A strain of Lactobacillus plantarum and its application in the co-production of fiber and lactic acid from ramie

Abstract

The present invention relates to a Lactobacillus plantarum strain and its use in the co-production of fiber and lactic acid from ramie, belonging to the field of microbial technology. The present invention provides Lactobacillus plantarum strain Y-16, which was deposited with the China Center for Type Culture Collection on October 25, 2024, with a deposit number of CCTCC No. M 20242334. Ramie enzymatic hydrolysate can be used as a raw material to convert soluble sugars therein into lactic acid with high conversion efficiency.

Description

Technical Field

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

[0002] Ramie is a significant source of natural fiber, a distinctive cash crop in my country, and an integral component of the country's agricultural economic development. Ramie fiber is widely used in textiles, rail transportation, aerospace, and medical applications. Ramie nanocellulose can be further applied in the preparation of leading materials such as biomass-based / bionic materials, nano- and micro-composite yarns, and plant-based fiber composites. Procuring high-quality fiber / cellulose is a crucial prerequisite for promoting the development of my country's ramie industry.

[0003] In addition to fiber, ramie bast also contains 25%-35% bound non-cellulosic material, primarily composed of pectin and hemicellulose. Removing this bound non-cellulosic material is essential for obtaining ramie fiber. Bioextraction methods include enzymatic and microbial extraction. Enzymatic extraction is the preferred method for producing stable nanocellulose due to its short extraction time, mild extraction conditions, simple process, and high reproducibility.

[0004] The enzymatic extraction of ramie nanofibers leaves a large amount of soluble sugars in the system, which is typically disposed of as waste, resulting in waste and environmental pollution. Bioconversion can convert these soluble sugars into high-value products, such as lactic acid. However, the enzymatic hydrolysate contains a wide variety of soluble sugars, and industrial lactic acid bacteria, which mostly use corn starch as a carbon source, struggle to fully convert them 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 strain of Lactobacillus plantarum and its application in the co-production of fiber and lactic acid from ramie. The ramie enzymatic hydrolysate can be used as raw material to convert the soluble sugars 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 raw material, which comprises: using ramie bast as raw material, obtaining ramie nanocellulose and an enzymatic hydrolysis solution containing soluble sugars through enzymatic hydrolysis, and adding the Lactobacillus plantarum strain Y-16 according to claim 1 to the enzymatic hydrolysis solution 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 the 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 hydrolysate containing soluble sugars, 15 g / L yeast powder, 5 g / L peptone, 0.5 g / L MgSO4.7H2O, and 10 g / L sterilized CaCO3 to adjust the 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 has screened 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, with a conversion rate of more than 80%, which is simple and easy to operate. The process of the present invention can simultaneously achieve 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] The following description sets forth numerous specific details to facilitate a thorough 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 may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations 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 raw ramie bast was chopped into 1-2cm pieces and crushed into nano-sized particles for later use. The pretreated ramie was placed in a conical flask containing 200mL of citric acid-sodium citrate buffer solution (pH 4.8, 0.05mol / L). Pectinase (enzyme activity 100,000U / g) was then added at varying dosages of 1g, 2g, 3g, 4g, and 5g. Enzymatic hydrolysis was performed at 50°C and 150rpm for 48h. The hydrolyzed solution was then vacuum filtered through a nano-sized filter membrane, and the filter residue was washed and placed in a 60°C oven until constant weight was achieved. The enzymatic weight loss was calculated, as 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 xylanase addition in the enzymatic preparation process of ramie nanocellulose

[0028] 10g of raw ramie bast was chopped into 1-2cm pieces and crushed into nano-sized particles for later use. The pretreated ramie was placed in a conical flask containing 200mL of citric acid-sodium citrate buffer solution (pH 4.8, 0.05mol / L). Pectin-xylanase (enzyme activity 50,000U / g) was then added at varying enzyme dosages of 1g, 2g, 3g, 4g, and 5g. Enzymatic hydrolysis was performed at 50°C and 150rpm for 48h. The hydrolyzate was then vacuum filtered through a nano-sized filter membrane, and the filter residue was washed and placed in a 60°C oven until constant weight was achieved. The enzymatic weight loss was calculated, as shown in Table 2.

[0029] Table 2 Optimization of β-mannanase addition amount

[0030]

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

[0032] 10g of raw ramie bast was chopped into 1-2cm pieces and crushed into nano-sized particles for later use. The pretreated ramie was placed in a conical flask containing 200mL of citric acid-sodium citrate buffer solution (pH 4.8, 0.05mol / L). β-mannanase (enzyme activity 60,000U / g) was then added at varying dosages of 1g, 2g, 3g, 4g, and 5g. Enzymatic hydrolysis was performed at 50°C and 150rpm for 48h. The hydrolyzed solution was then vacuum filtered through a nano-sized filter membrane, and the filter residue was washed and placed in a 60°C oven until constant weight was achieved. The enzymatic weight loss was calculated, as shown in Table 3.

[0033] Table 3 Optimization of β-glucanase addition amount

[0034]

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

[0036] 10g of raw ramie bast was chopped into 1-2cm pieces and crushed into nano-sized particles for later use. The pretreated ramie was placed in a conical flask containing 200mL of citric acid-sodium citrate buffer solution (pH=4.8, 0.05mol / L). β-glucanase (enzyme activity 20,000U / g) was then added at varying dosages of 1g, 2g, 3g, 4g, and 5g. Enzymatic hydrolysis was performed at 50°C and 150rpm for 48h. The hydrolyzed solution was then vacuum filtered through a nano-sized filter membrane, and the filter residue was washed and placed in a 60°C oven until constant weight was achieved. The enzymatic weight loss was calculated, as shown in Table 4.

[0037] Table 4 Optimization of xylanase addition amount

[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 raw ramie bast was chopped into 1-2cm pieces and crushed into nano-sized particles for later use. The pretreated ramie was placed in a conical flask containing 200mL of citric acid-sodium citrate buffer solution (pH 4.8, 0.05mol / L). Then, 4g of pectinase, 4g of β-mannanase, 2g of β-glucanase, and 4g of xylanase were added. Enzymatic hydrolysis was carried out at 50°C and 150rpm for 48h. The hydrolyzate was then vacuum filtered through a nano-sized filter membrane, and the filtrate was collected to obtain a hydrolyzate containing soluble sugars. The filter residue after enzymatic hydrolysis was washed and placed in a 60°C oven until constant weight was obtained to obtain crude ramie nanocellulose.

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

[0042] Samples were collected from pickled radish juice, pickles, and kimchi. The collected samples were placed in clean, sealed bags and stored at room temperature. Lactic acid-producing strains were promptly isolated in the laboratory. The samples were diluted with sterile water and spread onto CaCO3-bromocresol purple screening medium (30 g / L glucose, 5 g / L yeast extract, 4 g / L peptone, 0.5 g / L MgSO4.7H2O, 10 g / L CaCO3, 20 g / L agar, 1 mL of 1.6% bromocresol purple solution, pH 7.0). The medium was incubated at 37°C for 48 hours. Colonies with distinct yellow transparent zones and distinct colony morphologies were selected for further purification and identification.

[0043] The isolated lactic acid-producing strain was activated in MRS broth and then diluted and plated onto CaCO₃-bromocresol purple selection medium. The resulting colonies were picked and further propagated in MRS broth at 37°C, shaker speed of 180 rpm, and incubation time of 24 hours. The bacteria in the culture medium were purified using a gradient dilution and plating method until multiple single colonies with consistent morphology were obtained on CaCO₃-bromocresol purple solid plates.

[0044] MRS broth medium: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, CH3COONa·3H2O 5 g / L, diammonium hydrogen citrate 20 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, Tween-80 1 mL / L, glucose 20 g / L.

[0045] Lactic acid-producing bacteria from test tube slants or glycerol tubes were activated in MRS broth at 37°C for approximately 24 hours on a shaker at 180 rpm. The activated bacteria were transferred to CaCO₃-bromocresol purple solid medium by streaking and incubated in the dark at 37°C. After 24 hours of incubation, three single colonies were randomly selected from the plate and plated in MRS broth. Genomic DNA was extracted the next day according to the instructions of a bacterial genomic DNA kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China).

[0046] CaCO3-bromocresol purple solid medium: 30 g / L glucose, 5 g / L yeast extract, 4 g / L peptone, 0.5 g / L MgSO4·7H2O, 10 g / L CaCO3, 1 mL / L 1.6% bromocresol purple solution, 20 g / L agar, pH 7.0. The 1.6% bromocresol purple solution was prepared by dissolving 1.6 g bromocresol purple in 100 mL anhydrous ethanol.

[0047] Lactic acid-producing bacteria were identified using 16S rRNA gene sequence analysis. Genomic DNA was used as a template to amplify the 16S rRNA gene sequence using universal primers 27F and 1492R. A 50 μL PCR amplification system was established: 34 μL sterile double-distilled water, 5 μL 10× PCR buffer, 4.5 μL dNTPs, 2 μL 27F primer, 2 μL 1492R primer, 1.5 μL bacterial genomic template, and 1 μL rTaq DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China). PCR reactions were performed using a Bio-Rad PCR instrument with the following protocol: 94°C denaturation for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s; extension at 72°C for 10 min; and a stop cycle at 12°C for storage. PCR amplification results were verified by 1% agarose gel electrophoresis and visualized 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] ① Initial screening

[0050] After obtaining a single colony culture of a lactic acid-producing strain according to the activation method, 5 μL of the bacterial culture of each strain was spotted on a CaCO3-bromocresol purple solid medium, with three spots spotted on each plate for repetition. The plates were placed in a 37°C incubator and cultured 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. 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 ratios of transparent zones to colony zones.

[0055] ②Rescreening

[0056] The five strains screened were activated in 10 mL of MRS broth and cultured at 37°C for 24 h. 2% of the culture was then transferred to 200 mL of fermentation medium and cultured at 37°C and 180 rpm. After 48 h of fermentation, the fermentation broth was poured into a 50 mL centrifuge tube and centrifuged at 8000 rpm for 10 min to remove the bacteria and calcium carbonate. The supernatant was then transferred to a new 50 mL centrifuge tube. The centrifuged fermentation broth was then acid-hydrolyzed with an equal volume of 0.5 mol / L concentrated sulfuric acid solution. The supernatant was then centrifuged at 8000 rpm for 10 min to remove the calcium carbonate. The supernatant was then stored at -20°C. 16S rRNA gene sequence analysis was used to identify the lactic acid-producing bacteria. 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 from the initial screening were inoculated into a fermentation medium and fermented for 48 hours to produce lactic acid. The fermentation medium contained an enzymatic hydrolysate containing soluble sugars at a final concentration of 65-70 g / L, 15 g / L yeast extract, 5 g / L peptone, and 0.5 g / L MgSO4.7H2O. 10 g / L sterilized CaCO3 was also added to adjust the pH. Furthermore, a commercially available Lactobacillus plantarum BNCC194165 (commonly used in research and teaching) was used as a control to produce lactic acid. It was found that L. plantarum BNCC194165 exhibited poor growth and a lower bacterial concentration in the fermentation broth when using an enzymatic hydrolysate containing soluble sugars as a carbon source, compared to the five strains obtained from the initial screening. This may be due to the strain's low utilization of the enzymatic hydrolysate containing soluble sugars.

[0060] The lactic acid content in the fermentation broth was determined by high-performance liquid chromatography, and the results are shown in Table 7. The chromatographic column was a ZORBAX SB-Aq, 150×4.6 (5 μm) column (Merck, USA), with a flow rate of 0.5 mL / min, UV detection at 210 nm, an injection volume of 10 μL, and a mobile phase consisting of water (0.02 mol / L KH2PO4):acetonitrile = 99.5:0.5. The 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, ultimately selected from radish juice, exhibited excellent lactic acid conversion efficiency, using the soluble sugars in the ramie hydrolysate as a carbon source for fermentation to produce lactic acid. The other selected strains, however, exhibited lower conversion rates, likely because they were unable to convert various sugar components in the hydrolysate into lactic acid.

[0064] like Figure 1 As shown, in CaCO₃-bromocresol purple solid medium (microaerophilic culture at 37°C for 48 hours), Lactobacillus plantarum Y-16 forms yellow, round, raised colonies with neat edges and a smooth, moist surface. A distinct CaCO₃ dissolution zone with sharp edges is visible around the colonies, while the background medium remains dark purple. The bromocresol purple color reaction, resulting from acid production, indicates a local drop in pH below 5.0.

[0065] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for co-producing fiber and lactic acid from ramie, characterized in that: Ramie bast is used as raw material, and ramie nanocellulose and an enzymatic hydrolysate containing soluble sugar are obtained through enzymatic hydrolysis. Lactobacillus plantarum is added to the enzymatic hydrolysate containing soluble sugar to ferment and produce lactic acid. 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; the enzymatic hydrolysis specifically comprises: using pectinase, β-mannanase, β-glucanase and xylanase as enzyme preparations; the dosage ratio of the pectinase, β-mannanase, β-glucanase and xylanase is 2:2:1:

2.

2. The method according to claim 1, wherein The concentration of soluble sugar in the enzymatic hydrolyzate containing soluble sugar is 65-70 g / L.

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