Candida tropicalis and application thereof in white spirit brewing

Abstract

The invention relates to candida tropicalis and application thereof, the candida tropicalis is a candida tropicalis JSY83 strain, the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 20241913, and the preservation date is September 4, 2024. The strain has excellent ethanol, acetic acid and lactic acid tolerance, especially has more excellent lactic acid tolerance, and ensures the growth and functional effects of the strain in the actual white spirit brewing process; by inhibiting the growth of ester-producing yeast, reducing the accumulation of acetic acid, reducing the content of acetic acid in fermented grains and inhibiting the accumulation of ethyl acetate in the fermented grains, the method has important significance on improving the high-quality product rate and the liquor yield of raw liquor when being used for producing yeast for making hard liquor and Luzhou-flavor liquor.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial cultivation and relates to tropical Candida and application thereof in liquor brewing. Background Art

[0002] In recent years, with the in-depth study of liquor brewing flora, strain-enhanced fermentation has gradually become the main technical means to improve the quality of liquor. By adding functional microorganisms to regulate the succession and metabolism of the fermented grains microbial community, the purpose of improving the flavor and quality of liquor can be achieved. A large number of reports have been found in the liquor brewing system, but there are generally few studies on the direct use of Candida for liquor fermentation, and the role of these yeasts in liquor brewing is still unclear.

[0003] As one of the four major esters in Luzhou-flavor liquor, the ratio of ethyl acetate to ethyl caproate plays an important role in the typical flavor of Luzhou-flavor liquor. In recent years, due to the high content of ethyl acetate in the brewing process, even exceeding the content of ethyl caproate, the coordination and typicality of the liquor body have been reduced, which has restricted the improvement of the quality of Luzhou-flavor liquor.

[0004] Previous studies have mostly controlled the synthesis of ethyl acetate in liquor fermentation by adjusting the brewing process, such as increasing the pressure on the cellar, reducing dissolved oxygen in the cellar, and increasing the use of double-wheel bottoms, but the process adjustment is time-consuming and labor-intensive and the regulatory effect is limited. At the same time, liquor solid-state fermentation is usually carried out under various environmental stresses (lactic acid, ethanol, pH, etc.), especially in the later stage of fermentation when the environmental pressure is greater, which will significantly affect the growth and inhibition of exogenous strains. Therefore, if some strains with strong stress resistance can be screened and applied to liquor fermentation, and can effectively inhibit the synthesis of ethyl acetate, it is very meaningful for improving and enhancing the quality of liquor. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a tropical Candida and its application in liquor brewing. The tropical Candida has excellent tolerance to ethanol, acetic acid and lactic acid, can survive in the high-temperature koji making process and stably play a role in inhibiting the synthesis of ethyl acetate. The use of the strain in the production of Daqu and Luzhou-flavor liquor is of great significance for improving the high-quality rate and yield rate of the original liquor.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tropical Candida, wherein the tropical Candida is the tropical Candida (Candida tropicalis) JSY83 strain, with a preservation number of CCTCC NO: M 20241913 and a preservation date of September 4, 2024.

[0008] The present invention separates, screens and preserves a new tropical Candida that can inhibit the synthesis of ethyl acetate from the medium-high temperature pure wheat Daqu sample fermented for 10 days of Jinshiyuan Liquor Co., Ltd. The strain is sequenced and analyzed, and its 16SrDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is tropical Candida. The strain has excellent tolerance to ethanol, acetic acid and lactic acid, especially lactic acid tolerance, which ensures its growth and functional role in the actual liquor brewing process; it can reduce acetic acid accumulation, reduce the acetic acid content of fermented grains, and inhibit the accumulation of ethyl acetate in fermented grains by inhibiting the growth of ester-producing yeast, and its use in Daqu and Luzhou-flavor liquor production is of great significance to improving the quality rate and yield rate of raw liquor.

[0009] In a second aspect, the present invention provides a culture of Candida tropicalis according to the first aspect, wherein the culture is obtained by inoculating Candida tropicalis JSY83 strain into a sterile culture medium and culturing at 25-35°C (eg, 25°C, 28°C, 30°C, 32°C, 35°C, etc.).

[0010] In a third aspect, the present invention provides a microbial preparation for inhibiting the accumulation of ethyl acetate, wherein the strain in the microbial preparation comprises the Candida tropicalis JSY83 strain described in the first aspect.

[0011] Preferably, the microbial preparation is in the form of a solution or a powder.

[0012] Preferably, the microbial preparation further comprises a protective agent and / or a carrier material.

[0013] Preferably, the protective agent is selected from any one or a combination of at least two of skim milk, gelatin, gum arabic, dextran, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0014] Preferably, the carrier material is selected from any one of maltodextrin, starch, microcrystalline cellulose, calcium carbonate or calcium hydrogen phosphate, or a combination of at least two thereof.

[0015] In a fourth aspect, the present invention provides use of the tropical Candida described in the first aspect, the culture of tropical Candida described in the second aspect, or the microbial preparation described in the third aspect in liquor brewing or koji making.

[0016] In a fifth aspect, the present invention provides use of the Candida tropicalis described in the first aspect, the culture of the Candida tropicalis described in the second aspect, or the microbial preparation described in the third aspect in the preparation of an ester-producing yeast growth inhibitor.

[0017] In a sixth aspect, the present invention provides the tropical Candida described in the first aspect, the culture of tropical Candida described in the second aspect, or the microbial preparation described in the third aspect for use in reducing acetic acid accumulation and lowering the acidity of fermented grains during liquor fermentation.

[0018] In a seventh aspect, the present invention provides the tropical Candida described in the first aspect, the culture of tropical Candida described in the second aspect, or the microbial preparation described in the third aspect for use in inhibiting the accumulation of ethyl acetate during liquor fermentation.

[0019] In an eighth aspect, the present invention provides a method for screening Candida tropicalis according to the first aspect, the screening method comprising the following steps:

[0020] (1) Take a medium-high temperature pure wheat Daqu sample fermented for 10 days, mix it with sterile water, and shake it to obtain a bacterial suspension;

[0021] (2) After the bacterial suspension is graded diluted, the graded dilutions are spread on G25N hypertonic medium to obtain colonies; a single colony is picked and streaked on G25N hypertonic medium for culture, and the operation is repeated multiple times to obtain a single pure colony.

[0022] Preferably, the formula of the G25N hypertonic culture medium is: K2HPO4 0.5-1g, Czapek concentrate 5-10mL, yeast extract powder 1-5g, glycerol 200-300g, agar 10-15g, and distilled water 500-1000mL.

[0023] Preferably, the culture is cultured at 25-35°C (e.g., 25°C, 27°C, 28°C, 30°C, 35°C, etc.) for 24-72h (e.g., 24h, 36h, 48h, 50h, 62h, 72h, etc.).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention separates, screens and preserves a new tropical Candida that can inhibit the synthesis of ethyl acetate from the medium-high temperature pure wheat Daqu sample fermented for 10 days of Jinshiyuan Liquor Co., Ltd. The strain is sequenced and analyzed, and its 16SrDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is tropical Candida. The strain has excellent tolerance to ethanol, acetic acid and lactic acid, especially lactic acid tolerance, which ensures its growth and functional role in the actual liquor brewing process; it can reduce acetic acid accumulation, reduce the acetic acid content of fermented grains, and inhibit the accumulation of ethyl acetate in fermented grains by inhibiting the growth of ester-producing yeast, and its use in Daqu and Luzhou-flavor liquor production is of great significance to improving the quality rate and yield rate of raw liquor.

[0026] The tropical Candida involved in the present invention is classified and named as Candida tropicalis JSY83 Candida tropicalis JSY83, the preservation time is September 4, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241913. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The colony appearance and microscopic examination images of the 7 pure strain colonies screened out in Example 1;

[0028] Figure 2 is a schematic diagram of the phylogenetic tree of the seven yeast strains screened in Example 1;

[0029] Figure 3 This is a graph showing the evaluation results of the ethanol tolerance of strains JSY96, JSY89, and JSY83;

[0030] Figure 4 This is a graph showing the evaluation results of the tolerance of strains JSY96, JSY89, and JSY83 to acetic acid;

[0031] Figure 5 This is a graph showing the evaluation results of the lactic acid tolerance of strains JSY96, JSY89, and JSY83;

[0032] Figure 6 It is the test result diagram of ethyl acetate content in the system after the tropical Candida JSY83 strain was cultured in the Luzhou-flavor liquor fermentation system for 10 days and 30 days;

[0033] Figure 7 This is a graph showing the test results of the acetic acid content in the system after the tropical Candida JSY83 strain was cultured in the Luzhou-flavor liquor fermentation system for 10 days;

[0034] Figure 8 This is the test result of lactic acid content in the system after the tropical Candida JSY83 strain was cultured in the Luzhou-flavor liquor fermentation system for 10 days. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0036] The formula of the G25N hypertonic medium involved in the following examples is: K2HPO4 0.75g, Czapek concentrate 7.5mL, yeast extract powder 3.7g, glycerol 250g, agar 12g, and distilled water 750mL.

[0037] Example 1

[0038] Screening of Candida tropicalis JSY83 strain:

[0039] (1) 20 g of medium-high temperature pure wheat Daqu sample fermented for 10 days produced by Jiangsu Jinshiyuan Wine Co., Ltd. was selected and placed in a conical flask filled with 180 mL of sterile water, and shaken for 30 min to obtain a bacterial suspension;

[0040] (2) After the bacterial suspension was diluted 10 times, 10 -3 The dilution gradient was spread on G25N hypertonic medium and inverted for 2 days at 30°C to obtain colonies; a single colony was picked and streaked on G25N hypertonic medium for 4 times to obtain a single pure colony, which was named JSY199, JSY96, JSY89, JSY83, JSY30, JSY29, and JSY18. The microscopic examination results are shown in the following table. Figure 1 As shown, the results showed that all of them were yeast cell morphology.

[0041] Example 2

[0042] Identification of Candida tropicalis JSY83 strain:

[0043] The genomes of the seven yeast strains in Example 1 were extracted, and the yeast 26S rDNA D1 / D2 fragments NL1 and NL4 were used as amplification primers to amplify the 26S rDNA sequences from the genomic DNA of the yeast. The successfully amplified PCR products were sent to Shanghai Biotechnology for sequencing, and BLAST sequence comparison was performed in the NCBI database. The strain identification results are shown in Table 1, where the 26S rDNA sequence of the JSY83 strain is shown in SEQ ID No: 1. Using MEGAX software, the 26S rDNA regional sequences of the related strains with the highest homology were selected as reference objects according to the BLAST comparison results, and the phylogenetic tree of the seven yeast strains was constructed, as shown in Table 1. Figure 2 shown.

[0044] Table 1

[0045]

[0046] Example 3

[0047] Evaluation of the tolerance of Candida tropicalis JSY83 strain to ethanol, acetic acid and lactic acid:

[0048] In Example 1, a total of three tropical Candida strains JSY96, JSY89 and JSY83 were screened. In order to screen the optimal tropical Candida strain, in this example, the ethanol, acetic acid and lactic acid tolerance of the three tropical Candida strains were evaluated respectively.

[0049] (1) Comparison of ethanol tolerance: The three yeast strains were inoculated into YPD liquid medium and cultured on a shaking table at 30°C and 200 rpm for 24 h to prepare seed solution. The cultured seed solution was taken and then heated to 10 6 The bacterial solution was inoculated into YPD liquid medium with different ethanol contents (0%, 2%, 4% and 6%, v / v) at 30°C and 200 rpm for 24 h. The bacterial solution was diluted to a certain multiple and the OD was measured. 600nm The absorbance value is as follows Figure 3 The results showed that strains JSY96, JSY89 and JSY83 were all tolerant to 4% ethanol and their growth was almost unaffected; at 6% ethanol concentration, the biomass of strains JSY96, JSY89 and JSY83 decreased by 72.5%, 61.5% and 60.6%, respectively, and their growth was inhibited to a certain extent.

[0050] (2) Comparison of acetic acid tolerance: The seed solution was prepared in the same way as in (1); the cultured seed solution was taken and heated to 10 6 The bacterial solution was inoculated into YPD liquid medium with different acetic acid amounts (0, 0.05%, 0.1% and 0.2%, v / v) at 30°C and 200 rpm for 24 h on a shaking table. The bacterial solution was diluted to a certain multiple and the OD was measured. 600nm The absorbance value is as follows Figure 4 The results showed that strains JSY96, JSY89 and JSY83 were all tolerant to 0.05% acetic acid, and their growth was almost unaffected; at 0.1% acetic acid, the growth of the three strains was inhibited to a certain extent, but they could still grow; at 0.2% acetic acid, the growth of the three strains was significantly inhibited, and there was almost no growth. The acetic acid tolerance of the three strains was not much different.

[0051] (3) Comparison of lactic acid tolerance: The seed solution was prepared in the same way as in (1); the cultured seed solution was taken and heated to 10 6 The bacterial solution was inoculated into YPD liquid medium with different lactic acid contents (0, 1%, 2% and 4%, v / v) at 30°C and 200 rpm for 24 h. The bacterial solution was diluted to a certain multiple and the OD was measured. 600nm The absorbance value is as follows Figure 5 The results showed that at a lactic acid concentration of 4%, the growth of strains JSY96 and JSY89 was significantly inhibited, with almost no growth, while the growth of strain JSY83 was almost unaffected, indicating its strong lactic acid tolerance.

[0052] (4) The strain JSY83 was deposited and named as Candida tropicalis JSY83 on September 4, 2024. The depositor was China Center for Type Culture Collection (CCTCC), and the deposit address was Wuhan University, Wuhan, China. The deposit number was CCTCC NO: M 20241913.

[0053] Example 4

[0054] Evaluation of the inhibitory ability of Candida tropicalis JSY83 strain on the growth of ester-producing yeast:

[0055] Agar plate diffusion test: The Pichia kudriavzevii JSY199 strain and the Candida tropicalis JSY83 strain isolated in Example 1 were cultured at 30°C for 24 h in YPD liquid medium, counted under a microscope, and the cell concentration was adjusted to 1×10 4 CFU / mL; take 50μL of the adjusted concentration of JSY83 suspension and evenly spread it on the YPD solid medium, then inoculate 10μL of the adjusted concentration of JSY199 suspension at different positions on the plate; the control group is not inoculated with JSY83 suspension; all plates are cultured at 30℃ for 3 days, and the colony diameter is measured. The results show that the colony diameter of JSY199 alone is 4cm, while the colony diameter is reduced to 1.6cm when inoculated with JSY83, indicating that JSY83 has a significant inhibitory effect on the growth of ester-producing yeast.

[0056] Example 5

[0057] Application of tropical Candida JSY83 strain to reduce ethyl acetate content in the production of Luzhou-flavor liquor:

[0058] The fermentation system of Luzhou-flavor liquor includes four experimental groups, namely blank control group (control), group inoculated with tropical Candida JSY83 alone (C. tropicalis), group inoculated with Pichia pastoris JSY199 alone (P. kudriavzevii), and group inoculated with Pichia pastoris JSY199 and tropical Candida JSY83 together (C.t+Pk).

[0059] 4.8 kg of fermented grains to be put into the pool were collected directly from the workshop and divided into 24 portions, each of which was about 200 g. Each group had 6 portions and was inoculated according to the above method. The total amount of yeast inoculation was 10 6 CFU / kg; fermentation time 30 days, samples were taken at 10 and 30 days of fermentation to test the ethyl acetate content. The results are as follows Figure 6 shown.

[0060] The results showed that the ethyl acetate content in the four groups was 299 mg / kg, 116 mg / kg, 323 mg / kg, and 298 mg / kg, respectively, at 10 days of fermentation. Compared with the control group, the ethyl acetate content in the group inoculated with ester-producing Pichia pastoris JSY199 alone increased by 7.9%, while the ethyl acetate content in the group inoculated with tropical Candida JSY83 alone decreased by 61.2%, and the ethyl acetate content in the co-inoculation group remained basically unchanged. At 30 days of fermentation, the ethyl acetate content in the four groups was 544 mg / kg, 398 mg / kg, 589 mg / kg, and 488 mg / kg, respectively. Compared with the control group, the ethyl acetate content in the group inoculated with ester-producing Pichia pastoris JSY199 alone increased by 8.3%, while the ethyl acetate content in the group inoculated with tropical Candida JSY83 alone decreased by 26.8%, and the ethyl acetate content in the co-inoculation group decreased by 10.2%. The above results show that tropical Candida JSY83 effectively inhibits the synthesis of ethyl acetate during fermentation.

[0061] Samples were taken on the 10th day of fermentation to test the acetic acid and lactic acid content. The results are as follows: Figure 7 and Figure 8 As shown. The results showed that the acetic acid content in the group inoculated with tropical Candida JSY83 alone and the group co-inoculated decreased by 53.5% and 35.9%, respectively; the lactic acid content in the different treatment groups did not change significantly. As one of the important precursors for the synthesis of ethyl acetate, the reduction of acetic acid content is beneficial to reducing the synthesis of ethyl acetate in the esterase pathway. Studies have shown that an acidic environment is conducive to the accumulation of ethyl acetate, and the reduction of acetic acid content is conducive to the reduction of the acidity of the mash and the reduction of ethyl acetate accumulation.

[0062] Based on the above analysis results, the tropical Candida JSY83 involved in the present invention may inhibit the accumulation of ethyl acetate in the fermented grains during the fermentation process of Luzhou-flavor liquor by inhibiting the growth of ester-producing yeast, reducing the acetic acid content and lowering the acidity of the fermented grains, thereby achieving the expected regulatory effect.

[0063] Example 6

[0064] Application of tropical yeast JSY83 strain to reduce ethyl acetate content in medium-high temperature Daqu production:

[0065] Candida tropicalis JSY83 was inoculated into YPD medium and cultured in a shaking incubator at 30°C and 200 rpm for 2 days to prepare seed solution. The concentration of the bacterial solution was adjusted to 10 8CFU / ml, and was inoculated into the koji block as a functional strain, with an inoculation amount of 0.05% (v / m), and the control group was not inoculated. Samples were taken on the 0th, 3rd and 7th days of fermentation to detect the contents of acetic acid, lactic acid and ethyl acetate. The results showed that after 3 days of fermentation, no ethyl acetate was detected in the control group and the group inoculated with tropical Candida JSY83; after 7 days of fermentation, the ethyl acetate contents of the control group and the group inoculated with tropical Candida JSY83 were 1281mg / kg and 369mg / kg, respectively, indicating that tropical Candida JSY83 also played an inhibitory role in the medium and high temperature koji making process.

[0066] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0067] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0068] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A tropical Candida, characterized in that The tropical Candida is the tropical Candida (Candida tropicalis) JSY83 strain, with a preservation number of CCTCC NO: M 20241913 and a preservation date of September 4, 2024.

2. The culture of Candida tropicalis according to claim 1, characterized in that The culture is obtained by inoculating Candida tropicalis JSY83 strain into a sterile culture medium and culturing at 25-35°C.

3. A microbial preparation for inhibiting the accumulation of ethyl acetate, characterized in that: The strain in the microbial preparation includes the Candida tropicalis JSY83 strain according to claim 1.

4. The microbial preparation according to claim 3, characterized in that The dosage form of the microbial preparation includes solution or powder.

5. The microbial preparation according to claim 3 or 4, characterized in that: The microbial preparation also includes a protective agent and / or a carrier material.

6. The microbial preparation according to claim 5, characterized in that The protective agent is selected from any one or a combination of at least two of skim milk, gelatin, gum arabic, dextran, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol; The carrier material is selected from any one of maltodextrin, starch, microcrystalline cellulose, calcium carbonate or calcium hydrogen phosphate, or a combination of at least two thereof.

7. Use of the tropical Candida according to claim 1, the culture of tropical Candida according to claim 2, or the microbial preparation according to any one of claims 3 to 6 in liquor brewing or koji making.

8. Use of the Candida tropicalis according to claim 1, the culture of Candida tropicalis according to claim 2, or the microbial preparation according to any one of claims 3 to 6 in the preparation of an inhibitor for the growth of ester-producing yeast.

9. The tropical Candida according to claim 1, the culture of tropical Candida according to claim 2, or the microbial preparation according to any one of claims 3 to 6 is used for reducing acetic acid accumulation and lowering the acidity of fermented grains during liquor fermentation.

10. The Candida tropicalis according to claim 1, the culture of Candida tropicalis according to claim 2, or the microbial preparation according to any one of claims 3 to 6 is used for inhibiting the accumulation of ethyl acetate during the fermentation of liquor.

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