CN101870955A - Screening method of strain capable of simultaneously degrading malic acid and citric acid strain in fruit wine and application - Google Patents
Screening method of strain capable of simultaneously degrading malic acid and citric acid strain in fruit wine and application Download PDFInfo
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Abstract
The invention relates to a screening method of a strain capable of simultaneously degrading malic acid and citric acid strain in a fruit wine and an application, and is characterized in that the method comprises the following steps: collecting 2g of vineyard soil, and quantitatively adding the soil into a triangular flask containing 100mL of physiological saline solution; adding 8-12 sterile glass beads, and fully oscillating the flask and then standing; taking supernate and then respectively adding into a fluid enrichment medium which takes the malic acid with the concentration of 2g/L as the only carbon source for enrichment culture under the culture conditions of 28 DEG C and 150r/min; carrying out table shake culture and taking 24h as one circle; according to the above operations, after each circle is finished, transferring the cultured strain liquid into a fresh enrichment medium successively, wherein the concentration of the malic acid gradually increases from 2g/L, 4g/L, 6g/L and 8g/L to 10G/L at the same time; and selecting out the bacterial strain having better deacidification effect on the malic acid after preliminary screening and secondary screening. The strain in the invention has higher deacidification rate, can resist SO2 with higher concentration, has proper fermentation temperature, has degradation ratio over 80%, and can resist SO2 with the concentration of 450mg/L, the most proper culture temperature is 30 DEG C, and the fermentation temperature is moderate.
Description
Technical field
The present invention relates to the screening method and the application of oxysuccinic acid and citric acid bacterial strain in a kind of fruit wine of degrading simultaneously, is a kind of microorganism that utilizes---land Sheng Yisa yeast reduces the method for oxysuccinic acid and citric acid content in the fruit wine, belongs to food processing field.
Background technology
The quality of fruit wine depends primarily on fruit quality, in the bad time of weather condition (low temperature, high humidity) or owing to gather and often cause the quality of fruit raw material not demonstrate fully too early, causes that fruit maturity is not enough, acid content is high.The higher acid of content mainly is oxysuccinic acid and citric acid in the fruit.Oxysuccinic acid, the formal name used at school hydroxy-butanedioic acid mainly contains three kinds of existence forms, i.e. D-oxysuccinic acid, DL-oxysuccinic acid, L MALIC ACID, the oxysuccinic acid that nature exists all is a L MALIC ACID.L MALIC ACID mainly is present in the juice of jejune hawthorn, apple and grape fruit.Citric acid, formal name used at school 2-hydroxy propane-1,2, the 3-tricarboxylic acid, natural lemon acid is very wide in distributed in nature, mainly is present in the fruits such as plant such as lemon, oranges and tangerines, pineapple, hawthorn.Citric acid content changes to some extent along with the growing state of different Cultivars and plant.
The existence of free acid not only makes fruit wine have tart flavour, and can prevent that fruit wine from being polluted and taking place chemical transformation by harmful microorganism, keeps the stable of fruit wine quality.But organic acid is not The more the better, and the acidity of appropriateness is brought pure and fresh, tasty and refreshing and joyful sensation to the people, and the too high meeting of acidity makes mountain wine that sour and astringent sense, thick and stiff, the quality decline of vinosity be arranged.So in the fruit wine production process, tend to face the problem of falling acid.
The common sour microorganism that falls mainly comprises schizosaccharomyces pombe (Schisosaccharomyces pombe) and oxysuccinic acid-milk-acid bacteria.Schizosaccharomyces pombe decomposes oxysuccinic acid and generates ethanol and CO under the condition of being sick of
2This yeast has stronger anti-SO
2Performance, but breeding is slower with fermenting speed, the fermentation optimum temperuture is higher, and the fruit wine quality of using the schizosaccharomyces pombe pure-blood ferment to obtain has a strand musty, vinosity is not good.The malo-lactic fermentation bacterium is a metabolism of utilizing milk-acid bacteria, and the oxysuccinic acid that tart flavour is sharp-pointed changes into the soft lactic acid of mouthfeel, thereby reduces the acidity of wine.This method can improve the stability of fruit wine to microorganism, changes the content and the ratio of minor component in the wine, changes the concentration of aroma-producing substance, helps improving the local flavor of wine.But this method also is faced with many difficulties in the production practical application, for example operates waywardly, and milk-acid bacteria easily causes the multiple diseases of fruit wine.The land Sheng Yisa yeast that the present invention utilizes screening to obtain reduces the acidity of oxysuccinic acid and citric acid, has not yet to see report.
Summary of the invention
The object of the invention is to provide the screening method of oxysuccinic acid and citric acid bacterial strain in a kind of fruit wine of degrading simultaneously; By being that sole carbon source is done substratum and screened from the soil in vineyard and obtain can degrade microorganism---the land Sheng Yisa yeast of oxysuccinic acid and citric acid of a strain with the oxysuccinic acid.
Another object of the present invention is to provide a kind of bacterial strain---the application of land Sheng Yisa yeast oxysuccinic acid and citric acid in degraded fruit wine.
Technical scheme of the present invention is achieved in that oxysuccinic acid and citric acid bacterial strain in the fruit wine of degrading simultaneously, it is characterized in that: be that sole carbon source is done substratum screen acid-reducing bacterium from the soil in vineyard with the oxysuccinic acid, concrete screening method is as follows:
Gathering vineyard soil 2g is added in the triangular flask that fills the 100mL stroke-physiological saline solution, and add 8～12 sterile glass beads, fully leave standstill after the vibration, getting that supernatant liquor joins respectively with concentration again is that the oxysuccinic acid of 2g/L is to carry out enrichment culture in the liquid enrichment medium of sole carbon source, culture condition is 28 ℃, 150r/min, the shaking table shaking culture, with 24h is one-period, behind each end cycle, according to above operation, in the fresh enrichment medium of successively the bacterium liquid of being cultivated being transferred, malic acid concentration is by 2g/L simultaneously, 4g/L, 6g/L, 8g/L is incremented to 10g/L gradually; Select the bacterial strain preferably of growing, after the acid-reducing bacterium that screening is obtained carries out the separating for several times purifying with plate streak, picking list bacterium colony be linked on the YPD slant medium in 28 ℃ cultivate 24h after 4 ℃ of preservations.
The experimental example of land Sheng Yisa yeast application of oxysuccinic acid and citric acid in degraded fruit wine is as follows:
The fruit raw material is carried out the polygalacturonase of broken back adding 0.05% and the SO of 100mg/L
2Soak juice and carry out the disjoining pressure pressed juice with juice slag separating machine after 12 hours, access is fallen the effective bacterium of acid and is fallen acid-fermentation under 30 ℃ of conditions, the laggard smart fermentation of serving a round of liquor to the guests of fermentation 48h, zymamsis is to insert S. cervisiae under 20～25 ℃ of conditions, zymamsis is filtered, is clarified after finishing, and enters the ageing operation at last.
Acidity detection method: adopt NaOH volumetry (with oxysuccinic acid or citrometer).
Strain identification: by the efficient acid-reducing bacterium that filters out is carried out morphology, physio-biochemical characteristics and molecular biology identification.Final determine that screening obtains the land Sheng Yisa yeast (Issatchenkia terricola) that bacterial strain (called after S8) that a strain has efficient degradation oxysuccinic acid and citric acid ability belongs in the Issatchenkia (Issatchenkia) and plants.
The growth characteristics research of bacterial strain: with the incubation time is X-coordinate, OD
600Value is determined the thalli growth curve for ordinate zou.The initial pH value of culture temperature, substratum and the numerical value of shaking speed design different levels are tested, finally determined the suitableeest culture temperature, the suitableeest initial pH value of substratum and the suitableeest shaking speed of bacterial strain.
The tolerance studies of bacterial strain: to SO
2The numerical value of concentration, alcoholic strength and pH value design different levels is tested, and has determined that finally bacterial strain is to SO
2The tolerance of concentration, alcoholic strength and pH value.
Compared with prior art, the advantage and the effect that have of the present invention is as follows:
The land Sheng Yisa yeast that obtains of screening has the effect of degrade simultaneously oxysuccinic acid and citric acid among the present invention, and degradation rate reaches more than 80%, and this bacterial strain tolerance SO
2Concentration reaches 450mg/L, and the suitableeest culture temperature is 30 ℃, and leavening temperature is moderate.
Description of drawings
Colonial morphology on Fig. 1 wort agar substratum
The microscopic morphology of Fig. 2 bacterial strain S8 cell
The growth curve of Fig. 3 bacterial strain S8
The optimum temperuture of Fig. 4 bacterial strain S8 is determined
Determining of the optimal pH of Fig. 5 bacterial strain S8
Fig. 6 bacterial strain S8 is determining of the suitableeest shaking speed
Fig. 7 bacterial strain S8 is to SO
2The tolerance of concentration
Fig. 8 bacterial strain S8 is to the tolerance of alcoholic strength
Embodiment:
Embodiment 1:
The land Sheng Yisa yeast (Issatchenkia terricola) that obtains degrade oxysuccinic acid and citric acid are screened in utilization from the soil of vineyard.
The main medium of utilization of the present invention is:
Enrichment medium: (NH
4)
2SO
42g, KH
2PO
42.5g, FeSO
47H
2O 0.1g, yeast extract paste 0.5g, oxysuccinic acid adds as required, is used for 1L distilled water, gets the 100mL obtain solution and is sub-packed in the 250mL triangular flask, 115 ℃ of moist heat sterilization 30min.
Solid separation culture medium: (NH
4)
2SO
42g, KH
2PO
42.5g, FeSO
47H
2O 0.1g, yeast extract paste 0.5g, agar powder 20g is dissolved in the 800mL distilled water, and oxysuccinic acid adds in the 200mL distilled water as required, and 115 ℃ of moist heat sterilization 30min are cooled to 50～60 ℃, with the two mixing, fall dull and stereotyped.
Malt extract medium: by kilned malt system, concrete grammar is with reference to modern food microbiology experimental technique: Liu Hui chief editor, China Light Industry Press, 2006:265.
The YPD substratum: glucose 2%, yeast soak powder 1%, peptone 1%, natural pH, 121 ℃ of moist heat sterilization 20min.
The WL nutritional medium: yeast soaks powder 0.4%, Tryptones 0.5%, glucose 5%, KH
2PO
40.055%, KCl 0.0425%, anhydrous CaCl
20.0125%, FeCl
30.00025%, MgSO
40.0125%, MnSO
40.00025%.
1, bacterial screening
(1) enrichment culture: quantitatively be added to the soil that collects in the stroke-physiological saline solution, addend grain granulated glass sphere, fully leave standstill after the vibration, getting supernatant liquor, to join with oxysuccinic acid (malic acid concentration is 2g/L) respectively be in the liquid enrichment medium of sole carbon source, 28 ℃, 150r/min, shaking table shaking culture 24h.24h is an one-period, and behind each end cycle, according to above operation, the bacterium liquid of getting a certain amount of cultivation is successively transferred in the fresh enrichment medium, and switching is cultivated repeatedly continuously.Malic acid concentration is incremented to 10g/L gradually by 2g/L, 4g/L, 6g/L, 8g/L simultaneously.
(2) primary dcreening operation: after enrichment culture, get a certain amount of enrichment bacterium liquid, be diluted to 10 with physiological saline
-5, select 10
-3, 10
-4, 10
-5Extent of dilution is drawn the 0.5mL diluent respectively and is coated and contain on the Agar Plating that malic acid concentration is 10g/L 28 ℃ of constant temperature culture 24h.Typical single bacterium colony point of gained is planted on isolation medium, and the same terms is cultivated down.
(3) multiple sieve: the resulting single bacterium colony of primary dcreening operation is carried out the separating for several times purifying with plate streak, and picking list bacterium colony inserts the YPD slant culture and cultivates 4 ℃ of preservations behind the 24h based on 28 ℃.
2, sour characteristic research falls
With the WL substratum is basic medium, replaces glucose to do sole carbon source with 5g/L oxysuccinic acid or citric acid and cultivates, measure and fall sour rate, obtains a strain and falls the highest bacterial strain of sour rate, and its degraded oxysuccinic acid and citric acid rate are all up to more than 80%.
3, strain identification
(1) colonial morphology is observed: what will obtain falls the highest inoculation of sour rate on malt extract medium, cultivates 1～2d down at 30 ℃, the colony characteristics that forms is observed, as shown in Figure 1.
(2) cellular form is observed: adopt the cellular form of observation by light microscope bacterial strain, as shown in Figure 2.
(3) biochemical test is identified: bacterial strain is done various biochemical identification tests such as starch hydrolysis experiment, gelatin liquification test, M R test, nitrate reduction test, observe the utilize situation of bacterial strain to various carbon sources and nitrogenous source.
(4) molecular biology identification: the ITS conserved sequence of measuring this bacterial strain is formed, and compares with the correlated series among the Genbank, to determine the concrete kind of this bacterium.The result draws the ITS gene order of this bacterial strain and the homology of land Sheng Yisa zymic ITS gene order reaches 100%, therefore identify that this bacterial strain is land Sheng Yisa yeast (Issatchenkia terricola) kind in the Issatchenkia (Issatchenkia), called after S8.
4, the growth characteristics research of bacterial strain S8
(1) mensuration of growth curve
The activatory inoculation in the YPD liquid nutrient medium of sterilization, in 28 ℃, after shaking table is cultivated 12h under the 150r/min, is transferred in the YPD liquid nutrient medium with 2% inoculum size, measures OD
600Value, then in 28 ℃, shaking table is cultivated under the 150r/min, measures OD every 2h
600Value (doing blank test with the YPD nutrient solution that does not connect bacterium) is an X-coordinate with the incubation time, OD
600Value is determined the thalli growth curve, as shown in Figure 3 for ordinate zou.
(2) optimum growth temperature determines
With the activatory inoculation to the sterilization the YPD liquid nutrient medium in, get the bacteria suspension that is in logarithmic phase, be transferred in the 50mLYPD liquid nutrient medium with 2% inoculum size, respectively at 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, the 150r/min shaking table is cultivated, and gets bacterium liquid behind the cultivation 24h and measures OD
600(doing blank) with the YPD nutrient solution that does not connect bacterium, maximum OD
600Culture temperature under the value is optimum growth temperature, as shown in Figure 4.
(3) the initial pH value of the suitableeest growth determines
The pH value of regulating the YPD liquid nutrient medium with NaOH or HCl is respectively 3.5,4.0,4.5,5.0,5.5,6.0, get the bacteria suspension that is in logarithmic phase, inoculum size with 2% is inoculated in the YPD liquid nutrient medium of the above-mentioned different pH values of 50mL, place optimum growth temperature, the 150r/min shaking table is cultivated, and gets bacterium liquid behind the cultivation 24h and measures OD
600(doing blank) with the YPD nutrient solution that does not connect bacterium, maximum OD
600PH under the value is the suitableeest growth pH.As shown in Figure 5, the suitableeest culture temperature is 30 ℃.
(4) determining of the suitableeest shaking speed
Get the bacteria suspension that is in logarithmic phase, be transferred in the 50mLYPD liquid nutrient medium with 2% inoculum size, under optimum growth temperature, the suitableeest growth pH, shaking culture under 110r/min, 130r/min, 150r/min, 170r/min, 190r/min, 210r/min is got bacterium liquid behind the cultivation 24h and is measured OD respectively
600(doing blank) with the YPD nutrient solution that does not connect bacterium, maximum OD
600Shaking speed under the value can obtain the suitableeest shaking speed.As shown in Figure 6, the suitableeest shaking speed is 150r/min.
5, the tolerance test of bacterial strain S8
(1) bacterial strain is to SO
2Tolerance
Get the bacteria suspension that is in logarithmic phase, be inoculated in SO respectively with identical inoculum size
2Addition is 350mg/L, 400mg/L, and 450mg/L, in the 50mL YPD liquid nutrient medium of 500mg/L, 550mg/L, 600mg/L, 30 ℃, shaking culture 24h under the 150r/min gets bacterium liquid and measures OD
600(doing blank with the YPD nutrient solution that does not connect bacterium) determines that bacterial strain is to SO
2Tolerance.As shown in Figure 7, the maximum anti-SO of bacterial strain
2Concentration is 450mg/L.
(2) bacterial strain is to the tolerance of alcoholic strength
Get the bacteria suspension that is in logarithmic phase, be respectively in the 50mL YPD liquid nutrient medium of 2%, 4%, 5%, 6%, 8%, 10% (v/v) with the identical inoculum size dehydrated alcohol content of transferring respectively, 30 ℃, shaking culture 24h under the 150r/min gets bacterium liquid and measures OD
600(doing blank with the YPD nutrient solution that does not connect bacterium) determines the tolerance of bacterial strain to alcohol, and as shown in Figure 8, the maximum ethanol-tolerant degree of bacterial strain is 5%.
Claims (4)
1. the screening method of oxysuccinic acid and citric acid bacterial strain in the fruit wine of degrading simultaneously, it is characterized in that: effective bacterium of oxysuccinic acid and citric acid is land Sheng Yisa yeast (Issatchenkia terricola) kind of Issatchenkia (Issatchenkia) in the degraded fruit wine, concrete screening method is as follows: gather vineyard soil 2g and quantitatively be added in the triangular flask that fills the 100mL stroke-physiological saline solution, and add 8～12 sterile glass beads, fully leave standstill after the vibration, getting that supernatant liquor joins respectively with concentration again is that the oxysuccinic acid of 2g/L is to carry out enrichment culture in the liquid enrichment medium of sole carbon source, culture condition is 28 ℃, 150r/min, the shaking table shaking culture, with 24h is one-period, behind each end cycle, according to above operation, successively the bacterium liquid of being cultivated is transferred in the fresh enrichment medium, malic acid concentration is by 2g/L simultaneously, 4g/L, 6g/L, 8g/L is incremented to 10g/L gradually; By selecting behind primary dcreening operation and the multiple sieve oxysuccinic acid had the bacterial strain that better falls sour effect again;
The acid-reducing bacterium that screening is obtained---after land Sheng Yisa yeast carries out the separating for several times purifying with plate streak, picking list bacterium colony be linked on the YPD slant medium in 28 ℃ cultivate 24h after 4 ℃ of preservations.
2. according to the screening method of oxysuccinic acid and citric acid bacterial strain in a kind of fruit wine of degrading simultaneously shown in the claim 1, it is characterized in that described primary dcreening operation process is as follows: after enrichment culture, get a certain amount of enrichment bacterium liquid, be diluted to 10 with physiological saline
-5, select 10
-3, 10
-4, 10
-5Extent of dilution is drawn the 0.5mL diluent respectively and is coated and contain on the Agar Plating that tartaric acid concentration is 10g/L 28 ℃ of constant temperature culture 72h.Typical single bacterium colony point of gained is planted on isolation medium, and the same terms is cultivated down.
3. according to the screening method of oxysuccinic acid and citric acid bacterial strain in a kind of fruit wine of degrading simultaneously shown in the claim 1, it is characterized in that the described process of sieving again is as follows: the resulting single bacterium colony of primary dcreening operation is carried out the separating for several times purifying with plate streak, and picking list bacterium colony inserts the YEPD slant culture and cultivates 4 ℃ of preservations behind the 72h based on 28 ℃.
4. the application of bacterial strain---land Sheng Yisa yeast oxysuccinic acid and citric acid in degraded fruit wine.
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| CN103205369A (en) * | 2013-03-26 | 2013-07-17 | 江苏省农业科学院 | Novel brewing yeast strain with L-apple acid degrading property and application of novel brewing yeast strain |
| CN105543117A (en) * | 2016-01-31 | 2016-05-04 | 安徽楚井坊酒业有限公司 | Soybean dreg hydrolysate high-density yeast technology |
| CN110885731A (en) * | 2019-11-15 | 2020-03-17 | 山东省林业科学研究院 | Method for realizing deacidification of raspberry wine |
| CN111647481A (en) * | 2020-05-09 | 2020-09-11 | 阜阳师范大学 | Sweet strawberry wine production process capable of efficiently reducing sour taste of strawberry wine |
| CN112877226A (en) * | 2021-03-31 | 2021-06-01 | 中国农业大学 | Alcohol-resistant Issatchenkia terricola produced by ultraviolet mutation breeding and application thereof in wine brewing |
| CN113025455A (en) * | 2021-03-12 | 2021-06-25 | 四川大学 | Two-stage fermentation method of cider |
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2010
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205369A (en) * | 2013-03-26 | 2013-07-17 | 江苏省农业科学院 | Novel brewing yeast strain with L-apple acid degrading property and application of novel brewing yeast strain |
| CN103205369B (en) * | 2013-03-26 | 2014-07-16 | 江苏省农业科学院 | Novel brewing yeast strain with L-apple acid degrading property and application of novel brewing yeast strain |
| CN105543117A (en) * | 2016-01-31 | 2016-05-04 | 安徽楚井坊酒业有限公司 | Soybean dreg hydrolysate high-density yeast technology |
| CN110885731A (en) * | 2019-11-15 | 2020-03-17 | 山东省林业科学研究院 | Method for realizing deacidification of raspberry wine |
| CN111647481A (en) * | 2020-05-09 | 2020-09-11 | 阜阳师范大学 | Sweet strawberry wine production process capable of efficiently reducing sour taste of strawberry wine |
| CN113025455A (en) * | 2021-03-12 | 2021-06-25 | 四川大学 | Two-stage fermentation method of cider |
| CN113025455B (en) * | 2021-03-12 | 2023-08-08 | 四川大学 | Two-stage fermentation method of cider |
| CN112877226A (en) * | 2021-03-31 | 2021-06-01 | 中国农业大学 | Alcohol-resistant Issatchenkia terricola produced by ultraviolet mutation breeding and application thereof in wine brewing |
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Application publication date: 20101027 |