CN112111416B - Issatchenkia orientalis strain for whole-process green production of fruit wine and application thereof - Google Patents
Issatchenkia orientalis strain for whole-process green production of fruit wine and application thereof Download PDFInfo
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- CN112111416B CN112111416B CN202010525940.8A CN202010525940A CN112111416B CN 112111416 B CN112111416 B CN 112111416B CN 202010525940 A CN202010525940 A CN 202010525940A CN 112111416 B CN112111416 B CN 112111416B
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- fruit
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- A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
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Abstract
The invention relates to Issatchenkia orientalis (Issatchenkia orientalis), which is preserved in China general microbiological culture Collection center (CGMCC) 24.04.2020, with the preservation number of CGMCC No.19724 and is named as Issatchenkia orientalis WTB20042304. The strain can effectively utilize citric acid and tartaric acid, has the degradation capability on citric acid, lactic acid, formic acid and succinic acid in fruit wine, and has particularly remarkable degradation capability on citric acid; can effectively improve the content of active substances such as total phenols, total flavonoids and anthocyanidin, and improve the antioxidation level of the fruit wine. In addition, the strain has a certain inhibiting effect on botrytis cinerea. Therefore, the strain can be applied to storage and preservation of picked fruits or acid reduction and fermentation of fruit wine, and is an optimal strain for the whole process of green production of fruit wine.
Description
Technical Field
The invention relates to a functional yeast strain, in particular to Issatchenkia orientalis for green production of fruit wine in the whole process and application thereof.
Background
The fruit wine is wine which is prepared by fermenting saccharomycetes into alcohol by utilizing sugar of fruits, contains specific flavor and nutrient components of the fruits, and is brewed by the earliest human society. The fruit of the fruit wine can be made from the fruit of the Ficus eight-flower phyla, and the fruit is more ideal from kiwi fruit, waxberry, orange, grape, blueberry, red date, cherry, lychee, honey peach, persimmon, strawberry and the like. In fruit wine, most famous wines are popular in recent years because blueberries contain abundant anthocyanin which can resist free radicals and have the function of delaying senescence.
The brewing process of the fruit wine comprises the following steps: picking fresh fruit → sorting → crushing, removing stem → pulp → separating and extracting juice → clarifying → clear juice → fermenting → pouring barrel → storing wine → filtering → cold processing → mixing → filtering → finished product. The variety of the raw materials is one of the important factors for ensuring the quality of the fruit wine product, and the raw materials directly influence the sensory characteristics of the brewed fruit wine. Wherein, the fruits selected from the picked fresh fruits require the ripeness degree of the fruits to reach full ripeness, the sugar content of the fruit juice is high, and the fruits do not have mildew, rot, deterioration and plant diseases and insect pests. The brewed finished fruit wine has mellow, harmonious and palatable taste if the acid content is proper. Conversely, the palatability is poor, the sourness is too heavy, the wine juice is cloudy after being faded out, and the desire of consumers to buy is reduced. The acid in the fruit wine is partially brought by raw materials, such as tartaric acid in grapes, malic acid in apples, citric acid in waxberries and the like; there are also yeast metabolites produced during fermentation, such as acetic acid, butyric acid, lactic acid, succinic acid, etc. L-malic and tartaric acids are among the most prominent organic acids in wine and play a crucial role in wine brewing, including the organoleptic quality and physical, biochemical and microbiological stability of wine. One of the quality indexes of fruit wine products is to control the total amount of organic acids in the fruit wine.
In conclusion, in the brewing process of the fruit wine, the preservation of fresh fruits and the control of the total amount of organic acids in finished wine are two important links influencing the cost and the quality of the fruit wine.
According to analysis, the quality deterioration of fresh fruits and vegetables is influenced by a plurality of factors, but diseases are the most main reasons. Among them, rotting and deterioration caused by fungal diseases are the most serious factors in postharvest loss of fruits, main diseases of different fruits are different, grapes mainly cause powdery mildew due to Botrytis cinerea (Botrytis cinerea), and apples mainly cause Penicillium expansum (Penicillium expansum). At present, the common and main methods for preventing fresh fruits from being preserved are physical prevention and chemical agent prevention, the use of chemical pesticides not only causes pathogenic bacteria to generate drug resistance to reduce the sterilization effect, but also causes chemical agent residues to influence the health of people due to unclean treatment. Physical control methods (such as low-temperature storage) require special equipment, often consume energy, and are not favorable for retaining nutrient components in fresh fruits. In the prior art, acid reducing agent (edible alkali), anion exchange resin column acid reduction, low-temperature freezing acid reduction and the like are usually added into fruit wine, the former acid reducing mode influences the flavor and taste of the fruit wine due to the introduction of alkali, the latter acid reducing mode only influences the control of organic acid in the production process, the cost is high, the efficiency is low, and the control of the organic acid in the wine cannot be realized after filling is finished.
Compared with physical and chemical deacidification and biological deacidification, the biological deacidification method can reduce the acidity of the fruit wine, more importantly can increase the stability of the fruit wine and improve the quality of the wine, has small side effect, and is mostly developed by malic acid-lactic acid fermentation (MLF) which is fermented by lactic acid bacteria at present. Lactic acid bacteria produce malic acid-lactase, L-malic acid is changed into L-lactic acid and carbon dioxide under the catalysis of the malic acid, and compared with malic acid, lactic acid is softer, so that the acid reduction effect is achieved. However, lactic acid is also an organic acid, so that the method cannot effectively reduce the organic acid in the fruit wine and improve the mouthfeel of the fruit wine.
Disclosure of Invention
Technical problem to be solved
In order to solve the problems in the prior art, the invention provides Issatchenkia orientalis for green production of fruit wine in the whole process and application thereof. The strain not only has the biocontrol effect, but also has the inhibition effect on the postharvest pathogenic fungi of the fruits, can replace chemical bactericides to prevent and control postharvest diseases of the fruits, and keeps the fruits fresh to prevent the fruits from rotting and deteriorating; meanwhile, the strain has an excellent function of reducing organic acids such as citric acid, lactic acid, formic acid, acetic acid and the like in fruit wine, and can be used for fermenting and reducing the acid of the fruit wine. Especially when the fruit wine is blueberry fruit wine, the strain can also obviously improve the anthocyanin content in the blueberry fruit wine and almost completely degrade citric acid in the blueberry fruit wine.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
the applicant selects and purifies microorganisms in naturally fermented blueberry fruit wine by using a WL nutrient agar culture medium to obtain a strain which can effectively utilize citric acid and tartaric acid and has insensitive acidity reducing capability to alcohol concentration, confirms that the strain is Issatchenkia orientalis (I.orientalis), classifies and names the strain as I.orientalis WTB20042304, and deposits the strain in China general microbiological culture Collection center at 24.04.2020 with the preservation number of CGMCC No.19724.
Based on the screened yeast strains, the invention also provides the following technical scheme:
the invention also relates to application of the Issatchenkia orientalis strain I.orientalis WTB20042304 in storage and preservation of picked fruits or reduction of acidity of fruit wine. The method specifically comprises the following steps:
the first scheme comprises the following steps: a fresh fruit preservation method is characterized in that Issatchenkia orientalis I.orientalis WTB20042304 is prepared into bacterial suspension, and the bacterial suspension is used for spraying or dip-coating fresh fruits.
The specific method comprises the following steps: activating Issatchenkia orientalis I.orientalis WTB20042304, fermenting and culturing with YPD liquid culture medium, centrifuging to obtain thallus, cleaning thallus with sterile water to remove culture medium, and making into 1 × 10 6 CFU/mL～1×10 8 CFU/mL of bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a preservation box, sealing, and storing at normal temperature.
Preferably, the fresh fruit is grape or blueberry.
Preferably, the activating step is: taking a single colony on the solid culture medium, culturing in YPD liquid culture medium at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.
Scheme II: a method for reducing acid and fermenting fruit wine comprises the steps of inoculating Issatchenkia orientalis I.orientalis WTB20042304 before inoculating Saccharomyces cerevisiae into fruit juice, and then performing fermentation treatment.
Preferably, the fruit wine is blueberry fruit wine, and the fruit juice is blueberry fruit juice.
Preferably, the method is: crushing fresh blueberries, adjusting the components to obtain blueberry juice, adding Issatchenkia orientalis I.orientalis WTB20042304 into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing sugar degree adjustment, primary fermentation, filtration and post-fermentation to obtain the blueberry juice. The delayed addition of Saccharomyces cerevisiae can make the I.orientalis WTB20042304 added earlier survive in large amount and degrade the organic acid, such as citric acid, malic acid, etc. in the fruit juice. The sugar is added later, so that the problem that the acid reducing effect is lost because the Issatchenkia orientalis WTB20042304 preferentially utilizes the sugar is avoided.
Preferably, the bacterial adding amount of the Issatchenkia orientalis I.orientalis WTB20042304 is1 × 10 6 CFU/mL。
Preferably, the bacterial adding amount of the saccharomyces cerevisiae is1 × 10 6 CFU/mL, the sugar adding amount for regulating the sugar degree is 120g/L, the fermentation is carried out at the room temperature of 22-26 ℃, the stirring is carried out for 2 times every day, and after the fermentation is finished, the filtration is carried out, and the filtrate is the blueberry wine.
The Issatchenkia orientalis I.orientalis WTB20042304 comprises an activation treatment before use: taking single colony on solid culture medium, culturing in YPD liquid culture medium at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.
(III) advantageous effects
The invention has the beneficial effects that:
(1) According to the Issatchenkia orientalis I.orientalis WTB20042304 screened by the method, after the Issatchenkia orientalis I.orientalis WTB 5363 is cultured for 96 hours on a citric acid (citric acid is a unique carbon source) screening culture medium, the content of citric acid in the culture medium is reduced by 92.39%, which indicates that the Issatchenkia orientalis I.orientalis WTB 5363 can utilize citric acid; after the culture medium is cultured for 96 hours on a tartaric acid (tartaric acid is the only carbon source) screening culture medium, the content of tartaric acid in the culture medium is reduced by 38.84%, which indicates that the tartaric acid can be utilized. The strain is added into blueberry juice for fermentation, and no citric acid is detected at the end point of fermentation, which indicates that the strain has strong citric acid degradation capability. In addition, if the blueberry juice fermentation broth only added with saccharomyces cerevisiae is taken as a reference, and the blueberry juice fermentation broth added with saccharomyces cerevisiae and issatchenkia orientalis i.orientalis WTB20042304 is simultaneously added, the content of lactic acid is reduced by 36.17%, the content of formic acid is reduced by 24.39%, the content of acetic acid is reduced by 9.20%, and the content of succinic acid is reduced by 13.43% at the end point of fermentation. Therefore, the Issatchenkia orientalis WTB20042304 screened by the invention has the capacity of degrading citric acid, lactic acid, formic acid, acetic acid and succinic acid in fruit wine, wherein the capacity of degrading citric acid is particularly remarkable.
The Issatchenkia orientalis WTB20042304 is used as the acid-reducing yeast, can degrade most of organic acids in fruit wine, and more importantly can increase the stability of the fruit wine (can also stabilize the acidity after being bottled) and improve the quality of the wine.
(2) The screened Issatchenkia orientalis WTB20042304 is added into blueberry juice fermentation liquor, if the blueberry juice fermentation liquor only added with the saccharomyces cerevisiae is used as a reference, and the blueberry juice fermentation liquor added with the saccharomyces cerevisiae and the Issatchenkia orientalis I.orintalis WTB20042304 is added at the end of fermentation, the total phenols in the blueberry fruit wine are increased by 7.08%, the total flavones are increased by 2.93%, and the anthocyanin content is increased by 7.91%. Phenols, flavonoids and anthocyanins are important active substances in blueberry fruit wine, and have activities of resisting oxidation, scavenging free radicals, resisting variation and preventing canceration. DPPH free radical scavenging capacity is improved by 5.82%, FRAP total antioxidant capacity is improved by 9.58%, ABTS free radical scavenging capacity is improved by 5.53%, total reducing power is improved by 4.09%, and the improvement of fruit wine antioxidant capacity effectively prevents aging and pathological changes of organisms caused by aging. Therefore, the Issatchenkia orientalis I.orientalis WTB20042304 screened by the method can greatly improve the quality of blueberry fruit wine when being applied to brewing of the blueberry fruit wine.
(4) In the process of preparing the blueberry wine by fermentation, the sugar content at the fermentation end point proves that the fermentation process of the saccharomyces cerevisiae to the blueberry wine is not influenced after the issatchenkia orientalis I.orientalis WTB20042304 is added. Therefore, the blueberry juice can be normally fermented after the Issatchenkia orientalis I.orientalis WTB20042304 is added, the alcoholic strength is 11.73% at the end point of the fermentation, and SSC is 6.81 degrees Bx. When the blueberry juice fermentation broth added with only saccharomyces cerevisiae is used as a control (pH =3.61 at the end of fermentation), and the pH at the end of fermentation is 3.92 after the Issatchenkia orientalis I.orientalis WTB20042304 and saccharomyces cerevisiae are added, the Issatchenkia orientalis I.orientalis WTB20042304 of the invention is further proved to have obvious acid reduction effect.
(5) Taking the Issatchenkia orientalis I.orientalis WTB20042304 as a biocontrol bacterium, counting the morbidity of grapes inoculated with botrytis cinerea in advance, and obtaining a result of 42.22%; under the condition of the same concentration of the biocontrol bacteria, the morbidity of the existing biocontrol bacteria (the existing strain with the preservation number of CGMCC No. 14909) is 71.11 percent, thereby showing that the yeast strain has better inhibiting effect on the botrytis cinerea compared with the existing biocontrol bacteria.
Therefore, the Issatchenkia orientalis I.orientalis WTB20042304 can not only prevent the fresh fruits from being rotted and deteriorated after the fresh fruits are picked and ensure the freshness of the fresh fruits, but also play a role in reducing acid when the fruit juice is used for fermentation and brewing wine in the later period, the role in reducing acid is not influenced by the alcohol concentration, the normal fermentation process of the Saccharomyces cerevisiae is not influenced, the acid reducing effect can be realized on most kinds of organic acid in the fruit wine, the mouth feel and the taste phase of the fruit wine (the fruit wine with high acidity is turbid), the Issatchenkia orientalis I.orientalis WTB20042304 is a preferable strain which can be applied to the whole process of the fruit wine preparation process, and the acid reducing effect can still be realized in the fermentation process even if part of the sprayed biocontrol bacterium suspension in the fresh fruit preservation process is remained. In addition, the content of active substances such as phenols, flavonoids, anthocyanin and the like in the fruit wine can be increased, and the antioxidation and total reducing power are improved, so that the quality of the fruit wine is further improved.
Drawings
FIG. 1 is a photograph showing the morphological characteristics of the selected Issatchenkia orientalis I.orientalis WTB20042304 of the present invention.
FIG. 2 is a PCR electrophoresis detection map of total DNA of Issatchenkia orientalis I.orientalis WTB20042304 screened by the present invention.
FIG. 3 is a phylogenetic tree analysis of the selected Issatchenkia orientalis I.orientalis WTB20042304 of the present invention.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
1. Screening and identification of strains
(I) isolation of the Strain
Sampling the naturally fermented blueberry fruit wine at 0d, 3d, 6d, 9d, 12d and 15d respectively, diluting the wine sample liquid in a WL nutrient agar culture medium by 1000 times, 10000 times and 100000 times, paving the sample, culturing at the constant temperature of 26 ℃ for 48 hours to obtain a single bacterial colony, and performing two parallel experiments. And (3) selecting a single colony on a WL nutrient agar plate for separation and purification, culturing for 48h at a constant temperature of 26 ℃, selecting a suspected colony according to morphological characteristics of the yeast for microscopic examination, and carrying out streak separation on the WL plate to obtain a purified strain.
Biological characteristics of Issatchenkia orientalis I.orientalis WTB20042304
(1) Morphological characteristics
Referring to FIGS. 1A and 1B, A represents the colony morphology of WL nutrient agar [ fungal culture medium, 75gWL nutrient agar medium dissolved in 1000mL water, subpackaged, sterilized at 121 ℃ for 20min ] cultured at 26 ℃ for 48h, with a scale of 1cm; b represents the cell morphology observed under a 40-fold microscope (10 μm scale). The colony was observed to be irregular, white, flat, rough in shape; the cells were observed under a microscope to have an oval shape and a diameter of about 8 μm.
(1) Molecular biological identification
(1) Extracting total DNA of the strain by SDS boiling method, amplifying target fragment of 26s rDNA D1/D2 region by taking NL-1 and NL-4 as primers, and performing agarose gel electrophoresis analysis (electrophoresis conditions: 180V, 30min), wherein the result is shown in FIG. 2; and (4) detecting that the size of the target fragment is correct, and performing DNA sequencing on the sample.
(2) Homology alignment and evolutionary tree results
And (3) comparing the sequencing Gene sequence with the corresponding sequence of the known strain in the Gene Bank, finding the corresponding model strain in strainafo, and constructing a phylogenetic evolution tree according to the homology comparison result. As shown in fig. 3. The results show that: the strain and I.orientalis (EU 585754.1) have 100% confidence on the same branch and have close affinity (distance is 0), so that the strain is determined to be the Issatchenkia orientalis (I.orientalis).
2. Acid reducing property of Issatchenkia orientalis I.orientalis WTB20042304
(one) the acid reducing capability of Issatchenkia orientalis I.orientalis WTB20042304 is tested by using a culture medium with citric acid and tartaric acid as unique carbon sources
(1) Experimental Material
(1) The strain is as follows: issatchenkia orientalis I.orientalis WTB20042304, I.orientalis WBD 2 (blueberry wine fermented mash), I.orientalis WBD 3 (grape wine fermented mash), I.orientalis WBD 3 (blueberry wine fermented mash), I.orientalis WBD 4 (blueberry wine fermented mash), I.orientalis WBD 5 (hawthorn wine fermented mash) P.cactophia (preservation strain number CGMCC No. 14909)
(2) Drugs and reagents
TABLE 1 drugs and reagents
(3) Laboratory apparatus
TABLE 2 Main Instrument of the experiment
(4) Culture medium
Citric acid medium: 1g of citric acid, 1g of yeast extract, 2g of peptone and 0.1g of potassium dihydrogen phosphate were dissolved in 100mL of water and sterilized by autoclaving at 121 ℃ for 20min for further use.
Tartaric acid culture medium: 1g of tartaric acid, 1g of yeast extract, 2g of peptone and 0.1g of potassium dihydrogen phosphate were dissolved in 100mL of water and sterilized by autoclaving at 121 ℃ for 20min for further use.
(2) Experimental methods
(1) Respectively picking single strains of cultured Issatchenkia orientalis I.orientalis WTB20042304, I.orientalis WBD 2 (blueberry fruit wine fermented mash), I.orientalis WBD 3 (grape fruit wine fermented mash), I.orientalis WBD 3 (blueberry fruit wine fermented mash), I.orientalis WBD 4 (blueberry fruit wine fermented mash), I.orientalis WBD 5 (hawthorn fruit wine fermented mash) P.cactophia (preservation strain number CGMCC No. 14909), placing the single strains into a test tube filled with 5ml YPD culture medium, carrying out shake flask on a shaker for 16-24h to ensure the purity and the activity, taking out, and removing supernatantThe solution is washed by adding water and then centrifuged for 3 times, and finally 1ml of water is added for constant volume to prepare the bacterial suspension. Diluting the prepared bacterial suspension by 10 times, 100 times and 1000 times in gradient, selecting 1000 times, counting cells with a blood counting plate under a microscope, calculating the bacterial suspension concentration by a formula, and diluting to 2 × 10 6 CFU/mL。
(2) mu.L of the above-mentioned acid-addition medium (citric acid medium and tartaric acid medium) and 100. Mu.L of the bacterial suspension were added to a 96-well plate, and each strain was divided into three groups. And scanning the absorbance under 590nm at 0h, 24h, 48h and 72h respectively, calculating the difference value of the absorbance with 0h, reflecting the growth condition of the strains through the difference value of the absorbance, and further judging whether the strains can grow by using corresponding organic acid.
(3) Single acid reduction effect analysis
3 portions of citric acid medium and tartaric acid medium, 50mL each per bottle of medium, were prepared and sterilized for use. Respectively picking out single strains of the cultured 3 strains of bacteria, dropping the single strains into a test tube filled with 5mLYPD culture medium, shaking the test tube on a shaking table for 16-24h to ensure the purity and the activity of the single strains of bacteria, taking out the test tube, removing supernatant, adding water, centrifuging for 3 times, finally adding 1ml of water to fix the volume, preparing bacterial suspension, and counting blood cells. Adding 3 strains of yeast into corresponding triangular flasks respectively to make the final concentration of yeast be 10 6 And (3) placing the CFU/mL into a constant-temperature shaking table for shaking, sampling at 26 ℃ and 200rpm for 0h, 48h and 96h respectively, taking 2X 4mL each time, and measuring the content of the organic acid at the sampling time point respectively.
Adopting a liquid chromatograph to detect conditions:
a chromatographic column: agilent 5 TC-C18 (250X 4.6 mm), using diammonium hydrogen phosphate with pH of 2.4 as mobile phase, column temperature of 45 deg.C, flow rate of 0.8ml/min, and sample amount of 20 μ L, respectively centrifuging and diluting 20 times samples of culture medium containing citric acid and tartaric acid and organic acid culture medium after adding 3 strains of acid-reducing yeast, fermenting for 48h and 96h, and detecting with high performance liquid chromatography. Determining the retention time of the organic acid through the chromatogram results of the citric acid and tartaric acid standard substances, and comparing the retention time with the chromatographic peak area of each organic acid in the 0h culture medium to detect the acid reduction effect of each strain. Before testing, samples of 0h citric acid and 0h tartaric acid medium were first diluted 20 times with ultrapure water, loaded for testing, and then diluted and tested for 48h and 96h media, respectively.
Establishing a standard curve: weighing a certain amount of organic acid standard substance, putting the organic acid standard substance into a volumetric flask, diluting with ultrapure water to prepare a mother solution with the concentration of 10mg/ml, diluting with a mobile phase to the required concentration with the concentration gradient of 2.5mg/ml,2mg/ml,1mg/ml,0.4mg/ml,0.2mg/ml and 0.1mg/ml respectively, carrying out sample injection in sequence to obtain corresponding peak areas, and obtaining a corresponding linear regression equation by using a least square method.
(4) Results of the experiment
(1) The acid reducing effect by taking citric acid and tartaric acid as unique carbon sources is shown in the table 3-table 5:
TABLE 3 OD in citric acid relative to the start of the experiment 590 Increase value
TABLE 4 relative OD in tartaric acid at the beginning of the experiment 590 Increase in value
TABLE 5 content of 72h citric acid and tartaric acid
As can be seen from table 3, when the i.orientalis WTB20042304 strain was added to the citric acid medium (citric acid was the sole carbon source), the absorbance increased by 1.6 or more at 48h and 72h of culture, as can be seen from table 3. Higher than other strains. As can be seen from table 4, when the i.orientalis WTB20042304 strain was added to the tartaric acid medium (tartaric acid was the sole carbon source), the absorbance increased by 1.1 or more at 48h and 72h of culture. Higher than other strains. From table 5, it can be seen that the degradation rate of i.orientalis WTB20042304 to citric acid reaches 92.39%, and the degradation rate of other strains is less than 90%; the degradation rate of tartaric acid reaches 41.04%, and other strains are lower than 40%.
In conclusion, the strains are most potent in utilizing citric acid and tartaric acid.
Influence of I.orientalis WTB20042304 on organic acids and active substances in blueberry mash
(1) Experimental Material
(1) The strain is as follows: issatchenkia orientalis I.orientalis WTB20042304, I.orientalis WBD 2 (blueberry wine fermented mash), I.orientalis WBD 3 (grape wine fermented mash), I.orientalis WBD 3 (blueberry wine fermented mash), I.orientalis WBD 4 (blueberry wine fermented mash), I.orientalis WBD 5 (hawthorn wine fermented mash) P.cactophia (preservation strain number CGMCC No. 14909)
Blueberry: provided by the sunshine gift winery 2019, 1 month and 6 days, and the variety is Bei Lu
(2) Drugs and reagents
TABLE 6 drugs and reagents
(3) Laboratory apparatus
TABLE 7 main instruments of experiment
(2) Experimental methods
The blueberries are crushed and then distributed into 3 fermentation tanks, and 3.5 kg of the blueberries are placed in each fermentation tank.Respectively adding activated Issatchenkia orientalis WTB20042304 and Pichia capraphia BY35 (the existing strain and the preserved strain number are CGMCC No. 14909) into 2 fermentation tanks, wherein the addition amount is1 × 10 6 CFU/mL, control without any acid reducing yeast. Adding Saccharomyces cerevisiae at day 3, the concentration is also 1 × 10 6 CFU/mL, sugar addition 120g/L. The fermenter was placed at 22 ℃ for fermentation with 2 stirring times per day. Filtering after fermentation is finished, and sealing and storing the fruit wine filtrate.
(3) Blueberry fruit wine index determination
a. Measurement of physical and chemical indexes
And (3) pH value measurement: measured with a pH meter. SSC determination: measured with a glucometer. Alcohol content determination: measured with an alcohol meter. And (3) total sugar determination: phenol-sulfuric acid method, 1mL sample +0.5mL5% phenol solution +2.5mL concentrated sulfuric acid, mixing uniformly for 5min, taking out and cooling to room temperature, and measuring light absorption value at 490nm of a spectrophotometer. And (3) total acid determination: measured according to the national standard method.
b. Active substance assay
The determination of the total phenol content is carried out by the method described in Singleton [ Singleton V.L., rossi J.A.colorimetry of total phenolics with phospho-molar phosphoric acid reagents [ J ]. American Journal of Enlogology and Viticulture,1965, 16. The total flavone content is researched according to extraction conditions of antioxidant and free radical-scavenging active substances in sophora japonica [ J ] food industry science and technology, 2009 (12): 130-132 ], according to a method of Wang You liters and the like [ Zhu Yuyan, wang Yousheng, zhao Qian and the like ], rutin is used as a standard curve, and the total phenol content in mash is converted into the rutin content in each gram of sample. The anthocyanin content is determined by a method of Xu Kun [ Xu Kun. Production process research of dry blueberry wine [ D ]. Anhui university, 2014 ].
c. Detection of acid reducing effect of blueberry fruit wine
Preparing a mixed standard product: taking 10mg of solid standard substance and 100 μ L of liquid standard substance mother liquor, metering to 10ml with mobile phase, mixing, and storing in refrigerator at 4 deg.C in dark place. In use, 1ml of 45. Mu.L microporous membrane was taken with a syringe and tested in a liquid phase vial under optimal conditions.
Sample pretreatment: the samples were first diluted 20 times with ultrapure water, tested by loading, and then diluted and tested for 48h, 96h of culture medium, respectively.
Liquid phase conditions: a chromatographic column: agilent 5 TC-C18 (250X 4.6 mm), using diammonium hydrogen phosphate with pH of 2.4 as mobile phase, column temperature of 45 deg.C, flow rate of 0.8ml/min, and sample amount of 20 μ L, respectively centrifuging and diluting 20 times samples of culture medium containing citric acid and tartaric acid and organic acid culture medium after adding 2 strains of acid-reducing yeast, fermenting for 48h and 96h, and detecting with high performance liquid chromatography. Determining the retention time of the organic acid through the chromatogram results of the citric acid and tartaric acid standard substances, and comparing the retention time with the chromatographic peak area of each organic acid in the 0h culture medium to detect the acid reduction effect of each strain.
(4) Results of the experiment
(1) Variation of citric acid content
The citric acid content in the blueberry juice was high at 6.81g/L at the beginning of fermentation, and at the end of fermentation, no citric acid was detected in any of the remaining 2 groups except the control group (without the addition of the acid-reducing yeast).
Therefore, the yeast I.orientalis WTB20042304 has strong degradation capability on citric acid in blueberry wine.
(2) Change in lactic acid content
The lactic acid content at the beginning of the fermentation was very low, only 0.08g/L. As can be seen by comparing the lactic acid content at the end of fermentation with that at the beginning of fermentation, the Saccharomyces cerevisiae produces lactic acid during the fermentation process, so that the lactic acid content at the end of fermentation in the control group is 0.94g/L. Specific results are shown in table 8:
TABLE 8
In addition, when the screened yeast strains are added, the content of lactic acid at the fermentation end point is reduced by 36.17 percent compared with that of a control group, and the lactic acid reducing capability of the yeast strains is obviously better than that of the existing yeast strains and is also better than that of other experimental group strains.
(3) Variation of formic acid content
Formic acid detected in the blueberry juice at the beginning of fermentation is only 0.07g/L, and saccharomyces cerevisiae can generate more formic acid in the fermentation process, and the formic acid content of the contrast is 0.41g/L at the end of fermentation. By comparing the formic acid content at the end of the fermentation with that at the beginning of the fermentation, it can be seen that the saccharomyces cerevisiae produces formic acid during the fermentation. Specific results are shown in table 9:
TABLE 9
After the calcium Phila BY35 is added into the blueberry mash, the content of formic acid is increased, and it is supposed that other organic acids are decomposed to generate partial formic acid. The I.orientalis WTB20042304 screened by the invention has the capability of degrading formic acid, the content of the formic acid at the fermentation end point is 0.31g/L, which is reduced by 24.39 percent compared with a control group, and the degradation of the formic acid by other experimental groups I.orientalis is less than 10 percent.
(4) Variation of acetic acid content
The blueberry juice contains a very small amount of acetic acid which is only 0.15g/L at the beginning of fermentation, a certain amount of acetic acid can be generated by saccharomyces cerevisiae in the fermentation process, and the acetic acid content of a control group reaches 14.79g/L at the end of fermentation. The saccharomyces cerevisiae is easy to produce acetic acid in the fermentation process. The results are shown in Table 11.
Watch 10
After the I.orientalis WTB20042304 of the invention is added, the content of acetic acid is reduced by 9.20 percent compared with the control group, the degradation capacity of the I.orientalis WTB 5 and the I.orientalis WTB20042304 on the acetic acid is equivalent, the I.orientalis WTB2 is reduced by less than 5 percent compared with the control group, and the acetic acid accumulation of other strains is higher than that of the control group compared with all the strains.
(5) Change in succinic acid content
The blueberry juice contains a very small amount of succinic acid of only 0.05g/L at the beginning of fermentation, the saccharomyces cerevisiae can generate succinic acid in the fermentation process, and the succinic acid content of a control group at the end of fermentation is 0.67g/L. It can be seen by comparing the succinic acid content at the end of fermentation with that at the beginning of fermentation that the Saccharomyces cerevisiae produces succinic acid during the fermentation. The results are shown in Table 10.
TABLE 11
The addition of the i.orientalis WTB20042304 strain of the present invention reduced succinic acid by 13.43% relative to the control, while the accumulation of succinic acid by other strains relative to the control was greater than that of the control.
Therefore, the I.orientalis WTB20042304 has degradation effects on citric acid, lactic acid, formic acid, acetic acid and succinic acid in blueberry fruit wine fermentation, and the degradation effect on the citric acid is particularly obvious.
(6) Influence of orientalis WTB20042304 on active substances in blueberry fruit wine
The content of total phenols in the control group at the end of fermentation is 1.13mg/g, the content of total flavonoids is 1.05mg/g, and the content of anthocyanidin is 120.29mg/L.
Compared with a control group, the strain I.orientalis WTB20042304 is added, the total phenol content is increased by 10.37 percent and is higher than other strains, the total flavone content is increased by 2.93 percent and is higher than other strains, and the anthocyanin content is 7.91 percent and is higher than other strains.
TABLE 12
Compared with a control group, the antioxidant indexes of the strain I.orientalis WTB20042304 are increased, wherein DPPH is increased by 9.44%, FRAP is increased by 9.58%, ABTS is increased by 6.50%, and the total reducing power is increased by 15.92%. Wherein the increase of FRAP is slightly lower than that of strain I.orientalis WBD 4, but other antioxidant indexes are all higher than that of strain I.orientalis WBD 4.
(7) Effect of orientalis WTB20042304 on pH in blueberry wine
The blueberry fruit wine added with the acid-reducing yeast can be normally fermented, the alcoholic strength of all treatments at the end point of fermentation is 11.73%, and SSC is 6.87 degrees Bx. The pH of the control at the end of the fermentation was 3.61. After adding Issatchenkia orientalis I.orientalis WTB20042304, the fermentation end point pH was 3.92. The I.orientalis WTB20042304 of the invention has obvious acid reduction effect.
Biological control effect of Issatchenkia orientalis WTB20042304 on fresh fruit
(1) Experimental Material
(1) Strain: yeast: issatchenkia orientalis I.orientalis19724
Pathogenic bacteria: botrytis cinerea (Botrytis cinerea) for laboratory preservation
Fruit: grape, purchased from New places in Beijing
(2) Main instruments of the experiment are shown in Table 2
(2) Experimental method
Step 1: preparing yeast suspension as biocontrol bacteria suspension, comprising the following steps:
(1) activating the strain: 1 single colony was inoculated to YPD liquid medium on YPD plate and shake-cultured at 26 ℃ for 24 hours.
(2) And (3) collecting thalli: centrifuging, removing culture medium, washing with 1mL sterile water (blowing with pipette or shaking, mixing well), centrifuging at 12000r/min,4 deg.C, and 2 min. This operation was repeated three times. After the water is removed and the thalli are left, 1mL of sterile water is added and mixed evenly, and the mixture is diluted to a proper multiple.
(3) And (3) counting blood corpuscle plates: cover the glass slide first, add 10 uL bacterial suspension in the upper chamber and lower chamber separately, find the square with 10 times of mirror first, then change 40 times of mirror to count. After counting, diluting the suspension into a suspension with a target concentration.
And 2, step: preparing mould bacterial suspension
Adding 2mL sterile water into sterilized 10mL centrifuge tube, clamping mycelia on the surface of the culture medium with sterilized forceps, adding into the centrifuge tube, blowing and mixing with pipette to completely mix spores into water, filtering with filter cloth, counting with blood counting plate, and diluting to 5 × 10 4 Spore suspension at CFU/mL concentration.
And step 3: fruit preparation
Selecting bought grapes, placing the bought grapes with bruise and damage independently, cleaning the selected good fruits with consistent maturity and size with clear water, treating with sodium hypochlorite aqueous solution (0.5%) for 5min, placing the fruits into a cleaned box, pricking 15 grapes in each box, after the surface moisture of the grapes is dried, pricking 1 hole of each fruit with a pricking needle, and burning the inoculating needle one box at each time. After the grape sap had dried (about 4 hours), the experimental group was added with 20. Mu.L of yeast suspension of the desired concentration, and after 4 hours (essentially complete absorption of the yeast suspension) 20. Mu.L of pathogenic bacteria 5X 10 was added 4 CFU/mL spore suspension, control, also added 20. Mu.L of sterile water, and in the case of framing, two groups of water-wetted toilet paper rolls were placed in the frame.
And 4, step 4: statistics of disease (rot)
The statistical method of the rotting rate adopts an observation method, and the calculation formula is as follows: rotting rate (%) = rotted fruit count/total fruit count × 100%. The diameter of the lesion is measured by the cross method in mm.
(3) Results of the experiment
The incidence rate of grape is counted at 96h, 144h and 192h of grape inoculated with botrytis cinerea (B.cinerea), and the concentration of biocontrol bacteria is1 × 10 6 CFU/mL, sterile water blank, differentThe difference of disease condition between treatments is more obvious. The result shows that the incidence rate of adding the strain I.orientalis WTB20042304 of the invention is 42.22%; under the condition of the same concentration of the biocontrol bacteria, the morbidity of the existing biocontrol bacteria (the existing strain with the preservation number of CGMCC No. 14909) is 71.11 percent. The incidence rates of the I.orientalis WTB2 strain, the I.orientalis WTB 3 strain, the I.orientalis WTB 4 strain and the I.orientalis WTB 5 strain are respectively 70%, 93% and 90%. The concentration of biocontrol bacteria is1 multiplied by 10 6 The fruit is soaked by CFU/mL, the soaking incidence rate of the strain I.orientalis WTB20042304 is 43.00%, the incidence rate of the strain I.orientalis WTB2 is 63.00%, the incidence rate of the strain I.orientalis WTB 3 is 88.00%, the incidence rate of the strain I.orientalis WTB 4 is 88.00%, the incidence rate of the strain I.orientalis BD2 is 93.00%, and the incidence rate of the conventional biocontrol strain P.cactophila is 60%.
Therefore, compared with the existing biocontrol bacteria, the yeast strain disclosed by the invention has a better inhibiting effect on botrytis cinerea, and the biocontrol effects of different strains of the same strain are obviously different.
The comparison shows that the Issatchenkia orientalis I.orientalis WTB20042304 of the invention has certain inhibition effect on botrytis cinerea (B.cinerea). When the concentration of the biocontrol bacteria is increased to 1 x 10 8 The biocontrol effect of the Issatchenkia orientalis I.orientalis WTB20042304 bacterial suspension is not obviously increased (the incidence rate is 31.11%) at CFU/mL. Therefore, from the economical point of view, when fresh fruits are preserved using the suspension of Issatchenkia orientalis I.orientalis WTB20042304 of the present invention, it is preferable to set the concentration at 1X 10 6 CFU/mL。
Based on the above description, issatchenkia orientalis i.orientalis WTB20042304 (CGMCC No. 19724) has uniqueness in degrading citric acid, lactic acid, formic acid, acetic acid, etc. in fruit wine compared with other acid-reducing yeasts, and has excellent acid-reducing performance and wide adaptability. By reducing the content of organic acid in the fruit wine, the quality of the fruit wine such as taste, color and the like can be improved.
In addition, experiments also prove that in the process of preparing fruit wine by fermentation, the sugar content at the fermentation end point shows that the fermentation process of the saccharomyces cerevisiae is not influenced after the saccharomyces cerevisiae strain is added, and the ethanol concentration has no obvious influence on the capability of the saccharomyces cerevisiae strain to utilize organic acid.
When the fruit wine, particularly the blueberry fruit wine is prepared, issatchenkia orientalis I.orientalis WTB20042304 is added before saccharomyces cerevisiae is added, the alcoholic strength of all treatments at the fermentation end point is 11.73 percent, SSC is 6.87 degrees Bx, the pH value at the fermentation end point is higher and is 3.92, the contents of total phenols, total flavones and anthocyanin in the fruit wine are increased, and the oxidation resistance and the total reduction capability are improved.
In addition, the Issatchenkia orientalis I.orientalis WTB20042304 also proves that the invention has certain inhibition effect on botrytis cinerea.
Therefore, the Issatchenkia orientalis I.orientalis WTB20042304 can not only prevent the fresh fruits from being rotted and deteriorated after the fresh fruits are picked and ensure the freshness of the fresh fruits, but also play a role in reducing acid when the fruit juice is used for fermentation and brewing wine in the later period, the effect of reducing acid is not influenced by the alcohol concentration, the normal fermentation process of the Saccharomyces cerevisiae is not influenced, the effect of reducing acid can be achieved on most kinds of organic acid in the fruit wine, the taste and the quality of the fruit wine are improved (the wine juice with high acidity is turbid), the Issatchenkia orientalis I.orientalis WTB20042304 is a preferable strain for the whole-process green production of the fruit wine, and the biocontrol bacterium suspension sprayed during the fresh-keeping storage of the fresh fruits can still play the effect of reducing acid even if the bacteria are not cleaned.
Claims (10)
1. Issatchenkia orientalis (Issatchenkia orientalis) is preserved in China general microbiological culture Collection center (CGMCC) at 24.04.2020, with the preservation number of CGMCC No.19724.
2. Use of the issatchenkia orientalis of claim 1 for storage and freshness preservation of fruit after harvest or deacidification of fruit wine.
3. Use according to claim 2, characterized in that: the fruit is blueberry, and the fruit wine is blueberry fruit wine.
4. A fresh fruit preservation method is characterized by comprising the following steps: the Issatchenkia orientalis of claim 1 is prepared into a bacterial suspension, and the bacterial suspension is used for spraying or dip-coating fresh fruits.
5. The fresh-keeping method of claim 4, wherein: activating the Issatchenkia orientalis of claim 1, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, and making into 1 × 10 6 CFU/mL~1×10 8 CFU/mL of bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a preservation box, sealing, and storing at normal temperature.
6. The fresh-keeping method of fresh fruit according to claim 4 or 5, characterized in that: the fresh fruit is grape or blueberry.
7. A fruit wine deacidification fermentation method is characterized in that before saccharomyces cerevisiae is inoculated into fruit juice, the issatchenkia orientalis according to claim 1 is inoculated, and then fermentation treatment is carried out.
8. The fruit wine acid-reducing fermentation method according to claim 7, characterized in that: the fruit wine is blueberry fruit wine, and the fruit juice is blueberry fruit juice.
9. The acid-reducing fermentation method of fruit wine according to claim 7, characterized in that: crushing fresh blueberries, adjusting the components to obtain blueberry juice, adding the Issatchenkia orientalis into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and then carrying out the process flows of sugar degree adjustment, primary fermentation, filtration and post-fermentation.
10. The acid-reducing fermentation method of fruit wine according to claim 7, characterized in that: the adding amount of the Issatchenkia orientalis is1 multiplied by 10 6 CFU/mL。
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