A strain of Pichia kudrida and its application

Abstract

The present invention discloses a strain of Pichia kudrida and its application. The Pichia kudrida is Pichia kudrida ( Pichia Kudryavzevii )YK116, on March 17, 2023, was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.26823. The present invention also discloses the use of the kudni yeast in the simultaneous production of phenylacetaldehyde and 2-phenylethanol. The kudni yeast of the present invention ( Picea Kudryavzevi ) YK116 has high stress resistance, grows well in vinegar lees hydrolysate, has strong tolerance to inhibitors such as furfural and acetic acid in vinegar lees hydrolysate, and has the ability to degrade furfural, and can be used to ferment hydrolysate containing lignocellulose. It can be used to ferment phenylacetaldehyde and 2-phenylethanol, and can use cheap vinegar lees, which has huge emissions and causes serious environmental pollution, as raw materials. The method is simple to operate, has low production cost, is environmentally friendly and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to a strain of Pichia kudrida and application thereof. Background Art

[0002] Aromatic compounds account for a large proportion of fragrances, and traditional production methods include chemical synthesis and plant extraction that rely on petroleum resources. However, due to various issues, including oil depletion, environmental protection, global warming, and limited availability and yield of plants, biotechnology for producing aromatic compounds from renewable materials with the help of microorganisms has received great attention.

[0003] Phenylacetaldehyde and 2-phenylethanol in aromatic synthetics are important flavoring agents. Phenylacetaldehyde has a strong aroma of hosta flowers, and 2-phenylethanol has a rose scent. They are widely used in cosmetics, perfumes, food and other fields. At present, the industrial production methods of phenylacetaldehyde are all prepared by chemical synthesis, using phenyl compounds as raw materials to synthesize under high temperature and high pressure, which causes certain pollution to the environment. Microbial transformation has the advantages of sustainability, mild reaction conditions, and environmental friendliness, but due to metabolic flux problems, phenylacetaldehyde is difficult to be accumulated by microbial cells. At present, there are no research reports on the production of phenylacetaldehyde using microorganisms. 2-Phenylethanol is mainly synthesized by chemical synthesis, using phenyl compounds as raw materials under high temperature and high pressure, but the finished product contains carcinogenic byproducts such as styrene, and the price is low (US$5 per kilogram). The natural production method of 2-phenylethanol is mainly extracted from roses, but due to the seasonality and scarcity of raw materials, natural 2-phenylethanol is expensive (US$1,000 per kilogram) and cannot meet market demand. Research on the microbial synthesis of 2-phenylethanol mainly focuses on yeast and Escherichia coli. Compared with Escherichia coli, yeast strains have a synthetic pathway for 2-phenylethanol, and no exogenous gene introduction is required, which simplifies the complexity of strain construction. In addition, yeast strains have higher stress resistance and can more efficiently resist the growth inhibition of host strains caused by the external environment and metabolites. The pathways for yeast strains to synthesize 2-phenylethanol include the Ehrlich pathway and the phenylpyruvate synthesis pathway (also known as the de novo synthesis pathway). The Ehrlich pathway uses L-phenylalanine as a precursor and has high production costs; the phenylpyruvate synthesis pathway uses glucose as a precursor, but due to the many metabolic branches, the yield of 2-phenylethanol is low, and the yield of 2-phenylethanol in wild-type strains is mostly less than 200 mg / L. In addition, refined sugar is generally used as a carbon source to produce 2-phenylethanol. In order to further reduce production costs, it is necessary to find economically attractive production waste as a carbon source and a cheap nitrogen source to meet the growth of microorganisms and the production needs of target products.

[0004] Vinegar dregs are the main byproduct of traditional solid-state vinegar brewing, with an annual discharge of more than 3 million tons nationwide. The discarding of vinegar dregs will cause serious pollution to the air and soil, and is a waste of biological resources. Calculated on the basis of dry vinegar dregs, vinegar dregs mainly contain hemicellulose (16.22% to 38.90%), cellulose (22.96 to 34.91%), lignin (9.20 to 24.78%), crude protein (9.56 to 17.60%), various amino acids and inorganic salts, etc., and have high resource recovery value. Among them, crude protein can provide a nitrogen source for yeast growth after protease hydrolysis; the lignocellulose contained in vinegar dregs can generate glucose after hydrolysis to provide a carbon source for yeast growth. At present, the recycling technologies of vinegar dregs resources include anaerobic digestion to produce biogas, feed production, composting, soil / water remediation and fermentation to produce fuel ethanol and xylose, etc., but the added value is low and it is impossible to truly realize the efficient and comprehensive utilization of vinegar dregs resources. In addition, the methods for pretreatment of lignocellulose include steam explosion, acid treatment, alkali treatment and biodegradation. Among them, the acid treatment method has become the most commonly used method for pretreatment of lignocellulose raw materials due to its advantages such as good hydrolysis effect, simple equipment requirements and short extraction time. However, this method will produce harmful substances such as furfural, which seriously inhibits the growth of strains. Therefore, the lignocellulose raw materials after dilute acid treatment must be detoxified before use. However, detoxification will produce new pollutants and greatly increase production costs. Therefore, it is of great significance to maximize the value of resource recovery and develop environmentally friendly and cost-effective vinegar lees resource recovery technology. If a highly stress-resistant strain can be selected, it can produce high-value products such as phenylacetaldehyde and 2-phenylethanol from undetoxified vinegar lees hydrolyzate, which will have broad market prospects. Summary of the invention

[0005] Objectives of the invention: The first objective of the present invention is to provide a strain of Pichia kudriavzevii YK116 that has a strong tolerance to furfural and acetic acid and can decompose furfural. The second objective of the present invention is to provide a method for simultaneously producing phenylacetaldehyde and 2-phenylethanol using the above-mentioned Pichia kudriavzevii YK116.

[0006] Technical solution: The Pichia kudriavzevii strain described in the present invention is Pichia kudriavzevii YK116, which was deposited in the China General Microbiological Culture Collection Center on March 17, 2023, with a deposit number of CGMCC No.26823.

[0007] The invention also provides an application of the Pichia kudriavzevii YK116 in the simultaneous production of phenylacetaldehyde and 2-phenylethanol.

[0008] The present invention also provides a method for simultaneously producing phenylacetaldehyde and 2-phenylethanol using the above-mentioned Pichia kudriavzevii YK116, comprising the following steps:

[0009] (1) using the above-mentioned Pichia kudriavzevii YK116 to prepare a strain seed solution;

[0010] (2) The strain seed liquid is inoculated into vinegar lees hydrolyzate for fermentation to obtain phenylacetaldehyde and 2-phenylethanol.

[0011] Furthermore, the preparation of the strain seed solution in step 1) includes the preparation of a primary seed solution and a secondary seed solution.

[0012] Furthermore, the preparation of the primary seed solution specifically includes the following steps: picking a ring of activated Pichiakudriavzevii YK116 and inoculating it into 10 mL of seed culture medium, culturing at 28-40° C. and a rotation speed of 180-200 r / min for 10-18 hours.

[0013] Furthermore, the preparation of the secondary seed solution includes the following steps: inoculating the primary seed solution into 50 mL of seed culture medium at an inoculation rate of 2-10%, culturing at 28-40° C. and a rotation speed of 180-200 r / min for 10-18 hours.

[0014] Furthermore, the seed culture medium is 10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance is water.

[0015] Furthermore, the preparation steps of the vinegar grains hydrolyzate in step 2) include crushing the vinegar grains, acid pretreatment and enzyme hydrolysis.

[0016] Specifically, dry vinegar dregs are taken and mechanically crushed, passed through a 60-100 mesh sieve, and the crushed vinegar dregs are soaked in a 1-2% (w / v) dilute sulfuric acid aqueous solution with a solid-liquid ratio of 25-35% (w / w), soaked for 6-8 hours, and then heated at 120-140° C. for 1-2 hours. After cooling, 4-6 mol / L NaOH is used to adjust the pH to 4.5-6.0, and cellulase is added in an amount of 20-30 FPU / g dry vinegar dregs, the hydrolysis temperature is 45-55° C., the rotation speed is 180-200 r / min, and the hydrolysis is carried out for 60-72 hours. Finally, 4-6 mol / L NaOH is used to adjust the solution pH to 6.5-7.5, and neutral protease is added in an amount of 1500-3000 u / g dry vinegar dregs, the rotation speed is 180-200 r / min, and the hydrolysis is carried out at 45-55° C. for 3-4 hours, and then the undetoxified vinegar dregs hydrolyzate is obtained by centrifugation.

[0017] Furthermore, the fermentation culture described in step 2) specifically includes the following steps: inoculating the secondary seed liquid into 100 mL of fermentation medium at an inoculation rate of 2 to 10%, culturing at 28 to 40° C., at a rotation speed of 180 to 200 r / min, for 36 to 52 hours; the fermentation medium includes KH2PO4, MgSO4·7H2O and vinegar lees hydrolyzate.

[0018] Furthermore, the fermentation medium also includes one or more of yeast powder, peptone or L-phenylalanine.

[0019] Further, the fermentation medium is KH2PO4 5g / L, MgSO4·7H2O 0.5g / L, and the balance is vinegar lees hydrolyzate. The fermentation medium can also be yeast powder 5g / L, peptone 5g / L, KH2PO4 5g / L, MgSO4·7H2O 0.5g / L, and the balance is vinegar lees hydrolyzate. The fermentation medium can also be L-phenylalanine 5g / L, KH2PO4 5g / L, MgSO4·7H2O0.5g / L, and the balance is vinegar lees hydrolyzate.

[0020] In the method for producing 2-phenylethanol of the present invention, the carbon source in the fermentation medium is glucose generated by hydrolyzing cellulose in vinegar grains with cellulase; the nitrogen source in the fermentation medium is various nitrogen-containing compounds generated by hydrolyzing crude protein in vinegar grains with neutral protease and various amino acids originally in vinegar grains.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the Pichia kudriavzevii YK116 of the present invention has high stress resistance, grows well in vinegar lees hydrolyzate, has strong tolerance to inhibitors such as furfural and acetic acid present in the vinegar lees hydrolyzate, and has the ability to degrade furfural, and can be used to ferment hydrolyzate containing lignocellulose. It is used to ferment and generate phenylacetaldehyde and 2-phenylethanol, and can use vinegar lees, which are cheap, have huge emissions, and cause serious environmental pollution, as raw materials. The method is simple to operate, has low production cost, is environmentally friendly, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth of Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 in medium containing different concentrations of acetic acid;

[0023] Figure 2 The growth of Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 in culture medium containing different concentrations of furfural;

[0024] Figure 3The growth and furfural degradation of Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 in non-detoxified vinegar grains hydrolyzate;

[0025] Figure 4 Gas chromatogram (A) and mass spectra at 7.099 min (phenylacetaldehyde, B) and 8.144 min (2-phenylethanol, C) of the fermentation organic extract;

[0026] Figure 5 It represents the changes in the production of phenylacetaldehyde and 2-phenylethanol of the strain in the fermentation broth during the fermentation process. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] The Pichia kudriavzevii YK116 used in the following examples was isolated from the vinegar mash in the Zhenjiang aromatic vinegar production workshop and was deposited in the China General Microbiological Culture Collection Center on March 17, 2023, with the deposit address: Beijing, China, and the deposit number is CGMCC No.26823.

[0029] Example 1: Test of the acetic acid and furfural resistance of Pichia kudriavzevii YK116

[0030] In order to investigate the tolerance of Pichia kudriavzevii YK116 to acetic acid and furfural, the model strain of Saccharomyces cerevisiae BY4741 (ATCC 4040002) was selected as the control strain.

[0031] Seed culture medium: yeast powder 10g / L, protein 20g / L, glucose 20g / L, and the balance is water.

[0032] Acetic acid stress culture medium: the concentrations of acetic acid are 0, 5, 7, and 20 g / L respectively, and the concentrations of the other ingredients are yeast powder 10 g / L, protein 20 g / L, glucose 20 g / L, and the balance is water.

[0033] Furfural stress culture medium: the furfural concentrations were 0, 1, 2, 3, and 4 g / L, respectively; the concentrations of the other ingredients were 10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the remainder was water.

[0034] Pick a ring of activated Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 from the plate and inoculate into a 50 mL conical flask with a seed culture medium volume of 10 mL, place it in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min, and culture it continuously for 12 hours to obtain the first-level seed solution;

[0035] The first-level seed liquid of Pichia kudriavzevii YK116 and the model strain of Saccharomyces cerevisiae BY4741 were inoculated into a 250 mL conical flask with a seed culture medium liquid volume of 50 mL at a 5% inoculation rate, respectively, and placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min, and cultured continuously for 12 hours to obtain a second-level seed liquid;

[0036] The secondary seed liquid of the above-mentioned Pichia kudriavzevii YK116 and the Saccharomyces cerevisiae model strain BY4741 was inoculated into a 250 mL conical flask containing 50 mL acetic acid pressure medium or 50 mL furfural pressure medium at an inoculation rate of 5%, respectively, and placed in a shaking incubator at a temperature of 30° C. and a rotation speed of 180 r / min for continuous culture, and samples were taken regularly to detect the growth of the strains.

[0037] The results of the acetic acid tolerance test (such as Figure 1 As shown in Figure 2, when 7 g / L acetic acid was added to the culture medium, Pichia kudriavzevii YK116 grew slowly in the first 72 hours, but then grew rapidly, and the final biomass was no different from the biomass of strain YK116 without acetic acid. When 20 g / L acetic acid was added to the culture medium, the growth of strain YK116 was severely inhibited, but some strains still grew, and its biomass was 22.6% of the biomass without acetic acid. However, when 5 g / L acetic acid was added to the culture medium, the growth of the model strain of Saccharomyces cerevisiae BY4741 was severely inhibited, the growth lag period was as long as 72 hours, and the final biomass was only 15.8% of the biomass without acetic acid. When 7 g / L acetic acid was added to the culture medium, the growth of the model strain of Saccharomyces cerevisiae BY4741 was completely inhibited. This result shows that compared with the Saccharomyces cerevisiae model strain BY4741, Pichia kudriavzevii YK116 has a strong resistance to acetic acid inhibition, and its acetic acid tolerance reaches 20g / L.

[0038] The results of the furfural tolerance test (such as Figure 2As shown in the figure, when 2.0 g / L furfural was added to the culture medium, the growth of Pichia kudriavzevii YK116 was almost unaffected compared with the culture medium without furfural. When 3.0 g / L furfural was added, the growth of Pichia kudriavzevii YK116 was slow, but the final biomass was no different from the biomass of strain YK116 without furfural. When 4 g / L acetic acid was added to the culture medium, the growth of strain YK116 was severely inhibited, but some strains still grew, and its biomass was 16.7% of the biomass without furfural. However, when 1.0 g / L furfural was added to the culture medium, the growth of the model strain of Saccharomyces cerevisiae BY4741 was severely inhibited, and its final biomass was only 22.7% of the biomass without furfural. When 2.0 g / L furfural was added to the culture medium, the growth of the model strain of Saccharomyces cerevisiae BY4741 was completely inhibited. This result shows that compared with the Saccharomyces cerevisiae model strain BY4741, Pichia kudriavzevii YK116 has a strong anti-furfural inhibition performance, and its furfural tolerance capacity reaches 4.0g / L.

[0039] Example 2 Preparation of vinegar lees hydrolyzate

[0040] Take the vinegar mash sample after the fermentation of aromatic vinegar in the aromatic vinegar production workshop of Zhenjiang, dry it in a drying oven, crush it mechanically, pass it through an 80-mesh sieve, soak the crushed vinegar lees in a 1.5% (w / v) dilute sulfuric acid aqueous solution with a solid-liquid ratio of 30% (w / w), soak it for 6 hours and heat it at 121°C for 2 hours. After cooling, adjust the pH to 5.0 with 6mol / L NaOH, add cellulase in an amount of 25FPU / g dry vinegar lees, hydrolyze at 50°C, rotate at 200r / min, and hydrolyze for 72h. Finally, adjust the solution pH to 7.0 with 6mol / L NaOH, add neutral protease in an amount of 2000u / g dry vinegar lees, rotate at 200r / min, hydrolyze at 55°C for 3h, and then centrifuge to obtain the undetoxified vinegar lees hydrolyzate. HPLC detection shows that the undetoxified hydrolyzate contains 32.4g / L glucose, 8.3g / L acetic acid and 1.2g / L furfural.

[0041] Example 3 Comparison of the growth performance of Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 in non-detoxified vinegar lees hydrolyzate

[0042] Seed culture medium: yeast powder 10g / L, protein 20g / L, glucose 20g / L, and the balance is water.

[0043] Fermentation medium: KH2PO4 5 g / L, MgSO4·7H2O 0.5 g / L, the balance being the non-detoxified vinegar grains hydrolyzate prepared in Example 2.

[0044] Pick a ring of activated Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 (ATCC 4040002) from the plate and inoculate into a 50 mL conical flask containing 10 mL seed culture medium, place it in a shaking incubator at 30°C and 180 r / min, and culture continuously for 12 h to obtain the primary seed solution;

[0045] The first-level seed solutions of Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 were inoculated into 250 mL conical flasks containing 50 mL seed culture medium at an inoculation rate of 5%, respectively, and placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min, and cultured continuously for 12 hours to obtain the second-level seed solution;

[0046] The secondary seed liquid of the above-mentioned Pichia kudriavzevii YK116 and Saccharomyces cerevisiae model strain BY4741 was inoculated into a 500 mL conical flask containing 100 mL fermentation medium at an inoculation rate of 5%, and placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min for continuous culture, and samples were taken regularly to detect the growth of the strains.

[0047] The results are as follows Figure 3 As shown in the figure, Pichia kudriavzevii YK116 grew well in the non-detoxified vinegar lees hydrolyzate. The OD of the strain was 600 The furfural content in the hydrolyzate was reduced to 0 g / L. However, the cell growth of the model strain BY4741 of Saccharomyces cerevisiae was completely inhibited, indicating that Pichia kudriavzevii YK116 has high stress resistance and can use the undetoxified vinegar lees hydrolyzate to grow strains and then ferment to produce high-value compounds.

[0048] Example 4: Synthesis of phenylacetaldehyde and 2-phenylethanol by fermentation of undetoxified vinegar residue hydrolyzate by Pichia kudriavzevii YK116

[0049] (1) Implementation of the fermentation process

[0050] Pick a ring of activated Pichia kudriavzevii YK116 from the plate and inoculate it into a 50 mL conical flask containing 10 mL seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance is water), place it in a shaking incubator at 30°C and 180 r / min, and culture it continuously for 12 h to obtain the primary seed solution;

[0051] The first-level seed solution of Pichia kudriavzevii YK116 was inoculated into a 250-mL conical flask containing 50 mL of seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance was water) at a 5% inoculation rate, and the flask was placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min for continuous cultivation for 12 hours to obtain the second-level seed solution;

[0052] The secondary seed liquid of Pichia kudriavzevii YK116 was inoculated with 5% inoculum into a 500 mL conical flask containing 100 mL fermentation medium (KH2PO4 5 g / L, MgSO4·7H2O 0.5 g / L, the remainder being the undetoxified vinegar lees hydrolyzate prepared in Example 2), and the mixture was placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min for fermentation for 40 h.

[0053] (2) Qualitative analysis of phenylacetaldehyde and 2-phenylethanol

[0054] After the fermentation is completed, take 50mL of fermentation liquid and centrifuge at 10000r / min for 10min to collect the supernatant, add 20mL of pentane and cyclohexane mixture (2:1, v:v), and shake continuously to extract the phenylacetaldehyde and 2-phenylethanol in the supernatant into the organic phase. After separating the organic phase, add Na2SO4 to dry, and finally take 1μL of the organic phase for injection. Detection instrument: Agilent GC-MS. Detection conditions: HP-5ms chromatographic column, helium flow rate of 1.2mL / min, inlet temperature of 250℃, heating program is initially 50℃ for 1min, then increase the temperature to 230℃ at 10℃ / min, and finally maintain 230℃ for 5min. The results show ( Figure 4 ), Pichia kudriavzevii YK116 can use vinegar lees hydrolyzate to synthesize phenylacetaldehyde and 2-phenylethanol.

[0055] (3) Determination of the production of phenylacetaldehyde and 2-phenylethanol

[0056] During the fermentation process, samples were taken at regular intervals, and the sampled fermentation broth was centrifuged at 10,000 r / min for 10 min to collect the supernatant. The supernatant was filtered through a 0.22 μm microporous filter membrane and then detected by high performance liquid chromatography (HPLC, Agilent). The determination conditions were: RP-C18 column, detection wavelength 210 nm, mobile phase 50% methanol, flow rate 0.8 mL / min, column temperature 30 ° C, injection volume 20 μL. The results showed ( Figure 5 ), at the end of fermentation, the production of phenylacetaldehyde was 83.1 mg / L, and the production of 2-phenylethanol was 232.7 mg / L.

[0057] Example 5: Synthesis of phenylacetaldehyde and 2-phenylethanol by fermentation of undetoxified vinegar residue hydrolyzate by Pichia kudriavzevii YK116

[0058] (1) Implementation of the fermentation process

[0059] Pick a ring of activated Pichia kudriavzevii YK116 from the plate and inoculate it into a 50 mL conical flask containing 10 mL seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance is water), place it in a shaking incubator at 30°C and 180 r / min, and culture it continuously for 12 h to obtain the primary seed solution;

[0060] The first-level seed solution of Pichia kudriavzevii YK116 was inoculated into a 250-mL conical flask containing 50 mL of seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance was water) at a 5% inoculation rate, and the flask was placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min for continuous cultivation for 12 hours to obtain the second-level seed solution;

[0061] The secondary seed liquid of Pichia kudriavzevii YK116 was inoculated with 5% inoculum into a 500 mL conical flask containing 100 mL fermentation medium (yeast powder 5 g / L, peptone 5 g / L, KH2PO4 5 g / L, MgSO4.7H2O 0.5 g / L, and the balance was the non-detoxified vinegar lees hydrolyzate prepared in Example 2), and the mixture was placed in a shaking table at a temperature of 30°C and a rotation speed of 180 r / min for fermentation culture for 40 h. The fermentation was completed.

[0062] (2) Determination of the production of phenylacetaldehyde and 2-phenylethanol

[0063] During the fermentation process, samples were taken at regular intervals, and the sampled fermentation broth was centrifuged at 10,000 r / min for 10 min to collect the supernatant, which was filtered through a 0.22 μm microporous filter membrane and then detected by high performance liquid chromatography (HPLC, Agilent). Determination conditions: RP-C18 column, detection wavelength 210 nm, mobile phase 50% methanol, flow rate 0.8 mL / min, column temperature 30°C, injection volume 20 μL.

[0064] The results showed that the growth of the strain was significantly improved after adding yeast powder and peptone to the fermentation medium. At the end of the fermentation, the OD 600 was 18.14, and the yields of phenylacetaldehyde and 2-phenylethanol increased accordingly, reaching 164.8 mg / L and 388.5 mg / L, respectively.

[0065] Example 6 Synthesis of phenylacetaldehyde and 2-phenylethanol by fermentation of undetoxified vinegar residue hydrolyzate by Pichia kudriavzevii YK116

[0066] (1) Implementation of the fermentation process

[0067] Pick a ring of activated Pichia kudriavzevii YK116 from the plate and inoculate it into a 50 mL conical flask containing 10 mL seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance is water), place it in a shaking incubator at 30°C and 180 r / min, and culture it continuously for 12 h to obtain the primary seed solution;

[0068] The first-level seed solution of Pichia kudriavzevii YK116 was inoculated into a 250-mL conical flask containing 50 mL of seed culture medium (10 g / L yeast powder, 20 g / L protein, 20 g / L glucose, and the balance was water) at a 5% inoculation rate, and the flask was placed in a shaking incubator at a temperature of 30°C and a rotation speed of 180 r / min for continuous cultivation for 12 hours to obtain the second-level seed solution;

[0069] The secondary seed liquid of Pichia kudriavzevii YK116 was inoculated into a 500mL conical flask containing 100mL fermentation medium (L-phenylalanine 5g / L, KH2PO4 5g / L, MgSO4·7H2O 0.5g / L, the remainder being the undetoxified vinegar lees hydrolyzate prepared in Example 2) at an inoculum size of 5%, and the fermentation was carried out in a shaking table at a temperature of 30°C and a rotation speed of 180r / min for 40h, and the fermentation was completed.

[0070] (2) Determination of the production of phenylacetaldehyde and 2-phenylethanol

[0071] During the fermentation process, samples were taken at regular intervals, and the sampled fermentation broth was centrifuged at 10,000 r / min for 10 min to collect the supernatant, which was filtered through a 0.22 μm microporous filter membrane and then detected by high performance liquid chromatography (HPLC, Agilent). Determination conditions: RP-C18 column, detection wavelength 210 nm, mobile phase 50% methanol, flow rate 0.8 mL / min, column temperature 30°C, injection volume 20 μL.

[0072] The results showed that after adding L-phenylalanine, the synthetic precursor of phenylacetaldehyde and 2-phenylethanol, into the fermentation medium, the production of phenylacetaldehyde and 2-phenylethanol increased significantly, reaching 877.3 mg / L and 2632.7 mg / L, respectively.

Claims

1. A method of using vinegar residue hydrolyzate and Pichia kudriba ( Pichia kudriavzevii ) A method for synthesizing phenylacetaldehyde and 2-phenylethanol by fermentation of YK116, characterized in that: The following steps are involved: (1) Using Pichia kudrida Pichia kudriavzevii ) YK116 strain seed solution preparation; (2) inoculating the strain seed liquid into vinegar lees hydrolyzate for fermentation and culture to obtain phenylacetaldehyde and 2-phenylethanol; The Pichia kudrida Pichia kudriavzevii )YK116 was deposited in the China General Microbiological Culture Collection Center on March 17, 2023, with the deposit number CGMCC No. 26823.

2. The method according to claim 1, characterized in that The preparation of the strain seed solution in step 1) includes the preparation of the primary seed solution and the preparation of the secondary seed solution.

3. The method according to claim 2, characterized in that The preparation of the first-level seed solution specifically comprises the following steps: picking a ring of activated Pichia kudriavzevii YK116 was inoculated into 10 mL of seed culture medium and cultured at 28-40°C and 180-200 r / min for 10-18 h.

4. The method according to claim 2, characterized in that: The preparation of the secondary seed solution comprises the following steps: inoculating the primary seed solution into 50 mL of seed culture medium at an inoculation rate of 2-10%, culturing at 28-40° C. and a rotation speed of 180-200 r / min for 10-18 h.

5. The method according to claim 1, characterized in that Step 2) The preparation of the vinegar lees hydrolyzate comprises the steps of crushing the vinegar lees, acid pretreatment and enzyme hydrolysis.

6. The method according to claim 5, characterized in that The preparation steps of the vinegar lees hydrolyzate specifically include taking dried vinegar lees and mechanically crushing them, passing them through a 60-100 mesh sieve, soaking the crushed vinegar lees in a 1-2% (w / v) dilute sulfuric acid aqueous solution with a solid-liquid ratio of 25-35% (w / w), soaking for 6-8 hours, and then heating at 120-140 ° C for 1-2 hours; after cooling, adjusting the pH to 4.5-6.0 using 4-6 mol / L NaOH, adding cellulase in an amount of 20-30 FPU / g of dry vinegar lees, hydrolyzing at a temperature of 45-55 ° C, rotating at a speed of 180-200 r / min, and hydrolyzing for 60-72 hours; finally, adjusting the solution pH to 6.5-7.5 using 4-6 mol / L NaOH, adding neutral protease in an amount of 1500-3000 u / g of dry vinegar lees, rotating at a speed of 180-200 r / min, and hydrolyzing at 45-55 ° C for 3-4 After h, the non-detoxified vinegar grains hydrolyzate was obtained by centrifugation.

7. The method according to claim 1, characterized in that The fermentation culture described in step 2) specifically comprises the following steps: inoculating the secondary seed liquid into 100 mL of fermentation medium at an inoculation rate of 2-10%, culturing at 28-40°C and a rotation speed of 180-200 r / min for 36-52 h; the fermentation medium comprises KH2PO4, MgSO4·7H2O and vinegar lees hydrolyzate.

8. The method according to claim 7, characterized in that The fermentation medium further comprises one or more of yeast powder, peptone or L-phenylalanine.

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