Composite protective agent, preparation method of active dry yeast, active dry yeast and application

Abstract

The invention discloses a composite protective agent, a preparation method of active dry yeast, the active dry yeast and application, and belongs to the technical field of bioengineering and fermented food. The composite protective agent comprises a first-phase biological enhancer and a second-phase film-forming protective agent, the first-phase biological enhancer comprises palmitoleic acid and trehalose; the second-phase film-forming protective agent comprises trehalose; the first-phase biological enhancer is added when the yeast grows into the logarithmic growth later stage or the residual sugar of the fermentation liquor is reduced to 40-60% of the residual sugar of the initial fermentation liquor; and the second-phase film-forming protective agent is added before drying. The active dry yeast protective agent is applied to the aspect of active dry yeast preparation, the problem that an existing active dry yeast protective agent for the beer industry cannot prevent leakage of intracellular substances (such as K < + > and nucleotide) is solved, the ultra-high viable bacterium rate of 95% or above can be achieved, and the foam stability and ester flavor of beer are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and fermented food technology, and particularly relates to a compound protective agent, a method for preparing active dry yeast, active dry yeast and its applications. Background Technology

[0002] The current production process of active dry yeast for the beer industry mainly includes: high-density yeast cultivation, centrifugation, mixing of protective agents, granulation, and fluidized bed drying. Among these, the protective agent is usually mixed with the wet yeast sludge after cell collection and before drying. The components of the protective agent are mostly trehalose, sorbitan monostearate (SMS), etc.

[0003] However, existing protectants still have the following problems when used in the production of active dry yeast for the beer industry: 1. Protection only stays on the surface: In traditional "pre-drying mixing" processes, the protective agent only physically adheres to the cell surface; during rehydration, the cell membrane phospholipid layer is prone to undergoing a "gel-like" phase transition, leading to physical damage, and the protective agent cannot prevent intracellular substances (such as potassium) from being absorbed. + Leakage of nucleotides; 2. Insufficient endogenous stress resistance: Industrially cultured yeast often pursues biomass, resulting in low levels of endogenous trehalose and unsaturated fatty acids in cells, and poor resistance to drying and oxidation. 3. Flavor and foam defects: Damaged yeast is prone to producing a large amount of diacetyl (creamy / stale flavor) during fermentation, and ordinary preservatives cannot improve the foam retention of beer. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is that existing protectants for active dry yeast used in the beer industry cannot prevent intracellular substances (such as K...) from entering the cell. + This paper addresses the leakage of nucleotides and proposes a composite protectant that can achieve an ultra-high viable bacterial rate of over 95% and significantly improve the foam stability and ester aroma of beer. It also describes the preparation method of active dry yeast, the application of active dry yeast, and other related technologies.

[0005] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: The present invention provides a composite protective agent, comprising a first-phase bio-fortifier and a second-phase film-forming protective agent; the first-phase bio-fortifier comprises palmitoleic acid and trehalose; the second-phase film-forming protective agent comprises trehalose; the first-phase bio-fortifier is added when yeast enters the late logarithmic growth stage or when the residual sugar in the fermentation broth drops to 40-60% of the initial residual sugar in the fermentation broth; the second-phase film-forming protective agent is added before drying.

[0006] In some embodiments, the first-phase bio-enhancing agent accounts for 20-30% of the dry weight of the composite protective agent, and the second-phase film-forming protective agent accounts for 70-80% of the dry weight of the composite protective agent.

[0007] In some embodiments, the first-phase bio-enhancing agent includes palmitoleic acid, a nitrogen source signal, and a carbon source signal; the second-phase film-forming protective agent includes trehalose, fillers, antioxidants, auxiliary antioxidants, and emulsifiers.

[0008] In some embodiments, the nitrogen source signal is barley peptide, the carbon source signal is trehalose, the filler is barley peptide, the antioxidant is glutathione, the co-antioxidant is sodium ascorbate, and the emulsifier is sorbitan monostearate.

[0009] Another aspect of the present invention provides a method for preparing active dry yeast, using the composite protectant described in any of the above technical solutions as the protectant, comprising: conducting multi-stage expansion culture of brewing yeast, and in the last generation culture process, when the yeast growth enters the late logarithmic growth stage or the residual sugar in the fermentation broth drops to half of the initial residual sugar in the fermentation broth, adding a first-phase biofortifier to the fermenter.

[0010] In some embodiments, after the yeast with the first bio-fortifier is cultured, the yeast cells are collected to obtain fortified yeast slurry, and a second film-forming protectant is added to the fortified yeast slurry.

[0011] In some embodiments, yeast with added first-phase biofortifier is cultured for 4-6 hours to induce the yeast to integrate specific fatty acids into the cell membrane and accumulate intracellular trehalose; the yeast cells are collected by centrifugation, washed with water, and fortified yeast slurry with a solid content of 15-25% is obtained, and second-phase film-forming protectant is added to the fortified yeast slurry.

[0012] In some embodiments, a second-phase film-forming protectant is added to the enhanced yeast slurry; high-speed stirring emulsification is performed to form an external protective film; extrusion granulation is carried out, and the slurry is dried in a fluidized bed with three-stage variable temperature to a moisture content of 4%-6%.

[0013] The present invention also provides active dry yeast prepared by the method described in any of the above technical solutions, wherein the viability rate of the active dry yeast is >95%.

[0014] The present invention also provides the application of the active dry yeast described above in beer brewing, wherein the diacetyl content in the beer is not greater than 0.03 mg / L, the foam retention is greater than 290 seconds, and the isoamyl acetate content is not less than 2.25 mg / L.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a composite protectant. By limiting the composition of the first-phase biofortifier and the second-phase film-forming protectant, as well as the timing of their addition, a method for preparing "internal repair and external resistance" active dry yeast is provided. The first-phase biofortifier is added at the end of the culture period to induce the yeast to reorganize the cell membrane structure and accumulate intracellular stress-resistant substances. Combined with the film-forming embedding before drying (second-phase film-forming protectant), an ultra-high viable cell rate of over 95% is achieved, significantly improving the foam stability and ester aroma of beer. Attached Figure Description

[0016] Figure 1 Comparison of gas chromatographic (GC) images of fatty acid components in cell membranes; Figure 2 This is a comparison of cell membrane fluidity after rehydration (fluorescence polarization P-value). Detailed Implementation

[0017] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0018] The present invention provides a composite protective agent, comprising a first-phase bio-fortifier and a second-phase film-forming protective agent; the first-phase bio-fortifier comprises palmitoleic acid and trehalose; the second-phase film-forming protective agent comprises trehalose; the first-phase bio-fortifier is added when yeast enters the late logarithmic growth stage or when the residual sugar in the fermentation broth drops to 40-60% of the initial residual sugar in the fermentation broth; the second-phase film-forming protective agent is added before drying.

[0019] In the aforementioned composite protective agent, palmitoleic acid (C16:1) serves as a membrane lipid precursor, enhancing membrane fluidity; trehalose, added when yeast enters the late logarithmic growth stage or when the residual sugar in the fermentation broth drops to 40-60% of the initial residual sugar, acts as a carbon source signal, inducing intracellular trehalose synthase (TPS1) expression; and trehalose added before drying replaces water molecules during drying, forming a glassy protective state.

[0020] By "feeding" the yeast with palmitoleic acid during the culture period, the ratio of unsaturated fatty acids (C16:1 / C16:0) in the yeast cell membrane was significantly increased. This endogenous change in membrane structure is more resistant to cold shock and dry phase transition than external addition. Trehalose is added in two stages as both a component of the first-phase biofortifier and a component of the second-phase film-forming protectant, achieving dual trehalose protection. Not only is there trehalose encapsulation on the outside, but a high concentration of trehalose is also accumulated inside due to metabolic induction, achieving comprehensive protection from the inside out.

[0021] The above technical solution specifies that the first-phase biofortifier should be added when the yeast enters the late logarithmic growth stage or when the residual sugar in the fermentation broth drops to 40-60% of the initial residual sugar (preferably when the residual sugar in the fermentation broth drops to half of the initial residual sugar). The reason is that adding it at this time can act as a signal to induce the yeast to reorganize the cell membrane structure (integrating palmitoleic acid into the membrane to improve fluidity) and accumulate intracellular stress-resistant substances (inducing the expression of trehalose synthase to accumulate intracellular trehalose), thus making it more resistant to cold shock and drying phase transition than simply adding it externally. The second-phase film-forming protectant is added before drying, using trehalose to replace water molecules to form a glassy protective state during drying, and preventing the leakage of intracellular substances (such as K+ and nucleotides) through film formation and embedding. Combined with the enhancement of intracellular metabolism, it achieves comprehensive protection from the inside out.

[0022] In some embodiments, the first-phase bio-enhancing agent accounts for 20-30% of the dry weight of the composite protective agent, and the second-phase film-forming protective agent accounts for 70-80% of the dry weight of the composite protective agent.

[0023] The above technical solution limits the dosage of the first-phase bio-enhancer and the second-phase film-forming protectant because the first phase mainly acts as a "signal and precursor," and a lower dose (20-30%) is sufficient to meet the metabolic induction requirements and trigger the cell's "internal repair" mechanism (accumulation of intracellular trehalose and reconstruction of the cell membrane); while the second phase mainly acts as a "physical embedding and filling" mechanism, requiring a higher dose (70-80%) to provide sufficient matrix to form a complete external vitreous protective film ("external resistance") to buffer drying stress and effectively prevent intracellular leakage.

[0024] Understandably, the first-phase bio-enhancing agent can also account for any value within the range of 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% of the dry weight of the composite protective agent, and the second-phase film-forming protective agent can also account for any value within the range of 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% of the dry weight of the composite protective agent.

[0025] In some embodiments, the first-phase biofortifier includes palmitoleic acid, a nitrogen source signal, and a carbon source signal; the second-phase film-forming protectant includes trehalose, a filler, an antioxidant, a co-antioxidant, and an emulsifier. In some embodiments, the nitrogen source signal is barley peptide, the carbon source signal is trehalose, the filler is barley peptide, the antioxidant is glutathione, the co-antioxidant is sodium ascorbate, and the emulsifier is sorbitan monostearate.

[0026] In the above technical solution, barley peptides (low molecular weight) serve as a nitrogen source signal and simultaneously form a hydration layer in the cell wall; barley peptides also act as a filler to buffer drying stress; glutathione (GSH) acts as an antioxidant to scavenge free radicals. The introduction of barley peptides (partially entering the cell wall and partially existing extracellularly) improves beer foam retention by more than 20%.

[0027] Another aspect of the present invention provides a method for preparing active dry yeast, using the composite protectant described in any of the above technical solutions as the protectant, comprising: conducting multi-stage expansion culture of brewing yeast, and in the last generation culture process, when the yeast growth enters the late logarithmic growth stage or the residual sugar in the fermentation broth drops to half of the initial residual sugar in the fermentation broth, adding a first-phase biofortifier to the fermenter.

[0028] The above-mentioned method for preparing active dry yeast is a "internal repair and external resistance" method. By adding a bio-fortifier at the end of the culture period, the yeast is induced to reorganize its cell membrane structure and accumulate intracellular stress-resistant substances. Combined with the film-forming and embedding before drying, an ultra-high viable cell rate of over 95% is achieved, and the foam stability and ester aroma of beer are significantly improved.

[0029] The above technical solution involves first conducting multi-stage large-scale culture of Saccharomyces cerevisiae, followed by metabolic biofortification in the last generation ("biofortification" refers to the process of altering cell membrane lipid composition and intracellular solute concentration through metabolic pathways). This is to overcome the problem of insufficient stress resistance caused by low levels of endogenous trehalose and unsaturated fatty acids in cells due to high biomass. By adding fortifiers during a specific physiological window in the late logarithmic growth phase of the last generation, the yeast can be induced to shift its metabolic flow from "cell proliferation" to "accumulation of endogenous stress-resistant substances" (reconstructing cell membranes and synthesizing intracellular trehalose), thereby endowing the cells with stronger endogenous resistance to cold shock and dry phase transition before drying.

[0030] In some embodiments, after the yeast with the first-phase biofortifier is cultured further, the yeast cells are collected to obtain fortified yeast slurry, and a second-phase film-forming protectant is added to the fortified yeast slurry. In some embodiments, the yeast with the first-phase biofortifier is cultured for another 4-6 hours to induce the yeast to integrate specific fatty acids into the cell membrane and accumulate intracellular trehalose; the yeast cells are collected by centrifugation, washed with water, and fortified yeast slurry with a solid content of 15-25% is obtained, and a second-phase film-forming protectant is added to the fortified yeast slurry.

[0031] The above-described method for preparing active dry yeast enhances yeast activity and improves beer flavor by introducing metabolic regulators during the cultivation stage and surface-coating them during the drying stage. In some embodiments, a fortified yeast sludge with a solids content of 20% is obtained.

[0032] In some embodiments, a second-phase film-forming protectant is added to the enhanced yeast slurry; high-speed stirring emulsification is performed to form an external protective film; extrusion granulation is carried out, and the slurry is dried in a fluidized bed with three-stage variable temperature to a moisture content of 4%-6%.

[0033] The present invention also provides active dry yeast prepared by the method described in any of the above technical solutions, wherein the viability rate of the active dry yeast is >95%.

[0034] This invention also provides the application of the active dry yeast described above in beer brewing, wherein the diacetyl content in the beer is no more than 0.03 mg / L, the foam retention is greater than 290 seconds, and the isoamyl acetate content is no less than 2.25 mg / L. The ultra-high viable bacteria rate (>96%) results in extremely rapid fermentation initiation, rapid diacetyl reduction, and a significant increase in the production of ester aroma compounds.

[0035] To provide a clearer and more detailed description of the composite protective agent, the preparation method of active dry yeast, the active dry yeast and its applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0036] Example 1 1) Multi-stage expansion culture: The Saccharomyces cerevisiae strain is inoculated into the culture medium and expanded step by step.

[0037] 2) Last-generation metabolic enhancement: During the final generation of culture, when the residual sugar in the fermentation broth dropped to half of the initial residual sugar, a first-phase biofortifier (containing 0.15% palmitoleic acid, 0.5% barley peptide, and 1% trehalose by volume of the culture medium) was added, and the culture was continued for 5 hours to induce the yeast to integrate specific fatty acids into the cell membrane and accumulate intracellular trehalose.

[0038] 3) Cell separation and washing: Collect yeast cells by centrifugation, wash with water, and obtain fortified yeast slurry with a solid content of about 20%.

[0039] 4) Surface embedding treatment: Collect the bacterial cells and add a second-phase film-forming protectant (containing 2.5% trehalose, 1% barley peptide, 0.2% glutathione, and 1.2% SMS) to the yeast slurry; emulsify by high-speed stirring to form an external protective film.

[0040] 5) Granulation and drying: Extrusion granulation, followed by three-stage fluidized bed temperature-controlled drying to a moisture content of 5.2%.

[0041] Comparative Example 1 Same as Example 1, except that no incubation period enhancement was performed. After cell collection, all the above-mentioned protective agent components (including palmitoleic acid) were added at once.

[0042] Comparative Example 2 Similar to Comparative Example 1, except that SMS and sucrose were added only before drying.

[0043] Comparative Example 3 1) Without intensifying the culture period, add the first-phase biofortifier (containing 0.15g palmitole, 0.5g barley peptide, and 1.0g trehalose) and continue culturing for 5 hours; 2) Cell separation and washing: Collect yeast cells by centrifugation, wash with water, and obtain fortified yeast slurry with a solid content of about 20%.

[0044] 3) Surface embedding treatment: Collect 100g of bacterial cells (wet weight), and add a second-phase film-forming protectant (trehalose 2.5g, barley peptide 1.0g, glutathione 0.2g, SMS 1.2g) to the yeast slurry; emulsify by high-speed stirring to form an external protective film.

[0045] 4) Granulation and drying: Extrusion granulation, followed by three-stage fluidized bed temperature-controlled drying to a moisture content of 5.2%.

[0046] Performance testing Physiological parameters and microstructure of the dry yeast obtained in the examples and comparative examples were analyzed, and the results are shown in Table 1. The test methods were as follows: Rehydration viability: 10 mL of deionized water was added to yeast samples treated with different drying times, followed by rehydration treatment at 25℃ for 15 min. Methylene blue staining was used to quantitatively analyze changes in yeast cell viability. First, the dried and rehydrated yeast suspension was diluted to a suitable concentration. Then, 10 μL of methylene blue staining solution was added to 1 mL of yeast suspension, and staining was performed for 5 min. Cell counting was conducted using a five-point sampling method under an optical microscope (cell viability = number of viable cells / total number of cells × 100%).

[0047] Intracellular trehalose content: A trehalose standard solution with a concentration of 0.1 mg / mL was prepared, and solutions with trehalose concentrations of 0, 0.01, 0.02, 0.03, 0.04, 0.06, and 0.08 mg / mL were prepared. The intracellular trehalose content was determined according to the experimental method of Wang Zengmei et al. The absorbance versus trehalose content curve was fitted, yielding the linear equation y = 4.7455x + 0.0665, R0. 2 =0.9989, the sample was centrifuged at 8000 r / min for 5 min, and then the sample was measured according to the above method for trehalose determination.

[0048] Membrane unsaturation: Yeast culture cultured to the mid-to-late logarithmic growth stage (24 h) was centrifuged at 5000 rpm for 5 min at 4 °C, and the supernatant was discarded. The cell precipitate was washed 2-3 times with sterile distilled water to remove culture medium residue. The wet cells were collected and freeze-dried under vacuum to prepare dry cells, which were then ground into powder for later use. Approximately 50-100 mg of dry cells was weighed and placed in a pressure-resistant screw-capped glass test tube.

[0049] Extraction and methyl esterification of membrane fatty acids: Add 2 mL of 5% H₂SO₄-methanol solution to a test tube containing bacterial cells. Add 1 mL of toluene or n-hexane. Add 100 μL of an internal standard solution of known concentration (C17:0). Purge with nitrogen for 30 s to displace air from the tube and prevent fatty acid oxidation, then quickly tighten the cap. Heat the reaction in a 95-100°C water bath for 60-90 min, shaking vigorously every 20 min. After the reaction is complete, add 1 mL of n-hexane and 1 mL of saturated NaCl solution, and shake vigorously for 1 min to extract. Centrifuge at 2000 rpm for 5 min to separate the layers. Carefully transfer the upper organic phase (containing fatty acid methyl esters, FAMEs) to a new centrifuge tube. Add an appropriate amount of anhydrous sodium sulfate to dehydrate and dry, then proceed with analysis.

[0050] Gas chromatography (GC) detection conditions: Column: HP-88 highly polar capillary column (100m × 0.25mm × 0.2μm); Carrier gas: High-purity nitrogen (N2) or helium (He), flow rate 1.0mL / min, split ratio 10:1; Injector temperature: 250°C; Detector (FID) temperature: 280°C; Temperature program: Initial temperature 140°C, hold for 5 min; ramp to 240°C at a rate of 4°C / min; hold for 15 min; Injection volume: 1μL.

[0051] Qualitative and quantitative analysis: Qualitative analysis: The retention time of the chromatographic peak of the sample is compared with the retention time of the mixed standard of 37 fatty acid methyl esters to determine the type of each fatty acid (such as C16:0, C16:1, C18:0, C18:1, etc.).

[0052] Quantitative analysis: The relative percentage content of each fatty acid was calculated using the area normalization method. This represents the percentage of a specific fatty acid within the total fatty acid content. Unsaturation was calculated using the C16 / C16:1 ratio in yeast cell membranes.

[0053] Intracellular K +Leakage: Cell Preparation and Washing (Critical Steps) Culture yeast cells for 24 hours. Take a certain volume (20 mL) of bacterial suspension, centrifuge at 5000 rpm for 5 min at 4°C, and collect the cells. Resuspend the cells in sterile deionized water (DDW) and wash the precipitate three times. Resuspend the washed cells in an appropriate amount of sterile DDW and adjust the cell concentration to OD0.05. 600 =1.0, to prepare a cell suspension. Stress treatment and sampling grouping: Experimental group: Add the test drug or stress treatment to the cell suspension and incubate for a certain time (1h) under specified conditions. Negative control group: Add an equal volume of solvent to the cell suspension and incubate simultaneously. Total potassium control group: Take the untreated cell suspension and boil it in a boiling water bath for 15-20min to completely destroy the cell membrane and release all intracellular potassium ions. At the 12h time point, take 2mL of bacterial suspension from each group. Separation: Centrifuge at 8000rpm for 5min and carefully transfer the supernatant to a new clean test tube. Wavelength: 766.5nm; Slit width: 0.2-0.4nm; Lamp current: Sample determination: Measure the absorbance of the supernatant of each group of samples in sequence and substitute it into the standard curve to calculate the concentration. Data processing and calculation: The potassium ion leakage rate is calculated according to the following formula: Leakage (%) = ((C t —C0) / C total —C0))×100%. C t C0: Potassium ion concentration in the supernatant of the experimental group (stress group); C0: Potassium ion concentration in the supernatant of the negative control group (blank ... total Potassium ion concentration in the supernatant of the full potassium control group (maximum release).

[0054] Table 1. Physiological indicators and microstructure analysis of dried yeast

[0055] The membrane fatty acid ratio and fluorescence polarization degree of the dry yeast obtained in the examples and comparative examples were determined, and the results are as follows: Figure 1 , 2 As shown, by Figure 1 It was found that the process of this invention significantly improved the C16:1 ratio; by Figure 2 It can be seen that the P value of the present invention is the lowest, indicating that the membrane has the best fluidity.

[0056] The method for determining the membrane fatty acid ratio was as follows: Stearic acid, palmitic acid, and oleic acid were prepared separately using deionized water, with concentrations of stearic acid (5, 10, 15, 20, 25 mg / L), palmitic acid (5, 10, 15, 20, 25 mg / L), and oleic acid (50, 100, 150, 200, 250 mg / L). 2.5 mL of methanol-toluene (4:1, v:v) was added, and 200 μL of chloroacetyl was slowly added while mixing to catalyze the reaction. The esterification reaction was carried out at 100 °C for 1 h, followed by cooling to room temperature. 5 mL of 6% Na₂CO₃ and 1 mL of toluene were added, and the mixture was vortexed and incubated at 4 °C and 3000 rpm. Centrifuge for 20 min, take the upper layer (toluene-extracted organic phase), add 0.5 g / mL anhydrous sodium sulfate to remove water, shake thoroughly, and let stand for 1.5 h. After standing, residual powdery sodium sulfate should still be visible, rather than all being blocky crystals. Remove the solids through an organic membrane, and store in a 2 mL liquid chromatography vial at -20℃ for later use. Subsequently, following the method of Ma Xinfeng et al., GC-MS was used to determine the content of each component. Based on the peak time and peak area, the concentration of fatty acid components was determined. Take 10 mL of dried and rehydrated yeast cells, centrifuge and wash, sonicate to obtain cell membrane fragments, methylate them, and then determine the content of each fatty acid.

[0057] The method for determining fluorescence polarization was as follows: Yeast cells were cultured to the logarithmic growth phase (24 h). An appropriate amount of bacterial suspension was taken and centrifuged at 5000 rpm for 5 min at 4°C, and the supernatant was discarded. The cells were washed twice with sterile PBS buffer to remove background fluorescence interference from the culture medium. The cells were resuspended in PBS buffer, and the cell concentration was adjusted to achieve an absorbance (OD) of 600 nm. 600 Maintain the concentration between 0.25 and 0.35. Label the fluorescent probe with 4 mL of yeast suspension at the adjusted concentration in a test tube. Add 4 μL of DPH stock solution (to achieve a final DPH concentration of 2 μM, controlling the organic solvent content to <0.1% to avoid cell poisoning). Set up a blank control group: containing only yeast cell suspension at the same concentration without DPH. Incubate at 30°C (or the same as the culture temperature) in a constant-temperature shaker, away from light, for 30-60 min to ensure the probe is fully embedded in the membrane. For fluorescence polarization measurement, turn on the fluorescence spectrophotometer and preheat the xenon lamp for 20 min. Install the polarizer accessory. Parameter settings: Excitation wavelength: 360 nm; Emission wavelength: 430 nm. The calculation of fluorescence polarization (P) requires first calculating the instrument correction factor (G factor), used to correct for differences in the instrument's transmission efficiency for light with different polarization directions. G factor calculation: G = HV / I HH Fluorescence polarization degree (P): P = (I VV -G×I VH ) / I VV +G×IVH ).

[0058] Beer brewing was conducted using dry yeast from the examples and comparative examples. The brewing process was as follows: Yeast culture stored in -80℃ glycerol tubes was inoculated into YPD medium at a 1% inoculation rate and cultured on a shaker at 28℃ for 16 hours. The activated yeast was then transferred to YPD medium at a 1% inoculation rate and cultured on a shaker at 28℃ for 24 hours. The yeast used in the fermentation experiments was 12... ° The yeast was propagated in three stages using wort. The first stage was at 25℃ and 180 rpm for 18 hours; the second stage was at 18℃ and 120 rpm for 24 hours; and the third stage was at 11℃ with static incubation for 24 hours. The Erlenmeyer flask fermentation experiment used 12... ° P and 24 ° The wort was fermented at 11°C. The activated and expanded yeast was centrifuged at low temperature according to a 10... 6 Inoculation was performed using 1 inoculum per mL of Brix. Beer fermentation was carried out in 500 mL (300 mL wort) Erlenmeyer flasks with fermentation plugs. The weight of the fermentation apparatus was measured daily, and CO2 weight loss was calculated. The main fermentation was considered complete when the weight difference between two consecutive days dropped below 0.5 g. The results are shown in Table 2.

[0059] Table 2 Beer brewing application test (12°P wort)

Claims

1. A composite protective agent, characterized in that, It includes a first-phase bio-fortifier and a second-phase film-forming protectant; the first-phase bio-fortifier includes palmitoleic acid and trehalose; the second-phase film-forming protectant includes trehalose; the first-phase bio-fortifier is added when the yeast enters the late logarithmic growth stage or when the residual sugar in the fermentation broth drops to 40-60% of the initial residual sugar in the fermentation broth; the second-phase film-forming protectant is added before drying.

2. The composite protective agent according to claim 1, characterized in that, The first-phase bio-enhancing agent accounts for 20-30% of the dry weight of the composite protective agent, and the second-phase film-forming protective agent accounts for 70-80% of the dry weight of the composite protective agent.

3. The composite protective agent according to claim 1, characterized in that, The first-phase bio-fortifiers include palmitoleic acid, nitrogen source signal, and carbon source signal; the second-phase film-forming protectants include trehalose, fillers, antioxidants, auxiliary antioxidants, and emulsifiers.

4. The composite protective agent according to claim 3, characterized in that, The nitrogen source signal is barley peptide, the carbon source signal is trehalose, the filler is barley peptide, the antioxidant is glutathione, the co-antioxidant is sodium ascorbate, and the emulsifier is sorbitan monostearate.

5. A method for preparing active dry yeast, characterized in that, Using the composite protectant according to any one of claims 1-4 as the protectant, the method includes: conducting multi-stage expansion culture of brewing yeast, and in the last generation culture process, when the yeast growth enters the late logarithmic growth stage or the residual sugar in the fermentation broth drops to half of the initial residual sugar in the fermentation broth, adding a first-phase biofortifier to the fermenter.

6. The method for preparing active dry yeast according to claim 5, characterized in that, After the yeast with the first phase bio-fortifier was cultured, the yeast cells were collected and fortified yeast slurry was obtained. The second phase film-forming protectant was then added to the fortified yeast slurry.

7. The method for preparing active dry yeast according to claim 6, characterized in that, Yeast with added first-phase biofortifier was cultured for another 4-6 hours to induce the yeast to integrate specific fatty acids into the cell membrane and accumulate intracellular trehalose; the yeast cells were collected by centrifugation, washed with water, and a fortified yeast slurry with a solid content of 15-25% was obtained. A second-phase film-forming protectant was then added to the fortified yeast slurry.

8. The method for preparing active dry yeast according to claim 6, characterized in that, Add a second-phase film-forming protectant to the enhanced yeast slurry; emulsify by high-speed stirring to form an external protective film; granulate by extrusion and dry in a fluidized bed with three-stage variable temperature to a moisture content of 4%-6%.

9. The active dry yeast prepared by the method according to any one of claims 5-8, characterized in that, The viability rate of the active dry yeast is >95%.

10. The application of the active dry yeast according to claim 9 in beer brewing, characterized in that, The diacetyl content in beer shall not exceed 0.03 mg / L, the foam retention time shall be greater than 290 seconds, and the isoamyl acetate content shall not be less than 2.25 mg / L.

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