Production strain for efficiently producing L-lactic acid with high optical purity and application

Abstract

The invention relates to a production strain for efficiently producing L-lactic acid with high optical purity and application of the production strain, the production strain is lactobacillus rhamnosus Lrch1017, the production strain is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32079. The production strain can be used for efficiently producing L-lactic acid with high optical purity. The lactobacillus rhamnosus Lrch1017 is fermented for 12 hours at 37 DEG C and then is heated to 48 DEG C to be fermented for 7.5 hours, the total fermentation time is 19.5 hours, the L-lactic acid yield reaches 17% (W / V) or above, and the fermentation efficiency reaches 97%. The rhamnose lactic acid bacteria Lrch1017 is a strain which takes the shortest time when the yield of L-lactic acid reaches 170g / L or above at present, and the optical purity of the L-lactic acid produced by fermentation can reach 99.5% or above, so that the requirements of polymer-grade L-lactic acid are completely met.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a production strain for efficiently producing high-optically pure L-lactic acid and its application. Background Art

[0002] Polylactic acid (PLA) is a biodegradable polymer formed by polycondensation of lactic acid monomers. It is mainly obtained by polymerization reaction using lactic acid, a fermentation product of microorganisms, as a monomer. PLA is unanimously recognized by the industry as the most promising new "bio-based material" in the new century because its raw materials are renewable biological resources. In addition, products made of PLA have good gloss, transparency, feel and heat resistance, and also have certain bacteria resistance, flame retardancy and UV resistance, and have high gloss and processing performance; PLA also has the characteristics of non-toxicity, non-irritation and good biocompatibility. Therefore, PLA has broad market prospects and a wide range of uses. It is currently mainly used in clothing, construction, agriculture, forestry, papermaking, medical and health care and other fields. Polylactic acid mainly includes poly L-lactic acid (PLLA), poly D-lactic acid (PDLA) and poly DL-lactic acid (PDLLA), among which PLLA is the most widely used. More specifically, polylactic acid materials polymerized from L-lactic acid with an optical purity greater than 98% are widely used in food, medicine, pesticides, chemicals, etc.

[0003] However, during the fermentation process, as lactic acid continues to accumulate, the calcium lactate content in the fermentation broth continues to increase. In the later stage of fermentation, calcium lactate in the fermentation broth will continue to precipitate, and the fermentation is forced to terminate. Therefore, we need to improve the high temperature tolerance of the strain in order to delay the precipitation of calcium lactate; at the same time, we hope to increase the acid production rate and shorten the fermentation cycle while meeting production requirements, so as to reduce production costs. The solution strategies are: First, improve the temperature resistance of the fermentation strain and delay the precipitation time of calcium lactate. Second, increase the acid production rate of the fermentation strain and shorten the fermentation cycle. Summary of the invention

[0004] The purpose of the present invention is to provide a production strain for efficiently producing high optical purity L-lactic acid and its application.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a production strain for efficiently producing high optical purity L-lactic acid, wherein the production strain is Lactobacillus rhamnosus ( Lactobacillus rhamnosus )Lrch1017, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNo.32079.

[0006] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: the use of the production strain for efficiently producing high optical purity L-lactic acid in the fermentation preparation of efficiently producing high optical purity L-lactic acid.

[0007] The preferred technical solution is: inoculate Lactobacillus rhamnosus Lrch1017 into MRS solid medium, culture at 35-39°C, transfer to seed shake flask after 10-20 hours, expand culture, control the seed age at 14-16 hours, OD 600 4.5-6.5, pH4-5, to obtain seed liquid; inoculate the seed liquid into the fermentation medium, inoculate by flame inoculation, adopt the flow calcium hydroxide method, control the pH to 5.8-6.2, ferment at a speed of 100-200rpm, 35-39°C, raise the temperature to 48°C after 12h, and ferment for another 7.5h; after fermenting for 19.5h, the L-lactic acid output reaches 171.6 g / L, the sugar-acid conversion rate is 97% (g / g), and the L-lactic acid optical purity is 99.5%.

[0008] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The starting strain of the present invention is a recombinant rhamnosus bacillus Lr-ALTHT-DBC (the deposit number is CGMCC No. 19507 and the strain has been disclosed in CN 111500517 A). After physical mutagenesis and chemical mutagenesis are combined and multiple rounds of superimposed mutagenesis screening are performed, a mutagenized strain Lrch1017 is obtained. The fermentation cycle of the strain Lrch1017 is within 20 hours, and the L-lactic acid production can reach more than 170 g / L, and the optical purity of the L-lactic acid is more than 99%. A production strain for efficiently producing high optical purity L-lactic acid is provided for the industrial production of lactic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The chromatogram is a graph showing the optical purity (99.8%) of the fermentation product L-lactic acid when L-lactic acid is produced using the L-lactic acid producing strain Lrch1017 of the present invention. DETAILED DESCRIPTION

[0010] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0011] See also Figure 1. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0012] Deposit of biological material: Lactobacillus rhamnosus ( Lactobacillus rhamnosus )Lrch1017 was deposited on September 26, 2024 at the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCC No.32079.

[0013] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0014] Example 1: A strain for efficiently producing high optical purity L-lactic acid and its application 1. Strain mutagenesis and growth screening The Lr-ALTHT-DBC strain was activated by streaking and then transferred to MRS liquid medium. It was cultured at 45℃ and 150 rpm until OD=1. The bacterial solution was diluted with saline to OD=0.6 for atmospheric pressure room temperature plasma (ARTP) mutagenesis. Helium was used as the working gas of the plasma, with a power of 100 W, a gas flow of 10 slpm, and a treatment time of 120 s, 150 s, and 180 s. MRS medium was added for recovery at 37℃ and 150 rpm for 1 hour. The bacterial solution was graded diluted with sterile saline and spread on MRS+CaCO 3 solid plates. The plates were placed in a 48℃ incubator for culture until a single colony was formed and its calcium dissolution ring was observed.

[0015] About 500 single colonies with fast colony growth and large calcium dissolution zone were selected from the plate and transferred to 12 deep-well plates containing acid-producing fermentation medium to complete the fermentation test. The culture was carried out at 37℃ 150 rpm for 12 hours and then at 48℃ 150 rpm for 12 hours. The total fermentation time was 24 hours. After the fermentation, the lactic acid content of the fermentation liquid was detected, and the single clones with lactic acid production of more than 170 g / L were collected and named Lr-Ms-1 for the second round of mutagenesis screening.

[0016] Lr-Ms-1 was cultured in MRS liquid culture medium at 45℃ and 150 rpm until OD=1. The bacterial suspension was diluted with physiological saline to OD=0.6. Nitrosoguanidine (NTG) at a final concentration of 0.4~0.6 mg / mL was added to the bacterial suspension and incubated at 37℃ for 15-20min. After treatment, it was centrifuged at 5000rpm for 3min, the supernatant was removed, and the MRS liquid culture medium was washed 3 times. The bacterial suspension was gradiently diluted with sterile physiological saline and spread on MRS+CaCO3 solid plates. The plates were placed in a 48℃ incubator for culture until single colonies were formed and their calcium dissolution circles were observed.

[0017] About 300 single colonies with fast colony growth and large calcium dissolution circle were selected from the plate and transferred to 12 deep-well plates containing acid-producing fermentation medium to complete the fermentation test. The culture was carried out at 37°C 150 rpm for 12 hours and 48°C 150 rpm for 10 hours. The total fermentation time was 22 hours. After the fermentation, the lactic acid content of the fermentation liquid was detected, and the single clones with lactic acid production above 170g / L were collected and named Lr-Ms-2 for the third round of mutagenesis screening.

[0018] Lr-Ms-2 was cultured in MRS liquid medium at 45℃ and 150 rpm until OD=1. The bacterial suspension was diluted with physiological saline to OD=0.1 and UV mutagenesis was performed (5 mL of bacterial suspension was evenly spread on a 6 cm sterile plate and irradiated with a 30 W UV lamp at a distance of 20 cm for 45-60 s). The bacterial suspension was graded diluted with sterile physiological saline and spread on MRS+CaCO3 solid plates. The plates were placed in a 48℃ incubator and cultured until single colonies were formed and their calcium dissolution circles were observed.

[0019] About 200 single colonies with fast colony growth and large calcium dissolution circle were selected from the plate and transferred to 12 deep-well plates with acid fermentation medium to complete the fermentation test. The culture was carried out at 37℃ 150 rpm for 12 hours and then at 48℃ 150 rpm for 8 hours. The total fermentation time was 20 hours. After the fermentation, the lactic acid content of the fermentation liquid was detected. The monoclones with lactic acid production above 170g / L were: Lrch1109 lactic acid production 178.4 g / L, Lrch1017 lactic acid production 175.8 g / L, Lrch1114 lactic acid production 173.6 g / L, Lrch1169 lactic acid production 173.6 g / L, and rch1185 lactic acid production 170.2 g / L.

[0020] 2. Shake flask fermentation screening Shake flask fermentation screening: Five mutagenic strains, Lrch1109, Lrch1017, Lrch1114, Lrch1169, and rch1185, were tested in shake flask fermentation. The five strains were inoculated into MRS medium and cultured overnight at 37°C and 150 rpm to obtain OD 600 The seed solution was 12. Then, the seed solution was inoculated at a ratio of 10% (v / v) in 200 mL of acid-producing fermentation medium, and the strain was cultured in a shaking incubator at 37°C and 150 rpm for 12 hours to allow the strain to grow, and then the temperature was raised to 48°C, and the shaking incubator was continued at 150 rpm for 8 hours to obtain the fermentation liquid. The total fermentation time was 20 hours. After the fermentation, the total lactic acid production and the optical purity of L-lactic acid were determined by high performance liquid chromatography. As a result, when fermented at 48°C in a 200 mL shake flask, the lactic acid content in the fermentation liquid obtained by fermenting the Lrch1017 strain for 20 hours was 175.2 g / L, and the optical purity of L-lactic acid was 99.8%.

[0021] Comparative Example 1 The starting strain Lr-ALTHT-DBC was subjected to a shake flask fermentation experiment using the method in Example 2. The strain was cultured at 37°C and 150rpm for 12 hours to allow the strain to grow, then the temperature was raised to 48°C, and the culture was continued at 150rpm for 8 hours to obtain the fermentation broth. The total fermentation time was 20 hours. After the fermentation was completed, the total lactic acid production and the optical purity of L-lactic acid were determined by high performance liquid chromatography. The lactic acid content in the fermentation broth obtained after 20 hours of fermentation was 150.9 g / L, and the optical purity of L-lactic acid was 99.8%.

[0022] 3. Fermentation tank verification Seed preparation: Use MRS medium, activate Lrch1017 strain at 37℃, culture at 37℃, transfer to 1.5L seed shake flask after 14 hours, expand the culture, and control the seed age at 14-16 hours, OD 600 4.5-6.5, inoculation is possible only when pH is around 4.5 Fermentation culture: 30L fermentation tank, liquid volume is 15L. After preparing 15L of fermentation medium, perform actual digestion at 115℃ for 10 min. After controlling the temperature to 37℃, inoculate by flame inoculation, use the flow addition of calcium hydroxide, control pH6.0, and rotate at 150 rpm. After 12h at 37℃, heat to 48℃. Samples are taken every 3h, and lactic acid and residual sugar are detected by biosensor and HPLC. At 19.5h of fermentation, the L-lactic acid yield reached 171.6 g / L, the sugar-acid conversion rate was 97% (g / g), and the optical purity of L-lactic acid was 99.5%.

[0023] Based on rhamnosus Bacillus Lr-ALTHT-DBC, the present invention obtains strain Lrch1017 after combining physical mutagenesis and chemical mutagenesis and multiple rounds of superimposed mutagenesis screening. The strain can efficiently produce high optical purity L-lactic acid, providing an option for industrial production needs.

[0024] 1. Culture medium and reagents MRS+CaCO3 plate: MRS solid medium + 10g / L CaCO3; MRS liquid medium: peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, dipotassium hydrogen phosphate 2g, diammonium citrate 2g, anhydrous sodium acetate 5g, manganese sulfate 0.25g, magnesium sulfate 0.58g, Tween 80 1mL, distilled water 1000mL, pH 6.5, sterilized at 121℃ for 20min; MRS solid medium: add agar to the above MRS liquid medium, with the amount of agar being 18 g relative to 1000 mL of distilled water, adjust the pH to 6.5, and sterilize at 121°C for 20 min; Acidogenic fermentation medium: glucose 180 g / L, yeast extract 10 g / L, sodium acetate 2 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 · 7H 2 O 0.5 g / L, MnSO 4 0.2 g / L, Tween 80 1 ml / L, CaCO 3 90 g / L.

[0025] Fermentation medium: yeast powder 902 30g / L, glucose 230g / L, magnesium sulfate heptahydrate 0.2g / L, sodium acetate 5g / L, triammine citrate 2g / L, dipotassium hydrogen phosphate 2g / L, manganese sulfate 0.05g / L, Tween 80 1ml / L; Neutralizing agent: calcium hydroxide suspension.

[0026] 2. Detection of lactic acid in fermentation broth by HPLC Chromatograph: Agilent Technologies 1260 Infinity II; Detector: RID; Separation column: Aminex HPX-87H Column 300×7.8 mm; Mobile phase: 0.005 M sulfuric acid; Flow rate: 0.5 mL / min; Injection volume: 20 μL.

[0027] The retention time of lactic acid is about 14 min.

[0028] 3. Biosensor detection of glucose and L-lactic acid in fermentation broth Instrument: SBA-40E biosensor; Enzyme membrane: D-glucose enzyme membrane and L-lactate enzyme membrane; Injection volume: 25 μl.

[0029] 4. Detection of optical purity of lactic acid by HPLC Chromatograph: Agilent Technologies 1260 Infinity; Detector: wavelength 254 nm, sensitivity 0.32AUFS; Separation column: MCI GEL-CRS10 W (3u) 4.6 ID × 50mm; Mobile phase: 0.002 M copper sulfate; Flow rate: 0.5 mL / min; Injection volume: 20 μL.

[0030] The sample was diluted to a total lactic acid concentration of 0.5-1 g / L before testing. The retention time of D-lactic acid was about 11 min, and the retention time of L-lactic acid was about 13 min. The optical purity of L-lactic acid was calculated based on its peak area.

[0031] The present invention screens out a production strain that can efficiently produce high optical purity L-lactic acid. Mutation breeding of strains is to change the ability of strains to produce lactic acid from a genetic perspective, and is one of the more thorough solutions to improve the lactic acid production capacity and efficiency of strains. The present invention combines physical mutagenesis with chemical mutagenesis, and uses a multi-round superposition mutagenesis screening method. It can induce changes in the basic unit bases of genetic material or induce variations in chromosome structure under the condition of low-dose injection and light cell damage.

[0032] In the present invention, the starting strain is a recombinant rhamnosus bacillus Lr-ALTHT-DBC (the deposit number is CGMCC No. 19507 and the strain has been disclosed in CN 111500517 A). After physical mutagenesis and chemical mutagenesis are combined and multiple rounds of superimposed mutagenesis screening are performed, the mutagenized strain Lrch1017 is obtained. The fermentation cycle of strain Lrch1017 is within 20 hours, and the L-lactic acid production can reach more than 170 g / L, and the optical purity of L-lactic acid is more than 99%. A production strain for efficiently producing high optical purity L-lactic acid is provided for industrial production of lactic acid.

[0033] Compared with the Chinese invention "A strain of Lactobacillus rhamnosus with high L-lactic acid production and a method for fermenting cassava and sugarcane molasses to produce lactic acid" with application publication number CN102304480A, the present invention's Lactobacillus rhamnosus Lrch1017 ferments at 37°C for 12 hours and then heats up to 48°C for 7.5 hours, with a total fermentation time of 19.5 hours. The L-lactic acid production rate reaches more than 17% (W / V), and the fermentation efficiency reaches 97%. Lactobacillus rhamnosus Lrch1017 is currently the strain with the shortest time to produce L-lactic acid to more than 170g / L, and the optical purity of the L-lactic acid produced by fermentation can reach more than 99.5%, which fully meets the requirements of polymer-grade L-lactic acid.

[0034] The present invention starts from the point of increasing the acid production rate of the fermentation strain and shortening the fermentation cycle, and screens the strain to obtain Lactobacillus rhamnosus ( Lactobacillus rhamnosus )Lrch1017.

[0035] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A strain for efficiently producing high optical purity L-lactic acid, characterized in that: The production strain is Lactobacillus rhamnosus ( Lactobacillus rhamnosus )Lrch1017, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32079.

2. Use of the strain for efficiently producing highly optically pure L-lactic acid according to claim 1 in fermentation preparation of efficiently producing highly optically pure L-lactic acid.

3. The use of the strain for efficiently producing high optical purity L-lactic acid according to claim 2 in fermentation preparation of high optical purity L-lactic acid, characterized in that: Lactobacillus rhamnosus Lrch1017 was inoculated into MRS solid medium and cultured at 35-39°C. After 10-20 h, it was transferred to a seed shake flask for expansion. The seed age was controlled at 14-16 h, and the OD 600 4.5-6.5, pH 4-5, to obtain seed solution; The seed liquid was inoculated into the fermentation medium by flame inoculation, and the pH was controlled at 5.8-6.2 by flow-addition of calcium hydroxide. The fermentation was carried out at a rotation speed of 100-200 rpm and 35-39°C. After 12 hours, the temperature was raised to 48°C and fermented for another 7.5 hours. After 19.5 hours of fermentation, the L-lactic acid production reached 171.6 g / L, the sugar-acid conversion rate was 97% (g / g), and the optical purity of L-lactic acid was 99.5%.

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