CN111534434B - Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis - Google Patents
Freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis Download PDFInfo
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- CN111534434B CN111534434B CN202010595370.XA CN202010595370A CN111534434B CN 111534434 B CN111534434 B CN 111534434B CN 202010595370 A CN202010595370 A CN 202010595370A CN 111534434 B CN111534434 B CN 111534434B
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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
- C12N1/20—Bacteria; Culture media therefor
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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 discloses a freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis, belonging to the technical field of microorganisms. The invention provides a freeze-drying protective agent capable of remarkably improving the freeze-drying survival rate of bifidobacterium adolescentis, which comprises the components of sorbitol, raffinose and lactalbumin; when the freeze-drying protective agent is used for preparing bifidobacterium adolescentis freeze-dried powder, the freeze-drying survival rate of the bifidobacterium adolescentis is as high as 97.43 +/-4.98%.
Description
Technical Field
The invention relates to a freeze-drying protective agent and application thereof in freeze-drying bifidobacterium adolescentis, belonging to the technical field of microorganisms.
Background
Bifidobacterium adolescentis (Bifidobacterium adolescentis) is one of probiotics, and has abundant probiotic functions, including treating chronic diarrhea and constipation, resisting aging, reducing cholesterol, preventing steatohepatitis, relieving colitis, inhibiting periodontitis, etc. Due to the remarkable probiotic efficacy, bifidobacterium adolescentis is becoming the key point of research, development and production of people more and more. At present, bifidobacterium adolescentis is widely applied to the fields of food, medicine and the like.
The bifidobacterium adolescentis freeze-dried powder is the most common bifidobacterium adolescentis product in the market, is mainly obtained by freeze-drying bifidobacterium adolescentis, and has the advantages of long storage period, difficulty in pollution, convenience in transportation and storage, easiness in quality control and the like. Although freeze-drying causes little damage to bifidobacterium adolescentis in the process of preparing the bifidobacterium adolescentis freeze-dried powder, the bifidobacterium adolescentis can still suffer from various damages in the freeze-drying process, such as mechanical damage, solute damage, membrane damage, protein denaturation (including enzyme denaturation) and DNA damage, and the like.
However, currently, the freeze-drying protective agent developed specially for bifidobacterium adolescentis, which is a probiotic, is rare and has a poor effect, for example, in documents of xu hai yan, zhangyan, cao, and the like, optimization of culture medium of bifidobacterium adolescentis and screening of freeze-drying protective agent [ J ]. zootechnics and feed science, 2013,34(3):15-18 ], when zhangyan and the like use freeze-drying protective agent containing trehalose, skim milk powder, glycerol, sucrose and gelatin to freeze bifidobacterium adolescentis, the freeze-drying survival rate is only 57.6%; in the literature, "study of high-density culture method and lyophilization process of bifidobacterium adolescentis in white language [ D ]. Jilin agricultural university, 2006", when bifidobacterium adolescentis is lyophilized using a lyoprotectant containing skim milk, sucrose and mannitol, the lyophilization survival rate is only 73%. Therefore, it is urgently needed to find a freeze-drying protective agent capable of obviously improving the freeze-drying survival rate of the bifidobacterium adolescentis so as to improve the viable count of the bifidobacterium adolescentis in the freeze-dried powder of the bifidobacterium adolescentis.
Disclosure of Invention
[ problem ] to
The invention aims to provide a freeze-drying protective agent capable of remarkably improving the freeze-drying survival rate of bifidobacterium adolescentis.
[ solution ]
In order to solve the technical problem, the invention provides a lyoprotectant, and the components of the lyoprotectant comprise sorbitol, raffinose and lactalbumin.
In one embodiment of the present invention, the components of the lyoprotectant include, by mass, 0.1 to 99.8% sorbitol, 0.1 to 99.8% raffinose, and 0.1 to 30% whey protein.
In one embodiment of the invention, the components of the lyoprotectant include, by mass, 37.5% sorbitol, 37.5% raffinose and 25% whey protein.
The invention also provides a method for preparing the bifidobacterium adolescentis freeze-dried powder, which is to prepare the bifidobacterium adolescentis freeze-dried powder by using the freeze-drying protective agent.
In one embodiment of the invention, the method comprises the steps of centrifuging the bifidobacterium adolescentis bacterial liquid, and collecting bacterial sludge; resuspending the bacterial sludge in the lyoprotectant according to any one of claims 1-3 to obtain a resuspension solution; diluting the resuspension with water, normal saline or buffer solution to obtain a diluent; and (4) freeze-drying the diluent to obtain the bifidobacterium adolescentis freeze-dried powder.
In one embodiment of the invention, the mass ratio of the dry matter of the bacterial sludge to the freeze-drying protective agent is 1: 0.5-1.5.
In one embodiment of the invention, the mass ratio of dry matter of the bacterial sludge to the lyoprotectant is 1: 1.2.
In one embodiment of the invention, the content of dry matter in the dilution is 15-25% by mass.
In one embodiment of the present invention, the pH of the diluent is 6.0 to 7.0.
In one embodiment of the invention, the pH of the dilution is 6.5.
In one embodiment of the invention, the freeze-drying is done in a freeze-dryer, including prefreezing, primary drying, and secondary drying; the pre-freezing step is to control the temperature of the laminate to be reduced to-50 ℃ within 1 hour and keep the temperature for 4-6 hours; the primary drying is to control the temperature of the laminate to be raised to-30 ℃ within 1.3h and keep the temperature for 20-30 h; and the secondary drying is to control the temperature of the laminate to be increased to 25 ℃ within 1 hour and keep the temperature for 10-20 hours.
In one embodiment of the invention, said bifidobacterium adolescentis comprises bifidobacterium adolescentis GDMCC No.60706, bifidobacterium adolescentis GDMCC No.60707 and/or bifidobacterium adolescentis GDMCC No. 14395.
The invention also provides a bifidobacterium adolesentis freeze-dried powder prepared by the method.
The invention also provides the application of the freeze-drying protective agent or the method in freeze-drying bifidobacterium adolescentis.
[ advantageous effects ]
(1) The invention provides a freeze-drying protective agent capable of remarkably improving the freeze-drying survival rate of bifidobacterium adolescentis, which comprises the components of sorbitol, raffinose and lactalbumin; when the freeze-drying protective agent is used for preparing bifidobacterium adolescentis freeze-dried powder, the freeze-drying survival rate of the bifidobacterium adolescentis is as high as 97.43 +/-4.98%.
(2) The invention provides a method for preparing bifidobacterium adolescentis freeze-dried powder capable of obviously improving the freeze-drying survival rate of bifidobacterium adolescentis, which is to prepare the bifidobacterium adolescentis freeze-dried powder by using a freeze-drying protective agent containing sorbitol, raffinose and whey protein as components; when the freeze-dried powder of the bifidobacterium adolescentis is prepared by the method, the freeze-drying survival rate of the bifidobacterium adolescentis is as high as 97.43 +/-4.98%.
Detailed Description
The invention is further illustrated with reference to specific examples.
Sorbitol, raffinose, whey protein, trehalose, sucrose, skim milk powder referred to in the following examples were purchased from creative mix biotechnology limited; bifidobacterium adolescentis GDMCC No.60706, which is described in the following examples, is disclosed in patent application publication No. CN110331118A, deposited as GDMCC No. 60706; bifidobacterium adolescentis GDMCC No.60707 described in the following examples is described in patent application publication No. CN110432332A, deposited as GDMCC No. 60707; the bifidobacterium adolescentis GDMCC No.14395 referred to in the examples below is described in the patent application publication No. CN107699517A, deposited as GDMCC No. 14395.
The media involved in the following examples are as follows:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L, agar 20g/L and cysteine hydrochloride 0.5 g/L.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L and cysteine hydrochloride 0.5 g/L.
The detection methods referred to in the following examples are as follows:
the method for detecting the number of the live bifidobacterium adolescentis comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection lactic acid bacteria is adopted.
Example 1: preparation of lyophilized powder of Bifidobacterium adolescentis (Bifidobacterium adolescentis GDMCC No.60706)
The method comprises the following specific steps:
preparing 1-4 freeze-drying protective agents according to the formula in the table 1; drawing lines on MRS solid culture medium with inoculating loop dipped Bifidobacterium adolescentis GDMCCNo.60706 bacterial liquid in a bacteria-protecting tube, and culturing at 37 deg.C for 36h to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at a constant temperature of 37 ℃ for 12 hours to obtain a seed solution; inoculating the seed solution into a fresh MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at the constant temperature of 37 ℃ for 14h to the last logarithmic growth stage to obtain a bacterial solution; centrifuging the bacterial liquid at 8000g for 20min, and collecting bacterial sludge; washing the bacterial sludge with sterile normal saline for 2 times, and then suspending the bacterial sludge in a freeze-drying protective agent 1-4 to obtain a resuspension solution 1-4; diluting the resuspension with sterile water to obtain a diluent 1-4; adjusting the pH value of the diluent 1-4 to 6.5, and then (adjusting the pH value by hydrochloric acid) freeze-drying the diluent 1-4 to obtain bifidobacterium adolescentis freeze-dried powder 1-4; wherein the mass ratio of dry matter of the bacterial sludge to the freeze-drying protective agent is 1: 1.2; the content of dry matter in the diluent is 15%, 20% and 25% by mass, respectively; the freeze drying is completed in a freeze dryer (purchased from Telstar, Spain) and comprises prefreezing, primary drying and secondary drying, wherein the prefreezing is to control the temperature of the laminate to be reduced to-50 ℃ within 1h and keep for 4h, the primary drying is to control the temperature of the laminate to be increased to-30 ℃ within 1.3h and keep for 30h, and the secondary drying is to control the temperature of the laminate to be increased to 25 ℃ within 1h and keep for 20 h.
Detecting the viable count of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 1-4, and calculating the freeze-drying survival rate of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 1-4 (the detection result is shown in table 2);
wherein, the calculation formula of the freeze-drying survival rate is as follows:
as can be seen from tables 1-2, the lyophilized survival rate of bifidobacterium adolescentis in the lyophilized powder of bifidobacterium adolescentis prepared by using the lyophilized protectant containing 37.5% of sorbitol, 37.5% of raffinose and 25% of whey protein is highest, and is as high as 97.43 ± 4.98%.
TABLE 1 formulation of lyoprotectant 1-4
| Group of | Formulation (by mass) |
| Freeze-drying protective agent 1 | 37.5% sorbitol + 37.5% raffinose + 25% whey protein |
| Freeze-drying protective agent 2 | 37.5 percent of trehalose, 37.5 percent of cane sugar and 25 percent of skim milk powder |
| Freeze-drying protective agent 3 | 47.5% sorbitol + 37.5% raffinose + 15% whey protein |
| Freeze-drying protective agent 4 | 37.5% sorbitol + 47.5% raffinose + 15% whey protein |
TABLE 2 Freeze-drying survival rate of Bifidobacterium adolescentis in Bifidobacterium adolescentis freeze-dried powder 1-4
Example 2: preparation of bifidobacterium adolescentis freeze-dried powder
The method comprises the following specific steps:
on the basis of the embodiment 1, bifidobacterium adolescentis GDMCC No.60706 is replaced by bifidobacterium adolescentis GDMCCNo.60707, and bifidobacterium adolescentis freeze-dried powder 5-8 is obtained.
And detecting the viable count of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 5-8, and calculating the freeze-drying survival rate of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 5-8 (the detection result is shown in a table 3).
As can be seen from table 3, the lyophilized survival rate of bifidobacterium adolescentis in the lyophilized powder of bifidobacterium adolescentis prepared by using the lyophilized protectant containing 37.5% of sorbitol, 37.5% of raffinose and 25% of whey protein is the highest, and is as high as 92.45 ± 4.81%.
TABLE 3 Freeze-drying survival rate of Bifidobacterium adolescentis in Bifidobacterium adolescentis freeze-dried powder 5-8
Example 3: preparation of bifidobacterium adolescentis freeze-dried powder
The method comprises the following specific steps:
on the basis of the embodiment 1, bifidobacterium adolescentis GDMCC No.60706 is replaced by bifidobacterium adolescentis GDMCCNo.14395, and bifidobacterium adolescentis freeze-dried powder 9-12 is obtained.
And detecting the viable count of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 9-12, and calculating the freeze-drying survival rate of the bifidobacterium adolescentis in the bifidobacterium adolescentis freeze-dried powder 9-12 (the detection result is shown in table 4).
As can be seen from table 4, the lyophilized survival rate of bifidobacterium adolescentis in the bifidobacterium adolescentis lyophilized powder prepared by using the lyophilized protectant containing 37.5% of sorbitol, 37.5% of raffinose and 25% of whey protein is the highest, and is as high as 95.72 ± 4.01%.
TABLE 4 Freeze-drying survival rate of Bifidobacterium adolescentis in Bifidobacterium adolescentis freeze-dried powder 9-12
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for preparing Bifidobacterium adolescentisBifidobacterium adolescentis ) The freeze-dried powder method is characterized in that the method comprises the steps of centrifuging a bifidobacterium adolescentis bacterial liquid, and collecting bacterial sludge; re-suspending the bacterial sludge in a freeze-drying protective agent, wherein the mass ratio of dry matter of the bacterial sludge to the freeze-drying protective agent is 1: 0.5-1.5, and obtaining a re-suspension; diluting the resuspension with water, normal saline or buffer solution to obtain a diluent, wherein the content of dry matter in the diluent is 20-25%; freeze-drying the diluent to obtain bifidobacterium adolescentis freeze-dried powder;
the ingredients of the lyoprotectant were 37.5% sorbitol, 37.5% raffinose and 25% whey protein, or 47.5% sorbitol, 37.5% raffinose and 15% whey protein, or 37.5% sorbitol, 47.5% raffinose and 15% whey protein by mass.
2. The method for preparing the bifidobacterium adolescentis lyophilized powder of claim 1, wherein the pH of the diluent is 6.0-7.0.
3. A lyophilized powder of bifidobacterium adolescentis prepared using the method of claim 1 or 2.
4. Use of the method according to claim 1 or 2 for freeze-drying bifidobacterium adolescentis.
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| CN111534435B (en) * | 2020-06-30 | 2022-07-05 | 江南大学 | Freeze-drying protective agent capable of improving acid resistance of bifidobacteria and application thereof |
| CN113151115B (en) * | 2021-05-27 | 2023-07-04 | 江南大学 | Method for improving freeze-drying survival rate and storage stability of bifidobacteria |
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