CN115927025B - Saccharomyces cerevisiae ZB423 and application thereof - Google Patents
Saccharomyces cerevisiae ZB423 and application thereof Download PDFInfo
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Abstract
The invention relates to a saccharomyces cerevisiae (Saccharomyces cerevisiae) ZB423 with a preservation number of GDMCC NO:62791. the saccharomyces cerevisiae ZB423 is derived from pickled plum, and can produce aroma substances such as acetoin, ethyl acetate and the like. After salt-tolerant domestication is carried out on the strain, the strain has good salt tolerance, and can still produce fragrant substances under a high-salt environment (salt concentration is 20%). The strain of the invention can produce 3-octanone, amyl propionate, isoamyl acetate and other fragrant substances when used for brewing soy sauce, and can also improve the contents of acetoin, phenethyl acetate and phenethyl acetate in the soy sauce. The strain ZB423 can be used as a soy sauce fermentation functional strain to promote the fragrance of soy sauce, so that the soy sauce has unique flavor and a new product flavor soy sauce is developed.
Description
Technical Field
The invention belongs to the technical field of food fermentation, and particularly relates to a saccharomycete (Saccharomyces cerevisiae) ZB423 suitable for food fermentation and application thereof.
Background
Soy sauce is a liquid seasoning brewed by adding water, salt and various microorganisms into soybeans or defatted soybeans, wheat or bran, and has reddish brown color and delicious taste. The soy sauce has complex components, besides the components of salt, various amino acids, saccharides, organic acids, pigments, spices and the like, mainly has salty taste, also has delicate flavor, fragrance and the like, can increase and improve the taste of dishes, and can also increase or change the color of the dishes. Soy sauce has a long history in China, and in order to meet different market demands, soy sauce with flavor such as straw mushroom soy sauce, steamed fish soy sauce and fish sauce has been developed in the market, but the seasoning industry is still developing soy sauce with various flavors, for example: patent document CN201010149460.2 discloses a tea-flavored soy sauce, CN202110721855.3 discloses a soy sauce with seafood flavor; CN202010209057.8 discloses a soy sauce with smoke flavor, etc., however, various types of soy sauce appearing on the market are not enough to meet the increasing demands of people.
The composition of microbial community and its metabolites have a great influence on the quality and flavor of soy sauce in the soy sauce brewing process, and the species mainly comprise lactobacillus, aspergillus, saccharomycetes and other bacteria. The strain used for soy sauce brewing affects the flavor and quality of soy sauce finally presented, and soy sauce brewed by different strains presents different flavors. In the prior art, related reports on producing flavor substances by saccharomycetes exist, but the flavor substances produced by the existing saccharomycetes in the soy sauce fermentation process are single, and the requirements of brewing soy sauce with different flavors cannot be met. Therefore, development of a new yeast strain suitable for brewing soy sauce is a technical problem to be solved in the art for enriching flavor soy sauce.
Disclosure of Invention
Based on the above purpose, in order to enrich the variety of the flavor soy sauce, the inventor screens out a strain of saccharomycete capable of improving the flavor of the soy sauce from the pickled plums, so as to enrich the variety of the flavor soy sauce and meet the market demand. The invention provides a saccharomyces cerevisiae (Saccharomyces cerevisiae) ZB423. The strain can be used for fermenting soy sauce to generate unique aroma substances, so that the aroma of the soy sauce is improved.
In order to achieve the purpose of the invention, the invention adopts the following technical modes:
in a first aspect, the invention provides a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) ZB423, deposited at the Guangdong province microbiological strain collection center at 9 and 14 days 2022, deposited at the Guangzhou Mitsui No. 100 university building 59 under accession number GDMCC NO:62791, taxonomic name: saccharomyces cerevisiae, which is named Saccharomyces cerevisiae Saccharomyces cerevisiae ZB423 in the present invention. The saccharomyces cerevisiae ZB423 can be used for high-yield of acetoin, phenethyl acetate and other unique fragrant substances.
The growth form of the saccharomyces cerevisiae ZB423 colony provided by the invention is as follows: round ridges, clean edges, smooth surface, moist, opaque, creamy.
In a second aspect, the invention provides a fermentation inoculant comprising the Saccharomyces cerevisiae ZB423. The strain can be used for producing acetoin and phenethyl acetate aroma substances by fermentation and can be used as a production strain of the aroma substances.
In a third aspect, the invention provides the use of said Saccharomyces cerevisiae ZB423 in food fermentation.
In a fourth aspect, the invention provides an application of the saccharomyces cerevisiae ZB423 in preparing soy sauce or yellow wine.
In a fifth aspect, the invention provides an application of the saccharomyces cerevisiae ZB423 in improving fragrance of soy sauce or yellow wine.
The Saccharomyces cerevisiae ZB423 has good salt resistance, and is not only suitable for fermenting and brewing low-salt or salt-reduced soy sauce, but also suitable for brewing soy sauce in a high-salt environment. The embodiment of the invention proves that the saccharomyces cerevisiae ZB423 is used for fermenting the high-salt diluted soy sauce, so that the prepared soy sauce contains rich aroma substances such as acetoin, phenethyl acetate, 3-octanone, phenethyl acetate, amyl propionate, phenethyl alcohol, isoamyl acetate and the like, the overall aroma and the flavor of the soy sauce are improved, and the saccharomyces cerevisiae ZB423 is a food fermentation strain with an aroma enhancing function. Compared with a blank control group, the content of acetoin, phenethyl acetate and phenethyl alcohol is obviously improved, which shows that the strain ZB423 can be used for preparing soy sauce and can also improve the fragrance of the soy sauce.
In a sixth aspect, the invention provides a method for increasing the content of acetoin and phenethyl acetate in soy sauce or yellow wine, the method comprising the step of fermentation using the Saccharomyces cerevisiae ZB423.
In a seventh aspect, the present invention also provides a soy sauce preparation method, comprising the steps of:
s1, preparing yeast according to a conventional high-salt diluted soy sauce brewing method to obtain a soy sauce yeast material;
s2, mixing the soy sauce yeast material obtained in the step S1 with salt water to obtain soy sauce mash;
s3, inoculating the saccharomyces cerevisiae ZB423 into the soy sauce mash obtained in the S2 for normal-temperature fermentation;
s4, fermenting and maturing, and separating to obtain soy crude oil.
In certain embodiments, the preparation method of the present invention, wherein the salt concentration of the high-salt diluted soy sauce mash in step S2 is 10% -20% (w/v).
In certain embodiments, the methods of the present invention wherein the ambient fermentation temperature of step S3 is from 25℃to 40 ℃ (e.g., 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃).
In certain embodiments, the methods of the invention are described wherein step S3 Saccharomyces cerevisiae ZB423 is inoculated at a final concentration of 10 5 CFU/ml～10 7 CFU/ml (e.g. 10 5 CFU/ml、10 6 CFU/ml、10 7 CFU/ml)。
Compared with the prior art, the invention has the following beneficial effects:
(1) The saccharomyces cerevisiae ZB423 is derived from plum samples of plum wine, and can be used for high-yield aroma substances such as acetoin, ethyl acetate and the like.
(2) The saccharomyces cerevisiae ZB423 provided by the invention has good salt tolerance after salt tolerance domestication, and can still produce fragrant substances in a high-salt environment (salt concentration is 20%). The strain is used for brewing soy sauce, and the detection shows that unique fragrant substances such as 3-octanone, amyl propionate and isoamyl acetate are generated in the soy sauce, and the content of other fragrant substances such as acetoin, phenethyl acetate and ethyl phenylacetate is obviously improved. Therefore, the strain ZB423 can be used as a soy sauce fermentation functional bacterium to promote the fragrance of soy sauce, so that the soy sauce has unique flavor and develops soy sauce with new flavor.
(3) The saccharomyces cerevisiae ZB423 can also be used for brewing yellow wine, can improve the content of acetoin and phenethyl acetate aroma substances in the yellow wine, and improves the overall aroma and quality of the yellow wine.
Drawings
FIG. 1 is a graph of colonies of Saccharomyces cerevisiae ZB423 on a plate;
FIG. 2 is a flow chart of the process of the Saccharomyces cerevisiae ZB423 of the present invention for high salt dilute fermented soy sauce;
FIG. 3 is a graph showing the detection of aroma substances in soy sauce brewed by Saccharomyces cerevisiae ZB423 of the present invention;
FIG. 4 is a flow chart of the preparation process of the Saccharomyces cerevisiae ZB423 for yellow wine;
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the reagents, methods and apparatus employed in the present invention are conventional food-grade reagents, methods and apparatus in the art.
Unless otherwise indicated, the test conditions used in the examples of the present invention are conventional test conditions in the art. Reagents used in the examples of the present invention are all commercially available unless otherwise specified.
EXAMPLE 1 isolation and screening of the strains of interest
1. Bacterial strain primary screening
PDA medium: 20% of potato juice, 2% of glucose and 2% of agar.
YPD medium: 1% of yeast extract, 2% of peptone and 2% of glucose.
Removing cores from soaked plum in plum wine, homogenizing, crushing, taking 5g sample, adding 45ml sterile water to obtain suspension, and gradually diluting the suspension with sterile physiological saline to 10 -3 、10 -4 、10 -5 、10 -6 Preparing bacterial cell dilution liquid with different concentrations, counting under microscope, selecting 10 -5 And carrying out subsequent experiments on the bacterial dilution with the concentration gradient.
0.1ml of the above 10 was removed -5 The bacterial colony is selected to be round, raised, neat in edge, smooth in surface, moist, opaque and creamy, and single bacterial colony with the diameter of more than 1.0mm is further streaked and separated on a YPD flat plate, so that 5 bacterial strains with the bacterial colony diameter of more than 1.0mm are obtained. The primary strain diameters are shown in table 1:
TABLE 1 diameter of the primary strain
| Strain numbering | Colony diameter (mm) |
| 1# | 0.3 |
| 2# | 0.6 |
| 3# | 1.4 |
| 4# | 2.2 |
| 5# | 1.2 |
| 6# | 0.5 |
| 7# | 0.9 |
| 8# | 2.0 |
| 9# | 1.8 |
| 10# | 0.8 |
| 11# | 0.4 |
2. Salt tolerance domestication
In order to adapt to the high-salt environment of soy sauce fermentation, salt tolerance domestication experiments are carried out on the strain which is screened initially: the 5 strains (3#, 4#, 5#, 8#, 9#) which are screened at first are respectively inoculated into YPD liquid culture media with salt concentration of 5%, 8%, 10%, 12%, 15%, 18% and 20% (w/v) in sequence for domestication, and after salt-tolerant strains are obtained, colony diameters of the 5 strains are compared, the specific operation is as follows:
s1, respectively inoculating the 5 strains screened in the liquid YPD liquid culture medium with the salt concentration of 5%, and placing the liquid YPD liquid culture medium at 30 ℃ for shake culture at 200rpm for 2 days; inoculating the bacterial liquid dipped by an inoculating loop to YPD plate culture medium (the salt concentration is 5%) for streaking separation to obtain single colony;
s2, inoculating the single colony obtained in the step S1 into a liquid YPD liquid culture medium with the salt concentration of 8%, and placing the liquid YPD liquid culture medium at 30 ℃ for shake culture at 200rpm for 2 days; inoculating the bacterial liquid dipped by an inoculating loop to a PDA plate culture medium (the salt concentration is 8%) for streaking separation to obtain single colonies;
s3, inoculating the single colony obtained in the step S2 into a liquid YPD liquid culture medium with the salt concentration of 10%, and placing the liquid YPD liquid culture medium at 30 ℃ for shake culture at 200rpm for 2 days; inoculating the bacterial liquid dipped by an inoculating loop to a PDA plate culture medium (the salt concentration is 10%) for streaking separation to obtain single colonies;
s4, repeating the steps until single colonies are picked and inoculated into a liquid YPD liquid culture medium with the salt concentration of 20%, and placing the liquid YPD liquid culture medium at 30 ℃ for shake culture at 200rpm for 2 days; the single colony was obtained by streaking the PDA plate medium (20% salt concentration) with the inoculating loop dipped with the bacterial liquid. The diameters of the plate colonies after salt tolerance domestication of 5 strains are shown in table 2:
TABLE 2 diameter of salt tolerant strains
| Strain numbering | Colony diameter (salt concentration 20%, mm) |
| 3# | 0.5 |
| 4# | 1.1 |
| 5# | 0.8 |
| 8# | 1.2 |
| 9# | 1.1 |
As shown in Table 2, strains No. 4, no. 8 and No. 9 were excellent in salt tolerance, and thus 3 strains were selected for re-screening.
2. Bacterial strain re-screening
Strains 4# and 8# are respectively inoculated into YPD liquid culture medium with the salt concentration of 20%, and after the YPD liquid culture medium is placed at the constant temperature of 30 ℃ and cultured for 2 days at the constant speed of 200rpm, the contents of acetoin and phenethyl acetate in fermentation liquid are detected by adopting headspace extraction-gas phase-mass spectrometry (GC-MS).
Chromatographic conditions: 5ml of fermentation broth was taken in a 25ml sample bottle, extracted for 40min at a constant temperature of 55℃using a 75. Mu. MCAR/PDMS solid phase extraction head, and injected for GC-MS analysis. Chromatographic column: OV1701 quartz capillary column (60 m. Times.0.25 mm. Times.0.5 μm); sample inlet: 270 ℃, sample injection is not carried out in a split way; heating program: the temperature is kept at 40 ℃ for 5min, the temperature is raised to 100 ℃ at 2.5 ℃/min, and the temperature is raised to 250 ℃ at 20 ℃/min for 10min.
Mass spectrometry conditions: ion source temperature: 230 ℃; ion source: an EI source; electron energy: lev; four-stage bar temperature: 150 ℃; mass-to-charge ratio scan range: 25-450 amu; the results of the mass spectrogram search and detection by using NIST standard spectrum library are shown in Table 3:
TABLE 3 detection of aroma substances in fermentation broths of different strains (peak area X10) 6 )
| Strain | Acetoin | Phenyl ethyl acetate |
| 4# | 6.72 | 0.63 |
| 8# | 6.89 | 2.56 |
| 9# | 6.21 | 1.12 |
As can be seen from Table 3, the yield of acetoin and phenethyl acetate was highest in comparison with strain 8# and thus strain 8# was used as the objective strain of the present invention. Bacterial colony of strain 8# on PDA plate culture medium is milky white, raised, round, smooth in surface, regular in edge, opaque, creamy, 1.5-2.5 mm in diameter and shown in figure 1. And (3) identifying the 18srDNA gene sequence of the strain 8# strain, and determining that the strain 8# is a saccharomyces cerevisiae when the similarity with the saccharomyces cerevisiae (Saccharomyces cerevisiae) reaches 100%. The strain is preserved in the microorganism strain preservation center of Guangdong province, and the obtained preservation number is GDMCC No. 62791, and the taxonomic name is: saccharomyces cerevisiae, the invention names it: saccharomyces cerevisiae Saccharomyces cerevisiae ZB423.
Example 2 Saccharomyces cerevisiae ZB423 used in high salt dilute fermented Soy sauce
First-stage seed: activating ZB423 strain, inoculating the strain into YPD liquid culture medium containing 5% of salt, and shake culturing at 30 ℃ and 200rpm until logarithmic growth phase to obtain first-stage seeds.
Secondary seed: the first seed is inoculated into YPD liquid culture medium containing 10% of salt in an inoculum size of 3%, and shake culture is carried out at 30 ℃ and 200rpm until logarithmic growth phase, so as to obtain the second seed.
And (3) soy sauce fermentation: a starter (refer to figure 2 of the specification) is prepared according to a conventional method, and is prepared byAdding salt water into the material to prepare diluted soy sauce mash with salt content of 20%, inoculating ZB423 strain of the invention into the soy sauce mash for 0-1 day, and finally inoculating 10% of strain 6 CFU/mL. And simultaneously taking a fermentation tank which is not inoculated with the strain as a blank control group, and respectively inoculating the strains with the numbers of 4# and 9# as control groups. After the fermentation, the content of the aroma substances in each group of soy sauce crude oil samples was measured by the detection method of example 1, and the results are shown in table 4:
table 4: detection result of aroma substances of fermented crude oil
As can be seen from table 4, 1) the soy sauce aroma substances fermented by the saccharomyces cerevisiae ZB423 are more abundant than the blank, and the aroma substances not present in the control are newly added, for example: acetoin, 3-octanone, amyl propionate, isoamyl acetate; the content of the same aroma substances is significantly improved, for example: the phenethyl acetate is improved by 10 times, the phenethyl acetate is improved by 9 times, and the phenethyl alcohol is improved by 7 times. 2) Compared with the control groups 4# and 9# the new aroma substances were detected in soy sauce fermented with ZB 423: 3-octanone, ethyl phenylacetate and isoamyl acetate. It can be seen that although acetoin and phenethyl acetate were also produced in soy sauce fermented with strains 4# and 9#, the types of aroma substances were not as abundant as ZB423.
Acetoin and 3-octanone have pleasant milk flavor, phenethyl acetate and phenethyl acetate have honey flavor, and amyl propionate and isoamyl acetate have flower and fruit flavor. The phenethyl alcohol is an important mellow substance produced by the fermentation of the saccharomyces cerevisiae, has fragrant and sweet flower fragrance, can cooperate with the fragrance substances in the soy sauce to promote the comprehensive fragrance of the soy sauce, has excellent retention capability in liquid, and can permanently emit fragrance.
In order to verify the quality of soy sauce fermented by saccharomyces cerevisiae ZB423, 15 high-experience evaluators are summoned to form a sensory evaluation group, and the high-salt diluted soy sauce products prepared by ZB423 and a blank control group are comprehensively evaluated, wherein the indexes of sensory evaluation comprise aroma, delicate flavour, sweet taste, salty taste and sour taste, the score is 0-10, and the higher the score, the more outstanding the index and the better the taste. The results of sensory evaluation are shown in table 5:
table 5 sensory evaluation results of soy sauce
| Group of | Fragrance of fragrance | Fresh flavor | Sweet taste | Salty taste | Sour taste | Comprehensive scoring |
| Blank control | 5.2 | 6.5 | 5.3 | 8.1 | 7.8 | 6.6 |
| ZB423 fermentation group | 9.2 | 8.1 | 8.5 | 7.2 | 8.1 | 8.2 |
Remarks: the data in the table are the average value of the evaluation results of 15 evaluation persons, the score value of each index is 0-10, and the higher the score, the more outstanding the index.
Example 3 genetic stability detection
Inoculating the Saccharomyces cerevisiae ZB423 screened in the embodiment 1 to PAD slant culture medium for 10 generations, observing the growth condition of each generation of strain, fermenting and culturing the strain by the 1 st generation, 5 th generation and 10 th generation slant strains according to the method in the embodiment 1, and determining the content of aroma substances in fermentation liquor by adopting GC-MS after fermentation is finished, so as to judge the genetic stability of the strain. If the content error of the aroma substances measured in the fermentation liquor obtained by fermenting the 10-generation strain is within the error range of 10%, the strain is indicated to have good genetic stability, and the detection result is shown in the following table 6:
TABLE 6 results of test for stability of passage ZB423 of Yeast
| Detecting items | ZB423 generation 1 | ZB423 generation 5 | ZB423 generation 10 |
| Acetoin | 1.00 | 0.98 | 0.97 |
| Phenyl ethyl acetate | 1.00 | 0.99 | 0.96 |
Remarks: the 1 st generation of microzyme ZB431 is used as each index of 1.00, the 5 th generation and the 10 th generation are respectively converted into corresponding ratios.
As can be seen from the results of Table 6, compared with the generation 1, the contents of the aroma substances in the fermentation liquor of the generation 5 and generation 10 strain ZB423 are not changed significantly, which indicates that the strain ZB423 of the invention has good genetic stability and meets the production and use requirements.
Example 4 Saccharomyces cerevisiae ZB423 used for preparing yellow wine
Activating ZB423 strain, inoculating in YPD liquid culture medium, shake culturing at 30deg.C and 200rpm to logarithmic phase, and collecting thallus to obtain yeast of fermented yellow wine. Referring to the yellow wine preparation process (adopting a conventional preparation process) of fig. 4 in the specification, the yellow wine is prepared by fermenting yeast ZB423, and then the content of acetoin and ethyl acetate in a yellow wine sample is detected by GC-MS, so that ZB423 is not added as a blank control group, and the result is shown in Table 7:
table 7: detection result of acetoin and phenethyl acetate in yellow wine
As can be seen from Table 7, the use of Saccharomyces cerevisiae ZB423 to prepare yellow wine can increase the content of acetoin and phenethyl acetate in the yellow wine, and promote the fragrance of the yellow wine, which indicates that ZB423 can be used as a functional production strain of the yellow wine.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. A strain of saccharomyces cerevisiae (Saccharomyces cerevisiae) ZB423, wherein the saccharomyces cerevisiae ZB423 is deposited under the accession number GDMCC NO:62791.
2. a fermenting bacteria agent, which is characterized in that the bacteria agent comprises the saccharomyces cerevisiae ZB423 of claim 1.
3. The use of Saccharomyces cerevisiae ZB423 as claimed in claim 1 for the preparation of soy sauce or for brewing.
4. The use of saccharomyces cerevisiae ZB423 of claim 1 in enhancing the aroma of soy sauce or yellow wine.
5. A method for increasing the content of acetoin and phenethyl acetate in soy sauce or yellow wine, which comprises the step of fermenting soy sauce or yellow wine using saccharomyces cerevisiae ZB423 according to claim 1.
6. A soy sauce preparation method, which is characterized by comprising the following steps:
s1, preparing starter according to a conventional high-salt diluted soy sauce brewing method to obtain a soy sauce starter;
s2, mixing the soy sauce yeast material obtained in the step S1 with salt water to obtain soy sauce mash;
s3, inoculating the saccharomyces cerevisiae ZB423 of claim 1 into the soy sauce mash obtained in the S2 for normal-temperature fermentation;
s4, fermenting and maturing, and separating to obtain soy crude oil.
7. The soy sauce preparation method of claim 6, wherein the salt concentration of the brine in the step S1 is 10% to 20% (w/v).
8. The method for preparing soy sauce according to claim 6, wherein the fermentation temperature at room temperature in the step S3 is 25℃to 40 ℃.
9. The soy sauce preparation method of claim 6 wherein the step S3S Saccharomyces cerevisiae ZB423 is inoculated to a final concentration of 10 5 CFU/ml～10 7 CFU/ml。
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| CN114507610A (en) * | 2021-03-15 | 2022-05-17 | 佛山市海天(高明)调味食品有限公司 | Saccharomyces cerevisiae for producing sauce flavor and application thereof |
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| CN106929438A (en) * | 2016-11-18 | 2017-07-07 | 天津科技大学 | One plant height produces the saccharomyces cerevisiae and its construction method of Tetramethylpyrazine |
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| CN113637596A (en) * | 2021-08-24 | 2021-11-12 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
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