CN104877922A - Method for screening low-yield diacetyl beer yeast - Google Patents
Method for screening low-yield diacetyl beer yeast Download PDFInfo
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- CN104877922A CN104877922A CN201510258686.9A CN201510258686A CN104877922A CN 104877922 A CN104877922 A CN 104877922A CN 201510258686 A CN201510258686 A CN 201510258686A CN 104877922 A CN104877922 A CN 104877922A
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Abstract
The invention belongs to a method for screening low-yield diacetyl beer yeast. D-valine (D-Val) has a certain inhibition function on yeast growth. According to the method, 13 DEG P wort containing D-Val at certain concentration is adopted to domesticate yeast under appropriate conditions, strains which are sensitive to D-Val are screened from yeast paste, fermentation experiment is implemented to detect the diacetyl content of a sensitive strain, and the result shows that the reduction rate of diacetyl can reach 34.08%. The method is simple to operate and accurate and stable in result, and technical support is provided for research and development of the beer industry and high-quality beer production.
Description
Technical field
The present invention relates to a kind of screening method of low yield di-acetyl cereuisiae fermentum.
Background technology
Connect the main source that diones material is " raw vinosity " in beer, be also one of restricted index of beer maturation, mainly comprise di-acetyl and diacetylmethane, wherein particularly important with di-acetyl.Di-acetyl is compound that is volatile, that have intense stimulus, and it is the precursor of multiple fragrance material, is also the main flavor material of the milk-product such as butter, cheese.The threshold value of di-acetyl in beer, between 0.1-0.2mg/L, has the unhappy pungent taste of similar sour rice taste as content >0.5mg/L; The malty seemingly burnt is had as content >0.2mg/L.Outstanding light beer di-acetyl should control at below 0.06mg/L.Screening low yield di-acetyl cereuisiae fermentum not only effectively can reduce diacetyl content in the main ferment part of beer fermentation, also for the fast restore of ferment part di-acetyl after beer has established good basis.Therefore, adopt the screening low yield di-acetyl cereuisiae fermentum method simply effective, suitability is strong, quality beer will be produced for Beer Industry and bring the effect of getting twice the result with half the effort.
Di-acetyl is the alternative product of the nitrogenous substancess such as yeast synthesis α-amino-isovaleric acid, and its precursor α-acetylactis directly can generate di-acetyl by non-enzymatic oxidation decomposition reaction when accumulating excessive.α-acetylactis residual in finished beer after filling, reaction can generate di-acetyl under certain condition, cause the phenomenon of di-acetyl " bounce-back ", affect finished beer local flavor.Research shows, adds the pathways metabolism of excess valine meeting feedback inhibition α-acetylactis synthesis α-amino-isovaleric acid, thus reduces α-acetolactic generation, Diacetyl in Beer content is reduced in fermented liquid.Reducing diacetyl content report has a lot, but adds amino acid or relevant enzymes high cost in a large number in brewing industry production process, also can produce certain influence to the local flavor of beer; Though by gene recombination obtain α-amino-isovaleric acid, leucine, isoleucine auxotroph yeast strain method effectively can reduce diacetyl content, consider food-safety problem, rarely have factory to adopt this method to carry out suitability for industrialized production at present.
D-Val is the optically active isomer of Valine (L-Val), and experiment confirms, certain density D-Val has certain restraining effect to yeast growth metabolism.The yeast strain of screening to D-Val sensitivity, effectively can reduce α in beer fermentation-acetolactic accumulation, thus the di-acetyl reduced generates and solves the phenomenon of di-acetyl " bounce-back ".The low yield di-acetyl cereuisiae fermentum screening method that present method is set up, its advantage is the diacetyl content that effectively can reduce beer, overcomes di-acetyl " bounce-back " problem, has very strong theory and practice meaning to the research and development of quality beer with production.
Summary of the invention
The present invention mainly generates the pathways metabolism of di-acetyl and α-amino-isovaleric acid according to yeast α-acetylactis, utilize D-Val to the restraining effect of yeast growth metabolism, 13 ° of P wheat juice containing proper concn D-Val are adopted to tame yeast under proper condition, get yeast slurry to be diluted to certain multiple and to coat in wheat juice nutrient agar, screening D-Val sensitive strain carries out fermenting experiment, detect sensitive strain and starting strain diacetyl content, calculate the di-acetyl reduced rate of sensitive strain, evaluate the validity of this method with this.
Technical scheme
The present invention mainly generates the pathways metabolism of di-acetyl and α-amino-isovaleric acid according to yeast α-acetylactis, first arrange the wheat juice nutrient agar containing different concns gradient D-Val and L-Val, checking D-Val grows inhibited to yeast metabolism and draws growth-inhibiting curve.
Utilize D-Val to the restraining effect of yeast growth metabolism, the 13 ° of P wheat juice chosen containing proper concn D-Val tame yeast at 28 DEG C, in fermented liquid to D-Val comparatively sensitive strain precipitate from fermented liquid, relatively non-sensitive bacterial strain is then suspended in growth and breeding in fermented liquid, outwell high fermentation liquid and retain bottom sediment, from fermented liquid, domestication obtains the responsive yeast slurry of D-Val accordingly.
Get yeast slurry to be diluted to certain multiple and to coat in 13 ° of P wheat juice nutrient agars, contrast with 13 of proper concn D-Val and L-Val ° of P wheat juice nutrient agars, screening D-Val sensitive strain carries out fermenting experiment.
Measuring method with reference to di-acetyl in " beer analysis method " GB/T4928-2008 detects sensitive strain and starting strain diacetyl content, calculates the di-acetyl reduced rate of sensitive strain, evaluates the validity of this method with this.
Concrete operation method of the present invention:
1) get the activation of appropriate yeast slurry, contain in the triangular flask of 300mL13 ° of P wheat juice (containing D-Val500mg/L) with the inoculum size access of 10%, 28 DEG C of constant temperature quiescent culture 8-12h, now yeast has started to send out;
2) outwell the muddy fermented liquid in upper strata, only retain bottom yeast mud;
3) pour 300mL sterilized water (pH3.5) into and shake up rear standing 2-3h, non-sensitive yeast is soaked in pickling; Outwell the turbid liquid in upper strata, only retain bottom sediment yeast slurry;
4) 300mL13 ° of P wheat juice (containing L-Val500ppm) 28 DEG C of constant temperature culture 24h are poured into;
5) fermented liquid being diluted to proper concn coats in 13 ° of P wheat juice agar plates, 28 DEG C of constant temperature culture 24h;
6) be separated also preservation list bacterium colony, with sterile toothpick, it put access respectively containing in 13 ° of P wheat juice agar plates of following material (A: containing D-Val 500mg/L, B: nothing, C:L-Val 500mg/L), 28 DEG C of constant temperature culture 24h;
7) growing state of each bacterial strain on different flat board is observed every 6h;
8) sensitive strain is subject to D-Val and suppresses that bacterium colony is minimum does not even grow on A, maximum by L-Val promoter action bacterium colony on C.Therefore, choose successively on A, B, C flat board the ascending change of bacterium colony comparatively significantly bacterial strain carry out fermenting experiment as D-Val sensitive strain;
9) measuring method with reference to di-acetyl in " beer analysis method " GB/T4928-2008 detects D-Val sensitive strain and starting strain diacetyl content, calculates the di-acetyl reduced rate of sensitive strain.
Embodiment
Below by embodiment, the present invention is further illustrated, its objective is and better understand content of the present invention.
Embodiment
1. D-Val tests the restraining effect of cereuisiae fermentum:
1) (inoculum size is about 1.5 × 10 to connect appropriate yeast slurry from yeast inclined-plane
7cFU/mL) in containing in the test tube of 10mL 13 ° of P wheat juice nutrient solutions, shake up, 28 DEG C of activation culture 24h;
2) 13 ° of P wheat juice nutrient agars are sub-packed in 7 triangular flasks, every bottle of 100mL, sterilizing 20min at 115 DEG C.Then, before aseptic wheat juice nutrient agar does not also solidify respectively to each triangular flask in add final concentration be followed successively by 0,100,500,1000,1500,3000, the D-Val solution of 5000mg/L, flat board is made after shaking up, each flat board is all containing substratum 25mL, topple over little graduated cylinder, solidify rear for subsequent use;
3) preparation containing 13 ° of P wheat juice agar plates of same concentrations gradient L-Val as positive controls, preparation condition and principle the same;
4) get 1mL activation after yeast juice, after being diluted to finite concentration by gradient dilution method, getting 100 μ L successively and coat in different concns D-Val and L-Val flat board, each concentration gradient establish 3 parallel, then at 28 DEG C, cultivate 2-3d.
Experimental result is as follows:
Yeast lethality rate increases and stable rising with D-Val concentration, and when D-Val concentration reaches 5000mg/L, lethality rate is 40.61%; And along with the increase of control group L-Val concentration, yeast lethality rate is slow negative growth trend, reaches 5000mg/L in L-Val concentration, and lethality rate is-13.22%.As can be seen here, D-Val has certain restraining effect to yeast growth; On the contrary, L-Val only has faint promoter action to yeast growth, without obvious restraining effect.
2. the screening of D-Val sensitive strain and fermenting experiment:
1) get the activation of appropriate yeast slurry, contain in the triangular flask of 300mL 13 ° of P wheat juice (containing D-Val500mg/L) with the inoculum size access of 10%, 28 DEG C of quiescent culture 8-12h;
2) outwell the muddy fermented liquid in upper strata, only retain bottom yeast mud;
3) pour 300mL sterilized water (pH3.5) into and shake up rear standing 2-3h, non-sensitive yeast is soaked in pickling; Outwell the turbid liquid in upper strata, only retain bottom sediment yeast slurry;
4) 300mL13 ° of P wheat juice (containing L-Val 500mg/L) 28 DEG C of constant temperature culture 24h are poured into;
5) fermented liquid being diluted to proper concn coats in 13 ° of P wheat juice agar plates, 28 DEG C of constant temperature culture 24h;
6) be separated also preservation list bacterium colony, with sterile toothpick, it put access respectively containing in 13 ° of P wheat juice agar plates of following material (A: containing D-Val 500mg/L, B: nothing, C:L-Val 500mg/L), 28 DEG C of constant temperature culture 24h;
7) observe the growing state of each bacterial strain on different flat board every 6h, choose successively on A, B, C flat board the ascending change of bacterium colony comparatively significantly bacterial strain carry out fermenting experiment as D-Val sensitive strain;
8) after main ferment terminates, with reference to the measuring method distillation di-acetyl of di-acetyl in " beer analysis method " GB/T4928-2008, and utilize ultraviolet spectrophotometry, under 335nm UV-light, detect D-Val sensitive strain and starting strain diacetyl content, calculate the di-acetyl reduced rate of sensitive strain.
Experimental result is as follows:
Table 1 experimental strain (containing starting strain) diacetyl content and reduced rate thereof
| Strain number | Diacetyl content (mg/L) | Reduced rate (%) |
| KB | 0.12 | 0.00 |
| 1 | 0.10 | 14.11 |
| 2 | 0.09 | 25.09 |
| 3 | 0.17 | -41.82 |
| 4 | 0.11 | 8.11 |
| 5 | 0.12 | 2.12 |
| 6 | 0.10 | 17.10 |
| 7 | 0.10 | 15.11 |
| 8 | 0.14 | -19.85 |
| 9 | 0.11 | 12.11 |
| 10 | 0.20 | -63.80 |
| 11 | 0.12 | 3.70 |
| 12 | 0.08 | 30.09 |
| 13 | 0.11 | 8.11 |
| 14 | 0.10 | 13.11 |
| 15 | 0.11 | 12.11 |
| 16 | 0.08 | 34.08 |
As shown in table 1, have in 16 strain D-Val sensitive strains 13 strain diacetyl content comparatively starting strain decline to some extent, its positive mutation rate is 81.25%, and reduced rate maximum is 34.08%.
Claims (1)
1. a screening method for low yield di-acetyl cereuisiae fermentum, comprises the following steps:
Get the activation of appropriate yeast slurry, contain in the triangular flask of 300mL13 ° of P wheat juice (containing D-Val500mg/L) with the inoculum size access of 10%, 28 DEG C of constant temperature quiescent culture 8-12h;
Outwell the muddy fermented liquid in upper strata, only retain bottom yeast mud;
Pour 300mL sterilized water (pH3.5) into and shake up rear standing 2-3h, non-sensitive yeast is soaked in pickling, outwells the turbid liquid in upper strata, only retains bottom sediment yeast slurry;
Pour 300mL13 ° of P wheat juice (containing L-Val500ppm) 28 DEG C of constant temperature culture 24h into;
Fermented liquid being diluted to proper concn coats in 13 ° of P wheat juice agar plates, 28 DEG C of constant temperature culture 24h;
Be separated and preservation list bacterium colony, distinguished access successively containing in 13 ° of P wheat juice agar plates of following material (A: containing D-Val 500mg/L, B: nothing, C:L-Val 500mg/L) with sterile toothpick, 28 DEG C of constant temperature culture 24h;
The growing state of each bacterial strain on different flat board is observed every 6h;
Sensitive strain is subject to D-Val and suppresses that bacterium colony is minimum does not even grow on A, maximum by L-Val promoter action bacterium colony on C, choose successively on A, B, C flat board the ascending change of bacterium colony comparatively significantly bacterial strain carry out fermenting experiment as D-Val sensitive strain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154662A (en) * | 2020-01-07 | 2020-05-15 | 广州南沙珠江啤酒有限公司 | High-concentration beer strain and screening method thereof |
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Non-Patent Citations (6)
| Title |
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| DOUGLAS A GEE ET AL.: "A FLAVOUR MODEL FOR BEER FERMENTATION", 《J. INST. BREW.》 * |
| KRISTOFFER KROGERUS ET AL.: "Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort", 《APPL MICROBIOL BIOTECHNOL 》 * |
| 李秀婷: "《现代啤酒生产工艺》", 2013063, 中国农业大学出版社 * |
| 秦玉静 等: "啤酒生产中双乙酰形成的分子遗传学及其控制", 《工业微生物》 * |
| 范秀英 等: "啤酒酿造中双乙酞的代谢调控的研究进展", 《酿酒》 * |
| 韦函忠 等: "啤酒发酵中双乙酰的产生和α-乙酰乳酸脱羧酶的应用", 《广西轻工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111154662A (en) * | 2020-01-07 | 2020-05-15 | 广州南沙珠江啤酒有限公司 | High-concentration beer strain and screening method thereof |
| CN111154662B (en) * | 2020-01-07 | 2023-05-16 | 广州南沙珠江啤酒有限公司 | High-concentration beer strain and screening method thereof |
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Application publication date: 20150902 |