CN113621519B - Composite yeast freeze-drying protective agent - Google Patents
Composite yeast freeze-drying protective agent Download PDFInfo
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- CN113621519B CN113621519B CN202110920034.2A CN202110920034A CN113621519B CN 113621519 B CN113621519 B CN 113621519B CN 202110920034 A CN202110920034 A CN 202110920034A CN 113621519 B CN113621519 B CN 113621519B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of food processing, and particularly relates to a composite yeast freeze-drying protective agent. The freeze-drying protective agent is formed by compounding plant-derived beta-glucan, gamma-polyglutamic acid and mannitol. The invention provides a novel yeast freeze-drying protective agent, which can effectively reduce the damage to thalli in the freeze-drying process and improve the survival rate of the yeast.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a composite yeast freeze-drying protective agent.
Background
Vacuum cooling deviceFreeze drying is a drying method in which a material is frozen below the eutectic point temperature and then the moisture in the material is removed by sublimation under a low pressure. The freeze drying is a process of converting moisture in a material from a liquid state to a solid state and then from the solid state to a gas state, thereby realizing a drying process of the material. In the process, the bacteria are stimulated by the freezing process and the drying process, the bacteria are inevitably damaged, and the damage to the bacteria in the two processes can be effectively reduced by adding the protective agent. In terms of permeation, the protectant may be classified into a permeable protectant and a non-permeable protectant, wherein the permeable protectant undergoes hydration reaction by binding water inside the cells, thereby reducing the formation of intracellular ice crystals; the impermeable protective agent is attached to the outside of the cells to increase the viscosity of the protective system, thereby reducing the mechanical damage to the cells during lyophilization. For the bacterial cells, a protective agent is added to reduce the damage to the bacterial cells in the two processes, thereby improving the survival rate of the bacterial cells. Song Zhiyuan the research adopts skim milk powder 5%, mannitol 4% and sodium ascorbate 3% as freeze-drying protective agent of fruit and vegetable biological yeast strain, and the survival rate reaches 83.64%. Zhou Qiuyang and other researches show that when sorbitol is 5.43g/100mL, trehalose is 12.45g/100mL and sodium glutamate is 13.56g/100mL, the combined protection effect is best, and the survival rate of saccharomycetes can reach 84.21% +/-0.87%. Wang Hua and the like, when 14.15g/100mL of sucrose, 7.07g/100mL of L-sodium glutamate and 1.10g/100mL of polyethylene glycol are adopted as candida tropicalis freeze-drying protective agents, the freeze-drying survival rate is 82.73 percent. The CAHNYUAN research shows that when the human-like collagen 1.23%, the trehalose 11.50% and the glycerol 4.65% are used as the bifidobacterium lyoprotectant, the lyophilization survival rate is 88.23%. SHU study showed that with trehalose 13%, na 2 HPO 4 0.33 percent of composite low-temperature protective agent, 7.5 percent of lactose and 21 percent of skim milk powder, and the survival rate of the freeze-dried lactobacillus acidophilus is (93.9+/-0.12 percent). The development of new yeast protectants is essential for the protection of frozen yeasts.
Disclosure of Invention
The invention aims to provide a novel composite yeast freeze-drying protective agent which can effectively protect yeast in the freezing process and improve the survival rate of the yeast.
The technical scheme adopted by the invention is as follows:
a composite yeast freeze-drying protective agent is formed by compounding plant-derived beta-glucan, gamma-polyglutamic acid and mannitol.
Further, the freeze-drying protective agent comprises the following components in parts by mass: 6-7 parts of beta-glucan, 0.1-0.2 part of gamma-polyglutamic acid and 1-1.3 parts of mannitol.
Preferably, the freeze-drying protective agent comprises the following components in parts by mass: 6.56 parts of beta-glucan, 0.15 part of gamma-polyglutamic acid and 1.15 parts of mannitol.
The plant-derived beta-glucan is preferably oat beta-glucan and/or barley beta-glucan.
The yeast is preferably Saccharomyces cerevisiae.
According to the invention, the research shows that the addition of the beta-glucan can maintain the stability of a yeast cell membrane, improve the activity of antioxidant enzyme in the yeast cell and increase the content of intracellular trehalose, so that the survival rate of the yeast cell is improved. Gamma-polyglutamic acid as a macromolecular compound can protect cells in a "wrapping" manner, mannitol as a small molecule protects proteins from aggregation mainly by forming an amorphous structure, which promotes protein stabilization. The macromolecular compound can protect cells and promote the effect of the small molecular compound on the cells. However, it should be noted that when the mannitol content is higher than a certain value, the formed crystal structure has no protective effect on the protein, and even promotes the destruction of the cells by water molecules.
Compared with the prior art, the invention has the following advantages:
the invention provides a novel yeast freeze-drying protective agent, which can effectively reduce the damage to thalli in the freeze-drying process and improve the survival rate of the yeast.
Drawings
FIG. 1 shows the cell morphology of Saccharomyces cerevisiae after lyophilization, wherein a is the blank group and b is the group with the addition of the composite protectant.
Detailed Description
The following specific embodiments are used to illustrate the technical solution of the present invention, but the scope of the present invention is not limited thereto:
example 1
The yeast freeze-drying protective agent comprises the following components in parts by mass: 6 parts of beta-glucan, 0.17 part of gamma-polyglutamic acid and 1 part of mannitol. When in use, the beta-glucan is mixed according to the corresponding parts and then prepared into an aqueous solution, wherein the mass percentage concentration of the beta-glucan is 6%.
The strain is used for subsequent saccharomyces cerevisiae freeze-drying protection, and the corresponding survival rate of the saccharomyces cerevisiae is 87.96 percent. The obtained product is used for subsequent freeze-drying protection of Pichia pastoris (Pichia kudriavzevii IFM 53500), the survival rate after freeze-drying is 82.50%, and the survival rate after freeze-drying is 81.68% when the obtained product is used for freeze-drying protection of Wick ham yeast (Wickerhamomyces anomalus isolate MDY). However, the survival rate of the blank control group (group without added protective agent) of the three yeasts is only 3% -4%.
Example 2
The yeast freeze-drying protective agent comprises the following components in parts by mass: 6.56 parts of beta-glucan, 0.15 part of gamma-polyglutamic acid and 1.15 parts of mannitol. When in use, the beta-glucan is mixed according to the corresponding parts to prepare an aqueous solution, wherein the mass percentage concentration of the beta-glucan is 6.56%.
The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 90.69%. The obtained product is used for subsequent freeze-drying protection of Pichia pastoris (Pichia kudriavzevii IFM 53500), the survival rate after freeze-drying is 85.17%, and the survival rate after freeze-drying is 83.45% when the obtained product is used for freeze-drying protection of Wick ham yeast (Wickerhamomyces anomalus isolate MDY).
Comparative example 1
The yeast freeze-drying protective agent is prepared into an aqueous solution, and each 100g of the aqueous solution contains 5g of skim milk powder, 4g of mannitol, 3g of ascorbic acid and the balance of sterile distilled water. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 80.8%.
Comparative example 2
The yeast freeze-drying protective agent comprises 12.45g of trehalose, 13.56g of sodium glutamate, 5.43g of sorbitol and the balance of sterile distilled water according to 100 ml. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 73.77%.
Comparative example 3
The yeast freeze-drying protective agent comprises 1.10g of polyethylene glycol, 7.07g of L-sodium glutamate, 14.15g of sucrose and the balance of sterile distilled water according to 100 ml.
The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 72.77%.
Comparative example 4
The yeast freeze-drying protective agent is distilled water solution with the mass percentage concentration of 6 percent of beta-glucan. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 36.78%.
Comparative example 5
The yeast freeze-drying protective agent is distilled water solution with the mass percentage concentration of gamma-polyglutamic acid of 0.17 percent. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 36.03%.
Comparative example 6
The yeast freeze-drying protective agent is distilled water solution with the mass percentage concentration of mannitol of 1 percent. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 37.12%.
Comparative example 7
The yeast freeze-drying protective agent is a solution of beta-glucan and gamma-polyglutamic acid dissolved in distilled water, wherein the mass percentage concentration of the beta-glucan is 6%, and the mass percentage concentration of the gamma-polyglutamic acid is 0.17%. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 67.07%.
Comparative example 8
The yeast freeze-drying protective agent is a solution of beta-glucan and mannitol dissolved in distilled water, wherein the mass percentage concentration of the beta-glucan is 6%, and the mass percentage concentration of the mannitol is 1%. The strain was used for subsequent Saccharomyces cerevisiae protection, corresponding to a yeast survival rate of 69.52%.
Comparative example 9
The yeast freeze-drying protective agent is a solution of mannitol and gamma-polyglutamic acid dissolved in distilled water, wherein the mass percentage concentration of the gamma-polyglutamic acid is 0.17%, and the mass percentage concentration of the mannitol is 1%. The strain is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is 65.91%.
Comparative examples 10 to 17
The yeast freeze-drying protective agent is a solution of beta-glucan, mannitol and gamma-polyglutamic acid dissolved in distilled water, and the mass percentage concentration of the beta-glucan, the mannitol and the gamma-polyglutamic acid is shown in the following table. The yeast is used for subsequent Saccharomyces cerevisiae protection, and the corresponding yeast survival rate is shown in the following table.
Therefore, the composite freeze-drying protective agent of the comparative example has a better protective effect on saccharomyces cerevisiae than the composite freeze-drying protective agent of the invention.
The effect of the protective agent of examples and comparative examples was evaluated by the following methods.
Experimental method
1. Vacuum freeze drying process
The yeast system with sterile water was used as a blank and the yeast system with the protectant solutions of the different examples and comparative examples was used as a treatment.
The yeast system is obtained by the following method:
1) Saccharomyces cerevisiae
Dissolving a proper amount of Angel high activity dry yeast produced by Angel Yeast Co., ltd in sterile water, coating on YPD solid culture medium by dilution coating plate method, culturing at 37deg.C for 48 hr, picking single colony, culturing in YPD liquid culture medium for 20 hr, and centrifuging to obtain bacterial mud.
2) Acquisition of Wilkham's yeast and Pichia
10g of a sample of the aged dough (glycolytic aged dough, sampled in mountain areas and plain areas in Henan, west) was weighed, 100mL of sterilized distilled water was added to the ultra clean bench, homogenized for 2 minutes with a sterile homogenizer, and the suspension was drawn for ten-fold gradient dilution, taking 0.1mL of coated plates. 50mg/L chloramphenicol and 50mg/L ampicillin were added to YPD medium for isolated culture of yeast, respectively, and the culture was carried out in a constant temperature and humidity incubator at 30℃for 48 hours, respectively. And selecting a flat plate with colony number of 50-200 from each sample, randomly picking 15 single colonies from the flat plate, performing shake culture at 30 ℃ and 150rpm/min in YPD liquid culture medium for 15 hours for further activation culture, and identifying by using DNA sequencing to obtain the Wick ham yeast and Pichia anomala. And respectively inoculating the Wilkham yeast and the Pichia anomala into YPD liquid culture medium for expansion culture, and centrifuging after 20h of culture to obtain corresponding bacterial sludge.
Mixing the corresponding saccharomycete mud with sterile water or a protective agent solution according to the mass ratio of 1:2, balancing for 60min in a constant temperature box at 30 ℃, pre-freezing for 8h in a refrigerator at-18 ℃, rapidly putting the mixture into a vacuum freeze dryer after the pre-freezing is finished, and freeze-drying for 20h at the cold trap temperature of-75 ℃ and the vacuum degree of 145-155 mTorr to obtain the saccharomycete freeze-dried powder.
2. Calculation of Yeast survival Rate after lyophilization
Rehydrating the yeast freeze-dried powder obtained in the previous step to a pre-freeze-drying volume by using sterile physiological saline with the volume concentration of 0.85%, and then placing the pre-freeze-dried powder into a constant-temperature incubator at 30 ℃ for activation for 30min, wherein the freeze-drying survival rate of the yeast is determined by using a Mailan staining method, and the survival rate is calculated according to the following formula:
3. microstructure observation
The damage of the freeze-drying to the saccharomyces cerevisiae cells and the effect of the protective agent can be intuitively observed through fig. 1. Wherein, the partial cell deformation and rupture (indicated by arrow in fig. 1 a) of the group without added protective agent corresponds to a survival rate of only 3% after lyophilization. The composite protectant group (shown in figure 1b, corresponding to example 2) is added, so that the bacteria are complete in shape and full in cells, and the freeze-drying survival rate of the yeast can reach 90.69%. The result shows that the freeze-drying-rehydration process damages the integrity of the saccharomycete cell membrane, the composite protective agent can well maintain the permeation barrier and the structural integrity of the freeze-drying cell membrane, avoid the leakage of cell contents and realize the freeze-drying protection of saccharomycete.
Claims (3)
1. The composite yeast freeze-drying protective agent is characterized by being formed by compounding plant-derived beta-glucan, gamma-polyglutamic acid and mannitol; the freeze-drying protective agent comprises the following components in parts by mass: 6-7 parts of beta-glucan, 0.1-0.2 part of gamma-polyglutamic acid and 1-1.3 parts of mannitol; the yeast is Saccharomyces cerevisiae, pichia pastoris, and Wilkham's yeast.
2. The composite yeast lyoprotectant of claim 1, wherein the lyoprotectant comprises the following components in parts by mass: 6.56 parts of beta-glucan, 0.15 part of gamma-polyglutamic acid and 1.15 parts of mannitol.
3. The composite yeast lyoprotectant of any one of claims 1-2, wherein the plant-derived beta-glucan is oat beta-glucan and/or barley beta-glucan.
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