CN101603013A - A kind of yeast saccharomyces cerevisiae and application thereof - Google Patents
A kind of yeast saccharomyces cerevisiae and application thereof Download PDFInfo
- Publication number
- CN101603013A CN101603013A CNA2009100723546A CN200910072354A CN101603013A CN 101603013 A CN101603013 A CN 101603013A CN A2009100723546 A CNA2009100723546 A CN A2009100723546A CN 200910072354 A CN200910072354 A CN 200910072354A CN 101603013 A CN101603013 A CN 101603013A
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- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- yeast
- juice
- fruit wine
- sea buckthorn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
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- 235000019634 flavors Nutrition 0.000 description 1
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- 235000015203 fruit juice Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
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- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
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- 230000000192 social effect Effects 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- PODWXQQNRWNDGD-UHFFFAOYSA-L sodium thiosulfate pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[O-]S([S-])(=O)=O PODWXQQNRWNDGD-UHFFFAOYSA-L 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention discloses a kind of yeast saccharomyces cerevisiae and application thereof, what relate to is microorganism and brewing fruit wine technology, and is used for this bacterial strain with juice of Fructus Hippophae or Fructus Hippophae to be the preparation of the sea buckthorn fruit wine of raw material, to produce the fine sea buckthorn fruit wine.The separation source of yeast saccharomyces cerevisiae bacterial classification is the pericarp of sea buckthorn juice natural fermentation broth, Fructus Hippophae and pulp, sea-buckthorn orchard soil, and colony characteristics has film and do not have web for cultivate tube wall in malt juice liquid medium; The bacterium colony of growing on the wort agar solid medium is white, butteriness, surface smoothing and glossy, neat in edge.Separate the brand new strain that obtains after cultivating, screen, optimizing, can fully adapt to the higher acidity of juice of Fructus Hippophae, overcome the highly acid defective of wine active dry yeast incompatibility juice of Fructus Hippophae, strain fermentation speed is fast, and security is superior; With the sea buckthorn fruit wine that this yeast is produced, residual sugar is low, wine degree height, the smell of fruits is very sweet, and the wine body is plentiful, has unique sea buckthorn fruit wine style.
Description
One, technical field
What the present invention relates to is microorganism and brewing fruit wine technology, the application of particularly a kind of Wine brewing yeast strain and this bacterial strain.
Two, background technology
In brewing fruit wine, yeast is the prime mover of fermentation, obtain the high-level efficiency and the fine fruit wine product of fermentation, and good production bacterial classification will be arranged.The yeast kind that can fermented juice forms fruit wine is various, but different saccharomycetes to make fermentation speed, produces the wine ability and to generate useful by product different, and is also different to the adaptive faculty of yeasting.Because various fruit all have different characteristics, particularly juice of Fructus Hippophae has singularity such as acidity height again, still there is not sea buckthorn fruit wine quality yeast bacterium at present, and in sea buckthorn fruit wine fermentation normally used wine active dry yeast, it is not thorough to exist fermentation, sea buckthorn fruit wine residual sugar height, shortcoming such as the wine degree is low, fruital is thin.
Therefore, filter out the yeast that is fit to sea buckthorn fruit wine fermentation usefulness, the outstanding distinctive sea-buckthorn fragrance of product, overcome the highly acid defective of present employing wine active dry yeast incompatibility juice of Fructus Hippophae, for production provides good bacterial strain, the zymotechnique in conjunction with best can obtain the success of fermentative production, make high-grade sea buckthorn fruit wine product have vast market prospect and development potentiality, have very important economic implications, social effect and ecological benefits.
Three, summary of the invention
The purpose of this invention is to provide a kind of yeast saccharomyces cerevisiae, this yeast bacterial strain can effectively adapt to the sea buckthorn fruit wine fermentation, be used to overcome the highly acid defective of wine active dry yeast incompatibility juice of Fructus Hippophae, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preserving number is CGMCC No.3080.
The separation source of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3080 is the pericarp of sea buckthorn juice natural fermentation broth, Fructus Hippophae and pulp, sea-buckthorn orchard soil; Its colony characteristics: the cultivation tube wall does not have film in malt juice liquid medium is not had web; The bacterium colony of growing on the wort agar solid medium is white, butteriness, surface smoothing and glossy, neat in edge.
Another object of the present invention is that being used for juice of Fructus Hippophae or Fructus Hippophae is the preparation of the sea buckthorn fruit wine of raw material with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCCNo.3080, produces the fine sea buckthorn fruit wine.
Juice of Fructus Hippophae is a raw material, and its soluble solid content is 6%～8%; Fructus Hippophae is that raw material is then earlier squeezed the juice Fructus Hippophae, makes after the oil removing that to contain soluble solid content be 6%～8% juice of Fructus Hippophae, and then the preparation sea buckthorn fruit wine.
Preparation sea buckthorn fruit wine technical process is: juice of Fructus Hippophae → deoil → adjust composition → sterilization → inoculation → Primary Fermentation → secondary fermentation → fall acid → ageing → clarification → filtration → sterilization → finished product.
In the flow process, the initial pol of adjusting composition is 20～23%, and inoculum size is 8～12% (V/V), and main fermentation temperature is 23～26 ℃.
The positively effect that the present invention has: the yeast saccharomyces cerevisiae among the present invention (Saccharomyces cerevisiae) CGMCC No.3080, be from the pericarp and pulp of sea buckthorn juice natural fermentation broth, sea-buckthorn orchard soil, Fructus Hippophae, separate the brand new strain that obtains after cultivating, screen, optimizing, security is superior; This bacterial strain can fully adapt to the higher acidity of juice of Fructus Hippophae, and strain fermentation speed is fast, and with the sea buckthorn fruit wine that this yeast is produced, residual sugar is low, wine degree height, the smell of fruits is very sweet, and the wine body is plentiful, has unique sea buckthorn fruit wine style.
Four, description of drawings
Fig. 1 is the colonial morphology of yeast saccharomyces cerevisiae on the wort agar substratum;
Fig. 2 is a yeast saccharomyces cerevisiae vegetative cell form (* 400);
Fig. 3 is the yeast saccharomyces cerevisiae cellular form (* 400) of sprouting;
Fig. 4 is a yeast saccharomyces cerevisiae thecaspore form (* 400);
Fig. 5 is the fermenting power curve of yeast saccharomyces cerevisiae;
Fig. 6 is leavening temperature and the response surface figure (inoculum size=12%) of initial pol to the sea buckthorn fruit wine aesthetic quality;
Fig. 7 is leavening temperature and the isogram (inoculum size=12%) of initial pol to the sea buckthorn fruit wine aesthetic quality;
Fig. 8 is initial pol and inoculum size to sea buckthorn fruit wine aesthetic quality's response surface figure (leavening temperature=25 ℃);
Fig. 9 is initial pol and inoculum size to sea buckthorn fruit wine aesthetic quality's isogram (leavening temperature=25 ℃);
Figure 10 is leavening temperature and the inoculum size response surface figure (initial pol=20%) to the sea buckthorn fruit wine aesthetic quality.
Figure 11 is leavening temperature and the inoculum size isogram (initial pol=20%) to the sea buckthorn fruit wine aesthetic quality.
Preservation information:
Strain name: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3080
Preservation date: 2009 5 years 31 days
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC)
Deposit number: CGMCC No.3080
Five, embodiment
Strain name: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3080, carried out preservation in 2009 5 years 31 days in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at the BeiJing, China city, preserving number is CGMCC No.3080.
The present invention is described in further detail below by specific embodiment.
Embodiment 1: the isolation identification of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3080, biological characteristics and the application in the sea buckthorn fruit wine fermentation thereof
1, material
1.1 yeast separation source: the pericarp and the pulp of sea buckthorn juice fermented liquid (spontaneous fermentation), sea-buckthorn orchard soil, Fructus Hippophae.
1.2 reagent:
| Glucose | Biochemical reagents | Tianjin Da Mao chemical reagent factory |
| Peptone | Biochemical reagents | Beijing extensive and profound in meaning star biotechnology limited liability company |
| Yeast extract paste | Biochemical reagents | Beijing extensive and profound in meaning star biotechnology limited liability company |
| Agar | Biochemical reagents | Beijing extensive and profound in meaning star biotechnology limited liability company |
| ??KH 2PO 4 | Analytical pure | Tianjin Da Mao chemical reagent factory |
| ??K 2HPO 4 | Analytical pure | Tianjin Da Mao chemical reagent factory |
| Potassium metabisulfite | Analytical pure | Shanghai four He Wei chemical industry company limiteds |
| Ethanol | Analytical pure | East China, Shenyang City chemical reagent work |
| Actidione | Biochemical reagents | Sigma company |
| Ethamine | Biochemical reagents | Sigma company |
| Cadaverine | Biochemical reagents | Sigma company |
| Yeast extract paste | Biochemical reagents | Tianjin Da Mao chemical reagent factory |
| Peptone | Biochemical reagents | Beijing extensive and profound in meaning star biotechnology limited liability company |
| Saltpetre | Analytical pure | East China, Shenyang City chemical reagent work |
| Glucose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Sucrose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Maltose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Semi-lactosi | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Lactose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Raffinose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Cellobiose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Inositol | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Ribose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Erythritol | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Rhamnosyl | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Wood sugar | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Ribitol | Analytical pure | Beijing extensive and profound in meaning star biotechnology limited liability company |
| N.F,USP MANNITOL | Analytical pure | Beijing extensive and profound in meaning star biotechnology limited liability company |
| Zulkovsky starch | Analytical pure | Tianjin Da Mao chemical reagent factory |
| Pectinose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
| Trehalose | Analytical pure | Beijing Suo Laibao Science and Technology Ltd. |
1.3 laboratory apparatus
| The XSZ-4G biomicroscope | The optical instrument factory, Chongqing |
| The biochemical incubator of SPH-250 type | The gloomy reliable Instr Ltd. that tests in Shanghai |
| The full temperature vibrator of HZQ-QX | East, Harbin connection electronic technology development corporation, Ltd. |
| The AR2140 electronic balance | Plum Teller-Tuo benefit Instr Ltd. |
| Micropipet | The thermoelectric Instr Ltd. in Shanghai |
| BCN-1360 type Bechtop | Beijing Dong Lianhaer Instr Ltd. |
| DELTA320 type acidometer | Plum Teller-Tuo benefit Instr Ltd. |
| 79-1 magnetic force heating stirrer | Jintan City's rainbow is contained instrument plant |
| S108 type hand refractometer | Dimension photoelectric technology limited liability company is surveyed in Shanghai |
| LDZX-75KBS type vertical pressure steam sterilizer | Shenan Medical Appliances Factory, Shanghai |
| DH4000 type electro-heating standing-temperature cultivator | Tianjin Tai Site Instr Ltd. |
| T6 new millennium ultraviolet-visible pectrophotometer | The Beijing Puxi General Instrument Co., Ltd |
1.4 culture medium preparation
1.4.1 selective enrichment substratum: alcoholic strength 10% (V/V) liquid malt extract medium (adds 4 times to the water of 60 ℃ of malt weights, be incubated saccharification in 55～60 ℃ of water-bath, behind 3～4h, use filtered through gauze, filter with absorbent cotton after boiling again.Adjust pol 10%, add alcohol after the sterilization).
1.4.2 isolation medium: wort agar substratum.Add 4 times to 60 ℃ of water of malt weight, in 55～60 ℃ of water-baths, be incubated saccharification, behind 3～4h, use filtered through gauze, filter with absorbent cotton again after boiling, adjust pol 10%, add 1.5%～2% agar, natural pH value, it is standby to sterilize).
1.4.3 yeast preservation substratum: wort agar substratum (same isolation medium).
1.4.4 yeast extract paste peptone glucose (YPD) substratum: yeast extract paste 1g, peptone 2g, glucose 2g, distilled water 100mL.
1.4.5 Corn Meal Agar substratum: get the 12.5g yellow corn powder and add water 300mL, stir, kept about 60 ℃ about 1 hour, after filtered through gauze, add an amount of water and supply 300mL, add agar, heating makes it slowly to melt, and it is standby to sterilize.
1.4.6 produce the thecaspore substratum: yeast extract paste 0.3g, wort 0.3g, peptone 0.5g, glucose 1g, agar 2g, distilled water 100mL.
2, the separation of yeast saccharomyces cerevisiae, evaluation
2.1 strains separation is got in the liquid malt extract medium that an amount of sample joins sterilized alcoholic strength 10% (V/V), places biochemical incubator to cultivate 48h for 28 ℃.Microscopy had or not viable bacteria to exist in per 24 hours, if there is viable bacteria to exist, under aseptic condition, get 0.1～0.2mL yeast pregnant solution and coat in the wort solid plate, under 28 ℃ of conditions, cultivate about 2～3 days then, if after having bacterium colony to grow, single bacterium colony that selection has a products of typical yeast bacterium colony characteristics is rule in wort solid plate and is separated 2～3 times, microscopy is that wort solid inclined-plane is gone in inoculation behind the purebred yeast, and with the yeast numbering that separation obtains, 4 ℃ of refrigerators are preserved.
2.2 screening
2.2.1 Du Shi tubule fermentation method is adopted in the screening of yeast one-level, measures each bacterial strain gas deliverability, compares the ferment ability that rises of each saccharomycete, preliminary screening goes out to have the bacterial strain of good leavening property.The 13% wort adding of 10mL is had in the test tube of Du Shi tubule, after the sterilization cooling, with inoculum size 1 * 10
7Individual/mL inserts the yeast activation solution, and under 28 ℃ of conditions, static fermentation 48h observes and write down the aerogenesis time and the gas production rate of each bacterial strain, test repetition 3 times.
2.2.2 the juice of Fructus Hippophae fermentation method is adopted in the screening of yeast secondary, measures the fermentation capacity of each saccharomycete.At first the juice of Fructus Hippophae pol is adjusted to 20%, with inoculum size 1 * 10
7Individual/mL inserts the yeast activation solution, and under 28 ℃ of conditions, static fermentation 7 days is adopted the alcoholic strength of distillation method mensuration fermented liquid and carried out subjective appreciation, and test repeats 3 times.
2.2.3 the yeast three level screen adopts Du Shi tubule fermentation method, measures the patience of each saccharomycete to ethanol and sulfurous gas.Inoculum size is 1 * 10
7Individual/mL, the yeast activation solution is inserted respectively in 13% wort that contains different concentration ethanol (8%, 10%, 12%, 14%, 16%, 18%, 20%) and different concns sulfurous gas (60mg/L, 80mg/L, 100mg/L, 120mg/L, 140mg/L, 160mg/L, 180mg/L, 200mg/L), and inoculum size is 1 * 10
7Individual/mL, constant temperature culture is 4 days under 28 ℃ of conditions, observes and write down the aerogenesis situation of each bacterial strain, and test repeats 3 times.
2.3 bacterial strain preliminary evaluation
2.3.1 phenotypic characteristic
The solid culture feature; The liquid culture feature.
2.3.2 Physiology and biochemistry is identified
(1) carbohydrate fermentation test: this evaluation is tested as carbon source with glucose, maltose, semi-lactosi, lactose and sucrose respectively.A certain amount of above-mentioned sugar is not joined in the nitrogenous source basic medium that contains Durham's fermentation tube, make concentration reach 50mmol/L, the activated liquid of yeast is inoculated in the above-mentioned substratum, places under 25 ℃ of conditions and cultivate, about a week, and vibrate test tube every day, to help the precipitation yeast, check the bubble situation that produces simultaneously, with the negative control of doing of carbonaceous sources not, observe once again after 4 weeks, repeat 3 times.
(2) assimilation carbon source test: this evaluation is tested as sole carbon source with raffinose, erythritol, sucrose, Zulkovsky starch, ribitol, D-wood sugar, D-mannitol, cellobiose, L-arabinose, succsinic acid, trehalose, D-ribose, citric acid, L-lactose, L-rhamnosyl and inositol respectively.A certain amount of above-mentioned sugar is not joined in the nitrogenous source basic medium that contains Durham's fermentation tube, makes concentration reach 50mmol/L, filter (0.20 μ m) degerming, with the test tube of carbonaceous sources not as the blank of not growing.Be linked in the above-mentioned substratum after yeast is activated respectively, place under 25 ℃ of conditions and cultivate, in order to keep cultivation results stability, test tube is inclined expose maximum liquid wheat face, and vibrate test tube every day, observe muddy degree, 4 weeks of observation to the, count the positive to become turbid in the test tube, no muddiness is counted feminine gender, repeats 3 times.Observation caliber as a result: the test tube backsight is observed the saccharomycetic muddy degree of having grown so that the card of black line to be arranged, if black line is covered fully, the result is designated as +++; If can see discontinuous line segment, be designated as ++; If can see discontinuous line segment, be designated as+; If can see the whole piece black line, be designated as-, the expression yeast is not grown.
(3) assimilation nitrogenous source test: this evaluation is tested as only nitrogen source with saltpetre, ethamine and cadaverine respectively, in the carbon source basic medium, add and go into saltpetre, ethamine, cadaverine respectively, add-on is respectively 0.078%, ethamine 0.064%, cadaverine 0.068%, filter (0.20 μ m) degerming, be linked into respectively in the above-mentioned substratum after yeast is activated, place 25 ℃ to cultivate for 1 week.With the substratum that do not add nitrogenous source as blank, triplicate.Observation caliber is identical with assimilation carbon source test as a result, and muddy degree is ++ or +++, then be to grow, otherwise can not grow.
(4) no VITAMIN growth test: will not have (the 0.20 μ m) degerming after filtration of VITAMIN liquid nutrient medium, the yeast that the aseptic technique inoculation has activated, cultivate for 25 ℃ and observed in 7 days, be the error of avoiding causing owing to the inoculation mcg vitamin of sneaking into, can will cultivate 7 days initial incubation liquid as the zymic activation solution, taking a morsel is inoculated in another culture medium without vitamin test tube, cultivates 7 days in 25 ℃.Observation caliber such as carbon assimilation are tested as a result, if solution presents in the test tube +++, for can the required VITAMIN of synthetically grown; If ++, then be can not the required VITAMIN of synthetically grown.
(5) 100mg/kg actidione resistance test: dissolving 1g actidione is in 2.5mL acetone, in acetone soln, add 6.7g nitrogenous source basal culture medium, add the distilled water that contains 10g glucose, fully mixing after-filtration (0.20 μ m) degerming again, this solution contains actidione 10,000mg/kg.Get above-mentioned solution 0.05mL and join in the test tube that contains the 4.5mL sterilized water, make the substratum that contains the 100mg/kg actidione.To the yeast that inoculation of medium has activated, cultivated for 3 weeks for 25 ℃.Observation caliber is tested with carbon assimilation as a result.
(6) 37 ℃ of growth tests: will activate good yeast streak inoculation to the wort agar inclined-plane, and place 37 ℃ to cultivate 2～4 days, and if weak life is arranged, then go down to posterity once, and continue to cultivate at 37 ℃, if can grow, then expression can be 37 ℃ of growths down.
Concrete outcome sees Table 1, table 2, table 3, table 4 and table 5.
On the basis of repeatedly sampling, separate with line through enrichment culture, be divided into from obtaining yeast 108 strains, classify by its source, from yeast 40 strains of soil; Yeast 68 strains from Fructus Hippophae and fruit juice.
Yeast is in the test tube that contains the Du Shi tubule, and 28 ℃ fermented 48 hours, and the energy aerogenesis reaches the yeast that approximates the full volume of Du Shi tubule 15 strains.15 saccharomycetes that primary dcreening operation is obtained are inoculated into respectively in 20% the sea-buckthorn, in 28 ℃ of fermentations 7 days, measure its alcoholic strength, and carry out subjective appreciation.Fermenting, the result is after 7 days, and the fermented wine precision has 3 strains greater than the yeast of 10% (V/V), and alcoholic strength has 4 strains between 9%～10%, between 8%～9% 6 strains is arranged, and is lower than 8% 2 strains that have.Alcoholic strength is respectively yeast SJY78 from the sea-buckthorn orchard soil greater than 3 saccharomycetes of 10% (V/V), from the SJY64 of seabuckthorn fruit peel, from the SJY1 in the sea buckthorn juice natural fermentation broth.In this 3 saccharomycete, be 10.1% (V/V) from the sea buckthorn fruit wine alcoholic strength of yeast SJY78 after Primary Fermentation in 7 days of sea-buckthorn orchard soil, though the sea-buckthorn flavor is arranged, pained slightly; And the sea buckthorn fruit wine aroma of brewageing from the SJY64 of seabuckthorn fruit peel and from the SJY1 in the sea buckthorn juice natural fermentation broth is strong, the fruital that keeps sea-buckthorn preferably, the sea buckthorn fruit wine alcoholic strength of bacterial strain SJY64 after Primary Fermentation in 7 days is 10.3% (V/V), bacterial strain SJY1 alcoholic strength after Primary Fermentation in 7 days can reach 11.4% (V/V), and special taste, typicalness is good, compare product wine height with the wine active dry yeast of present industrial application, carry out follow-up test so classify bacterial strain SJY1 as aimed strain, and deliver to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) and carry out survival test and preservation, preserving number: CGMCC No.3080.
Table 1 is the saccharomycetic patience test-results of screening, shows in the experimental result of ethanol-tolerant, sulfurous gas and pH value for the examination bacterial classification, and the alcoholic strength that yeast SJY1 can anti-18%, but need just can make Du Shi tubule aerogenesis reach full volume by 3.5d, can the SO of anti-200mg/L
2, it should be noted that it can tolerate the acidity of pH value 2.5; SJY64 can anti-14% alcoholic strength, the 2d aerogenesis can reach the full volume of Du Shi tubule under this condition, it can the SO of anti-160mg/L
2, the acidity of tolerance pH value 3.0; SJY78 can anti-14% alcoholic strength, need 3d to reach the full volume of Du Shi tubule by aerogenesis.It can the SO of anti-140mg/L
2, the acidity of tolerance pH value 3.0.This shows that yeast SJY1 is fit to the strong requirement of sea buckthorn fruit wine yeast acid resistance very much.
The saccharomycetic patience test-results of table 1 screening
| Bacterial strain | Ethanol-tolerant | Anti-SO 2(mg/L) | Anti-pH value |
| ??SJY1 ??SJY64 ??SJY78 | ??18％ ??14％ ??14％ | ??200 ??160 ??140 | ??2.5 ??3.0 ??3.0 |
Table 2 carbohydrate fermentation test-results
| Fermenting carbohydrate | ??SJY1 | Yeast saccharomyces cerevisiae |
| Glucose saccharose maltose semi-lactosi lactose | ??+ ??+ ??+ ??+ ??- | ??+ ??V ??V ??V ??- |
Annotate: "+" expression positive reaction, "-" expression negative reaction, " V " represents variable reactive.
Table 3 assimilation carbon source test-results
| The assimilation carbon source | ??SJY1 | Yeast saccharomyces cerevisiae |
| Raffinose cellobiose inositol D-ribose erythritol L-rhamnosyl L-lactose D-wood sugar ribitol citric acid succsinic acid N.F,USP MANNITOL Zulkovsky starch L-arabinose trehalose sucrose | ??+ ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??+ ??+ | ??V ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??V ??V ??- ??V ??V |
Annotate: "+" expression positive reaction, "-" expression negative reaction, " V " represents variable reactive.
Table 4 assimilation nitrogenous source test-results
| The assimilation nitrogenous source | ??SJY1 | Yeast saccharomyces cerevisiae |
| Potassium salt acid diethylamide hydrochloric acid cadaverine | ??- ??- ??- | ??- ??- ??- |
Annotate: "+" expression positive reaction, " " expression negative reaction, " V " expression variable reactive.
Other test-results of table 5
| Test | ??SJY1 | Yeast saccharomyces cerevisiae |
| The growth of 37 ℃ of growths of no VITAMIN growth cycloheximide | ??+ ??+ ??- | ??V ??V ??- |
Annotate: "+" expression positive reaction, "-" expression negative reaction, " V " expression variable reactive.
According to observation to yeast morphology, syngenesis mode, sugar fermentation, assimilation carbon source, assimilation nitrogenous source, the growth of no VITAMIN, 37 ℃ of growths and anti-cycloheximide test-results, and the special work in contrast J.A. Barney, Hu Ruiqing translates " saccharomycetic feature and identification handbook " (press of Qingdao Marine University, 1991), can identify that SJY1 is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) to saccharomycetic description.
3, colony characteristics
The cultivation tube wall does not have film in malt juice liquid medium is not had web; The bacterium colony of growing on the wort agar substratum is white, butteriness, surface smoothing and glossy, neat in edge.
4, the form of microscopically
Fig. 1 provides the colonial morphology of Saccharomyces Cerevisiae in S JY1 on the wort agar substratum.
Fig. 2 provides Saccharomyces Cerevisiae in S JY1 vegetative cell form (* 400), Fig. 3 provides the Saccharomyces Cerevisiae in S JY1 cellular form (* 400) of sprouting, as shown in the figure, vegetative cell is oval, the cell size is (3.38～5.81) * (4.35～7.74) μ m, and the vegetative propagation mode is monolateral sprouts or sprout in both sides.
Fig. 4 provides Saccharomyces Cerevisiae in S JY1 thecaspore form (* 400), does not form pseudohypha on the Corn Meal Agar substratum.Have thecaspore to form, each ascus contains 1～4 thecaspore, and spore is rounded.
5, the main fermentation character of Saccharomyces Cerevisiae in S JY1 is identified
5.1 saccharomycetic fermenting power is measured
Adopt CO
2Weight-loss method.The bacterial classification that activation is good inserts in the sterilized 10% wheat juice body substratum, respectively at 23 ℃, 28 ℃, 33 ℃, 38 ℃, and constant temperature culture 96h, timing in per 12 hours is weighed and is write down weightlessness.With CO
2Weight loss is an ordinate zou, and fermentation time is an X-coordinate, draws the fermenting power curve.
5.2 saccharomycetic coherency is measured
Yeast is inoculated in the malt juice liquid medium, cultivated 5 days for 25 ℃, get nutrient solution and be loaded in the centrifuge tube with 3500r/min, centrifugal 15min collects yeast cell, with sterilized water washing 2～3 times, abandoning supernatant takes by weighing 1g bacterium mud in graduated centrifuge tube, adds the acetate buffer (pH4.5) of 10mL, 20 ℃ leave standstill 20min after, shake centrifuge tube 5min yeast cell is evenly suspended again, leave standstill again, the sedimentary milliliter of yeast cell number during record 10min.
Fig. 5 provides the fermenting power curve of Saccharomyces Cerevisiae in S JY1, and as shown in Figure 5, free Saccharomyces Cerevisiae in S JY1 is in the 12nd～24 hour, and fermenting speed is fast, and the average fermentation speed in the time of 28 ℃ is 0.38g/h, and carbonic acid gas weightlessness is 9.8g, reaches maximum.
In addition, this value of the basis of Saccharomyces Cerevisiae in S JY1 illustrates that its coherency is strong for 3.4mL, helps the clarification after this sea buckthorn fruit wine fermentation ends.
The application of Saccharomyces Cerevisiae in S JY1 in the sea buckthorn fruit wine fermentation.
1, material and equipment
1.1 material
Academy of agricultural sciences, Heilongjiang Province berry institute produces juice of Fructus Hippophae, juice of Fructus Hippophae soluble solid content 8%; White sugar, food grade; Free Saccharomyces Cerevisiae in S JY1.
Yeast preservation substratum: wort agar substratum (the same); The activated yeast substratum: juice of Fructus Hippophae adds isopyknic water, adjusts pol to 15%.
1.2 main agents
| Potassium metabisulfite | Analytical pure | Shishewei Chemical Co., Ltd., Shanghai |
| Hydrochloric acid | Analytical pure | East China, Shenyang City chemical reagent work |
| Sulfothiorine | Analytical pure | Tianjin Hedong District red rock chemical reagent work |
| Zulkovsky starch | Analytical pure | Tianjin Da Mao chemical reagent factory |
| Copper sulfate | Analytical pure | Tianjin Da Mao chemical reagent factory |
| Seignette salt | Analytical pure | Tianjin section close europeanized reagent development centre |
| Vitamins C | Standard substance | Chinese Medicine Shanghai chemical reagents corporation |
| Oxalic acid | Analytical pure | Tianjin chemical reagent three factories |
| Sodium Nitrite | Analytical pure | Tianjin Hedong District red rock chemical reagent work |
| Aluminum nitrate | Analytical pure | Tianjin Hedong District red rock chemical reagent work |
| Rutin | Standard substance | Nat'l Pharmaceutical ﹠ Biological Products Control Institute |
| Lime carbonate | Analytical pure | Tianjin Milky Way chemical reagent factory |
| Polyvinylpyrrolidone (PVPP) | Analytical pure | Tianjin Da Mao chemical reagent factory |
1.3 key instrument
| The XSZ-4G biomicroscope | The optical instrument factory, Chongqing |
| The biochemical incubator of SPH-250 type | The gloomy reliable Instr Ltd. that tests in Shanghai |
| The full temperature vibrator of HZQ-QX | East, Harbin connection electronic technology development corporation, Ltd. |
| The AR2140 electronic balance | Plum Teller-Tuo benefit Instr Ltd. |
| Micropipet | The thermoelectric Instr Ltd. in Shanghai |
| BCN-1360 type Bechtop | Beijing Dong Lianhaer Instr Ltd. |
| DELTA320 type acidometer | Plum Teller-Tuo benefit Instr Ltd. |
| WS108 type hand refractometer | Dimension photoelectric technology limited liability company is surveyed in Shanghai |
| LDZX-75KBS type vertical pressure steam sterilizer | Shenan Medical Appliances Factory, Shanghai |
| ??T 6The type ultraviolet-visible pectrophotometer | The general universal apparatus equipment company of analysing in Beijing |
| The Agilent1200 high performance liquid chromatograph | Anjelen Sci. ﹠ Tech. Inc |
2, experimental technique
2.1 sea buckthorn fruit wine making method
2.1.1 technical process:
Juice of Fructus Hippophae → deoil → adjust composition → sterilization → inoculation → Primary Fermentation → secondary fermentation → fall acid → ageing → clarification → filtration → sterilization → finished product
2.1.2 key points for operation: get juice of Fructus Hippophae, after centrifugal the deoiling,, after the conventional sterilization, add the SO of 80mg/L according to the initial pol of requirement of experiment adjustment (replacing sugar degree with soluble solid herein)
2, inserting the cell concentration of cultivating 24h during inoculation on request is 2.0 * 10
8The Saccharomyces Cerevisiae in S JY1 liquid of cfu/mL.25 ℃ of main fermentation temperatures ferment and remove the wine pin when residual sugar no longer obviously reduce, and behind room temperature secondary fermentation 7d, adopt 2.0g/L lime carbonate to fall acid, then ageing 30d in 15～18 ℃ of wine cellars.Take out the back and clarify, carry out subjective appreciation and physico-chemical analysis after the filtration with 0.4g/L polyvinylpyrrolidone (PVPP).
2.2 Saccharomyces Cerevisiae in S JY1 fermentation sea buckthorn fruit wine experimental design
On the experiment of single factor basis, choose leavening temperature (x
1), initial pol (x
2), inoculum size (x
3) 3 factors are as the investigation factor, are dependent variable (Y) with the sensory evaluation scores of wine sample, the optimal processing parameter of this Saccharomyces Cerevisiae in S JY1 fermentation sea buckthorn fruit wine is studied in design response surface analysis experiment (seeing Table 6).
Table 6 response surface analysis empirical factor coding and water-glass
Annotate: X
1=(x
1-25)/3; X
2=(x
2-20)/2; X
3=(x
3-12)/2
2.3 sea buckthorn fruit wine sensory evaluation scores method
The sea buckthorn fruit wine sensory evaluation scores is carried out according to table 7.
Table 7 sea buckthorn fruit wine sensory evaluation scores standard
2.4 sea buckthorn fruit wine physical and chemical index measuring method
Soluble solid is measured: the hand refractometer method; Alcoholic strength is measured: the Ebullioscope method; Reducing sugar test: Fehlings reagent; Titration of total acidity: acid base titration; Vitamins C is measured: with reference to " vitamins C in high effective liquid chromatography for measuring protective foods and the beverage " article method that people such as the Zhang Pei of Tianjin Disease Prevention And Control Center delivered on " Chinese food health magazine " the 4th phase in 2008, permaphase ODS C
18Reverse-phase chromatographic column (250mm * 4.6mm, 5 μ m), moving phase 0.1% oxalic acid solution detects wavelength 262nm; Determination of total flavonoids: " microwave-assisted extracts Pasania cuspidata tender leaf flavones technical study " the article method that waits the people on " Transactions of the Chinese Society of Agricultural Engineering " 2007 the 2nd phases, to deliver by force with reference to South China Science ﹠ Engineering University's light industry and Foodstuffs Academy Dong Hua, with the rutin is standard specimen, adopts Sodium Nitrite-aluminum nitrate development process.
2.5 data statistic analysis
The data biometrics SAS (8.0) statistical software analyzes on computers.
3, result
3.1 the response surface experimental result is according to the experimental design of table 6, the response surface experimental result sees Table 8.
Experimental design of table 8 response surface analysis and result
| Sequence number | ??X 1 | ??X 2 | ??X 3 | ??Y |
| ??1 ??2 ??3 ??4 ??5 | ??-1 ??-1 ??1 ??1 ??0 | ??-1 ??1 ??-1 ??1 ??-1 | ??0 ??0 ??0 ??0 ??-1 | ??78 ??72 ??70 ??80 ??85 |
| ??6 ??7 ??8 ??9 ??10 ??11 ??12 ??13 ??14 ??15 | ??0 ??0 ??0 ??-1 ??1 ??-1 ??1 ??0 ??0 ??0 | ??-1 ??1 ??1 ??0 ??0 ??0 ??0 ??0 ??0 ??0 | ??1 ??-1 ??1 ??-1 ??-1 ??1 ??1 ??0 ??0 ??0 | ??76 ??82 ??80 ??78 ??84 ??76 ??75 ??89 ??86 ??88 |
According to the experimental result of table 8, be response value with the sensory evaluation scores of sea buckthorn fruit wine, carry out statistical study through SAS 8.0 statistical softwares, the regression equation that obtains is:
Y＝87.666667+0.625000X
1+0.625000X
2-2.750000X
3-7.583333X
1 2+4.000000X
2X
1-5.083333X
2 2-1.750000X
3X
1+1.750000X
3X
2-1.833333X
3 2。
The analysis of variance table of table 9 regression model
| Source of variation | Degree of freedom | Sum of squares | All square | The F value | The P value |
| The model residual error is lost and is intended a pure error summation | ??9 ??5 ??3 ??2 ??14 | ??446.016667 ??8.916667 ??4.250000 ??4.666667 ??454.933334 | ??49.557407 ??1.783333 ??1.416667 ??2.333333 | ??27.79 ? ??0.61 | ??0.0010 ? ??0.6709 |
The fail-safe analysis of this model can be considered from variance analysis and definite coefficient two aspects.Table 9 is analysiss of variance table of table 3 experimental result, the P (F＞F of model
α)=0.0010＜0.01, so regression model is extremely remarkable, model determines that coefficient is R
2=0.9804, lose and intend P=0.6709＞0.05, show that the mistake plan is not remarkable, this model is stable, illustrates that model and actual match get fine.In the experimental design scope, can effectively predict and analyze the condition of brewageing of sea buckthorn fruit wine.
The significance analysis table of table 10 factor
| The source | Degree of freedom | Regression coefficient | Standard deviation | The t value | Significance |
| ??X 1??X 2??X 3??X 1 2??X 2X 1??X 2 2??X 3X 1??X 3X 2??X 3 2 | ??1 ??1 ??1 ??1 ??1 ??1 ??1 ??1 ??1 | ??0.625000 ??0.625000 ??-2.750000 ??-7.583333 ??4.000000 ??-5.083333 ??-1.750000 ??1.750000 ??-1.833333 | ??0.472141 ??0.472141 ??0.472141 ??0.694972 ??0.667708 ??0.694972 ??0.667708 ??0.667708 ??0.694972 | ??1.32 ??1.32 ??-5.82 ??-10.91 ??5.99 ??-7.31 ??-2.62 ??2.62 ??-2.64 | ??0.2429 ??0.2429 ??0.0021 ??0.0001 ??0.0019 ??0.0007 ??0.0470 ??0.0470 ??0.0461 |
Table 10 is the significance analysis table of the factor, and by table as seen, in once, inoculum size is extremely remarkable to sea buckthorn fruit wine aesthetic quality's influence, and in the quadratic term, square influence of initial pol, leavening temperature is extremely remarkable, and square influence of inoculum size is remarkable; A mutual X
2X
1Influence extremely remarkable, X
3X
1, X
3X
2Influence significantly.According to regression equation, use SAS software and draw response surface chart and isogram thereof, can reflect the variation tendency of each factor intuitively to the response value influence.
Fig. 6 is leavening temperature and the response surface figure (inoculum size=12%) of initial pol to the sea buckthorn fruit wine aesthetic quality; Fig. 7 is leavening temperature and the isogram (inoculum size=12%) of initial pol to the sea buckthorn fruit wine aesthetic quality; Fig. 8 is initial pol and inoculum size to sea buckthorn fruit wine aesthetic quality's response surface figure (leavening temperature=25 ℃); Fig. 9 is initial pol and inoculum size to sea buckthorn fruit wine aesthetic quality's isogram (leavening temperature=25 ℃); Figure 10 is leavening temperature and the inoculum size response surface figure (initial pol=20%) to the sea buckthorn fruit wine aesthetic quality; Figure 11 is leavening temperature and the inoculum size isogram (initial pol=20%) to the sea buckthorn fruit wine aesthetic quality.In conjunction with Fig. 1～shown in Figure 11, can predict under steady state the maximum of sea buckthorn fruit wine sensory evaluation scores, X according to regression model
1=0.128550, X
2=-0.030078, X
3=-0.825709, the actual value corresponding with it is respectively: 25.4 ℃ of leavening temperatures, and initial pol 19.9%, inoculum size 10.3% (V/V), prediction sensory evaluation scores with this understanding is 88.8 minutes.
3.2 the sea buckthorn fruit wine physical and chemical index is analyzed
Under optimum level, be 25.4 ℃ of leavening temperatures, initial pol 19.9%, inoculum size 10.3% (V/V), utilize same bacterial classification, identical raw material and technology brew sea buckthorn fruit wine after, carry out the physical and chemical index analysis: sea buckthorn fruit wine wine degree is 11.2% (V/V, 20 ℃), be higher than the wine degree that uses the wine active dry yeast fermentation, using the wine degree of wine active dry yeast fermentation is 7.5% (V/V, 20 ℃).Reducing sugar is 3.5gL in the sea buckthorn fruit wine of ageing after 30 days
-1Total acid content is 7.4gL
-1Subjective appreciation must be divided into 91 fens; Vitamin C content is 16.5mg100mL
-1, content of total flavone is 2.4mgmL
-1Wine sample behind the clarification filtration, wine body clear, glossy, be golden yellow, fruital, aroma are strong, and the wine body is plentiful, has unique sea buckthorn fruit wine style.
Embodiment 2: the preparation of sea buckthorn fruit wine
Raw material: Fructus Hippophae is squeezed the juice, and makes juice of Fructus Hippophae after the oil removing, and soluble solid content 6% in the juice of Fructus Hippophae.
The fruit wine preparation process condition is 23 ℃ of leavening temperatures, initial pol 21%, and inoculum size 8% (V/V), other is with embodiment 1.
The fruit wine of preparation is analyzed its physical and chemical index and is: sea buckthorn fruit wine wine degree is 11.5% (V/V, 20 ℃).Reducing sugar is 4.0gL in the sea buckthorn fruit wine of ageing after 30 days
-1Total acid content is 7.4gL
-1Subjective appreciation must be divided into 90 fens; Vitamin C content is 17.0mg100mL
-1, content of total flavone is 2.5mgmL
-1Wine sample after ageing, clarification, the filtration, wine body clear, glossy, be golden yellow, fruital, aroma are strong, and the wine body is plentiful, has unique sea buckthorn fruit wine style.
Embodiment 3: the preparation of sea buckthorn fruit wine
Raw material: juice of Fructus Hippophae, soluble solid content 7%.
The fruit wine preparation process condition is 26 ℃ of leavening temperatures, initial pol 23%, and inoculum size 12%V/V, other is with embodiment 1.
The fruit wine physico-chemical analysis index of preparation is: sea buckthorn fruit wine wine degree is 12.3% (V/V, 20 ℃).Reducing sugar is 4.1gL in the sea buckthorn fruit wine of ageing after 30 days
-1Total acid content is 7.6gL
-1Subjective appreciation must be divided into 92 fens; Vitamin C content is 18.2mg100mL
-1, content of total flavone is 2.8mgmL
-1Wine sample after ageing, clarification, the filtration, clear, glossy, be golden yellow, fruital, aroma are strong, and the wine body is plentiful, and is sour-sweet tasty and refreshing, has unique typical sea buckthorn fruit wine style.
Claims (5)
1, a kind of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No.3080.
2, yeast saccharomyces cerevisiae according to claim 1 (Saccharomyces cerevisiae) CGMCCNo.3080, it is characterized in that: described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCCNo.3080, its colony characteristics cultivate tube wall in malt juice liquid medium do not have film and do not have web; The bacterium colony of growing on the wort agar solid medium is white, butteriness, surface smoothing and glossy, neat in edge.
3, the described yeast saccharomyces cerevisiae of a kind of claim 1 (Saccharomyces cerevisiae) CGMCCNo.3080, it is characterized in that: it is the sea buckthorn fruit wine of raw material that this yeast saccharomyces cerevisiae is used to produce with juice of Fructus Hippophae or Fructus Hippophae.
4, yeast saccharomyces cerevisiae according to claim 3 (Saccharomyces cerevisiae) CGMCCNo.3080, the technical process that is used to produce sea buckthorn fruit wine is: juice of Fructus Hippophae → deoil → adjust composition → sterilization → inoculation → Primary Fermentation → secondary fermentation → fall acid → ageing → clarification → filtration → sterilization → finished product, it is characterized in that: the initial pol of adjusting composition in the technical process is 20～23%, inoculum size is 8～12% (V/V), and main fermentation temperature is 23～26 ℃.
5, according to claim 3 or 4 described yeast saccharomyces cerevisiaes (Saccharomyces cerevisiae) CGMCCNo.3080, it is characterized in that: soluble solid content is 6～8% in the juice of Fructus Hippophae.
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