Lactobacillus plantarum and application of lactobacillus plantarum in co-production of fibers and lactic acid from ramie

Abstract

The invention relates to lactobacillus plantarum and application thereof in co-production of fibers and lactic acid from ramie, and belongs to the technical field of microorganisms. The invention provides a lactobacillus plantarum strain Y-16 which is preserved in the China Center for Type Culture Collection on October 25, 2024, and the preservation number of the lactobacillus plantarum strain Y-16 is CCTCC No: M 20242334. The ramie enzymatic hydrolysate can be used as a raw material, soluble sugar in the ramie enzymatic hydrolysate is converted into lactic acid, and the conversion efficiency is high.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to a plant lactobacillus strain and application thereof in the co-production of fiber and lactic acid from ramie. Background Art

[0002] Ramie is an important source of natural fiber, a unique economic crop in my country, and an indispensable part of my country's agricultural economic development. Ramie fiber is widely used in textiles, rail transportation, aerospace, and medical fields. Ramie nanocellulose can be further used in the preparation of leading materials such as biomass-based / bionic materials, nano-micro composite yarns, and plant fiber composite materials. Obtaining high-quality fiber / cellulose is an important prerequisite for promoting the development of my country's ramie industry.

[0003] In addition to fiber, ramie bast also contains 25%-35% bonded non-cellulose substances, which are mainly composed of pectin and hemicellulose. Stripping bonded non-cellulose substances is the only way to obtain ramie fiber. Biological extraction is divided into two ways: enzyme extraction and microbial extraction. Enzymatic extraction has the characteristics of short time consumption, mild extraction conditions, simple process and good repeatability. It is the preferred method for preparing nanocellulose with stable performance.

[0004] The system after enzymatic extraction of ramie nanofibers contains a large amount of soluble sugars, which are usually treated as waste liquid, causing waste and environmental pollution. Biotransformation can transform these soluble sugars into high-value products, such as lactic acid. However, there are many types of soluble sugars in the enzymatic hydrolyzate, and most industrial lactic acid bacteria use corn starch as a carbon source, which is difficult to completely convert into lactic acid.

[0005] In view of this, it is necessary to screen out a lactic acid bacterium that can convert lactic acid using multiple types of sugars as raw materials. Summary of the invention

[0006] Based on the above technical problems existing in the prior art, the present invention provides a plant lactobacillus and its application in the co-production of fiber and lactic acid from ramie. Ramie enzymatic hydrolysate can be used as raw material to convert the soluble sugar therein into lactic acid with high conversion efficiency.

[0007] The invention screens out a Lactobacillus plantarum strain from sour radish juice and names it as Y-16.

[0008] Biological deposit information

[0009] It was deposited in the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072) on October 25, 2024, with the deposit number: CCTCC No: M 20242334.

[0010] The present invention also provides a method for co-producing fiber and lactic acid using ramie as a raw material, which comprises: using ramie bast as a raw material, obtaining ramie nanocellulose and an enzymatic hydrolyzate containing soluble sugars by enzymatic hydrolysis, and adding the plant lactobacillus plantarum strain Y-16 as claimed in claim 1 into the enzymatic hydrolyzate containing soluble sugars to ferment and produce lactic acid.

[0011] Preferably, the enzymatic hydrolysis comprises: using pectinase, β-mannanase, β-glucanase and xylanase as enzyme preparations.

[0012] Preferably, the usage ratio of pectinase, β-mannanase, β-glucanase and xylanase is 2:2:1:2.

[0013] Preferably, the soluble sugars include: ribose, mannose, glucuronic acid, rhamnose, glucose, galacturonic acid, xylose, galactose, fucose and arabinose.

[0014] Preferably, the conversion rate of lactic acid is greater than 80%.

[0015] Preferably, the fermentation to produce lactic acid comprises: an enzymatic hydrolyzate containing soluble sugar, 15 g / L yeast powder, 5 g / L peptone, MgSO 4 .7H 2 O 0.5g / L, and 10g / L sterilized CaCO 3 Adjust pH.

[0016] Preferably, the concentration of soluble sugar in the enzymatic hydrolyzate containing soluble sugar is 65-70 g / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention screened the strains that can directly use the enzymatic hydrolysate after ramie nanocellulose extraction for fermentation, and the concentration of lactic acid produced can reach 30.51g / L, and the conversion rate can reach more than 80%, which is simple and easy. The process of the present invention can simultaneously realize the extraction of ramie nanocellulose and the biosynthesis of lactic acid, and can maximize the comprehensive utilization of ramie bast, and convert the waste enzymatic hydrolysate after ramie nanocellulose extraction into high-value lactic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the appearance of the Y-16 strain.

[0020] Figure 2 The present invention is a flow chart of the method for co-producing fiber and lactic acid using ramie as raw material. DETAILED DESCRIPTION

[0021] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0022] Unless otherwise specified, the reagents and instruments used in the present invention are common commercial products.

[0023] Experimental Example 1 Optimization of the amount of pectinase added in the enzymatic preparation process of ramie nanocellulose

[0024] 10g of ramie bast raw hemp was cut into pieces of 1-2cm and crushed into nano-scale particles for later use. The pre-treated hemp was placed in a conical flask containing 200mL, pH=4.8, 0.05mol / L citric acid-sodium citrate buffer solution, and then pectinase (enzyme activity 100000U / g) was added. The enzyme addition amount was set to 1g, 2g, 3g, 4g and 5g, and enzymolysis was performed at 50°C and 150rpm for 48h. The enzymolysis solution was then vacuum filtered with a nano-scale filter membrane, and the filter residue was washed and placed in a 60°C oven to dry until the mass was constant. The enzymolysis weight loss rate was calculated, and the results are shown in Table 1.

[0025] Table 1 Optimization of pectinase addition

[0026] Pectinase addition amount (g / L) 5 10 15 20 25 Weight loss rate 19.70％ 23.30％ 23.10％ 24.30％ 24.25％ Reducing sugar content (g / L) 13.11 29.94 28.00 44.63 42.23

[0027] Experimental Example 2 Optimization of the amount of xylanase added in the enzymatic preparation process of ramie nanocellulose

[0028] 10g of ramie bast raw hemp was cut into pieces of 1-2cm and crushed into nano-scale particles for later use. The pre-treated hemp was placed in a conical flask containing 200mL, pH=4.8, 0.05mol / L citric acid-sodium citrate buffer solution, followed by the addition of pectin xylanase (enzyme activity 50000U / g), the enzyme addition amount was set to 1g, 2g, 3g, 4g and 5g, and the enzymolysis was carried out at 50°C and 150rpm for 48h. The enzymolysis solution was then vacuum filtered with a nano-scale filter membrane, the filter residue was washed and placed in a 60°C oven, and dried to constant weight, and the enzymolysis weight loss rate was calculated, and the results are shown in Table 2.

[0029] Table 2 Optimization of β-mannanase addition

[0030]

[0031] Experimental Example 3 Optimization of the amount of β-mannanase added in the enzymatic preparation process of ramie nanocellulose

[0032] 10g of ramie bast raw hemp was cut into pieces of 1-2cm and crushed into nano-scale particles for later use. The pretreated hemp was placed in a conical flask containing 200mL, pH=4.8, 0.05mol / L citric acid-sodium citrate buffer solution, and then β-mannanase (enzyme activity 60000U / g) was added, and the enzyme addition amount was set to 1g, 2g, 3g, 4g and 5g, and enzymolysis was carried out at 50°C and 150rpm for 48h. The enzymolysis solution was then vacuum filtered with a nano-scale filter membrane, and the filter residue was washed and placed in a 60°C oven to dry to constant weight, and the enzymolysis weight loss rate was calculated. The results are shown in Table 3.

[0033] Table 3 Optimization of β-glucanase addition

[0034]

[0035] Experimental Example 4 Optimization of the amount of β-glucanase added in the enzymatic preparation process of ramie nanocellulose

[0036] 10g of ramie bast raw hemp was cut into pieces of 1-2cm and crushed into nano-scale particles for later use. The pre-treated hemp was placed in a conical flask containing 200mL, pH=4.8, 0.05mol / L citric acid-sodium citrate buffer solution, and then β-glucanase (enzyme activity 20000U / g) was added, and the enzyme addition amount was set to 1g, 2g, 3g, 4g and 5g, and enzymolysis was performed at 50°C and 150rpm for 48h. The enzymolysis solution was then vacuum filtered with a nano-scale filter membrane, and the filter residue was washed and placed in a 60°C oven to dry until the mass was constant, and the enzymolysis weight loss rate was calculated. The results are shown in Table 4.

[0037] Table 4 Optimization of xylanase addition

[0038] Xylanase addition amount (g / L) 5 10 15 20 25 Weight loss rate 10.00％ 11.60％ 17.60％ 19.20％ 19.00％ Reducing sugar content (g / L) 2.02 4.66 10.26 13.19 13.16

[0039] Example 1 Preparation of ramie nanocellulose and acquisition of soluble sugar

[0040] like Figure 2 As shown, 10g of ramie bast raw hemp is cut into pieces of 1-2cm and crushed into nano-scale particles for later use. The pretreated hemp is placed in a conical flask containing 200mL, pH=4.8, 0.05mol / L citric acid-sodium citrate buffer solution, followed by the addition of 4g pectinase, 4g β-mannanase, 2g β-glucanase, and 4g xylanase, and enzymolysis is performed at 50°C and 150rpm for 48h. The enzymolysis solution is then vacuum filtered with a nano-scale filter membrane, and the filtrate is collected to obtain an enzymolysis solution containing soluble sugars; the filter residue after enzymolysis is washed and placed in a 60°C oven and dried to constant weight to obtain ramie nanocellulose crude product.

[0041] Experimental Example 5 Isolation of lactic acid producing strains

[0042] Take samples from pickled radish juice, pickles and kimchi, put them in clean sealed bags, store them at room temperature, and isolate lactic acid-producing strains in the laboratory in time. Dilute the samples with sterile water and apply them on CaCO 3 -bromocresol purple screening medium (glucose 30g / L, yeast powder 5g / L, peptone 4g / L, MgSO 4 .7H 2 O 0.5g / L, CaCO 3 10 g / L, agar 20 g / L, 1.6% bromocresol purple solution 1 mL, pH 7.0), incubated at 37°C incubator for 48 h, and colonies with obvious yellow transparent circles and different colony morphologies were selected for further purification and identification.

[0043] The isolated lactic acid-producing strains were activated in MRS broth medium and then diluted and plated on CaCO 3 -bromocresol purple screening medium, the grown colonies were picked up and further cultured in MRS broth medium, the culture temperature was 37°C, the shaking speed was 180r / min, and the culture time was 24h. The bacteria in the culture medium were purified by gradient dilution and coating until they were in CaCO 3 -Multiple single colonies with consistent colony morphology were obtained on the bromocresol purple solid plate.

[0044] MRS broth medium: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, K 2 HPO 4 2 g / L, CH 3 COONa·3H 2 O 5g / L, diamine hydrogen citrate 20g / L, MgSO 4 7H 2 O 0.2g / L, MnSO 4 ·4H 2 O 0.05g / L, Tween-80 1mL / L, glucose 20g / L.

[0045] The lactic acid-producing bacteria in the test tube slant or glycerol storage tube were activated in MRS broth medium at 37°C for about 24 hours and at a shaking speed of 180 r / min. The activated bacteria were transferred to CaCO by streaking. 3 -bromocresol purple solid medium, inverted and incubated in the dark at 37°C. After 24 h of incubation, three single colonies were randomly picked from the plate and placed in MRS broth medium. The next day, genomic DNA was extracted according to the instructions of the bacterial genomic kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China).

[0046] CaCO3 - Bromocresol purple solid medium: glucose 30g / L, yeast powder 5g / L, peptone 4g / L, MgSO 4 7H 2 O0.5g / L, CaCO 3 10 g / L, 1.6% bromocresol purple solution 1 mL / L, agar 20 g / L, pH 7.0, wherein the preparation of 1.6% bromocresol purple solution is as follows: 1.6 g bromocresol purple is dissolved in 100 mL anhydrous ethanol.

[0047] The 16S rRNA gene sequence analysis method was used to identify lactic acid bacteria. The genomic DNA was used as a template to amplify the 16S rRNA gene sequence in the genome using universal primers 27F and 1492R. A 50μL PCR amplification system was established: 34μL sterile double distilled water, 5μL 10× PCR buffer, 4.5μL dNTP, 2μL 27F primer, 2μL 1492R primer, 1.5μL bacterial genome template, 1μL rTaqDNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). The PCR reaction was performed using a Bio-Rad PCR instrument, and the reaction program was as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 90s, 30 cycles; 72℃ extension for 10min; 12℃ stop program and save. The PCR amplification results were detected by 1% agarose gel electrophoresis, and the PCR amplification results were observed and photographed using a gel imaging system. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results of the strains were compared using the BLAST tool on NCBI.

[0048] Example 2 Screening of lactic acid producing strain Lactobacillus plantarum Y-16

[0049] ①Preliminary screening

[0050] After obtaining the single colony culture solution of lactic acid-producing strains according to the activation method, 5 μL of bacterial solution of different strains was inoculated on CaCO 3 -Bromcresol purple solid culture medium, 3 spots were inoculated on each plate for replication, and the plates were placed in a 37°C incubator for 48 h. The diameters of the colony circle and the transparent circle were then measured, and the ratio of the transparent circle to the colony circle diameter was calculated. The results are shown in Table 5. The strains with a larger ratio of the transparent circle to the colony circle diameter were selected for rescreening.

[0051] Table 5 Diameters of colony circles and transparent circles of different acid-producing strains

[0052]

[0053]

[0054] The preliminary screening results showed that strains Y-23, Y-16, Y-10, Y-20 and Y-6 were the five bacterial strains with the largest ratio of transparent zone to colony circle.

[0055] ②Rescreening

[0056] The five strains obtained from the initial screening were activated in 10 mL MRS broth medium, cultured at 37 ° C for 24 h, and then transferred to 200 mL fermentation medium, cultured at 37 ° C, 180 r / min, and fermented for 48 h. The fermentation broth was poured into a 50 mL centrifuge tube, centrifuged at 8000 rpm for 10 min to remove the bacteria and calcium carbonate, and then the supernatant was transferred to a new 50 mL centrifuge tube. The fermentation broth after centrifugation was added with an equal volume of 0.5 mol / L concentrated sulfuric acid solution for acid hydrolysis, centrifuged at 8000 rpm for 10 min to remove calcium carbonate, and the supernatant was stored at -20 ° C. The lactic acid-producing bacteria were identified by 16S rRNA gene sequence analysis, and the results are shown in Table 6.

[0057] Table 6 16S rRNA gene sequence similarity of lactic acid bacteria

[0058]

[0059] The five strains obtained in the initial screening were inoculated into the fermentation medium and fermented for 48 hours to produce lactic acid. The fermentation medium: the final concentration of the enzymatic hydrolyzate containing soluble sugar was 65-70g / L, yeast powder was 15g / L, peptone was 5g / L, MgSO 4 .7H 2 O 0.5g / L, and 10g / L sterilized CaCO 3 Adjust pH. In addition, commercially available Lactobacillus plantarum BNCC194165 (this strain is commonly used in research and teaching) was used as a control to carry out lactic acid fermentation. It was found that when L. plantarum BNCC194165 used the enzymolysis solution containing soluble sugar as the carbon source, the growth of L. plantarum BNCC194165 was poorer than that of the five strains obtained in the initial screening, and the bacterial concentration in the fermentation liquid was lower. This may be related to the low utilization rate of the enzymolysis solution containing soluble sugar by this strain.

[0060] The content of lactic acid in the fermentation broth was determined by high performance liquid chromatography, and the results are shown in Table 7. The chromatographic column was ZORBAX SB-Aq, 150×4.6 (5 μm) from Merck, USA, with a flow rate of 0.5 mL / min, a UV detection wavelength of 210 nm, an injection volume of 10 μL, and a mobile phase of water (0.02 mol / L KH 2 PO 4 ):acetonitrile = 99.5:0.5, column temperature was 30°C.

[0061] Table 7 Comparison of lactic acid production capacity

[0062]

[0063] As shown in Table 7, the Y-16 strain finally screened from the radish juice has a good lactic acid conversion effect, and can use the soluble sugar in the ramie hydrolysate as a carbon source to ferment and produce lactic acid. The conversion rate of other screened strains is not high, which may be because the various sugar components in the hydrolysate cannot be converted into lactic acid by these strains.

[0064] like Figure 1 As shown, in CaCO 3 -In bromocresol purple solid medium (37℃ microaerobic culture for 48 hours), Lactobacillus plantarum Y-16 formed yellow round raised colonies with neat edges and smooth and moist surface. Obvious CaCO 3 The dissolution zone has sharp edges and the background medium remains dark purple. The bromocresol purple color reaction caused by acid production indicates that the local pH drops below 5.0.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Lactobacillus plantarum strain Y-16, deposited in the China Center for Type Culture Collection on October 25, 2024, with the deposit number: CCTCC No: M 20242334.

2. A method for co-producing fiber and lactic acid from ramie, characterized in that: Ramie bast is used as a raw material, ramie nanocellulose and an enzymatic hydrolyzate containing soluble sugar are obtained through enzymatic hydrolysis, and the Lactobacillus plantarum strain Y-16 as claimed in claim 1 is added to the enzymatic hydrolyzate containing soluble sugar to ferment and produce lactic acid.

3. The method according to claim 2, characterized in that The enzymolysis specifically comprises: using pectinase, β-mannanase, β-glucanase and xylanase as enzyme preparations.

4. The method according to claim 3, characterized in that The dosage ratio of the pectinase, beta-mannanase, beta-glucanase and xylanase is 2:2:1:

2.

5. The method according to claim 2, characterized in that: The soluble sugars include: ribose, mannose, glucuronic acid, rhamnose, glucose, galacturonic acid, xylose, galactose, fucose and arabinose.

6. The method according to claim 2, characterized in that The conversion rate of lactic acid is greater than 80%.

7. The method according to claim 2, characterized in that The fermentation process for producing lactic acid comprises: an enzymatic hydrolyzate containing soluble sugar, 15 g / L yeast powder, 5 g / L peptone, 0.5 g / L MgSO4.7H2O, and 10 g / L sterilized CaCO3 is added to adjust pH.

8. The method according to claim 7, characterized in that The concentration of soluble sugar in the enzymatic hydrolyzate containing soluble sugar is 65-70 g / L.

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