CN100500838C - Preparation method and application of fermentation production for acid urase for wine using intestinal bacillus - Google Patents
Preparation method and application of fermentation production for acid urase for wine using intestinal bacillus Download PDFInfo
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- CN100500838C CN100500838C CNB2006100966351A CN200610096635A CN100500838C CN 100500838 C CN100500838 C CN 100500838C CN B2006100966351 A CNB2006100966351 A CN B2006100966351A CN 200610096635 A CN200610096635 A CN 200610096635A CN 100500838 C CN100500838 C CN 100500838C
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Abstract
This invention relates to a preparation and application of producing wine-used acid urea enzyme with enteric bacilli fermenting, and it belongs to enzyme preparation and brewing wine additives technology field. The cell is produced and collected with Enterobacter sp. 9043C12 in this invention and the cell breakage is conducted by grinding with Lysozyme and liquid nitrogen, then wine-used acid urea enzyme is obtained by alcohol fractional precipitation. This invention also relates its application that the removal proportion of urea of yellow wine will reach 70% by adding this acid urea enzyme into yellow wine after 24 hours.
Description
Technical field
A kind of preparation method and application with enterobacteria fermentative production acid urease for wine, specifically utilize the enterobacteria fermentative production to collect thalline, with the method for N,O-Diacetylmuramidase and liquid nitrogen grinding with the somatic cells fragmentation, pass through ethanol precipitation, obtain the acid urease for wine goods, belong to zymin, wine brewing additive technology field.
Background technology
After confirming that the urethanum in the alcoholic beverage had carcinogenesis in 1985, cause the great attention of the World Health Organization and various countries.In December, 1985, Canadian government has taken the lead in issuing the limit standard of urethanum in all kinds of wine.Afterwards, states such as U.S.'s wine industry association, Japan have also successively formulated the limit standard of urethanum in the wine.
Both at home and abroad to urethanum in the wine studies show that the main path that urethanum forms in the wine is the urea approach, the urethanum in China's yellow rice wine and the Japanese sake 90% or more is to derive from that contained urea and alcoholic acid react in the wine:
And the main source of urea is in the wine:
(1) produces by microbial metabolism, i.e. the urea that produces by the arginine metabolism.
(2) contained urea in the nutrient solution composition of raw and auxiliary material and brewing process.
After having studied the factor that influences urethanum formation, find that urea concentration, alcohol concn etc. is topmost factor.The various countries scholars have carried out deep research to the method that reduces urethanum in the wine, by the urea content in removal or the reduction wine, to reach the purpose of control urethane ester content.The main direction of research is:
1. the yeast of seed selection high-yield urea;
2. suitably control fermentation condition;
3. add an amount of acid urease for wine in the wine.
By contrast, it is the easiest, practical to add acid urease for wine in wine, also is method commonly used at present, and this method has significant advantage:
1. do not change the processing condition of former brewing wine, do not influence ordinary production.
2. do not change the flavor characteristics of former wine.This is because addition is little, and urase is through frying in shallow oil inactivation after drinking.
3. the degree height of urethanum in the reduction wine can reach 90%.
China is the country of year liquor output maximum in the world, and very long wine brewing history is arranged, and not only output is big, and wide in variety, and many products are enjoyed higher reputation in the world.The shao-hsing rice wine of China has market widely in Japan and South East Asia, but in 1987, Japan relevant department proposes urethanum content overproof (greater than 100 μ g/L) in the shao-hsing rice wine, in recent years, on the shao-hsing rice wine export trade, run into the problem of urethanum again, in order to reduce the content of urethanum in the wine, shao-hsing rice wine group still needs from Japanese import acid urease for wine so far.Therefore, for people health, also in order more to export goods and earn foreign currency, the acid urease for wine of research and production China oneself has become the task of top priority.
Summary of the invention
The objective of the invention is to screen a strain and can produce the microorganism that is fit to the acid urease that wine uses, by cytoclasis, with biochemical separation means such as ethanol precipitations, the purer acid urease for wine of final acquisition, the whole process flow yield is 45%, the ratio of enzyme is lived and is 138u/mg, optimum temperuture is 75 ℃, the temperature-stable scope is below 75 ℃, optimal pH 5.5, stable pH range 4.0～6.5.This enzyme is added in the yellow rice wine,, avoid the generation of urethanum, play the back modification by removing urea.
Technical scheme of the present invention: enterobacteria is inserted in the fermention medium that contains sugar, add nitrogenous source and nutritive substance simultaneously, regulate fermention medium pH with hydrochloric acid, fermentation obtains thalline, with the method for N,O-Diacetylmuramidase and liquid nitrogen grinding with the somatic cells fragmentation, use ethanol precipitation, obtain the intracellular enzyme acid urease for wine.Technology is:
A ferments by enterobacteria, collects thalline:
(1) starting strain: adopt enterobacteria (Enterobacter sp.) 9043C
12This bacterial strain is open at " Zhejiang Agricultural Univ's journal " 22 (5): 493～498,1996.
(2) fermention medium is formed: sugared 10g/L, nitrogenous source 16g/L, nutritive substance 10g/L, NaCl5g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 2g/L, urea 3g/L, MnSO
40.1g/L, NiSO
40.1g/L, transfer pH5.0～5.5 with hydrochloric acid, the nutritive substance of being addressed is soya-bean cake hydrolyzed solution and/or corn steep liquor.
(3) fermentation condition: inoculum size 6%, initial pH5.0～5.5,30～34 ℃ of constant temperature leave standstill and cultivate 28h.
(4) collect thalline: with fermented liquid with 8000r/min, 4 ℃ of centrifugal 10min after, abandon supernatant, with thalline with twice of deionized water wash.
B carries out cytoclasis with N,O-Diacetylmuramidase and liquid nitrogen grinding:
(1) N,O-Diacetylmuramidase is handled: thalline is dissolved among the high osmotic buffer SMM by mass volume ratio 1:5, and the add-on of N,O-Diacetylmuramidase is 0.6mg/mL, 30 ℃ of water bath processing 13h.Behind 8000r/min, 4 ℃ of centrifugal 10min, redissolve the precipitation of centrifugal collection with citric acid-sodium citrate buffer solution of pH5.5, behind 8000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor.
(2) liquid nitrogen grinding: after N,O-Diacetylmuramidase is handled back collection supernatant liquor, the precipitation of remainder is added an amount of liquid nitrogen, grind fast, after treating the liquid nitrogen volatilization, add an amount of liquid nitrogen once more and grind, three times repeatedly, with the thalline after citric acid-sodium citrate buffer solution redissolution grinding of pH5.5, behind 8000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor.
The C ethanol precipitation
(1) merge the supernatant liquor of collecting among the B and add-20 ℃ of ethanol, making the ethanol final concentration is 30% to carry out alcohol precipitation, behind 5000r/min, 4 ℃ of centrifugal (I) 10min, abandons precipitation, gets supernatant liquor.
(2) get supernatant liquor and add-20 ℃ of ethanol, making the ethanol final concentration is 50% to carry out alcohol precipitation, behind 5000r/min, 4 ℃ of centrifugal (II) 10min, gets precipitation, abandons supernatant.
(3) precipitation adds the citric acid of a small amount of pH5.5-sodium citrate buffer solution dissolving, behind 10000r/min, 4 ℃ of centrifugal (III) 10min, abandons precipitation, gets supernatant, is acid urease enzyme liquid.
The application of D acid urease in yellow rice wine
The dosage that acid urease is pressed 0.05u/mL is adding in the yellow rice wine below 30 ℃, and 24h urea clearance reaches more than 70%.
The enzyme definition of living: under normal pressure, 37 ℃, the condition of pH4.5, the enzyme amount that every min decomposing urea produces 1 μ mol ammonium ion is 1 enzyme unit that lives.
Urea measuring method in the yellow rice wine is used the method for Diacetylmonoxime, is under the condition of 527nm at wavelength, as blank, measures the OD value of urea standardized solution with ultrapure water, and maps the drawing standard curve with the corresponding concentration of OD value.Measure the OD value of yellow rice wine under the same conditions, again by its content of regression equation calculation.
Beneficial effect of the present invention: the fermentative production acid urease for wine can enlarge, extracting method enlarges production easily, and this acid urease can be stablized in wine and plays a role urea clearance height, in yellow rice wine, the urea clearance can reach more than 70% behind the adding acid urease for wine.The present invention can not change the processing condition of former brewing wine, does not change the local flavor of wine, is the method for urea in the present comparatively ideal removal wine.
Description of drawings
Fig. 1 acid urease for wine preparation technology synoptic diagram.
Embodiment
Embodiment 1
With enterobacteria (Enterobacter sp.) 9043C
12The inclined-plane inserts seed liquor, after the enlarged culturing, inserts in the 20L fermention medium by inoculum size 6% again, and fermention medium is formed as described in the specification sheets.Initial pH5.0～5.5,30～34 ℃ leaves standstill and cultivates 28h, and 4 ℃ of centrifugal 10min under 8000r/min collect thalline 80g fermented liquid, behind twice of 2L deionized water wash, in the 400mlSMM high osmotic buffer, redissolve, add N,O-Diacetylmuramidase 240mg, 30 ℃ of water bath processing 13h.The following 4 ℃ of centrifugal 10min of 8000r/min condition redissolve the precipitation of centrifugal collection with citric acid-sodium citrate buffer solution of 400mLpH5.5, behind the following 4 ℃ of centrifugal 10min of 8000r/min condition, collect supernatant liquor.Remaining precipitation adds an amount of liquid nitrogen, grinds fast, after treating the liquid nitrogen volatilization, add an amount of liquid nitrogen once more and grind, three times repeatedly, with the thalline after citric acid-sodium citrate buffer solution redissolution grinding of 400mLpH5.5, behind the following 4 ℃ of centrifugal 10min of 8000r/min, collect supernatant liquor.The 800mL supernatant of twice collection is merged, add 342.7mL-20 ℃ ethanol sedimentation, behind the following 4 ℃ of centrifugal 10min of 5000r/min condition, get supernatant adding 457.3mL-20 ℃ ethanol again and carry out alcohol precipitation, behind the following 4 ℃ of centrifugal 10min of 5000r/min condition, get precipitation, precipitation adds citric acid-sodium citrate buffer solution dissolving of 80mLpH5.5, behind the following 4 ℃ of centrifugal 10min of 10000r/min condition, obtains honest and upright and thrifty 80mL, be spissated acid urease crude enzyme liquid, get 2g enzyme powder after the lyophilize.Add 0.69mg enzyme powder in the 100mL yellow rice wine, urea content drops to 10.3mg/L from 35.2mg/L to measure the urea clearance is 70.6% behind the 24h.
Claims (5)
1, a kind of preparation method with enterobacteria fermentative production acid urease for wine, it is characterized in that comprising the steps: enterobacteria is inserted in the fermention medium that contains sugar, add nitrogenous source and nutritive substance simultaneously, regulate fermention medium pH with hydrochloric acid, fermentation obtains thalline; Method with N,O-Diacetylmuramidase and liquid nitrogen grinding is carried out cytoclasis; Use ethanol precipitation, obtain its intracellular enzyme acid urease for wine; Its technology is:
A ferments by enterobacteria, collects thalline:
(1) starting strain: adopt enterobacteria (Enterobacter sp.) 9043C
12
(2) fermention medium is formed: sugared 10g/L, nitrogenous source 16g/L, nutritive substance 10g/L, NaCl5g/L, sodium acetate 2g/L, dipotassium hydrogen phosphate 2g/L, urea 3g/L, MnSO
40.1g/L, NiSO
40.1g/L, transfer pH5.0～5.5 with hydrochloric acid, described nutritive substance is soya-bean cake hydrolyzed solution and/or corn steep liquor;
(3) fermentation condition: inoculum size 6%, initial pH5.0～5.5,30～34 ℃ of constant temperature leave standstill and cultivate 28h;
(4) collect thalline: with fermented liquid with 8000r/min, 4 ℃ of centrifugal 10min after, abandon supernatant, with thalline with twice of deionized water wash;
B carries out cytoclasis with N,O-Diacetylmuramidase and liquid nitrogen grinding:
(1) N,O-Diacetylmuramidase is handled: thalline is dissolved among the high osmotic buffer SMM by mass volume ratio 1:5, the add-on of N,O-Diacetylmuramidase is 0.6mg/mL, 30 ℃ of water bath processing 13h, behind 8000r/min, 4 ℃ of centrifugal 10min, redissolve the precipitation of centrifugal collection with citric acid-sodium citrate buffer solution of pH5.5, behind 8000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor;
(2) liquid nitrogen grinding: after N,O-Diacetylmuramidase is handled back collection supernatant liquor, the precipitation of remainder is added an amount of liquid nitrogen, grind fast, after treating the liquid nitrogen volatilization, add an amount of liquid nitrogen once more and grind, three times repeatedly, grind the precipitation that collect the back with citric acid-sodium citrate buffer solution redissolution of pH5.5, behind 8000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor;
C. use ethanol precipitation:
(1) merge the supernatant liquor of collecting among the B, add-20 ℃ of ethanol, making the ethanol final concentration is 30% to carry out alcohol precipitation, behind 5000r/min, 4 ℃ of centrifugal 10min, abandons precipitation, gets supernatant liquor;
(2) get supernatant liquor and add-20 ℃ of ethanol, making the ethanol final concentration is 50% to carry out alcohol precipitation, behind 5000r/min, 4 ℃ of centrifugal 10min, gets precipitation, abandons supernatant;
(3) precipitation adds the citric acid of a small amount of pH5.5-sodium citrate buffer solution dissolving, behind 10000r/min, 4 ℃ of centrifugal 10min, abandons precipitation, gets supernatant, is acid urease liquid.
2, method according to claim 1 is characterized in that used hydrochloric acid is the HCl of 30% concentration.
3, method according to claim 1 is characterized in that described sugar is glucose.
4, method according to claim 1 is characterized in that described nitrogenous source is one or more in soya-bean cake hydrolyzed solution, corn steep liquor, urea, the ammonium sulfate.
5, the application of the acid urease for wine of the method for claim 1 preparation is characterized in that acid urease is adding in the yellow rice wine below 30 ℃ by the dosage of 0.05u/mL, and the urea clearance reaches more than 70% behind the 24h.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102051355B (en) * | 2010-12-03 | 2012-08-22 | 江南大学 | Preparation method and application of immobilized acidic urease membrane |
| CN102978065A (en) * | 2012-12-05 | 2013-03-20 | 浙江大学 | Brewing method for restraining generation of ethyl carbamate in conventional yellow rice wine |
| CN102994487B (en) * | 2012-12-11 | 2014-03-19 | 四川绵竹剑南春酒厂有限公司 | Production method of acid urease |
| CN102994398B (en) * | 2012-12-11 | 2014-04-16 | 四川绵竹剑南春酒厂有限公司 | Urease high-yielding Aspergillus sydowii and application thereof |
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5006468A (en) * | 1987-11-17 | 1991-04-09 | Suntory Limited | Novel urease and method of producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5006468A (en) * | 1987-11-17 | 1991-04-09 | Suntory Limited | Novel urease and method of producing the same |
Non-Patent Citations (4)
| Title |
|---|
| Purification,characterization,and application of an acid ureasefrom Arthrobacter mobilis. Katsuro Miyagawa et al.Journal of Biotechnology,Vol.68 . 1999 |
| Purification,characterization,and application of an acid ureasefrom Arthrobacter mobilis. Katsuro Miyagawa et al.Journal of Biotechnology,Vol.68 . 1999 * |
| 黄酒用脲酶产生菌9043C12发酵条件和酶学特性的研究. 吴伟祥等.浙江农业大学学报，第22卷第5期. 1996 |
| 黄酒用脲酶产生菌9043C12发酵条件和酶学特性的研究. 吴伟祥等.浙江农业大学学报，第22卷第5期. 1996 * |
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