CN101831386A - Method for screening salt-tolerant aroma-producing yeasts - Google Patents
Method for screening salt-tolerant aroma-producing yeasts Download PDFInfo
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- CN101831386A CN101831386A CN200910037818A CN200910037818A CN101831386A CN 101831386 A CN101831386 A CN 101831386A CN 200910037818 A CN200910037818 A CN 200910037818A CN 200910037818 A CN200910037818 A CN 200910037818A CN 101831386 A CN101831386 A CN 101831386A
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Abstract
The invention discloses a method for screening salt-tolerant aroma-producing yeasts and a process thereof. The method is used for screening a good strain suitable for a high-salt dilute-state fermentation process by performing mutation breeding on an aspergillus oryzae 3.042 starting strain in a process of preparing a soy sauce through high-salt dilute-state fermentation; and the method comprises the following steps of: adopting a 15 W ultraviolet lamp to irradiate a bacterial suspension from a position 30 cm away from the bacterial suspension so as to mutagenize a strain; primarily screening a strain which is superior to the starting strain by using a transparent ring method; putting the strain into a yeast production room to perform synchronous cultivation with produced yeasts, and performing re-screening; and finally, performing domestication culture on the yeasts for multiple times, then performing flat plate separation on the yeast, and purifying screened strains to obtain high-quality salt-tolerant aroma-producing yeasts.
Description
One, technical field:
The invention belongs to field of food industry, particularly relate to a kind of screening method and technology of salt-tolerant aroma-producing yeasts.
Two, background technology:
Soy sauce is to be raw material with grain and oil and its byproduct, makes through making method, has the fluid flavoring that special color, smell and taste help lend some impetus to appetite, and consumption is very big.During soy sauce brewing, utilize Institute of Micro-biology's excretory plurality of enzymes that raw material is acted synergistically, thereby zymotechnique is very important to quality of sauce in soy sauce brewing, the quality of zymotechnique will directly influence quality of soy sauce and raw material availability.Fermentation process is a lot, can be divided into low-salt solid-state fermentation and high-salt dilute two classes of fermenting at present basically: low salt solid state fermentation, because fermentation time is short, raw material availability is low, become Qu Zhiliang undesirable, product specification is not high, and taste, fragrance etc. far can not satisfy the needs of people's life.The high-salt dilute zymotechnique has overcome all drawbacks and the product defects that traditional workshop-based production brings, and at aspects such as medical support, nutritive value, taste color and lusters leading superiority is arranged all.The soy sauce that China sells in the market is many based on low grade products, raising along with people's living standard, people more and more pay attention to the local flavor and the nutritive value of soy sauce, so the high-salt dilute zymotechnique is undoubtedly the place trend of sauce fermentation technology, and the bacterial classification that the high-salt dilute zymotechnique at first needs, thereby salt-tolerant aroma-producing yeasts strain improvement and screening technology also just become the critical process of the development of making high-grade soy sauce industry, is each soy sauce enterprise and each people's of correlative study mechanism research emphasis.、
The national at present soy sauce brewing enterprise more than 90% makes 3.042 aspergillus oryzae bacterial classifications with Shanghai and makes soy sauce, utilize aspergillus oryzae 3.042 to be the bacterial classification that sets out, mutagenic and breeding goes out a bacterial classification that is fit to the high-salt dilute zymotechnique that present China generally uses, change its genetic construction and function, by screening, from diversified varient, filter out output height, salt-tolerant aroma-producing yeasts bacterial classification mutant strain that proterties is good, and find out performance this mutant strain optimal medium and culture condition.The soy sauce that is improved becomes the content of bent neutral protease vigor, soy sauce finished product total nitrogen and amino-acid nitrogen, the salt-tolerant aroma-producing yeasts bacterial classification of premium propertiess such as the outward appearance that raising makes soy sauce, taste, and since this technology have speed fast, bring notable results, cost is lower, convenient feasible, therefore have huge market application foreground.
Three, summary of the invention:
The objective of the invention is at the many shortcomings that exist in traditional soy sauce production, obtain a kind of screening method and technology with the fragrant salt tolerant yeast of efficient life.
The present invention takes following technical scheme:
(1) sets out and cultivate before the bacterial classification, aspergillus oryzae is inserted fresh, in 30 ℃ incubator, cultivate 75 ℃, the preparation bacteria suspension.
(2) adopt the ultraviolet lamp 30cm place of 15W to shine bacteria suspension, mutagenesis bacterial classification.
(3) being determined by experiment the used substratum of aspergillus oryzae bacterial classification primary dcreening operation is that double-deck casein flat board, primary dcreening operation are selected the transparent circle method for use, is sieved to the bacterial classification better than the bacterial classification that sets out.
(4) bacterial classification that primary dcreening operation is obtained, imitation enterprise produces bent room, contains song with plastic crate, is placed on to produce bent room and produce bent synchronized culture to carry out multiple sieve, makes laboratory method and produces actual combining.
(5) will obtain cultivating through repeatedly taming in the yeast access fermention medium, plate isolation makes the strain screening purifying.
Embodiment;
Embodiment one:
Aspergillus oryzae is inserted fresh inclined-plane, in 30 ℃ incubator, cultivate 75h, to the spore maturation.The preparation bacteria suspension washes slant pore with the physiological saline after the sterilization, pours in the physiological saline after the 45ml that granulated glass sphere is housed sterilizes, and vibration disperses spore.Get the above bacteria suspension 5ml for preparing in the plate of diameter 9cm.Ultraviolet lamp is opened preheating 20min, to stablize light wave.The plate that fills bacteria suspension is placed on from 15W ultraviolet ray 30cm place.Open the ware lid, be exposed to the time of irradiation 5min under the UV-light, stir, make every effort to make cell evenly to absorb ultraviolet light wave while shining.Improve mutation rate.Get the bacteria suspension of 0.1ml after uviolizing and be coated with ware, and compare ware with the bacteria suspension that non-irradiated with ultraviolet radiation is crossed, after the cultivation, picking colony screens waiting.Get granulated glass sphere 20-30 grain, put into the 250ml triangular flask, add 0.85% physiological saline 50ml, tampon beyond the Great Wall, oilpaper wrapping, normal-pressure sterilization, cooling back aseptic technique.Get a ring spore from the strain inclined plane of rejuvenation to be purified and put into triangular flask, vibration 30min.Get test tube number, every pipe adds 9ml 0.85% physiological saline, tampon beyond the Great Wall, and the oilpaper wrapping is sterilized through 0.1MPa 20min.Carry out gradient dilution after the cooling, make the sub-suspension of embracing of different concns at last.Being determined by experiment the used substratum of aspergillus oryzae bacterial classification primary dcreening operation is that double-deck casein flat board, primary dcreening operation are selected the transparent circle method for use, guarantees have superiority all relative with colony diameter of k value, screens the bacterial classification better than the bacterial classification that sets out.The bacterial classification that will come out with transparent circle method primary dcreening operation again, carry out the triangular flask enlarged culturing, can carry out the triangular flask enlarged culturing one time in order to go up similar condition with production, 32 ℃ of 72 hour times cultivate, and connect soya bean, flour then and make bent, detect the egg enzyme activity, contain bent with plastic crate, Cheng Quhou 68mm or 34mm are placed on the Quchi and produce bent synchronized culture, the salt-tolerant aroma-producing yeasts proteinase activity that multiple sieve the is got bacterial classification height that all sets out.To obtain cultivating through repeatedly taming in the yeast access fermention medium, plate isolation makes the strain screening purifying, and through performance detects, and promptly obtains best salt-tolerant aroma-producing yeasts bacterial classification.
Embodiment two:
Aspergillus oryzae is inserted fresh inclined-plane, in 31 ℃ incubator, cultivate 73h, to the spore maturation.The preparation bacteria suspension washes slant pore with the physiological saline after the sterilization, pours in the physiological saline after the 45ml that granulated glass sphere is housed sterilizes, and vibration disperses spore.Get the above bacteria suspension 6ml for preparing in the plate of diameter 9cm.Ultraviolet lamp is opened preheating 23min, to stablize light wave.The plate that fills bacteria suspension is placed on from 15W ultraviolet ray 30cm place.Open the ware lid, be exposed to the time of irradiation 5min under the UV-light, stir, make cell evenly absorb ultraviolet light wave, improve mutation rate while shining.Get the bacteria suspension of 0.15ml after uviolizing and be coated with ware, and compare ware with the bacteria suspension that non-irradiated with ultraviolet radiation is crossed, after the cultivation, picking colony screens waiting.Get granulated glass sphere 20-30 grain, put into the 250ml triangular flask, add 0.85% physiological saline 50ml, tampon beyond the Great Wall, oilpaper wrapping, normal-pressure sterilization, cooling back aseptic technique.Get a ring spore from the strain inclined plane of rejuvenation to be purified and put into triangular flask, vibration 30min.Get test tube number, every pipe adds 10ml 0.85% physiological saline, tampon beyond the Great Wall, and the oilpaper wrapping is sterilized through 0.1MPa 20min.Carry out gradient dilution after the cooling, make the sub-suspension of embracing of different concns at last.Being determined by experiment the used substratum of aspergillus oryzae bacterial classification primary dcreening operation is that double-deck casein flat board, primary dcreening operation are selected the transparent circle method for use, guarantees have superiority all relative with colony diameter of k value, screens the bacterial classification better than the bacterial classification that sets out.The bacterial classification that will come out with transparent circle method primary dcreening operation, carry out the triangular flask enlarged culturing, can carry out the triangular flask enlarged culturing one time in order to go up similar condition with production, cultivated in 75 hours for 32 ℃, connect soya bean, flour then and make bent, detect the egg enzyme activity, contain bent with plastic crate, Cheng Quhou 68mm or 34mm are placed on the Quchi and produce bent synchronized culture, the salt-tolerant aroma-producing yeasts proteinase activity that multiple sieve the is got bacterial classification height that all sets out.To obtain cultivating through repeatedly taming in the yeast access fermention medium, plate isolation makes the strain screening purifying, and through performance detects, and promptly obtains best salt-tolerant aroma-producing yeasts bacterial classification.
Embodiment three:
Aspergillus oryzae is inserted fresh inclined-plane, in 33 ℃ incubator, cultivate 70h, to the spore maturation.The preparation bacteria suspension washes slant pore with the physiological saline after the sterilization, pours in the physiological saline after the 45ml that granulated glass sphere is housed sterilizes, and vibration disperses spore.Get the above bacteria suspension 5ml for preparing in the plate of diameter 9cm.Ultraviolet lamp is opened preheating 25min, to stablize light wave.The plate that fills bacteria suspension is placed on from 15W ultraviolet ray 30cm place.Open the ware lid, be exposed under the UV-light and shine the regular hour, stir, make every effort to make cell evenly to absorb ultraviolet light wave while shining.Improve mutation rate.Get the bacteria suspension of 0.3ml after uviolizing and be coated with ware, and compare ware with the bacteria suspension that non-irradiated with ultraviolet radiation is crossed, after the cultivation, picking colony screens waiting.Get granulated glass sphere 20-30 grain, put into the 250ml triangular flask, add 0.85% physiological saline 50ml, tampon beyond the Great Wall, oilpaper wrapping, normal-pressure sterilization, cooling back aseptic technique.Get a ring spore from the strain inclined plane of rejuvenation to be purified and put into triangular flask, vibration 35min.Get test tube number, every pipe adds 9ml 0.85% physiological saline, tampon beyond the Great Wall, and the oilpaper wrapping is sterilized through 0.15MPa 20min.Carry out gradient dilution after the cooling, make the sub-suspension of embracing of different concns at last.Being determined by experiment the used substratum of aspergillus oryzae bacterial classification primary dcreening operation is that double-deck casein flat board, primary dcreening operation are selected the transparent circle method for use, guarantees have superiority all relative with colony diameter of k value, screens the bacterial classification better than the bacterial classification that sets out.The bacterial classification that will come out with transparent circle method primary dcreening operation, carry out the triangular flask enlarged culturing, can carry out the triangular flask enlarged culturing one time in order to go up similar condition with production, 30 ℃ of 80 hour times cultivate, and connect soya bean, flour then and make bent, detect the egg enzyme activity, contain bent with plastic crate, Cheng Quhou 68mm or 34mm are placed on the Quchi and produce bent synchronized culture, the salt-tolerant aroma-producing yeasts proteinase activity that multiple sieve the is got bacterial classification height that all sets out.To obtain cultivating through repeatedly taming in the yeast access fermention medium, plate isolation makes the strain screening purifying, and through performance detects, and promptly obtains best salt-tolerant aroma-producing yeasts bacterial classification.
Embodiment four:
Aspergillus oryzae is inserted fresh inclined-plane, in 34 ℃ incubator, cultivate 68h, to the spore maturation.The preparation bacteria suspension washes slant pore with the physiological saline after the sterilization, pours in the physiological saline after the 48ml that granulated glass sphere is housed sterilizes, and vibration disperses spore.Get the above bacteria suspension 8ml for preparing in the plate of diameter 9cm.Ultraviolet lamp is opened preheating 30min, to stablize light wave.The plate that fills bacteria suspension is placed on from 15W ultraviolet ray 28cm place.Open the ware lid, be exposed under the UV-light and shine the regular hour, stir, make every effort to make cell evenly to absorb ultraviolet light wave while shining.Improve mutation rate.Get the bacteria suspension of 0.1ml after uviolizing and be coated with ware, and compare ware with the bacteria suspension that non-irradiated with ultraviolet radiation is crossed, after the cultivation, picking colony screens waiting.Get granulated glass sphere 20-30 grain, put into the 250ml triangular flask, add 0.85% physiological saline 60ml, tampon beyond the Great Wall, oilpaper wrapping, normal-pressure sterilization, cooling back aseptic technique.Get a ring spore from the strain inclined plane of rejuvenation to be purified and put into triangular flask, vibration 30min.Get test tube number, every pipe adds 9ml 0.85% physiological saline, tampon beyond the Great Wall, and the oilpaper wrapping is sterilized through 0.15MPa 15min.Carry out gradient dilution after the cooling, make the sub-suspension of embracing of different concns at last.Being determined by experiment the used substratum of aspergillus oryzae bacterial classification primary dcreening operation is that double-deck casein flat board, primary dcreening operation are selected the transparent circle method for use, guarantees have superiority all relative with colony diameter of k value, screens the bacterial classification better than the bacterial classification that sets out.The bacterial classification that will come out with transparent circle method primary dcreening operation, carry out the triangular flask enlarged culturing, can carry out the triangular flask enlarged culturing one time in order to go up similar condition with production, 32 ℃ of 75 hour times cultivate, and connect soya bean, flour then and make bent, detect the egg enzyme activity, contain bent with plastic crate, Cheng Quhou 68mm or 34mm are placed on the Quchi and produce bent synchronized culture, the salt-tolerant aroma-producing yeasts proteinase activity that multiple sieve the is got bacterial classification height that all sets out.To obtain cultivating through repeatedly taming in the yeast access fermention medium, plate isolation makes the strain screening purifying, and through performance detects, and promptly obtains best salt-tolerant aroma-producing yeasts bacterial classification.
Claims (5)
1. method for screening salt-tolerant aroma-producing yeasts is characterized in that: utilize aspergillus oryzae 3.042 to be the bacterial classification that sets out, the mutagenic and breeding screening obtains adapting to the excellent species of high-salt dilute zymotechnique.
2. a kind of method for screening salt-tolerant aroma-producing yeasts according to claim 1 is characterized in that: described mutagenic and breeding is to adopt the ultraviolet lamp of 15W to shine bacteria suspension and the mutagenesis bacterial classification at the 30cm place.
3. a kind of method for screening salt-tolerant aroma-producing yeasts according to claim 1 is characterized in that: primary dcreening operation is to be double-deck casein flat board with substratum in the described screening technology, uses the transparent circle method, obtains the bacterial classification better than the bacterial classification that sets out.
4. a kind of method for screening salt-tolerant aroma-producing yeasts according to claim 1 is characterized in that: described multiple sieve is the bacterial classification that primary dcreening operation is obtained, and imitation enterprise produces bent room, contains bent with plastic crate, is placed on and produces bent room and produce that bent synchronized culture carries out.
5. a kind of method for screening salt-tolerant aroma-producing yeasts according to claim 1, it is characterized in that: described salt-tolerant aroma-producing yeasts bacterial classification is that the multiple yeast that sieves is inserted in the fermention medium through repeatedly domestication cultivation, plate isolation obtains the strain screening purifying.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250780A (en) * | 2011-06-27 | 2011-11-23 | 四川剑南春（集团）有限责任公司 | Pichia sydowiorum and application thereof |
| CN103205368A (en) * | 2013-01-08 | 2013-07-17 | 浙江省致中和生物健康食品研究院 | Domestication method for high temperature-resistant ethanol-tolerant aroma-producing yeast |
| CN106947757A (en) * | 2016-12-13 | 2017-07-14 | 广东轻工职业技术学院 | A kind of preparation method of the high ester yield of resistance to high salt yeasty fusant |
-
2009
- 2009-03-11 CN CN200910037818A patent/CN101831386A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250780A (en) * | 2011-06-27 | 2011-11-23 | 四川剑南春（集团）有限责任公司 | Pichia sydowiorum and application thereof |
| CN103205368A (en) * | 2013-01-08 | 2013-07-17 | 浙江省致中和生物健康食品研究院 | Domestication method for high temperature-resistant ethanol-tolerant aroma-producing yeast |
| CN103205368B (en) * | 2013-01-08 | 2014-10-15 | 浙江省致中和生物健康食品研究院 | Domestication method for high temperature-resistant ethanol-tolerant aroma-producing yeast |
| CN106947757A (en) * | 2016-12-13 | 2017-07-14 | 广东轻工职业技术学院 | A kind of preparation method of the high ester yield of resistance to high salt yeasty fusant |
| CN106947757B (en) * | 2016-12-13 | 2020-05-01 | 广东轻工职业技术学院 | Preparation method of high-salt-resistance high-ester-yield yeast fusant |
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Application publication date: 20100915 |