Strain for degrading 5-hydroxymethylfurfural and application thereof

Abstract

The invention provides a bacterial strain for degrading 5-hydroxymethylfurfural and application of the bacterial strain, and belongs to the technical field of microorganisms. The strain is bacillus subtilis BLCY-SI-001, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.24941, and the strain has the characteristic of tolerance to 5-hydroxymethylfurfural, has the capability of degrading the 5-hydroxymethylfurfural in functional sugar, is sensitive to a plurality of common antibacterial drugs, and has relatively high safety.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain for degrading 5-hydroxymethylfurfural and application thereof. Background Art

[0002] 5-Hydroxymethylfurfural (5-HMF) is a furan compound that contains a furan ring, an aldehyde group, and a hydroxymethyl group in its structure. It can be upgraded to higher value-added chemicals, advanced fuels, and biopolymers through oxidation, reduction, rehydration, and etherification. Carbohydrates such as fructose and glucose can be used as raw materials for the preparation of 5-HMF and are often used as food raw materials and auxiliary materials for food processing. During food processing, fructose, glucose, etc. can be converted into 5-HMF through Maillard reaction and caramelization reaction. Recent studies have shown that high concentrations of 5-HMF ingested from food will have many adverse effects on human health. Such as irritation of mucous membranes, skin, upper respiratory tract, and eyes, and the promotion of neurodegenerative diseases, diabetes, and cardiovascular diseases. In addition, 5-HMF can also be converted into various compounds, such as chloromethylfurfural, sulfomethylfurfural, and formic acid, which have adverse effects on the body. Therefore, the food industry needs to strictly control the content of 5-HMF to prevent excessive furfural intake from damaging human health.

[0003] In the field of functional sugar production, there are many products that use glucose and fructose as raw materials, and a large amount of furfural will be produced during the production process, such as polydextrose, allulose and crystalline fructose. The national standard of the People's Republic of China also limits its 5-HMF content: the furfural content in polydextrose is less than 0.05% (w / %), and the furfural content in crystalline fructose is less than 0.32 (by absorbance). In this field, the removal of furfural usually depends on the effectiveness of functional resins. However, the resin often requires a large amount of acid, alkali and water during the regeneration process, which not only consumes a lot of material resources, but also the 5-HMF contained in the discharged wastewater will also pollute the water quality and soil. Therefore, in this field, an efficient and environmentally friendly method for degrading 5-HMF is needed to solve the current shortcomings of 5-HMF in production. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a strain for degrading 5-hydroxymethylfurfural and its application, wherein the strain has 5-hydroxymethylfurfural tolerance and obvious degradation ability for 5-hydroxymethylfurfural.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a strain for degrading 5-hydroxymethylfurfural, wherein the strain is Bacillus subtilis ( Bacillus subtilis)BLCY-SI-001, the deposit number is CGMCC No.24941.

[0006] Preferably, the colonies of the strain are milky white, have irregular edges, are Gram-positive, and have spores.

[0007] The invention provides a bacterial liquid for degrading 5-hydroxymethylfurfural. The bacterial liquid comprises a bacterial suspension of the Bacillus subtilis BLCY-SI-001.

[0008] Preferably, the bacterial suspension of Bacillus subtilis BLCY-SI-001 is a bacterial suspension cultured to the logarithmic growth phase.

[0009] The invention provides a method for preparing the bacterial liquid, comprising the following steps: inoculating Bacillus subtilis BLCY-SI-001 into LB broth medium for culturing to obtain the bacterial liquid.

[0010] Preferably, the formula of the LB broth medium is: 8.0-12.0 parts by weight of tryptone, 3.0-7.0 parts by weight of yeast extract powder, 8.0-12.0 parts by weight of sodium chloride, 0.8-1.1 L of water, pH 6.8-7.2.

[0011] Preferably, the culture temperature is 28-32°C, the culture speed is 180-220 rpm, and the culture time is 7-9 h.

[0012] The present invention also provides application of the strain or the bacterial solution or the bacterial solution obtained by the preparation method in degrading 5-hydroxymethylfurfural.

[0013] The present invention also provides application of the strain or the bacterial solution or the bacterial solution obtained by the preparation method in degrading 5-hydroxymethylfurfural in functional sugars.

[0014] The present invention also provides a method for degrading 5-hydroxymethylfurfural, wherein the strain or the bacterial solution or the bacterial solution obtained by the preparation method is inoculated into a sample containing 5-hydroxymethylfurfural.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to isolate Bacillus subtilis ( Bacillus subtilis )BLCY-SI-001, with the deposit number of CGMCC No.24941, has the characteristics of tolerance to 5-hydroxymethylfurfural, the ability to degrade 5-hydroxymethylfurfural, and is sensitive to a variety of common antimicrobial drugs, and has high safety.

[0016] Further, the Bacillus subtilis of the present invention ( Bacillus subtilisBLCY-SI-001 has the ability to degrade 5-hydroxymethylfurfural in functional sugars and can be used for the degradation of 5-hydroxymethylfurfural in functional sugars. It is expected to be applied in the production process of functional sugars to reduce dependence on functional resins, reduce the loss of regenerated acid, alkali and water, reduce costs and be environmentally friendly.

[0017] Biological deposit information Bacillus subtilis BLCY-SI-001, classified as Bacillus subtilis Bacillus subtilis , Latin name Bacillus subtilis , deposited on May 23, 2022 at the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.24941. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the screening result of 5-HMF-resistant strains.

[0019] Figure 2 Phylogenetic tree for identification of BLCY-SI-001 strain.

[0020] Figure 3 This is the 5-HMF tolerance test of Bacillus subtilis BLCY-SI-001.

[0021] Figure 4 This is the test of 5-HMF degradation effect of Bacillus subtilis BLCY-SI-001.

[0022] Figure 5 To detect the degradation effect of 5-HMF in polydextrose. DETAILED DESCRIPTION

[0023] The present invention provides a strain for degrading 5-hydroxymethylfurfural, wherein the strain is Bacillus subtilis ( Bacillus subtilis )BLCY-SI-001, the deposit number is CGMCC No.24941. The Bacillus subtilis of the present invention ( Bacillus subtilis ) BLCY-SI-001 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 23, 2022, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The Bacillus subtilis BLCY-SI-001 described in the present invention is derived from the soil near the wastewater discharge outlet of the workshop of Shandong Bailong Chuangyuan Biotechnology Co., Ltd. The colony of the Bacillus subtilis BLCY-SI-001 is milky white, with irregular edges, is Gram-positive, and has spores.

[0024] The present invention also provides a bacterial solution for degrading 5-hydroxymethylfurfural, the bacterial solution comprising the bacterial suspension of Bacillus subtilis BLCY-SI-001. The bacterial suspension of Bacillus subtilis BLCY-SI-001 of the present invention is a bacterial suspension cultured to the logarithmic growth phase.

[0025] The present invention also provides a method for preparing a bacterial solution for degrading 5-hydroxymethylfurfural, comprising the following steps: inoculating Bacillus subtilis BLCY-SI-001 into an LB broth medium for culture to obtain a bacterial solution. The formula of the LB broth medium of the present invention is: 8.0-12.0 parts by weight of tryptone, 3.0-7.0 parts by weight of yeast extract powder, 8.0-12.0 parts by weight of sodium chloride, 0.8-1.1L of water, pH 6.8-7.2; the formula of the LB broth medium is preferably 10.0 parts by weight of tryptone, 5.0 parts by weight of yeast extract powder, 10.0 parts by weight of sodium chloride, 1.0L of water, pH 7.0. The culture temperature of the present invention is 28-32°C, preferably 30°C; the rotation speed of the culture is 180-220rpm, preferably 200rpm; the culture time is 7-9h, preferably 8h.

[0026] The present invention also provides application of the strain or the bacterial solution or the bacterial solution obtained by the preparation method in degrading 5-hydroxymethylfurfural.

[0027] The present invention also provides the use of the strain or the bacterial solution or the bacterial solution obtained by the preparation method in degrading 5-hydroxymethylfurfural in functional sugars. The functional sugars of the present invention include but are not limited to polydextrose, psicose and crystalline fructose.

[0028] The present invention also provides a method for degrading 5-hydroxymethylfurfural, wherein the strain or the bacterial solution or the bacterial solution obtained by the preparation method is inoculated into a sample containing 5-hydroxymethylfurfural. The inoculation amount of the bacterial solution of the present invention is 2-8% (v / v%), preferably 5% (v / v%).

[0029] In the present invention, unless otherwise specified, all reagents or culture media are commercially available products well known to those skilled in the art.

[0030] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 1. Initial screening of strains: The collected and packaged soil samples near the wastewater discharge outlet of the workshop of Shandong Bailong Chuangyuan Biotechnology Co., Ltd. were placed in a clean workbench that had been disinfected in advance. 1g of soil sample was taken and diluted to 10mL with 0.85% sterile saline, and then diluted to 10 in a gradient. -6 , take 10 -3 , 10 -4 , 10 -5 , 10 -6 1 mL of the gradient dilution was spread on LB agar medium and cultured in a 38°C constant temperature incubator for 48-72 hours. After the culture was completed, single colonies with large differences in colony morphology were selected and continued to be streaked until no foreign bacteria appeared under microscopic examination. Five strains with large differences in colony morphology were screened out, and the purified strains were stored in a -80°C refrigerator using glycerol tubes. The formula of LB agar medium (g / L) is: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 15.0 g of agar, 1 L of water, pH 7.0.

[0032] 2. Strain rescreening: The five purified strains screened above were inoculated into LB broth containing 80 mM 5-HMF, cultured at 30°C and 200 rpm for 24 h, and the OD was measured using a spectrophotometer. 600 . strain OD 600 The higher the value, the stronger the strain's tolerance to the environment. 600 The value was used as the re-screening index, that is, the strain with the highest OD value was considered to be tolerant to 5-HMF. The LB broth medium (g / L) formula was: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, 1 L water, pH 7.0.

[0033] The results are as follows Figure 1 As shown, the results show that the OD of strain BLCY-SI-001 among the five strains 600 The highest value, with the ability to tolerate 5-HMF ( Figure 1 ).

[0034] 3. Strain morphology observation and strain identification Strain morphology observation: Through observation of the colony morphology of strain BLCY-SI-001, it was found that the colony of the strain was milky white with irregular edges; after Gram staining, it was found that the strain was Gram-positive and had spores under oil microscope observation.

[0035] Molecular biological identification of strains: Gene fragments were extracted and PCR amplified using the steps described in the bacterial genome extraction kit (DP302, Beijing Tiangen Biochemical Technology Co., Ltd.). The amplification reaction system was 50 μL, including 1.0 μL template DNA, 2 μL upstream primer 27F, 2 μL downstream primer 1492R, 10 μL 5×PCR Buffer, 4 μL DNTP (2.5 mmol / L), 1 μL TaqDNA Polymerase (5 U / μL), and ultrapure water to 50 μL. The 16S rRNA universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID No. 1) and 1492R: 5′-CGGTTACCTTGTTACGACTT-3′ (SEQ ID No. 2) were used for amplification. The PCR reaction program was: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ extension for 100s; after 30 cycles, 72℃ extension for 5min. The samples were stored at 4℃ and tested by 1.0% agarose gel electrophoresis to see if the desired fragment was amplified. The PCR products were sent to Shanghai Paisono Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database by Blast sequence alignment, and the strain phylogenetic tree was drawn by MEGA11 software ( Figure 2 ). The strain was identified as Bacillus subtilis ( Bacillus subtilis ).

[0036] Example 2 1. Determination of tolerance of Bacillus subtilis BLCY-SI-001 to 5-HMF Use an inoculation loop to inoculate the selected Bacillus subtilis BLCY-SI-001 (2-3 loops) into LB broth medium and culture at 30°C and 200 rpm until the logarithmic growth phase (8 h). 600 =2.93). The bacterial solution was inoculated into LB broth containing 50, 100, 150, 200, and 250 mM 5-HMF at an inoculum volume of 5% (v / v%), and cultured at 30°C and 200 rpm for 24 h. The OD was measured using a spectrophotometer. 600 , to determine the strain's tolerance to furfural. Figure 3 The results showed that when the 5-HMF concentration in the culture medium reached 250 mM, the strain hardly grew, so it was believed that Bacillus subtilis BLCY-SI-001 could tolerate up to 200 mM 5-HMF.

[0037] 2. Degradation efficiency of 5-HMF by Bacillus subtilis BLCY-SI-001 Bacillus subtilis BLCY-SI-001 was cultured to the logarithmic growth phase (the culture method was the same as step 1 of this embodiment). The bacterial solution was inoculated into LB broth containing 40 mM 5-HMF at an inoculum of 5%, and cultured at 30°C and 200 rpm. The 5-HMF content in the culture medium was detected every 6 hours. The 5-HMF content was detected using the method described in national standard GB 28351-2012, and the furfural degradation efficiency of the strain was calculated. Figure 4 The results showed that the furfural degradation efficiency could reach over 90% after 12 h of culture, and further extending the culture time to 18 h could completely degrade the furfural in the culture medium.

[0038] Example 3 1. Drug sensitivity test and safety evaluation of Bacillus subtilis BLCY-SI-001 Heat and dissolve 21g of clarified MH broth medium in 1L pure water, and dispense into test tubes. Except for the first tube which is 4mL, the rest are 2mL, and sterilize at 121℃ for 15min. Number the sterilized test tubes, add antibiotics to the first test tube to make its concentration 128mg / L, draw 2mL into the second test tube, mix well, and then draw 2mL into the third test tube, and follow the same operation until the thirteenth test tube. Discard the 2mL solution in the thirteenth test tube to keep the volume consistent with the first 12 test tubes. According to this operation, the concentration of antibiotics in the first to thirteenth test tubes is 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg / L, and the fourteenth and fifteenth test tubes do not contain antibiotics.

[0039] The minimum inhibitory concentration (MIC) was determined by broth dilution method: Bacillus subtilis BLCY-SI-001 was cultured to the logarithmic growth phase (the culture steps were the same as step 1 in Example 1), and the bacterial solution concentration was adjusted to 10 7 CFU / mL, 0.1mL per tube is inoculated into the 1st to 14th test tubes, and the test tubes are observed after 24 hours of culture. The lowest drug concentration that inhibits the visible growth of the strain to be tested is the minimum inhibitory concentration of the drug for the strain to be tested. Antimicrobial drugs include amoxicillin, penicillin, tetracycline, chloramphenicol, erythromycin, and vancomycin (GPN3F, Thermo Fisher Scientific, USA). The test results are displayed as sensitive, intermediate, resistant, and undetermined.

[0040] The test results showed that Bacillus subtilis BLCY-SI-001 was sensitive to most antimicrobial drugs, which preliminarily proved that the strain had a certain degree of safety (Table 1).

[0041] Table 1 Results of drug sensitivity test

[0042] 2. Degradation efficiency of furfural in polydextrose by Bacillus subtilis BLCY-SI-001 After culturing Bacillus subtilis BLCY-SI-001 to the logarithmic growth phase (the culturing steps are the same as step 1 of Example 1), it is inoculated into a polydextrose solution with a solid content of 30% at an inoculum size of 5%, and cultured at 30° C. and 200 rpm. Samples are taken every 6 hours for detection, and the measurement index is 5-HMF content.

[0043] The 5-HMF content was determined according to the steps of GB 25541-2010 "National Food Safety Standard Food Additive Polydextrose": 1 g of polydextrose sample was weighed, dissolved in water and diluted to 100 mL, and the absorbance at 283 nm was measured after thorough mixing. The 5-HMF content was calculated according to X=0.749×A / C, where X is the 5-HMF content, A is the absorbance of the sample at 283 nm, and C is the sample concentration.

[0044] like Figure 5 The results showed that the 5-HMF content in the solution decreased by 85.31% after 18 hours of cultivation, and 5-HMF was no longer detected in the solution after 24 hours of fermentation. It can be seen that Bacillus subtilis BLCY-SI-001 can be used to remove furfural from polydextrose.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain for degrading 5-hydroxymethylfurfural, characterized in that: The strain is Bacillus subtilis ( Bacillus subtilis )BLCY-SI-001, the deposit number is CGMCC No.24941.

2. The strain according to claim 1, characterized in that The colonies of the strain are milky white, have irregular edges, are Gram-positive, and have spores.

3. A bacterial solution for degrading 5-hydroxymethylfurfural, characterized in that: The bacterial solution comprises a bacterial suspension of the strain according to claim 1 or 2.

4. The bacterial solution according to claim 3, characterized in that The bacterial suspension of Bacillus subtilis BLCY-SI-001 is a bacterial suspension cultured to the logarithmic growth phase.

5. The method for preparing bacterial liquid according to claim 3 or 4, characterized in that: The method comprises the following steps: inoculating Bacillus subtilis BLCY-SI-001 into LB broth medium for cultivation to obtain bacterial liquid.

6. The preparation method according to claim 5, characterized in that: The formula of the LB broth medium is: 8.0-12.0 parts by weight of tryptone, 3.0-7.0 parts by weight of yeast extract powder, 8.0-12.0 parts by weight of sodium chloride, 0.8-1.1 L of water, and pH 6.8-7.

2.

7. The preparation method according to claim 5, characterized in that: The culture temperature is 28-32° C., the culture rotation speed is 180-220 rpm, and the culture time is 7-9 hours.

8. Use of the strain according to claim 1 or 2, the bacterial solution according to claim 3 or 4, or the bacterial solution obtained by the preparation method according to any one of claims 5 to 7 in degrading 5-hydroxymethylfurfural.

9. Use of the strain according to claim 1 or 2, the bacterial solution according to claim 3 or 4, or the bacterial solution obtained by the preparation method according to any one of claims 5 to 7 in degrading 5-hydroxymethylfurfural in functional sugars.

10. A method for degrading 5-hydroxymethylfurfural, characterized in that: The strain according to claim 1 or 2, the bacterial solution according to claim 3 or 4, or the bacterial solution obtained by the preparation method according to any one of claims 5 to 7 is inoculated into a sample containing 5-hydroxymethylfurfural.

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