CN117327584A

CN117327584A - Freeze-drying protective agent for salmonella, preparation method and application of freeze-drying protective agent

Info

Publication number: CN117327584A
Application number: CN202311299756.6A
Authority: CN
Inventors: 熊家军; 吕志远; 刘丽娟; 史月军; 韦唯; 杨翔淞; 舒永兰
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2023-10-09
Filing date: 2023-10-09
Publication date: 2024-01-02

Abstract

The application relates to a freeze-drying protective agent for salmonella, a preparation method and application of a freeze-dried product. The lyoprotectant is at least two selected from maltose, glucose, trehalose, glycerol, sodium glutamate, yeast extract, and sodium ascorbate. The freeze-dried product comprises salmonella which is mixed with the freeze-drying protective agent and freeze-dried to form a solid sample. The freeze-dried product is convenient to store and transport, has very high bacterial survival rate, and improves the activity efficiency of the bacterial strain.

Description

Freeze-drying protective agent for salmonella, preparation method and application of freeze-drying protective agent

Technical Field

The application relates to the technical field of salmonella sample preparation, in particular to a salmonella freeze-drying protective agent, a salmonella freeze-drying product preparation method and application.

Background

Salmonella is the causative agent of salmonellosis and belongs to the family Enterobacteriaceae, gram-negative enterobacteria. The biochemical reaction has important reference significance for identifying the genus bacteria (see table). The gelatin is not liquefied, urea is not decomposed, indole is not produced, lactose and sucrose are not fermented, glucose, mannitol, maltose and Wei Ya sugar can be fermented, most of acid and gas are produced, and few acid and gas are produced. VP test negative, lysine decarboxylase. The G+C content of the DNA is 50 to 53%. Is not strong against heat and can be killed at 60 ℃ for 15 minutes. Survive in water for 2-3 weeks. In 5% carbolic acid, 5 minutes of death. The genus bacteria are divided into 4 subgenera according to biochemical reaction. Subgenera i is salmonella typical and most common for biochemical reactions; subgenera II and IV are Salmonella whose biochemical reaction is atypical; subgenera III is Salmonella arizona.

Common salmonella include salmonella typhi, salmonella gallinarum, salmonella suis, salmonella choleraesuis, salmonella paratyphi a, salmonella typhi and salmonella sendai. Salmonella has a complex antigen structure, and generally salmonella has three antigens, a thallus (O) antigen, a flagella (H) antigen, and a surface antigen (capsular or envelope antigen).

For the investigation of this genus, it is often necessary to preserve it, for example, at low temperature, glycerol, however, these preservation methods have a negative effect on the activity of the strain and the activation of the strain also becomes a burden on the use of the strain.

Disclosure of Invention

The vacuum freeze drying technology is the most effective means for preserving the stability of biological samples, is relatively mature, can carry out batch production on products, has stable effect, has the characteristics of convenient preservation, strong storage stability and convenient transportation of freeze-dried products, and is widely applied to the production in the fields of protein, probiotics, vaccine and the like. The inventor of the application develops a preparation which can be added into salmonella bacteria liquid for freeze-drying protection in the freeze-drying process, and can form a salmonella freeze-dried product with stable performance. The freeze-dried products are convenient to store and transport, have very high bacterial survival rate and improve the activity efficiency of the bacterial strain. Therefore, the embodiment of the application at least discloses the following technical scheme:

(1) A freeze-drying protective agent for salmonella, which is selected from at least two of maltose, glucose, trehalose, glycerol, sodium glutamate, yeast extract and sodium ascorbate.

(2) A lyophilized product of salmonella comprising (1) the lyoprotectant and salmonella mixed with the lyoprotectant and freeze-dried to form a solid sample.

(3) A method for preparing salmonella lyophilized product comprises:

preparing salmonella bacteria liquid and the lyoprotectant according to (1), wherein the lyoprotectant is an aqueous solution;

and after the salmonella bacteria liquid and the freeze-drying protective agent are mixed, prefreezing, maintaining the first temperature, heating and maintaining the second temperature in sequence.

(4) The application of the freeze-drying protective agent (1) in preparing salmonella freeze-dried products.

Drawings

Fig. 1 is an appearance of salmonella C500 lyophilizate provided in example 1.

Fig. 2 is an appearance of salmonella C500 lyophilizate provided in comparative example 13.

Fig. 3 is an appearance of salmonella C500 lyophilized product provided in comparative example 14.

Fig. 4 is an appearance of salmonella C500 lyophilizate provided in comparative example 15.

Fig. 5 is an appearance of salmonella C500 lyophilizate provided in comparative example 16.

Fig. 6 is an appearance of salmonella C500 lyophilizate provided in comparative example 17.

Fig. 7 is an appearance of salmonella C500 lyophilizate provided in comparative example 18.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

In the present application, the term "part" means only part by weight, and specifically any weight may be used as 1 part, such as 0.001g, 0.01g, 0.05g, 0.1g, 0.5g, 1g, 10g, 100g, 1000g, 5000g, 10000g, etc.

In the application, the yeast extract is also called yeast taste agent, and is a brown yellow soluble paste or pale yellow powdery pure natural product prepared by adopting edible yeast with rich protein content as raw material according to the regulation of Chinese pharmacopoeia and adopting modern biological high and new technologies of autolysis, enzymolysis, separation, concentration and the like to degrade protein, nucleic acid and the like in yeast cells and refining.

The embodiment discloses a salmonella freeze-drying protective agent which is at least two selected from maltose, glucose, trehalose, glycerol, sodium glutamate, yeast extract and sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight, 200-1200 parts glucose, 200-1200 parts trehalose, 50-300 parts glycerol, 50-300 parts sodium glutamate, 200-1200 parts yeast extract, and 30-180 parts sodium ascorbate.

In some embodiments, glucose is optionally in any of 200-1200 parts, e.g., 200 parts, 300 parts, 400 parts, 500 parts, 600 parts, 700 parts, 800 parts, 900 parts, 1000 parts, 1100 parts, 1200 parts. In some embodiments, trehalose is optionally in any of 200-1200 parts, e.g., 200 parts, 300 parts, 400 parts, 500 parts, 600 parts, 700 parts, 800 parts, 900 parts, 1000 parts, 1100 parts, 1200 parts.

In some embodiments, the glycerol is optionally in any of 50-300 parts, e.g., 50 parts, 100 parts, 150 parts, 200 parts, 250 parts, 300 parts.

In some embodiments, sodium glutamate is optionally in any of 50-300 parts, e.g., 50 parts, 100 parts, 150 parts, 200 parts, 250 parts, 300 parts.

In some embodiments, the yeast extract is optionally in any of 200-1200 parts, e.g., 200 parts, 300 parts, 400 parts, 500 parts, 600 parts, 700 parts, 800 parts, 900 parts, 1000 parts, 1100 parts, 1200 parts.

In some embodiments, sodium ascorbate is optionally any of 30-180 parts, for example 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, and 180 parts.

In some embodiments, the lyoprotectant comprises, in parts by weight: 200 parts of glucose, 200 parts of trehalose, 50 parts of glycerol, 50 parts of sodium glutamate, 200 parts of yeast extract and 30 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight: 400 parts of glucose, 400 parts of trehalose, 100 parts of glycerol, 100 parts of sodium glutamate, 400 parts of yeast extract and 60 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight: 600 parts of glucose, 600 parts of trehalose, 150 parts of glycerol, 150 parts of sodium glutamate, 600 parts of yeast extract and 90 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight: 800 parts of glucose, 800 parts of trehalose, 200 parts of glycerol, 200 parts of sodium glutamate, 800 parts of yeast extract and 120 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight: 1000 parts of glucose, 1000 parts of trehalose, 250 parts of glycerol, 250 parts of sodium glutamate, 1000 parts of yeast extract and 150 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises, in parts by weight: 1200 parts of maltose, 1200 parts of trehalose, 300 parts of glycerol, 300 parts of sodium glutamate, 1200 parts of yeast extract and 180 parts of sodium ascorbate.

In some embodiments, the lyoprotectant comprises 500-900 parts maltose, 80-200 parts glycerol, and 300-500 parts yeast extract in parts by weight.

In these embodiments, the maltose is optionally present in any of 500 to 900 parts, such as 500 parts, 600 parts, 700 parts, 800 parts, and 900 parts, for example, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649 650 parts, 651 parts, 652 parts, 653 parts, 654 parts, 655 parts, 656 parts, 657 parts, 658 parts, 659 parts, 660 parts, 661 parts, 662 parts, 663 parts, 664 parts, 665 parts, 666 parts, 667 parts, 668 parts, 669 parts, 670 parts, 671 parts, 672 parts, 673 parts, 674 parts, 675 parts, 676 parts, 677 parts, 678 parts, 679 parts, 680 parts, 681 parts, 682 parts, 683 parts, 684 parts, 685 parts, 686 parts, 687 parts, 688 parts, 689 parts, 690 parts, 691 parts, 692 parts, 693 parts, 694 parts, 695 parts, 696 parts, 697 parts, 698 parts, 699 parts.

In these embodiments, the glycerin is optionally any of 80-900 parts, such as 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, 130 parts, 140 parts, 150 parts.

In these embodiments, the yeast extract is optionally in any of 300 to 500 parts, such as 300 parts, 400 parts, and 500 parts; for example, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399.

In some embodiments, the lyoprotectant comprises 600 parts maltose, 100 parts glycerol, and 400 parts yeast extract in parts by weight.

In some embodiments, the lyoprotectant comprises 900 parts maltose, 150 parts glycerol, and 400 parts yeast extract in parts by weight.

In some embodiments, the lyoprotectant further comprises water as a solvent.

The embodiment also discloses a salmonella lyophilizate which comprises the lyoprotectant provided by the embodiment above and salmonella which is mixed with the lyoprotectant and subjected to freeze drying to form a solid sample.

In some embodiments, the salmonella is divided into biochemical reactions, including type i, type II, type III, and type IV salmonella.

In some embodiments, the salmonella is selected from the group consisting of salmonella typhi, salmonella gallinarum, salmonella suis, salmonella choleraesuis, salmonella paratyphi a, salmonella typhi, and salmonella sendai; optionally, the salmonella is salmonella choleraesuis C500.

The embodiment also discloses a preparation method of the salmonella freeze-dried product, which comprises the following steps:

preparing salmonella bacteria liquid and the lyoprotectant provided by the embodiment, wherein the lyoprotectant is an aqueous solution;

and after the salmonella bacteria liquid and the freeze-drying protective agent are mixed, pre-freezing, maintaining the first temperature, heating and maintaining the second temperature.

In some embodiments, the pre-freezing comprises: and (3) reducing the temperature of the mixture of the salmonella bacteria liquid and the freeze-drying protective agent to-50 to-40 ℃ at normal pressure and room temperature at a cooling rate of 0.5-1.5 ℃/min.

In some embodiments, the pre-freezing comprises: and (3) reducing the temperature of the mixture of the salmonella bacteria liquid and the freeze-drying protective agent to-45 ℃ at the normal pressure and room temperature at the cooling rate of 1.0 ℃/min.

In some embodiments, the first temperature maintaining comprises: and (3) keeping the mixture of the salmonella bacteria liquid and the freeze-drying protective agent at the temperature of between 50 ℃ below zero and 40 ℃ below zero for 2 to 5 hours under normal pressure.

In some embodiments, the first temperature maintaining comprises: and (3) maintaining the mixture of the salmonella bacteria liquid and the freeze-drying protective agent at the temperature of-45 ℃ under normal pressure for 3 hours.

In some embodiments, the heating comprises: and (3) raising the temperature of the mixture of the salmonella bacteria liquid and the freeze-drying protective agent to 5-15 ℃ at the temperature rising rate of 0.2-1.5 ℃/min under the pressure of less than or equal to 15 Pa.

In some embodiments, the heating comprises: and (3) raising the temperature of the mixture of the salmonella bacteria liquid and the freeze-drying protective agent to 5-15 ℃ at the temperature rising rate of 0.2-1.5 ℃/min under the pressure of less than or equal to 10 Pa.

In some embodiments, the heating comprises: and (3) the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 15 ℃ at the temperature rising rate of 0.5 ℃/min under the pressure of less than or equal to 15 Pa. For example, the first temperature resulting from the first temperature maintaining stage is raised to 15℃and the first temperature is-50 to-40℃or-45 ℃.

In some embodiments, the heating comprises: and (3) the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 10 ℃ at the temperature rising rate of 1.0 ℃/min under the pressure of less than or equal to 15 Pa. For example, the first temperature obtained from the first temperature maintaining stage is raised to 10℃and the first temperature is-50 to-40℃or-45 ℃.

In some embodiments, the heating comprises: firstly, the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to-5 ℃ at the heating rate of 1.0 ℃/min under the pressure of less than or equal to 15Pa, and then heated to 15 ℃ at the heating rate of 0.5 ℃/min under the pressure of less than or equal to 15 Pa. For example, the first temperature resulting from the first temperature maintaining stage is raised to 15℃and the first temperature is-50 to-40℃or-45 ℃.

In some embodiments, the second temperature maintaining comprises: and (3) keeping the mixture of the salmonella bacteria liquid and the freeze-drying protective agent for 2-5 hours at the temperature of 5-15 ℃ under the pressure of less than or equal to 15 Pa.

In some embodiments, the second temperature maintaining comprises: and (3) keeping the mixture of the salmonella bacteria liquid and the freeze-drying protective agent for 2-5 hours at 15 ℃ under the pressure of less than or equal to 15 Pa.

The embodiment also discloses application of the freeze-drying protective agent provided by the embodiment in preparation of salmonella freeze-dried products.

In some embodiments, lyoprotectants as shown in table 1 are provided as aqueous solutions, table 1 showing the solute composition therein, where the concentrations are both mass per volume percent (g/100 mL).

Table 1 (bacterial survival rate expressed as mean and standard deviation, and marked with significant differences, n=4)

Examples	Solute composition	Bacterial storage rate
			Example 1	6% maltose, 1.0% glycerol, 4% yeast extract	79.09±2.44％a
Example 2	9% maltose, 1.5% glycerol, 4% yeast extract	76.39±3.64％a
			Comparative example 1	3% maltose	42.81±1.26％b
Comparative example 2	6% maltose	43.14±3.54％b
			Comparative example 3	9% maltose	54.63±4.19％b
Comparative example 4	12% maltose	54.84±6.91％b
			Comparative example 5	15% maltose	55.72±8.48％b
Comparative example 6	18% maltose	51.63±1.75％a
			Comparative example 7	3% Yeast extract	33.85±3.42％c
Comparative example 8	6% Yeast extract	42.31±5.86％b
			Comparative example 9	9% Yeast extract	45.47±6.55％b
Comparative example 10	12% Yeast extract	51.72±6.55％b
			Comparative example 11	15% yeast extract	49.8±6.93％b
Comparative example 12	18% yeast extract	47.97±3.28％b
			Comparative example 13	0.2% Glycerol	14.32±1.85％d
Comparative example 14	0.4% Glycerol	17.60±3.57％d
			Comparative example 15	0.6% Glycerol	16.15±2.09％d
Comparative example 16	0.8% Glycerol	16.30±2.93％d
			Comparative example 17	1.0% Glycerol	15.06±2.80％d
Comparative example 18	1.2% Glycerol	12.27±2.55％d

Wherein the step of resuscitating the strain comprises:

collecting Salmonella choleraesuis C500 (pVAX-S/GnRH-2 a/KISS 1-asd) strain stored in-80deg.C refrigerator, preserving in China center for type culture collection (preservation number: CCTCC NO: M2020055) at 3 month and 19 days in 2020, standing at room temperature for 3-5 min to naturally dissolve, burning, cooling, inoculating, dipping a small amount of bacterial liquid on LB solid plate, drawing lines in regions, and culturing at 37deg.C with air-permeable sealing film for 48 hr. Larger single colonies were selected and inoculated onto new LB solid plates for plate secondary purification and cultured at 37℃for 24 hours. The larger single colony is selected and inoculated on a new LB solid plate (four to five single colonies are enlarged), and the single colony is cultured for 24 hours at 37 ℃ and then is cultured for 24 hours at room temperature, so that four to five single colonies with common sources are obtained, and the single colony is stored at the temperature of 4 ℃. One single colony after expansion is randomly selected, a small amount of colony is selected and inoculated into a 50mL centrifuge tube filled with 30mL LB culture medium for culturing for 7 hours at 37 ℃ at 220 r/min.

Wherein the step of strain culture comprises: the whole single drop was picked up with a cooled inoculating loop after firing and inoculated into a 50mL centrifuge tube containing 30mL LB medium at 220rpm and 37℃for 7 hours to prepare a seed solution. 5ml of the cultured bacterial liquid was taken, and the bacterial concentration (OD 600) was measured by a spectrophotometer and blank correction was performed with LB medium.

Wherein, the preparation steps of the concentrated bacterial liquid comprise: according to the OD600 of 1%, inoculating the seed solution into a 500ml conical flask filled with 400ml LB liquid culture medium according to the inoculation proportion, culturing for 14 hours at 220rpm and 37 ℃, performing expansion culture, subpackaging the cultured bacterial solution into corresponding centrifuge tubes, temporarily storing for 3 hours at 4 ℃, centrifuging for 10 minutes at 5000rpm and 4 ℃, discarding the supernatant, re-suspending the bacterial solution by using sterile PBS (pH: 7.2-7.4) with the volume of 1/20 of the original bacterial solution, fully and uniformly mixing, taking 500 mu L for measuring the viable bacteria concentration, temporarily storing the rest sample in a refrigerator at 4 ℃, and waiting for the next operation.

Wherein the step of freeze-drying the sample comprises: the lyoprotectants provided in examples and comparative examples of Table 1, respectively, were dispensed into sterile 7mL penicillin bottles (750. Mu.L/vial) or 2mL chromatographic sample bottles (200. Mu.L/vial). Taking bacterial liquid which is 20 times concentrated, subpackaging the bacterial liquid into sterile 7mL penicillin bottles or 2mL chromatographic sample injection bottles according to the volume ratio of 1:1, and lightly blowing and uniformly mixing. Each test was repeated in parallel with 6 to 40 replicates of each group according to grouping conditions and required amount, marked with label paper, and then placed in a refrigerator at 4 ℃ for cold stress promotion for about 12 hours. And (3) lightly blowing and uniformly mixing the sample subjected to stress treatment, and then placing the sample in a freeze dryer or a refrigerator for pre-freezing. And (3) uniformly placing the samples on a partition board of a freeze dryer as far as possible, freeze-drying according to a set freeze-drying program, sealing the freeze-dried samples, and temporarily storing the freeze-dried samples in a refrigerator at the temperature of minus 20 ℃. The lyophilization procedure included: pre-freezing: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is cooled to-45 ℃ at normal pressure and room temperature of 20 ℃ at a cooling rate of 1.0 ℃/min; first temperature maintenance: maintaining the mixture of the salmonella bacteria liquid and the freeze-drying protective agent at the normal pressure and the temperature of-45 ℃ for 5 hours; heating: firstly, the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to-5 ℃ at the heating rate of 1.0 ℃/min under the pressure of less than or equal to 15Pa, and then heated to 15 ℃ at the heating rate of 0.5 ℃/min under the pressure of less than or equal to 15 Pa; and (3) maintaining the second temperature: and (3) treating the mixture of the salmonella bacteria liquid and the freeze-drying protective agent for 5 hours at 15 ℃ under the pressure of less than or equal to 15 Pa.

The bacterial survival rate detection method comprises the following steps:

taking 3 samples stored in a refrigerator at the temperature of minus 20 ℃, placing the samples in each group for 5min at room temperature, adding an equal volume of sterile PBS for rehydration, blowing and mixing uniformly, collecting the samples into a 2ml centrifuge tube, carrying out gradient concentration dilution after mixing uniformly upside down, selecting proper dilution factors for flat plate coating, coating 4 flat plates on each group, calculating the viable bacteria concentration of the freeze-dried samples after rehydration, and calculating the bacterial storage rate according to the viable bacteria concentration of the concentrated bacterial liquid, wherein the calculation mode is as follows:

bacterial yield = 100% ×complex horizontal plate count x dilution x 2/(bacterial suspension plate count x dilution)

As a result, as shown in Table 1, the bacterial count was lower than that of examples 1 or 2 using maltose (comparative examples 1 to 6), yeast extracts (comparative examples 7 to 12) or glycerin (comparative examples 13 to 18) alone as lyoprotectants. Thus, the glycerol and the yeast extract are respectively a permeability protective agent and a protein protective agent, and the maltose is a semi-permeability protective agent, so that the freeze-drying protective effect of the glycerol and the yeast extract has better complementary effect with that of the maltose. The freeze-drying protective agent provided in the embodiments 1 and 2 is more beneficial to preparing salmonella freeze-dried products.

In addition, as shown in fig. 1, glycerin alone is used as a lyoprotectant, and the obtained salmonella lyophilized product has poor appearance and cannot form a stable solid appearance.

In some embodiments, lyoprotectants and lyophilization processes are provided as shown in table 2, where table 2 shows the solute composition (solvent is sterile water) and the lyophilization process, where the concentrations are both mass/volume percent (g/100 mL).

In the examples and comparative examples provided in Table 2, the steps and methods of strain recovery, strain culture and bacterial survival rate were the same as those of the examples provided in Table 1. The sample lyophilization steps in the examples and comparative examples provided in table 2 were the same as the examples provided in table 1 except that the lyophilization process was shown in table 2. The lyoprotectant in table 2 consisted of 6% maltose, 1.0% glycerol, 4% yeast extract.

As can be seen from Table 2, comparative example 19 uses less pre-freezing time and heating time, and comparative example 20 uses a faster pre-freezing rate, and thus has a lower bacterial count, than examples 3 to 5. The pre-freezing speed is an important aspect affecting the bacterial survival rate after lyophilization. The faster the cooling speed at the same temperature is, the smaller the volume of the formed ice crystals is, and the more the quantity is; the slower the cooling rate, the greater the volume of ice crystals formed. Excessive ice crystals are more likely to cause mechanical damage to cells, so that cell membranes are broken to cause bacterial damage, meanwhile, water molecules in cells are seriously leaked out, and the osmotic pressure of cytoplasm is increased to cause osmotic pressure damage to cells. However, at higher freezing rates, the number of ice crystals formed is large, the particles are small, and when the water in the cell does not always permeate out of the cell in time, the ice crystals are formed in the cell, so that cell membranes and organelles are damaged. The temperature rising speed can also have direct influence on the water sublimation speed and whether the water sublimation causes cell damage in the frozen solid freeze-dried product.

Table 2 (bacterial survival rate expressed as mean and standard deviation, and marked with significant differences, n=4)

Based on this, the present application divides the lyophilization process into four stages, prefreezing, first temperature maintaining, warming and second temperature maintaining, and the lyophilization process thus formed is more conducive to preservation of salmonella. In addition, comparative example 21, which uses a higher first temperature maintenance than examples 3 to 5, had insufficient pre-freezing, had insufficient numbers of ice crystals formed from water, and small particles, and still had adverse effects on the shelf life and bacterial survival rate of the final lyophilized product due to the fact that a small amount of water was still present in the final lyophilized product due to the sufficient sublimation of water therein.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims

1. A freeze-drying protective agent for salmonella, which is selected from at least two of maltose, glucose, trehalose, glycerol, sodium glutamate, yeast extract and sodium ascorbate.

2. The lyoprotectant of claim 1, comprising, in parts by weight:

200-1200 parts of glucose;

200-1200 parts of trehalose;

50-300 parts of glycerol;

50-300 parts of sodium glutamate;

200-1200 parts of yeast extract; and

30-180 parts of sodium ascorbate.

3. The lyoprotectant according to claim 2,

optionally, the lyoprotectant comprises the following components in parts by weight: 200 parts of glucose, 200 parts of trehalose, 50 parts of glycerol, 50 parts of sodium glutamate, 200 parts of yeast extract and 30 parts of sodium ascorbate;

optionally, the lyoprotectant comprises the following components in parts by weight: 400 parts of glucose, 400 parts of trehalose, 100 parts of glycerol, 100 parts of sodium glutamate, 400 parts of yeast extract and 60 parts of sodium ascorbate;

optionally, the lyoprotectant comprises the following components in parts by weight: 600 parts of glucose, 600 parts of trehalose, 150 parts of glycerol, 150 parts of sodium glutamate, 600 parts of yeast extract and 90 parts of sodium ascorbate;

optionally, the lyoprotectant comprises the following components in parts by weight: 800 parts of glucose, 800 parts of trehalose, 200 parts of glycerol, 200 parts of sodium glutamate, 800 parts of yeast extract and 120 parts of sodium ascorbate;

optionally, the lyoprotectant comprises the following components in parts by weight: 1000 parts of glucose, 1000 parts of trehalose, 250 parts of glycerol, 250 parts of sodium glutamate, 1000 parts of yeast extract and 150 parts of sodium ascorbate;

optionally, the lyoprotectant comprises the following components in parts by weight: 1200 parts of glucose, 1200 parts of trehalose, 300 parts of glycerol, 300 parts of sodium glutamate, 1200 parts of yeast extract and 180 parts of sodium ascorbate.

4. The lyoprotectant of claim 1, comprising, in parts by weight: 500-900 parts of maltose, 80-200 parts of glycerol and 300-500 parts of yeast extract.

5. The lyoprotectant of claim 4, optionally comprising, in parts by weight: 600 parts of maltose, 100 parts of glycerol, and 400 parts of yeast extract;

optionally, the lyoprotectant comprises the following components in parts by weight: 900 parts of maltose, 150 parts of glycerol and 400 parts of yeast extract.

6. A lyophilized preparation of salmonella comprising the lyoprotectant of any one of claims 1-5 and salmonella mixed with the lyoprotectant and lyophilized to form a solid sample.

7. The lyoprotectant of claim 5, wherein the salmonella is biochemically divided into type i, type II, type III and type IV salmonella; alternatively, the salmonella is selected from the group consisting of salmonella typhi, salmonella gallinarum, salmonella suis, salmonella choleraesuis, salmonella paratyphi a, salmonella typhi, and salmonella sendai; optionally, the salmonella is salmonella choleraesuis C500.

8. A method for preparing salmonella lyophilized product comprises:

preparing salmonella bacteria liquid and the lyoprotectant according to any one of claims 1-5, wherein the lyoprotectant is an aqueous solution;

9. The method of preparing according to claim 8, optionally, the prefreezing comprising: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is cooled to-50 to-40 ℃ at normal pressure at a cooling rate of 0.5-1.5 ℃/min;

optionally, the pre-freezing comprises: the temperature reduction rate of the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is reduced to-45 ℃ at the normal pressure of 1.0 ℃/min;

optionally, the first temperature maintaining includes: maintaining the mixture of the salmonella bacteria liquid and the freeze-drying protective agent at the temperature of between 50 ℃ below zero and 40 ℃ below zero for 2 to 5 hours under normal pressure;

optionally, the first temperature maintaining includes: maintaining the mixture of the salmonella bacteria liquid and the freeze-drying protective agent at the normal pressure and the temperature of-45 ℃ for 3 hours;

optionally, the heating comprises: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 5-15 ℃ at the temperature rising rate of 0.2-1.5 ℃/min under the pressure of less than or equal to 15 Pa;

optionally, the heating comprises: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 5-15 ℃ at the temperature rising rate of 0.2-1.5 ℃/min under the pressure of less than or equal to 10 Pa;

optionally, the heating comprises: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 15 ℃ at the temperature rising rate of 0.5 ℃/min under the pressure of less than or equal to 15 Pa;

optionally, the heating comprises: the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to 10 ℃ at the temperature rising rate of 1.0 ℃/min under the pressure of less than or equal to 15 Pa;

optionally, the heating comprises: firstly, the mixture of the salmonella bacteria liquid and the freeze-drying protective agent is heated to-5 ℃ at the heating rate of 1.0 ℃/min under the pressure of less than or equal to 15Pa, and then heated to 15 ℃ at the heating rate of 0.5 ℃/min under the pressure of less than or equal to 15 Pa;

optionally, the second temperature maintaining includes: maintaining the mixture of the salmonella bacteria liquid and the freeze-drying protective agent for 2-5 hours at the temperature of 5-15 ℃ under the pressure of less than or equal to 15 Pa;

optionally, the second temperature maintaining includes: and (3) keeping the mixture of the salmonella bacteria liquid and the freeze-drying protective agent for 2-5 hours at 15 ℃ under the pressure of less than or equal to 15 Pa.

10. Use of the lyoprotectant according to any one of claims 1-5 for the preparation of a salmonella lyophilizate.