A Portuguese corynespora yeast and its application in liquor brewing

Abstract

The invention relates to the technical field of liquor brewing, and specifically discloses a Portuguese Clavispora yeast and an application thereof in liquor brewing. The Portuguese Clavispora yeast has a preservation number of GDMCC No: 61758 and is named Clavispora lusitaniae YJ18. The Portuguese Clavispora yeast has a very excellent ability to produce ethyl lactate and ethyl acetate, and is applied to the brewing of rice-flavor liquor to effectively increase the content of ethyl lactate and ethyl acetate in the rice-flavor liquor.

Description

Technical Field

[0001] The invention relates to the technical field of liquor brewing, and in particular to a Portuguese corynespora yeast and application thereof in liquor brewing. Background Art

[0002] There are three production methods for rice-flavor liquor: solid fermentation, semi-solid fermentation, and liquid fermentation. The semi-solid fermentation method uses rice as raw material and Xiaoqu as a saccharification and fermentation agent. In the early stage of fermentation, the fermented grains are solid and mainly saccharified, with a small amount of alcohol produced. In the later stage, the fermented grains are diluted with water to a semi-solid state for alcohol fermentation. Finally, it is made through distillation, aging, blending and other processes.

[0003] Liquor is composed of water, ethanol and other trace components that contribute to aroma and flavor, of which water and ethanol account for 98% to 99% of the total, while trace components account for only 1% to 2%. There are many types of trace components in liquor, which can be divided into alcohols, aldehydes, acids, esters, ketones, acetal compounds, aromatic compounds and pyrazines according to their chemical properties. Among the flavor components of liquor, alcohols are the main source of the sweetness of liquor and the flavor enhancer, acetaldehyde and acetal can coordinate the aroma and modify the flavor of liquor, and an appropriate amount of organic acid can make the aroma and taste of liquor fuller, and esters are the main components of the aroma of liquor.

[0004] Prior art research shows that compared with other types of liquor, rice-flavor liquor has relatively fewer flavor components, and its prominent main aroma components include ethyl lactate, ethyl acetate and β-phenylethanol. Therefore, increasing the content of ester substances in rice-flavor liquor is of great significance for improving the flavor of rice-flavor liquor.

[0005] The present invention discloses a brewing yeast and its application in liquor brewing in the previous invention patent CN 114181845 A. The contents of ethyl lactate and ethyl acetate in the volatile components of the rice-flavor liquor obtained by using the brewing yeast in cooperation with the old eight-foot liquor koji are 17.44% and 5.69%, respectively. However, the brewing yeast disclosed in the patent must cooperate with the old eight-foot liquor koji to prepare the rice-flavor liquor with high ethyl lactate and ethyl acetate contents; however, the rice-flavor liquor prepared by using the brewing yeast and other liquor kojis does not have high contents of ethyl lactate and ethyl acetate; and the old eight-foot liquor koji is produced by the old eight-foot liquor factory in Pingyuan County, Meizhou City, and its sales volume in the market is limited, thus limiting the large-scale production of other liquor factories.

[0006] In summary, providing an ester-producing yeast that can prepare rice-flavored liquor with high ethyl lactate and ethyl acetate contents without the use of a specific distiller's yeast has a broader application value. Summary of the invention

[0007] In order to solve at least one of the above technical problems existing in the prior art, the present invention first provides a Portuguese corynespora yeast.

[0008] The Portuguese coryneform yeast has a preservation number of GDMCC No: 61758 and is named Clavisporalusitaniae YJ18.

[0009] The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 30, 2021; the collection address is No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0010] The invention also provides application of the Portuguese corynespora yeast in liquor brewing.

[0011] Preferably, the Portuguese Corynespora yeast is used in liquor brewing to increase the ester content of liquor.

[0012] Preferably, the liquor is rice-flavor liquor.

[0013] Preferably, the ester substances are ethyl lactate and ethyl acetate.

[0014] The invention also provides a method for brewing rice-flavor liquor, wherein the method adopts the Portuguese corynespora yeast of the invention in the brewing process of the rice-flavor liquor.

[0015] Preferably, the Portuguese corynespora yeast of the present invention is used in the fermentation.

[0016] Beneficial effects: The present invention provides a new Portuguese corynosporum yeast, which has very excellent ability to produce ethyl lactate and ethyl acetate and its ability to produce ethyl lactate and ethyl acetate is significantly higher than that of brewer's yeast disclosed in patent CN 114181845 A.

[0017] In particular, in the brewing process of rice-flavor liquor, the Portuguese corynosporium yeast can prepare rice-flavor liquor with high ethyl lactate and ethyl acetate contents without the need for cooperating with specific distiller's yeast; and the rice-flavor liquor prepared by using the Portuguese corynosporium yeast has higher ethyl lactate and ethyl acetate contents than the rice-flavor liquor prepared by using the brewer's yeast disclosed in patent CN 114181845 A. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis diagram of the amplified products of 8 yeast strains with different colony morphologies isolated.

[0019] Figure 2 This is the phylogenetic tree of 8 yeast strains with different colony morphologies isolated. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to specific examples, but the examples do not limit the present invention in any form.

[0021] Example 1 Isolation and identification of Portuguese corynespora yeast

[0022] (1) Sample processing

[0023] Weigh 5.00g of Yingjinqianjiuqu and Shaofuqu samples respectively, add them into a conical flask containing 45mL of sterile saline, shake at 200r / min for 30min, and mix thoroughly. Take 1mL of sample solution, add it into a test tube containing 9mL of McFarland medium, and shake and culture at 28℃ and 120r / min for 24h.

[0024] (2) Isolation and purification of yeast

[0025] The dilution factor is 10 -4 , 10 -5 , 10 -6 and 10 -7 100 μL of each of the four gradient sample dilutions were spread on a Bengal red medium plate and cultured at 28°C for 48 hours. Observe the colony morphology, pick a typical single yeast colony with a moist and smooth surface, streak it on a PDA medium plate with bromocresol green, and invert it for 48 hours at 28°C. Pick a colony with a larger yellow circle for plate streak purification, select a single colony with good growth, number it, and inoculate it in YEPD medium, and culture it at 28°C and 120r / min for 24 hours. After culture, store it at -80°C with 20% glycerol (bacterial solution: 20% glycerol = 1:1) for use. Finally, a total of 8 yeast strains with different colony morphologies were isolated.

[0026] (3) Identification of yeast

[0027] 3.1 Morphological identification: Stain the yeast with methylene blue and examine under a microscope.

[0028] 3.2 Molecular Biology Identification:

[0029] Identification method: The yeast genomic DNA extraction kit was used to extract the DNA of 8 yeast strains with different colony morphologies, and then the universal primers were used to amplify the 26S rDNA sequence, purified and sequenced; then the 26S rDNA sequence was compared in the database to perform molecular identification of the screened strains. The universal primer sequence was referenced to the literature "Peng Jun, Yang Tuanyuan, Liu Pulin, et al. Comparison of yeast diversity and fermentation characteristics of oriental yeast in fermented grains and surrounding soils of liquor brewing [J]. China Brewing, 2019, 38(08): 77-84)", and was synthesized by Thermo Fisher Scientific (China) Co., Ltd., with a concentration of 20 μmol / L and stored at -20°C. The PCR amplification reaction system is shown in Table 1, and the PCR amplification program conditions are shown in Table 2. The obtained PCR product was purified using a DNA product purification kit and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the National Center for Biotechnology Information database by BLAST, and the phylogenetic tree was constructed using MEGA X software.

[0030] Table 1. PCR amplification reaction system

[0031]

[0032]

[0033] Table 2. PCR amplification program conditions

[0034]

[0035] Identification results: 8 yeast strains with different colony morphology, the electrophoresis of their PCR amplification products is shown in Figure 1. Figure 1 It can be seen that the bands of PCR amplification of all strains are clear and have no tailing phenomenon, which can be used for sequencing. Compared with the marker, the size of the DNA molecule amplified by PCR is between 500 and 750 bp.

[0036] The PCR products were sequenced and the results were compared with the National Center for Biotechnology Information database by BLAST. The strain identification results are shown in Table 3. Using MEGAX software, the 26S rDNA region sequence of the most homologous related strain was selected as the reference object based on the BLAST comparison results, and the phylogenetic tree of the eight strains was constructed. Figure 2 .

[0037] Table 3 Figure 1 and Figure 2The labeled strain YJ18 is the Portuguese Clavispora lusitaniae YJ18 described in the present invention.

[0038] Table 3. Results of homology analysis of strain 26S rDNA sequences

[0039]

[0040]

[0041] From Table 3 and Figure 2 It can be seen from the homology analysis results that the strain with the deposit number of GDMCC No: 61758 of the present invention has 100% homology with Clavispora lusitaniae YM26322; therefore, it is named Clavispora lusitaniae YJ18.

[0042] The strain with the deposit number of GDMCC No: 61758 is named Clavispora lusitaniae YJ18 Portuguese Clavispora yeast, and its main morphological characteristics are as follows: the cell morphology is oval, the single-end budding is obvious, and asexual reproduction is performed; the colony characteristics are that the colony in the solid culture medium is milky white, sticky, has smooth and neat edges, a flat surface, and reflects light under light.

[0043] Experimental Example 1 Effect of Glucose Matrix Environment on Clavispora lusitaniae YJ18

[0044] 100 mL of glucose fermentation medium (glucose mass fraction was 12%) was added to a 250 mL conical flask and yeast seed solution was inoculated (the amount of yeast seed solution added was 10% of the volume of glucose fermentation medium; the concentration of yeast in the yeast seed solution was 1×10 7 CFU / mL), and fermented at 28℃ for 3 days. The ester substances in the fermentation broth were analyzed by headspace solid phase microextraction-gas chromatography-mass spectrometry.

[0045] The yeast seed liquid tested was the yeast seed liquid containing Clavispora lusitaniae YJ18.

[0046] The specific method of headspace solid phase microextraction-gas chromatography-mass spectrometry technology for analyzing ester substances in fermentation broth is as follows: the extraction needle (50 / 30μm DVB / CAR / PDMS) is activated at 250℃ for 10min and set aside. 7mL of fermentation broth is drawn into a 25mL sample bottle, 1.4g of NaCl and a magnetic rotor are added, and an aluminum cap is covered; then the sample is placed on a magnetic stirring heater and preheated at 45℃ for 5min; the activated extraction needle is inserted and adsorption extraction is performed for 50min; then the extraction needle is inserted into the injection port of the GC-MS instrument and analyzed at 250℃ for 5min. GC conditions: DB-WAX UI chromatographic column (30m×0.25mm×0.25μm) is used, Agilent, USA. The injection port temperature is 250℃, no splitting, and helium is used as the carrier gas. Temperature program: starting temperature 40°C, hold for 2 min, increase to 100°C at 5°C / min, hold for 10 min, increase to 200°C at 10°C / min, hold for 10 min. MS conditions: EI ionization source; ion source temperature: 230°C; electron energy: 70 eV; acquisition mode: full scan; mass scan range (m / z): 50-550.

[0047] The experimental results show that the total amount of ethyl lactate and ethyl acetate produced by Clavispora lusitaniae YJ18 in a glucose matrix environment is 14.45 mg / L, which is significantly higher than 11.26 mg / L of the saccharomyces cerevisiae disclosed in CN 114181845 A (see paragraph 47 of its specification). It can be seen that the Clavispora lusitaniae YJ18 provided by the present invention has excellent ability to produce ethyl lactate and ethyl acetate, and its ability to produce ethyl lactate and ethyl acetate is significantly higher than that of the saccharomyces cerevisiae disclosed in patent CN114181845 A.

[0048] Embodiment 2 The brewing method of semi-solid rice-flavor liquor

[0049] (1) Washing rice: Weigh 250 g of rice that does not have a moldy smell and rinse it twice.

[0050] (2) Steaming rice: Put the washed rice into a bowl, add 190 mL of water, place in a high pressure sterilizer, and steam at 115°C for 15 min. The rice grains should be elastic but not sticky, and cooked but not mushy.

[0051] (3) Mixing the koji and making a nest: After the rice has cooled, add the Taiwan Rhizopus suspension (the weight of the Taiwan Rhizopus suspension added is 1% of the weight of the rice), stir well, put into a 2L fermentation tank, make a nest, seal it with gauze, and put it into a biochemical incubator for saccharification at 34°C for 48 hours.

[0052] (4) Fermentation: After saccharification, add Clavispora lusitaniae YJ18 bacterial suspension (12 mL Clavispora lusitaniae YJ18 bacterial suspension was added per 100 g rice, and the yeast suspension concentration was 1×10 7 CFU / mL), add 120% (120 mL of water per 100 g of rice) purified water, stir evenly, seal with plastic wrap, and ferment at 28°C for 21 days.

[0053] (5) Distillation: After fermentation, add 250 mL of water to the mash and distill to obtain liquor;

[0054] The Taiwan Rhizopus suspension is prepared by the following method: taking a Rhizopus slant cultured at 30° C. for 5 days, adding the spores on the slant into 10 mL of sterile water to prepare a Rhizopus spore suspension.

[0055] Experimental Example 2: Detection method of flavor substances in rice-flavor liquor prepared

[0056] (1) Preparation steps of standard solution: accurately pipette 2.0 mL each of methanol, n-propanol, n-butanol, isobutanol, n-pentanol, isopentanol, n-octanol, β-phenylethanol, ethyl acetate, isopentyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl benzoate, diethyl succinate, phenylethyl acetate, ethyl palmitate, ethyl oleate, ethyl lactate, acetaldehyde, acetal, furfural, benzaldehyde, acetophenone, acetic acid, octanoic acid, capric acid, and nonanoic acid, place them in a 100 mL volumetric flask, dilute to volume with 60% ethanol, mix well, and obtain standard stock solutions of each component; accurately pipette 0 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 4.0 mL of the standard stock solutions of each component, respectively, place them in 100 mL sample bottles, dilute to volume with 60% ethanol, and obtain standard solutions of different concentration gradients of each component;

[0057] (2) Steps for establishing a standard curve: Accurately pipette 10.0 mL of the standard solution of each component with different concentration gradients, and add 0.10 mL of the internal standard solution to each component; load the sample separately and detect each component by gas chromatography; establish a standard curve for each component with the concentration ratio of each component to the internal standard as the abscissa and the peak area ratio of each component to the internal standard as the ordinate;

[0058] (3) The steps for determining the content of volatile flavor substances in rice-flavor liquor are as follows: accurately aspirate 10.0 mL of rice-flavor liquor, add 0.10 mL of internal standard solution, mix well, load the sample, and use gas chromatography to detect the peak area ratio of each volatile flavor substance to the internal standard; substitute the peak area ratio of each volatile flavor substance to the internal standard into the standard curve of each component to obtain the concentration of each volatile flavor substance.

[0059] The internal standard solution described in steps (2) and (3) is prepared by the following method: accurately aspirate 2.0 mL of n-pentyl acetate, dilute to 100 mL with 60% (v / v) ethanol solution, mix well, and prepare an internal standard solution with a volume fraction of 2%.

[0060] The gas chromatography conditions for establishing the standard curve in step (2) are the same as the gas chromatography conditions for determining the content of volatile flavor substances in rice-flavor liquor in step (3).

[0061] The gas chromatography conditions are as follows: a DB-WAX UI chromatographic column (30m×0.25mm×0.25μm, Agilent, USA); an injection port temperature of 220°C, an injection volume of 1μL, a split ratio of 100:1, a flow rate of 1mL / min, and an FID detector temperature of 220°C; a hydrogen (H2): air (O2): tail gas (N2) of 40:400:25; and a gas chromatography gradient temperature program of 30°C, maintaining for 6min, increasing the temperature to 40°C at 2°C / min, maintaining for 2min, increasing the temperature to 100°C at 5°C / min, maintaining for 10min, increasing the temperature to 200°C at 10°C / min, and maintaining for 10min.

[0062] The standard curves and retention times of the volatile flavor substances obtained by the detection method are shown in Table 4. The content of ethyl lactate and ethyl acetate in the volatile flavor substances in the rice-flavor liquor prepared in Example 2 was tested by the detection method, and the detection results are shown in Table 5.

[0063] Table 4 Standard curves and retention times of volatile flavor substances

[0064]

[0065]

[0066] Table 5. Content of ethyl lactate and ethyl acetate in volatile components of rice-flavor liquor

[0067]

[0068]

[0069] It can be seen from the experimental data in Table 5 that the contents of ethyl lactate and ethyl acetate in the volatile components of the rice-flavored liquor prepared by the Portuguese Corynespora yeast of the present invention are 20.58% and 7.73%, respectively, which are significantly higher than the rice-flavored liquor prepared by the brewer's yeast disclosed in patent CN114181845 A (which are 17.44% and 5.69%, respectively, as specifically recorded in Table 5 in paragraph 76 of its specification).

[0070] It can be seen that the rice-flavored liquor with high ethyl lactate and ethyl acetate contents can be prepared by using the Portuguese corynosporium yeast of the present invention without cooperating with a specific old eight-foot distiller's yeast; and the rice-flavored liquor prepared by using the Portuguese corynosporium yeast has higher ethyl lactate and ethyl acetate contents than the rice-flavored liquor prepared by using the brewer's yeast disclosed in patent CN 114181845 A.

Claims

1. A Portuguese corynespora yeast, whose deposit number is GDMCC No: 61758, and is named Clavisporalusitaniae YJ18.

2. Use of the Portuguese corynespora yeast according to claim 1 in liquor brewing.

3. The use according to claim 2, characterized in that: The Portuguese corynespora yeast is used in improving the ester content of liquor in liquor brewing.

4. The use according to claim 3, characterized in that: The liquor is rice-flavor liquor.

5. The use according to claim 3, characterized in that: The ester substances are ethyl lactate and ethyl acetate.

6. A method for brewing rice-flavor liquor, characterized in that: The Portuguese corynespora yeast according to claim 1 is used in the brewing process of rice-flavor liquor.

7. The brewing method of rice-flavor liquor according to claim 6, comprising the steps of washing rice, steaming rice, mixing koji, fermenting and distilling, characterized in that: The Portuguese coryneformis yeast according to claim 1 is used in the fermentation.

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