Portuguese williomia yeast yjht233 and application thereof

Abstract

The present application relates to the technical field of industrial microorganism, and particularly discloses a yeast strain and application thereof. The present application provides a Clavispora lusitaniae YJHT233, which is obtained by spaceflight mutagenesis breeding, can tolerate 9% alcohol, the minimum tolerance pH is 2.5 of acidic environment, can tolerate 12% high NaCl system, 55% high glucose system and 44℃ high temperature environment, and has excellent ester production capacity. The strain is used for brewing rice-flavor liquor or yellow rice wine, the total content of ester substances in the obtained product is greatly improved, the content of main volatile ester substances such as ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl octanoate and ethyl lactate is significantly improved, the overall flavor is harmonious and pleasant, the fruity aroma is rich and the levels are rich.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Clavispora lusitaniae YJHT233 and application thereof. BACKGROUND

[0002] Yellow rice wine, as a traditional Chinese beverage, has a production history of more than 3000 years. Yellow rice wine is mainly made of glutinous rice and water, with the addition of distiller's yeast and saccharifying agent, and is brewed under the action of more than 100 kinds of microorganisms. Yellow rice wine is famous for its rich nutrition and health care, and is highly praised by experts, because it is rich in active peptides, amino acids, organic acids, vitamins, polyphenols, esters and other beneficial components.

[0003] The flavor characteristics of yellow rice wine are derived from aroma substances. When the content of a certain aroma substance exceeds the threshold value, the corresponding odor is easy to be perceived. The complex flavor of yellow rice wine mainly comes from three aspects: ① the inherent aroma substances in the fermentation raw materials such as rice and wheat starter; ② the aroma substances produced by microbial metabolism during fermentation; and ③ the aroma components formed by oxidation, esterification and Maillard reaction during the aging process of yellow rice wine. It has been found that esters are one of the main skeleton components of the aroma of yellow rice wine. As an important source of the main aroma of yellow rice wine, more than 40 kinds of esters such as ethyl acetate, ethyl butyrate, diethyl succinate, isoamyl acetate, ethyl octanoate and ethyl lactate have been commonly found and confirmed, which give yellow rice wine rich fruit aroma and floral aroma.

[0004] The fermentation process is the key to affect the flavor quality of rice wine, which mainly involves the joint action of distiller's yeast and environmental microorganisms. Yeast is one of the most active microbial groups in the fermentation process of wine, and can be divided into Saccharomyces cerevisiae (producing alcohol) and non-Saccharomyces cerevisiae (producing esters). Non-Saccharomyces cerevisiae is rich in species, and its metabolic products and secreted enzymes have important influence on the aroma and flavor of brewed wine. In recent years, it has been found that some non-Saccharomyces cerevisiae can synthesize esters, higher alcohols and other aroma compounds using fermentation raw materials, increase the complexity of flavor, and improve the flavor of products, but most of the strains have poor alcohol tolerance, acid tolerance and osmotic pressure tolerance, which seriously restricts the large-scale fermentation application. SUMMARY

[0005] Based on the above technical problems, the main purpose of the present application is to overcome the shortcomings of the prior art, and to provide a Clavispora lusitaniae YJHT233 strain which can produce esters and has strong alcohol tolerance, high osmotic pressure tolerance, high temperature tolerance and acid tolerance. The strain can significantly increase the content of esters in wine and improve the flavor of products when used for brewing, and has good application value.

[0006] In order to achieve the above-mentioned purpose, the inventors have conducted in-depth research and completed the present application scheme after repeated research and demonstration, as follows.

[0007] In a first aspect, the present application provides a Clavispora lusitaniae strain YJHT233, characterized in that the Clavispora lusitaniae YJHT233 has been preserved in the Guangdong Microbial Culture Collection Center on December 21, 2021, and the preservation number is GDMCC No: 62152.

[0008] The strain colony is milky white and creamy, with smooth edges and flat surface, and reflects light under illumination.

[0009] In a second aspect, the present application provides a fermentation agent, characterized in that the fermentation agent comprises the above-mentioned Clavispora lusitaniae YJHT233.

[0010] In a third aspect, the present application provides application of the above-mentioned Clavispora lusitaniae YJHT233 or fermentation agent in brewing.

[0011] Further, the application is to improve the content of ester substances in wine.

[0012] Further, the brewing is brewing of rice-flavor liquor or yellow rice wine.

[0013] In a fourth aspect, the present application provides a preparation method of rice-flavor liquor or yellow rice wine, which comprises the step of adding the above-mentioned Clavispora lusitaniae YJHT233 or fermentation agent to the wine mash for fermentation.

[0014] Further, the addition amount of the Clavispora lusitaniae YJHT233 is 10 5 CFU / mL-10 8 CFU / mL.

[0015] Further, the fermentation temperature is 28-32℃.

[0016] Further, the fermentation time is 10-20 days.

[0017] In a fifth aspect, the present application provides a rice-flavor liquor or yellow rice wine prepared using the above-mentioned Clavispora lusitaniae YJHT233 or fermentation agent.

[0018] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0019] 1. The Portuguese Clavispora lusitaniae YJHT233 provided by the present application is obtained by spaceflight mutagenesis breeding, can tolerate 9% alcohol, the minimum tolerance pH is 2.5 of acidic environment, can tolerate 12% high NaCl system, 55% high glucose system and 44℃ high temperature environment, has excellent ester production capacity, compared with the original strain, the content of ethyl caprylate, ethyl butyrate, ethyl lactate and ethyl benzoate is increased by 1-3.25 times, and the content of ethyl acetate and ethyl heptanoate is also increased by more than 20%.

[0020] 2. The Portuguese Clavispora lusitaniae YJHT233 provided by the present application can significantly increase the content of main volatile esters and beta-phenylethanol in rice-flavor liquor fermentation, wherein the increase rate of main flavor substances such as ethyl acetate, ethyl hexanoate, ethyl acetate, ethyl nonanoate, ethyl caprylate and beta-phenylethanol is more than 10%, the fermented rice-flavor liquor has harmonious and pleasant flavor, and the fruity aroma is rich.

[0021] 3. The Portuguese Clavispora lusitaniae YJHT233 provided by the present application has obvious promotion effect on ester flavor substances in fermented yellow rice. Among them, ethyl phenyl acetate, ethyl benzoate, diethyl succinate, ethyl butyrate, ethyl decanoate, ethyl laurate, ethyl palmitate, ethyl nonanoate, ethyl caprylate and ethyl lactate are greatly improved, the total ester content is increased by more than one time, reaches 45.401 mg / L, and the fermented yellow rice has rich aroma and rich levels. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a colony morphology diagram of the Portuguese Clavispora lusitaniae YJHT233;

[0023] Figure 2 It is a flavor chromatogram of fermented yellow rice fermented by active dry yeast;

[0024] Figure 3 It is a flavor chromatogram of fermented yellow rice fermented by YJHT233;

[0025] The Portuguese Clavispora lusitaniae YJHT233 provided by the present application is preserved in the Guangdong Microbial Digital Culture Collection Center on December 21, 2021, the address is No. 59, Building 5, Guangzhou Institute, No. 100, Martyrs Road, Guangzhou, Guangdong Province, and the preservation number is GDMCC No: 62152; the strain was received and registered by the preservation center on December 21, 2021, and was detected as a living strain by the preservation center on December 21, 2021. DETAILED DESCRIPTION

[0026] For the purposes of the present invention, a more complete description will be provided in the following. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention is more thorough and complete.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0028] Unless specifically mentioned, the reagents, methods and apparatuses employed in the present invention are the conventional food-grade reagents, methods and apparatuses in the art.

[0029] The terms used in the specification of the present invention are only for the purpose of describing the specific embodiments of the present invention and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0030] The culture media used in the specification of the present invention are as follows:

[0031] Malt extract medium (malt extract powder 13%)

[0032] GPYM medium (glucose 4%, peptone 0.5%, yeast extract powder 0.5%, malt extract 0.5%, MgSO4·7H2O 0.1%)

[0033] High glucose substrate medium (glucose 10%, peptone 2%, yeast extract powder 1%)

[0034] Molasses substrate medium (molasses 20BX°, (NH4)2SO4 0.1%, KH2PO4 0.1%, MgSO4 0.04%)

[0035] Starch substrate medium ((NH4)2SO4 0.25%, KH2PO4 0.25%, soluble starch 2%)

[0036] High protein substrate medium (soybean powder 5%)

[0037] Example 1 Screening of strains

[0038] (1) Ester-producing functional strain screening: 25 g of rice-flavor liquor koji sample was added into a triangular flask containing 225 ml of sterile water, and incubated at 150 rpm / min for 15 min. Gradient dilution was performed according to a ratio of 1:10. 100 μL of bacterial suspension at different dilution ratios was taken and spread on a sample dilution plate. The plate was incubated at 28°C for 48 h. The colony morphology was observed, and typical yeast single colonies with a smooth and wet surface were picked and inoculated on a PDA plate containing bromocresol green. The plate was incubated at 28°C for 48 h. The colonies with larger yellow rings were picked and purified by plate streaking. The purified yeast colonies were inoculated in malt juice medium, GPYM medium, high glucose medium, molasses medium, starch medium and high protein medium, and incubated at 28°C for 48 h. The aroma of the yeast culture was evaluated, and the strain with the best ester-producing ability was selected as the ester-producing functional strain, which was named YJ26.

[0039] (2) Mutagenesis: The single colony of the ester-producing functional strain YJ26 was placed in a cryogenic tube and carried by a spacecraft into space orbit for 67 h. The orbit was a large elliptical orbit with a height of 324-7970 km. During the period, it passed through the Van Allen radiation belt with an inclination of 42.5° and a cabin temperature of (20±5) °C. After returning to the ground, the bacterial solution was diluted to 10 -1 and stored in a new centrifuge tube at -80°C for future use.

[0040] (3) Target strain screening: 200 μL of the preserved bacterial solution was added to 5 mL of YPD liquid medium for recovery, and incubated at 28°C and 150 r / min for 48 h. 500 μL of the recovered bacterial solution was added to YPD liquid medium containing 9% ethanol, and incubated at 28°C and 150 r / min for 48 h. 200 μL of the bacterial solution was spread on YPD solid medium, and incubated at 28°C for 48 h. 0.4% TTC dye and 9% ethanol were added to the sterilized and cooled YPD liquid medium, mixed well, and then dispensed into a 48-well plate at 2 mL per well. All single colonies on the YPD solid plate were inoculated into the 48-well plate, and incubated at 28°C and 150 r / min for 48 h. The strains with deeper red color in the fermentation broth and pleasant odor were selected as the target strains.

[0041] (4) Identification and preservation of target strain: The strain was identified by 26S rDNA sequencing as Clavispora lusitaniae, and was named YJHT233. The colony morphology of the strain is shown in FIG. 1. Figure 1As shown, the colony is cream-colored and creamy, with smooth edges and a flat surface that reflects light. It was preserved at the Guangdong Microbial Culture Collection Center on December 21, 2021, with the accession number GDMCC No: 62152.

[0042] Example 2 Performance test of Portuguese Williopsis saturnus YJHT233

[0043] (1) Ester production ability test of Portuguese Williopsis saturnus YJHT233:

[0044] After activation of the YJHT233 strain, 10% of the seed liquid was inoculated into the wort medium. After 72h of fermentation culture, GC-MS was used to determine the proportion of main ester substances in the fermentation broth. YJ26 strain was used as a control, and the results are recorded in Table 1.

[0045] Gas chromatography conditions: DB-WAX UI chromatographic column (30m x 0.25mm x 0.25um), USA Agilent company, injection port temperature 220℃, injection volume 1μL, split ratio 100:1, flow rate 1mL / min, FID detector temperature 220℃, hydrogen (H2): air (O2): tail gas (N2) 40:400:25. Temperature program: initial temperature 30℃, hold for 2min, increase to 100℃ at 5℃ / min, hold for 10min, increase to 200℃ at 10℃ / min, hold for 10min.

[0046] Table 1 Main ester content in fermentation broth (mg / L)

[0047] Esters YJHT233 YJ26 Increased proportion Ethyl acetate 53 44 20％ Ethyl butyrate 151 44 243％ Ethyl heptanoate 66 53 25％ Ethyl lactate 27 8 238％ Ethyl octanoate 119 28 325％ Ethyl benzoate 14 7 100％

[0048] Table 1 shows that YJHT233 has excellent ester production ability. Compared with YJ26 fermentation broth, the main ester components of YJHT233 wort fermentation broth are greatly improved, especially ethyl octanoate, ethyl butyrate, ethyl lactate and ethyl benzoate, with an increase of 1-3.25 times in content. The content of ethyl acetate and ethyl heptanoate also increased by more than 20%.

[0049] (2) Tolerance test of Portuguese Williopsis saturnus YJHT233:

[0050] After activation of the Portuguese Williopsis saturnus YJHT233 single colony, 10% of the seed liquid was inoculated into YPD medium containing different concentrations of alcohol, glucose, NaCl and different pH, and cultured at different temperature gradients of 28℃-50℃, 150r / min for 72h. The turbidity of the culture under each condition was observed, and the OD 600 of the turbid broth was measured to test the tolerance of YJHT233.

[0051] The results show that the maximum alcohol tolerance of the strain YJHT233 is 9%vol, the minimum pH tolerance is 2.5, the maximum NaCl tolerance is 12%, the maximum glucose tolerance is 55%, and the maximum temperature tolerance is 44℃.

[0052] Example 3 Application of P. portuguese YJHT233 in rice-flavor liquor

[0053] Take 250g of northeast selected rice and wash it, then add 190mL of mineral water and cook it in a pressure cooker for 20min. After the rice is cooked and cooled, take 1% of the rice-flavor liquor starter from Guangdong region and evenly spread it on the cooled rice. Mix the starter and rice evenly and scatter it into granules, then pour it into a 500mL clean wide-mouth bottle, and place it in a constant temperature incubator at 34℃ for 40h. Take 0.3% of fermentation powder (liquor cake powder), add 10% of P. portuguese YJHT233 liquid, and add 300mL of water at the same time. Ferment it in a constant temperature incubator at 28℃ for 10d. At the same time, set up a control group with YJ26 added. The final concentration of P. portuguese YJHT233 or YJ26 is controlled at 10 6 -10 7 CFU / mL. After the fermentation of the liquor is completed, pour the fermented rice into a distillation apparatus, add 300mL of water, and distill 300mL of liquor. Use the method described in Example 2 to determine the content of various ester substances in the liquor, and the results are recorded in Table 2.

[0054] Table 2 Content of flavor substances in rice-flavor liquor (mg / L)

[0055] Flavour substances YJ26 fermentation YJHT233 fermentation Increased proportion Ethyl acetate 17.7 22.2 25.4％ Ethyl lactate 5.7 6.2 8.8％ Ethyl butyrate 142.6 156.7 9.9％ Ethyl hexanoate 367.6 440.3 19.8％ Isoamyl acetate 318.4 364.1 14.4％ Ethyl nonanoate 26.4 33.8 28.0％ Ethyl octanoate 51.1 56.7 11.0％ Beta-phenylethanol 57.6 71.4 24.0％

[0056] As shown in Table 2, when P. portuguese YJHT233 is applied in rice-flavor liquor, the content of ethyl acetate, ethyl nonanoate, and β-phenylethanol increases by more than 20%, which is 25.4%, 28.0%, and 24.0%, respectively. Compared with other types of liquor, the types of flavor components in rice-flavor liquor are relatively few, and ethyl acetate and β-phenylethanol are the main aroma components. In addition, the content of ethyl lactate, ethyl butyrate, ethyl hexanoate, ethyl isovalerate, and ethyl octanoate in rice-flavor liquor brewed by P. portuguese YJHT233 also increases to different degrees, with an increase of 8.8%-19.8%. The increase in the content of these ester substances and β-phenylethanol main flavor substances makes the aroma of rice-flavor liquor pleasant, which is of great significance to improving the aroma of rice-flavor liquor.

[0057] Example 4 Application of P. portuguese YJHT233 in yellow rice wine

[0058] 1. Preparation of yellow rice wine

[0059] (1) Rice liquefaction: according to the ratio of rice to water 1:1-3.5 (w / w), a certain amount of rice and water was weighed, 0.5‰-2‰ (w / w) liquefaction enzyme was added, the temperature was raised to 90-98℃, and the whole process was kept for 60-90 min with sufficient stirring to avoid sticking to the pot, to prepare rice liquefaction juice.

[0060] (2) Activation of Pachysolen portugaliensis YJHT233: one spoon of Pachysolen portugaliensis YJHT233 slurry was inoculated into 100 mL YPD liquid medium, and incubated at 30℃, 200 r / min for 48 h. 1% inoculation was performed in the rice liquefaction juice cooled to room temperature, and incubated in a 30℃ incubator for 1 day. The concentration of the bacterial solution was adjusted to 10 6 -10 8 CFU / mL for standby.

[0061] (3) Saccharification: rice liquefaction juice was prepared according to step (1), and cooled with cold water to 60-70℃, 0.5‰-2‰ (w / w) saccharification enzyme was added, and incubated for 1-2 h with sufficient stirring to avoid sticking to the pot.

[0062] (4) Cooling and feeding: the saccharification mash was cooled to 28-35℃, and the water evaporated during liquefaction and saccharification was supplemented by weighing. 1 kg of fermentation mash was added with the following materials according to the mass ratio, one group of samples was added with 3%-8% of production yellow rice wine wheat starter, 1‰-6‰ active dry yeast; another group of samples was added with 3%-8% of production yellow rice wine wheat starter, 1‰-5‰ active dry yeast and 1%-10% Pachysolen portugaliensis YJHT233 culture solution, and the final concentration of Pachysolen portugaliensis YJHT233 was 10 5 -10 8 CFU / mL.

[0063] (5) Fermentation: fermentation was carried out at 28-32℃ for 15-20 days.

[0064] (6) Pressing: perlite was used as a filter aid for filtration, and clear rice was collected.

[0065] (7) Wine boiling: sterilization was carried out at 70-90℃ for 10-40 min to obtain yellow rice wine. The yellow rice wine fermented by active dry yeast and YJHT233 was the test group, and the yellow rice wine fermented by active dry yeast was the control group.

[0066] 2. Detection and analysis

[0067] The physicochemical indexes and volatile flavor substance content of the test group yellow rice wine and the control group yellow rice wine were detected, and sensory evaluation was carried out, and the results were recorded in Tables 3-5.

[0068] Table 3 Physicochemical indexes of fermented yellow rice wine

[0069] Alcohol %vol Total acids g / L Amino acid nitrogen g / L pH Control group 16.0 5.5 0.56 4.83 Test group 16.6 5.61 0.62 4.81

[0070] The results are shown in Table 3, the total acid, alcohol content, amino acid nitrogen content of the rice wine fermented by adding YJHT233 yeast is higher than that of the rice wine fermented by active dry yeast, the pH value is lower, and each physical and chemical index meets the requirements of the national standard of traditional rice wine.

[0071] Table 4 Key flavor substance content of fermented rice wine (mg / L)

[0072] Flavour substances Control group Test group Increased proportion Phenylethyl acetate 0.039 0.087 123％ Ethyl benzoate 0.076 0.17 124％ Diethyl succinate 0.086 0.197 129％ Ethyl butyrate 0.087 0.195 124％ Ethyl decanoate 1.385 3.127 126％ Ethyl laurate 0.117 0.24 105％ Ethyl palmitate 0.223 0.448 101％ Ethyl hexanoate 1.869 3.032 62％ Ethyl linoleate 0.665 0.951 43％ Ethyl nonanoate 0.166 0.345 108％ Ethyl octanoate 0.104 0.224 115％ Ethyl valerate 0.340 0.404 19％ Ethyl lactate 0.055 0.122 122％ Ethyl propionate 0.0000 0.039 - Total esters 21.753 45.401 109％

[0073] The results are shown in Table 4, the rice wine fermented by adding Portuguese Williopsis sp. YJHT233 has a substantial increase in ester flavor substances compared with the rice wine fermented by active dry yeast. Ester is an important source of the main aroma of rice wine, which gives rice wine rich fruit and flower aroma. The content of phenyl ethyl acetate and ethyl nonanoate with rose flower aroma in the rice wine inoculated with YJHT233 increases by 123% and 108% respectively; the content of ethyl benzoate and diethyl succinate with mild fruit aroma increases by 1.24% and 1.29% respectively. The content of ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl valerate with pineapple aroma increases by 124%, 62%, 115% and 19% respectively; and ethyl propionate with pineapple aroma is introduced into rice wine for the first time by YJHT233. The content of ethyl palmitate and ethyl lactate with butter aroma increases by 101% and 122% respectively, the content of ethyl laurate, ethyl linoleate and ethyl decanoate with light and lively fruit aroma increases by 105%, 43% and 126% respectively, and the total ester content increases by 109%. It can be seen that YJHT233 yeast can greatly increase the content of flower and fruit aroma substances in rice wine fermentation, and has obvious promoting effect on the increase of key ester flavor substance content of rice wine.

[0074] Table 5 Sensory evaluation of fermented rice wine

[0075]

[0076]

[0077] The results are shown in Table 5, the rice wine fermented by adding Portuguese Williopsis sp. YJHT233 has better sensory aroma and taste than the rice wine fermented by active dry yeast, and has more abundant flower and fruit aroma.

[0078] In summary, the physicochemical indexes of the rice wine fermented by the Portuguese Williopsis sp. YJHT233 meet the requirements of the national standard of traditional rice wine, and the aroma is better than that of the traditional rice wine, and the amount of ester flavor substances is significantly improved, wherein the amount of phenyl ethyl acetate, ethyl benzoate, diethyl succinate, ethyl butyrate, ethyl decanoate, ethyl laurate, ethyl palmitate, ethyl hexanoate / ethyl linoleate, ethyl nonanoate, ethyl octanoate, ethyl valerate and ethyl lactate is greatly improved, the total ester content is doubled, reaches 45.401 mg / L, and the rice wine with rich aroma and rich layers can be obtained.

[0079] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims.

Claims

1. A strain of the species Starmerella portuguese (formerly known as Starmerella portuguesae) YJHT233, characterized in that, Clavispora lusitaniae ) YJHT233, characterized in that, The preservation number of the Clavispora lusitaniae YJHT233 is GDMCC No: 62152.

2. A fermentation inoculant characterized in that, The fermentation agent comprises the Clavispora lusitaniae YJHT233 of claim 1.

3. The Clavispora lusitaniae YJHT233 of claim 1 or the fermentation agent of claim 2 is applied in brewing.

4. Use according to claim 3, wherein the compound is ###0002### The application is to increase the content of ester substances in the wine.

5. The use according to claim 3, wherein the compound is ###0002### The brewing is brewing of rice-flavor liquor or yellow rice wine.

6. A rice-flavor liquor or yellow rice wine preparation method, characterized in that, The method comprises the step of adding the Clavispora lusitaniae YJHT233 of claim 1 or the fermentation agent of claim 2 to the wine mash for fermentation.

7. The production method according to claim 6, wherein The addition amount of the Portuguese Williopsis sp. YJHT233 is 10 5 CFU / mL—10 8 CFU / mL.

8. The production method according to claim 6, wherein The fermentation temperature is 28-32℃.

9. The production method according to claim 6, wherein The fermentation time is 10-20 days.

10. A rice-flavor liquor or yellow wine, characterized in that, The wine is prepared by using the Clavispora lusitaniae YJHT233 of claim 1 or the fermentation agent of claim 2.

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