CN107287135A - A kind of malolactic fermentation bacterial strain and its cultural method and application - Google Patents
A kind of malolactic fermentation bacterial strain and its cultural method and application Download PDFInfo
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- CN107287135A CN107287135A CN201710532012.2A CN201710532012A CN107287135A CN 107287135 A CN107287135 A CN 107287135A CN 201710532012 A CN201710532012 A CN 201710532012A CN 107287135 A CN107287135 A CN 107287135A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G1/00—Preparation of wine or sparkling wine
- C12G1/02—Preparation of must from grapes; Must treatment and fermentation
- C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a kind of malolactic fermentation bacterial strain and its cultural method and application, the bacterial strain is that the bacterial strain is the bacterial strains of Lab2 1, the bacterial strain is screened the Oneococcus onei of malolactic fermentation by the present inventor from epidermis, 200 plants of bacterial strain carries out primary dcreening operation and secondary screening after mutagenesis, using the test method, the 1 plant mutant bacterial strain filtered out such as Morphological Identification, Physiology and biochemistry identification, the screening of culture medium, the screening of fermentation system pH value, total acidometric titration, high performance liquid chromatography.The present invention is solved terminated the problem that color and luster falls sharply, volatile acid increases using import lactic bacteria strain malolactic fermentation in the past.
Description
Technical field
The present invention relates to bacterial strain field, and in particular to a kind of malo-lactic fermentation bacterial strain and its cultural method and application.
Background technology
At present, high-activity lactic acid bacteria at home also without professional production wine production, Wine Enterprises can only be from
External import lactic acid bacteria.By the project implementation, environmental condition is to thalline during studying grape wine malo-lactic fermentation
Content in terms of influence, Malic Metabolism rule and the energetic supersession of growth.Select with native characteristic, original producton location wind
The Lactic Acid Bacteria strain of lattice, improving the breeding rate and deliverability of lactic bacteria strain has good social benefit.
The content of the invention
In view of the above-mentioned problems, the present invention from epidermis by according to grape variety and soil characteristic, screening malic acid-lactic acid
The Oneococcus onei of fermentation, 200 plants of bacterial strain carries out primary dcreening operation and secondary screening after mutagenesis, identified using Morphological Identification, Physiology and biochemistry,
The test method such as the screening of culture medium, the screening of fermentation system pH value, total acidometric titration, high performance liquid chromatography, is finally successfully screened
Go out 1 plant mutant bacterial strain：Lab2-1 bacterial strains.
To achieve the above object, the technical scheme taken of the present invention is：
A kind of malo-lactic fermentation bacterial strain, the bacterial strain is Lab2-1 bacterial strains.
The cultural method of above-mentioned malo-lactic fermentation bacterial strain, comprises the following steps：
In a clean container, 100 liters of distilled water and 100 liters of grape juices, adjustment temperature to 20 are added.C, adds and promotees
Enter agent to stir, be then slowly added to Lab2-1 bacterial strains, it is stirring while adding, it is allowed to uniform, spreads cultivation 24 at a temperature of 18-22
Hour, when detecting that the liquid bacterium colony that spreads cultivation reaches 107-108cfu/ml, obtaining can 100 tons of extra dry red wine Fructus Vitis viniferae wine base malic acid of pilot scale-lactic acid hair
The ferment liquid that spreads cultivation.
A kind of above-mentioned malo-lactic fermentation bacterial strain can be used for grape wine malo-lactic fermentation, specifically include following step
Suddenly：
Lab2-1 bacterial strains and saccharomycete are linked into the pulp of fermentation to be leached simultaneously, start two fermentation process, just
18-20 DEG C of beginning start-up temperature, after alcoholic fermentation starts 24 hours, addition lactic acid bacteria spreads cultivation liquid, and fermentation temperature keeps 18-20
DEG C, detect that 24-26 DEG C of heating terminates to alcoholic fermentation during malic acid≤0.8g/L after 7 days, 18-20 DEG C is cooled to, in this temperature
Under proceed malo-lactic fermentation, make malic acid decompose fully and completely after with pericarp separation.
The invention has the advantages that：
By the grape wine of Lab2-1 strain fermentations of mutagenesis screening, its malic acid-rotational rate of lactic acid is 38.83%, fermentation
The colourity (OD values) of wine can reach 14 afterwards, and the soft index of wine is 6.95, and the phase interaction between microorganism is kept during fermentation
With and balance, it is quick to suppress that rotten microbiologic population is caused.Gained time for malo-lactic fermentation, so as to save
Save the cost caused by warming temperature.Pilot product indices meet national standard, and which part limits index higher than country
Standard, this product volatile acid content 0.42g/L limits index (≤1.2g/L) requirement, grape wine less than international and national standard
Bacteriological stability increase.
Brief description of the drawings
Fig. 1 is the standard items chromatogram of mixed acid (malic acid and lactic acid).
Fig. 2 is malic acid standard curve.
Fig. 3 is lactate standard curve.
Fig. 4 is the content analysis of malic acid in fermentation period.
Fig. 5 is the change of grape wine malic acid, lactic acid and total acid in fermentation period.
Fig. 6 is the change of lactic acid content in fermentation period.
Fig. 7 is the variation tendency (concentration mg/L) of different fermentations period claret total sugar concentration
Fig. 8 is the change of the tannin content in grape wine sample in fermentation period.
Fig. 9 is the chromatogram of grape wine sample.
Figure 10 is the chromatogram of grape wine sample citric acid mark-on.
Figure 11 is the variation tendency of grape wine citric acid during the fermentation.
Figure 12 is the process chart of grape wine malo-lactic fermentation of the embodiment of the present invention.
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
Embodiment
Amplification increases the determination of bacterium condition：Amplification increases 1000 grams of bacterium number amount, in a clean container, adds 100 liters of steamings
Distilled water and 100 liters of grape juices, adjustment temperature add accelerator and stirred to 20 DEG C, then will utilize the good breast of mutagenesis screening
Sour bacterium is slowly added to, stirring while adding, is allowed to uniform, is spread cultivation at a temperature of 18-22 24 hours, detects that the liquid bacterium colony that spreads cultivation reaches
, can 100 tons of extra dry red wine Fructus Vitis viniferae wine base malo-lactic fermentation liquid that spread cultivation of pilot scale during 107-108cfu/ml.
Coefficient correlation is all higher than 0.9995, correlation in respective concentration range and under chromatographic condition for malic acid, lactic acid
Well.According to obtained standard curve, the peak area of respective substance in grape wine sample can be substituted into corresponding linear equation
Obtain the concentration of respective substance in grape wine.The high performance liquid chromatographs of Agilent 1100 are linear to the analysis method of object
, still analyzed from external standard method, as a result see Fig. 1, Fig. 2, Fig. 3.
The concentration range of claret malic acid during the fermentation is 6.97144-8.00600g/L, lactic acid it is dense
Degree is in the range of 3.82726-5.22338g/L.The content of grape wine malic acid during the fermentation becomes in decline as seen from Figure 5
Gesture, and lactic acid is in rising trend.The malic acid that its changing rule may be interpreted as in 1. grape wine derives from grape, the decline of leading portion
By glycolysis during mainly due to alcoholic fermentation.From fermentation the 11st day to fermentation the 25th day this section of plateau be probably by
Malic acid is decomposed in saccharomycete and has reached threshold value, and malic acid concentration no longer declines.After a period of time malic acid content declines afterwards
Reach plateau, enter mainly due to grape wine after malo-lactic fermentation, lactic acid bacteria decompose malic acid and produce lactic acid and
It is caused.To sum up, a reduction part for malic acid content during the fermentation is due to glycolysis, and another part is due to lactic acid bacteria
Decomposition and reduce.2. the lactic acid in grape wine is not to derive from grape, but the product of fermentation.Grape wine is in fermentation process
Yeast metabolism produces a small amount of breast during the rising of leading portion is mainly due to primary fermentation in middle lactic acid content variation tendency line
Acid, rear a period of time lactic acid content rises mainly due to entering after malo-lactic fermentation, and lactic acid bacteria decomposes malic acid and produced
Caused by lactogenesis acid.
(3) time of extra dry red wine Fructus Vitis viniferae wine base apple-milk fermentation inoculation：It is determined that suitable inoculation time is to ensure apple-breast
Fermentation is smoothed out, and malic acid avoids that tartaric acid is decomposed and color and luster falls sharply as far as possible in the case of being totally converted.Therefore, selection connects
The Best Times planted, first selection is inoculated with during alcoholic fermentation；Second selection is after alcoholic fermentation terminates
It is inoculated with immediately；Volatile acid, malic acid, pH value, chromaticity index corresponding to the fermentation ends analysis different vaccination time, it is determined that
Feasible inoculation method is gone out and the time is as follows：
Alcoholic fermentation starts inoculation：Mutagenic strain and saccharomycete are linked into the pulp of fermentation to be leached simultaneously, started
Two fermentation process, 18-20 DEG C of initial starting temperature, after alcoholic fermentation starts 24 hours, addition lactic acid bacteria spreads cultivation liquid, fermentation
Temperature keeps detecting that 24-26 DEG C of heating terminates to alcoholic fermentation during malic acid≤0.8g/L after 18-20 DEG C, 7 days.To make apple
Acid-lactic fermentation proceeds, therefore uses delay and skin slag time of contact, is cooled to 18-20 DEG C, proceeds at this temperature
Malo-lactic fermentation, makes malic acid be separated after decomposing fully and completely with pericarp.To adapt to the smooth of malo-lactic fermentation
Carry out.
Alcoholic fermentation terminates inoculation：Soon terminate former wine in alcoholic fermentation to separate with pericarp, gained former wine addition lactic acid bacteria
Spread cultivation liquid, enters after stirring and malo-lactic fermentation is carried out in oak barrel, and it is the 95% of container to enter barrelage amount, seal bucket,
By wine cellar temperature control at 22-24 DEG C, malic acid, which is completely converted into lactic fermentation, to be terminated.
It can be seen that by Fig. 6-Fig. 7, sugared changes of contents can divide five stages：First stage, 13 days before the fermentation phase is entered
Sugared content steadily declines；Second stage, was raised suddenly to the 15th day sugared content, and the sugared content rise in wine liquid is even more than wine liquid
Initial sugared content；Sugared content in phase III, the 15th day to the 23rd day sample, which is drastically reduced, almost levels off to 0；4th
Period is the content of sugar and raised suddenly；5th period is to start to fermentation ends for the 25th day, and sugared content has of short duration rise
After tend to be steady decline, by 6.05-3.98mg/L, meet the standard of claret.
The percentage composition variation tendency of tannin can be divided into four-stage：First stage, into preceding 7 days tannin hundred of fermentation phase
Divide content in a slight decrease；Second stage, from playing for the 7th day the 9th day for fermentation, the rise of tannin percentage composition；Phase III, fermentation
Mid-term, i.e., the 9th are day by the 19th day, and tannin content is decrease continuously to minimum value；Fourth stage, from the 19th day to the 31st day, tannin
Second of rise of percentage composition, then basically reaches a plateau.The changing rule of claret during the fermentation
Reason is probably：
Tannin content is reduced.Because tannin is primarily present in the carpopodium of grape, pericarp, fermentation just starts, tannin content
Higher, it could be speculated that being mixed into a large amount of grape branch stalks when making, tannin directly can be dissolved into wine liquid, with the progress of fermentation, Portugal
Grape branch stalk is sifted out, and tannin content is in a slight decrease.
Tannin content is raised.With the progress of fermentation, a part derives from the tannin of grape, i.e. catechotannia, condensation list
Peaceful or burnt catechotannia, also referred to as endogenous tannin is leached out during vinous fermentation from grape pomace and seed.But leaching
The process carried is slower, therefore the anthocyanidin generally in grape wine just starts just to add tannin when leaching, typically in fermentation
Startup is a few days ago added, if the time of addition too late, can not have due condensation, to ensure tannin rapidly and anthocyanidin
It is combined, it is ensured that the quality and color and luster of wine.
Tannin content starts to be gradually reduced.This stage existing tannin from grape is in Fermentation of Grape Wine from fruit
Gradually it is leached out in skin and grape pip, increases tannin content in zymotic fluid, has the synthesis of tannin and other materials anti-again
Answer and be consumed.
The analysis of citric acid content in grape wine.In the case where determining the optimum chromatogram condition of citric acid, by different fermentations period
Claret sample difference sample introduction, obtain the high-efficient liquid phase chromatogram of grape wine sample, such as Fig. 9.
It can show that the retention time of citric acid is under chromatographic condition according to the chromatogram of Figure 10 citric acid standard items
4.9min left and right.Due to the interference of other materials in grape wine, the retention time of citric acid can be with citric acid standard in grape wine
The retention time of solution disagrees, and is not to fit like a glove.Therefore, it is very necessary to carry out mark-on experiment, i.e., in grape
Add certain density citric acid standard liquid in wine sample, obtained chromatogram and the corresponding grape wine for not having an adding citric acid titer
Sample carries out paired observation.
The data of citric acid concentration in grape wine.The peak of citric acid in grape wine sample chromatogram is integrated, lemon is obtained
The peak area of lemon acid.Then according to citric acid standard curve：Y=1437.5x+45.365, (wherein y is the peak face at citric acid peak
Product, x is the concentration g/L of citric acid in grape wine) concentration of citric acid is calculated, it is listed in table 1：
The claret different fermentations period citric acid concentration of table 1
The claret citric acid concentration that table 1 can be seen that the kind measured is less than 0.15g/L in earlier fermentation,
Ferment the later stage, its concentration range is between 0.07-0.3g/L.
The variation tendency of citric acid in Fermentation of Grape Wine
It can be seen from figure 11 that the citric acid content change that claret is generated during the fermentation can be divided into four ranks
Section：
(1) with the progress of alcoholic fermentation, citric acid content is gradually increasing, and the speed risen is more slow, until hair
Ferment reaches maximum 0.18702g/L on the 13rd day.
(2) enter after malic-lactic acid fermentation, citric acid content has declined since fermentation reaches peak on the 13rd day, to fermentation
Reach minimum value within 18th day.
(3) when citric acid content is seldom, 0.23091g/L is increased to suddenly within the 19th day in fermentation.
(4) since fermentation the 19th day, citric acid content is gradually decreased, and in slow downward trend and is tended towards stability.
The alcoholic fermentation startup of table 2 and closure component comparative analysis data
As can be seen from the above table, the inoculating lactic acid bacterium after alcoholic fermentation 24 hours is started, now to lactobacillus-fermented temperature
It must carry out being easy to control under conditions of 18-20 DEG C, now improve malo-lactic fermentation speed.Protect during fermentation
The interaction between microorganism and balance are held, it is quick to suppress that rotten microbiologic population is caused.For malo-lactic fermentation
Gain time, so as to save the pollution of cost and reduction to the Nature caused by warming temperature；Chemical analysis is reduced to survey
Fixed number of times and cost；Making the management in wine cellar becomes more Energy and comfort；Malo-lactic fermentation can be terminated earlier, from
And the trial test that can carry out new wine earlier brings help to sale.
(5) alcoholic fermentation terminates inoculation：Alcoholic fermentation terminates inoculation malo-lactic fermentation and starts slow, fermentation period
It is long, easily cause activity and the breeding of the wild-type strain of denaturalization phenomenon, cause volatile acid to increase, the phenomenon that color and luster falls sharply.
(6) by implementing this project, key processing equipment and detection device and French Oak bucket is purchased, has been drawn in oak
Malo-lactic fermentation and ageing stability indicator are carried out in bucket.
Malo-lactic fermentation and the requirement of ageing ageing standard in the oak barrel of table 3
The technical indicator of table 6
| Project | Index before curing | Index after curing |
| Alcoholic strength (20 DEG C) %VOL | 11-12 | 12-13 |
| Volatile acid (with acetometer) g/l | ≤1.1 | ≤0.7 |
| Sugar-free extract g/l | ≥16 | ≥18 |
| Free sulur dioxide mg/l | ≤50 | ≤30 |
| Total sulfur dioxide mg/l | ≤150 | ≤100 |
| pH | ≤3.8 | ≤3.6 |
| Colourity (0D values) | ≤6 | ≥10 |
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (4)
1. a kind of malo-lactic fermentation bacterial strain, it is characterised in that the bacterial strain is Lab2-1 bacterial strains.
2. a kind of cultural method of malo-lactic fermentation bacterial strain as claimed in claim 1, it is characterised in that including as follows
Step：
In a clean container, 100 liters of distilled water and 100 liters of grape juices are added, adjustment temperature adds accelerator to 20 DEG C
Stir, be then slowly added to Lab2-1 bacterial strains, it is stirring while adding, it is allowed to uniform, is spread cultivation at a temperature of 18-22 24 small
When, when detecting that the liquid bacterium colony that spreads cultivation reaches 107-108cfu/ml, obtaining can 100 tons of extra dry red wine Fructus Vitis viniferae wine base malo-lactic fermentation of pilot scale
With the liquid that spreads cultivation.
3. a kind of application of malo-lactic fermentation bacterial strain as claimed in claim 1, it is characterised in that for grape wine apple
Tartaric acid-lactic fermentation.
4. a kind of application of malo-lactic fermentation bacterial strain as claimed in claim 3, it is characterised in that comprise the following steps：
Lab2-1 bacterial strains and saccharomycete are linked into the pulp of fermentation to be leached simultaneously, starts two fermentation process, initially opens
18-20 DEG C of dynamic temperature, after alcoholic fermentation starts 24 hours, addition lactic acid bacteria is spread cultivation liquid, and fermentation temperature is kept for 18-20 DEG C, 7 days
Detect that 24-26 DEG C of heating terminates to alcoholic fermentation during malic acid≤0.8g/L afterwards, be cooled to 18-20 DEG C, continue at this temperature
Malo-lactic fermentation is carried out, malic acid is separated after decomposing fully and completely with pericarp.
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| CN105112318A (en) * | 2015-07-02 | 2015-12-02 | 西北农林科技大学 | Malic acid-lactic acid bacteria and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011582A1 (en) * | 1994-10-12 | 1996-04-25 | Warner-Lambert Company | Fruit juice center-filled chewing gum |
| WO2001060183A2 (en) * | 2000-02-17 | 2001-08-23 | Welch Foods, Inc. | Calcium-fortified, grape-based products and methods for making them |
| CN102168027A (en) * | 2010-11-08 | 2011-08-31 | 福建省农业科学院农业工程技术研究所 | New strain J4 for biofermentation of fruit wine and application thereof |
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| CN102925384A (en) * | 2012-10-11 | 2013-02-13 | 福建省农业科学院农业工程技术研究所 | High-activity strain capable of realizing malolactic conversion |
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