CN116376711A - Preparation method of apple wine base, fermentation strain and application thereof - Google Patents
Preparation method of apple wine base, fermentation strain and application thereof Download PDFInfo
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- saccharomyces cerevisiae
- aspergillus niger
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- 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
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- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
- C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
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Abstract
The invention discloses a preparation method of apple wine base, a fermentation strain and application thereof. The invention takes red Fuji apples and red glutinous rice as raw materials, and takes high-yield saccharifying enzyme strain Aspergillus niger (Aspergillus niger) PWY-03 and strain Saccharomyces cerevisiae (Saccharomyces cerevisiae) YD-01 with strong alcoholic fermentation capacity as fermentation strains of apple wine base, so as to prepare the novel apple wine base beverage with rich wine aroma and unique wind style. The red glutinous rice is used in the raw materials of the apple wine base, so that the aroma of the apple wine base is improved, the aroma components of the apple wine base are enriched, the apple wine base has unique flavor, and meanwhile, no foreign substances are added in the whole fermentation process, so that the apple wine base is safe and convenient.
Description
Technical Field
The invention relates to a preparation method of apple wine base, a fermentation strain and application thereof, and belongs to the technical field of wine brewing.
Background
The increase of the apple yield promotes the development of the apple deep processing industry, but the apple deep processing proportion in China is lower, the fresh food, the concentrated apple juice and the fruit juice beverage products are mainly used, the added value of the products is lower, and the products are single. Cider (order) is an alcoholic beverage fermented from apple juice, is called apple wine base without any processing, is one of apple deep processing products, and is deeply favored by consumers due to unique taste and rich nutrition and health care value. At present, the cider is the world second largest fermented fruit wine which is inferior to the grape wine, and along with the improvement of the living standard of people and the deepening of the healthy living concept, the cider has a potential huge market prospect in China.
The brewing process of the apple wine base is a complex microorganism metabolic process and is limited by factors such as raw material varieties, fermentation strains, fermentation process and the like, so that the apple wine product in the market has single taste and good quality. At present, common cider beverages in the market mainly use single apple juice as a raw material, so that the raw material selection of production enterprises is limited in the fermentation process, the fermentation process is not optimized completely, the fermentation strain is single, and the produced cider is low in quality, bad in taste and insufficient in aroma. CN102021102a discloses a preparation process of apple fermented compound aroma distilled spirit, comprising: (1) pressing apple juice: (2) adding wine-making yeast, and fermenting at low temperature to obtain apple wine base; (3) sealing and storing; (4) distillation; (5) ageing; (6) freezing; and (7) preparing finished wine. The apple juice is prepared by fermenting, storing in an oak barrel, distilling, and ageing in a micro-oxygen device, so that the content of methanol and fusel oil is greatly reduced, and the brewed white spirit has faint scent taste and is not drunk. CN105524797a discloses a cider and a brewing process thereof, which sequentially carry out raw material treatment, crushing pulping, enzymolysis clarification, yeast fermentation and clarifier clarification on apples, and the prepared cider has high clarity, less impurities, good taste and no headache after drinking. CN111304036a discloses an appetizing summer-heat relieving sugar-heart apple wine base and a high-efficiency deep processing technology, which comprises the following steps: (1) preparing a raw slurry; (2) detoxification treatment of patulin; (3) inoculating Saccharomyces cerevisiae and accurately fermenting; (4) enzymolysis of residues; (5) clarifying and filtering; (6) ageing; (7) After adsorption and clarification, canning, aiming at the phenomenon of turbidity in the later storage period of the cider, the problem of adsorption of the nanofiber modified resin with multiple stimulus responsivity is solved. The process still has the problem of single apple juice as a raw material, single strain and single taste of apple wine products.
Based on the current development status of apple wine base, the invention takes red Fuji apples and red glutinous rice as main raw materials, and adopts aspergillus niger PWY-03 saccharification and fermentation and Saccharomyces cerevisiae YD-01 alcoholic fermentation, thereby not only improving the fragrance of apple wine and enriching the fragrance components of apple wine, but also having simple and efficient fermentation mode. At present, no report is made on the process for preparing apple wine base and apple wine by taking red Fuji apples and red glutinous rice as main raw materials.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention takes red Fuji apples and red glutinous rice as raw materials, aspergillus niger PWY-03 and saccharomyces cerevisiae YD-01 as fermentation strains, prepares the novel apple wine with rich wine fragrance and unique style, and has the advantages of simple and efficient fermentation mode, no need of adding any exogenous substances in the whole fermentation process, safety and convenience.
The invention firstly separates a high-yield saccharifying enzyme strain Aspergillus niger (Aspergillus niger) PWY-03 and a strain Saccharomyces cerevisiae (Saccharomyces cerevisiae) YD-01 with strong alcoholic fermentation capability from rotten apples stored in a cold storage as fermentation strains of apple wine base; the strain is preserved in China general microbiological culture Collection center (CGMCC) at 1-12 of 2023, and the preservation numbers are CGMCC No.40486 and CGMCC No.26465 respectively. The preservation address is North Chen Silu No.1 and No. 3 in the Chaoyang area of Beijing city.
The second purpose of the invention is to provide a preparation method of apple wine base, which comprises the following steps:
(1) Preparation of fermentation inoculant
Fermenting Aspergillus niger PWY-03 by a solid fermentation medium, and then drying and crushing to obtain a solid fermentation inoculant (raw powder) of Aspergillus niger PWY-03 with a spore content of more than or equal to 20 hundred million/g; fermenting the saccharomyces cerevisiae YD-01 by a large amount of fermentation culture medium to obtain a saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum with the bacterial activity of more than or equal to 200 hundred million/mL;
(2) Pretreatment of raw materials
The raw materials are as follows: 95+/-1 parts of fresh red Fuji apples and 5+/-1 parts of red glutinous rice;
soaking fructus Mali Pumilae, peeling, removing core, and crushing; grinding red glutinous rice in advance; fully mixing the two materials and then placing the mixture in a saccharification tank;
(3) Saccharification
Then inoculating Aspergillus niger PWY-03 solid fermentation inoculant according to the mass ratio of 1-2%, and saccharifying for 5-7 d at 25-30 ℃;
(4) Alcohol fermentation
After saccharification is finished, inoculating a Saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum according to the mass ratio of 0.05-0.1%, and keeping the temperature at 25-30 ℃ for continuous fermentation for 12-15 d; after the alcoholic fermentation is completed, the temperature is reduced to 8-15 ℃, the fermentation is continued for 5-7 d, and the filtration is carried out; settling, secondary filtering and sterilizing to obtain the high-quality apple wine base. The alcohol content of the wine base is stabilized between 20 and 22 percent vol.
Further, the preparation method of the Aspergillus niger PWY-03 solid fermentation inoculant comprises the following steps: activating Aspergillus niger PWY-03, inoculating the activated Aspergillus niger into a PDB culture medium, and vibrating at a constant temperature of 25-30 ℃ for 3-5 d to obtain seed liquid; then inoculating the seed liquid into a solid fermentation culture medium according to the mass ratio of 10-20%, fermenting at the fermentation temperature of 26-28 ℃ and the fermentation humidity of more than 85%, drying and crushing after the fermentation is finished to obtain the aspergillus niger PWY-03 solid fermentation microbial inoculum (raw powder) with the spore content of more than or equal to 20 hundred million/g.
Wherein, the solid fermentation culture medium (mass ratio): 30% of starch, 8% of wheat bran, 25% of rice hull powder, 0.5% of glucose and 0.5% of CaCO 3 3 percent of magnesium sulfate 0.2 percent, potassium dihydrogen phosphate 0.15 percent, red glutinous rice flour 33.15 percent, water content controlled between 55 and 60 percent and pH between 6 and 5.5.
Further, the preparation method of the Saccharomyces cerevisiae YD-01 liquid fermentation inoculant comprises the following steps: activating Saccharomyces cerevisiae YD-01, inoculating the activated Saccharomyces cerevisiae YD-01 into YPD liquid culture medium, and carrying out shaking culture at a constant temperature of 25-30 ℃ for 12-14 h to prepare YD-01 shake flask seed liquid; inoculating seed liquid into a fermentation tank filled with a primary seed liquid culture medium according to the volume ratio of 0.3-0.8%, and fermenting and culturing for 10-12 h at 25-30 ℃ to prepare YD-01 primary seed liquid; the first-stage seed liquid is inoculated into a fermentation tank filled with a large amount of fermentation medium according to the volume ratio of 3-8%, and is fermented and cultured for 20-22 hours at the temperature of 25-30 ℃ to obtain the saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum with the bacterial activity of more than or equal to 200 hundred million/mL.
The formula (mass ratio) of the first-stage seed liquid culture medium is as follows: glucose 1%, peptone 1.5%, beef powder 0.3%, sodium chloride 0.5%, and water the balance, pH 6.5-7.0.
A large amount of fermentation medium formula (mass ratio): 1.5% of edible glucose, 1.5% of fish peptone, 1.5% of yeast extract powder, 0.05% of magnesium sulfate, 0.02% of monopotassium phosphate and the balance of water, wherein the pH value is 6.5-7.0.
The third object of the invention is to provide the high-quality cider, which is characterized in that the apple wine base prepared by the method is added with fresh concentrated apple juice for blending, and the high-quality cider with the alcohol content of 3.5% vol is prepared through the links of filtering, sedimentation, secondary filtering and sterilization.
The invention has the technical effects that:
1. the invention screens high-yield saccharifying enzyme strain Aspergillus niger PWY-03 and strain Saccharomyces cerevisiae YD-01 with strong alcoholic fermentation capacity as fermenting strain of apple wine base through experiments, wherein saccharifying enzyme activity of Aspergillus niger PWY-03 can reach 170.67U/mL, and apple juice added with Saccharomyces cerevisiae YD-01 is CO after fermentation for 72 hours 2 The accumulated weight loss can reach 61.6g/L, thereby providing good fermentation conditions for the preparation of the apple wine base；
2. The red glutinous rice is used in the raw materials of the apple wine base, so that the aroma of the apple wine base is improved, the aroma components of the apple wine base are enriched, and the apple wine base has unique flavor.
3. The apple wine base preparation process has simple and efficient fermentation mode and pure microorganism fermentation; meanwhile, no exogenous substances can be added in the whole fermentation process, so that the method is safe and convenient.
4. The high-quality cider with 3.5% vol made of the apple wine base is typically perfect and unique in style.
Drawings
FIG. 1 is a colony morphology of Aspergillus niger (Aspergillus niger) PWY-03;
FIG. 2 shows the fermentation broth CO of Aspergillus niger (Aspergillus niger) PWY-03 2 Accumulating a weightlessness map;
FIG. 3 is a colony morphology of Saccharomyces cerevisiae (Saccharomyces cerevisiae) YD-01.
Detailed Description
In order to clearly illustrate the technical features of the inventive solution, the present invention will be described below with reference to specific embodiments. The protective scope of the invention is not limited to the embodiments. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.
Example 1: screening and identification of high-yield saccharifying enzyme strain PWY-03
The embodiment provides relevant experiments of separating and purifying the strain PWY-03, detecting the activity of the saccharifying enzyme and identifying the species.
1. Isolation and purification of Strain PWY-03
(1) Separating the source: the high-yield saccharifying enzyme strain PWY-03 is separated from rotten apples stored in a refrigerator.
(2) Separation time and place: the water science innovation development limited company is separated in the Shandong on 9 and 19 days 2022;
(3) Separation and purification steps: the method is obtained by adopting a tissue separation method, and comprises the following specific steps:
dissecting the rotten apples with a dissecting knife and other tools under the aseptic condition, inoculating dissected small blocks on a PDA culture medium containing chloramphenicol, and culturing for 2-4 d under the constant temperature condition of 30 ℃; according to the morphological characteristics (including shape, color, edge, texture and the like) of different bacterial colonies, inoculating different bacterial strains on a PDA culture medium again, culturing for 2-4 d at a constant temperature of 30 ℃, picking up one piece of bacterial strain to be inoculated in a PDA inclined surface test tube after no pollution is observed, culturing for 2-4 d at a constant temperature of 30 ℃, and preserving at 4 ℃. Using this procedure, a total of 10 fungi, accession numbers PWY-01 to 10, were isolated.
PDA medium: 5.0g/L of potato soaked powder, 20g/L of glucose and 15-20 g/L of agar.
PDA medium containing chloramphenicol: 5.0g/L of potato soaked powder, 20g/L of glucose, 0.1g/L of chloramphenicol and 15-20 g/L of agar.
2. Screening of high-yield saccharifying enzyme Strain PWY-03
(1) And (3) primary screening: the purified fungi are respectively inoculated on a primary screening culture medium, cultured for 48 hours at the constant temperature of 30 ℃, taken out, dripped with iodine solution, the diameter D of a transparent ring and the diameter D of a colony are observed, the ratio of D to D is calculated, strains PWY-03 and PWY-05 (refer to Table 1) with the function of producing saccharifying enzyme are primarily screened out, and PWY-03 and PWY-05 are simultaneously used as primary strains for re-screening.
TABLE 1 results of preliminary screening of saccharifying enzyme-producing strains
Primary screening of the culture medium: 1% of peptone, 0.5% of NaCl, 0.5% of beef extract, 2% of agar powder, 2% of soluble starch and the balance of water.
(2) And (3) re-screening: respectively inoculating the strains PWY-03 and PWY-05 obtained by primary screening into a fermentation medium, and performing shaking fermentation for 48 hours at 30 ℃ and 150 r/min; centrifuging the bacterial liquid at 4 ℃ for 20min at 5000r/min, and taking the supernatant as crude enzyme liquid. Saccharification force detection was performed by referring to the method specified in QB/T4257-2011. Strain PWY-03 was found to have a saccharifying enzyme of 170.67U/mL, which was significantly higher than PWY-05 (see Table 2).
TABLE 2 high-yield saccharifying enzyme Strain re-screening results
Note that: the different letters indicate significant differences (P < 0.05).
Fermentation medium: bran 40g, magnesium sulfate 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, water 1L, and high pressure steam sterilization at 121 ℃ for 30min.
3. Identification of Strain PWY-03
Inoculating the strain PWY-03 on a PDA plate, and culturing at a constant temperature of 30 ℃ for 72 hours; the average growth rate of hypha of the strain is found to be 21.16mm/d; the bacterial colony is elliptical, and the edge hypha is white and gradually turns into gray; the flora is large and loose, the surface protrusions are plush-shaped, the edges are rough, and the middle part of the spore grows to be black brown (see figure 1).
Activating strain PWY-03, inoculating the strain onto PDB culture medium, shaking and fermenting at 30deg.C for 72 hr, collecting upper mycelium, drying with filter paper, grinding with liquid nitrogen into powder, extracting fungus genome DNA by CTAB method, and sequencing.
The rDNA gene sequence determination result (ITS 1 region) of the strain is as follows (SEQ No. 1):
GCGGAAGGATCATTACCGAGTGCTGGGTCCTTCGGGGCCCAACCTCCCACCCGTGCTTACCGTACCCTGTTGCTTCGGCGGGCCCGCCTTCGGGCGGCCCGGGGCCTGCCCCCGGGACCGCGCCCGCCGGAGACCCCAATGGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGATACCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTTCTCCCCTCCAGCCCCGCTGGTTGTTGGGCCGCGCCCCCCCGGGGGCGGGCCTCGAGAGAAACGGCGGCACCGTCCGGTCCTCGAGCGTATGGGGCTCTGTCACCCGCTCTATGGGCCCGGCCGGGGCTTGCCTCGACCCCCAATC
the strain PWY-03 is identified as Aspergillus niger (Aspergillus niger) by morphology and molecular biology, and is preserved in the China general microbiological culture Collection center, and the preservation date is that: and 2023, 1 month and 12 days, wherein the preservation number is CGMCC No.40486.
Example 2: screening and identification of Excellent fermentation Strain YD-01
The embodiment provides related experiments of separation, purification, physical and chemical property detection of gas production and the like of the fermentation strain YD-01 and species identification.
1. Isolation and purification of Strain YD-01
(1) Separating the source: the fermentation strain YD-01 is separated from rotten apples stored in a refrigerator.
(2) Separation time and place: is separated from Shandong Water technology Innovative development Limited laboratory at 9 and 27 days 2022.
(3) Separation and purification steps: the preparation method is separated by adopting a dilution coating method, and comprises the following specific steps:
cleaning rotten apples, and fully mashing; weighing 25g, placing in a triangular flask containing 225mL of sterile physiological saline, and uniformly oscillating to prepare bacterial suspension; under aseptic conditions, adding absolute ethyl alcohol into sterilized apple juice culture medium according to the amounts of 0, 3%, 5%, 8%, 10% and 12% respectively to prepare an enrichment culture medium; respectively taking 9mL of enrichment culture mediums with different concentrations into test tubes, and inoculating 1mL of bacterial suspension, and culturing for 2-3 d at the constant temperature of 28 ℃; the cultured sample was subjected to gradient dilution, and a suitable dilution (10 -4 、10 -5 、10 -6 ) And (3) performing YPD solid coating and separating saccharomycetes, culturing at the constant temperature of 28 ℃, after bacterial colonies grow out, selecting single bacterial colonies with typical saccharomyces cerevisiae bacterial colony characteristics, further streaking and separating, observing the single bacterial colonies into pure species by a microscope, respectively transferring the pure species into YPD inclined planes, culturing in a culture box at the temperature of 28 ℃ for 12-24 hours, and refrigerating in a refrigerator at the temperature of 4 ℃ for standby. By the method, 12 strains with Saccharomyces cerevisiae characteristic strains are separated, and the numbers are YD-01 to YD-12 respectively.
Apple juice medium: apple 100g, peptone 2g, sucrose 20g, water 1L, pH nature.
Enrichment medium: based on apple juice culture medium, different amounts of absolute ethyl alcohol are added.
YPD solid medium: 20g of peptone, 20.0g of glucose, 10.0g of yeast extract powder, 15-20 g of agar, 1000mL of water and pH of 6.5+/-0.2.
YPD liquid medium: 20g of peptone, 20.0g of glucose, 10.0g of yeast extract powder, 1000mL of water and pH of 6.5+/-0.2.
2. Screening of Excellent fermentation Strain YD-01
(1) Gas production capability test
Activating the isolated 12 yeasts in YPD liquid medium, and preparing into 1.0X10 s by dilution method 6 CFU·mL -1 Standard bacterial liquid; 0.5mL of standard bacterial liquid is inoculated into a test tube containing 10mL of YPD liquid culture medium (containing Du Shixiao tubes), and each strain is repeated 3 times; culturing at 28 deg.c for 1-2 d, observing Du Shixiao pipe gas producing condition, determining the fermentation capacity of each strain and sieving out strain with excellent gas producing performance.
As a result, it was found that after 48 hours of culture, du Shixiao tubes inoculated with YD-01, YD-05 and YD-06 were foam-filled (see Table 3), and light wine flavor was also smelled at these test tube ends. This experiment demonstrates that YD-01, YD-05 and YD-06 are capable of faster fermentation of sugar to ethanol and carbon dioxide.
Table 3:48h Du Shixiao pipe gas production condition
Note that: "+" indicates that the gas production is less than or equal to 1/4 of the Du's tubule volume, "++" indicates that the gas production is about 1/2 of the Du's tubule volume, "+++" indicates that the gas production rate is approximately equal to 3/4 of the volume of the Du's tubule, 3/4 Du's the volume of the small tube is equal to the volume of the small tube.
(2) Fermentation ability test
First, 1.0X10 of YD-01, YD-05 and YD-06 were prepared by the above-mentioned method, respectively 6 CFU·mL -1 Standard bacterial liquid; inoculating apple juice with 2% of inoculation amount by volume ratio, performing sealed fermentation in a triangular flask, and using CO 2 The fermentation capacity of each strain in different fermentation time periods is detected by a weightlessness method. The discovery is as follows: after fermentation for 72h, adding fermentation liquor CO of YD-01 2 The maximum accumulated weight loss is 61.6g/L (refer to figure 2), which shows that the YD-01 has strong fermentation capacity; after 12h of YD-01 addition, CO 2 The amount of the produced product is fast and continuously high, which indicates that YD-01 has stronger fermentation capability.
3. Identification of fermentation Strain YD-01
(1) Microbiological characteristics: the strain is cultured in NA culture medium for 72h, the colony is round, the surface is moist and smooth, sticky, milky white, slightly convex in the middle and 3-4 mm in diameter (see figure 3).
(2) Molecular biological properties: the 16S rDNA gene sequence of this strain was determined as follows (SEQ-2).
TGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCT
The strain YD-01 is identified as saccharomyces cerevisiae (Saccharomyces cerevisiae) through morphology and molecular biology, and the strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date: and 2023, 1 month and 12 days, wherein the preservation number is CGMCC No.26465.
Example 3: preparation of high-yield saccharifying enzyme strain Aspergillus niger PWY-03 solid fermentation inoculant
Solid fermentation medium (mass ratio): 30% of starch, 8% of wheat bran, 25% of rice hull powder, 0.5% of glucose and 0.5% of CaCO 3 3 percent of magnesium sulfate 0.2 percent, potassium dihydrogen phosphate 0.15 percent, red glutinous rice flour 33.15 percent, water content controlled between 55 and 60 percent and pH controlled between 6 and 5.5.
Aspergillus niger PWY-03 solid fermentation process:
(1) Strain activation: inoculating Aspergillus niger PWY-03 stored in a refrigerator at 4 ℃ on a PDA flat plate, and culturing for 2-3 d at 28 ℃ for activation; during the period, the bacteria are observed, and if the bacteria are infected, purification is needed.
(2) Seed liquid preparation: inoculating the activated Aspergillus niger PWY-03 bacterial cake in the step (1) into a PDB culture medium, and vibrating at a constant temperature of 28 ℃ and 120r/min for 3-5 d to obtain seed liquid.
(3) Fermentation of a solid culture medium: inoculating the seed liquid prepared in the step (2) into a solid culture medium according to 15% of inoculum size, fully uniformly mixing and paving the seed liquid with the thickness of 8-10 cm, maintaining the fermentation humidity above 85%, and maintaining the temperature at 26-28 ℃; after the hypha is fully grown, lightly turning over, and keeping the culture medium block shape as much as possible; after the spores grow fully, lightly turning again, reducing the air humidity, keeping for more than 1-2 d, and finishing fermentation.
(4) Raw powder acquisition: drying in a fluidized bed after fermentation, fully crushing after the completion of the drying, and sieving with a 60-80 mesh sieve to obtain raw powder, wherein the spore content of the raw powder is not less than 20 hundred million/g. The raw powder is Aspergillus niger PWY-03 solid fermentation inoculant.
Example 4: preparation of excellent fermentation strain Saccharomyces cerevisiae YD-01 liquid fermentation inoculant
The formula (mass ratio) of the first-stage seed liquid culture medium is as follows: glucose 1%, peptone 1.5%, beef powder 0.3%, sodium chloride 0.5%, and water the rest.
A large amount of fermentation medium formula (mass ratio): 1.5% of edible glucose, 1.5% of fish peptone, 1.5% of yeast extract powder, 0.05% of magnesium sulfate, 0.02% of potassium dihydrogen phosphate and the balance of water.
The preparation process of the Saccharomyces cerevisiae YD-01 liquid fermentation inoculant comprises the following steps:
(1) Shake flask seed liquid acquisition: scribing Saccharomyces cerevisiae YD-01 in YPD solid culture medium under aseptic condition, culturing at 28 deg.C for 20-24 hr, and observing the presence or absence of bacteria; inoculating the annular lawn into YPD liquid culture medium with inoculating loop after activation, and culturing at 28 deg.C and 150r/min under shaking for 12-14 hr to obtain YD-01 shake flask seed liquid;
(2) Obtaining primary seed liquid: injecting water according to 60-70% of the volume of the tank body, feeding according to the formula of the first-stage seed liquid, adjusting pH to about 7.0 after the raw materials are completely dissolved, adding a defoaming agent according to 0.05%, and sterilizing for 30min at 121 ℃ under 0.1-0.12 MPa; after sterilization, the pH is required to be ensured to be between 6.5 and 7.0, the seed liquid is inoculated into a fermentation tank (500L) according to the inoculation amount of 0.5 percent, and the fermentation culture is carried out for 10 to 12 hours under the conditions of 28 ℃ and 120r/min and the aeration ratio of 1.2vvm, and then the seed liquid is YD-01 first-grade seed liquid;
(3) And (3) carrying out mass fermentation culture: injecting water according to 60-70% of the volume of the tank body, feeding according to a large amount of fermentation medium formula, adjusting pH to about 7.0 after the raw materials are completely dissolved, adding a defoaming agent according to 0.05%, and sterilizing for 30min at 121 ℃ under 0.1-0.12 MPa; after sterilization, the pH value is ensured to be between 6.5 and 7.0, the first-stage seed liquid is inoculated into a fermentation tank (2000L) according to the inoculation amount of 5 percent, and the fermentation culture is carried out for 20 to 22 hours under the conditions of 28 ℃ and 120r/min and the ventilation ratio of 1.2vvm, and the viable count is above 200 hundred million/mL at the moment, thus the YD-01 liquid fermentation microbial inoculum is obtained.
Example 5: preparation method of apple wine base
The method for sectional fermentation by adopting Aspergillus niger PWY-03 solid fermentation inoculant and Saccharomyces cerevisiae YD-01 liquid fermentation inoculant takes fresh red Fuji apples and red glutinous rice as raw materials, no exogenous components are added, and the novel apple wine base is obtained by pure natural fermentation, and the specific method is as follows:
(1) Weighing raw materials; weighing 95 parts of fresh apples and 5 parts of red glutinous rice according to the weight ratio;
(2) Material treatment and saccharification; soaking fructus Mali Pumilae for 24 hr, peeling, removing core, and crushing; grinding red glutinous rice in advance; placing the mixture in a saccharification tank after fully mixing, inoculating Aspergillus niger PWY-03 solid fermentation inoculant according to an inoculum size of 1.5%, and saccharifying for 5-7 d at 28 ℃;
(3) Fermenting with alcohol; after saccharification is completed, inoculating a Saccharomyces cerevisiae YD-01 liquid fermentation inoculant according to 0.1% of inoculation amount, keeping the temperature at 25-30 ℃, and continuing fermentation for 12-15 d;
(4) After the alcoholic fermentation is completed, the temperature is reduced to 8-15 ℃, the fermentation is continued for 5-7 d, and the filtration is carried out;
(5) Settling, secondary filtering and pasteurizing to obtain the high-quality apple wine base with the alcohol content of 20-22%vol.
To demonstrate the ability to produce high quality cider base wine using red glutinous rice and the preparation method of the present invention, the following comparative test was performed based on example 5:
comparative example 1:
comparative example 1 differs from example 5 in that: the fermentation raw material is fresh red Fuji apples, and red glutinous rice is not added.
Comparative example 2:
comparative example 2 differs from example 5 in that: the fermentation raw material is 95 parts of fresh red Fuji apples and 5 parts of Kangyou 188 (indica type three-line hybrid rice).
Comparative example 3:
comparative example 3 differs from example 5 in that: the Saccharomyces cerevisiae is YD-05.
Comparative example 4:
comparative example 4 differs from example 5 in that: the Saccharomyces cerevisiae is YD-06.
Different quality apple wine bases are prepared according to the method described in the embodiment 5, and the basic physicochemical properties of the different quality apple wine bases are detected according to the general technical requirements of QB/T5476-2020 fruit wine, and the method mainly comprises the following steps: the alcohol content, total sugar, dry extract and volatile acid are measured by the method specified in GB/T15038-2006.
The apple wine base obtained in example 5 has the highest alcohol content of 20-22%vol (refer to Table 4); the apple base wine obtained in example 5 has higher alcoholic strength than comparative examples 1 and 2, indicating that the ingredients contained in red glutinous rice are more suitable for cider brewing (see Table 4).
The apple wine base obtained in example 5 has higher alcoholic strength than that of comparative examples 3 and 4 (refer to Table 4), which shows that Aspergillus niger PWY-03 and Saccharomyces cerevisiae YD-01 have excellent fermentation ability and stable effect.
The highest dry extract content of the cider base wine obtained in example 5 was 43.3g/L (see Table 4), which indicates that the cider wine contains a large amount of non-sugar substances, has a mellow and thick aroma and is high in quality.
The total sugar and volatile acid content of the cider base wine obtained in example 5 meet the standards specified by QB/T5476-2020, which shows that the fermentation process in this test is well controlled and the process described in example 5 is feasible.
TABLE 4 partial physicochemical index of different quality apple wine base
To further demonstrate the feasibility of the cider base fermentation process described in example 5, the content of aroma substances contained in the cider base obtained in example 5 and comparative examples 1 to 4 was determined by GC-MS analysis technique, and the method mainly comprises: lipid, acid and aldehyde ketone species, the results are shown in Table 5. Analysis shows that the total amount of lipid substances, aldehyde ketone substances and aroma substances of the cider raw wine prepared by the process described in the example 5 is obviously higher than that of cider raw wine produced by other processes, and further proves that the fermentation process of the cider raw wine described in the example 5 is feasible.
TABLE 5 fragrance content of different quality apple wine base
Example 6: preparation and screening of high-quality cider
Based on the process described in example 5, the apple wine base with 20-22%vol is prepared by adding fresh concentrated apple juice (the clarified juice is concentrated 8 times by vacuum concentration equipment), blending, filtering, settling, secondary filtering and sterilizing, and the apple wine with different alcoholicity (1.5%vol, 3.5%vol, 5.5%vol and 7.5%vol) is prepared. The sensory evaluation of the 4 types of cider prepared according to the standard specified by national standard GB/T15038-2006 is mainly carried out in terms of color, aroma, taste and the like, and finally a high-quality cider, namely, the high-quality cider with the alcohol content of 3.5 percent vol, is screened out.
The evaluation criteria are referred to in Table 6.
TABLE 6 cider sensory scoring criteria
The 4-section cider thus prepared was evaluated by 10 professionals and scored according to the table 6 standard, and the results are shown in table 7. It was found that cider with an alcohol content of 3.5% vol gave a higher scoring result than other alcohol content cider, regardless of color, aroma, taste and representativeness, which met the first criterion. This result demonstrates that, based on the preparation of cider base according to the cider base fermentation process described in example 5, the addition of concentrated cider juice for blending, 3.5% vol cider is the most preferred choice.
Table 7 cider sensory scores of different alcoholicity
Claims (10)
1. A strain with high yield of diastase is Aspergillus niger (Aspergillus niger) PWY-03 with a preservation number of CGMCC No.40486.
2. A strain with strong alcoholic fermentation capability is Saccharomyces cerevisiae (Saccharomyces cerevisiae) YD-01 with a preservation number of CGMCC No.26465.
3. The preparation method of the apple wine base is characterized by adopting aspergillus niger PWY-03 as claimed in claim 1 for saccharification and fermentation and Saccharomyces cerevisiae YD-01 as claimed in claim 2 for alcoholic fermentation, and specifically comprises the following steps:
(1) Preparation of fermentation inoculant
Fermenting, drying and crushing Aspergillus niger PWY-03 to obtain Aspergillus niger PWY-03 solid fermentation microbial inoculum with spore content more than or equal to 20 hundred million/g; fermenting the saccharomyces cerevisiae YD-01 to obtain a saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum with the bacterial activity of more than or equal to 200 hundred million/mL;
(2) Pretreatment of raw materials
The raw materials are as follows: 95+/-1 parts of fresh red Fuji apples and 5+/-1 parts of red glutinous rice;
soaking fructus Mali Pumilae, peeling, removing core, and crushing; grinding red glutinous rice in advance; fully mixing the two materials and then placing the mixture in a saccharification tank;
(3) Saccharification
Then inoculating Aspergillus niger PWY-03 solid fermentation inoculant according to the mass ratio of 1-2%, and saccharifying for 5-7 d at 25-30 ℃;
(4) Alcohol fermentation
After saccharification is finished, inoculating a Saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum according to the mass ratio of 0.05-0.1%, and keeping the temperature at 25-30 ℃ for continuous fermentation for 12-15 d; after the alcoholic fermentation is completed, the temperature is reduced to 8-15 ℃, the fermentation is continued for 5-7 d, and the filtration is carried out; settling, secondary filtering and sterilizing to obtain apple wine base.
4. The method for preparing apple wine base according to claim 3, wherein the method for preparing the Aspergillus niger PWY-03 solid fermentation inoculant comprises the following steps: activating Aspergillus niger PWY-03, inoculating the activated Aspergillus niger into a PDB culture medium, and vibrating at a constant temperature of 25-30 ℃ for 3-5 d to obtain seed liquid; then inoculating the seed liquid into a solid fermentation medium according to the mass ratio of 10-20%, fermenting at the fermentation temperature of 26-28 ℃ and the fermentation humidity of more than 85%, drying and crushing after fermentation to obtain the aspergillus niger PWY-03 solid fermentation microbial inoculum with the spore content of more than or equal to 20 hundred million/g.
5. The method for preparing apple wine base according to claim 4, wherein the solid fermentation medium comprises the following components in mass ratio: 30% of starch, 8% of wheat bran, 25% of rice hull powder, 0.5% of glucose and 0.5% of CaCO 3 3%, magnesium sulfate 0.2%, monopotassium phosphate 0.15%, red glutinous rice flour 33.15%; the water content is controlled to 55-60% and the pH is controlled to 6-5.5.
6. The method for preparing apple wine base according to claim 3, wherein the method for preparing the Saccharomyces cerevisiae YD-01 liquid fermentation inoculant comprises the following steps: activating Saccharomyces cerevisiae YD-01, inoculating the activated Saccharomyces cerevisiae YD-01 into YPD liquid culture medium, and carrying out shaking culture at a constant temperature of 25-30 ℃ for 12-14 h to prepare YD-01 shake flask seed liquid; inoculating seed liquid into a fermentation tank filled with a primary seed liquid culture medium according to the volume ratio of 0.3-0.8%, and fermenting and culturing for 10-12 h at 25-30 ℃ to prepare YD-01 primary seed liquid; the first-stage seed liquid is inoculated into a fermentation tank filled with a large amount of fermentation medium according to the volume ratio of 3-8%, and is fermented and cultured for 20-22 hours at the temperature of 25-30 ℃ to obtain the saccharomyces cerevisiae YD-01 liquid fermentation microbial inoculum with the bacterial activity of more than or equal to 200 hundred million/mL.
7. The method for preparing apple wine base according to claim 6, wherein the primary seed liquid culture medium comprises the following components in mass ratio: glucose 1%, peptone 1.5%, beef powder 0.3%, sodium chloride 0.5%, and water the balance, pH 6.5-7.0.
8. The method for preparing apple wine base according to claim 6, wherein the mass fermentation medium comprises the following components in mass ratio: 1.5% of edible glucose, 1.5% of fish peptone, 1.5% of yeast extract powder, 0.05% of magnesium sulfate, 0.02% of monopotassium phosphate and the balance of water, wherein the pH value is 6.5-7.0.
9. Apple wine base prepared by the method of any one of claims 3-8, having an alcohol content of 20-22%vol.
10. The high-quality cider is characterized in that the apple wine base is prepared by adding fresh concentrated apple juice, filtering, settling, secondary filtering and sterilizing, and the high-quality cider with the alcohol content of 3.5% vol is prepared.
Priority Applications (1)
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