Strain for producing mannitol and method for producing mannitol by using strain

Abstract

The invention provides a strain for producing mannitol and a method for producing mannitol by using the strain, and belongs to the technical field of food biology. According to the present invention, the fermentation lactobacillus fermentum SK50.004 is screened from the pickled vegetables, and the strain is used for the fermentation production of the mannitol; by optimizing the carbon source composition and inorganic salt components in the fermentation medium, the yield of mannitol produced by fermentation of the strain at 37-40 DEG C under the conditions that the concentration of CuSO45H2O is 10 mg / L and the total sugar concentration is 265 g / L (glucose is 80 g / L and fructose is 185 g / L) reaches 160 g / L, the maximum productivity is 4.21 g / L / h, byproducts in the fermentation process are reduced, the production efficiency is remarkably improved, and the production cost is reduced. And a reliable technical support is provided for industrial efficient production of mannitol.

Description

Technical Field

[0001] This invention relates to a mannitol-producing strain and a method for producing mannitol, belonging to the field of food biotechnology. Background Technology

[0002] Mannitol (C6H) 14 Mannitol (O6) is a hexose sugar alcohol that crystallizes as colorless or white needle-like or rhomboid crystals, possessing a refreshing sweet taste and low hygroscopicity. Due to its high safety and sweetness (approximately 50%–70% that of sucrose), it is widely used in the food, pharmaceutical, and chemical industries, such as as a sweetener, diuretic, food additive, and excipient in pharmaceutical preparations. In recent years, with the surge in demand for functional foods and low-sugar products, the global market size of mannitol has maintained an average annual growth rate of 6%–8%. However, traditional natural extraction and chemical synthesis processes are limited by unstable raw material supply, high costs, and drawbacks such as high temperature and pressure, high energy consumption, and the production of numerous byproducts, hindering the sustainability of its large-scale production.

[0003] Biosynthesis is considered the most promising approach for the green production of mannitol due to its mild process conditions, low by-product generation, and wide availability of raw materials (renewable carbon sources such as glucose and fructose). Currently, microbial fermentation and enzyme catalysis are the two major biosynthetic strategies. Enzyme catalysis achieves efficient conversion through multi-enzyme coupling or whole-cell catalysis, but it still faces challenges in industrialization, such as cofactor regeneration and enzyme stability. In contrast, microbial fermentation, utilizing lactic acid bacteria and yeast to metabolize substrates into mannitol under mild conditions, offers advantages such as high metabolic specificity, high conversion rate, and easy product separation, and has become a research hotspot.

[0004] Currently, the microorganisms used for mannitol fermentation mainly include bacteria, yeasts, and fungi. Bacterial fermentation for mannitol production is primarily carried out by lactic acid bacteria. Based on different hexose metabolic pathways, lactic acid bacteria fermentation is divided into homolactic fermentation and heterolactic fermentation. Both can synthesize mannitol, but heterolactic fermentation has a stronger mannitol synthesis capacity than homolactic fermentation, but it also suffers from lower volumetric productivity. Using yeast to produce mannitol generally results in lower space-time yields than heterolactic fermentation, and there is also the issue of mannitol reuse during the fermentation process. Compared to bacteria and yeast, mannitol production using filamentous fungi has a longer cycle time, generally lower yields, and insufficient industrial competitiveness. To improve the efficiency of industrial-scale mannitol production, screening high-yielding strains and optimizing fermentation conditions are key to mannitol fermentation production. This invention aims to promote the efficient and green production of mannitol by screening strains and optimizing the fermentation process. Summary of the Invention

[0005] The purpose of this invention is to provide a novel fermentation-promoting Lactobacillus (Lactobacillus mucinus) Limosiactobacillus fermentumSK50.004 was deposited at the China Center for Type Culture Collection on November 19, 2025, with accession number CCTCC NO: M20252591.

[0006] The present invention also provides a microbial preparation containing the aforementioned *Lactobacillus fermentans* SK50.004.

[0007] In one embodiment, the number of *Lactobacillus fermentans* SK50.004 in the microbial preparation is ≥1×10⁻⁶. 7 CFU / g.

[0008] The present invention also provides a method for producing mannitol using the fermented mucus lactis SK50.004.

[0009] In one embodiment, the method includes seed culture of the fermenting Lactobacillus mucinus SK50.004 and fermentation to produce mannitol in a medium containing glucose and fructose.

[0010] In one embodiment, the culture medium contains Cu 2+ .

[0011] In one embodiment, the CuSO4•5H2O in the culture medium is ≤20 mg / L.

[0012] In one embodiment, the glucose content in the culture medium is 60-80 g / L, and the fructose content is 135-185 g / L.

[0013] In one embodiment, the fermentation is carried out by static fermentation at 30°C to 40°C for 24 to 72 hours.

[0014] In one embodiment, the seed culture is carried out in MRS medium at 35-40°C for 8-14 h.

[0015] In one implementation, the method specifically comprises: (1) Seed culture: In MRS medium, static culture at 37℃ for 8-14 h; (2) Fermentation culture: Ferment in fermentation medium at 40±1℃ for 24~48 h.

[0016] In one embodiment, the fermentation medium contains: glucose 50-100 g / L, fructose 100-200 g / L, peptone 1-10 g / L, yeast extract 1-20 g / L, MgSO4•7H2O 0-0.5 g / L, K2HPO4 0.1-2.0 g / L, MnSO4•H2O 0.01-0.1 g / L, CuSO4•5H2O 0-0.1 g / L, and pH 3.5-6.5.

[0017] The present invention also provides the use of fermenting *Lactobacillus mucinus* or the method thereof in the preparation of mannitol or mannitol-containing products.

[0018] Beneficial effects: This invention relates to a fermenting mucilage lactobacillus strain screened from kimchi and naturally selected through high sugar content. Limosilactobacillus fermentum SK50.004. Under anaerobic or slightly aerobic conditions, at pH 5.0, a temperature of 30℃~40℃, a CuSO4•5H2O concentration of 10 mg / L, and a total sugar concentration of 265 g / L (80 g / L glucose and 185 g / L fructose), this strain achieved a mannitol yield of 160 g / L after 38 h of fermentation, with a space-time production efficiency of 4.19 g / L / h. Simultaneously, this strain produced few byproducts during fermentation, with lactic acid and acetic acid yields of 14.24 g / L and 5.00 g / L, respectively.

[0019] Preservation of biological materials A mannitol-producing strain, classified as *Lactobacillus fermentatus* (… Limosilactobacillus leaven SK50.004 was deposited on November 19, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252591. Attached Figure Description

[0020] Figure 1 The results of BLAST alignment of the 16S rDNA sequence of strain SK50.004 are shown.

[0021] Figure 2 strains in a 3L fermenter Limosilactobacillus fermentum Fermentation culture of SK50.004 under the conditions of glucose 50 g / L and fructose 100 g / L.

[0022] Figure 3 For different Cu² + strains at concentrations Limosilactobacillus fermentum Shake-flask fermentation culture of SK50.004.

[0023] Figure 4 strains in a 3L fermenter Limosilactobacillus fermentum Fermentation culture of SK50.004 under the conditions of glucose 50 g / L, fructose 100 g / L, and CuSO4·5H2O 10 mg / L.

[0024] Figure 5 For 3L fermentation Limosilactobacillus fermentum Fermentation culture of SK50.004 at different sugar concentrations. (A) Total sugar concentration of 195 g / L (glucose to fructose mass ratio of 2.25); (B) Total sugar concentration of 220 g / L (glucose to fructose mass ratio of 2.14); (C) Total sugar concentration of 265 g / L (glucose to fructose mass ratio of 2.31).

[0025] Figure 6 The effects of different metal ions on the growth of the strain and the fermentation production of mannitol. Detailed Implementation

[0026] The following is fermenting *Lactobacillus mucinus* (… Limosilactobacillus fermentum The examples provided illustrate the fermentation production of glycine using SK50.004, but the scope of this invention is not limited to the listed examples. Various modifications can be made to implement the invention without altering its essential points. Furthermore, the scope of this invention extends to equivalent ranges.

[0027] The culture media involved in the following examples are as follows: MRS liquid culture medium: peptone 10 g / L, yeast extract 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 1 mL / L, pH 6.5, sterilized at 118℃ for 20 min. MRS solid medium: Add 15 g / L agar to the MRS liquid medium.

[0028] Fermentation medium: glucose 50 g / L, fructose 100 g / L, peptone 5 g / L, yeast extract 10 g / L, MgSO4•7H2O 0.4 g / L, K2HPO4 2.0 g / L, MnSO4•H2O 0.02 g / L, pH 5.0.

[0029] Methods for determining glucose, fructose, and mannitol: HPLC was used for determination: 1 mL of fermentation broth was boiled at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm membrane. The amount of mannitol produced and the amount of glucose and fructose consumed were determined by HPLC. HPLC detection conditions: differential refractive index detector; Sugar-Pak I column (Waters, USA), column temperature 85℃; mobile phase 50 mg / L EDTA•2Ca aqueous solution, flow rate 0.4 mL / min; injection volume 10 μL.

[0030] Example 1: Limosilactobacillus fermentum Separation, screening and identification of SK50.004 A certain amount of sauerkraut sample was mixed with an appropriate amount of MRS liquid medium and incubated at 37℃ for 24 h for enrichment. 20 μL of the enriched solution was spread onto MRS solid medium plates and incubated at 37℃ for 48–72 h. Single colonies of different morphologies were selected for purification. After activation twice with liquid medium, the strain was inoculated into fermentation medium and incubated at 37℃ for 24 h. The fermentation broth was boiled for 10 minutes to terminate fermentation, centrifuged at 12000 r / min for 5 min, filtered through a 0.22 μm membrane, and the mannitol production was detected by HPLC. Strains with high mannitol production were selected as target strains.

[0031] 16S rDNA sequencing revealed that the 16S rDNA sequence of strain SK50.004 was 1412 bases long. BLAST alignment of this sequence yielded the following results: Figure 1 As shown, this strain is related to Limosilactobacillus fermentum CECT562 (AJ575812) showed the highest homology, reaching 99.86%. Based on cell morphology, physiological and biochemical characteristics, and referring to the Bacterial Identification Manual, along with 16S rDNA sequence alignment results, SK50.004 was identified as *Lactobacillus fermentum* (…). Limosilactobacillus fermentum It has been deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC No. M 20252591.

[0032] Example 2 Limosilactobacillus fermentum SK50.004 was cultured in a 3L fermenter under conditions of 50 g / L glucose and 100 g / L fructose. The fermentation steps are as follows: (1) Seed culture: Select fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum A single colony of SK50.004 was incubated in 5 mL of MRS liquid medium at 37°C for 12 h. The bacterial culture was then inoculated into 100 mL of MRS liquid medium at a 2% (v / v) inoculation rate and incubated at 37°C for 12 h as a seed culture (OD 2.520).

[0033] (2) Fermentation culture: The activated seed culture was inoculated at a rate of 10% (v / v) into a fermentation medium with a total sugar concentration of 150 g / L (50 g / L glucose, 100 g / L fructose, 5 g / L peptone, 10 g / L yeast extract, 0.4 g / L MgSO4•7H2O, 2.0 g / L K2HPO4, 0.02 g / L MnSO4•H2O, pH 5.0). The pH was controlled at 5.0 using 5 M NaOH, the fermentation temperature was set at 40℃, and the stirring speed was 50~100 rpm for 24 h.

[0034] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was boiled at 100 °C for 10 min to terminate fermentation and inactivate cells. The mixture was then centrifuged at 12000 r / min for 5 min. The supernatant was filtered through a 0.22 μm membrane. The production of mannitol and the consumption of glucose and fructose were determined by HPLC. The results showed that after 22 h of fermentation, all glucose was consumed, with only 0.54 g / L of fructose remaining. The concentration of mannitol in the product reached 72.18 g / L. Figure 2 The maximum productivity is 3.28 g / L / h.

[0035] Example 3: Different Cu 2+ concentration Limosilactobacillus fermentum Shake-flask fermentation culture of SK50.004 The fermentation steps are as follows: (1) Seed culture: Select fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum A single colony of SK50.004 was cultured in 5 mL of MRS liquid medium at 37°C for 12 h as a seed culture (OD was 2.180). (2) Fermentation culture: The bacterial culture was inoculated at an inoculum volume of 10% (v / v) into different Cu concentrations. 2+ Fermentation media containing CuSO4•5H2O at concentrations of 0, 5, 10, and 20 mg / L were incubated at 40°C for 24 h to evaluate the Cu content. 2+ Effects on the fermentation performance of strain SK50.004.

[0036] The results are as follows Figure 3 As shown, when the addition amount of CuSO4•5H2O was 10 mg / L, the mannitol concentration produced by strain SK50.004 during fermentation was the highest, reaching 10.22 g / L, and the OD600 value was the highest at 4.22. This was compared to the result without CuSO4•5H2O. 2+Compared with the original fermentation medium, the mannitol yield increased by 8% compared with the original yield (9.44 g / L).

[0037] Example 4: In a 3L fermenter Limosilactobacillus fermentum Fermentation culture of SK50.004 under the conditions of glucose 50 g / L, fructose 100 g / L, and CuSO4·5H2O 10 mg / L The fermentation steps are as follows: (1) Seed culture: Select fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum A single colony of SK50.004 was incubated in 5 mL of MRS liquid medium at 37°C for 12 h. The bacterial culture was then inoculated into 100 mL of MRS liquid medium at a 2% (v / v) inoculation rate and incubated at 37°C for 12 h as a seed culture (OD 2.640).

[0038] (2) Fermentation culture: The activated seed culture was inoculated at a rate of 10% (v / v) into a fermentation medium (50 g / L glucose, 100 g / L fructose, 5 g / L peptone, 10 g / L yeast extract, 0.4 g / L MgSO4•7H2O, 2.0 g / L K2HPO4, 0.02 g / L MnSO4•H2O, 10 mg / L CuSO4•5H2O, pH 5.0) with a CuSO4•5H2O addition of 10 mg / L. The pH was controlled at 5.0 using 5 M NaOH, the fermentation temperature was set at 40℃, and the stirring speed was 50~100 rpm for 24 h.

[0039] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was boiled at 100 °C for 10 min to terminate fermentation and inactivate cells. The mixture was then centrifuged at 12000 r / min for 5 min. The supernatant was filtered through a 0.22 μm membrane. The amount of mannitol produced and the consumption of glucose and fructose were determined by HPLC. The results showed that after 22 h of fermentation, the concentration of mannitol reached 84.00 g / L. Figure 4 ). Compared with Example 2 (without Cu² added) + Compared to the original fermentation medium, the yield of mannitol increased by 16%.

[0040] Example 5: In a 3L fermenter Limosilactobacillus fermentum Fermentation culture of SK50.004 strain at different sugar concentrations The fermentation steps are as follows: (1) Seed culture: Select fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentumSK50.004 single colony was incubated in 5 mL of MRS liquid medium at 37°C for 12 h; the bacterial culture was then inoculated into 100 mL of MRS liquid medium at a 2% (v / v) inoculation rate and incubated at 37°C for 12 h as seed culture (OD 2.840).

[0041] (2) Fermentation culture: The activated seed culture was inoculated into 1L of fermentation medium A, B and C containing different sugar concentrations at an inoculation rate of 10%~15% (v / v). The pH was controlled at 5.0 using 5M NaOH, the fermentation temperature was set at 40℃, and the stirring speed was 50~100 rpm for 24~48 h.

[0042] Fermentation medium A: glucose 60 g / L, fructose 135 g / L, peptone 5 g / L, yeast extract 10 g / L, MgSO4•7H2O 0.4 g / L, K2HPO4 2.0 g / L, MnSO4•H2O 0.02 g / L, CuSO4•5H2O 10 mg / L, pH 5.0.

[0043] Fermentation medium B: glucose 70 g / L, fructose 150 g / L, peptone 5 g / L, yeast extract 10 g / L, MgSO4•7H2O 0.4 g / L, K2HPO4 2.0 g / L, MnSO4•H2O 0.02 g / L, CuSO4•5H2O 10 mg / L, pH 5.0.

[0044] Fermentation medium C: glucose 80 g / L, fructose 185 g / L, peptone 5 g / L, yeast extract 10 g / L, MgSO4•7H2O 0.4 g / L, K2HPO4 2.0 g / L, MnSO4•H2O 0.02 g / L, CuSO4•5H2O 10 mg / L, pH 5.0.

[0045] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 6001 mL of fermentation broth was boiled at 100 °C for 10 min to terminate fermentation and inactivate cells. The mixture was centrifuged at 12000 r / min for 5 min. The supernatant was filtered through a 0.22 μm membrane. The amount of mannitol produced and the amount of glucose and fructose consumed were determined by HPLC. The results showed that when the substrate concentrations were 195 g / L (glucose 60 g / L, fructose 135 g / L), 220 g / L (glucose 70 g / L, fructose 150 g / L), and 265 g / L (glucose 80 g / L, fructose 185 g / L), the highest mannitol yields of the strain were 126.1 g / L (29 h), 132.6 g / L (31 h), and 160 g / L (38 h), respectively. The fructose utilization rates were 93.41%, 88%, and 87%, respectively, and the maximum productivity was 4.35 g / L / h, 4.28 g / L / h, and 4.21 g / L / h, respectively. The lactic acid content was 10.58, 12.64, and 14.24 g / L, respectively, and the acetic acid content was 4.24, 4.56, and 5.00 g / L, respectively. Figure 5 Therefore, it can be seen that when the substrate concentration is 265 g / L (80 g / L glucose and 185 g / L fructose), the mannitol yield in the fermentation broth is the highest, reaching 160 g / L, with a maximum productivity of 4.21 g / L / h. Simultaneously, this strain produces fewer byproducts during fermentation, with lactic acid and acetic acid contents of 14.24 g / L and 5.00 g / L, respectively. Compared to Example 4 (substrate concentration of 150 g / L), the mannitol yield is increased by 90.47%.

[0046] Comparative Example 1: The specific implementation method is the same as in Example 3, except that 5 mg CuSO4•5H2O is replaced with equal amounts of CoCl2•2H2O, FeSO4•7H2O, MgSO4•7H2O, and ZnSO4•7H2O, respectively. The results are as follows: Figure 6 As shown, Co 2+ Fe 2+ Mg 2+ and Zn 2+ Its promoting effect on mannitol production is not as good as that of Cu. 2+ .

[0047] Comparative Example 2: Fermentation was carried out in 250 mL shake flasks using *Lactobacillus fermentans* SK50.004 and *Lactobacillus fermentans* CGMCC 1.2029 (obtained from the China General Microbiological Culture Collection Center), both screened in Example 1. The fermentation steps are as follows: (1) Seed culture: A single colony of fermenting Lactobacillus mucinus was inoculated into 5 mL of MRS liquid medium and cultured at 37℃ for 12 h; the bacterial culture was inoculated into 100 mL of MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 12 h as seed culture. (2) The activated seed culture was inoculated at a rate of 10% (v / v) into a fermentation medium with a total sugar concentration of 150 g / L (50 g / L glucose, 100 g / L fructose, 5 g / L peptone, 10 g / L yeast extract, 0.4 g / L MgSO4•7H2O, 2.0 g / L K2HPO4, 0.02 g / L MnSO4•H2O). The culture was incubated at 40℃ for 24 h. The mannitol yield in the fermentation broth was then measured. The results showed that the commercially available fermented Lactobacillus mucilaginosus CGMCC 1.2029 did not produce mannitol, while the yield of SK50.004 was 48.68 g / L.

[0048] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Fermented Lactobacillus mucinus ( Limosiactobacillus fermentum SK50.004 was deposited at the China Center for Type Culture Collection on November 19, 2025, with accession number CCTCC NO: M 20252591.

2. A microbial preparation comprising the Limosiactobacillus fermentum SK50.004 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The number of L. fermentum SK50.004 in the microbial preparation is ≥ 1 x 10 7 CFU / g.

4. A process for the preparation of mannitol, characterized in that, Use of the Limosiactobacillus fermentum SK50.004 of claim 1 for the fermentation production of mannitol.

5. The method of claim 4, wherein, The method comprises fermenting the Limosiactobacillus fermentum SK50.004 in a culture medium containing glucose and fructose to produce mannitol.

6. The method of claim 5, wherein, The glucose content in the culture medium is 60-80 g / L, and the fructose content is 135-185 g / L.

7. The method according to claim 5 or 6, characterized in that, The medium contains Cu 2+ .

8. The method of any one of claims 4-7, wherein, The fermentation is carried out at 30-40℃ for 24-72 h.

9. The method of any one of claims 4-8, wherein, The method comprises the following steps: (1) Seed culture: static culture at 35-37℃ in MRS medium for 8-14 h; (2) Fermentation culture: inoculate the seed liquid prepared in step (1) into a fermentation medium, and ferment at 39-41℃ for 24-48 h.

10. Use of the Limosiactobacillus fermentum SK50.004 of claim 1 or the method of any one of claims 4-9 for the production of mannitol or a product containing mannitol.

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