阅读说明：本技术 一种复合酵母冻干保护剂 (Composite yeast freeze-drying protective agent ) 是由 李�真 朱畇昊 艾志录 姬生鑫 范会平 索标 于 2021-08-11 设计创作，主要内容包括：本发明属于食品加工技术领域,特别涉及一种复合酵母冻干保护剂。所述冻干保护剂由植物源的β-葡聚糖、γ-聚谷氨酸、甘露醇复合构成。本发明提供了一种新型的酵母冻干保护剂,能有效地减少菌体在冷冻干燥过程中受到的损伤,提高酵母的存活率。(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 beta-glucan, gamma-polyglutamic acid and mannitol which are plant sources. The invention provides a novel yeast freeze-drying protective agent which can effectively reduce the damage of thalli in the freeze-drying process and improve the survival rate of yeast.)

1. The composite yeast freeze-drying protective agent is characterized by being prepared by compounding plant-derived beta-glucan, gamma-polyglutamic acid and mannitol.

2. The composite yeast freeze-drying protective agent as claimed in claim 1, wherein 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.

3. The composite yeast freeze-drying protective agent as claimed in claim 2, wherein 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.

4. The composite yeast lyoprotectant of any one of claims 1 to 3, wherein said beta-glucan of vegetable origin is oat beta-glucan and/or barley beta-glucan.

5. The composite yeast lyoprotectant of claim 4, wherein said yeast is Saccharomyces cerevisiae.

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 freeze drying is a drying method in which the material is frozen to a temperature below the eutectic point and then water is removed from the material by sublimation under a low pressure. The freeze drying is a process of firstly converting the moisture in the material from a liquid state to a solid state and then converting the moisture from the solid state to a gas state, thereby realizing the drying process of the material. In the processThe thallus is stimulated by two processes of freezing and drying and inevitably damaged, and the damage of the thallus in the two processes can be effectively reduced by adding the protective agent. In terms of permeation, protective agents can be divided into permeable protective agents and impermeable protective agents, wherein the permeable protective agents undergo hydration reaction by entering the interior of cells to combine with water, thereby reducing the formation of intracellular ice crystals; the impermeable protective agent is attached to the outside of the cell, so that the viscosity of the protective system is improved, and the mechanical damage to the cell in the freeze-drying process is reduced. 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. In the remote research of Song Shi, 5% of skim milk powder, 4% of mannitol and 3% of sodium ascorbate are used as freeze-drying protective agents of fruit and vegetable biocontrol yeast strains, and the survival rate reaches 83.64%. The study of the autumn-yang and the like shows that when the sorbitol is 5.43g/100mL, the trehalose is 12.45g/100mL and the sodium glutamate is 13.56g/100mL, the combined protection effect is the best, and the survival rate of the microzyme can reach 84.21% +/-0.87%. When 14.15g/100mL of sucrose, 7.07g/100mL of L-sodium glutamate and 1.10g/100mL of polyethylene glycol are adopted by Wanghua and the like as the candida tropicalis freeze-drying protective agent, the freeze-drying survival rate is 82.73%. The CAHNYUAN research shows that the freeze-drying survival rate is 88.23% when 1.23% of human-like collagen, 11.50% of trehalose and 4.65% of glycerol are used as the bifidobacterium freeze-drying protective agent. The SHU study showed that the trehalose was 13% and Na₂HPO₄0.33 percent, 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)%. The development of new yeast protectants is essential for the protection of frozen yeast.

Disclosure of Invention

The invention aims to provide a novel composite yeast freeze-drying protective agent which can effectively protect yeast in a 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 prepared by compounding beta-glucan, gamma-polyglutamic acid and mannitol which are plant sources.

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 cryoprotectant 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 beta-glucan of vegetable origin is preferably oat beta-glucan and/or barley beta-glucan.

The yeast is preferably saccharomyces cerevisiae.

According to the invention, researches show that the addition of the beta-glucan can maintain the stability of the yeast cell membrane, improve the activity of antioxidant enzyme in the yeast cell and increase the content of intracellular trehalose, thereby improving the survival rate of the yeast cell. Gamma-polyglutamic acid as a macromolecular compound can protect cells in a wrapping mode, mannitol as a small molecule is mainly used for protecting proteins from aggregation by forming an amorphous structure, and mannitol in the amorphous structure can promote protein stability. 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 amount of mannitol added is higher than a certain value, the formed crystal structure has no protective effect on the protein, and even promotes the destruction of the cell 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 of thalli in the freeze-drying process and improve the survival rate of yeast.

Drawings

FIG. 1 shows the cell morphology of lyophilized Saccharomyces cerevisiae, wherein a is blank group and b is added composite protectant group.

Detailed Description

The technical solution of the present invention is illustrated by the following specific examples, 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 corresponding parts to prepare an aqueous solution, wherein the mass percentage concentration of the beta-glucan is 6%.

When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae freeze-drying protection, the corresponding survival rate of the yeast is 87.96%. It was used for the subsequent lyophilization protection of Pichia pastoris (Pichia kudriavzevii IFM 53500) with a survival rate of 82.50% after lyophilization, and for the lyophilization protection of Hanjim yeast Exo (Wickerhamomyces anomalus isolate MDY2) with a corresponding survival rate of 81.68%. However, the survival rate of the blank control group (without 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 corresponding parts to prepare an aqueous solution, wherein the mass percentage concentration of the beta-glucan is 6.56%.

When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast is 90.69%. It was used for the subsequent lyophilization protection of Pichia pastoris (Pichia kudriavzevii IFM 53500) with a survival rate of 85.17% after lyophilization, and for the lyophilization protection of Hanjim yeast Exo (Wickerhamomyces anomalus isolate MDY2) with a corresponding survival rate of 83.45%.

Comparative example 1

The yeast freeze-drying protective agent is prepared into aqueous solution, 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. When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast 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. It was used for subsequent saccharomyces cerevisiae protection, corresponding to a yeast survival rate of 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 cane sugar and the balance of sterile distilled water according to 100 ml.

It was used for subsequent saccharomyces cerevisiae protection, corresponding to a yeast survival rate of 72.77%.

Comparative example 4

The yeast freeze-drying protective agent is a distilled water solution with the mass percentage concentration of 6 percent of beta-glucan. It was used for subsequent saccharomyces cerevisiae protection, corresponding to a yeast survival rate of 36.78%.

Comparative example 5

The yeast freeze-drying protective agent is a distilled water solution with the mass percentage concentration of 0.17 percent of gamma-polyglutamic acid. When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast is 36.03%.

Comparative example 6

The yeast freeze-drying protective agent is a distilled water solution with the mass percentage concentration of mannitol of 1%. When used for subsequent saccharomyces cerevisiae protection, the corresponding yeast survival rate is 37.12%.

Comparative example 7

The yeast freeze-drying protective agent is a solution formed by dissolving beta-glucan and gamma-polyglutamic acid 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%. When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast is 67.07 percent.

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%. When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast is 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%. When the recombinant yeast strain is used for subsequent saccharomyces cerevisiae protection, the corresponding survival rate of the yeast is 65.91 percent.

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 concentrations of the beta-glucan, the mannitol and the gamma-polyglutamic acid are shown in the following table. The product is used for subsequent saccharomyces cerevisiae protection, and the corresponding survival rate of the yeast is detailed in the following table.

Therefore, the composite freeze-drying protective agent of the comparative example is not as good as the composite freeze-drying protective agent of the invention in the protection effect on the saccharomyces cerevisiae.

The following methods were used to evaluate the effects of the protective agents of the examples and comparative examples.

Experimental methods

1. Vacuum freeze drying process

The yeast system added with sterile water is taken as a blank control group, and the yeast system added with the protective agent solution of different examples and comparative examples is taken as a treatment group.

The yeast system is obtained by the following method:

1) saccharomyces cerevisiae

Dissolving appropriate amount of Angel high activity dry yeast produced by Angel Yeast GmbH in sterile water, coating on YPD solid culture medium by dilution coating plate method, culturing at 37 deg.C for 48 hr, selecting single colony, performing amplification culture in YPD liquid culture medium, culturing for 20 hr, and centrifuging to obtain bacterial sludge.

2) Acquisition of Hanm's yeast Exo and Pichia pastoris

Weighing 10g of old fermented dough sample (yeast type old fermented dough, sampling in mountainous and plain areas in the west of Henan), adding 100mL of sterilized distilled water into an ultra-clean workbench, homogenizing for 2 minutes in an aseptic homogenizer, sucking suspension, diluting in ten times of gradient, and coating 0.1mL of flat plate. 50mg/L chloramphenicol and 50mg/L ampicillin were added to YPD medium separately cultured with yeast, and cultured in constant temperature and humidity incubator at 30 deg.C for 48 hr. Selecting a plate with the colony number of 50-200 from each sample, randomly selecting 15 single colonies from the plate, performing shake culture at 150rpm/min for 15h in a YPD liquid culture medium at 30 ℃, further performing activation culture, and identifying by using DNA sequencing to obtain abnormal Wilkhem yeast and Pichia pastoris. Then inoculating the abnormal yeast Wickhamia and the Pichia pastoris into a YPD liquid culture medium respectively for amplification culture, and centrifuging after culturing for 20h to obtain corresponding bacterial sludge.

Mixing the corresponding yeast paste with sterile water or a protective agent solution according to a mass ratio of 1:2, balancing in a 30 ℃ constant temperature cabinet for 60min, then pre-freezing in a-18 ℃ refrigerator for 8h, quickly putting the pre-frozen yeast paste into a vacuum freeze-drying machine after pre-freezing, and freeze-drying for 20h under the conditions of a cold trap temperature of-75 ℃ and a vacuum degree of 145-155 mToor to obtain the yeast freeze-dried powder.

2. Calculation of the viability of the Yeast bacteria after lyophilization

Rehydrating the yeast freeze-dried powder obtained in the previous step with sterile physiological saline with the volume concentration of 0.85% to the volume before freeze-drying, putting the yeast freeze-dried powder into a 30 ℃ constant temperature incubator for activation for 30min after dissolution, and determining the freeze-drying survival rate of the yeast by using a methylene blue staining method, wherein the survival rate calculation formula is as follows:

3. microstructure observation

The damage of the freeze-drying to the saccharomyces cerevisiae cells and the effect of the protective agent can be visually observed through fig. 1. The cell deformation and rupture (indicated by arrows in fig. 1 a) of the part without the protective agent group are only 3% of the survival rate of the freeze-dried yeast. The added compound protective agent group (figure 1b, corresponding to example 2) has complete thallus morphology and full cells, and the freeze-drying survival rate of the yeast can reach 90.69%. The result shows that the freeze-drying-rehydration process destroys the integrity of the cell membrane of the yeast, and the composite protective agent can well maintain the permeability barrier and the structural integrity of the cell membrane of the freeze-dried thallus, avoid the leakage of cell contents and realize the freeze-drying protection of the yeast.