CN110468050B - Freeze-drying method for improving survival rate of lactobacillus plantarum by utilizing polysaccharide - Google Patents
Freeze-drying method for improving survival rate of lactobacillus plantarum by utilizing polysaccharide Download PDFInfo
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Abstract
The invention provides a freeze-drying method for improving survival rate of lactobacillus plantarum by utilizing polysaccharide, which comprises the following steps: activating the strain to obtain a single bacterial colony; inoculating and culturing the single colony to obtain a seed solution; transferring the seed liquid to an MRS liquid culture medium for amplification culture to obtain an amplification culture liquid; centrifuging the enlarged culture solution to obtain thallus precipitate; and washing the thallus precipitate with PBS buffer solution, then suspending in polysaccharide protective agent solution, transferring to a container, and freeze-drying, wherein the strain is Lactobacillus plantarum AR113 or Lactobacillus plantarum WCFS1, and the polysaccharide protective agent solution is soybean polysaccharide solution or Arabic gum solution. According to the freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide, the survival rate of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 after being rehydrated after freeze-dried is obviously improved, a small molecular protective agent can be replaced by a macromolecular compound, and the range and the types of the protective agent of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 are widened.
Description
Technical Field
The invention belongs to the field of freeze drying, and particularly relates to a freeze drying method for improving the survival rate of lactobacillus plantarum by using polysaccharide.
Background
The lactobacillus plantarum belongs to probiotics, can be effectively planted in human intestinal tracts, can inhibit the proliferation of harmful bacteria, protect the intestinal tracts and the like, and can effectively reduce cholesterol, improve immunity and the like. Therefore, at present, the lactobacillus plantarum is not only widely applied to fermentation products, but also has great potential in the aspects of functional foods and clinical application. However, storage of lactobacillus plantarum is a difficult problem. In order to stabilize unstable products of probiotic species, the water content of the stored samples must be reduced to reach a state of dormancy.
Freezing is a unit operation that freezes water, thereby reducing the water content of the sample to a resting state. However, maintaining and transporting the sample in a frozen state is expensive and may result in a loss of value to the product. Alternatively, the sample may be dried in air by high processing temperatures, but conventional drying methods can cause changes in the physical and chemical properties of the sample. Freeze-drying, in turn, combines the advantages of freezing and drying to provide a dry, highly active, shelf stable, and easily dissolvable product. Freeze-drying is now widely used for the preservation of lactic acid bacteria and is one of the most effective methods for preserving biological materials.
Freeze-drying has many advantages, but it causes some physiological damage due to the exposure of cells to extreme environmental stresses, thereby reducing cell viability and functional activity. The major damage during freeze-drying can be attributed to changes in cell membrane integrity, fluidity, and structure of sensitive proteins, among others. The characteristics of small cell volume and large specific surface area determine the characteristic of high water permeability of a cell membrane. In the freeze drying process, when the temperature is reduced and the vacuum degree is increased, the water in the cells is frozen and evaporated, and the solutes, electrolytes and the like in the cells are gradually concentrated, so that the cells are excessively dehydrated, severely shrink and deform, and even die; in addition, when the electrolyte is highly concentrated, the higher structure of some electrolyte-sensitive proteins in the cell is changed, especially key enzymes related to metabolism lose their physiological functions, so that the regulation of physiological metabolism is disordered, and the freeze-drying survival rate of the cell is reduced.
To increase the resistance of cells to lyophilization, damage from lyophilization is often mitigated by the use of protective agents. There are many kinds of protective agents, but at present, small molecular saccharides or alcohols are mainly used. For example, dimitrellou et al protect Lactobacillus casei with trehalose, carvalho et al protect Lactobacillus delbrueckii with sucrose, sorbitol, etc. Most of these saccharides are reducing saccharides, which may induce a destructive Maillard reaction, affect the stability of the sample, and the like. Therefore, non-reducing sugars such as sucrose and trehalose are widely used as cryoprotectants. The protective effect of the non-reducing sugars is mainly attributed to the fact that the non-reducing sugars have higher glass transition temperatures, particularly trehalose, can remarkably reduce the formation of ice crystals inside and outside cells at low temperature, and stabilize phospholipid bilayer and membrane protein structures. Compared with a micromolecular carbohydrate protective agent, the polysaccharide protective agent has better stability, does not interact with cells, can be used as natural dietary fiber and has multiple functions.
In the freeze drying process, different protective agents have great difference on the protective effects of different species of strains, and how to research a specific high-efficiency protective agent aiming at a specific strain is very important for the preservation of the strain.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a freeze-drying method for improving the survival rate of lactobacillus plantarum using a polysaccharide.
The invention provides a freeze-drying method for improving the survival rate of lactobacillus plantarum by utilizing polysaccharide, which is characterized by comprising the following steps of: step 1, activating strains to obtain single colonies; step 2, inoculating and culturing the single bacterial colony to obtain a seed solution; step 3, transferring the seed liquid to an MRS liquid culture medium for amplification culture to obtain an amplification culture liquid; step 4, centrifuging the expanded culture solution to obtain thallus precipitates; and 5, washing the thallus precipitate with PBS buffer solution, then suspending the thallus precipitate in polysaccharide protective agent solution, transferring the thallus precipitate into a container, and freeze-drying, wherein the strain in the step 1 is lactobacillus plantarum AR113 or lactobacillus plantarum WCFS1, and the polysaccharide protective agent solution in the step 5 is soybean polysaccharide solution or Arabic gum solution.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: wherein, the concentration of the soybean polysaccharide solution is 1 to 10 percent.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: wherein, the concentration of the Arabic gum solution is 1-3%.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: the strain activation method comprises the step of repeatedly marking and activating lactobacillus plantarum in a solid MRS culture medium for 2-5 times.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: the method for single colony inoculation culture comprises the steps of picking activated single colonies, and carrying out inoculation culture for 12-16 h.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: wherein the condition of the expanded culture in the step 3 is culture for 12 to 16 hours at the temperature of between 35 and 40 ℃.
The freeze-drying method for improving the survival rate of the lactobacillus plantarum by using the polysaccharide provided by the invention can also have the following characteristics: wherein, the freeze-drying parameters are as follows: precooling at-45 to-35 ℃ for 2 to 4 hours, heating to-30 to-25 ℃ at the rate of 0.8 to 1.5 ℃/min for primary drying, keeping the time for 700 to 900 minutes, then heating to 20 to 25 ℃ at the rate of 0.8 to 1.5 ℃/min for secondary drying, keeping the time for 2 to 3 hours, keeping the temperature of a cold trap at-85 to-70 ℃ and keeping the vacuum degree of 10 to 30Pa.
The invention also provides an application of the polysaccharide protective agent in improving the freeze-drying survival rate of lactobacillus plantarum, which is characterized in that the polysaccharide protective agent is soybean polysaccharide or Arabic gum, and the lactobacillus plantarum is lactobacillus plantarum AR113 or lactobacillus plantarum WCFS1.
Action and Effect of the invention
According to the freeze-drying method for improving the survival rate of the lactobacillus plantarum by utilizing the polysaccharide, which is disclosed by the invention, the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 are protected by adopting the soybean polysaccharide or the Arabic gum as a protective agent, so that the cell membrane damage of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 in the freeze-drying process is effectively reduced. Therefore, the survival rate of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 after being rehydrated after freeze-dried is remarkably improved, and the macromolecular compound can be used for replacing a micromolecular protective agent, so that the range and the types of the protective agent of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 are widened.
In addition, the soybean polysaccharide and the Arabic gum belong to polysaccharides, and have wide sources and low prices.
Drawings
FIG. 1 is a flow chart of a freeze-drying method for increasing survival rate of Lactobacillus plantarum using polysaccharides according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the survival rate of Lactobacillus plantarum AR113 in examples 1 to 3 of the present invention and comparative examples 1 to 6; and
FIG. 3 is a schematic diagram showing the survival rate of Lactobacillus plantarum WCFS1 in examples 4 to 6 of the present invention and comparative examples 7 to 12.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the freeze-drying method for improving the survival rate of lactobacillus plantarum by using polysaccharide is specifically described below with reference to the following embodiments and the accompanying drawings.
All chemical reagents in the examples and the comparative examples are chemically pure and purchased from national pharmaceutical group chemical reagents, ltd.
FIG. 1 is a flow chart of the freeze-drying method for increasing the survival rate of Lactobacillus plantarum using polysaccharides according to the present invention.
As shown in FIG. 1, a freeze-drying method for improving the survival rate of Lactobacillus plantarum using a polysaccharide comprises the following steps:
The specific operation is as follows: the strain is repeatedly streaked and activated for 3 times in a solid MRS culture medium to obtain a single colony. Wherein the strain is Lactobacillus plantarum AR113 or Lactobacillus plantarum WCFS1.
And 2, inoculating and culturing the single colony to obtain a seed solution.
The specific operation is as follows: and (4) selecting the activated single colony, inoculating and culturing for 12-16 h to obtain a seed solution.
And 3, transferring the seed liquid to an MRS liquid culture medium for amplification culture to obtain an amplification culture liquid. The condition of the enlarged culture is that the culture is carried out for 12 to 16 hours at the temperature of between 35 and 40 ℃.
The specific operation is as follows: transferring the seed liquid to an MRS liquid culture medium for amplification culture, and performing amplification culture at 37 ℃ for 12-16 h to obtain an amplification culture liquid.
And 4, centrifuging the enlarged culture solution to obtain a thallus precipitate.
The specific operation is as follows: adjusting and enlarging the bacteria concentration of the culture solution by using an enzyme-labeling instrument to ensure that the OD of the culture solution is 600 And =1, collecting the bacterial cell precipitate by a centrifugal tube under the condition that the centrifugal force is 4000rpm to obtain the bacterial cell precipitate.
And 5, washing the thallus precipitate by using PBS buffer solution, then suspending the thallus precipitate in polysaccharide protective agent solution, transferring the thallus precipitate to a container, and freezing and drying the thallus precipitate. Wherein, the polysaccharide protective agent solution is soybean polysaccharide solution with the concentration of 1-10% or Arabic gum solution with the concentration of 1-3%.
The freeze-drying parameters were: precooling at-45 to-35 ℃ for 2 to 4 hours, heating to-30 to-25 ℃ at the rate of 0.8 to 1.5 ℃/min for primary drying, keeping the time for 700 to 900 minutes, then heating to 20 to 25 ℃ at the rate of 0.8 to 1.5 ℃/min for secondary drying, keeping the time for 2 to 3 hours, keeping the temperature of a cold trap at-85 to-70 ℃ and keeping the vacuum degree of 10 to 30Pa.
The specific operation is as follows: the thalli sediment is washed by PBS buffer solution for 2 times and then respectively resuspended in different protective agent solutions, and the concentration of the thalli suspension is 10 9 cfu/mL, and transferred to a penicillin bottle for freeze-drying. The freeze-drying program was set up as follows: precooling at-40 deg.C for 3h, heating to-30 deg.C at a rate of 1 deg.C/min for primary drying for 800min, heating to 25 deg.C at a rate of 1 deg.C/min for secondary drying for 2h, cooling at-80 deg.C, and vacuum degree of 20Pa.
The species sources used in the following examples and comparative examples are as follows:
lactobacillus plantarum AR113: china Committee for culture Collection of microorganisms general microbiological culture Collection center (CGMCC) No.13909.
Lactobacillus plantarum WCFS1: purchased from ATCC (American type culture collection).
The MRS medium used in the following examples and comparative examples had the following composition:
MRS culture medium: 10.0g of peptone, 2.0g of dipotassium phosphate, 10.0g of beef extract powder, 5.0g of yeast extract, 0.25g of manganese sulfate, 5.0g of anhydrous sodium acetate, 20.0g of glucose, 2.0g of diamine citrate, 0.58g of magnesium sulfate, 801mL of tween and 1000mL of deionized water. Wherein, 2% agar is required to be added into the solid culture medium, and the liquid culture medium is not added.
The media were sterilized at 115 ℃ for 20min before use.
The formulations of the solutions used in the following examples or comparative examples were as follows:
10% sorbitol protectant solution: 10.0g of sorbitol and 100mL of deionized water;
10% mannitol protectant solution: 10.0g of mannitol and 100mL of deionized water;
10% mannose protectant solution: 10.0g of mannose and 100mL of deionized water;
10% trehalose protectant solution: 10.0g of trehalose and 100mL of deionized water;
10% sucrose protectant solution: 10.0g of sucrose and 100mL of deionized water;
1% gum arabic protectant solution: 1.0g of Arabic gum and 100mL of deionized water;
1% soy polysaccharide protectant solution: 1.0g of soybean polysaccharide and 100mL of deionized water;
10% soy polysaccharide protectant solution: 10.0g of soybean polysaccharide and 100mL of deionized water;
PBS buffer solution: 0.24g of monopotassium phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride, and the volume is adjusted to 1000mL by using water.
In the protective agent, sorbitol, mannitol and mannose belong to small molecular protective agents; trehalose and sucrose belong to non-reducing disaccharide protectors; arabic gum and soybean polysaccharide belong to polysaccharide protective agents.
PBS buffer solution was used as reference solution for blank control.
< example 1>
In the embodiment, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is 1% acacia gum solution.
< example 2>
In the embodiment, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is 1% soybean polysaccharide solution.
< example 3>
In this example, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is 10% soybean polysaccharide solution.
< example 4>
In the embodiment, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 1% Arabic gum solution.
< example 5>
In the embodiment, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 1% soybean polysaccharide solution.
< example 6>
In the embodiment, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 10% soybean polysaccharide solution.
The following is a comparative example of the present invention, which is identical to the examples except for the species of the seed and the protective agent.
< comparative example 1>
In the comparative example, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is PBS solution.
< comparative example 2>
In the control example, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is 10% sorbitol solution.
< comparative example 3>
In the control example, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is 10% mannitol solution.
< comparative example 4>
In the comparative example, the selected strain is Lactobacillus plantarum AR113, and the protective agent solution is a 10% mannose solution.
< comparative example 5>
In the comparative example, the selected strain is Lactobacillus plantarum AR113, and the protective agent solution is 10% trehalose solution.
< comparative example 6>
In the comparative example, the selected strain is lactobacillus plantarum AR113, and the protective agent solution is a 10% sucrose solution.
< comparative example 7>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is PBS solution.
< comparative example 8>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 10% sorbitol solution.
< comparative example 9>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 10% mannitol solution.
< comparative example 10>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is a 10% mannose solution.
< comparative example 11>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 10% trehalose solution.
< comparative example 12>
In the control example, the selected strain is lactobacillus plantarum WCFS1, and the protective agent solution is 10% sucrose solution.
< test example >
After freezing for 48h, adding PBS into the freeze-dried bacterial powder of the examples 1 to 6 and the comparative examples 1 to 12 for rehydration; then the bacterial liquid is diluted to a proper gradient and coated, and the bacterial liquid is cultured for 36 hours at 37 ℃ and counted. The experiment was repeated 3 times, 3 replicates each time. According to N 2 /N 0 X 100% calculation of the Freeze-drying survival Rate, N 0 Is the number of cells before freezing, N 2 Cell number after freeze-drying. The test results are shown in fig. 2, fig. 3 and table 1.
FIG. 2 is a schematic diagram showing the survival rate of Lactobacillus plantarum AR113 in examples 1 to 3 and comparative examples 1 to 6 of the present invention.
FIG. 3 is a schematic diagram showing the survival rate of Lactobacillus plantarum WCFS1 in examples 4 to 6 of the present invention and comparative examples 7 to 12.
TABLE 1 rehydration cell survival after freeze drying
Data are presented as mean ± standard deviation (n = 3). Different letters indicate significant differences (p <0.05, duncan).
As shown in fig. 2, fig. 3 and table 1, for lactobacillus plantarum AR113, sucrose and 10% soybean polysaccharide protective agent were the best, and the two protective agent solutions significantly improved the freeze-drying survival rate of AR113, which was improved by about 50% compared to PBS. And secondly, 1% of Arabic gum, 1% of soybean polysaccharide, trehalose, mannose, sorbitol and other protective agents improve the freeze-drying survival rate of AR113 by about 10-50% compared with PBS. Mannitol was the least effective and showed no significant difference compared to PBS. This shows that the macromolecular and micromolecular saccharide protective agents have significant effects on improving the freeze-drying survival rate of lactobacillus plantarum AR113, while the alcohol protective effects are significantly lower than those of saccharides. The protective effect of 10% of soybean polysaccharide is equivalent to that of sucrose, so that a macromolecular compound can be used for replacing a small molecular protective agent.
For the lactobacillus plantarum WCFS1, two protective agents, namely sucrose and 1% Arabic gum, have the most remarkable protective effect on the lactobacillus plantarum WCFS1, and the freeze-drying survival rate can be improved to be more than 70%. And secondly, protective agents such as trehalose, 10% of soybean polysaccharide, mannose and 1% of soybean polysaccharide improve the freeze-drying survival rate of the WCFS1 to about 40-60% compared with PBS. Followed by sorbitol, there was no significant difference between the two compared to PBS. Mannitol was the least effective and the freeze-drying survival rate was significantly lower than PBS. The results show that the macromolecular and micromolecular saccharide protective agents have obvious effects on improving the freeze-drying survival rate of the lactobacillus plantarum WCFS1, and the protective effect of alcohols is obviously lower than that of saccharides. The protective effect of 1% gum arabic is comparable to that of sucrose, so that a macromolecular compound can be used instead of a small molecule protective agent.
Effects and effects of the embodiments
According to the freeze-drying method for improving the survival rate of the lactobacillus plantarum by utilizing the polysaccharide, which is disclosed by the embodiment of the invention, the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 are protected by adopting the soybean polysaccharide or the Arabic gum as a protective agent, so that the cell membrane damage of the lactobacillus plantarum AR113 and the WCFS1 in the freeze-drying process is effectively reduced. Therefore, the survival rate of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 after freeze drying is obviously improved, and the macromolecular compound can be used for replacing a micromolecular protective agent, so that the range and the types of the protective agent of the lactobacillus plantarum AR113 and the lactobacillus plantarum WCFS1 are widened.
In addition, the soybean polysaccharide and the Arabic gum belong to polysaccharides, and have wide sources and low prices.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (6)
1. A freeze-drying method for improving the survival rate of lactobacillus plantarum by utilizing polysaccharide is characterized by comprising the following steps:
step 1, activating strains to obtain single colonies;
step 2, inoculating and culturing the single colony to obtain a seed solution;
step 3, transferring the seed liquid to an MRS liquid culture medium for amplification culture to obtain an amplification culture liquid;
step 4, centrifuging the enlarged culture solution to obtain a thallus precipitate;
step 5, washing the thallus precipitate by PBS buffer solution, then suspending the thallus precipitate in polysaccharide protective agent solution, transferring the thallus precipitate into a container, freezing and drying the thallus precipitate,
wherein the strain in the step 1 is lactobacillus plantarum AR113 or lactobacillus plantarum WCFS1,
the polysaccharide protective agent solution in the step 5 is soybean polysaccharide solution or Arabic gum solution,
the concentration of the soybean polysaccharide solution is 1 to 10 percent,
the concentration of the Arabic gum solution is 1% -3%.
2. The freeze-drying method for improving survival rate of lactobacillus plantarum using polysaccharide according to claim 1, wherein:
the strain activation method comprises the step of repeatedly marking and activating lactobacillus plantarum in a solid MRS culture medium for 2-5 times.
3. The freeze-drying method for improving survival rate of lactobacillus plantarum using a polysaccharide according to claim 1, wherein:
the single colony inoculation culture method comprises the step of selecting activated single colonies for inoculation culture for 12-16 h.
4. The freeze-drying method for improving survival rate of lactobacillus plantarum using a polysaccharide according to claim 1, wherein:
wherein the condition of the expanded culture in the step 3 is culture for 12 to 16 hours at the temperature of between 35 and 40 ℃.
5. The freeze-drying method for improving survival rate of lactobacillus plantarum using polysaccharide according to claim 1, wherein:
wherein the freeze-drying parameters are as follows: precooling at-45 to-35 ℃ for 2 to 4 hours, heating to-30 to-25 ℃ at the rate of 0.8 to 1.5 ℃/min for primary drying for 700 to 900 minutes, then heating to 20 to 25 ℃ at the rate of 0.8 to 1.5 ℃/min for secondary drying for 2 to 3 hours, keeping the temperature of a cold trap at-85 to-70 ℃ and keeping the vacuum degree at 10 to 30Pa.
6. The application of a polysaccharide protective agent as a unique effective component in improving the freeze-drying survival rate of lactobacillus plantarum is characterized in that the polysaccharide protective agent is soybean polysaccharide or Arabic gum, the lactobacillus plantarum is lactobacillus plantarum AR113 or lactobacillus plantarum WCFS1, the concentration of a soybean polysaccharide solution is 1% -10%, and the concentration of an Arabic gum solution is 1% -3%.
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| CN111304134B (en) * | 2020-03-26 | 2023-03-31 | 上海理工大学 | Lactobacillus plantarum capable of effectively relieving diabetes |
| CN115261276A (en) * | 2022-08-08 | 2022-11-01 | 安徽农业大学 | Method for improving activity of lactobacillus plantarum and preparation of composite microbial inoculum |
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| CN110468050A (en) | 2019-11-19 |
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