CN108384728B - Saccharomyces cerevisiae and application thereof - Google Patents
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- CN108384728B CN108384728B CN201810226937.9A CN201810226937A CN108384728B CN 108384728 B CN108384728 B CN 108384728B CN 201810226937 A CN201810226937 A CN 201810226937A CN 108384728 B CN108384728 B CN 108384728B
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Abstract
The invention discloses saccharomyces cerevisiae and application thereof, and belongs to the technical field of fermentation. Saccharomyces cerevisiae FS-ZJQ has been deposited in China general microbiological culture Collection center at 12 months and 14 days in 2017 with the preservation number of CGMCC No. 15063. The method is used for brewing the fingered citron vinegar alcohol, can adapt to the fermentation environment of fingered citron juice, and has strong alcohol production capacity, high fermentation speed and excellent flavor.
Description
Technical Field
The invention relates to saccharomyces cerevisiae and application thereof, and belongs to the technical field of fermentation.
Background
Saccharomyces cerevisiae (Saccharomyces cerevisiae) belongs to the kingdom of eukaryotes, kingdom fungi, phylum Ascomycota, subdivision Saccharomycotina, class Semiascomycetes, order Saccharomycophyllae, genus Saccharomyces in taxonomic classification. The saccharomyces cerevisiae colony is round, glossy, flat and neat in edge, and the cultured cells in the wort are in a small circle or oval shape. The growth temperature is 28-30 ℃, and the optimum pH value is 4-5. Saccharomyces cerevisiae is the most widely related yeast to human, not only because it is traditionally used for making bread, steamed bread and other food and brewing wine, but also as a eukaryotic model organism in modern molecular and cellular biology, and its function is equivalent to prokaryotic model organism Escherichia coli. Saccharomyces cerevisiae is the most commonly used biological species in fermentation. The cells of Saccharomyces cerevisiae are spherical or ovoid, 5-10 μm in diameter. The reproduction method is budding reproduction.
The citrus of the family Rutaceae of the genus Buddha, similar to citrus, grapefruit, etc., has a close relationship with citron, lemon, etc., and is currently cultivated in Zhejiang, Sichuan, Guangdong, Fujian, etc. The fingered citron contains abundant mineral elements and a plurality of physiological active substances, and particularly the limonin compounds contained in the fingered citron have anticancer, antibacterial, antimalarial, antiviral and insecticidal effects and other pharmacological effects on human bodies, so the fingered citron is a medicinal and edible crop. At present, the fingered citron is mainly used as landscape and medicinal materials, and the variety of deep-processed products is less. The research firstly utilizes the fingered citron juice as the raw material to obtain the fruit vinegar beverage through alcoholic fermentation and acetic acid fermentation. The purpose is to keep the medicinal and functional nutrient components of the fingered citron in the product, endow a plurality of beneficial metabolites produced in the fermentation process, and be more beneficial to the absorption of human body. The alcohol fermentation is the first step of producing the fruit vinegar, the accumulation of alcohol and good aroma play an important role in the quality of the finished fruit vinegar, and meanwhile, in order to avoid the influence of limonin on the fermentation capacity of yeast, the screening of a yeast strain which can adapt to the fermentation environment of the fingered citron, has strong alcohol production capacity, high initiation speed and good flavor has important significance in the industrial production of the fingered citron fruit vinegar.
Disclosure of Invention
The invention aims to provide a Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is preserved in China general microbiological culture Collection center (CGMCC) within 12 months and 14 days in 2017, wherein the preservation number is CGMCC No.15063, and the preservation address is Beijing university No.1 North road, Naja district, China academy of sciences microbial research institute.
The second purpose of the invention is to provide the culture method of the saccharomyces cerevisiae CGMCC No.15063, which is to inoculate the saccharomyces cerevisiae CGMCC No.15063 into YEPD liquid culture medium for culture.
In one embodiment of the present invention, the YEPD medium is: 5-20 g/L yeast powder, 10-30 g/L peptone and 10-30 g/L glucose, and the pH is natural.
The third purpose of the invention is to provide a method for producing the finger citron wine by using the saccharomyces cerevisiae, which is to inoculate the saccharomyces cerevisiae CGMCC No.15063 in the alcohol fermentation stage of the finger citron wine.
In one embodiment of the present invention, the inoculation is carried out in an amount of 4 to 6%.
The fourth purpose of the invention is to provide the fingered citron wine prepared by the saccharomyces cerevisiae CGMCC No. 15063.
The fifth purpose of the invention is to provide the application of the saccharomyces cerevisiae CGMCC No.15063 in food.
In one embodiment of the invention, the application comprises preparing fruit juice or alcoholic beverage with fingered citron as a fermentation raw material.
The sixth purpose of the invention is to provide a microbial agent containing the saccharomyces cerevisiae CGMCC No. 15063.
In one embodiment of the present invention, the microbial agent is a solid microbial agent or a liquid microbial agent.
The invention has the beneficial effects
The saccharomyces cerevisiae CGMCC No.15063 provided by the invention is used for brewing the fingered citron wine in the alcohol stage, can adapt to the fermentation environment of the fingered citron juice, and has strong alcohol production capacity, high initiation speed and excellent flavor.
Biological material preservation
The Saccharomyces cerevisiae FS-ZJQ provided by the invention is stored in the China general microbiological culture Collection center (CGMCC) within 12 months and 14 days in 2017, the storage number is CGMCC No.15063, the storage address is No. 3 of Beijing university Hokko No.1 of sunward time, China academy of sciences.
Drawings
FIG. 1 is an electropherogram of the result of amplification of 18S rDNA; m, marker; 1, CGMCC No. 15063;
FIG. 2 shows the growth of different yeasts on finger citron juice agar plates;
FIG. 3 shows the growth of Saccharomyces cerevisiae FS-ZJQ at different initial sugar concentrations;
FIG. 4 shows the growth of Saccharomyces cerevisiae FS-ZJQ at different initial pH values;
FIG. 5 shows the growth of Saccharomyces cerevisiae FS-ZJQ with different initial alcoholic strength.
Detailed Description
YEPD medium: 10g/L yeast powder, 20g/L peptone, 20g/L glucose, natural pH, distilled water preparation, and sterilization at 115 ℃ for 15 min.
The analysis method comprises the following steps: and (4) carrying out detection analysis on the related indexes according to a general analysis method for GBT 15038-.
In order to facilitate the reading of the technical scheme of the invention, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.12647 is named as Saccharomyces cerevisiae FS-ZJQ.
Example 1: screening and identification of Saccharomyces cerevisiae FS-ZJQ
1. Seed taking: naturally fermenting the fingered citron juice until a great amount of foam appears in the fermentation liquid.
2. Inoculating, enriching and culturing: the fermentation broth was aspirated, inoculated with 5% inoculum size in YEPD medium containing 100. mu.L of 10% AMP solution, and cultured at 28 ℃ at 150r/min for 1 day.
3. Pure strain separation, namely diluting the enriched bacterial suspension by 5 gradients, taking 100 mu L of the bacterial suspension, coating a flat plate, carrying out inverted culture for 2 days at 28 ℃, selecting single bacterial colonies with better morphology from the cultured flat plate, carrying out two-stage three-zone streak separation and microscopic examination, identifying by ① bacterial colony characteristic observation, selecting bacterial strains which are about 2.0mm in diameter, round, smooth in surface, milky and the like and accord with the bacterial colony characteristics of saccharomycetes through direct observation by naked eyes, ② individual morphology observation, namely observing by an optical microscope, selecting bacterial strains which are oval, 1-5 mu m x (5-30) mu m in size and have obvious bud cells in visual field, selecting 85 bacterial strains, and inoculating the bacterial strains into a test tube filled with 10mL of finger citron juice, wherein two bacterial strains are parallel.
4. Primary screening: uses Angel active dry yeast powder as reference, and uses the growth ability of bacterial strain and the fragrance component of fermentation liquor as indexes to eliminate the yeast with no obvious fragrance or bad smell and poor fermentation ability. 29 strains were kept.
5. Secondary screening: by taking Angel active dry yeast powder as a reference and taking alcoholic strength and aroma sensory evaluation as measurement indexes, yeasts with too low alcoholic strength of fermentation liquor and too poor evaluation results are eliminated. 7 strains were kept.
6. And (3) third-stage screening: and (3) selecting Angel active dry yeast powder as a reference, and selecting the slow-rise yeast as a measurement index. 3 strains were kept.
7. Four-stage screening: fermenting the 3 strains of bacteria into finger citron wine respectively, measuring the organic acid content of the finished wine, and reserving 1 strain with the maximum non-volatile acid content.
8. And (3) strain identification: extracting strain genome DNA, and amplifying the genome DNA by using yeast ITS universal primer pair forward primer ITS1 and reverse primer ITS4 under the following reaction conditions: pre-denaturation at 94 ℃ for 5min was followed by the following cycles: denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 40s, extension at 72 ℃ for 60s, and 35 cycles; extension at 72 ℃ for 10 min. The result is good after 2% agarose gel electrophoresis. The PCR amplification product was sent to Biotechnology engineering (Shanghai) Ltd for sequencing, and the sequencing result was subjected to BLAST sequence alignment on NCBI website to determine Saccharomyces cerevisiae, which was named as Saccharomyces cerevisiae FS-ZJQ. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 14 days in 2017, the preservation number is CGMCC NO.15063, and the preservation address is the institute of microbiology of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, south China, Kyoho, Beijing.
Example 2 analysis of major flavor Components in Saccharomyces cerevisiae FS-ZJQ fermentation broth
After FS-ZJQ fermentation, compared with Angel wine yeast, the fermentation liquor has the terpene compound with higher relative content, the terpene compound types include α -terpinene (citrus and lemon fragrance), α -phellandrene (mint fragrance), β -phellandrene (green fragrance) and other 9 ester compounds with relative content of 6.6%, which are respectively hexyl acetate (fresh and sweet fruit fragrance), ethyl caprylate (fruit fragrance), geranyl acetate (rose fragrance), phenethyl acetate (rose fragrance) and other 12 ester types and quantity, the types and quantity of the ester determine the flavor and aroma of the fruit wine, the FS-ZJQ yeast and Angel wine yeast can adapt to the volatile ingredients and the aroma capacity of the wine, and the aroma capacity of the wine is better than that of the Angel wine yeast, and the aroma capacity of the wine is ZJQ.
TABLE 1 analysis of the major flavor components in Saccharomyces cerevisiae FS-ZJQ fermentation broth
Example 3 growth comparison of Saccharomyces cerevisiae FS-ZJQ with other yeasts on finger citron juice agar plates
Activating yeast strain FS-ZJQ and other yeasts, and spreading on finger citron juice agar plate with concentration of 1.5 × 108Diluting to 10/mL-5Coating is carried out, and growth is observed after 2 d. As can be seen from FIG. 2, different yeasts grow on the bergamot juice plate in different ways. (a) The number of colonies of (b) (c) was. + -. 155, + -121, + -95, respectively, and the size of the colonies in (a) was significantly larger than that of the colonies of (b) (c), while no yeast colonies were grown on (d) (e). Therefore, FS-ZJQ can be adapted to the fingered citron juice, and the growth condition of the yeast is better than that of other yeasts such as Angel wine yeast.
Example 4 verification of the tolerance of Saccharomyces cerevisiae FS-ZJQ
1. Sugar tolerance: inoculating 5% of the extract into 12, 15, 18, 21, and 24 ° Bx fructus Citri Sarcodactylis juice, respectively, culturing at 28 deg.C for 3d, sampling every 12h, and measuring absorbance at 600nm with ultraviolet spectrophotometer. As can be seen from FIG. 3, the growth rate of yeast increases with increasing sugar concentration when the initial sugar concentration is lower than 21 ℃ Bx and then becomes stable when the sugar concentration is higher than 21 ℃ Bx during 0-24 h. From this, FS-ZJQ was found to have a strong sugar tolerance.
pH tolerance: the pH of the bergamot juice is about 4.2, so the tolerance of FS-ZJQ at pH <4 is considered. The pH was set to five gradients of 2, 2.5, 3, 3.5, 4, respectively. Inoculating 5% fructus Citri Sarcodactylis juice with sugar degree of 18 ° Bx, culturing at 28 deg.C for 3d, sampling every 12h, and measuring absorbance at 600nm with ultraviolet spectrophotometer. As can be seen from fig. 4, the growth of yeast cells was inhibited when the initial pH was <3, and the inhibitory effect was significant when the pH was 2. This is because the strong acid conditions may cause some damage to the cells, resulting in loss of physiological activity. When the initial pH is between 3 and 4, the growth trends of the yeasts are integrally similar, and the growth speeds of the yeasts are both fast and slow. Therefore, FS-ZJQ has acid resistance at pH 3-4, and meets the requirement of acid fruit juice alcohol fermentation on pH.
3. Alcohol tolerance: respectively setting initial alcoholic strength (% v.v)-1) Inoculating 6-7 at 5% inoculum size in 18 ° Bx fructus Citri Sarcodactylis juice, culturing at 28 deg.C for 3d, sampling every 12 hr, and measuring absorbance at 600nm with ultraviolet spectrophotometer. As can be seen from FIG. 5, the alcohol content is higher than 14% v.v-1At this time, yeast growth was inhibited. When the alcoholic strength is 5% v.v-1、8％v·v-1、 11％v·v-1The growth trends of the yeasts are similar. The yeast grows slowly within 0-24 h, and grows obviously faster and then tends to be stable within 24-48 h. This is because the alcohol in the initial medium exerts a stress on the growth of the yeast, which grows into the lag phase. After 24h, the yeast enters the logarithmic growth phase, the cell concentration is increased, and the alcohol accumulation inhibits the growth of the yeast, so the yeast grows slowly after 36h and finally tends to be stable. From this, it is found that FS-ZJQ has an alcohol content of less than 11% v.v-1Has stronger tolerance.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized in that the Saccharomyces cerevisiae (Saccharomyces cerevisiae) is preserved in China general microbiological culture collection center of China general microbiological culture Collection center in 2017, 12 months and 14 days, the preservation number is CGMCC No.15063, and the preservation address is No. 3 of West Lu No.1 of the sunward district in Beijing.
2. The method for culturing Saccharomyces cerevisiae CGMCC No.15063 according to claim 1, wherein the method comprises culturing Saccharomyces cerevisiae CGMCC No.15063 by inoculating it into YEPD liquid medium.
3. The method according to claim 2, wherein said YEPD medium is: 5-20 g/L yeast powder, 10-30 g/L peptone and 10-30 g/L glucose, and the pH is natural.
4. The method for producing bergamot wine by using saccharomyces cerevisiae of claim 1, wherein saccharomyces cerevisiae CGMCC No.15063 is inoculated in the alcohol fermentation stage of the bergamot wine.
5. A finger citron wine prepared by the saccharomyces cerevisiae CGMCC No.15063 as the claim 1.
6. The use of Saccharomyces cerevisiae CGMCC No.15063 of claim 1 in food.
7. The use according to claim 6, comprising the preparation of a fruit juice or alcoholic beverage starting from fingered citron.
8. A microbial agent comprising Saccharomyces cerevisiae CGMCC No.15063 as claimed in claim 1.
9. The microbial agent according to claim 8, wherein the microbial agent is a solid microbial agent or a liquid microbial agent.
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| CN114149933B (en) * | 2021-11-08 | 2023-06-02 | 泸州老窖股份有限公司 | Saccharomyces cerevisiae LJ-1 and application thereof |
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