Method for protecting saccharomycetes freeze-dried microbial inoculum based on low-liquefied starch
By treating yeast with a low-liquefaction starch freeze-drying protectant, the problems of difficulty in preserving yeast in liquid state and easy inactivation in solid state are solved, thereby improving the survival rate and stress resistance of yeast and broadening its application range.
Patent Information
- Application Number
- CN202511432405.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-09
- Publication Date
- 2026-02-17
AI Technical Summary
The existing technology of adding yeast in liquid state has problems such as large strain volume, easy contamination, difficult preservation, inconvenient transportation, and decreased strain activity. In addition, solid yeast is easily inactivated under adverse conditions, which limits its application range.
Low-liquefaction starch was used as a freeze-drying protectant. Yeast freeze-dried inoculum was prepared by starch enzymatic hydrolysis and freeze-drying to improve the survival rate of yeast.
It improved the survival rate of freeze-dried yeast inoculum to over 60%, significantly higher than the 30% survival rate without the addition of a protectant, thus enhancing the yeast's resistance to stress and its application range.
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Figure CN121538077A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of microbial preparation, and particularly relates to a protection method of a yeast freeze-dried bacterial agent based on low-liquefied starch. BACKGROUND
[0002] When the yeast and other microorganisms are used, they are mainly added in liquid state or solid state. When the yeast and other microorganisms are added in liquid state to the material to be fermented, there are some disadvantages such as large volume of bacterial species, easy contamination, difficult storage and difficult transportation. In addition, there is a problem of activity reduction of the bacterial species in the storage process of the liquid bacterial species, and even in a low-temperature environment, the problem still exists. Meanwhile, the components of the culture medium in the liquid bacterial species can bring good or bad taste to the material to be fermented. The solid state is more conducive to long-term high-activity storage of the bacterial species, and the bacterial species are not easy to be contaminated by other bacteria. Therefore, how to produce high-activity solid bacterial species is a key problem to be solved in the field of microbial application, and has far-reaching significance for exploring the practical popularization and application of microorganisms.
[0003] In addition, the yeast and other microorganisms do not have spore structure, and the resistance performance is relatively poor, so that they are more likely to be inactivated when they face unfavorable environments such as high temperature, low humidity, high sugar content, high acidity and nutrient deficiency, which greatly restricts the application of the yeast and other microorganisms. Therefore, exploring how to improve the tolerance of the yeast and other microorganisms to the unfavorable conditions can greatly increase their application range, and has important significance for their deep application in the future in the production of fruit wine and other aspects.
[0004] At present, there are few reports on the freeze-drying protection method of the yeast by using low-liquefied starch without adding additional protective agents, which can provide a new research idea for further widening the production method of high-activity solid yeast bacterial agent. SUMMARY
[0005] In view of the above problems existing in the prior art, the present application provides a freeze-drying protective agent, which is obtained by centrifugation, freeze-drying of starch solution after enzymolysis by amylase for 20-200 min.
[0006] In some embodiments, the starch solution is prepared by dissolving starch in a buffer solution.
[0007] In some embodiments, the starch is selected from rice starch, corn starch, wheat starch, sweet potato starch, potato starch or cassava starch; in some embodiments, the use amount of the starch and the buffer solution is 1-10 g of starch per 100 mL of buffer solution; in some embodiments, the use amount of the starch and the buffer solution is 4-6 g of starch per 100 mL of buffer solution.
[0008] In some embodiments, the buffer solution is a PBS buffer solution.
[0009] In some embodiments, the amount of α-amylase used for the enzymatic hydrolysis of the starch solution is 50-150 mg α-amylase per 100 mL of starch solution; in some embodiments, the amount of α-amylase used for the enzymatic hydrolysis of the starch solution is 60-75 mg α-amylase per 100 mL of starch solution; in some embodiments, the enzymatic hydrolysis temperature is 35-40 ℃; in some embodiments, the enzymatic hydrolysis temperature is 36-38 ℃; in some embodiments, the enzymatic hydrolysis time is 90-150 min.
[0010] In some embodiments, the α-amylase has an enzyme activity of 10-50 U / mg; in some embodiments, the α-amylase has an enzyme activity of 35 U / mg.
[0011] In some embodiments, the centrifugation speed is 2000-8000 rpm, and the centrifugation time is 1-15 min; in some embodiments, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 2-10 min; in some embodiments, the centrifugation speed is 4500-5000 rpm, and the centrifugation time is 4-6 min.
[0012] In one aspect, the present application provides a method for protecting yeast, comprising the following steps: The freeze-drying protective agent is added to the resuspended yeast cells, and after standing, the supernatant is removed to obtain a paste, and the paste is subjected to freeze-drying.
[0013] In some embodiments, the amount of low-liquefied starch added is (2.0-3.5) × 10 14 CFU of bacteria are added with 2.5-7.5 g of low-liquefied starch; in some embodiments, the amount of low-liquefied starch added is (2.5-3.2) × 10 14 CFU of bacteria are added with 3.0-5.0 g of low-liquefied starch.
[0014] In some embodiments, the method further comprises a pre-freezing step before freeze-drying; in some embodiments, the pre-freezing step is that the paste is pre-frozen at -80~-60 ℃ for 11~13 h.
[0015] In some embodiments, the resuspended yeast cells are prepared by the following method: yeast cells are cultured to obtain a bacterial solution, the bacterial cells are collected by centrifugation, and after washing, the bacterial cells are resuspended to obtain a resuspended yeast solution.
[0016] In some embodiments, the freeze-drying conditions are: Under an ambient vacuum of ≤5 Pa, the temperature was increased from -45 ℃ to 0 ℃ at a rate of 5-10 ℃ / h, and maintained at each intermediate temperature point for 2-3 h. The temperature was then increased from 0 ℃ to 25-27 ℃ at a rate of 2-5 ℃ / h, and maintained at each intermediate temperature point for 2-3 h. Finally, the temperature was maintained at 25-27 ℃ for 15-25 h.
[0017] In one specific implementation, the freeze-drying conditions are as follows: Under an ambient vacuum of ≤5 Pa, the following conditions were met: -45 ℃ for 2 h, -35 ℃ for 2 h, -25 ℃ for 2 h, -15 ℃ for 2 h, -5 ℃ for 2 h, 0 ℃ for 2 h, 2 ℃ for 3 h, 5 ℃ for 3 h, 7 ℃ for 2 h, 10 ℃ for 3 h, 12 ℃ for 3 h, 15 ℃ for 3 h, 18 ℃ for 3 h, 20 ℃ for 3 h, 23 ℃ for 3 h, 25 ℃ for 3 h, and 27 ℃ for 20 h.
[0018] In some embodiments, the yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces Cerevisiae ) or Kudria zweibichi yeast ( Pichia kudriavzevii ).
[0019] The brewing yeast was named *Saccharomyces cerevisiae*. Saccharomyces Cerevisiae XJ-YSSCE010-1 was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31174.
[0020] The *Kudriazwibich yeast* was named *Kudriazwibich yeast*. Pichia kudriavzevii XJ-YPPKU067-1 was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31175.
[0021] In one aspect, the present invention provides the application of the aforementioned freeze-drying protectant in yeast freeze-drying or yeast lyophilization inoculum.
[0022] The freeze-drying protectant provided by this invention has a simple process and mild conditions. Compared with directly preparing yeast into freeze-dried yeast, the total number of yeast cells surviving in the yeast freeze-dried agent prepared by the method provided by this invention increases significantly, reaching more than 60%. The yeast cell survival rate of the yeast freeze-dried agent without any freeze-drying protectant is only about 30%, so the yeast cell survival rate is doubled. Attached Figure Description
[0023] Figure 1 The effects of different wheat starch addition amounts and hydrolysis times on the survival rates of freeze-dried inoculum of Saccharomyces cerevisiae and Pichia kudrica.
[0024] The preservation information of the *Saccharomyces cerevisiae* and *Pichia pastoris* of this invention is as follows: The brewer's yeast strain of this invention is named XJ-YSSCE010-1, and its classification name is: brewer's yeast ( Saccharomyces Cerevisiae It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31174. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC. The name of the depository is China General Microbiological Culture Collection Center (CGMCC).
[0025] The *Pichia kudriezvichi* strain of this invention is named XJ-YPPKU067-1, and its classification name is: *Pichia kudriezvichi* (…). Pichia kudriavzevii It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31175. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC. The name of the depository is China General Microbiological Culture Collection Center (CGMCC). Detailed Implementation
[0026] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.
[0027] The CFU (Colony-Forming Unit) mentioned in this invention refers to a colony formed by the growth and reproduction of a single bacterial cell or multiple bacterial cells aggregated on a solid culture medium. It is called a colony-forming unit and is a unit for calculating the number of bacteria or fungi. CFU / mL refers to the total number of microorganisms contained in each milliliter of sample. The microbial counting method that uses CFU / mL as the unit of microbial counting is the plate colony counting method.
[0028] The survival rate calculation method in this invention is: the survival rate is the ratio of the total number of microorganisms in 1 / 15 of the mass of the freeze-dried bacterial agent after freeze-drying to the total number of microorganisms in 1 / 15 of the volume of the bacterial solution before freeze-drying, and the total number of microorganisms is expressed in CFU.
[0029] The preservation information of the brewing yeast of this invention is as follows: The brewer's yeast strain of this invention is named XJ-YSSCE010-1, and its classification name is: brewer's yeast ( Saccharomyces Cerevisiae It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31174. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC. The name of the depository is China General Microbiological Culture Collection Center (CGMCC).
[0030] The preservation information of the *Pichia pastoris* strain of the present invention is as follows: The *Pichia kudriezvichi* strain of this invention is named XJ-YPPKU067-1, and its classification name is: *Pichia kudriezvichi* (…). Pichia kudriavzevii It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31175. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC. The name of the depository is China General Microbiological Culture Collection Center (CGMCC).
[0031] In the embodiments of the present application, the materials and reagents used include: The wheat starch was purchased from Xinliang Grain and Oil Processing Co., Ltd. α-Amylase was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number A800731-500 g; The glucose was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 63005518; The peptone was purchased from Beijing Aoboxing Biotechnology Co., Ltd., product number 01-001; The yeast extract powder was purchased from OXOID, product number LP0021B; Disodium hydrogen phosphate was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 20040618; Sodium dihydrogen phosphate was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 20040818; PBS buffer is prepared by dissolving 8.191 g / L disodium hydrogen phosphate and 5.075 g / L sodium dihydrogen phosphate in distilled water; YPD liquid culture medium was prepared by autoclaving 10 g / L yeast extract powder, 20 g / L peptone and 20 g / L glucose with distilled water at 115 °C for 15 min.
[0032] In the embodiments of the present application, the instruments and equipment used include: The fermentation tank was purchased from Eppendorf AG in Germany, model BF-120; The magnetic stirrer heater was purchased from IKA GmbH, Germany, model IKARH basic 2; The centrifuge was purchased from Shanghai Anting Scientific Instrument Factory, model GL-21C; The shaking table was purchased from Tianjin Laiboteri Company, model ZQPL-200. The ultra-low temperature freezer was purchased from Haier Co., Ltd., model DW-86L338J; The freeze dryer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., model SCIENTZ-30YG / A.
[0033] Example 1 1. Preparation of yeast resuspended bacteria solution: Saccharomyces cerevisiae and Saccharomyces kudriazvibichii were inoculated into 5 L fermenters containing YPD liquid medium. The fermenter temperature was set to 28 ℃ and the stirring speed was set to 300 rpm. The yeast culture was obtained after 3 days of cultivation. Yeast cells were collected by centrifuging two yeast cultures at 4400–4600 rpm for 4–6 min. The yeast cells were washed twice with PBS buffer. After washing, the cells were centrifuged again at 4400–4600 rpm for 4–6 min to collect the washed yeast cells. The washed yeast cells were then resuspended in PBS buffer until the number of *Saccharomyces cerevisiae* cells in the resuspended solution reached 1.85 × 10⁻⁶. 13 The CFU / mL count of Pichia kudrica reached 1.91 × 10⁻⁶ CFU / mL. 13 CFU / mL.
[0034] 2. Preparation of low liquefied starch: 100 g of wheat starch was dissolved in 2000 mL of PBS buffer, and then 1.429 g of α-amylase was added. The mixture was placed in a magnetic stirrer and hydrolyzed at 36-38 °C for 90 min to obtain a wheat starch hydrolysate. The wheat starch hydrolysate was centrifuged at 4500-5000 rpm for 4-6 min to obtain hydrolyzed wheat starch. The hydrolyzed wheat starch was transferred to a freeze dryer and freeze-dried for 2-4 days to obtain low-liquefaction starch. The moisture content of the low-liquefaction starch was measured to be reduced to 3-6%.
[0035] 3. Preparation of yeast freeze-dried bacteria agent: Take 15 mL of the resuspension of Saccharomyces cerevisiae and Saccharomyces kudriazvibichthys and place them in sterile 50 mL centrifuge tubes. Add 5 g of low-liquefaction starch, place them on a shaker, and shake at 220-240 rpm for 170-190 min to mix. After shaking, remove the tubes and let them stand for 110-130 min. Discard the supernatant to obtain yeast low-liquefaction starch paste. The yeast low-liquefaction starch paste was pre-frozen in an ultra-low temperature freezer (-80~-60 ℃) for 11~13 h, and then transferred to a freeze dryer for freeze drying for 2~4 days. After freezing, lyophilized Saccharomyces cerevisiae and lyophilized Saccharomyces kudricazvichii were obtained. The water content of the lyophilized agents was found to be reduced to 3~6%.
[0036] 4. Survival rate of yeast in freeze-dried bacteria agent: The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of *Saccharomyces cerevisiae* in the corresponding mass of lyophilized bacterial agent was found to be 1.34 × 10⁻⁶. 13 The total CFU count of Pichia kudriazines was 1.15 × 10⁻⁶. 13 CFU, the survival rates for the two were 66.84% and 66.64%, respectively.
[0037] The freeze-drying conditions described in the examples are as follows: -45 ℃ vacuum freezing for 2 h, -35 ℃ vacuum freezing for 2 h, -25 ℃ vacuum freezing for 2 h, -15 ℃ vacuum freezing for 2 h, -5 ℃ vacuum freezing for 2 h, 0 ℃ vacuum freezing for 2 h, 2 ℃ vacuum freezing for 3 h, 5 ℃ vacuum freezing for 3 h, 7 ℃ vacuum freezing for 2 h, 10 ℃ vacuum freezing for 3 h, 12 ℃ vacuum freezing for 3 h, 15 ℃ vacuum freezing for 3 h, 18 ℃ vacuum freezing for 3 h, 20 ℃ vacuum freezing for 3 h, 23 ℃ vacuum freezing for 3 h, 25 ℃ vacuum freezing for 3 h, 27 ℃ vacuum freezing for 20 h, with a vacuum degree ≤ 5 Pa.
[0038] Example 2 Except for the yeast resuspension, the number of Saccharomyces cerevisiae reached 2.01 × 10⁻⁶. 13 The CFU / mL count of Pichia kudrica reached 1.72 × 10⁻⁶. 13 In the preparation of low-liquefaction starch, except for the enzymatic hydrolysis time of wheat starch at 36~38 ℃ for 120 min, the rest of the operation is the same as in Example 1.
[0039] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agents was found to be 1.13 × 10⁻⁶. 13 The total CFU count of Pichia kudrica was 1.18 × 10⁻⁶. 13CFU, the survival rates for the two were 60.73% and 61.79%, respectively.
[0040] Example 3 Except for the yeast resuspension, the number of Saccharomyces cerevisiae reached 2.01 × 10⁻⁶. 13 The CFU / mL count of Pichia kudrica reached 1.72 × 10⁻⁶. 13 In the preparation of low-liquefaction starch, except for the enzymatic hydrolysis time of wheat starch at 36~38 ℃ for 150 min, the rest of the operation is the same as in Example 1.
[0041] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agents was found to be 9.68 × 10⁻⁶. 12 The total CFU count of Pichia kudrica was 1.03 × 10⁻⁶. 13 CFU, the survival rates for the two were 48.21% and 59.99%, respectively.
[0042] Example 4 Except for the yeast resuspension, the number of Saccharomyces cerevisiae reached 2.01 × 10⁻⁶. 13 The CFU / mL count of Pichia kudrica reached 1.72 × 10⁻⁶. 13 In the preparation of low-liquefaction starch, wheat starch was enzymatically hydrolyzed at 36-38 °C for 120 min. In the preparation of yeast freeze-dried inoculum, except for the addition of 7.5 g of low-liquefaction starch, the other operations were the same as in Example 1.
[0043] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agents was found to be 8.60 × 10⁻⁶. 12 The total CFU count of Pichia kudrica was 9.58 × 10⁻⁶. 12 CFU, the survival rates for the two were 42.83% and 55.57%, respectively.
[0044] Example 5 Except for the yeast resuspension, the number of Saccharomyces cerevisiae reached 2.01 × 10⁻⁶. 13 The CFU / mL count of Pichia kudrica reached 1.72 × 10⁻⁶. 13 In the preparation of low-liquefaction starch, wheat starch was enzymatically hydrolyzed at 36-38 °C for 120 min. In the preparation of yeast freeze-dried inoculum, except for the addition of 3 g of low-liquefaction starch, the other operations were the same as in Example 1.
[0045] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agent was found to be 1.24 × 10⁻⁶.13 The total CFU count of Pichia kudriazines was 1.01 × 10⁻⁶. 13 CFU, the survival rates for the two were 61.69% and 58.71%, respectively.
[0046] Comparative Example 1 Except for not adding low-liquefied starch during the preparation of the yeast freeze-dried inoculum, the other operations are the same as in Example 1.
[0047] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agents was found to be 5.87 × 10⁻⁶. 12 The total CFU count of Pichia kudriazines was 6.30 × 10⁻⁶. 12 CFU, the survival rates for the two were 31.71% and 33.05%, respectively.
[0048] Comparative Example 2 Except for replacing 5 g of low-liquefaction starch with 5 g of wheat starch in the preparation of the freeze-dried yeast inoculum, the rest of the operation is the same as in Example 1.
[0049] The two lyophilized bacterial agents were counted using the dilution plating method, and the total number of Saccharomyces cerevisiae in the corresponding mass of lyophilized bacterial agents was found to be 8.92 × 10⁻⁶. 12 The total CFU count of Pichia kudrica was 9.06 × 10⁻⁶. 12 CFU, the survival rates for the two were 48.14% and 47.56%, respectively.
Claims
1. A freeze-drying protectant, characterized in that, The freeze-drying protectant is obtained by enzymatic hydrolysis of starch solution with amylase for 20-200 min, followed by centrifugation and freeze-drying.
2. The freeze-drying protectant according to claim 1, characterized in that, The starch solution was prepared by dissolving starch in a buffer solution; Preferably, the starch is selected from rice starch, corn starch, wheat starch, sweet potato starch, potato starch, or cassava starch; Preferably, the amount of starch and buffer solution used is 1-10 g of starch per 100 mL of buffer solution; Preferably, the amount of starch and buffer solution used is 4-6 g of starch per 100 mL of buffer solution; Preferably, the buffer solution is PBS buffer.
3. The freeze-drying protectant according to claim 1, characterized in that, The dosage of α-amylase for hydrolyzing starch solution is: 50-150 mg of α-amylase per 100 mL of starch solution; Preferably, the amount of α-amylase used to hydrolyze the starch solution is: 60-75 mg of α-amylase per 100 mL of starch solution; Preferably, the enzymatic hydrolysis temperature is 35 ℃-40 ℃; Preferably, the enzymatic hydrolysis time is 90-150 min.
4. The freeze-drying protectant according to claim 1, characterized in that, The centrifugation speed is 2000-8000 rpm, and the centrifugation time is 1-15 min; Preferably, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 2-10 min; Preferably, the centrifugation speed is 4500-5000 rpm and the centrifugation time is 4-6 min.
5. A method for protecting yeast, characterized in that, Includes the following steps: The freeze-drying protectant according to any one of claims 1-4 is added to the resuspended yeast cells, and after standing, the supernatant is removed to obtain a paste, which is then freeze-dried.
6. The method according to claim 5, characterized in that, The amount of the freeze-drying protectant added is (2.0-3.5) × 10 14 Add 2.5-7.5 g of freeze-drying protectant to CFU cells; Preferably, the amount of the freeze-drying protectant added is (2.5-3.2) × 10 14 Add 3.0-5.0 g of freeze-drying protectant to the CFU cells.
7. The method according to claim 5, characterized in that, The freeze-drying process includes a pre-freezing step, which involves placing the paste at -80 to -60 ℃ for 11 to 13 hours.
8. The method according to claim 5, characterized in that, The resuspension of yeast cells is prepared by culturing yeast to obtain a bacterial solution, centrifuging the bacterial solution to collect the cells, washing the cells and resuspending them to obtain the resuspension of yeast cells.
9. The method according to claim 5, characterized in that, The yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces Cerevisiae ) or Kudria zweibichi yeast ( Pichia kudriavzevii ).
10. The use of the freeze-drying protectant according to any one of claims 1-4 in yeast freeze-drying or yeast lyophilization inoculum.