Rhodotorula sp. And application thereof
By screening out the red yeast Rhodotorula dairenensis S9 from the bean paste, the safety and environmental problems of existing ethyl phenylacetate synthesis are solved, and the simple and efficient production of ethyl phenylacetate is achieved for food fermentation and fragrance production, which enhances the food flavor.
Patent Information
- Application Number
- CN202510626333.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
The existing synthesis method of ethyl phenylacetate has problems such as safety hazards, environmental pollution, high cost, cumbersome steps and difficulty in applying it to food production.
A salt-resistant and acid-resistant red yeast Rhodotorula dairenensis S9 was screened, isolated and purified from the bean paste, which was used for fermentation and production of ethyl phenylacetate, and was used for food fermentation and fragrance production.
It achieves safe, simple and efficient production of ethyl phenylacetate, enhances the flavor of foods such as bean paste, soy sauce and bread, and provides an environmentally friendly synthesis method.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of microorganisms, and particularly relates to a high-salt-resistant, acid-resistant red yeast (Rhodotorula dairenensis) that produces ethyl phenylacetate and an application thereof. Background Art
[0002] Ethyl phenylacetate has a strong, sweet honey aroma. It is commonly used in the production of synthetic fragrances, such as various floral fragrances, as a solvent and fragrance adjuvant. It is also used in organic synthesis and as a pesticide and pharmaceutical intermediate. Current synthesis methods include the following: 1. Reaction of benzyl cyanide with ethanol in the presence of sulfuric acid; 2. Direct esterification of phenylacetic acid with ethanol; 3. Hydrolysis of phenylacetamide followed by esterification; 4. Other methods include indirect conversion to benzaldehyde and organometallic reagents.
[0003] However, existing synthetic methods for ethyl phenylacetate generally suffer from significant drawbacks. The sodium cyanide method, relying on highly toxic raw materials and producing cyanide-containing wastewater, poses serious safety and environmental risks. Furthermore, the byproducts are foul-smelling and yields are unstable. The direct esterification method requires the use of highly corrosive concentrated sulfuric acid, which can easily induce side reactions such as dehydration and carbonization, resulting in complex post-processing and difficult wastewater disposal. The phenylacetamide hydrolysis-esterification method is cumbersome, with overall yields limited by the multi-step reaction process. The Friedel-Crafts acylation method requires stringent anhydrous conditions and highly toxic reagents (such as liquid bromine), making industrialization difficult and energy-intensive. Other methods, such as indirect conversion to benzaldehyde or organometallic reagents, are costly and lengthy. Furthermore, most methods face common challenges, such as limited catalyst selection, strong acid corrosion of equipment, and difficulty in product purification, hindering their application in food production.
[0004] Therefore, there is an urgent need to find an environmentally friendly, simple, safe, efficient and stable method for producing ethyl phenylacetate so that it can be applied in more fields. Summary of the Invention
[0005] In order to develop a new source of ethyl phenylacetate, the present invention screened, separated and purified a salt-tolerant and acid-tolerant red yeast that produces ethyl phenylacetate from broad bean paste, and used the same.
[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:
[0007] In a first aspect, the present invention provides Rhodotorula dairenensis S9, deposited with CCTCC No. M 2025465 on March 13, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei 430072, China. The strain is designated Rhodotorula dairenensis S9.
[0008] The nucleotide sequence of the ITS region of the red yeast S9 is shown in SEQ ID NO: 1.
[0009] SEQ ID NO: 1
[0010] .
[0011] Among them, the colony and cell characteristics of the above-mentioned red yeast S9 are: the colony edges are regular, red, the center is raised, the surface is smooth, and the surface is moist and easy to pick up.
[0012] The growth temperature of the red yeast S9 is 24-36°C, and the optimal growth temperature is 30°C.
[0013] The growth pH of the red yeast S9 is 2.0-7.0, and the optimal growth pH is 4.0-6.0.
[0014] Wherein, the volume concentration of the growth salt of the red yeast S9 is 5% to 20%.
[0015] In a second aspect, the present invention provides a microbial agent comprising the fermentation liquid, seed liquid or bacterial cells of the above-mentioned red yeast S9.
[0016] In a third aspect, the present invention provides the use of the above-mentioned red yeast S9 or microbial agent in the production of ethyl phenylacetate, ethyl caprate or ethyl laurate.
[0017] In a fourth aspect, the present invention provides the use of the above-mentioned red yeast S9 or microbial agent in the preparation of broad bean paste, soy sauce, fermented black beans, bread or liquor fermentation.
[0018] The liquor is selected from at least one of the following types: Luzhou-flavor liquor, Maotai-flavor liquor, Fen-flavor liquor and mixed-flavor liquor.
[0019] In a fifth aspect, the present invention provides a method for producing ethyl phenylacetate by metabolizing the above-mentioned red yeast S9 or microbial agent, which comprises the following steps:
[0020] After the red yeast S9 or microbial agent is activated, it is inoculated into the fermentation raw materials and cultured and fermented.
[0021] Wherein, the inoculation amount of the red yeast S9 or microbial agent is 0.3% to 1% (v / v).
[0022] The fermentation temperature is 24-36°C, preferably 30°C.
[0023] Wherein, the fermentation time is 20 to 40 days.
[0024] In a sixth aspect, the present invention provides a method for isolating, screening and identifying the above-mentioned red yeast S9, which comprises the following steps:
[0025] The fermentation of fermented fermented fermented soybean paste was carried out by preparing a sample bacterial suspension, which was enriched and separated to screen out the strain with the highest ethyl phenylacetate production. The red yeast S9 was identified by combining morphological, physiological and biochemical characteristics and / or molecular biology.
[0026] The fermentation medium is YPD medium, which includes 20.0 g / L glucose, 150.0 g / L NaCl, 20.0 g / L peptone, 10.0 g / L yeast extract powder, and 20.0 g / L solid culture medium plus agar.
[0027] Beneficial Effects: The present invention screened, isolated, and purified a salt- and acid-tolerant red yeast strain (Rhodotorula dairenensis) S9 from fermented broad bean paste (doubanjiang). The strain is deposited with CCTCC NO: M 2025465. This strain exhibits high-temperature, acid-, and salt-resistance. Therefore, when used in fermentation of broad bean paste, it not only metabolizes and produces a variety of esters but also increases its ethyl phenylacetate production from 104.86 μg / kg to 522.11 μg / kg. This Rhodotorula S9, when used in the food fermentation industry, not only enhances the flavor of fermented foods such as broad bean paste, soy sauce, and bread, but also provides a new, simple, environmentally friendly, and safe method for synthesizing ethyl phenylacetate, promising broad prospects in food applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 The morphological diagram of the red yeast S9 strain in Example 1 (a) and the phylogenetic tree diagram in Example 2 (b);
[0029] Figure 2 This is a diagram of the synthesis of ethyl phenylacetate by red yeast S9 in YPD medium in Example 1;
[0030] Figure 3 The results of the effects of different temperatures and pH on red yeast S9 in Example 3 (a: temperature, b: pH) and the growth curve under different salt concentration conditions (c);
[0031] Figure 4 This is a graph showing the effect of red yeast S9 on the volatile compound content of broad bean paste after 30 days of fermentation in Example 4; a: total volatile compound quantity, b: total volatile compound content, c: ethyl phenylacetate content.
[0032] Description of the deposit of the strain of the present invention:
[0033] The present invention provides a strain of red yeast (Rhodotorula dairenensis) S9, with a deposit number of CCTCCNO: M2025465. The deposit date is March 13, 2025, and the collection center is China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The strain is designated as Rhodotorula dairenensis S9. DETAILED DESCRIPTION
[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.
[0035] In one embodiment of the present invention, a salt-tolerant and acid-tolerant red yeast strain (Rhodotorula dairenensis) S9, which produces ethyl phenylacetate, was screened, isolated, and purified from fermented broad bean paste. The strain was deposited with the China Center for Type Culture Collection (CCTCC) on March 13, 2025, at the Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.
[0036] Based on the regular circular, red, centrally raised colony shape, and the fact that it was easily lifted when moist, and the nucleotide sequence of its ITS region was shown in SEQ ID NO: 1, it was identified as red yeast and classified as Rhodotorula dairenensis S9.
[0037] In one embodiment of the present invention, the red yeast S9 is salt-resistant, high-temperature-resistant and acid-resistant, with a growth temperature of 24-36°C, an optimal growth temperature of 30°C, a growth pH of 2.0-7.0, an optimal growth pH of 4.0-6.0, and a growth salt volume concentration of 5%-20%.
[0038] In one embodiment of the present invention, fermentation of Rhodotorula S9 can produce a variety of volatile substances, particularly high yields of ethyl phenylacetate. Therefore, this strain and microbial agents containing it can be used in the preparation of broad bean paste, soy sauce, fermented black beans, bread, or in the fermentation of white wine.
[0039] In a specific embodiment of the present invention, red yeast S9 is fermented in broad bean paste to produce a variety of flavor substances, especially ethyl phenylacetate, ethyl caprate, and ethyl laurate.
[0040] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.
[0041] The culture medium involved in the embodiment is as follows:
[0042] YPD medium: glucose 20.0 g / L, NaCl 150.0 g / L, peptone 20.0 g / L, yeast extract powder 10.0 g / L, solid medium plus agar 20.0 g / L, 121°C, sterilize for 20 min.
[0043] Example 1: Isolation, screening and purification of red yeast S9
[0044] (1) Enrichment and separation
[0045] After sterilization, 10 g of fermented bean paste (fermented fermented bean paste provided by Sichuan Fan Sao Guang Food Co., Ltd. and aged for one month) was weighed into a 250 mL conical flask containing 90 mL of saline (15% NaCl). The sample was shaken in a shaker at 30°C and 200 r / min for 30 min. The supernatant was diluted to 10% with sterile saline (15% NaCl). -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Evenly spread 100 μL of the dilution onto YPD solid medium and incubate inverted in a 30°C constant temperature incubator for 2–4 days. Pick a large number of single colonies with different morphologies and streak them onto YPD solid medium. After multiple isolation and purification, pick 65 colonies, inoculate them onto slant medium, incubate at 30°C for 36 hours, and store in a 4°C refrigerator for later use.
[0046] (2) Fermentation screening
[0047] Individual strains were inoculated into sterilized YPD medium and cultured at 30°C and 200 rpm for 48 h. The culture fluid in each flask was then centrifuged at 4°C and 8000 rpm for 20 min. The supernatant was collected and tested for ethyl phenylacetate synthesis. Table 1 shows the strains that produced ethyl phenylacetate. Strain S9 was screened for the highest ethyl phenylacetate production, with a yield of 5.1 μg / mL.
[0048] Table 1 Content of ethyl phenylacetate in fermentation broth of each strain
[0049] strains Ethyl phenylacetate (μg / mL) S1 1.4 S4 0.2 S6 4.5 S7 4.1 S8 3.9 S9 5.1 S19 2.3 S20 4.2 S21 4.3 S46 0.2 S47 0.3 S48 0.9 S63 0.3 S64 0.9 S65 4.2
[0050] Detection of ethyl phenylacetate: Detection was performed using a gas chromatograph using the following method.
[0051] GC: Chromatographic column: DB-624 (60m×0.32mm×1.80u), split injection, split volume ratio of 50:1; hydrogen flow rate: 30mL / min; nitrogen flow rate: 30mL / min; air flow rate: 300mL / min; inlet temperature: 160℃; pressure: 63.9302kPa.
[0052] Heating program: starting temperature 60°C, hold constant for 2 min, then increase to 90°C at 30°C / min, hold for 2 min, then increase to 150°C at 30°C / min, hold for 2 min; FID detector temperature: 200°C.
[0053] Preparation of reference solution: Accurately weigh 3 mg of ethyl phenylacetate and internal standard (methyl heptanoate) into a 100 mL volumetric flask, dilute to the mark with methanol, and shake well.
[0054] Linearity Assessment: 1, 2, 4, 6, and 8 mL of ethyl phenylacetate standard were measured in 100 mL volumetric flasks. 5 mL of internal standard was added to each flask. Each flask was accurately weighed, diluted to the mark with methanol, and shaken well. Under the above chromatographic conditions, 1 μL of each flask was injected into the gas chromatograph. The chromatogram was recorded and the results were repeated three times. A standard curve was plotted with the mass of ethyl phenylacetate as the abscissa and the peak area ratio of ethyl phenylacetate to the internal standard as the ordinate. The linear regression equation for ethyl phenylacetate was y = 0.845x - 0.0123, with R² = 0.9934, indicating a good linear relationship between the mass concentration of ethyl phenylacetate and the peak area ratio within the range of 4.5 to 45 μg / mL.
[0055] (3) Synthesis of ethyl phenylacetate in YPD medium
[0056] The screened strain S9 was inoculated into sterilized YPD medium and cultured at 30°C and 200 rpm. 5 mL of culture solution was collected on days 1, 2, 4, and 6. The culture solution was centrifuged at 4°C and 8000 rpm for 50 min, and the supernatant was collected to detect the ethyl phenylacetate content. Figure 2 As shown in the figure, ethyl phenylacetate in the culture medium reached its peak on the 2nd day and still had a high concentration of ethyl phenylacetate on the 6th day, indicating that strain S9 has a good ability to produce ethyl phenylacetate.
[0057] The above process is to isolate, purify and functionally identify the strains, and the purpose is to screen out the strains with high ethyl phenylacetate production. Among them, the colonies of strain S9 are regular round, red, with a raised center, and are easy to pick up when wet ( Figure 1 a).
[0058] Example 2: ITS molecular identification of Rhodotorula S9
[0059] The ITS region DNA of the screened strain S9 was detected, and the ITS gene sequence was compared and homology analyzed in the NCBI and BLAST systems. The phylogenetic tree was constructed according to the neighbor-joining method. Figure 1 As shown in Figure 2b, the results showed that strain S9 clustered on the same phylogenetic branch as Rhodotorula dairenensis strain A2W1-063. Combined with its colony and cellular characteristics, the strain was identified as belonging to Rhodotorula dairenensis, with regular red edges, a central raised area, and a smooth, moist, and easily lifted surface. The ITS region sequence of strain S9 is shown in SEQ ID NO:1.
[0060] Strain S9 was deposited with the China Center for Type Culture Collection (CCTCC) on March 13, 2025, at the Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei 430072, China, with the accession number: CCTCC NO: M2025465. The taxonomic designation is: Rhodotorula dairenensis S9.
[0061] Example 3: Analysis of growth characteristics of red yeast S9
[0062] (1) Drawing of the optimal growth temperature curve under different salt concentration conditions
[0063] The red yeast S9 was inoculated at 5% (V / V) into 100 mL of YPD medium with different salt contents (5% and 15%), and cultured at 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, and 36°C at 200 r / min for 48 h. The OD600 nm value of the culture solution was measured, and the YPD medium without bacterial solution was used as a blank control. The results are shown in Figure 2. Figure 3 As shown in a, the OD600 of the fermentation broth at two salt concentrations showed a trend of first increasing and then decreasing with increasing temperature. The OD600 was the largest at 30℃, indicating that 30℃ was the optimal growth temperature for strain S9.
[0064] (2) Drawing of optimal growth pH curve under different salt concentration conditions
[0065] The red yeast S9 was inoculated into 100 mL of YPD medium (5% and 15%) with different salt contents at pH 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 at 5% (V / V), and cultured at 30°C and 200 r / min for 48 h. The OD600 value of the bacterial solution was measured, and the YPD medium without bacterial solution was used as a blank control. The results are shown in Figure 2. Figure 3 As shown in Figure b, strain S9 has some growth ability at pH values between 2 and 7, with optimal growth at pH values between 4 and 6. At 15% salt concentration, the OD600 at the same pH was lower than at 5%. Overall, pH 4 was associated with the best growth. Strain S9 was also able to grow at pH 2, indicating a certain degree of acid tolerance.
[0066] (3) Plotting growth curves under different salt concentrations
[0067] The red yeast S9 was inoculated into YPD medium with different salt contents (5% and 15%) at 5% (V / V), pH 5, 30°C, 200 r / min, and the OD600 value of the bacterial solution was measured every 8 hours. Figure 3As shown in Figure c, at 5% salt concentration, the logarithmic phase of strain S9 lasted from 8 to 48 hours, with the stationary phase occurring after 48 hours. However, at 15% salt concentration, the logarithmic phase was delayed to 24 to 64 hours, with the stationary phase occurring after 64 hours. At 15% salt concentration, the OD600 value of strain S9 during the stationary phase was greater than 2, indicating that it thrived in a high-salt environment.
[0068] Example 4: Analysis of Volatile Compounds Produced in Doubanjiang Fermentation
[0069] 4.1 Test method
[0070] Seed preparation: strain S9 was cultured in 10% NaCl YPD medium until the logarithmic growth phase, and then the cells were washed twice with sterile saline containing 10% NaCl to prepare a seed solution with an OD of 1.
[0071] The seed liquid was added to fermented fermented doubanjiang (fermented broad bean paste) at a ratio of 0.5% (v / v). Uninoculated fermented doubanjiang served as a blank control, with triplicate replicates for each treatment. Fermentation was carried out at 30°C for 30 days, with samples collected on the 30th day for volatile analysis. Headspace solid-phase microextraction (SPM) was used to extract the samples, and the volatile compound content was determined using gas chromatography-mass spectrometry (GC-MS).
[0072] The headspace solid-phase microextraction method described above involves placing 2 g of ground sample in a 20 mL headspace vial, adding 20 μL of a 50 μg / mL internal standard solution of methyl heptanoate diluted in methanol, and immediately sealing the vial. After incubation at 60°C for 10 minutes, the extraction head is inserted and the fiber is pushed out. Extraction is continued for 50 minutes, followed by 5 minutes of desorption in the GC inlet.
[0073] The above GC conditions are as follows: chromatographic column: DB-WAX capillary column (60m×0.32mm×0.25μm); injection port temperature is 250°C; carrier gas is high-purity helium (1.0mL / min), and injection is carried out in splitless mode; programmed temperature conditions are as follows: initial temperature 40°C, maintained for 3min; then increased to 150°C at 3°C / min; then increased to 240°C at 6°C / min, maintained for 10min; EI ion source is used, and electron energy is 70eV; ion scanning range is 33-400m / z; ion source temperature is 230°C; interface temperature is 250°C.
[0074] 4.2 Effect of strain S9 on volatile compounds in fermentation of bean paste
[0075] After 30 days of constant temperature fermentation, the amount and content of volatile substances in the fermented bean paste after adding strain S9 increased significantly compared with the control group ( Figure 4a and b), the number of volatiles increased from 77 to 92, and the total content of volatiles increased from 18487.94μg / Kg to 27580.79μg / Kg. Among them, esters and acids increased exponentially, ethyl decanoate increased from 372.66μg / Kg to 1136.54μg / Kg, and ethyl laurate increased from 591.00μg / Kg to 960.19μg / Kg. At the same time, the content of ethyl phenylacetate also increased significantly ( Figure 4 c), increased from 104.86μg / Kg to 522.11μg / Kg.
Claims
1. Rhodotorula dairenensis, characterized in that: The deposit number is: CCTCC NO: M2025465.
2. A microbial agent, characterized in that: A fermentation liquid, seed liquid or bacterial cell containing the red yeast of claim 1.
3. Use of the red yeast rice according to claim 1 or the microbial agent according to claim 2 in producing ethyl phenylacetate, ethyl caprate or ethyl laurate.
4. Use of the red yeast according to claim 1 or the microbial agent according to claim 2 in the preparation of broad bean paste, soy sauce, fermented black beans, bread or liquor fermentation.
5. The use according to claim 4, characterized in that: The liquor is selected from at least one of the group consisting of Luzhou-flavor liquor, Maotai-flavor liquor, Fen-flavor liquor and mixed-flavor liquor.
6. A method for producing ethyl phenylacetate by metabolism, characterized in that: The method comprises the following steps: activating the red yeast of claim 1 or the microbial agent of claim 2, inoculating the activated red yeast rice into fermentation raw materials, and culturing and fermenting the activated red yeast rice.
7. The method according to claim 6, characterized in that: The inoculation amount of the red yeast or microbial agent is 0.3% to 1%.
8. The method according to claim 6, wherein: The fermentation temperature is 24-36°C.
9. The method according to claim 6, wherein: The fermentation time is 20 to 40 days.
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