A method for preparing a high-cell-density active yeast

Abstract

The application belongs to the technical field of bioengineering, and specifically discloses a preparation method of high-cell-number active yeast, which takes the cell number per unit dry weight as an evaluation index for screening of active yeast strains, and obtains dominant strains with high cell number through preliminary screening and rescreening; then, high-density culture fermentation is optimized through carbon source, nitrogen source, phosphorus source, metal ions and vitamins, and high-density accumulation of the biomass of the strains is realized through flow feeding; temperature induction treatment is introduced in the late stage of high-density fermentation to promote the increase of intracellular trehalose content, so that the amount of exogenous trehalose is reduced under the premise of ensuring the dry survival rate; and finally, high-activity yeast products are obtained through the Span60 assisted fluidized bed drying process. The application cooperatively optimizes from the source strain screening to the terminal drying, and solves the technical problems of disconnection between the strain screening index and the final active cell number, high cost of exogenous protective agent, and low dry survival rate in the traditional technology.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the preparation of active yeast, particularly a method for preparing high-cell-count active yeast, with cell count per unit dry weight as the evaluation index, from strain screening, intracellular trehalose-induced accumulation to fluidized bed drying. Background Technology

[0002] Active yeast, as an important microbial preparation, has wide applications in feed additives, food fermentation agents, agricultural microecological preparations, and functional food ingredients. The evaluation indicators for its product quality are the number of live cells and the drying survival rate; the number of live cells can evaluate the product's effectiveness and economic benefits.

[0003] However, existing active yeast preparation technologies have the following prominent problems: First, traditional yeast strain screening often uses biomass, fermentation performance, or a single metric as evaluation indicators, neglecting the strain's potential for cell accumulation per unit dry weight. This means that even if a high biomass is obtained, it may not necessarily be converted into a product with a high number of viable cells, resulting in a significant discrepancy between screening indicators and the final product.

[0004] Second, existing high-density culture processes mostly focus on optimizing biomass yield, lacking research on cells in the later stages of culture, particularly neglecting the accumulation of intracellular protective substances (such as trehalose). This leads to severe cell damage during the subsequent drying process and a low survival rate after drying.

[0005] Third, while traditional hot air drying processes combined with a single exogenous protective agent (such as trehalose) can improve the survival rate to some extent, they also have problems such as large amounts of exogenous protective agents (usually 10-15%), high costs, and low drying survival rates (usually below 80%).

[0006] Therefore, a technical system should be established that integrates strain screening and drying optimization to significantly increase the number of live cells in active yeast products while reducing production costs. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing high-cell-count, viable yeast. This invention uses cell count per unit dry weight as the core indicator for initial strain screening, and combines high-density fermentation, intracellular trehalose-induced accumulation, low-exogenous protective agent formulation, and fluidized bed drying processes to obtain a viable cell count of 3.0 × 10⁻⁶. 10 High-activity yeast products with cells / g or higher.

[0008] This invention is achieved through the following technical solution: A method for preparing high-cell-count, active yeast includes the following steps: (1) Strain screening: After the preserved active yeast strains are activated and cultured, they are first screened by cell concentration and then screened again by the number of cells per unit dry weight. The strain with the highest number of cells per unit dry weight is selected as the target strain. (2) Fermentation culture: After the target strain is propagated by seed culture, it is inoculated into synthetic culture medium and carried out high-density fed-batch fermentation culture in a fermenter; (3) Intracellular trehalose-induced accumulation: When the cell biomass reaches more than 180 g / L, adjust the fermentation system temperature to 30-40℃, control the pH to 5.0-6.0, and reduce the stirring speed and aeration rate at the same time, and induce the accumulation for 1.0-2.0 h. (4) Protectant treatment: After induction, the cells are collected by centrifugation and yeast sludge is prepared. The obtained yeast sludge is mixed evenly with the compound protectant solution and left to stand at room temperature to allow the protectant to fully act on the cell surface. (5) Fluidized bed drying: The yeast mud treated with the protectant is made into granules, and after adding Span60 emulsifier, it is dried in a fluidized bed to obtain the active yeast product.

[0009] A further improvement to the present invention is as follows: The culture medium used in step (1) for the initial screening and rescreening is YPD medium; Furthermore, the inoculum size for the activation culture is 1-3%, the temperature is 25-35℃, the rotation speed is 120-240 r / min, and the time is 20-30 h; Furthermore, the target strain has a cell count per unit dry weight ≥ 2.49 × 10⁻⁶. 10 cells / g, cell diameter ≤6.0um.

[0010] Furthermore, the seed expansion culture in step (2) is a three-stage seed expansion culture, all using YPD medium, with the following amounts: 50 mL for the first stage, 300 mL for the second stage, and 600 mL for the third stage. The culture temperature is 25-35℃, the rotation speed is 170-190 r / min, and the time is 12 h, 12 h, and 20 h respectively.

[0011] Furthermore, the synthetic culture medium in step (2) consists of a base culture medium and a supplementary culture medium. The base culture medium consists of the following components in the following weight ratio: glucose 3-6%, ammonium sulfate 0.5-1.0%, dipotassium hydrogen phosphate 0.1-0.2%, corn steep liquor 1-3%, appropriate amounts of metal ions and vitamins; the metal ions include Mg 2+ Zn 2+ Fe 2+ Mn 2+ Cu 2+ K +The vitamins mentioned include VB1, VB5, VB7, and VB3; the substrate culture medium is added to the tank before fermentation for inoculation of the seed liquid, with an inoculation amount of 6-10%. The fed culture medium consists of the following components at the following concentrations: 250-350 g / L glucose solution, 25-30 g / L ammonium sulfate, 13-16 g / L dipotassium hydrogen phosphate solution, and appropriate amounts of nutrients; the fed culture medium is added to the tank during high-density culture at a rate of 90-120 mL.

[0012] Furthermore, the concentration of metal ions in the substrate culture medium is: Mg 2+ 3u mmol / L, Zn 2+ 5ummol / L, Fe 2+ 5ummol / L, Mn 2+ 10ummol / L, Cu 2+ 1ummol / L, K + 10 ummol / L; Furthermore, the concentrations of the vitamins are VB1 10 μmol / L, VB5 10 μmol / L, VB7 0.5 μmol / L, and VB3 50 μmol / L.

[0013] Furthermore, the fermentation culture in step (2) is carried out at a temperature of 25-35℃, a pH of 3.8-4.2, a rotation speed of 100 r / min, and an aeration rate of 1-6 L / min.

[0014] Furthermore, the stirring speed in step (3) is 140-200 rpm and the aeration rate is 4-6 L / min.

[0015] Furthermore, the compound protective agent solution described in step (4) uses deionized water as a solvent and contains 8.0-12.0 wt% trehalose, 10.0-11.0 wt% skim milk powder, and 0.08-0.12 wt% glutathione; Furthermore, the mass ratio of the compound protective agent solution to the yeast sludge is 1-3:1.

[0016] Furthermore, the particle size of the particles in step (5) is 1.0-2.0 mm; the amount of Span60 emulsifier added is 0.5-1.5 wt% of the dry weight of yeast on a cell dry basis.

[0017] Furthermore, the inlet air temperature for drying in step (5) is 45-55℃, and the time is 35-45 min.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention is the first to use “cells per unit dry weight” as the core indicator for screening active yeast strains, which ensures the preparation of high cell count products from the source of the strains and can solve the problem of the disconnect between traditional screening indicators and the final number of live cells. (2) This invention establishes a synthetic culture medium system with optimized carbon source, nitrogen source, phosphorus source, metal ions and vitamins. Combined with fed-batch feeding, it achieves high-density accumulation of bacterial biomass of 197 g / L. (3) The present invention innovatively introduces temperature-induced treatment in the later stage of high-density fermentation, which promotes the increase of intracellular trehalose content by 53.7%, thereby reducing the amount of exogenous trehalose by more than 30% while ensuring the survival rate of dried trehalose, and significantly reducing the cost of protective agent; (4) This invention introduces Span60 emulsifier into the yeast fluidized bed drying process. By improving material dispersibility and interfacial state, and in conjunction with a low-temperature (50°C) fluidized bed drying process, the survival rate of the dried cells is increased from 75% in traditional hot air drying to over 92%, and the final number of viable cells exceeds 3.0 × 10⁻⁶. 10 The cell / g ratio is significantly better than existing technologies. Attached Figure Description

[0019] Figure 1 This describes the high-density fermentation process of microbial cells in a 5 L fermenter. Detailed Implementation

[0020] This invention provides a method for preparing high-cell-count active yeast. In specific embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available. The invention will now be described in detail with reference to specific embodiments.

[0021] In this invention, the cell number per unit dry weight (Cell Number per Unit Dry Weight) refers to the number of cells contained in a unit mass of cellular dry matter. It is commonly used to estimate the total number of cells in a population when the total biomass (dry weight) and the average dry weight per cell are known, or when a stable conversion relationship exists between the two. The specific calculation formula is as follows: Cells per unit dry weight = Cell concentration / Biomass dry weight; The culture medium involved in the specific embodiments of the present invention is as follows: YPD medium: 1% yeast extract, 2% peptone, 2% glucose, autoclaved at 115℃ for 20 min.

[0022] YPD solid medium: Add 2% agar to YPD liquid medium and autoclave at 115℃ for 20 min.

[0023] Fermentation tank bottom culture medium: glucose 6%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.16%, metal ion combination, vitamin combination, 3% corn steep liquor. Specifically, the concentration of metal ions in the bottom culture medium is: Mg 2+ 3u mmol / L, Zn 2+ 5ummol / L, Fe 2+ 5ummol / L, Mn 2+ 10ummol / L, Cu 2+ 1ummol / L, K + 10 μmmol / L; the concentrations of vitamins were VB1 10 μmol / L, VB5 10 μmol / L, VB7 0.5 μmol / L, and VB3 50 μmol / L.

[0024] Fermentation tank feed medium: 300 g / L glucose, adjusted to 1.2 L, sterilized at 115℃ for 20 min. 30 g / L ammonium sulfate and 14.8 g / L dipotassium hydrogen phosphate were simultaneously adjusted to 0.6 L. Urea 27.3 g / L was not sterilized and was filtered through a 0.22 μm filter membrane for sterilization before use.

[0025] Compound protective agents: trehalose 11.66%, skim milk powder 10.68%, GSH 0.105%.

[0026] Example 1: Preparation of high cell number active yeast (1) Strain screening 1.1 Strain activation Four live yeast strains (S1, S2, S3, and S4) stored at -80℃ were activated in 50 / 250 mL YPD liquid medium, then streaked onto YPD solid plates using a sterile inoculation loop and incubated at 30℃ for 24–36 h. Single colonies were then picked and inoculated into 50 / 250 mL YPD liquid medium and incubated in a shaker at 30℃ and 200 r / min for 12 h to obtain the seed culture.

[0027] 1.2 Initial Screening Experiment The seed culture was inoculated at a rate of 2% (v / v) into 50 / 250 mL of YPD liquid medium and cultured in a constant temperature shaker at 30℃ and 200 r / min for 24 h. Three replicates were set up for each strain. After culture, cell concentration was measured and ranked according to the average value. The results are shown in Table 1. Based on the data in the table, the two strains with the highest cell concentration, S2 and S4, were selected for the secondary screening experiment.

[0028] Table 1 Initial Screening Results S1 <![CDATA[2.52±0.18 c ]]> S2 <![CDATA[3.25±0.11 a ]]> S3 <![CDATA[2.14±0.31 d ]]> S4 <![CDATA[2.89±0.07 b ]]> 1.3 Secondary screening experiment The dominant strains S2 and S4 were used for secondary screening. After the culture was completed, 10 mL of culture medium was centrifuged at 8000 r / min for 10 min, washed, and dried to constant weight. The biomass, cell diameter, and number of cells per unit dry weight were measured. The results are shown in Table 2.

[0029] Table 2 Results of Rescreening S2 <![CDATA[13.05±0.23 a ]]> <![CDATA[5.82±0.41 a ]]> <![CDATA[2.49±0.03 a ]]> S4 <![CDATA[12.89±0.33 a ]]> <![CDATA[6.37±0.52 a ]]> <![CDATA[2.22±0.04 b ]]> Table 2 shows that there was no significant difference in biomass between S2 and S4, but the number of cells per unit dry weight of S2 was significantly higher than that of S4 (P<<0.05), and the cell diameter was also smaller. Therefore, S2 was selected as the target strain for subsequent preparation of high-cell-count, active yeast.

[0030] (2) High-density fermentation culture Yeast strain S2, stored at -80℃, was activated in 50 / 250 mL YPD liquid medium and then streaked onto YPD solid plates using a sterile inoculation loop. The plates were incubated at 30℃ for 24-36 h. Single colonies were picked from the YPD solid plates and inoculated into 50 / 250 mL YPD liquid medium, then cultured at 30℃ and 180 r / min in a shaker for 12 h to obtain the primary seed culture. The primary seed culture was then transferred to 300 / 1000 mL YPD liquid medium at a 2% (v / v) inoculation rate and cultured at 30℃ and 180 r / min in a shaker for 12 h to obtain the secondary seed culture. Finally, the secondary seed culture was transferred to 600 / 2000 mL YPD liquid medium at an 8% (v / v) inoculation rate and cultured at 30℃ and 180 r / min in a shaker for 20 h to obtain the tertiary seed culture.

[0031] The substrate culture medium was added to the fermenter, and the obtained tertiary seed culture was inoculated at an 8% inoculum rate into a 5 L automated fermenter (working volume 3.5 L). Fermentation parameters were controlled as follows: temperature: 30℃, pH: 4.0 (automatic alkali adjustment), fermentation speed: initial 100 rpm, adjusted according to the strain growth, with a range below 210 rpm, and aeration rate: 1-6 L / min. During fermentation, feed medium (90-120 mL) was added via a fed-batch method. Yeast biomass was analyzed during fermentation, and the results are shown below. Figure 1 .

[0032] Depend on Figure 1It can be seen that in the early stage of fermentation (1–2 h), the cell biomass remained relatively stable at around 40 g / L, indicating that the yeast was in the adaptation phase. At this time, the cells mainly adapted to the fermentation environment and activated related metabolic enzyme systems, resulting in a slow growth rate. As the culture time increased, the cell biomass began to increase significantly after 3 h, indicating that the yeast gradually entered the logarithmic growth phase. During 3–14 h, the biomass rapidly increased from 50 g / L to 188 g / L, showing a rapid growth trend, indicating that yeast cells can maintain a high growth rate under optimized culture medium and sufficient nutrient conditions. After 14 h, the cell growth rate gradually slowed down, and the rate of biomass growth decreased significantly. By 16 h, the biomass reached 192 g / L, then gradually stabilized, reaching 197 g / L at 18 h. This stage indicates that the culture system gradually entered the steady growth phase, and cell growth was limited by factors such as nutrient consumption and the accumulation of metabolic products. Overall, under the fed-batch feeding strategy and corn steep liquor nutrient supplementation, the yeast biomass ultimately reached 197 g / L, which is basically consistent with the design target of 200 g / L. This indicates that the culture medium system and fermentation process can effectively support high-density yeast growth and meet the expected requirements for high-density culture.

[0033] (3) Intracellular trehalose-induced accumulation The fermentation broth (biomass approximately 192 g / L, intracellular trehalose content 57.7 ± 2.2 mg / g DCW) was collected after 16 h of fermentation. The fermentation system temperature was adjusted to 35℃, pH controlled at 5.0, and stirring speed (140-200 rpm) and aeration rate (4-6 L / min) were controlled. Samples were taken after 1.5 h of induction. The intracellular trehalose content was measured to be 88.7 ± 3.2 mg / g DCW, an increase of 53.7% compared to before induction fermentation.

[0034] (4) Protective agent treatment After induction, the fermentation broth was centrifuged at 8000 r / min for 5 min at 4℃, the supernatant was discarded, and the mixture was washed 2-3 times with sterile distilled water to collect the wet yeast slurry. The obtained yeast slurry was mixed with the compound protective agent solution at a mass ratio of 2:1 and allowed to stand at room temperature to allow the protective agent to fully act on the cell surface; the survival rate was verified to be 75.06%.

[0035] (5) Granulation and fluidized bed drying Take the wet yeast mud after step (4), make it into uniform particles with a particle size of 1.0-2.0 mm, add 1.0% (based on cell dry basis) of Span60 emulsifier and then perform fluidized bed drying with an air inlet temperature of 50℃ and a drying time of 40 min to obtain active yeast product.

[0036] (6) Finished product inspection After drying, the product's moisture content, dry survival rate, and viable cell count were measured. The results showed a moisture content of 4.98%, a dry survival rate of 92.34%, and a viable cell count of 3.05 × 10⁻⁶. 10 cells / g; Rehydration performance: Rehydration with sterile distilled water at 30℃ for 10 min, rehydration survival rate >95%; Storage stability: After 90 days of sealed storage at 4℃, viable cell count retention rate >85%.

[0037] Example 2: Validation of Exogenous Trehalose Reduction The difference between this embodiment and embodiment 1 is that the compound protective agent formula used in step (4) is: 8.00% trehalose + 10.68% skim milk powder + 0.105% GSH. Other operations are roughly the same as in embodiment 1, and will not be repeated here.

[0038] After intracellular induction, even with the exogenous trehalose reduced to 8%, the survival rate still reached 73.42%, which was not significantly different from Example 1. This indicates that intracellular trehalose accumulation can effectively compensate for the loss of protective effect caused by the reduction of exogenous trehalose, achieving a 31.4% reduction in exogenous trehalose dosage.

[0039] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing high-cell-count active yeast, characterized in that, Includes the following steps: (1) Strain screening: After the preserved active yeast strains are activated and cultured, they are first screened by cell concentration and then screened again by the number of cells per unit dry weight. The strain with the highest number of cells per unit dry weight is selected as the target strain. (2) Fermentation culture: After the target strain is propagated by seed culture, it is inoculated into synthetic culture medium and carried out high-density fed-batch fermentation culture in a fermenter; (3) Intracellular trehalose-induced accumulation: When the cell biomass reaches more than 180 g / L, adjust the fermentation system temperature to 30-40℃, control the pH to 5.0-6.0, and control the stirring speed and aeration rate at the same time, and induce the accumulation for 1.0-2.0 h. (4) Protectant treatment: After induction, the cells are collected by centrifugation and yeast sludge is prepared. The obtained yeast sludge is mixed evenly with the compound protectant solution and left to stand at room temperature to allow the protectant to fully act on the cell surface. (5) Fluidized bed drying: The yeast mud treated with the protectant is made into granules, and after adding Span60 emulsifier, it is dried in a fluidized bed to obtain the active yeast product.

2. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The culture medium used in step (1) for the initial screening and rescreening is YPD medium; And / or, the inoculum size for the activation culture is 1-3%, the temperature is 25-35℃, the rotation speed is 120-240 r / min, and the time is 20-30 h; And / or, the target strain has a cell count per unit dry weight ≥ 2.49 × 10⁻⁶. 10 cells / g, cell diameter ≤6.0um.

3. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The seed propagation in step (2) is a three-stage seed propagation, all using YPD medium, with the following amounts: 50 mL for stage 1, 300 mL for stage 2, and 600 mL for stage 3. The cultivation temperature is 25-35℃, the rotation speed is 170-190 r / min, and the time is 12 h, 12 h, and 20 h respectively.

4. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The synthetic culture medium in step (2) consists of a base culture medium and a supplementary culture medium. The base culture medium is composed of the following components in the following weight-back ratio: The ingredients include 3-6% glucose, 0.5-1.0% ammonium sulfate, 0.1-0.2% dipotassium hydrogen phosphate, 1-3% corn steep liquor, appropriate amounts of metal ions and vitamins; the metal ions include Mg. 2+ Zn 2+ Fe 2+ Mn 2+ Cu 2+ K + The vitamins mentioned include VB1, VB5, VB7, and VB3; the substrate culture medium is added to the tank before fermentation for inoculation of the seed liquid, with an inoculation amount of 6-10%. The fed culture medium consists of the following components at the following concentrations: 250-350 g / L glucose solution, 25-30 g / L ammonium sulfate, 13-16 g / L dipotassium hydrogen phosphate solution, and appropriate amounts of nutrients; the fed culture medium is added to the tank during high-density culture at a rate of 90-120 mL.

5. The method for preparing a high-cell-count active yeast according to claim 4, characterized in that: The concentration of metal ions in the substrate culture medium is: Mg 2+ 3u mmol / L, Zn 2+ 5ummol / L, Fe 2+ 5ummol / L, Mn 2+ 10ummol / L, Cu 2+ 1ummol / L, K + 10 ummol / L; And / or, the concentrations of the vitamins are VB1 10 μmol / L, VB5 10 μmol / L, VB7 0.5 μmol / L, and VB3 50 μmol / L.

6. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The fermentation culture in step (2) is carried out at a temperature of 25-35℃, a pH of 3.8-4.2, a rotation speed of 100 r / min, and an aeration rate of 1-6 L / min.

7. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The stirring speed in step (3) is 140-200 rpm and the aeration rate is 4-6 L / min.

8. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The compound protective agent solution in step (4) uses deionized water as a solvent and contains 8.0-12.0 wt% trehalose, 10.0-11.0 wt% skim milk powder, and 0.08-0.12 wt% glutathione; And / or, the mass ratio of the compound protective agent solution to the yeast sludge is 1-3:

1.

9. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The particle size of the particles in step (5) is 1.0-2.0 mm; the amount of Span60 emulsifier added is 0.5-1.5 wt% of the dry weight of yeast on a cell dry basis.

10. The method for preparing a high-cell-count active yeast according to claim 1, characterized in that: The inlet air temperature for drying in step (5) is 45-55℃, and the time is 35-45 min.

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