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CN-118460389-B - Non-saccharomyces cerevisiae and preparation and application of immobilized yeast capsules thereof

CN118460389BCN 118460389 BCN118460389 BCN 118460389BCN-118460389-B

Abstract

The invention provides non-saccharomyces cerevisiae which is Wick ham yeast Wickerhamomycesanomalus with a preservation number of CGMCC No.25528, a preservation unit of China general microbiological culture Collection center with a preservation date of 2022, 8 months and 12 days. The preparation method of the non-saccharomyces cerevisiae immobilized yeast capsule is also provided, the immobilized yeast capsule prepared under the optimal condition has high hardness and good permeability, can improve ethanol tolerance, and can be inoculated and fermented with saccharomyces cerevisiae synchronously. Meanwhile, the invention also provides application of the immobilized yeast capsule in brewing wine. The immobilized yeast capsule brewing method can greatly improve the total amount of volatile substances in the wine, and improve the aroma complexity of the wine, thereby improving the quality of the wine.

Inventors

  • WANG JUN
  • HAN JING

Assignees

  • 山西农业大学

Dates

Publication Date
20260505
Application Date
20230714
Priority Date
20220929

Claims (7)

  1. 1. A method for preparing an immobilized yeast capsule by using non-saccharomyces cerevisiae is characterized in that the non-saccharomyces cerevisiae is Wick ham yeast Wickerhamomyces anomalus with a preservation number of CGMCC No. 25528, a preservation unit is China general microbiological culture Collection center with a preservation date of 2022 and 8 months and 12 days, and a 26S rDNA nucleotide sequence of the non-saccharomyces cerevisiae is shown as SEQ ID NO: 1, and the method comprises the following steps: S1, preparing bacterial suspension, namely inoculating frozen non-saccharomyces cerevisiae into YPD liquid culture medium under aseptic condition, culturing for 48 hours at the temperature of 28 ℃, repeating three times of subculture for activation, centrifuging at the temperature of 4 ℃ and the rotating speed of 4500r/min, diluting the collected thalli with sterile normal saline, preparing bacterial suspension with the concentration of 10 10 cfu/mL, and refrigerating at the temperature of 4 ℃ for later use; S2, preparing an immobilized yeast capsule, namely taking out 10mL of bacterial suspension for cooling in the S1, naturally recovering the bacterial suspension to room temperature, adding the bacterial suspension into 1L of sodium alginate solution with the mass fraction of 2%, uniformly stirring the solution to ensure that the total number of bacterial clusters in each milliliter of solution is 10 8 cfu, then dripping 2L of sterilized calcium chloride solution with the mass fraction of 2%, uniformly stirring the solution, solidifying the solution for 0.5h to form immobilized capsule particles, washing the immobilized capsule particles with sterile physiological saline for 3 times, transferring the immobilized capsule particles into 3L of chitosan solution with the mass fraction of 1.5%, oscillating the immobilized capsule particles for 3h, and washing the immobilized yeast capsule with the sterile physiological saline for 3 times, wherein the viscosity of the sodium alginate solution is 115 mPa.s, and the molecular weight of chitosan in the chitosan solution is 25 Da.
  2. 2. The method for preparing immobilized yeast capsules from non-Saccharomyces cerevisiae according to claim 1, wherein the YPD liquid medium in S1 is prepared from the following raw materials, by mass, 2% glucose, 2% peptone and 1% yeast extract, the balance being water, and sterilizing at 121 ℃ for 20 min.
  3. 3. The method for preparing immobilized yeast capsules from non-saccharomyces cerevisiae according to claim 1, wherein the ethanol tolerance of the immobilized yeast capsules in S2 is 9% vol.
  4. 4. The method for preparing immobilized yeast capsules from non-Saccharomyces cerevisiae according to claim 2, wherein the average particle size of the immobilized yeast capsules in S2 is 2.301-3.829 mm.
  5. 5. Use of an immobilized yeast capsule prepared by the method of any one of claims 1-4, wherein the immobilized yeast capsule is used for brewing wine to increase the content of volatile substances; the method for brewing the wine comprises the following steps: S1, removing stems of grapes, crushing, directly loading into a fermentation tank or squeezing, loading into the fermentation tank to obtain grape juice, loading the grape juice into the fermentation tank, adding sulfurous acid to enable the concentration of the sulfurous acid to be 60mg/L, standing for 4h, adding pectase to enable the final concentration of the pectase to be 20mg/L, and fermenting for 48 hours at the temperature of 4 ℃ to obtain grape fermentation liquor; S2, after the grape fermentation broth obtained in the step S1 is restored to room temperature, inoculating immobilized yeast capsules with the inoculation amount of 10 6 cfu/mL into the grape fermentation broth, fermenting 24-h, inoculating activated commercial Saccharomyces cerevisiae with the inoculation amount of 10 6 cfu/mL, fully stirring, standing and fermenting at 18 ℃ or 26 ℃, sealing a fermentation tank by a fermentation plug, filtering and separating out a mixture of the immobilized yeast capsules and grape seeds when the ethanol concentration reaches 9%vol, obtaining grape mash, fermenting the grape mash under the action of free yeast in the liquid until the residual sugar amount in the grape wine is less than 2g/L, and stopping fermenting, and filtering residues to obtain the grape wine.
  6. 6. The use according to claim 5, wherein when the grape is a grape variety for brewing red wine in S1, the grape is split, directly fed into the fermenter and fed into the fermenter with a volume fraction of 80%, and when the grape is a grape variety for brewing white wine in S1, the grape is split, pressed and fed into the fermenter with a volume fraction of 90%.
  7. 7. The use according to claim 5, wherein the temperature of the resting fermentation in S2 is 26 ℃ when the grape in S1 is a grape variety for brewing red wine and the temperature of the resting fermentation in S2 is 18 ℃ when the grape in S1 is a grape variety for brewing white wine.

Description

Non-saccharomyces cerevisiae and preparation and application of immobilized yeast capsules thereof Technical Field The invention belongs to the technical field of fermented glutinous rice, and particularly relates to non-saccharomyces cerevisiae and preparation and application of immobilized yeast capsules thereof. Background At present, wine enterprises in different production areas throughout the country and even worldwide have serious identity in the wine brewing process, and the characteristics and individuality of the wine products are not outstanding due to single commercial saccharomyces cerevisiae fermentation. In recent years, research has shown that non-Saccharomyces cerevisiae plays a vital role in wine brewing, and that non-Saccharomyces cerevisiae has changed from an undesirable yeast associated with spoilage of wine to a microorganism that can improve the aromatic properties of wine, and that non-Saccharomyces cerevisiae has been a hotspot in wine research in its continuous fermentation in conjunction with Saccharomyces cerevisiae strains. At present, about 20 non-Saccharomyces cerevisiae (non-Saccharomyces) of genus Schizosaccharomyces (Schizosaccharomyces), hansenula (Hanseniaspora), pichia (Pichia), issatchenkia (ISSATCHENKIA), klebsiella (Kloeckera), candida (Candida), saccharomyces cerevisiae (Metschnikowia), hansenula (Hansenula), and, cryptococcus, zygosaccharomyces, rhodotorula, and the like, which are non-yeast strains capable of increasing acidity, aromatic complexity, glycerol content, ethanol reduction, mannoprotein, rhodotorula, and the like, Anthocyanin and polysaccharide concentration and even bioactive compounds can be produced, wherein the pH is changed by organic acid generated by ① non-saccharomyces cerevisiae to realize the increase of anthocyanin color, the released metabolite or high expression of hydroxycinnamate decarboxylase activity can promote the formation of stable pigment, the formation of pyran anthocyanin and polymeric pigment can be enhanced, for example, the schizosaccharomyces pombe increases the release of extracellular pyruvic acid, the higher the extracellular release amount of pyruvic acid is, the higher the generation amount of stable pyran pigment is, ② non-saccharomyces cerevisiae can also release zymosan, the process of wine residue Ageing (AOL) is accelerated, the wine structure is improved, the existence of ③ non-saccharomyces cerevisiae can lead to the reduction of alcohol concentration, and terpenes, The concentration of esters, higher alcohols, glycerol, acetaldehyde, acetic acid and succinic acid increases, the presence of specific enzymes in ④ non-Saccharomyces cerevisiae, such as non-Saccharomyces cerevisiae non-encoded glycosidases, allows the non-volatile precursors to be enzymatically hydrolyzed to release aroma substances, thereby affecting flavor compounds, and ⑤ non-Saccharomyces cerevisiae also produces a killer to control wine spoilage microorganisms and their unpleasant taste. thus, the important role of non-Saccharomyces cerevisiae in wine production is increasingly prominent. At present, commercial non-saccharomyces cerevisiae strains at home and abroad comprise Mei-Ji-chi yeast (Metschnikowiapulcherrima), de-Bu-have-spore torula (Torulaspora delbrueckii), heat-resistant Kluyveromyces (LACHANCEA THERMOTOLERANS), kluyveromyces pichia (Pichia kluyvery), schizosaccharomyces pombe (Schizosaccharomycespombe) and the like, but the abnormal Wick-han yeast (Wickerhamomyces anomalus) has not been commercially applied to wine. In the co-fermentation process of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae, first, direct interactions exist between yeast cells, such as physical contact and quorum sensing between microorganisms. The volatile matter content of the mixed fermentation of kluyveromyces thermotolerans and saccharomyces cerevisiae is significantly changed compared with that of the non-contact mixed fermentation, and obviously, the metabolism of the yeast is changed by cell-cell contact. Some quorum sensing molecules, such as tyrosol, tryptophan and phenethyl alcohol, also participate in yeast-yeast interactions and affect yeast growth. Second, yeast strains produce a killer and kill yeasts to which they are sensitive. Albergaria et al (2010) found that the 2-10kDa protein of Saccharomyces cerevisiae CCMI 885 fermentation supernatant had bacteriostatic, even bactericidal, effects on some strains of Kluyveromyces, torulaspora and Hansenula. In addition, in the ethanol fermentation process, as the concentration of ethanol increases, stress is generated on the growth of yeast cells, and the yeast cells can generate corresponding stress response for survival and growth to cope with the stress, wherein the coping mechanism is that the yeast cells have tolerance to the ethanol. non-Saccharomyces cerevisiae has the disadvantage of poor ethanol tolerance, generally below 5% ethanol concentration. Therefore, there is a nee