.
The invention firstly provides Lactococcus for producing L-lactic acid, wherein the L-lactic acid bacteria are Lactococcus X1(Lactococcus sp.X1) which are preserved in China general microbiological culture Collection center (CGMCC) with the address of the institute of microbiology of China academy of sciences No. 3 of North West Lu No. 1 institute of North China, Chaozhou, the date of preservation is 2022 years, 1 month and 19 days, and the preservation number is CGMCC No. 24341.
The main biological characteristics of the high-yield L-lactic acid bacteria are as follows:
morphological characteristics of thallus: the shape is oval, (0.5-1.2) mum x (0.5-1.5) mum, and clusters appear in a liquid culture medium without forming spores;
physiological and biochemical characteristics: the growth can be carried out in both aerobic and anaerobic culture media, but the growth is better in the anaerobic culture medium; the growth temperature range is 25-37 ℃, and the optimal growth temperature is 30 ℃; the growth pH range is 4-7, and glucose, sucrose, cellobiose, arabinose and xylose can be used as carbon sources.
The invention also provides the fermentation medium for producing the L-lactic acid bacteria, which comprises the following components: 1g/L of yeast powder, NH 4 Cl 5g/L, carbon source 5g/L, KH 2 PO 4 1.5g/L，K 2 HPO 4 ×3H 2 O2.9 g/L, sodium acetate 1g/L, triammonium citrate 0.5g/L, MgCl 2 ×6H 2 O 0.2g/L，CaCl 2 ×2H 2 O 75mg/L，MnSO 4 0.05g/L, cysteine 0.5g/L, CaCO 3 KHCO at 50% (w/w) of the carbon source content 3 0.05M。
Further, the carbon source comprises one or more of glucose, sucrose, cellobiose, arabinose, xylose.
Further, the carbon source is glucose.
The invention also provides a microbial agent, which contains the lactococcus X1.
The invention also provides application of the lactococcus X1 or the microbial agent in fermentation production of L-lactic acid.
Further, the application is that lactococcus X1 or a microbial agent is fermented to produce the L-lactic acid by utilizing the fermentation culture medium or a culture medium containing a high-concentration carbon source.
The invention also provides application of the lactococcus X1 or the microbial agent in producing L-lactic acid by saccharifying and hydrolyzing straws synchronously with cellulase or cellulose degradation microorganisms.
The invention also provides a method for producing L-lactic acid with high yield, which is any one of the following methods:
(1) inoculating lactococcus X1 or a microbial agent into the fermentation culture medium or the fermentation culture medium containing a high-concentration carbon source;
(2) lactococcus X1 or a microbial agent is mixed with cellulase or cellulose degradation microorganisms and inoculated into a culture medium taking straws as a carbon source.
The invention also provides application of the lactococcus X1 or the microbial agent in production of water-soluble vitamins.
Further, the water-soluble vitamins include vitamin C, vitamin B2 and biotin.
The invention also provides application of the lactococcus X1 or the microbial agent in food or feed.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel lactococcus X1, the fermentation type of which is homolactic fermentation, in the homolactic fermentation process, lactococcus X1 can ferment 1mol of glucose into 2mol of lactic acid, and the theoretical value of the conversion rate of the lactic acid is close to 100%.
In the practical application process, when 10g/L glucose is degraded for one day, the yield of lactic acid reaches 100%, and no acetic acid and other byproducts are generated. And the content of L-lactic acid in the produced lactic acid is as high as 95.1%, and the optical purity is high. When a high-concentration carbon source is adopted for fermentation, lactococcus X1 can also achieve high lactic acid yield, the carbon source is 50g/L glucose, and the lactic acid yield reaches 99.1% in three days of fermentation; on the other hand, when the carbon source was sucrose (100 g/L), the lactic acid yield reached 79.8% after 9 days. Therefore, compared with the prior art, the lactococcus X1 has good lactic acid production effect and is beneficial to large-scale production of L-lactic acid in industry.
The lactococcus X1 can be synchronously hydrolyzed and fermented with cellulase or cellulose degradation microorganisms to avoid the inhibition of the excessively high sugar substrate concentration on the fermentation of the lactic acid bacteria strain and simplify the process. In addition, the lactococcus X1 can also be used for synthesizing water-soluble vitamins, such as vitamin C, vitamin B2 and biotin, and is a probiotic.
Drawings
FIG. 1 is an electron micrograph of lactococcus X1.
FIG. 2 is a phylogenetic tree of lactococcus X1 constructed by the Neighbor-joining method.
FIG. 3 is a graph showing the results of the effect of fermentation media containing different neutralizing agents on lactococcus X1 fermentation.
FIG. 4 is a graph showing the results of the effect of fermentation media containing different nitrogen sources on the fermentation of lactococcus X1.
FIG. 5 is a graph showing the results of the effect of fermentation media containing different carbon sources on lactococcus X1 fermentation.
FIG. 6 is a graph showing the results of the effect of high-concentration carbon sources on Lactococcus sp.X1 fermentation, wherein a is 50g/L glucose and b is 100g/L sucrose.
FIG. 7 is a diagram of the simultaneous hydrolysis of X1 and cellulase and the production of lactic acid by fermented straw.
FIG. 8 is a secondary mass spectrum of the ion peaks of three vitamins in the fermentation broth, wherein a is biotin, B is vitamin B2, and C is vitamin C.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the accompanying drawings and specific embodiments, which are described herein for illustrative purposes only and are not meant to be limiting.
The fermentation medium related in the invention comprises the following components: yeast powder 1g, NH 4 Cl 5g, carbon source 5g, KH 2 PO 4 1.5g，K 2 HPO 4 ×3H 2 O2.9 g, sodium acetate 1g, ammonium citrate tribasic 0.5g, MgCl 2 ×6H 2 O 0.2g，CaCl 2 ×2H 2 O 75mg，MnSO 4 0.05g, cysteine 0.5g/L, CaCO 3 2.5g，KHCO 3 0.05M, distilled water 1L. Wherein the carbon source comprises one or more of glucose, sucrose, cellobiose, arabinose, xylose.
As for the solid culture medium, 15g/L of agar is additionally added on the basis of the fermentation culture medium.
Example 1: obtaining strains
The high-efficiency homolactic lactococcus X1 of the present embodiment is obtained by screening from the intestinal tract of Laptotermes formosanus, and the specific separation and screening method is as follows:
washing and crushing the ivy white ant workers (collected from suburb of Suzhou, Jiangsu province) to obtain intestinal homogenate, adding 1mL of homogenate supernatant into 15mL of the fermentation medium (carbon source is 5g/L xylose), placing the mixture into a shaker at 30 ℃ for anaerobic culture, taking 1mL of culture after the culture medium is turbid, transferring the culture into 15mL of new liquid culture medium, and repeating the process for four times. Then using PBS buffer solution to perform gradient dilution of 10 times on the culture, fully shaking up, absorbing 50 mu L of diluent, respectively coating the diluent on a solid culture medium plate (prepared by adding 2% agar into the fermentation culture medium), performing anaerobic culture at 30 ℃ for 24h, tracking and detecting the lactic acid production capacity, screening the strain with the optimal L-lactic acid production capacity, further performing streak purification until pure strain is obtained, and storing the strain at 20% glycerol and 80 ℃ below zero, wherein the strain is named lactococcus X1.
The final identification of lactococcus X1 by molecular biology method is as follows:
morphological characteristic identification:
as shown in FIG. 1, lactococcus X1 is in the form of an oval, (0.5 to 1.2) μm.times. (0.5 to 1.5) μm, forms a pair or a short chain in a liquid medium, and does not form spores.
Genetic characterization (strain 16S rDNA sequence):
the lactococcus X1 screened by the method above was amplified using bacterial universal primers, namely the forward primer Eubac27F and the reverse primer Eubac27F, the specific method is as follows:
forward primer Eubac 27F: 5-AGAGTTTGATC-CTGGCTCAG-3(SEQ ID NO: 1);
reverse primer: eubac 1492R: 5-GGTTACCTTGTTACGACTT-3(SEQ ID NO: 2);
mixing 23 μ L of 1 × sPfu Master Mix (Beijing Bomai biology), 0.5 μ L of each primer forward and reverse primer, and 1 μ L of bacterial solution, performing PCR amplification, sequencing to obtain 16S rDNA gene sequence (SEQ ID NO:3) of lactococcus X1, and searching, comparing and analyzing the result of the 16S rDNA sequence of the strain and the 16S rDNA sequence of the similar strain in NCBI database by BLAST method. Multiple sequence alignments were performed by Mega (Version7.0) software and a phylogenetic tree as shown in FIG. 2 was constructed using the Neighbor-join method. Through comparison and analysis, the strain is lactococcus, is named as lactococcus X1, is preserved in China general microbiological culture Collection center (CGMCC), is addressed to the institute of microbiology of China academy of sciences, No. 3, West Lu No. 1 Hospital, North Cheng, south China, in Beijing, and has the preservation date of 2022 years, 1 month and 19 days and the preservation number of CGMCC No. 24341. 3, SEQ ID NO:
GAACGCTTGATTGTCACCGAAGCTTGCTTCACCGACAAACAAGAGTAGCGAACGGGTGAGTAACGCGTGGGTAACCTACCTTATAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGGAATTGCATAATTTCTATTTAAAAGTGTCTATTGGTACACTATGAGATGGACCCGCGTTGTATTAGCTAGTTGGTAGTGTAAAGGACTACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACGTGTGAGAGAGTAGAAAGTTCTCACAGTGACGGTATCTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTAATAAGTCTGATGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGTTAGACTTGAGTACAGTAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGACTGTGACTGACACTGAGGCTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGTTGTTTGGGGCTATCCAGCCCTAAGTGACGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTATGACCGTCTTAGAGATAAGATTTCCCTTCGGGGCAAAGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTCGCAAGCCAGTGATGGTTAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAATACCCAAAGCCGGTGGGCTAACCA
example 2:
in this example, the influence of the fermentation of lactococcus X1 screened in example 1 under different medium components was examined, and the fermentation medium was optimized by using this as a basis, and the specific optimization method is as follows:
(1) effect of fermentation media containing different neutralizers on lactococcus X1 fermentation:
when the neutralizer is not added into the fermentation medium, the yield of the lactic acid is not high, probably because the pH of the fermentation medium is low due to the lactic acid generated after fermentation, and the growth of thalli is influenced, therefore, on the basis of the fermentation medium, a carbon source is replaced by 10g/L glucose, and then CaCO with different concentrations is respectively used 3 、CaCO 3 And KHCO 3 、KHCO 3 Preparing a new fermentation medium by using the neutralizing agent as 10g/L CaCO 3 、5g/L CaCO 3 And 0.05M KHCO 3 、5g/L CaCO 3 And 0.65M KHCO 3 、5g/L CaCO 3 And 0.75M KHCO 3 、0.1M KHCO 3 、0.13M KHCO 3 、0.15M KHCO 3 。
Respectively transferring lactococcus X1 into 15mL of different prepared fermentation media according to the inoculation amount of 1%, carrying out constant-temperature oscillation anaerobic culture at 30 ℃ and 180rpm for 24h, respectively making three parallel samples, measuring the yield of lactic acid, and investigating the influence of different neutralizers on fermentation.
FIG. 3 is a graph showing the effect of fermentation media containing different neutralizing agents on lactococcus X1 fermentation, as can be seen from 10g/L CaCO 3 The effect of the catalyst is poor, relatively speaking, 5g/L CaCO 3 And 0.05M KHCO 3 As a regulator, the yield of lactic acid is up to 96.4-99.1%, and the parallel error is low, so that for experiments with a small system, 50% of the content of carbon source is used as CaCO 3 The dosage of the composition is 0.05M KHCO additionally added 3 To adjust the pH.
The content of L-lactic acid produced by fermenting 10g/L glucose with lactococcus X1 is measured by using an L-lactic acid content detection kit (Shanghai's Production), and the ratio of the content of L-lactic acid to the content of total lactic acid (measured by liquid chromatography) is the percentage content of L-lactic acid. The ratio of L-lactic acid can reach 95.1 percent through calculation, which indicates that the strain mainly produces L-lactic acid.
(2) Effect of fermentation media containing different nitrogen sources on lactococcus X1 fermentation:
in order to investigate the influence of different nitrogen sources on the fermentation, the carbon source in the basic culture medium was replaced with 10g/L glucose, and the nitrogen source was replaced with yeast powder, yeast powder and NH of different concentrations 4 Cl to prepare a new fermentation medium. Wherein the nitrogen source in each fermentation medium is 1g/L yeast powder, 1g/L yeast powder and 1-5 g/L NH respectively 4 Cl, 2g/L yeast powder, 3g/L yeast powder, 4g/L yeast powder and 5g/L yeast powder.
Lactococcus X1 was transferred to 15mL of each of the different fermentation media prepared as described above at an inoculum size of 1%, and anaerobically cultured at 30 ℃ and 180rpm for 24 hours under constant temperature shaking, and the effect of different nitrogen sources on fermentation was tested, and the results of the investigation are shown in FIG. 4.
FIG. 4 is a graph showing the effect of fermentation media containing different nitrogen sources on lactococcus X1 fermentation, and it can be seen from the graph that 1-5 g/L yeast powder dosage has little effect on lactic acid yield, and 1g/L yeast powder plus 5g/L NH 4 Cl can approach the peak lactic acid production. Thus, 1g/L yeast powder and 5g/L NH 4 Cl is used as a nitrogen source, so that the yield of lactic acid is high, the cost is low, and the fermentation cost is greatly reduced.
(3) Effect of fermentation media containing different carbon sources on lactococcus X1 fermentation:
in order to examine the influence of different carbon sources on the fermentation, 10g/L of cellobiose, sucrose, xylose and arabinose were used as carbon sources to replace the carbon sources of the basal medium, and a new fermentation medium was prepared.
Lactococcus X1 was transferred to 15mL of each of the fermentation media prepared as described above at an inoculum size of 1%, anaerobically cultured at 30 ℃ and 180rpm for 24 hours under constant temperature shaking, the yield of lactic acid was measured, and the effect of different carbon sources on fermentation was examined, with the results shown in FIG. 5.
FIG. 5 is a graph showing the effect of fermentation media containing different carbon sources on the fermentation of lactococcus X1, and it can be seen from the graph that lactococcus X1 can efficiently utilize sucrose, cellobiose and a part of two five-carbon sugars, xylose and arabinose, to produce lactic acid, in addition to glucose.
In this example, the ability of lactococcus X1 to ferment a high-concentration carbon source was also examined using 50g/L glucose and 100g/L sucrose as carbon sources. On the basis of a fermentation culture medium, 50g/L glucose and 100g/L sucrose are respectively used for replacing the original carbon source, 1g/L yeast powder and 5g/L NH are used 4 Cl replaces the original nitrogen source, and calcium carbonate with the dosage of 50% (w/w) carbon source is used as a neutralizer to prepare a new fermentation medium.
Lactococcus X1 was transferred to 50mL of each of the fermentation media prepared as described above at an inoculum size of 1%, anaerobically cultured at 30 ℃ under constant temperature shaking at 180rpm, and the yield of lactic acid was measured, periodically sampled and monitored, and the results of the measurement are shown in FIG. 6.
FIG. 6 is a graph showing the effect of high concentration carbon source on lactococcus X1 fermentation, and it can be seen that when the carbon source is 50g/L glucose, the fermentation can be completely degraded after three days, and the lactic acid yield is as high as 99.1%; when sucrose (100 g/L) was used as a carbon source, the lactic acid yield reached 79.8% after 9 days. This shows that lactococcus X1 can efficiently ferment high-concentration carbon sources of 50-100 g/L to produce lactic acid.
Example 3:
in this example, the commercial cellulase CTec2(Sigma) and lactococcus X1 were mixed to examine the ability of simultaneous hydrolysis and fermentation of straw to produce lactic acid, and the specific examination steps are as follows:
(1) mixing straws and 7% sodium hydroxide solution in a ratio of 1:7(w/v), carrying out water bath reaction at 86 ℃ for 1h, washing with water until the straws are neutral, and then drying to obtain alkali-treated straws.
(2) Cellulase was assayed using BCA protein quantification kit (shanghai) to determine cellulase dosage.
(3) Respectively synchronously hydrolyzing and fermenting corn straws with different dosages of cellulase and X1 to produce lactic acid:
adding 50g/L of alkali-treated straw instead of glucose into 10mL of the fermentation medium to serve as a carbon source to prepare a culture medium, respectively adding 10 or 20mg/g of corn cob cellulase after sterilization, transferring lactococcus X1 into the fermentation medium according to 1% of inoculation amount, carrying out anaerobic culture at constant temperature of 35 ℃ and 180rpm, and periodically sampling to detect the yield of lactic acid.
As shown in FIG. 7, under the condition of adding different dosages of cellulase, the yield of lactic acid of the corn straws after 192 hours of anaerobic fermentation in lactococcus X1 reaches 306mg/g and 336mg/g respectively, and the cellulose cannot hydrolyze xylan, so the lactic acid is mainly formed by converting cellulose hydrolysis products of glucose and cellobiose. This shows that lactococcus can degrade corn stalk synchronously with cellulase to produce lactic acid, and increasing cellulase dosage properly can raise lactic acid yield.
In the embodiment, the capacity of lactococcus X1 and cellulose degradation microorganisms for co-culturing and directly fermenting straws to produce lactic acid is also considered, and the specific investigation steps are as follows:
50g/L of alkali-treated straws are added into 10mL of liquid culture medium to prepare a new fermentation culture medium. Then, simultaneously inoculating anaerobic cellulose degrading bacteria Ruminococcus albus JCM 14654 (deposited in JCM bacterial bank in Japan) or Ruminococcus cellulolyticus DSM 5812 (deposited in DSMZ bacterial bank in Germany) and lactococcus X1 into a culture medium according to the inoculation amount of 1 percent, carrying out constant-temperature oscillation anaerobic culture at 35 ℃ and 180rpm, periodically sampling and detecting the yield of lactic acid, and finding that the fermented straws have obvious lactic acid accumulation under two co-culture conditions through liquid chromatography.
Example 4:
in the example, the ability of lactococcus X1 to produce water-soluble vitamins was examined, and the specific examination steps are as follows:
the components of the fermentation liquid after anaerobic culture in example 2 were analyzed by liquid chromatography-mass spectrometry (HPLC-MS), an appropriate amount of the fermentation liquid was put into a centrifuge tube and centrifuged at 12000rpm for 2min, and the supernatant was filtered through a 0.22 μm filter membrane and filtered through a 0.2M H filter membrane 2 SO 4 Adjusting pH to below 4.0, and performing on-line analysis.
The instrument adopts a liquid chromatography ion trap mass spectrometer (American Sammer fly), and the chromatographic conditions are as follows: agilent Eclipse Plus C18 column (2.1 mm. times.100 mm, 1.8 μm). 0.08% formic acid water solution is used as a mobile phase A, and 0.08% formic acid methanol is used as a mobile phase B. Elution procedure: 0-6 min, 99% A; 99-5% of A for 6-15 min; 15-24 min, 5% -50% A; 24-27 min, 50% -99% A; 27-30 min, 99% A. Flow rate: 0.2 mL/min -1 The sample injection amount is as follows: 10 μ L, column temperature: at 40 deg.C, the wavelength of the ultraviolet detector is 266 nm. Mass spectrum conditions: electrospray positive ion mode scan, electrospray voltage: 5500V; atomizer pressure: 0.345 MPa; air curtain pressure: 0.069 MPa; ion source temperature: 550 ℃; spray gas pressure: 344.74 kPa; auxiliary gas pressure: 344.74kPa, mass to charge ratio: 100-500.
In an ESI positive ion mode, [ M + H ] is obtained] + Peak, mass-to-charge ratio of vitamin C (177.1), vitamin B2(377.15) and biotin (245.10) was extracted by analyzing the mass spectrum results to obtain corresponding chromatographic peaks, and secondary mass spectrum under the molecular ion peak was extracted as shown in fig. 8.
FIG. 8 is a secondary mass spectrogram of three vitamins in fermentation broth under ion peaks, and the fragment ions in the secondary mass spectrogram of FIG. 8 are compared with the secondary fragments in database HMDB/MassBank to preliminarily confirm that the fermentation broth contains vitamin C, vitamin B2 and biotin, which indicates that lactococcus X1 can simultaneously produce the three vitamins and is a probiotic.
In conclusion, compared with the prior art, the Lactococcus sp.X1 has good lactic acid production effect and low nutritional requirement, and is beneficial to large-scale industrial production of L-lactic acid. And the Lactococcus sp.X1 can be used for synchronously hydrolyzing and fermenting straws with cellulase or cellulose degradation microorganisms, so that the inhibition effect of overhigh sugar substrate concentration on the fermentation of lactic acid bacteria strains can be avoided, equipment can be fully utilized, the fermentation period is shortened, the yield of lactic acid is improved, and the cost is saved. Furthermore, Lactococcus sp.X1 can also be used for synthesizing water-soluble vitamins, such as vitamin C, vitamin B2 and biotin, and is a probiotic.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Sequence listing
<110> university of Jiangsu
<120> lactococcus capable of highly producing L-lactic acid and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
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agagtttgat cctggctcag 20
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ggttaccttg ttacgactt 19
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<213> Artificial Sequence (Artificial Sequence)
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gaacgcttga ttgtcaccga agcttgcttc accgacaaac aagagtagcg aacgggtgag 60
taacgcgtgg gtaacctacc ttatagcggg ggataactat tggaaacgat agctaatacc 120
gcataacaat gggaattgca taatttctat ttaaaagtgt ctattggtac actatgagat 180
ggacccgcgt tgtattagct agttggtagt gtaaaggact accaaggcga tgatacatag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc ggcaatgggg gcaaccctga ccgagcaacg ccgcgtgagt 360
gaagaaggtt ttcggatcgt aaaactctgt tgttagagaa gaacgtgtga gagagtagaa 420
agttctcaca gtgacggtat ctaaccagaa agggacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtcccaag cgttgtccgg atttattggg cgtaaagcga gcgcaggtgg 540
tttaataagt ctgatgtaaa aggcagtggc tcaaccattg tatgcattgg aaactgttag 600
acttgagtac agtagaggag agtggaattc catgtgtagc ggtgaaatgc gtagatatat 660
ggaggaacac cggtggcgaa agcggctctc tggactgtga ctgacactga ggctcgaaag 720
cgtgggtagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctag 780
ttgtttgggg ctatccagcc ctaagtgacg cagcaaacgc attaagcact ccgcctgggg 840
agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc cctatgaccg 960
tcttagagat aagatttccc ttcggggcaa aggtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat tgttagttgc 1080
catcattaag ttgggcactc tagcgagact gccggtaata aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggttggtac 1200
aacgagtcgc aagccagtga tggttagcta atctcttaaa gccaatctca gttcggattg 1260
taggctgcaa ctcgcctaca tgaagtcgga atcgctagta atcgcggatc agcacgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaatac 1380
ccaaagccgg tgggctaacc a 1401

Claims (10)

1. The Lactococcus capable of producing L-lactic acid is Lactococcus X1(Lactococcus sp.X1) which is preserved in China general microbiological culture Collection center (CGMCC) and is deposited at the institute of microbiology of China academy of sciences No. 3, Xilu No. 1 institute of China, North Kyoto-Yang district, Beijing, with the preservation date of 2022 years, 1 month and 19 days and the preservation number of CGMCC No. 24341.
2. A lactococcus lactis bacterium according to claim 1, wherein said fermentation medium for producing L-lactic acid bacteria comprises: 1-5 g/L of yeast powder and NH 4 Cl 1-5 g/L, carbon source 5-10 g/L, KH 2 PO 4 1.5g/L，K 2 HPO 4 ×3H 2 O2.9 g/L, sodium acetate 1g/L, triammonium citrate 0.5g/L, MgCl 2 ×6H 2 O 0.2g/L，CaCl 2 ×2H 2 O 75mg/L，MnSO 4 0.05g/L, cysteine 0.5g/L, CaCO 3 KHCO accounting for 50-100% (w/w) of the carbon source 3 0.01～0.05M。
3. A lactococcus bacterium producing L-lactic acid according to claim 2, wherein said carbon source comprises one or more of glucose, sucrose, cellobiose, arabinose, xylose; in the fermentation medium, 1g/L of yeast powder and NH 4 Cl 5g/L，CaCO 3 KHCO at 50% (w/w) of the carbon source content 3 0.05M。
4. A microbial agent comprising the L-lactic acid-producing lactococcus X1 according to claim 1.
5. Use of lactococcus X1 according to claim 1 or a microbial agent according to claim 4 for the fermentative production of L-lactic acid.
6. The use according to claim 5, wherein said use is the production of L-lactic acid by fermentation of lactococcus X1 according to claim 1 or a microbial agent according to claim 4 in a fermentation medium or a medium containing a high concentration of a carbon source.
7. Use of lactococcus X1 according to claim 1 or a microbial agent according to claim 4 for simultaneous saccharification and hydrolysis of straw to produce L-lactic acid together with cellulase or a cellulose-degrading microorganism.
8. A method for producing L-lactic acid, comprising any one of:
(1) inoculating lactococcus X1 according to claim 1 or the microbial agent according to claim 4 into the fermentation medium or the medium containing a high concentration of a carbon source according to claim 3;
(2) mixing lactococcus X1 as defined in claim 1 or the microbial agent as defined in claim 4 with cellulase or a cellulose-degrading microorganism, and inoculating the mixture into a fermentation medium using straw as a carbon source.
9. Use of lactococcus X1 according to claim 1 or a microbial agent according to claim 4 for the production of water-soluble vitamins; the water-soluble vitamins include vitamin C, vitamin B2 and biotin.
10. Use of lactococcus X1 according to claim 1 or a microbial agent according to claim 4 in food or feed.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101768610A (en) * 2010-03-05 2010-07-07 广东绿百多生物科技有限公司 Method for efficiently cultivating lactobacillus and producing lactic acid
CN109536409A (en) * 2018-12-17 2019-03-29 吉林中粮生化有限公司 A kind of resistance is high and using the Pedicoccus acidilacticii strain of several kinds of carbon source and the method for producing lactic acid using the bacterial strain
CN109868235A (en) * 2019-01-28 2019-06-11 佛山市海天(高明)调味食品有限公司 One plant of Lactococcus lactis ZF625 and its application
CN113980853A (en) * 2021-11-12 2022-01-28 河南省科学院生物研究所有限责任公司 Lactococcus garvieae WBT0008 capable of producing lactic acid at high yield and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101768610A (en) * 2010-03-05 2010-07-07 广东绿百多生物科技有限公司 Method for efficiently cultivating lactobacillus and producing lactic acid
CN109536409A (en) * 2018-12-17 2019-03-29 吉林中粮生化有限公司 A kind of resistance is high and using the Pedicoccus acidilacticii strain of several kinds of carbon source and the method for producing lactic acid using the bacterial strain
CN109868235A (en) * 2019-01-28 2019-06-11 佛山市海天(高明)调味食品有限公司 One plant of Lactococcus lactis ZF625 and its application
CN113980853A (en) * 2021-11-12 2022-01-28 河南省科学院生物研究所有限责任公司 Lactococcus garvieae WBT0008 capable of producing lactic acid at high yield and application thereof

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