CN114181845A - Saccharomyces cerevisiae and application thereof in white spirit brewing - Google Patents
Saccharomyces cerevisiae and application thereof in white spirit brewing Download PDFInfo
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Abstract
The invention relates to the technical field of liquor brewing, and particularly discloses saccharomyces cerevisiae and application thereof in liquor brewing. The saccharomyces cerevisiae has a preservation number of GDMCC No: 61757, named Saccharomyces cerevisiae CM 15. The saccharomyces cerevisiae has excellent capacity of producing ethyl lactate and ethyl acetate, and can be applied to brewing rice-flavor liquor, so that the content of ethyl lactate and ethyl acetate in the rice-flavor liquor can be effectively improved.
Description
Technical Field
The invention relates to the technical field of liquor brewing, in particular to saccharomyces cerevisiae and application thereof in liquor brewing.
Background
The production method of the rice-flavor liquor can be divided into three methods: solid state fermentation, semi-solid state fermentation, and liquid state fermentation. The semi-solid fermentation method for producing rice-flavor liquor is characterized by that it uses rice as raw material, uses Xiaoqu as saccharification leaven, makes the fermented grains in early stage of fermentation be solid state, mainly makes saccharification and also has a small quantity of alcohol production, and adds water in later stage to make alcoholic fermentation, and makes them pass through the processes of distillation, ageing and blending, etc.
The white spirit is composed of water, ethanol and other fragrant and tasty trace components, wherein the water and the ethanol account for 98-99% of the total amount, and the trace components only account for 1-2%. The types of trace components in the white spirit are quite rich, and the white spirit can be divided into alcohols, aldehydes, acids, esters, ketones, acetal compounds, aromatic compounds, pyrazine compounds and the like according to chemical properties. In the flavor components of the white spirit, alcohol substances are main sources and flavor promoters of the alcohol and sweet white spirit, acetaldehyde, acetal and the like can coordinate the fragrance and modify the flavor of the white spirit, a proper amount of organic acid can ensure that the fragrance and the taste of the white spirit are fuller, and ester substances are main components of the fragrance of the white spirit.
The prior art research shows that: compared with other types of white spirits, the rice-flavor white spirits have relatively few types of flavor components, and the main fragrance components of the rice-flavor white spirits comprise ethyl lactate, ethyl acetate, beta-phenylethyl alcohol and the like. Therefore, the improvement of the content of the esters in the rice-flavor liquor is of great significance for improving the flavor of the rice-flavor liquor.
Disclosure of Invention
In order to solve at least one of the above technical problems in the prior art, the present invention firstly provides a saccharomyces cerevisiae.
The saccharomyces cerevisiae has a preservation number of GDMCC No: 61757, named Saccharomyces cerevisiae CM 15. The strain is preserved in Guangdong province microorganism strain preservation center in 2021, 6 months and 30 days; the preservation address is No. 100 of the first-fierce middle route in Guangzhou city, Guangdong province.
The inventor finds in research that the screened Saccharomyces cerevisiae CM15 has very excellent ethyl lactate and ethyl acetate producing capability, and particularly has very excellent ethyl lactate and ethyl acetate producing capability in a glucose matrix environment; the capacity for producing ethyl lactate and ethyl acetate was much better than that of conventional Hansenula anomala and the honey-conjugated yeast LGL-1.
The invention also provides application of the saccharomyces cerevisiae in liquor brewing.
Preferably, the saccharomyces cerevisiae is used for improving the content of ester substances in white spirit in cooperation with the distiller's yeast.
The Saccharomyces cerevisiae CM15 has excellent capacity of producing ethyl lactate and ethyl acetate; therefore, the Saccharomyces cerevisiae CM15 is applied to brewing of the rice-flavor liquor, and the contents of ethyl lactate and ethyl acetate in the rice-flavor liquor can be effectively improved. In particular, Saccharomyces cerevisiae CM15 is brewed in cooperation with distiller's yeast; which can greatly improve the content of ethyl lactate and ethyl acetate in the rice-flavor liquor.
Preferably, the white spirit brewing comprises the steps of rice washing, rice steaming, starter propagation, fermentation and distillation.
Further preferably, saccharomyces cerevisiae is added during the fermentation step.
Further preferably, the saccharomyces cerevisiae is added in the form of a saccharomyces cerevisiae suspension; the ratio of the addition amount of the saccharomyces cerevisiae suspension to the rice amount is 8-12 mL: 100g of the total weight of the mixture; the concentration of the saccharomyces cerevisiae in the saccharomyces cerevisiae suspension is 1-2 multiplied by 107CFU/mL；
Most preferably, the saccharomyces cerevisiae is added as a suspension of saccharomyces cerevisiae; the ratio of the addition amount of the saccharomyces cerevisiae suspension to the rice dosage is 10 mL: 100g of the total weight of the mixture; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1 × 107CFU/mL；
Further preferably, the distiller's yeast is added in the step of mixing the distiller's yeast and the nest; the distiller's yeast is old eight feet distiller's yeast.
Further preferably, the added weight of the old eight feet distiller's yeast is 1-1.5% of the weight of the rice.
Most preferably, the added weight of old eight feet koji is 1.2% of the weight of rice.
Has the advantages that: the invention provides a brand-new saccharomyces cerevisiae; the saccharomyces cerevisiae has very excellent capacity of producing ethyl lactate and ethyl acetate, and particularly has very excellent capacity of producing ethyl lactate and ethyl acetate in a glucose matrix environment; the saccharomyces cerevisiae is applied to brewing of rice-flavor liquor, and the content of ethyl lactate and ethyl acetate in the rice-flavor liquor can be effectively improved.
Drawings
FIG. 1 is an electrophoresis chart of the amplified products of 8 isolated yeast strains with different colony morphologies.
FIG. 2 is a phylogenetic tree of 8 isolated yeast strains with different colony morphologies.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to these examples in any way.
In the embodiment, the old eight-chi distiller's yeast is used for brewing the old eight-chi rice wine in the old eight-chi distillery of plain county of Meizhou city, and the type is as follows: LBCJQ-5; the roasted bran koji is the koji used for brewing the Changle roasted rice wine by Guangdong Changle roasted wine industry, and the types of the koji are as follows: CLSJQ-8; the hawk-money distiller's yeast is a distiller's yeast used for brewing three-flower wine by Guangzhou hawk-money three-flower wine industry Limited company, and the type is as follows: YJQJQ-3. Hansenula anomala, CICC1651, purchased from China center for culture Collection of Industrial microorganisms. Honey-conjugated yeast LGL-1: the laboratory self-screening is preserved in China typical collection center with the preservation number of CCTCCM 2015545.
Example 1 isolation and characterization of Saccharomyces cerevisiae
(1) Sample processing
Weighing 5.00g of the chickpea koji and the burnt bran koji respectively, adding into a triangular flask filled with 45mL of sterile physiological saline, oscillating at 200r/min for 30min, and fully and uniformly mixing. 1mL of the sample solution was added to a test tube containing 9mL of Maifanitum culture medium, and subjected to shaking culture at 28 ℃ and 120r/min for 24 hours.
(2) Separation and purification of yeast
The suction dilution multiple is 10-4、10-5、10-6And 10-7Each 100. mu.L of 4-gradient sample dilutions was plated on a Bengal red medium plate and incubated at 28 ℃ for 48 h. Observing colony morphology, sequentially picking typical yeast single colonies with wet and smooth surfaces, streaking on a PDA culture medium plate added with bromocresol green, and performing inverted culture at 28 ℃ for 48 h. And selecting the colony with a larger yellow circle for plate streaking purification, selecting a single colony with better growth, inoculating the single colony in a YEPD culture medium, and carrying out shake culture at 28 ℃ and 120r/min for 24 h. After culturing, 20% glycerol (bacterial solution: 20% glycerol ═ 1:1) was used and stored at-80 ℃ for further use. In total, 8 yeast strains with different colony morphologies were isolated.
(3) Identification of Yeast
3.1 morphological identification: and (4) dyeing and microscopic examination is carried out on the yeast by using a methylene blue dye solution.
3.2 molecular biology identification:
the identification method comprises the following steps: 8 separated yeast strains with different colony morphologies are extracted by a yeast genome DNA extraction kit, and the screened strains are identified by 26S rDNA sequence analysis. The primers are synthesized by Saimer Feishale science (China) Limited, and stored at-20 ℃ with the concentration of 20 mu mol/L, compared with the diversity of yeasts in peripheral soil and the fermentation characteristics of Issatchenkia orientalis (J) and the primers are synthesized by Saimer Feishale science (China). The PCR amplification reaction system and the PCR amplification program conditions are shown in Table 1 and Table 2, respectively. And purifying the obtained PCR product by adopting a DNA product purification kit, and sending the purified PCR product to Shanghai biological engineering Co. BLAST alignment analysis was performed on the sequencing results with the National Center for Biotechnology Information database, and phylogenetic trees were constructed using MEGA X software.
TABLE 1 PCR amplification reaction System
TABLE 2 PCR amplification procedure conditions
And (3) identification result: electrophoresis patterns of PCR amplification products of 8 yeasts with different colony morphologies are shown in figure 1. As can be seen from FIG. 1, PCR amplified bands of all strains are clearer, and no dragging phenomenon exists, so that the PCR amplified bands can be used for sequencing. Compared with a Marker, the size of the DNA molecule obtained by PCR amplification is 500-750 bp.
The PCR products were sequenced, and the results were compared with the products by BLAST and with the National Center for Biotechnology Information database to obtain the strain identification results shown in Table 3. And (3) adopting MEGAX software, selecting a related strain 26SrDNA region sequence with the highest homology as a reference object according to the BLAST comparison result, and constructing a phylogenetic tree of 8 strains by using the region sequence as the reference object, wherein the phylogenetic tree is shown in figure 2.
The strain CM15 identified in Table 3, FIG. 1 and FIG. 2 is a Saccharomyces cerevisiae strain named as Saccharomyces cerevisiae CM15 according to the present invention.
TABLE 3 results of sequence homology analysis of strain 26S rDNA
As can be seen from the results of homology analysis in Table 3 and FIG. 2, the deposit number of the present invention is GDMCC No: 61757 having a homology of 99.65% with Saccharomyces cerevisiae CM 15; it was thus named Saccharomyces cerevisiae CM 15.
The preservation number of the invention is GDMCC No: 61757 Saccharomyces cerevisiae CM15, the main morphological characteristics of which are as follows: the cell shape is round or oval, sclerotium is in the cell, aseptic silk is generated, and budding reproduction is carried out; the colony is characterized by showing milk white colony in a solid culture medium, smooth surface, reflection under illumination and luster.
Experimental example 1 Effect of glucose substrate Environment on Saccharomyces cerevisiae CM15
Adding 100mL of glucose fermentation culture medium (glucose mass fraction is 12%) into a 250mL conical flask, respectively inoculating yeast seed solution (the addition amount of yeast seed solution is 10% of the volume of glucose fermentation culture medium; the concentration of yeast in yeast seed solution is 1 × 10%7CFU/mL), fermented at 28 ℃ for 3 d. And analyzing the ester substances in the fermentation liquor by adopting a headspace solid phase microextraction-gas chromatography-mass spectrometry technology.
The tested yeast seed liquid is respectively yeast seed liquid containing Saccharomyces cerevisiae CM15 and yeast seed liquid containing abnormal Hansenula; a yeast seed liquid containing Saccharomyces meliloti LGL-1.
The specific method for analyzing the esters in the fermentation liquor by the headspace solid-phase microextraction-gas chromatography-mass spectrometry technology comprises the following steps: the extraction needle (50/30 μm DVB/CAR/PDMS) was activated at 250 ℃ for 10min for use. Sucking 7mL of fermentation liquid into a 25mL sample bottle, adding 1.4g of NaCl and a magnetic rotor, and covering an aluminum cover; then placing the sample on a magnetic stirring heater, and preheating for 5min at 45 ℃; inserting activated extraction needle, and performing adsorption extraction for 50 min; then the extraction needle is inserted into the sample inlet of the GC-MS instrument and is analyzed for 5min at 250 ℃. GC conditions were as follows: DB-WAX UI column (30 m.times.0.25 mm.times.0.25 μm), Agilent Inc. was used. The injection port temperature is 250 ℃, no shunt is carried out, and helium is used as a carrier gas. Temperature rising procedure: the initial temperature is 40 deg.C, the temperature is maintained for 2min, the temperature is raised to 100 deg.C at 5 deg.C/min, the temperature is maintained for 10min, the temperature is raised to 200 deg.C at 10 deg.C/min, and the temperature is maintained for 10 min. MS conditions: an EI ionization source; ion source temperature: 230 ℃; electron energy: 70 eV; an acquisition mode: full scanning; mass scan range (m/z): 50-550.
The experimental results show that: the total amount of ethyl lactate and ethyl acetate produced by Saccharomyces cerevisiae CM15 in a glucose matrix environment was 11.26 mg/L; the total amount of the ethyl lactate and the ethyl acetate produced by the Hansenula anomala is only 1.04 mg/L; the total amount of ethyl lactate and ethyl acetate produced by the honey-conjugated yeast LGL-1 is only 2.95 mg/L. Therefore, the Saccharomyces cerevisiae CM15 provided by the invention has excellent capacity of producing ethyl lactate and ethyl acetate; its ability to produce ethyl lactate and ethyl acetate in a glucose-based environment is much better than that of conventional Hansenula anomala and the honey-conjugated yeast LGL-1.
Example 2 brewing method of semi-solid rice-flavor liquor
(1) Rice washing: 250g of rice without musty smell was weighed and washed twice.
(2) And (3) steaming rice: filling the cleaned rice in a basin, adding 190mL of water, placing in an autoclave, and steaming at 115 ℃ for 15 min; the steamed rice should be non-undercooked, elastic but not sticky, and cooked but not rotten.
(3) Mixing with yeast and building a nest: spreading the cooked rice, adding the old eight feet distiller's yeast (the added weight of the old eight feet distiller's yeast is 1.2% of the weight of the rice), stirring, placing into 2L fermentation tank, laying nest, sealing with gauze, placing into biochemical incubator, and saccharifying at 34 deg.C for 48 h.
(4) Fermentation: after the saccharification is finished, adding Saccharomyces cerevisiae CM15 bacterial suspension (10 mL of Saccharomyces cerevisiae CM15 bacterial suspension is added per 100g of rice, and the concentration of the yeast suspension is 1 × 107CFU/mL), adding 120% (per 100g rice adding 120mL water) purified water, stirring well, sealing with preservative film, fermenting at 28 deg.C for 21 d.
(5) And (3) distillation: after fermentation, 250mL of water is added to the mash, and the mash is distilled to obtain wine.
Example 3 brewing method of semi-solid rice-flavor Chinese liquor
The brewing method is the same as that of example 2; the difference is that the eagle money distiller's yeast with the same dosage as the old eight feet distiller's yeast is added in the step (3); the remaining steps and the condition parameters were the same as in example 2.
Example 4 brewing method of semi-solid rice-flavor Chinese liquor
The brewing method is the same as that of example 2; the difference is that the same amount of the burnt bran koji as the old eight feet of distiller's yeast is added in the step (3); the remaining steps and the condition parameters were the same as in example 2.
Comparative example 1 brewing method of semi-solid rice-flavor liquor
The brewing method is the same as that of example 2; except that no Saccharomyces cerevisiae CM15 bacterial suspension was added in step (4); the remaining steps and the condition parameters were the same as in example 2.
Comparative example 2 brewing method of semi-solid rice-flavor liquor
The brewing method is the same as that of example 2; except that, in the step (4), the same amount of the suspension of Hansenula anomala as that of the suspension of Saccharomyces cerevisiae CM15 was added in place of the suspension of Saccharomyces cerevisiae CM 15; the remaining steps and the condition parameters were the same as in example 2.
Comparative example 3 brewing method of semi-solid rice-flavor liquor
The brewing method is the same as that of example 2; except that in step (4) the same amount of a suspension of LGL-1 strain of honey-conjugated yeast as the suspension of Saccharomyces cerevisiae CM15 was added in place of the suspension of Saccharomyces cerevisiae CM 15; the remaining steps and the condition parameters were the same as in example 2.
Experimental example 2 method for detecting flavor substances in rice-flavor liquor prepared in preparation of rice-flavor liquor
(1) Preparing a standard solution: accurately sucking 2.0mL of each of methanol, n-propanol, n-butanol, isobutanol, n-pentanol, isoamyl alcohol, n-octanol, beta-phenylethyl alcohol, ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl benzoate, diethyl succinate, ethyl acetate, ethyl palmitate, ethyl oleate, ethyl lactate, acetaldehyde, acetal, furfural, benzaldehyde, acetophenone, acetic acid, octanoic acid, decanoic acid and nonanoic acid, placing the mixture into a 100mL volumetric flask, fixing the volume by using ethanol with the volume fraction of 60%, and uniformly mixing to obtain a standard stock solution of each component; accurately sucking standard stock solutions of each component, wherein the standard stock solutions are respectively placed in 100mL sample bottles in an amount of 0mL, 0.25mL, 0.5mL, 1.0mL, 2.0mL and 4.0mL, and performing constant volume with 60% ethanol to obtain standard solutions of each component with different concentration gradients;
(2) and (3) standard curve establishing: accurately sucking 10.0mL of standard solutions with different concentration gradients of each component, and adding 0.10mL of internal standard solutions respectively; loading the sample respectively, and detecting each component by gas chromatography; establishing a standard curve of each component by taking the concentration ratio of each component to the internal standard substance as a horizontal coordinate and the peak area ratio of each component to the internal standard substance as a vertical coordinate;
(3) the content determination method of the volatile flavor substances in the rice-flavor liquor comprises the following steps: accurately sucking 10.0mL of rice-flavor liquor, adding 0.10mL of internal standard solution, uniformly mixing, loading, and detecting the peak area ratio of each volatile flavor substance to the internal standard substance by using gas chromatography; and substituting the peak area ratio of each volatile flavor substance to the internal standard substance into each component standard curve to obtain the concentration of each volatile flavor substance.
The internal standard solution described in the steps (2) and (3) is prepared by the following method: accurately sucking 2.0mL of n-amyl acetate, diluting to 100mL by using 60% (v/v) ethanol solution, uniformly mixing, and preparing an internal standard solution with the volume fraction of 2%.
And (3) establishing a standard curve in the step (2) and determining the gas chromatography condition in the step (3) to be the same as the gas chromatography condition in the step of determining the content of the volatile flavor substances in the rice-flavor liquor.
The gas chromatography conditions are as follows: DB-WAX UI column (30 m.times.0.25 mm.times.0.25 μm, Agilent Corp., USA); the sample inlet temperature is 220 ℃, the sample injection amount is 1 mu L, the split ratio is 100:1, the flow rate is 1mL/min, and the temperature of an FID detector is 220 ℃; hydrogen (H)2): air (O)2): tail blowing (N)2) 40:400: 25; the gradient temperature program of the gas chromatography is as follows: the initial temperature is 30 ℃, the temperature is kept for 6min, the temperature is raised to 40 ℃ at the speed of 2 ℃/min, the temperature is kept for 2min, the temperature is raised to 100 ℃ at the speed of 5 ℃/min, the temperature is kept for 10min, the temperature is raised to 200 ℃ at the speed of 10 ℃/min, and the temperature is kept for 10 min.
The standard curve and retention time of each volatile flavor obtained by this assay are shown in table 4. The content of ethyl lactate and ethyl acetate in volatile flavor substances in the rice-flavor liquor prepared in the examples 2-4 and the comparative examples 1-3 is tested by the detection method, and the detection result is shown in table 5.
TABLE 4 Standard curves and Retention times for the volatile flavors
TABLE 5 content of ethyl lactate and ethyl acetate in Rice-flavor Chinese liquor in volatile components
As can be seen from the experimental data in Table 5, the contents of ethyl lactate and ethyl acetate in the volatile components of the rice-flavor liquor prepared in example 2 are 17.44% and 5.69%, respectively, which are much larger than the rice-flavor liquor prepared in comparative example 1 by only using the old eight feet of distiller's yeast without adding Saccharomyces cerevisiae CM 15. This shows that the addition of Saccharomyces cerevisiae CM15 in the fermentation step can greatly increase the contents of ethyl lactate and ethyl acetate in the rice-flavor liquor.
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-flavor liquor prepared in example 2 are much higher than those of examples 3 and 4. This indicates that: the content of ethyl lactate and ethyl acetate can be further greatly improved by the cooperation of Saccharomyces cerevisiae CM15 and old eight feet distiller's yeast; the improvement degree of the content of the ethyl lactate and the ethyl acetate is far greater than that of the Saccharomyces cerevisiae CM15 which is cooperated with other distiller's yeast.
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-flavor liquor prepared in example 2 are much higher than those of comparative examples 2 and 3. This shows that the addition of other yeasts in the fermentation step can not effectively increase the contents of ethyl lactate and ethyl acetate in the rice-flavor liquor; the content of ethyl lactate and ethyl acetate in the rice-flavor liquor can be greatly improved only by adding Saccharomyces cerevisiae CM15 in the fermentation step.
Claims (10)
1. Saccharomyces cerevisiae with the preservation number GDMCC No: 61757, named Saccharomyces cerevisiae CM 15.
2. Use of the saccharomyces cerevisiae according to claim 1 in the brewing of white spirit.
3. The use of claim 2, wherein the saccharomyces cerevisiae is used in combination with distiller's yeast to increase the content of esters in white spirit.
4. The use according to claim 2 or 3, characterized in that said brewing of white spirit comprises steps of washing rice, steaming rice, mixing with yeast and building up pits, fermentation and distillation.
5. Use according to claim 4, characterized in that Saccharomyces cerevisiae is added during the fermentation step.
6. The use according to claim 5, wherein the Saccharomyces cerevisiae is added as a suspension of Saccharomyces cerevisiae; the ratio of the addition amount of the saccharomyces cerevisiae suspension to the rice amount is 8-12 mL: 100g of the total weight of the mixture; the concentration of the saccharomyces cerevisiae in the saccharomyces cerevisiae suspension is 1-2 multiplied by 107CFU/mL。
7. The use according to claim 6, wherein the Saccharomyces cerevisiae is added as a suspension of Saccharomyces cerevisiae; the ratio of the addition amount of the saccharomyces cerevisiae suspension to the rice dosage is 10 mL: 100g of the total weight of the mixture; the concentration of Saccharomyces cerevisiae in the Saccharomyces cerevisiae suspension is 1 × 107CFU/mL。
8. Use according to claim 3, characterized in that the addition of koji during the step of topping is carried out; the distiller's yeast is old eight feet distiller's yeast.
9. The use according to claim 8, wherein the old eight feet koji is added in an amount of 1 to 1.5% by weight based on the weight of the rice.
10. Use according to claim 9, characterized in that the old eight feet koji is added in an amount of 1.2% by weight of the rice.
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