CN115960757B - Lactic acid bacteria capable of tolerating low pH to produce L-lactic acid and application thereof - Google Patents
Lactic acid bacteria capable of tolerating low pH to produce L-lactic acid and application thereof Download PDFInfo
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- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title claims abstract description 54
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims description 68
- 239000004310 lactic acid Substances 0.000 title claims description 33
- 235000014655 lactic acid Nutrition 0.000 title claims description 33
- 241000894006 Bacteria Species 0.000 title description 16
- 230000012010 growth Effects 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims description 47
- 230000004151 fermentation Effects 0.000 claims description 47
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 24
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims description 15
- 244000199885 Lactobacillus bulgaricus Species 0.000 claims description 13
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims description 13
- 239000001888 Peptone Substances 0.000 claims description 13
- 108010080698 Peptones Proteins 0.000 claims description 13
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 13
- 240000008042 Zea mays Species 0.000 claims description 13
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 13
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 13
- 229940041514 candida albicans extract Drugs 0.000 claims description 13
- 235000005822 corn Nutrition 0.000 claims description 13
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 235000019319 peptone Nutrition 0.000 claims description 13
- 239000012138 yeast extract Substances 0.000 claims description 13
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 12
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 12
- 229940099596 manganese sulfate Drugs 0.000 claims description 12
- 239000011702 manganese sulphate Substances 0.000 claims description 12
- 235000007079 manganese sulphate Nutrition 0.000 claims description 12
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 12
- 239000001632 sodium acetate Substances 0.000 claims description 12
- 235000017281 sodium acetate Nutrition 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- 229920000136 polysorbate Polymers 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 3
- 238000012262 fermentative production Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 17
- 241000186660 Lactobacillus Species 0.000 abstract description 15
- 229940039696 lactobacillus Drugs 0.000 abstract description 15
- 238000000034 method Methods 0.000 abstract description 9
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 abstract description 8
- BRPQOXSCLDDYGP-UHFFFAOYSA-N calcium oxide Chemical compound [O-2].[Ca+2] BRPQOXSCLDDYGP-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000292 calcium oxide Substances 0.000 abstract description 6
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 abstract description 6
- 238000006386 neutralization reaction Methods 0.000 abstract description 6
- 239000002253 acid Substances 0.000 abstract description 5
- 239000002699 waste material Substances 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 description 35
- 229960000448 lactic acid Drugs 0.000 description 30
- 230000003287 optical effect Effects 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 238000011218 seed culture Methods 0.000 description 11
- 238000012258 culturing Methods 0.000 description 10
- 238000002513 implantation Methods 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 239000007787 solid Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000010884 ion-beam technique Methods 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 239000004626 polylactic acid Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 5
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 229920000747 poly(lactic acid) Polymers 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- 238000000861 blow drying Methods 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000003912 environmental pollution Methods 0.000 description 3
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- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 229930182843 D-Lactic acid Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000002053 acidogenic effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 229940022769 d- lactic acid Drugs 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000006068 polycondensation reaction Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a lactobacillus capable of tolerating low pH to produce L-lactic acid and application thereof, wherein the low pH-tolerant lactobacillus strain is classified as Lactobacillus bulgaricusLactobacillus bulgaricus) QSE-1993 was deposited in China Center for Type Culture Collection (CCTCC) at 18.2.2019, with deposit number: cctccc No. M2019095. Under the acid culture condition under the normal culture condition, no calcium oxide is needed to be added for neutralization, and the growth can be normally carried out under the condition that the pH is as low as 2-3, so that the cost is reduced, the pollution of waste calcium sulfate to the environment is reduced, and the method can be applied to mass industrialized production.
Description
Technical Field
The invention relates to a lactobacillus capable of tolerating low pH to produce L-lactic acid, belonging to the technical field of microbial fermentation.
Background
Lactic acid bacteria are a generic term for bacteria that can produce large amounts of lactic acid using fermentable sugars without decomposing proteins, and are generally non-motile, non-spore-forming gram-positive bacteria that can grow under anaerobic, facultative anaerobic conditions. Lactic acid bacteria are widely distributed in nature, exist in the alimentary tracts of people and animals, and have high application value in the fields of industry, agriculture and the like. In most lactic acid bacteria cells of homolactic fermentation type, lactic acid is the final product of the glycolytic pathway, which lactic acid bacteria can use to obtain the required energy.
Polylactic acid (PLA) is a biodegradable polymer obtained by polycondensation of lactic acid monomers, and is mainly obtained by polymerization of lactic acid, which is a fermentation product of microorganisms, as a monomer. As polylactic acid, three kinds of polylactic acid (PLLA), poly D-lactic acid (PDLA) and poly DL-lactic acid (PDLLA) are mainly included, among which PLLA is the most widely used scale. More specifically, polylactic acid materials polymerized from L-lactic acid having an optical purity of more than 98% are widely used in foods, medicines, agricultural chemicals, chemical industry, and the like.
Currently, L-lactic acid can be obtained by chemical synthesis and microbial fermentation methods. The chemical synthesis method has the troublesome problems of environmental pollution, high cost, complex technology, low optical purity and the like, and cannot meet the actual application needs. In contrast, the microbial fermentation method has the advantages of low production cost, mild conditions, high optical purity of the product and the like, so that the industrial production of L-lactic acid is mostly carried out by the microbial fermentation method. At present, L-lactic acid fermentation bacteria cannot tolerate low pH, have low resistance to the stress of a culture environment, and can be successfully fermented by adding calcium oxide to the pH of 6.0 in the fermentation production process, but a large amount of calcium sulfate waste is generated later, so that huge pollution is brought to the environment.
In this regard, in the lactic acid fermentation industry, the low pH resistance of the strain is remarkably improved, and more L-lactic acid is produced and accumulated, so that the strain is more suitable for industrial production and has positive research significance.
Disclosure of Invention
The invention aims to provide a lactobacillus strain capable of resisting low pH to produce L-lactic acid, which can obviously improve the tolerance of the original strain to low pH environment, and the produced L-lactic acid has high optical purity and little environmental pollution in the production process.
Low pH resistant lactic acid bacterial strain, its classification is named as Lactobacillus bulgaricus @Lactobacillus bulgaricus) QSE-1993 was preserved in China Center for Type Culture Collection (CCTCC) at 18 months 2 in 2019, with preservation address of university of Wuhan, china, and preservation number: cctccc No. M2019095.
The strain Lactobacillus bulgaricus QSE-1993 is obtained by mutagenesis of an original strain QSE-1 collected from Sichuan, the strain QSE-1 is inoculated into an MRS culture medium for activation for two generations by using a plate scribing mode, the strain QSE-1 is cultured to OD=1 at 37 ℃, sterile water is used for dilution to OD=0.6, the strain is coated on a plate after bacteria, ion beam implantation is carried out after no cell overlap is observed under a blow-drying microscopic condition under a sterile condition, nitrogen ion beams are selected, the implantation condition is 10-30KeV, and the implantation dosage is 15x10 13 ～35x10 13 ions/cm 3 The treatment time was 120s. After the injection is finished, taking out the flat plate to use sterile waterEluting. Spread on MRS plate, and culture activated in anaerobic environment. After a period of cultivation, the strain is inoculated into MRS solid culture medium with the pH adjusted for cultivation for 72 hours, and the process is repeated for a plurality of times, so that the strain with good growth condition is observed and screened. Preparing cell suspension from the strain obtained by the previous step, and adjusting the concentration to 10 -6 /ml. 0.1ml of cell suspension is coated on a sterilized flat plate, and nitrogen ion beam implantation is carried out after blow-drying under the conditions of 20-30 KeV with the implantation dosage of 15x10 14 ～35x10 14 ions/cm 3 The injection time was 120s. After the injection is finished, the plate is eluted by sterile water, and the eluted bacterial liquid is coated on a solid MRS culture medium and is reversely cultured for 24 hours at 37 ℃.
Inoculating the strain obtained by mutagenesis into a seed culture medium, and culturing for 10-16 h at 37 ℃, wherein the liquid filling amount of a shake flask is 10% -30% (v/v). Inoculating the bacterial liquid in the seed culture medium to MRS liquid culture medium, and placing the bacterial liquid and the anaerobic bag into an anaerobic tank for culturing for 48 hours. Inoculating the strain into a solid culture medium with the pH adjusted, and selecting the strain with good growth condition. Repeating the re-screening step until the target strain is screened out.
The lactobacillus with low pH resistance obtained by screening is named as Lactobacillus bulgaricus QSE-1993, grows well in MRS agar culture medium, and the observed strain is round or slightly irregular, and the colony is off-white, opaque, smooth in surface, slightly raised and rough in edge; is a gram positive bacillus, and has no sporulation.
The lactic acid bacteria capable of resisting low pH in the invention are facultative anaerobes, the proper growth temperature is 35-45 ℃, and the more proper growth temperature range is 30-37 ℃. Can grow normally under the low pH condition, the lowest pH value can reach 2-3, and the capability of the lactic acid bacteria for resisting the external bad environment is effectively improved.
The culture method of the lactobacillus bulgaricus QSE-1993 comprises the following steps:
1) Plate culture: inoculating lactobacillus bulgaricus QSE-1993 to a flat culture medium for culture at the temperature of 35-45 ℃ for 36-48 hours;
2) Slant culture: inoculating the lactic acid bacteria cultured by the flat-bed culture medium in the step 1) to a slant culture medium for culture, wherein the culture temperature is 35-45 ℃ and the culture time is 36-48 h;
3) Seed culture: inoculating the slant culture of the lactic acid bacteria in the step 2) to a seed culture medium for culture, wherein the culture temperature is 35-45 ℃ and the culture time is 10-20 hours;
4) Fermentation culture: inoculating the lactic acid bacteria cultured in the step 3) to a fermentation culture medium for culturing, wherein the inoculum size is 5% -15% (V/V), the fermentation temperature is 35-37 ℃, and the fermentation time is 24-120h.
The plate culture medium comprises the following components in mass: 10g/L peptone, 10g/L yeast extract, 5g/L sodium acetate, 2. 2g/L dipotassium hydrogen phosphate, 0.58 g/L magnesium sulfate, 2. 2g/L diammonium citrate, 20. 20g/L corn steep liquor, 5. 5g/L ammonium sulfate, 0.25. 0.25 g/L manganese sulfate, and 801mL tween.
The fermentation temperature is 35-37 ℃ and the fermentation time is 36-70 hours.
The slant culture medium comprises 2% of agar, 10g/L of peptone, 10g/L of yeast extract, 5g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 0.58 g/L of magnesium sulfate, 2g/L of diammonium citrate, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.25 g/L of manganese sulfate and 801mL of Tween.
The fermentation temperature is 35-37 ℃ and the fermentation time is 36-48 hours.
The seed culture medium comprises 10g/L of peptone, 10g/L of yeast extract, 0.5g/L of sodium acetate, 0.2g/L of diammonium citrate, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferric sulfate, 0.2g/L of dipotassium hydrogen phosphate and 50g/L of corn steep liquor.
The fermentation temperature is 35-45 ℃ and the fermentation time is 10-20h.
The fermentation medium comprises 10g/L of peptone, 10g/L of yeast extract, 0.5g/L of sodium acetate, 0.2g/L of diammonium citrate, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferric sulfate, 0.2g/L of dipotassium hydrogen phosphate and 120g/L of corn steep liquor.
The fermentation temperature is 35-37 ℃ and the fermentation time is 24-36h.
The application of the low-pH-resistant lactic acid bacterial strain in the fermentation production of L-lactic acid.
In nutrient cultivationCulturing Lactobacillus bulgaricus in culture mediumLactobacillus bulgaricus) QSE-1993, fermentation to produce L-lactic acid.
The nutrient medium is MRS liquid medium and comprises the following components: 10g/L peptone, 10g/L yeast extract, 5g/L sodium acetate, 2. 2g/L dipotassium hydrogen phosphate, 0.58 g/L magnesium sulfate, 2. 2g/L diammonium citrate, 20. 20g/L corn steep liquor, 5. 5g/L ammonium sulfate, 0.25. 0.25 g/L manganese sulfate, and 801mL tween.
The fermentation temperature is 30-37 ℃ and the fermentation time is 24-36 hours.
The lactobacillus bulgaricus isLactobacillus bulgaricus) QSE-1993 was inoculated into the nutrient medium at a ratio of 5-20% v/v, and no further addition of calcium oxide was required for neutralization during fermentation.
The strain of the invention has the capability of resisting low pH environment, has good genetic stability, improves the capability of producing L-lactic acid, obviously improves the optical purity of the product, and has the yield of 180-200g/L and the optical purity of 99.8 percent in 24-36 hours of production period. The mutagenized lactobacillus can normally grow under the condition of pH being as low as 2-3 without adding calcium oxide for neutralization under the condition of acidic culture.
The beneficial effects are that: compared with a wild strain, the lactobacillus QSE-1993 obtained by screening through an ion beam engineering technology effectively improves the low pH environment resistance of the strain, has good genetic stability and the L-lactic acid production capacity, remarkably improves the optical purity of the product, has the production period of 24-36 hours and the yield of 180-200g/L, and has the optical purity of 99.8 percent. After the mutagenesis of the lactobacillus, calcium oxide is not needed to be added for neutralization under the acid culture condition under the normal culture condition, the lactobacillus can normally grow under the condition that the pH is as low as 2-3, the cost is reduced, the pollution of waste calcium sulfate to the environment is reduced, and the lactobacillus can be put into mass industrialized production, so that the lactobacillus has great research significance and economic value.
Detailed Description
Example 1 mutagenesis and Low pH screening of strains
Starting strain screening:
use of strain QSE-1 in FlatThe method comprises the steps of inoculating to MRS culture medium for activating for two generations in a plate scribing mode, culturing at 37 ℃ until OD=1, diluting to OD=0.6 by using sterile water, coating on a plate after bacteria, blow-drying and microscopic observation under the sterile condition, performing ion beam implantation after no cell overlap, selecting nitrogen ion beam, wherein the implantation condition is 10-30KeV, and the implantation dosage is 15x10 13 ～35x10 13 ions/cm 3 The treatment time was 120s. After the end of the injection, the plate was removed and eluted with sterile water. Spread on MRS plate, and culture activated in anaerobic environment. After a period of incubation, the strain was inoculated into a conditioned MRS solid medium at ph=3.2 and incubated for 72 hours, and this procedure was repeated several times, and strains with good growth were observed and selected.
And (3) re-screening:
preparing cell suspension from the strain obtained by the previous step, and adjusting the concentration to 10 -6 /ml. 0.1ml of cell suspension is coated on a sterilized flat plate, and nitrogen ion beam implantation is carried out after blow-drying under the conditions of 20-30 KeV with the implantation dosage of 15x10 14 ～35x10 14 ions/cm 3 The injection time was 120s. After the injection is finished, the plate is eluted by sterile water, and the eluted bacterial liquid is coated on a solid MRS culture medium and is reversely cultured for 24 hours at 37 ℃.
Inoculating the strain obtained by mutagenesis into a seed culture medium, and culturing for 10-16 h at 37 ℃, wherein the liquid filling amount of a shake flask is 10% -30% (v/v). Inoculating the bacterial liquid in the seed culture medium to MRS liquid culture medium, and placing the bacterial liquid and the anaerobic bag into an anaerobic tank for culturing for 48 hours. Inoculating the strain into a solid culture medium with the pH adjusted, and selecting the strain with good growth condition. The re-screening step was repeated until the target strain QSE-1993 was selected.
The formula of MRS culture medium is (each component and content in 1000 ml): 10g of peptone, 4 g of yeast extract, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 2g of diammonium citrate, 20g of corn steep liquor, 5g of ammonium sulfate, 0.25 g of manganese sulfate and 1mL of Tween 80; 18g of agar (which is not added in the case of liquid medium).
Formula of seed culture medium (each component and content in 1000 ml): dipotassium phosphate 2g, triammonium citrate 2g, anhydrous sodium acetate 5g, manganese sulfate heptahydrate 0.25 g, magnesium sulfate heptahydrate 0.58 g, peptone 10g, yeast extract 5g, corn steep liquor 20g, ammonium sulfate 5g, tween 801ml, agar 20g.
The bacterial strain obtained by screening is positive in gram staining, negative in catalase test, white in colony morphology, smooth in surface, slightly raised, rough in edge and obvious in bacterial strain morphology, and a small number of bacterial strain morphologies are circular.
Determination of the low pH resistance of the screening strains:
the mutant strain QSE-1993 and the original strain QSE-1 are respectively placed in MRS culture mediums with pH values of 2, 3 and 4 for culture at 30 ℃, growth conditions are observed after 36 hours, the survival rate of the selected strain is higher than that of the original strain and reaches more than 90% under each group of pH conditions, and the growth state of the surviving strain is better than that of the original strain.
Example 2
This example illustrates the identification and genetic stability of strains
Identification of strains: on MRS solid culture medium, bacterial colony is off-white, round or irregular, surface raised, middle smooth, and edge obviously rough, and the proper growth temperature range of bacterial body is 30-37 deg.C.
Subculture experiments: inoculating lactobacillus obtained by screening into MRS solid culture medium, culturing at 37deg.C under anaerobic condition for 36 hr, inoculating into solid culture medium with pH adjusted to 2, 3, and 4, culturing for 36 hr, and recording strain survival rate of different generations.
The survival rates of the different strains are shown in the table:
| algebra (algebra) | Survival rate |
| 1 | 97.56% |
| 2 | 96.87% |
| 3 | 97.12% |
| 4 | 98.25% |
| 5 | 96.95% |
| 6 | 97.26% |
| 7 | 97.42% |
The experimental result shows that the mutant strain QSE-1993 has better passage stability after five continuous passages and can be used as a strain for further research and development.
Example 3: culture method of lactobacillus bulgaricus QSE-1993
This example illustrates a method of culturing lactobacillus bulgaricus QSE-1993 comprising the steps of:
1) Plate culture: inoculating Lactobacillus bulgaricus QSE-1993 to a plate culture medium for culture at 37 ℃ for 36h;
2) Slant culture: inoculating the lactobacillus cultured by the flat-bed culture medium in the step 1) to a slant culture medium for culture, wherein the culture temperature is 37 ℃ and the culture time is 36 hours;
3) Seed culture: inoculating the slant culture of the lactobacillus in the step 2) to a seed culture medium for culture, wherein the culture temperature is 37 ℃ and the culture time is 10 hours;
4) Fermentation culture: inoculating the lactic acid bacteria cultured in the step 3) to a fermentation medium for culturing, wherein the inoculum size is 10% (V/V), the fermentation temperature is 37 ℃, and the fermentation time is 24-120h.
The plate culture medium comprises the following components in mass: 10g/L peptone, 10g/L yeast extract, 5g/L sodium acetate, 2. 2g/L dipotassium hydrogen phosphate, 0.58 g/L magnesium sulfate, 2. 2g/L diammonium citrate, 20. 20g/L corn steep liquor, 5. 5g/L ammonium sulfate, 0.25. 0.25 g/L manganese sulfate, and 801mL tween.
The slant culture medium comprises 2% of agar, 10g/L of peptone, 410g/L of yeast extract, 5g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 0.58 g/L of magnesium sulfate, 2g/L of diammonium citrate, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.25 g/L of manganese sulfate and 801mL of Tween.
The seed culture medium comprises 10g/L of peptone, 10g/L of yeast extract, 0.5g/L of sodium acetate, 0.2g/L of diammonium citrate, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferric sulfate, 0.2g/L of dipotassium hydrogen phosphate and 50g/L of corn steep liquor.
The fermentation medium comprises 10g/L of peptone, 10g/L of yeast extract, 0.5g/L of sodium acetate, 0.2g/L of diammonium citrate, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferric sulfate, 0.2g/L of dipotassium hydrogen phosphate and 120g/L of corn steep liquor.
Example 4: strain fermentation production of L-lactic acid
The L-lactic acid producing strain QSE-1993 was inoculated into MRS liquid medium and cultured overnight at 37℃and 150rpm to obtain a seed liquid. Seed solution was inoculated in a ratio of 10% (v/v) in 200mL of an acidogenic fermentation medium, and the strain was subjected to shaking fermentation at 150rpm for 36 hours, and after the fermentation was completed, the lactic acid content and optical purity were measured by high performance liquid chromatography.
The formula of the acidogenic fermentation medium is as follows: 10g/L of peptone, 10g/L of yeast extract, 0.5g/L of sodium acetate, 0.2g/L of diammonium citrate, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferric sulfate, 0.2g/L of dipotassium hydrogen phosphate and 120g/L of corn steep liquor.
The fermentation temperature is 35 ℃.
As a result of the measurement, under the condition of enlarging the culture scale, the original strain can ferment and produce 121.5g/L of lactic acid in 70 hours, the sugar acid conversion rate is more than 95%, the optical purity of the obtained product L-lactic acid is 97.2%, the lactic acid produced by fermenting the strain after mutagenesis in 70 hours is 203.2g/L (67% increase), the sugar acid conversion rate is more than 95%, and the optical purity of the obtained product L-lactic acid is more than 99.8%.
Example 4: fermentation production of lactic acid at low pH
L-lactic acid-producing strain QSE-1993 and original strain QSE-1 were each cultured in a shake flask fermentation medium (YE medium) having a pH of 3.8 without neutralization with calcium carbonate for 48 hours, and lactic acid production was compared.
The YE medium formula is: 15g/L yeast powder, 100g/L glucose and 1L distilled water.
The result shows that the bacterial growth and lactic acid yield of the mutant strain are obviously improved compared with those of the original strain after the mutant strain is fermented and cultured for 48 hours under the condition of pH 3.8, the bacterial density of the original strain is 1.69, the lactic acid yield is only 1.59g/L, and the acid yield of the mutant strain reaches 4.995g/L and is 3.14 times that of the original strain.
High performance liquid chromatography detects lactic acid in fermentation broth:
chromatograph: agilent Technologies 1260 Infinity II;
the detector: RID;
separation column: aminex HPX-87H Column 300X 7.8 mm;
mobile phase: 0.005M sulfuric acid;
flow rate: 0.5mL/min;
sample injection amount: 20. Mu.L. The lactic acid retention time was about 14 min.
High performance liquid chromatography for detecting optical purity of lactic acid
Chromatograph: agilent Technologies 1260 Infinicity;
the detector: wavelength 254nm, sensitivity 0.32AUFS;
separation column: MCI GEL-CRS 10W (3 u) 4.6 ID. Times.50 nm;
mobile phase: 0.002M copper sulfate
Flow rate: 0.5mL/min;
sample injection amount: 20. Mu.L.
And diluting the sample to the total lactic acid concentration of 0.5-1g/L, and detecting. The retention time of D-lactic acid was about 11min, and the retention time of L-lactic acid was about 13min, and the optical purity of L-lactic acid was calculated from the peak area thereof.
The above study shows that the strain of the invention is significantly superior to the original strain without mutagenesis in terms of both the L-lactic acid production rate and the optical purity of the product (67% increase in production rate and 99% increase in optical purity). The invention provides a novel strain with low fermentation cost, high L-lactic acid production speed and high optical purity of the product, the strain can effectively resist a low pH environment, and the step of using calcium oxide for neutralization in the production fermentation process is reduced, so that the produced calcium sulfate waste is reduced, the purpose of reducing environmental pollution is achieved, and better potential selection is provided for the industrial microbial fermentation production of L-lactic acid.
Claims (7)
1. Low pH resistant lactic acid bacterial strain, its classification is named as Lactobacillus bulgaricus @Lactobacillus bulgaricus) QSE-1993, which is preserved in China Center for Type Culture Collection (CCTCC), with the preservation number: cctccc No. M2019095.
2. The low pH tolerant lactic acid bacterial strain according to claim 1, wherein the strain is capable of normal growth under low pH conditions, wherein the low pH conditions are pH 2-3.
3. Use of the low pH tolerant lactic acid bacterial strain according to claim 1 for the fermentative production of L-lactic acid.
4. The use according to claim 3, wherein lactobacillus bulgaricus is cultivated in a nutrient mediumLactobacillus bulgaricus) QSE-1993, fermentation to produce L-lactic acid.
5. The use according to claim 4, wherein the nutrient medium is an MRS liquid medium comprising the following components: 10g/L peptone, 410g/L yeast extract, 5g/L sodium acetate, 2. 2g/L dipotassium hydrogen phosphate, 0.58 g/L magnesium sulfate, 2. 2g/L diammonium citrate, 20g/L corn steep liquor, 5g/L ammonium sulfate, 0.25 g/L manganese sulfate, and 801mL Tween.
6. The use according to claim 4, wherein the fermentation temperature is 30-37 ℃ and the fermentation time is 24-36 hours.
7. The use according to claim 4, wherein the lactobacillus bulgaricus is @ or @ isLactobacillus bulgaricus) QSE-1993 was inoculated into the nutrient medium in a ratio of 5-20% v/v.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6875601B1 (en) * | 1998-05-22 | 2005-04-05 | Compagnie Gervais Danone | Mutant Lactobacillus bulgaricus strains free from β-galactoside activity |
| CN103215199A (en) * | 2013-03-13 | 2013-07-24 | 内蒙古农业大学 | Lactobacillus bulgaricus bacterial strain with post-acidification delaying effect and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6875601B1 (en) * | 1998-05-22 | 2005-04-05 | Compagnie Gervais Danone | Mutant Lactobacillus bulgaricus strains free from β-galactoside activity |
| CN103215199A (en) * | 2013-03-13 | 2013-07-24 | 内蒙古农业大学 | Lactobacillus bulgaricus bacterial strain with post-acidification delaying effect and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 保加利亚乳杆菌乳酸高产菌株的紫外诱变选育;李小平;魏玲玲;张慧发;霍乃蕊;;山西农业大学学报(自然科学版);20100131(01);88-90 * |
| 利用乳清培养基生产乳品发酵剂的研究;李艾黎;代敏;霍贵成;;食品科学;20060415(04);34-36 * |
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