CN113846030A - Vacuum freeze drying process of chicken intestine source probiotic lactobacillus preparation - Google Patents
Vacuum freeze drying process of chicken intestine source probiotic lactobacillus preparation Download PDFInfo
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Abstract
The invention discloses a vacuum freeze-drying process of a chicken intestine-derived probiotic lactobacillus preparation; according to the invention, a certain protection measure is adopted before freeze drying, and the skimmed milk powder, trehalose and fructo-oligosaccharide are selected as proper composite protective agents, wherein the composite protective agents comprise, by weight, 6-18% of the skimmed milk powder, 6-18% of the trehalose, 1-9% of the fructo-oligosaccharide and the balance of distilled water; mixing the bacterial sludge and the protective agent according to the weight ratio of 1:4-9, fully and uniformly mixing, and subpackaging into freeze-drying bottles; after pre-freezing in a refrigerator and freeze-drying treatment in a vacuum freeze-drying machine, the damage to cells in the freezing and drying processes can be reduced to the greatest extent and effectively avoided, and the survival rate of thalli can be effectively improved; simultaneously, when mixing bacterial and protective agent, go up and down the processing repeatedly with the material for the material has good mixed effect in a compounding section of thick bamboo, and the contact of bacterial mud and protective agent is more abundant even, is favorable to improving the activity of bacterial.
Description
Technical Field
The invention relates to the technical field of biological preparations, in particular to a vacuum freeze-drying process of a chicken intestine-derived probiotic lactic acid bacteria preparation.
Background
In recent years, the demand of lactobacillus preparations is increasing year by year on the market, the requirements on the viable count quality and the storage effect of the lactobacillus preparations are stricter and stricter, and the preservation and preparation of the lactobacillus preparations are important guarantees and preconditions for the lactobacillus preparations to exert a plurality of effects. Today, the rapid development of technology has made the vacuum freeze-drying technology an effective method for preparing bacterial preparations and preserving microorganisms, which is based on the principle of freezing water in the material into ice and then removing the water by sublimation of the ice.
During the freeze-drying process of microorganisms, the microbial cells and components are generally damaged, for example, protein denaturation and inactivation, membrane permeability change, DNA damage, pH dynamic imbalance and biological membrane fatty acid component change; the key technology for preparing the probiotic lactobacillus preparation lies in the selection and use of a freeze-drying protective agent, and the single protective agent in the prior art has limited protective effect on freeze-dried lactobacillus when in use, so that the survival rate of strains cannot be improved to the maximum extent; meanwhile, in the traditional vacuum freeze drying process, when the strains and the reagent are mixed, the mixing effect is not uniform, and the activity of the strains can be influenced, so that the design of the vacuum freeze drying process of the chicken intestine-derived probiotic lactobacillus preparation is necessary.
Disclosure of Invention
The invention solves the problem of providing a vacuum freeze-drying process of a chicken intestine-derived probiotic lactobacillus preparation, wherein a certain protective measure is adopted before freeze-drying, and skimmed milk powder, trehalose and fructo-oligosaccharide are selected as proper composite protective agents, and the composite protective agents comprise 6-18 wt% of skimmed milk powder, 6-18 wt% of trehalose, 1-9 wt% of fructo-oligosaccharide and the balance of distilled water; mixing the bacterial sludge and the protective agent according to the weight ratio of 1:4-9, fully and uniformly mixing, and subpackaging into freeze-drying bottles; after pre-freezing in a refrigerator and freeze-drying treatment in a vacuum freeze-drying machine, the damage to cells in the freezing and drying processes can be reduced to the greatest extent and effectively avoided, and the survival rate of thalli can be effectively improved; simultaneously, when mixing bacterial and protective agent, the material that will need the mixing through the inside compounding subassembly of dust-proof box mixes, through going up and down the processing repeatedly with the material for the material has good mixed effect in the inside of compounding section of thick bamboo, and the contact of bacterial mud and protective agent is more abundant even, thereby is favorable to improving the activity of bacterial.
In order to achieve the purpose, the invention adopts the following technical scheme:
the vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation comprises the following steps:
step S1, taking the screened pediococcus pentosaceus strain out of a refrigerator at the temperature of-80 ℃, inoculating the strain to an MRS solid culture medium by using an inoculating loop, and culturing in a standing incubator at the temperature of 16-42 ℃ overnight; selecting a revived single colony from the cultured MRS solid culture medium by using an inoculating loop, inoculating the single colony into a test tube containing 30mLMRS liquid culture medium, culturing in a shaking incubator at the temperature of 16-42 ℃ and the rotation speed of 160-200rpm overnight, and using the single colony for preparing bacterial suspension;
step S2, transferring the fermentation liquor into a sterilized 50mL centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation, pouring out the supernatant, weighing the thallus and obtaining bacterial sludge of the lactic acid bacteria for later use; subjecting the centrifuged bacterial sludge of lactic acid bacteria to a series of gradient dilutions, i.e. 106-108Diluting, sucking 200 mul with a gun, spreading on MRS solid culture medium, culturing overnight at 16-42 deg.C, and determining viable count before freeze-drying;
step S3, selecting skimmed milk powder, trehalose and fructo-oligosaccharide as raw materials to prepare a protective agent, wherein the protective agent comprises, by weight, 6-18% of the skimmed milk powder, 6-18% of the trehalose, 1-9% of the fructo-oligosaccharide and the balance of distilled water, and sterilizing the protective agent for later use after preparation;
step S4: proportioning the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 according to the weight ratio of 1:4-9, fully and uniformly mixing the bacterial sludge and the protective agent through uniformly mixing equipment, and subpackaging the mixture into freeze-dried bottles;
and step S5, placing the prepared mixed liquid of the bacterial sludge and the protective agent into a refrigerator at 4 ℃ for pre-freezing for 1-3h, then placing the mixed liquid into a refrigerator at-20 ℃ for pre-freezing for 2-24h, placing the pre-frozen sample into a vacuum freeze dryer for freeze-drying, wherein the freeze-drying time is 14-24h, the pressure is below 200pa, the temperature is-50 ℃ to-60 ℃, and taking out the freeze-dried sample and placing the freeze-dried sample into a refrigerator at 4 ℃ for preservation.
As a further scheme of the invention: in the step S1, the mixture ratio of the protective agent in the step S3 is 18% of skimmed milk powder, 12% of trehalose, 5% of fructo-oligosaccharide and the balance of distilled water. .
As a further scheme of the invention: in the step S4, the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 are mixed according to the weight ratio of 1: 6.
As a further scheme of the invention: in step S2, a fixed angle rotary head centrifuge is selected, and the working conditions of the centrifuge are 5000-.
As a further scheme of the invention: in step S4, mixing equipment includes mixing platform, dust proof box, observation chamber door, activestandby power supply, compounding subassembly, storage box and material box, mixing platform 'S top terminal surface one side fixed mounting has the dust proof box, the central fixed mounting of top terminal surface of dust proof box has activestandby power supply, the inboard of dust proof box is provided with the compounding subassembly, there is the observation chamber door terminal surface one side of dust proof box through hinge fixed mounting, mixing platform' S top terminal surface opposite side fixed mounting has the storage box, and the top terminal surface fixed mounting who stores the box has the material box.
As a further scheme of the invention: the mixing component comprises mounting bases, electric guide rails, limit switches, guide rail sliders, connecting supports, fastening frames, a protective tank, a mixing cylinder and a mounting tank cover, wherein the two mounting bases are respectively and fixedly mounted at two sides of the interior of the dust-proof box on the top end surface of the mixing platform, the electric guide rails are fixedly mounted in the center of the top end surface of the mounting bases, the top ends of the electric guide rails are in fit connection with the inner wall of the top of the dust-proof box, the limit switches are fixedly mounted at the top and the bottom of the electric guide rails, the guide rail sliders are mounted at one side of the outer wall of the two electric guide rails, the connecting support is mounted between the two guide rail sliders, the fastening frame is fixedly mounted at one side of the end surface of the connecting support, the top and the bottom of the fastening frame are fixedly connected with the guide rail sliders, the protective tank is fixedly mounted in the center of the end surface of the connecting support, and the mixing cylinder is inserted and mounted in the protective tank, the top terminal surface spiro union of protection jar is installed the installation cover, the terminal surface central authorities fixed mounting of installation cover has sealed backing plate, and the inboard top terminal surface laminating of sealed backing plate and compounding section of thick bamboo is connected.
As a further scheme of the invention: the center of the end face of the top of the inner side of the dust-proof box is fixedly provided with anti-collision rubber through a screw, and the anti-collision rubber is positioned right above the top of the mounting tank cover.
As a further scheme of the invention: the periphery of the end face of the bottom of the blending platform is fixedly provided with support grabbing feet through screws, and the centers of the end faces of the two sides of the blending platform are fixedly provided with carrying grab handles through screws.
The invention has the beneficial effects that: according to the invention, a certain protection measure is adopted before freeze drying, and the skimmed milk powder, trehalose and fructo-oligosaccharide are selected as proper composite protective agents, wherein the composite protective agents comprise, by weight, 6-18% of the skimmed milk powder, 6-18% of the trehalose, 1-9% of the fructo-oligosaccharide and the balance of distilled water; mixing the bacterial sludge and the protective agent according to the weight ratio of 1:4-9, fully and uniformly mixing, and subpackaging into freeze-drying bottles; after pre-freezing in a refrigerator and freeze-drying treatment in a vacuum freeze-drying machine, the damage to cells in the freezing and drying processes can be reduced to the greatest extent and effectively avoided, and the survival rate of thalli can be effectively improved; simultaneously, when mixing bacterial and protective agent, the material that will need the mixing through the inside compounding subassembly of dust-proof box mixes, through going up and down the processing repeatedly with the material for the material has good mixed effect in the inside of compounding section of thick bamboo, and the contact of bacterial mud and protective agent is more abundant even, thereby is favorable to improving the activity of bacterial.
Drawings
FIG. 1 is a line graph showing the effect of concentration of skim milk powder on the freeze-drying survival rate in the present invention;
FIG. 2 is a line graph showing the effect of trehalose concentration on the freeze-drying survival rate in the present invention;
FIG. 3 is a line graph showing the effect of fructooligosaccharide concentration on the freeze-drying survival rate in the present invention;
FIG. 4 is an overall perspective structural view of the kneading apparatus of the present invention;
FIG. 5 is an overall front view of the blending apparatus of the present invention;
FIG. 6 is an overall cross-sectional view of the blending apparatus of the present invention;
illustration of the drawings: 1. a blending platform; 2. a dust-proof box; 3. an observation box door; 4. a main power supply and a standby power supply; 5. a mixing assembly; 6. a storage box; 7. a material box; 51. mounting a base; 52. an electric rail; 53. a limit switch; 54. a guide rail slider; 55. connecting a bracket; 56. a fastening frame; 57. a protective tank; 58. a mixing barrel; 59. installing a tank cover; 510. and (3) anti-collision rubber.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Specific examples are given below.
Example 1:
referring to fig. 1-3, the vacuum freeze-drying process of the chicken intestine-derived probiotic lactobacillus preparation comprises the following steps:
step S1, taking the screened pediococcus pentosaceus strain out of a refrigerator at the temperature of-80 ℃, inoculating the strain to an MRS solid culture medium by using an inoculating loop, and culturing in a standing incubator at the temperature of 37 ℃ overnight; selecting a revived single colony from the cultured MRS solid culture medium by using an inoculating loop, inoculating the single colony into a test tube containing 30 mM MRS liquid culture medium, culturing in a shaking incubator at 37 ℃ and 180rpm overnight, and preparing bacterial suspension;
step S2, transferring the fermentation liquor into a sterilized 50mL centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation, pouring out the supernatant, weighing the thallus and obtaining bacterial sludge of the lactic acid bacteria for later use; subjecting the centrifuged bacterial sludge of lactic acid bacteria to a series of gradient dilutions, i.e. 106-108Diluting, sucking 200 μ L with a gun, spreading on MRS solid culture medium, culturing overnight at 37 deg.C, and determining viable count before freeze drying;
wherein, a fixed angle rotor centrifuge is selected, and the working conditions of the centrifuge are 6000rpm and 20 min;
step S3, selecting skim milk powder, trehalose and fructo-oligosaccharide as raw materials to prepare a protective agent, wherein the protective agent comprises 18 weight percent of skim milk powder, 12 weight percent of trehalose, 5 weight percent of fructo-oligosaccharide and the balance of distilled water, and sterilizing the protective agent for later use after preparation;
step S4: proportioning the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 according to the weight ratio of 1:4-9, fully and uniformly mixing the bacterial sludge and the protective agent through uniformly mixing equipment, and subpackaging the mixture into freeze-dried bottles; the preparation method comprises the following steps of selecting skimmed milk powder, trehalose and fructo-oligosaccharide as raw materials of a protective agent;
referring to fig. 4-6, the blending device includes a blending platform 1, a dust-proof box 2, an observation box door 3, a main power supply 4, a material mixing component 5, a storage box 6 and a material box 7, the dust-proof box 2 is fixedly mounted on one side of the top end face of the blending platform 1, the main power supply 4 is fixedly mounted in the center of the top end face of the dust-proof box 2, the material mixing component 5 is arranged on the inner side of the dust-proof box 2, the observation box door 3 is fixedly mounted on one side of the end face of the dust-proof box 2 through a hinge, the storage box 6 is fixedly mounted on the other side of the top end face of the blending platform 1, and the material box 7 is fixedly mounted on the top end face of the storage box 6.
The mixing component 5 comprises a mounting base 51, an electric guide rail 52, a limit switch 53, a guide rail sliding block 54, a connecting support 55, a fastening frame 56, a protective tank 57, a mixing barrel 58 and a mounting tank cover 59, wherein the two mounting bases 51 are respectively and fixedly mounted on two sides of the top end surface of the mixing platform 1 inside the dust-proof box 2, the electric guide rail 52 is fixedly mounted in the center of the top end surface of the mounting base 51, the top end of the electric guide rail 52 is attached to the inner wall of the top of the dust-proof box 2, the limit switch 53 is fixedly mounted on the top and the bottom of the electric guide rail 52, the guide rail sliding block 54 is mounted on one side of the outer wall of the two electric guide rails 52, the connecting support 55 is mounted between the two guide rail sliding blocks 54, the fastening frame 56 is fixedly mounted on one side of the end surface of the connecting support 55, the top and the bottom of the fastening frame 56 are fixedly connected with the guide rail sliding block 54, the protective tank 57 is fixedly mounted in the center of the end surface of the connecting support 55, a mixing cylinder 58 is inserted and installed inside the protective tank 57, the top end face of the protective tank 57 is screwed with an installation tank cover 59, a sealing cushion plate is fixedly installed in the center of the end face of the installation tank cover 59, and the inner side of the sealing cushion plate is attached and connected with the top end face of the mixing cylinder 58;
the center of the top end face of the inner side of the dust-proof box 2 is fixedly provided with an anti-collision rubber 510 through a screw, and the anti-collision rubber 510 is positioned right above the top of the mounting tank cover 59; the mounting tank cover 59 is prevented from colliding with the inner wall of the top of the dust-proof box 2 when rising, and the protection effect is improved;
the periphery of the end surface of the bottom of the blending platform 1 is fixedly provided with ground gripping support legs through screws, and the centers of the end surfaces of the two sides of the blending platform 1 are fixedly provided with carrying grab handles through screws; the carrying grab handle is convenient for carrying and moving the blending platform 1, and the ground support foot is convenient for supporting the blending platform, so that the stability of the whole structure is improved;
when the device is used, the device can be conveyed to a proper use position through the conveying grab handles on the two sides of the blending platform 1, the blending platform 1 is supported through the ground grabbing support legs at the bottom of the blending platform 1, materials to be blended are blended through the blending component 5 in the dust-proof box 2, and the materials are stored through the storage box 6 and the material box 7; after the observation box door 3 is opened, the mounting box cover 59 is taken down from the top of the protective box 57, the mixing cylinder 58 is taken out from the interior of the protective box 57, the materials to be mixed are placed in the mixing cylinder 58, then the mixing cylinder 58 is reset, the mounting box cover 59 is covered and fixed at the top of the protective box 57, so that the sealing cushion plate is attached to the top of the mixing cylinder 58, the materials are blocked in the mixing cylinder 58, the observation box door 3 is closed, the main power supply 4 supplies power to the electric guide rail 52 and the limit switches 53 at the top of the mounting base 51, the guide rail slide block 54 can reciprocate between the two limit switches 53 and move rapidly, as the connecting bracket 55 is connected with the guide rail slide block 54 through the fastening bracket 56, when the guide rail slide block 54 moves up and down, the protective box 57 and the mixing cylinder 58 can be driven to move up and down through the connecting bracket 55, and the protective box 57 repeatedly moves up and down along with the protective box 57, the materials in the mixing cylinder 58 can be uniformly mixed, after the materials are uniformly mixed for a period of time, the mixing cylinder 58 can be taken out and subpackaged into the subpackaging bottles, the subpackaging bottles are placed through the material box 7, and the subpackaging bottles can also be placed through the storage box 6; the anti-collision rubber 510 can avoid the collision between the installation tank cover 59 and the inner wall of the top of the dust-proof box 2 when the installation tank cover 59 is lifted, so that the protection effect is improved;
step S5, placing the prepared mixed solution of the bacterial sludge and the protective agent into a refrigerator at 4 ℃ for pre-freezing for 1h, then placing the mixed solution into a refrigerator at-20 ℃ for pre-freezing for 2h, placing the pre-frozen sample into a vacuum freeze dryer for freeze-drying, wherein the freeze-drying time is 14h, the pressure is 80pa, the temperature is-55 ℃, and taking out the freeze-dried sample and placing the freeze-dried sample into the refrigerator at 4 ℃ for preservation;
freeze-drying protective agent single-factor experiment:
taking out the blank control group and the freeze-dried sample of the experimental group added with the skimmed milk powder protective agent, carrying out rehydration activation, and counting 10 viable bacteria by a viable bacteria counting method-5～10-7Counting the bacterial suspension in the dilution region, sucking 200 mu L of the bacterial suspension, coating the bacterial suspension on an MRS solid culture medium, performing static culture in an incubator at 37 ℃ for 24 hours, performing three parallel experiments, and then calculating the average value of the bacterial suspension to obtain the number of freeze-dried viable bacteria; calculating the survival rate by using a freeze-drying survival rate calculation formula:
table 1: freeze-drying survival rate of skimmed milk powder as protective agent
Taking out the freeze-dried samples of the blank control group and the experimental group added with the trehalose protective agent together, carrying out rehydration activation, and counting 10 viable bacteria by a viable bacteria counting method-5～10-7Counting the bacterial suspension in the dilution region, sucking 200 mu L of the bacterial suspension, coating the bacterial suspension on an MRS solid culture medium, performing static culture in an incubator at 37 ℃ for 24 hours, performing three parallel experiments, and then calculating the average value of the bacterial suspension to obtain the number of freeze-dried viable bacteria; calculating the survival rate by using a freeze-drying survival rate calculation formula:
table 2: freeze-drying survival rate of trehalose as protective agent
Taking out the blank control group and the freeze-dried sample of the experimental group added with the fructo-oligosaccharide protective agent together, carrying out rehydration activation, and then carrying out a viable countNumber method, pair 10-5～10-7Counting the bacterial suspension in the dilution region, sucking 200 mu L of the bacterial suspension, coating the bacterial suspension on an MRS solid culture medium, performing static culture in an incubator at 37 ℃ for 24 hours, performing three parallel experiments, and then calculating the average value of the bacterial suspension to obtain the number of freeze-dried viable bacteria; calculating the survival rate by using a freeze-drying survival rate calculation formula:
table 3: freeze-drying survival rate of fructo-oligosaccharide as protective agent
According to the single-factor experiment result, the following results are obtained: the highest survival rate concentration of the skimmed milk powder as the protective agent is 15 percent, and the survival rate reaches 66.87 percent; when the trehalose is used as a freeze-drying protective agent, the concentration with the best protective effect is 9 percent, and the survival rate reaches 51.29 percent; the highest survival rate concentration of the fructo-oligosaccharide as a protective agent is 5 percent, and the survival rate reaches 57.07 percent;
orthogonal experiment:
three factors of three protective agents, namely skimmed milk powder, trehalose and fructo-oligosaccharide, with good protection effect in the freeze-drying process are selected to design an orthogonal experiment; adding a protective agent according to the concentration ratio, uniformly mixing with the bacterial sludge, and respectively measuring and calculating the number of viable bacteria before and after freeze-drying; according to the orthogonal experiment result, the optimal proportion of the composite protective agent is skim milk powder: concentration 18%, trehalose: concentration 12%, fructo-oligosaccharide: the concentration is 5%, and the survival rate reaches the highest, and is 93.6%.
Example 2:
referring to fig. 1-3, the vacuum freeze-drying process of the chicken intestine-derived probiotic lactobacillus preparation comprises the following steps:
step S1, taking the screened pediococcus pentosaceus strain out of a refrigerator at the temperature of-80 ℃, inoculating the strain to an MRS solid culture medium by using an inoculating loop, and culturing in a standing incubator at the temperature of 37.5 ℃ overnight; selecting a revived single colony from the cultured MRS solid culture medium by using an inoculating loop, inoculating the single colony into a test tube containing 30 mM MRS liquid culture medium, culturing in a shaking incubator at 37.5 ℃ and 180rpm overnight, and preparing bacterial suspension;
step S2, transferring the fermentation liquor into a sterilized 50mL centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation, pouring out the supernatant, weighing the thallus and obtaining bacterial sludge of the lactic acid bacteria for later use; subjecting the centrifuged bacterial sludge of lactic acid bacteria to a series of gradient dilutions, i.e. 106-108Diluting, sucking 200 μ L with a gun, spreading on MRS solid culture medium, culturing overnight at 40 deg.C, and determining viable count before freeze drying;
wherein, a fixed angle rotor centrifuge is selected, and the working conditions of the centrifuge are 7000rpm and 15 min;
step S3, selecting skim milk powder, trehalose and fructo-oligosaccharide as raw materials to prepare a protective agent, wherein the protective agent comprises 15 weight percent of skim milk powder, 10 weight percent of trehalose, 4 weight percent of fructo-oligosaccharide and the balance of distilled water, and sterilizing the protective agent for later use after preparation;
step S4: proportioning the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 according to the weight ratio of 1:4-9, fully and uniformly mixing the bacterial sludge and the protective agent through uniformly mixing equipment, and subpackaging the mixture into freeze-dried bottles;
and step S5, placing the prepared mixed liquid of the bacterial sludge and the protective agent into a 4 ℃ refrigerator for pre-freezing for 1.5h, then placing the mixed liquid into a-20 ℃ refrigerator for pre-freezing for 5h, placing the pre-frozen sample into a vacuum freeze dryer for freeze-drying, wherein the freeze-drying time is 18h, the pressure is below 200pa, the temperature is-60 ℃, and taking out the freeze-dried sample and placing the freeze-dried sample into a 4 ℃ refrigerator for storage.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation is characterized by comprising the following steps of:
step S1, taking the screened pediococcus pentosaceus strain out of a refrigerator at the temperature of-80 ℃, inoculating the strain to an MRS solid culture medium by using an inoculating loop, and culturing in a standing incubator at the temperature of 16-42 ℃ overnight; selecting a revived single colony from the cultured MRS solid culture medium by using an inoculating loop, inoculating the single colony into a test tube containing 30mLMRS liquid culture medium, culturing in a shaking incubator at the temperature of 16-42 ℃ and the rotation speed of 160-200rpm overnight, and using the single colony for preparing bacterial suspension;
step S2, transferring the fermentation liquor into a sterilized 50mL centrifuge tube, placing the centrifuge tube into a centrifuge for centrifugation, pouring out the supernatant, weighing the thallus and obtaining bacterial sludge of the lactic acid bacteria for later use; subjecting the centrifuged bacterial sludge of lactic acid bacteria to a series of gradient dilutions, i.e. 106-108Diluting, sucking 200 mul with a gun, spreading on MRS solid culture medium, culturing overnight at 16-42 deg.C, and determining viable count before freeze-drying;
step S3, selecting skimmed milk powder, trehalose and fructo-oligosaccharide as raw materials to prepare a protective agent, wherein the protective agent comprises, by weight, 6-18% of the skimmed milk powder, 6-18% of the trehalose, 1-9% of the fructo-oligosaccharide and the balance of distilled water, and sterilizing the protective agent for later use after preparation;
step S4: proportioning the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 according to the weight ratio of 1:4-9, fully and uniformly mixing the bacterial sludge and the protective agent through uniformly mixing equipment, and subpackaging the mixture into freeze-dried bottles;
and step S5, placing the prepared mixed liquid of the bacterial sludge and the protective agent into a refrigerator at 4 ℃ for pre-freezing for 1-3h, then placing the mixed liquid into a refrigerator at-20 ℃ for pre-freezing for 2-24h, placing the pre-frozen sample into a vacuum freeze dryer for freeze-drying, wherein the freeze-drying time is 14-24h, the pressure is below 200pa, the temperature is-50 ℃ to-60 ℃, and taking out the freeze-dried sample and placing the freeze-dried sample into a refrigerator at 4 ℃ for preservation.
2. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 1, wherein in the step S3, the ratio of the protective agent is 18% of skimmed milk powder, 12% of trehalose, 5% of fructo-oligosaccharide and the balance of distilled water.
3. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 1, wherein in the step S4, the bacterial sludge obtained in the step S2 and the protective agent obtained in the step S3 are mixed according to a weight ratio of 1: 6.
4. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation as claimed in claim 1, wherein in the step S2, a fixed angle head centrifuge is selected, and the operating conditions of the centrifuge are 5000-.
5. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 1, it is characterized in that in the step S4, the blending device comprises a blending platform (1), a dust-proof box (2), an observation box door (3), a main power supply (4), a material mixing component (5), a storage box (6) and a material box (7), a dust-proof box (2) is fixedly arranged on one side of the top end surface of the blending platform (1), the center of the top end surface of the dust-proof box (2) is fixedly provided with a main power supply (4), a material mixing component (5) is arranged on the inner side of the dust-proof box (2), an observation box door (3) is fixedly arranged on one side of the end surface of the dust-proof box (2) through a hinge, a storage box (6) is fixedly arranged on the other side of the top end surface of the blending platform (1), and the top end surface of the storage box (6) is fixedly provided with a material box (7).
6. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 5, wherein the mixing component (5) comprises mounting bases (51), electric guide rails (52), limit switches (53), guide rail sliders (54), a connecting bracket (55), a fastening frame (56), a protective tank (57), a mixing barrel (58) and a mounting tank cover (59), the two mounting bases (51) are respectively and fixedly mounted on the top end face of the blending platform (1) and located on two sides of the interior of the dust-proof box (2), the electric guide rails (52) are fixedly mounted in the centers of the top end faces of the mounting bases (51), the top ends of the electric guide rails (52) are attached to the inner wall of the top of the dust-proof box (2), the limit switches (53) are fixedly mounted at the top and the bottom of the electric guide rails (52), the guide rail sliders (54) are mounted on one side of the outer wall of the two electric guide rails (52), and install linking bridge (55) between two rail block (54), terminal surface one side fixed mounting of linking bridge (55) has fastening frame (56), and the top and the bottom of fastening frame (56) all with rail block (54) fixed connection, the terminal surface central authorities fixed mounting of linking bridge (55) has protection jar (57), and the inside grafting of protection jar (57) installs mixing barrel (58), the top terminal surface spiro union of protection jar (57) installs installation cover (59), the terminal surface central authorities fixed mounting of installation cover (59) has the sealing pad, and the inboard of sealing pad is connected with the top terminal surface laminating of mixing barrel (58).
7. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 6, wherein the center of the top end surface of the inner side of the dust-proof box (2) is fixedly provided with the anti-collision rubber (510) through a screw, and the anti-collision rubber (510) is positioned right above the top of the mounting tank cover (59).
8. The vacuum freeze-drying process of the chicken intestine-derived probiotic lactic acid bacteria preparation according to claim 5, wherein the periphery of the bottom end surface of the blending platform (1) is fixedly provided with the ground grabbing feet through screws, and the centers of the two side end surfaces of the blending platform (1) are fixedly provided with the carrying grab handles through screws.
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