Additive for improving fermentation yield of vanillin synthesized by microbiological method and application

Abstract

The invention provides application of tributyrin as an additive in improving the fermentation yield of vanillin synthesized by a microbiological method and an additive for improving the fermentation yield of vanillin synthesized by the microbiological method, and belongs to the technical field of additives. According to the invention, tributyrin is used as an additive for improving the fermentation yield of the vanillin synthesized by the microbiological method, and the toxicity of the vanillin to thalli can be reduced, so that the fermentation yield of the vanillin is effectively improved. The method is suitable for all microorganisms, and the adding volume proportion and the adding time after fermentation are not limited.

Description

Technical Field

[0001] The present invention relates to the technical field of additives, and in particular to an additive for improving the fermentation yield of vanillin synthesized by microbial method, wherein the additive is tributyrin, and application of the tributyrin as an additive in improving the fermentation yield of vanillin synthesized by microbial method. Background Art

[0002] Vanillin, also known as vanillin, is an organic compound. There are three production methods: one is extracted from vanilla beans of the orchid plant; one is chemical synthesis; and one is microbial synthesis, which is further divided into microbial fermentation, microbial whole-cell catalysis and microbial enzyme method.

[0003] Vanillin is also known as vanillin and vanillin. Its structural formula is as follows:

[0004]

[0005] Vanillin, chemically known as 4-hydroxy-3-methoxybenzaldehyde, is the main component of vanilla products and one of the most widely used seasonings in the world, with a wide range of applications in the food, fragrance, pharmaceutical and chemical industries. In particular, vanillin is used as a high-grade flavor additive in the food industry, with high sweetness intensity and creamy vanilla flavor.

[0006] Traditional methods for producing vanillin include plant extraction and chemical synthesis. Typically, plant extraction uses ethanol as a solvent to extract vanillin from vanilla pods. The traditional planting, harvesting and maintenance of vanilla plants is time-consuming, and the yield is extremely low, resulting in high prices for natural vanillin, limiting industrial applications.

[0007] At present, 95% of vanillin on the market is produced by chemical synthesis with lignin and guaiacol as the main raw materials. Vanillin can be obtained by hydrolyzing lignin under alkaline conditions and then oxidizing it, with a yield of about 10%. However, chemical synthesis has the disadvantages of complex process, expensive raw materials, high energy consumption, poor substrate specificity, low stereoselectivity, and high pollution. In addition, the amount of synthetic vanillin used in food is strictly restricted by the European Expert Committee.

[0008] The growing demand for sustainable and natural vanillin has stimulated interest in developing alternative, low-cost and bio-based methods. Bioconversion methods mimic the way plants produce vanillin by utilizing enzyme / microbial catalysts and inexpensive natural substrates, meeting food safety requirements.

[0009] Biosynthesized vanillin has attracted wide attention due to its potential in the food industry due to its economical nature compared to natural extraction and its environmental and health-friendly characteristics compared to chemical synthesis. The biosynthesis of vanillin mainly includes synthesis from precursors such as ferulic acid, isoeugenol and eugenol, or de novo synthesis from glucose[7]. Among them, eugenol, isoeugenol and ferulic acid are the main substrates. The vanillin produced after bioconversion is called "natural vanillin", which is superior to chemically synthesized vanillin in both price and quality.

[0010] However, studies have shown that vanillin has an inhibitory effect on microorganisms such as Escherichia coli, Bacillus subtilis, yeast, Rhizopus nigromaculus and Mucor, and its minimum inhibitory concentration (MIC) is 0.23%, 0.26%, 0.14%, 0.14% and 0.03%, respectively. Therefore, when using microorganisms to synthesize vanillin, the bactericidal activity of vanillin leads to low yields of vanillin synthesized by bacteria, which seriously limits the application of this method in industry. In order to eliminate product toxicity and cell load, scientists have done a lot of research, including the following methods:

[0011] 1. Multi-layer dynamic control strategy. The method is to balance cell growth and product formation by implementing a multi-layer dynamic control strategy to reduce the toxicity of the product to microorganisms. Advantages: It can effectively regulate metabolic flux and optimize the balance between cell growth and product accumulation. Disadvantages: The technical implementation is relatively complex and requires fine control.

[0012] 2. Fine-tune the ratio of biosynthetic enzymes. The method is to adjust the ratio of biosynthetic enzymes and regulate metabolic flux to reduce product toxicity. Advantages: It can be achieved through genetic engineering and has high operability. Disadvantages: It requires precise calculation and adjustment of enzyme ratios.

[0013] 3. Add resin to absorb vanillin. The method is to add resin to the fermentation system, absorb vanillin through the resin, reduce the concentration of vanillin in the aqueous phase, and thus reduce the toxic inhibition of microorganisms. Advantages: The resin has strong adsorption capacity and can effectively reduce the toxicity of the product. Disadvantages: The resin is expensive and may break during use. It may also absorb impurities and affect the purity of the final product.

[0014] 5. Chassis cell gene modification. The method is to introduce exogenous genes into chassis cells to convert vanillin into non-toxic or less toxic derivatives, such as vanillin β-D-glucoside. Advantages: It can fundamentally solve the problem of product toxicity and improve the tolerance of microorganisms to vanillin. Disadvantages: UDP-glucosyltransferase is difficult to express and fold in prokaryotes, and the enzyme activity is weak, which limits the application of this method.

[0015] 6. High-throughput screening to obtain resistant mutants. The method is to use high-throughput screening technology to obtain mutants resistant to vanillin to improve the tolerance of microorganisms to vanillin. Advantages: It can quickly screen out resistant mutants and improve the tolerance of microorganisms to a certain extent. Disadvantages: Random mutagenesis may have side effects and affect the growth and vitality of microorganisms.

[0016] Although there are various solutions to the toxicity of vanillin products during the biosynthesis process, each has its own advantages and disadvantages, and there is still a lack of efficient and feasible methods that can be applied industrially, which limits the industrial application of microbial synthesis of vanillin. Developing new methods to reduce toxicity and increase its yield is an urgent and very challenging task. Summary of the invention

[0017] In view of this, in order to solve the technical problem of low vanillin yield in the prior art, on the one hand, the present invention provides the use of tributyrin as an additive in increasing the fermentation yield of vanillin synthesized by microbial method, reducing the toxicity of vanillin to bacteria, thereby effectively increasing the fermentation yield of vanillin. It is applicable to all microorganisms, and the volume ratio of its addition and the time of addition after fermentation are not limited.

[0018] To achieve the above object, the present invention provides the following technical solutions:

[0019] Application of tributyrin as an additive in improving the fermentation yield of vanillin synthesized by microbial method.

[0020] Preferably, the tributyrin is suitable for all microorganisms in the microbiological process.

[0021] Preferably, the microorganism includes but is not limited to Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Streptomyces, Amycolatopsis, and Bacillus fusiformi.

[0022] Preferably, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is any volume ratio.

[0023] Preferably, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is 10%-100%.

[0024] Preferably, the tributyrin is added at any time during the microbial fermentation cycle.

[0025] Preferably, the tributyrin is added within 6-18 hours after microbial fermentation.

[0026] Preferably, the microbial method includes microbial fermentation method, microbial whole-cell catalysis method and microbial enzyme method.

[0027] On the other hand, the present invention provides an additive for improving the fermentation yield of vanillin synthesized by microbial method, wherein the additive is tributyrin.

[0028] Preferably, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is any volume ratio, and the preferred addition time is any time within the microbial fermentation cycle.

[0029] The application of tributyrin provided by the present invention as an additive in improving the fermentation yield of vanillin synthesized by microbial method can reduce the toxicity of vanillin to bacteria, thereby effectively improving the fermentation yield of vanillin. It is applicable to all microorganisms, and the volume ratio of its addition and the time of addition after fermentation are not limited. It can also reduce the fermentation cost of vanillin, indicating that it has important economic value and broad application prospects in the fermentation production of vanillin. DETAILED DESCRIPTION

[0030] The invention provides application of tributyrin as an additive in improving the fermentation yield of vanillin synthesized by a microbial method.

[0031] It should be pointed out that the vanillin mentioned in the present invention is vanillin synthesized by microbial method. As long as microbial cells and vanillin exist at the same time, they are within the protection scope of the present invention.

[0032] The tributyrin of the present invention is used as an additive in improving the fermentation yield of synthesizing vanillin by a microbial method, wherein the applicable microorganisms, the added volume and the time of adding the tributyrin after fermentation are not limited.

[0033] In other words, in the present invention, the tributyrin is applicable to all microorganisms in the microbiological method.

[0034] In the present invention, the microorganisms include but are not limited to Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Streptomyces, Amycolatopsis, and Bacillus fusiformi.

[0035] In the present invention, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method can be any volume ratio.

[0036] In the present invention, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is preferably 10%-100%, more preferably 40%, which volume is a percentage of the fermentation medium.

[0037] In the present invention, the tributyrin is added at any time during the microbial fermentation cycle.

[0038] In the present invention, the tributyrin is preferably added within 6-18 hours after microbial fermentation, and more preferably within 12 hours after fermentation.

[0039] In the present invention, the microbial method includes microbial fermentation method, microbial whole-cell catalysis method and microbial enzyme method.

[0040] The present invention provides a method for synthesizing vanillin by the microbial method, which is as follows:

[0041] Escherichia coli is cultured in LB seed medium at pH 7.0±0.1 and 37±1°C for 8±1 hours, and then transferred to XLS-3 liquid fermentation medium. Fermentation is carried out for 12 hours under the conditions of pH 7.0±0.1, culture temperature 30±1°C, and DO 30%. Then, tributyrin is added to the XLS-3 liquid fermentation medium at a volume ratio of preferably 40%, and the fermentation culture is continued for 24±2 hours to obtain a fermentation broth containing vanillin.

[0042] Among them, the composition and content of the LB seed culture medium are: 10g peptone, 5g yeast extract, 10g sodium chloride (NaCl), and 1000ml distilled water; the composition and content of the XLS-3 liquid fermentation culture medium are: 6.78g disodium hydrogen phosphate (Na2HPO4), 3.0g potassium dihydrogen phosphate (KH2PO4), 0.5g sodium chloride (NaCl), 1.0g ammonium chloride (NH4Cl), 0.241g magnesium sulfate (MgSO4), 0.011g calcium chloride (CaCl2), 5g / L yeast extract, 110mg VB1, 10g glucose, and 1000ml distilled water.

[0043] The present invention also provides a method for using tributyrin to increase the fermentation yield of vanillin synthesized by microbial method, which is as follows:

[0044] (1) Take 1 mL of the bacterial suspension of Escherichia coli XLS24-4 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0045] (2) The above shake flask was shaken and cultured in a shaker at 37°C and 220 rpm for 6-8 h.

[0046] (3) The E. coli XLS24-4 culture medium in the shake flask was transferred to a 2 L fermenter containing 1000 mL of XLS-3 liquid fermentation medium at a volume ratio of 5%, and fermented at 30° C., pH 7.0, and 30% dissolved oxygen.

[0047] (4) After 12 hours of cultivation, 400 mL of tributyrin (40% of the volume of the fermentation medium) that had been sterilized in advance was added to the fermentation medium according to 40% of the fermentation volume. Fermentation was continued for 30 hours at 30°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0048] (5) The fermentation medium without tributyrin was used as the control group.

[0049] (6) During the fermentation process, the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0050] (7) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0051] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0052] On the other hand, the present invention provides an additive for improving the fermentation yield of vanillin synthesized by microbial method, wherein the additive is tributyrin.

[0053] In the present invention, the volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is any volume ratio, and the preferred addition time is any time within the microbial fermentation cycle.

[0054] The technical solution of the present invention is explained clearly and in detail below in conjunction with specific embodiments.

[0055] Example 1

[0056] Effects of different addition ratios on the production of vanillin by genetically engineered Escherichia coli (E. coli).

[0057] (1) Take 1 mL of the bacterial suspension of Escherichia coli XLS24-4 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0058] (2) The shake flask was shaken and cultured in a shaker at 37°C and 220 rpm for 6-8 h.

[0059] (5) The E. coli XLS24-4 culture liquid in the above shake flask was transferred to a 2L fermenter containing 1000mL XLS-3 liquid fermentation medium at a volume ratio of 5%, and fermentation was carried out at 30°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to be less than 5g / L.

[0060] (6) After culturing for 12 hours, tributyrin that had been sterilized in advance was added to the fermentation medium according to different volume ratios of the fermentation medium as shown in Table 1. Fermentation was continued for 30 hours at 30° C., pH 7.0, and 30% dissolved oxygen.

[0061] (7) The fermentation medium without tributyrin was used as the control group.

[0062] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0063] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0064] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0065] The experimental results are shown in Table 1.

[0066] Table 1 Effect of adding different volumes of tributyrin on the fermentation yield of vanillin.

[0067]

[0068]

[0069] From the data in Table 1, we can see that without the addition of tributyrin, the content of vanillin in the Escherichia coli fermentation broth was 10.5±0.2g / L. When tributyrin was added to the XLS-3 liquid medium, the fermentation yield of vanillin was improved to varying degrees. When the volume ratio of tributyrin added reached 40%, the fermentation yield of vanillin in the fermentation broth reached the highest, i.e., 17.2±0.2g / L, which was increased by 1.6 times. However, when the volume ratio of tributyrin added was further increased to 60%, the fermentation yield of vanillin decreased, which was 15% lower than the final concentration of 40%, but it was still higher than that without the addition of tributyrin. When the volume ratio of tributyrin added was further increased to 100%, the fermentation yield of vanillin showed a trend of gradual decrease.

[0070] Example 2

[0071] Effect of different addition times on the production of vanillin by genetically engineered Escherichia coli (E. coli).

[0072] (1) Take 1 mL of the bacterial suspension of Escherichia coli XLS24-4 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0073] (2) The shake flask was shaken and cultured in a shaker at 37°C and 220 rpm for 6-8 h.

[0074] (5) The E. coli XLS24-4 culture medium in the shake flask was transferred to a 2 L fermenter containing 1000 mL of XLS-3 liquid fermentation medium at a volume ratio of 5%, and fermented at 30°C, pH 7.0, and 30% dissolved oxygen.

[0075] (6) At 0 hours, 6 hours, 12 hours, 18 hours, 36 hours and 72 hours of fermentation, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium at 40% of the volume of the fermentation medium. Fermentation was continued for 30 hours at 30° C., pH 7.0 and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to be less than 5 g / L.

[0076] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0077] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0078] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0079] The experimental results are shown in Table 2.

[0080] Table 2 Effect of different addition times of tributyrin on the fermentation yield of vanillin.

[0081] Adding time (h) Vanillin fermentation yield (g / L) 0 5.5±0.2 6 13.4±0.1 12 17.2±0.2 18 14.1±0.3 36 10.5±0.2 72 8.7±0.6

[0082] From the data in Table 2, we can see that adding 40% tributyrin by volume of the fermentation medium at different fermentation times can increase the yield of vanillin in E. coli fermentation to varying degrees. In particular, when tributyrin was added at a fermentation time of 12 hours, the yield of vanillin in the fermentation broth was the highest, reaching 17.2±0.2 g / L.

[0083] Example 3

[0084] Effect of tributyrin addition on vanillin production by genetically engineered bacteria Corynebacterium glutamicum (C. glutamicum).

[0085] (1) Take 1 mL of the bacterial suspension of Corynebacterium glutamicum Cg24-1 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0086] (2) Shake the flask in a shaker at 32°C and 220 rpm until the absorbance at 600 nm (A600) is 10-12.

[0087] (5) The C. glutamicum Cg24-1 bacterial solution in the shake flask was transferred to a 2L fermenter containing 1000mL of liquid fermentation medium at a volume ratio of 10%, and fermentation was carried out at 32°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to be less than 5g / L.

[0088] (6) After culturing for 12 hours, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium, and the fermentation was continued for 30 hours at 32° C., pH 7.0, and 30% dissolved oxygen.

[0089] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0090] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0091] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0092] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0093] The experimental results are shown in Table 3.

[0094] Table 3 Effect of adding tributyrin on the production of vanillin by Corynebacterium glutamicum fermentation.

[0095] Group Vanillin fermentation yield (g / L) Control group 5.5±0.2 Experimental Group 9.4±0.1

[0096] From the data in Table 3, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Corynebacterium glutamicum was 5.5±0.2 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 71%, reaching 9.4±0.1 g / L. This indicates that the addition of tributyrin has a promoting effect on the production of vanillin by Corynebacterium glutamicum fermentation.

[0097] Example 4

[0098] Effect of tributyrin addition on vanillin production by genetically engineered yeast Saccharomyces cerevisiae.

[0099] (1) Take 1 mL of the bacterial suspension of Saccharomyces cerevisiae Sc24-5 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0100] (2) The culture was shaken in a shaker at 30°C and 220 rpm until the absorbance at 600 nm (A600) was 5-6.

[0101] (5) The Saccharomyces cerevisiae Sc24-5 culture medium in the shake flask was transferred to a 2 L fermentation tank containing 1000 mL of liquid fermentation medium at a volume ratio of 10%, and fermented at 30° C., pH 7.0, and 30% dissolved oxygen.

[0102] (6) After culturing for 12 hours, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium. Fermentation was continued for 30 hours at 32°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0103] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0104] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0105] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0106] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0107] The experimental results are shown in Table 4.

[0108] Table 4 Effect of adding tributyrin on the production of vanillin by Saccharomyces cerevisiae fermentation.

[0109] Group Vanillin fermentation yield (g / L) Control group 4.5±0.2 Experimental Group 6.4±0.1

[0110] From the data in Table 4, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Saccharomyces cerevisiae was 4.5±0.2 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 42% to 6.4±0.1 g / L. This indicates that the addition of tributyrin has a promoting effect on the production of vanillin by Saccharomyces cerevisiae fermentation.

[0111] Example 5

[0112] Effect of adding tributyrin on the production of vanillin by genetically engineered Pichia pastoris.

[0113] (1) Take 1 mL of the bacterial suspension of Pichia pastoris Pp24-3 stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0114] (2) The culture was shaken in a shaker at 30°C and 220 rpm until the absorbance at 600 nm (A600) was 5-6.

[0115] (5) The Pichia pastoris Pp24-3 bacterial solution in the above shake flask was transferred to a 2 L fermentation tank containing 1000 mL of liquid fermentation medium at a volume ratio of 10%, and fermented at 30° C., pH 7.0, and 30% dissolved oxygen.

[0116] (6) After culturing for 12 hours, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium. Fermentation was continued for 30 hours at 30°C, pH 7.0, and 30% dissolved oxygen. Glycerol and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0117] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0118] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0119] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0120] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0121] The experimental results are shown in Table 5.

[0122] Table 5 Effect of adding tributyrin on the production of vanillin by Saccharomyces cerevisiae fermentation.

[0123] Group Vanillin fermentation yield (g / L) Control group 4.2±0.2 Experimental Group 6.3±0.4

[0124] From the data in Table 5, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Saccharomyces cerevisiae was 4.2±0.2 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 50%, reaching 6.3±0.4 g / L. This indicates that the addition of tributyrin has a promoting effect on the production of vanillin by Pichia pastoris.

[0125] Example 6

[0126] Effect of adding tributyrin on the fermentation production of vanillin by genetically engineered Streptomyces.

[0127] (1) Take 1 mL of Streptomyces ST24-6 bacterial suspension stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0128] (2) The shake flask was cultured in a shaker at 30°C and 220 rpm for 12 h.

[0129] (5) The Streptomyces ST24-6 bacterial solution in the above shake flask was transferred to a 2L fermenter containing 1000mL of liquid fermentation medium at a volume ratio of 10%, and fermented at 30°C, pH 7.0, and 30% dissolved oxygen.

[0130] (6) After 12 hours of culture, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium. Fermentation was continued for 30 hours at 30°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0131] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0132] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0133] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0134] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0135] The experimental results are shown in Table 6.

[0136] Table 6 Effect of adding tributyrin on the fermentation production of vanillin by Streptomyces ST24-6.

[0137] Group Vanillin fermentation yield (g / L) Control group 3.4±0.2 Experimental Group 5.3±0.3

[0138] From the data in Table 6, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Saccharomyces cerevisiae was 3.4±0.2 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 56% to 5.3±0.3 g / L. This indicates that the addition of tributyrin has a promoting effect on the production of vanillin by Streptomyces cerevisiae.

[0139] Example 7

[0140] Effect of tributyrin addition on vanillin production by genetically engineered Amycolatopsis bacteria.

[0141] (1) Take 1 mL of the bacterial suspension of Amycolatopsis spp. At24-13 strain stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0142] (2) The culture was shaken in a shaker at 30°C and 220 rpm until the absorbance at 600 nm (A600) was 5-6.

[0143] (5) The bacterial solution of Amycolatopsis simulans At24-13 in the shake flask was transferred to a 2 L fermentation tank containing 1000 mL of liquid fermentation medium at a volume ratio of 10%, and fermented at 30° C., pH 7.0, and 30% dissolved oxygen.

[0144] (6) After 12 hours of culture, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium. Fermentation was continued for 30 hours at 30°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0145] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0146] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0147] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0148] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0149] The experimental results are shown in Table 7.

[0150] Table 7 Effect of adding tributyrin on the fermentation production of vanillin by Amycolatopsis sp. At24-13.

[0151] Group Vanillin fermentation yield (g / L) Control group 6.4±0.2 Experimental Group 9.3±0.1

[0152] From the data in Table 7, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Amycolatopsis was 6.4±0.2 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 56% to 9.3±0.1 g / L, indicating that the addition of tributyrin has a promoting effect on the production of vanillin by Amycolatopsis.

[0153] Example 8

[0154] Effect of adding tributyrin on the fermentation production of vanillin by genetically engineered bacteria of Clostridium fusiformi.

[0155] (1) Take 1 mL of the bacterial suspension of Clostridium bacterium Bf24-13 stored at -80°C and inoculate it into a 250 mL shake flask containing 50 mL of seed culture medium.

[0156] (2) The culture was shaken in a shaker at 30°C and 220 rpm until the absorbance at 600 nm (A600) was 5-6.

[0157] (5) The Clostridium bacterium Bf24-13 bacterial solution in the above shake flask was transferred to a 2 L fermentation tank containing 1000 mL of liquid fermentation medium at a volume ratio of 10%, and fermented at 30° C., pH 7.0, and 30% dissolved oxygen.

[0158] (6) After 12 hours of culture, 400 mL of tributyrin that had been sterilized in advance was added to the fermentation medium. Fermentation was continued for 30 hours at 30°C, pH 7.0, and 30% dissolved oxygen. Glucose and ferulic acid were added to the feed stream to control the concentration to less than 5 g / L.

[0159] (7) The fermentation with tributyrin added as above was used as the experimental group, and the fermentation without tributyrin added was used as the control group.

[0160] (8) During the fermentation process, 1 mL of the sample was diluted 50 times with dimethyl sulfoxide and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic filter membrane and transferred to an HPLC sample bottle to detect the product content.

[0161] (9) The vanillin content in the fermentation broth was detected by HPLC. The chromatographic column was a C18 column (ZORBAX SB-C18, 4.6×150 mm, 3.5 μm, Agilent, US). The detection conditions were as follows: injection volume 5 μl; mobile phase flow rate: 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was a methanol solution. A multi-step gradient elution method was used, that is, the concentration of mobile phase B increased from 5% to 50% from 0 to 15 min; the concentration of mobile phase B decreased from 50% to 5% from 15 to 16 min; the concentration of mobile phase B was maintained at 5% from 16 to 20 min, and the detection wavelength was 281 nm.

[0162] (10) The yield of vanillin was calculated based on the relationship curve between the absorption peak area and vanillin.

[0163] The experimental results are shown in Table 8.

[0164] Table 8 Effect of adding tributyrin on the fermentation production of vanillin by Clostridium Bf24-13.

[0165] Group Vanillin fermentation yield (g / L) Control group 4.2±0.3 Experimental Group 5.4±0.2

[0166] From the data in Table 8, we can see that without the addition of tributyrin, the content of vanillin in the fermentation broth of Clostridium was 4.2±0.3 g / L. When tributyrin was added to the fermentation medium, the fermentation yield of vanillin increased by 28% to 5.4±0.2 g / L. This indicates that the addition of tributyrin has a promoting effect on the production of vanillin by Clostridium fermentation.

[0167] The above are only preferred specific implementations of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and improved concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of tributyrin as an additive in increasing the yield of vanillin produced by microbial fermentation.

2. The use according to claim 1, characterized in that: The tributyrin is suitable for all microorganisms in microbiological methods.

3. The use according to claim 2, characterized in that: The microorganisms include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Streptomyces, Amycolatopsis, and Bacillus fusiformi.

4. The use according to claim 1, characterized in that: The volume of the tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method can be any volume ratio.

5. The use according to claim 4, characterized in that: The volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method is 10%-100%.

6. The use according to claim 1, characterized in that: The tributyrin is added at any time during the microbial fermentation cycle.

7. The use according to claim 6, characterized in that: The tributyrin is added within 6-18 hours after microbial fermentation.

8. The use according to any one of claims 1 to 7, characterized in that: The microbial method includes microbial fermentation method, microbial whole cell catalysis method and microbial enzyme method.

9. An additive for increasing the fermentation yield of vanillin synthesized by microbial method, characterized in that: The additive is tributyrin.

10. The additive for increasing the fermentation yield of vanillin synthesized by microbial method according to claim 9, characterized in that: The volume of tributyrin added to the liquid fermentation medium for synthesizing vanillin by microbial method can be any volume ratio, and the preferred adding time is any time within the microbial fermentation cycle.

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