Freeze-drying protective additive for lactic acid bacteria, preparation method and application of freeze-drying protective additive

Abstract

The invention belongs to the technical field of freeze-drying of lactic acid bacteria, and particularly relates to a freeze-drying protective agent for lactic acid bacteria, a preparation method and application. The invention provides a freeze-drying protective agent for lactic acid bacteria, which comprises the following components in parts by weight: 12-20 parts of dried skim milk, 12-20 parts of synanthrin and 8-16 parts of trehalose. Wherein the skimmed milk powder is beneficial to keeping the structure of lactic acid bacteria stable and maintaining the cell morphology of the lactic acid bacteria; the synanthrin is cross-linked with proteins in the trehalose and the skimmed milk powder to form a wall material, so that the lactic acid bacteria can be better protected; the trehalose can be combined with protein to form a hydrated membrane, phase change of phospholipid, protein denaturation and ice crystal generation can be inhibited under the action of hydrogen bonds, and the stability of cell membranes can be maintained. When the freeze-drying protective agent is used for freeze-drying lactic acid bacteria, the survival rate of the lactic acid bacteria is high, and the activity of the lactic acid bacteria in gastrointestinal tracts can be improved.

Description

Technical Field

[0001] The present application belongs to the technical field of freeze-drying of lactic acid bacteria, and specifically relates to a freeze-drying protective agent for lactic acid bacteria, a preparation method and an application. Background Art

[0002] Freeze-drying of lactic acid bacteria is a biological preservation technique that uses low-temperature freezing and vacuum drying to remove moisture from the bacteria and maintain their activity. Freeze-drying avoids high temperatures and oxidation, effectively maintaining the stability and activity of the bacteria. The freeze-dried lactic acid bacteria powder is convenient for packaging, storage, and transportation, and is widely used in food, medicine, and other fields. However, existing freeze-drying protectants focus on the survival rate of lactic acid bacteria, but lack a freeze-drying protectant that simultaneously improves both the survival rate and the gastrointestinal tolerance of lactic acid bacteria. Summary of the Invention

[0003] The present invention provides a freeze-dried protective agent for lactic acid bacteria, a preparation method and an application thereof. The prepared freeze-dried protective agent has high survival rate and gastrointestinal tolerance for lactic acid bacteria.

[0004] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0005] The invention provides a freeze-drying protective agent for lactic acid bacteria, which comprises, by weight, 12 to 20 parts of skim milk powder, 12 to 20 parts of inulin and 8 to 16 parts of trehalose.

[0006] Preferably, the mixture further comprises 44 to 68 parts by weight of water.

[0007] The present invention provides a method for preparing the freeze-dried protective agent described in the above technical solution, comprising: mixing the skimmed milk powder, inulin and trehalose to obtain the freeze-dried protective agent.

[0008] The present invention provides the use of the lyoprotectant described in the above technical solution or the lyoprotectant prepared by the preparation method described in the above technical solution in the following 1) and / or 2):

[0009] 1) Improve the freeze-dried survival rate of lactic acid bacteria;

[0010] 2) Increase the number of viable lactic acid bacteria in the gastrointestinal tract.

[0011] Preferably, the lactic acid bacteria include Lactobacillus plantarum.

[0012] Preferably, increasing the number of viable lactic acid bacteria in the gastrointestinal tract includes increasing the gastrointestinal tolerance of lactic acid bacteria, and said increasing the gastrointestinal tolerance of lactic acid bacteria includes increasing the survival rate of lactic acid bacteria in gastric juice and / or increasing the survival rate of lactic acid bacteria in intestinal juice.

[0013] The invention provides a method for preparing freeze-dried bacterial powder, comprising mixing the freeze-dried protective agent with lactic acid bacteria and then drying the mixture.

[0014] Preferably, the application form of the lactic acid bacteria includes a bacterial suspension.

[0015] Preferably, the volume ratio of the lyoprotectant to the bacterial suspension is 1:1.

[0016] Preferably, the drying method includes sequentially performing pre-freezing and vacuum freeze drying.

[0017] Beneficial Effects of the Invention: The present invention provides a lyoprotectant for lactic acid bacteria, comprising, by weight, 12-20 parts of skim milk powder, 12-20 parts of inulin, and 8-16 parts of trehalose. Skim milk powder contains a large amount of protein, which effectively forms a protective layer on the surface of lactic acid bacteria cells. This helps maintain the structural stability and morphology of the lactic acid bacteria, protecting them from mechanical damage during freeze-drying. It also protects against free radical damage caused by freeze-drying and reduces crystallization of the cells during freeze-drying. Inulin can also lower the relative humidity of the outer shell, reduce water loss, maintain the glassy state of the outer shell, and form hydrogen bonds with proteins, thereby enhancing the stability of the lactic acid bacteria. Trehalose also combines with proteins to form a hydration film. Through hydrogen bonding, it inhibits phospholipid phase transitions, protein denaturation, and ice crystal formation, helping to maintain cell membrane stability. Furthermore, during freeze-drying, trehalose forms a glassy outer shell, reducing mechanical damage and stabilizing cell morphology. Inulin is cross-linked with trehalose, skim milk powder protein, etc. to form a wall material, which can better protect lactic acid bacteria.

[0018] When the freeze-drying protective agent is used to freeze-dry lactic acid bacteria, the survival rate of the lactic acid bacteria and the gastrointestinal tolerance are high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the freeze-drying survival rate of strain MRS1026.4 under different freeze-drying protective agents;

[0020] Figure 2 This is a scanning electron microscope image of skim milk powder;

[0021] Figure 3 is a scanning electron micrograph of trehalose;

[0022] Figure 4 This is a scanning electron microscope image of inulin;

[0023] Figure 5 This is a scanning electron micrograph of skim milk powder and inulin;

[0024] Figure 6 This is a scanning electron micrograph of skim milk powder and trehalose;

[0025] Figure 7 is a scanning electron micrograph of trehalose and inulin;

[0026] Figure 8 This is a graph showing the effect of different skim milk powder concentrations on the survival rate of MRS1026.4; Figure 8 The skimmed milk powder is skimmed milk powder;

[0027] Figure 9 This is a graph showing the effect of different concentrations of inulin on the freeze-drying survival rate of MRS1026.4;

[0028] Figure 10 This is a graph showing the effect of different trehalose concentrations on the survival rate of MRS1026.4;

[0029] Figure 11 is the contour map of the interaction between skim milk powder and trehalose;

[0030] Figure 12 This is the response surface diagram of the interaction between skim milk powder and trehalose;

[0031] Figure 13 is the contour map of the interaction between skim milk powder and inulin;

[0032] Figure 14 This is the response surface diagram of the interaction between skim milk powder and inulin;

[0033] Figure 15 is the contour map of the interaction between trehalose and inulin;

[0034] Figure 16 This is the response surface diagram of the interaction between trehalose and inulin.

[0035] Biological deposit information

[0036] Lactobacillus plantarum MRS1026.4 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on April 11, 2025. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 34169. DETAILED DESCRIPTION

[0037] The invention provides a freeze-drying protective agent for lactic acid bacteria, which comprises, by weight, 12 to 20 parts of skim milk powder, 12 to 20 parts of inulin and 8 to 16 parts of trehalose.

[0038] As another optional embodiment, the freeze-drying protective agent of the present invention includes, by weight: 12 to 20 parts of skim milk powder, 12 to 20 parts of inulin, 8 to 16 parts of trehalose and 44 to 68 parts of water.

[0039] As an optional embodiment, the freeze-drying protective agent provided by the present invention includes skim milk powder, which includes 12 to 20 parts by weight, or 13 to 18 parts. In the present invention, the skim milk powder contains a large amount of protein that can effectively form a protective layer on the surface of lactic acid bacteria cells, helping to maintain the stability of the lactic acid bacteria structure, maintain the morphology of lactic acid bacteria cells, help lactic acid bacteria cells resist mechanical damage caused by freeze drying, and also avoid free radical damage caused by freeze drying, reducing the crystallization of lactic acid bacteria cells during freeze drying.

[0040] As an optional embodiment, the lyoprotectant provided by the present invention includes inulin, which is present in an amount of 12 to 20 parts by weight, or 13 to 16 parts by weight. In the present invention, the inulin can reduce the relative humidity of the wall material shell, reduce water loss, maintain the wall material in a glassy state, form hydrogen bonds with proteins, and enhance the stability of lactic acid bacteria.

[0041] As an optional embodiment, the freeze-drying protective agent provided by the present invention includes trehalose, which is calculated by weight and includes 12 to 20 parts, or alternatively 13 to 16 parts. In the present invention, the trehalose is cross-linked with inulin and the like to better protect the lactic acid bacteria. At the same time, trehalose can form a glassy shell on the outside of the lactic acid bacteria during freeze-drying, reducing mechanical damage and stabilizing cell morphology.

[0042] The freeze-drying protectant provided by the present invention can improve the survival rate and gastrointestinal tolerance of the freeze-dried lactic acid bacteria. The freeze-dried lactic acid bacteria have a high survival rate in artificial gastric juice and artificial intestinal juice.

[0043] The present invention provides a method for preparing the lyoprotectant described in the above technical solution, which is obtained by mixing the skim milk powder, inulin, and trehalose. As an optional embodiment, the lyoprotectant of the present invention is prepared into a solution and then used. As an optional embodiment, water is used as a solvent when preparing the solution. As an optional embodiment, the lyoprotectant of the present invention is obtained by mixing the skim milk powder, inulin, trehalose, and water. The present invention does not specifically limit the mixing method, and conventional methods can be used.

[0044] The present invention provides the use of the freeze-drying protectant described in the above technical solution in improving the freeze-drying survival rate of lactic acid bacteria.

[0045] The present invention provides the use of the lyoprotectant described in the above technical solution to increase the viable count of lactic acid bacteria in the gastrointestinal tract. As an optional embodiment, the increasing the viable count of lactic acid bacteria in the gastrointestinal tract of the present invention includes improving the gastrointestinal tolerance of the lactic acid bacteria, and the increasing the gastrointestinal tolerance of the lactic acid bacteria includes increasing the survival rate of the lactic acid bacteria in gastric juice and / or increasing the survival rate of the lactic acid bacteria in intestinal juice.

[0046] As an optional embodiment, the lactic acid bacteria of the present invention include Lactobacillus plantarum MRS1026.4. The protective agent of the present invention is more suitable for Lactobacillus plantarum MRS1026.4.

[0047] The present invention provides a method for applying a freeze-dried protective agent or preparing freeze-dried bacterial powder. The freeze-dried protective agent is mixed with lactic acid bacteria and then dried.

[0048] As an optional embodiment, the application form of the lactic acid bacteria of the present invention includes a bacterial suspension.

[0049] As an optional embodiment, the method for preparing the bacterial suspension of the present invention comprises the following steps: inoculating the Lactobacillus plantarum MRS1026.4 into a culture medium for culturing to obtain a culture solution; and centrifuging, washing and resuspending the culture solution to obtain a bacterial suspension.

[0050] As another optional embodiment, the present invention inoculates the plant lactobacillus MRS1026.4 into a liquid MRS culture medium for resuscitation culture to obtain a seed liquid. As an optional embodiment, the present invention does not specifically limit the method of inoculation, and a conventional method can be used. As an optional embodiment, the temperature of the resuscitation culture of the present invention is 33°C to 39°C, or 35 to 37°C; in a specific embodiment of the present invention, the temperature of the resuscitation culture is 33, 34, 35, 36, 37, 38 or 39°C. The time of the resuscitation culture is 22 to 26 hours, or 23 to 25 hours. In a specific embodiment of the present invention, the time of the resuscitation culture is 22, 23, 24, 25 or 26 hours. The rotation speed of the resuscitation culture is 170 to 200 rpm, more preferably 180 rpm.

[0051] As another optional embodiment, the present invention inoculates the seed liquid into a liquid MRS culture medium for expansion culture to obtain a culture solution. As an optional embodiment, the inoculation amount of the inoculation of the present invention is 1% to 3% of the volume of the culture medium, more preferably 2%. The present invention has no special limitation on the method of inoculation, and a conventional method can be used. As an optional embodiment, the temperature of the expansion culture is 33°C to 38°C, or 35 to 37°C; in a specific embodiment of the present invention, the temperature of the expansion culture is 33, 34, 35, 36, 37 or 38°C. The time of the expansion culture of the present invention is 22 to 26h, more preferably 24h. In a specific embodiment of the present invention, the time of the expansion culture is 22, 23, 24, 25 or 26h. The rotation speed of the expansion culture of the present invention is 170 to 190rpm, and in a specific embodiment of the present invention, the rotation speed of the culture is 170, 180 or 190rpm. As an optional embodiment, the composition of each 1L of the liquid MRS culture medium includes: 10g protein Chen, 5g beef powder, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate and 1000ml distilled water.

[0052] The culture fluid is obtained, and the culture fluid is centrifuged to obtain bacterial cells, and the bacterial cells are washed and resuspended to obtain a bacterial suspension. The present invention does not specifically limit the parameters of the centrifugation, and conventional methods can be used. The solvent used for washing and resuspending in the present invention includes PBS buffer. The OD value of the bacterial suspension in the present invention is 600 The effective viable bacteria count of Lactobacillus plantarum MRS1026.4 in the bacterial suspension is ≥1.0×10 6 CFU / mL.

[0053] As an optional embodiment, the present invention mixes the lyophilization protectant and the bacterial suspension in a volume ratio of 1:1 and then dries.

[0054] As an optional embodiment, the drying method of the present invention includes pre-freezing and vacuum freeze-drying performed sequentially. The pre-freezing temperature of the present invention is -85 to -75°C, more preferably -80°C, and the pre-freezing time is 1.5 to 2.5 hours, more preferably 2 hours. The vacuum freeze-drying temperature of the present invention is -60 to -50°C, more preferably -55°C, the vacuum degree is <1 mbar, and the time is 48 hours.

[0055] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] 1 Experimental strain: Lactobacillus plantarum MRS1026.4, deposit number: CGMCC NO: 34169.

[0058] 2 Freeze-drying of lactic acid bacteria

[0059] 2.1 Six lyoprotectants, including skim milk powder, lactose, sucrose, inulin, trehalose, and glycerol, were screened. Lactobacillus plantarum MRS1026.4 was freeze-dried with each lyoprotectant at a 15% volume ratio, and the survival rates were compared. Protectant solutions 1 to 6 were prepared with skim milk powder, lactose, sucrose, inulin, trehalose, and glycerol at a 10% mass concentration, respectively. Each protectant solution contained one lyoprotectant.

[0060] The specific process is as follows:

[0061] Activation: Remove strain MRS1026.4 from the -80°C freezer and dissolve at room temperature. Inoculate 1 mL of glycerol stock into 20 mL of MRS medium and incubate on a shaker at 37°C, 180 rpm for 24 hours to resuscitate the strain. Inoculate 20 mL of MRS medium at a 3% volume ratio of the revived culture solution and incubate on a shaker at 37°C, 180 rpm for 24 hours to create the activated culture solution.

[0062] The activated bacterial suspension was centrifuged at 10,000 × g for 30 min, and the bacteria were washed 2 to 3 times with PBS buffer solution and resuspended to obtain a bacterial suspension. The OD value of the bacterial suspension was 0. 600 =1.0.

[0063] Protective agent solutions 1 to 6 were added to the obtained MRS1026.4 bacterial suspension at a volume ratio of 1:0.15, and the mixture was thoroughly shaken on a constant temperature shaker at 180 rpm and 37°C for 15 min to obtain a bacterial suspension-protective agent solution. The bacterial suspension-protective agent solution was placed in a -80°C ultra-low temperature refrigerator for pre-freezing for 2 h, and then freeze-dried at -55°C and <1 mbar for 48 h to obtain MRS1026.4 freeze-dried bacterial powder.

[0064] Method for determining the survival of lactic acid bacteria after freeze-drying: Weigh 1g of freeze-dried bacterial powder and perform a plate count to obtain the number of viable cells after freeze-drying. Perform a plate count on the MRS1026.4 bacterial suspension to obtain the initial number of viable cells.

[0065] Survival rate of lactic acid bacteria freeze-dried:

[0066] Survival rate (%) = m1 / m0×100%, where m1 is the number of viable bacteria after freeze-drying; m0 is the initial number of viable bacteria.

[0067] The results of freeze-drying survival rate of strain MRS1026.4 under different freeze-drying protective agents are shown in Figure 1 Among them, skim milk powder, trehalose and inulin freeze-dried lactic acid bacteria had the highest survival rates, which were 43.21%, 48.36% and 41.27% respectively.

[0068] Observation of skim milk powder and inulin: MRS1026.4 bacterial suspension and preservative solution 7 were added in a volume ratio of 1:0.1, respectively. The mixture was thoroughly shaken on a constant temperature shaker at 180 rpm and 37°C for 15 minutes to obtain a bacterial suspension-preservative solution. The bacterial suspension-preservative solution was pre-frozen in a -80°C ultra-low temperature freezer for 2 hours. The solution was then freeze-dried at -55°C and <1 mbar for 48 hours to obtain freeze-dried MRS1026.4 bacterial powder. The resulting freeze-dried MRS1026.4 bacterial powder was observed using a scanning electron microscope.

[0069] The composition of the protective agent solution 7 is as follows: the mass concentration of skim milk powder in the protective agent solution 7 is 10%, and the mass concentration of inulin is 10%.

[0070] Observation of skim milk powder and trehalose: MRS1026.4 bacterial suspension and protective agent solution 8 were added in a volume ratio of 1:0.1, respectively. The mixture was thoroughly shaken on a constant temperature shaker at 180 rpm and 37°C for 15 minutes to obtain a bacterial suspension-protectant solution. The bacterial suspension-protectant solution was pre-frozen in a -80°C ultra-low temperature freezer for 2 hours, and then freeze-dried at -55°C and <1 mbar for 48 hours to obtain freeze-dried MRS1026.4 bacterial powder. The resulting freeze-dried MRS1026.4 bacterial powder was observed using a scanning electron microscope.

[0071] The composition of the protective agent solution 8 is as follows: the mass concentration of skim milk powder in the protective agent solution 8 is 10%, and the mass concentration of trehalose is 10%.

[0072] Preparation of trehalose and inulin: MRS1026.4 bacterial suspension and preservative solution 9 were added in a volume ratio of 1:0.1, respectively. The mixture was thoroughly shaken on a constant temperature shaker at 180 rpm and 37°C for 15 minutes to obtain a bacterial suspension-preservative solution. The bacterial suspension-preservative solution was pre-frozen in a -80°C ultra-low temperature freezer for 2 hours, and then freeze-dried at -55°C and <1 mbar for 48 hours to obtain freeze-dried MRS1026.4 bacterial powder. The resulting freeze-dried MRS1026.4 bacterial powder was observed using a scanning electron microscope.

[0073] The composition of the protective agent solution 9 is as follows: the mass concentration of inulin in the protective agent solution 9 is 10%, and the mass concentration of trehalose is 10%.

[0074] Scanning electron microscopy observation of different freeze-drying protective agents Figures 2 to 7 ,in, Figure 2This is a scanning electron microscope image of skim milk powder. The skim milk powder appears irregular and flake-like. Excessive gaps can cause lactic acid bacteria to fall off. Figure 3 This is a scanning electron microscope image of trehalose. The surface of trehalose is rough and granular. Figure 4 This is a scanning electron microscope image of inulin. The surface of inulin is smooth with many wrinkles. Figure 5 This is a scanning electron microscope image of skim milk powder and inulin. Figure 5 Compared to Figure 4 The surface wrinkles were significantly reduced, indicating that skim milk powder can fill the wrinkles on the surface of inulin and increase the attachment area of ​​lactic acid bacteria; Figure 6 This is a scanning electron microscope image of skim milk powder and trehalose. Figure 6 Compared to Figure 3 The surface is smoother, with fewer particles and still small gaps, indicating that skim milk powder and trehalose can form a dense film on the surface of strain MRS1026.4; Figure 7 This is a scanning electron microscope image of trehalose and inulin. Figure 7 The surface particles are reduced and the wrinkles are alleviated.

[0075] 2.2 Freeze-drying of lactic acid bacteria

[0076] Activation: Remove strain MRS1026.4 from the -80°C freezer and dissolve at room temperature. Inoculate 1 mL of glycerol stock into 20 mL of MRS medium and incubate on a shaker at 37°C, 180 rpm for 24 hours to resuscitate the strain. Inoculate 20 mL of MRS medium at a 3% volume ratio of the revived culture solution and incubate on a shaker at 37°C, 180 rpm for 24 hours to create the activated culture solution.

[0077] The activated bacterial suspension was centrifuged at 10,000 × g for 30 min, and the bacteria were washed 2 to 3 times with PBS buffer solution and resuspended to obtain a bacterial suspension. The OD value of the bacterial suspension was 0. 600 =1.0.

[0078] The protective agent solution was added to the bacterial suspension at a volume ratio of 1:1, and the mixture was thoroughly shaken in a constant temperature shaker at 180 rpm and 37°C for 15 min to obtain a bacterial suspension-protectant solution. The bacterial suspension-protectant solution was placed in a -80°C ultra-low temperature freezer for pre-freezing for 2 h, and then freeze-dried at -55°C and <1 mbar for 48 h to obtain freeze-dried bacterial powder.

[0079] 2.3 Preliminary study on the effect of skim milk powder concentration on survival rate

[0080] Prepare a certain concentration of trehalose, skim milk powder and inulin solution, mix thoroughly to obtain a protective agent solution, and set aside.

[0081] Protective agent 1: The mass concentration of trehalose in the protective agent 1 solution is 10%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 4%.

[0082] Protective agent 2: The mass concentration of trehalose in the protective agent 2 solution is 10%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 8%.

[0083] Protective agent 3: The mass concentration of trehalose in the protective agent 3 solution is 10%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 12%.

[0084] Protective agent 4: The mass concentration of trehalose in the protective agent 4 solution is 10%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 16%.

[0085] Protective agent 5: The mass concentration of trehalose in the protective agent 5 solution is 10%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 20%.

[0086] Protective agents 1 to 5 were used to prepare freeze-dried bacterial powder according to the method in 2.2, and the survival rate of strain MRS1026.4 in the freeze-dried bacterial powder was analyzed.

[0087] Skim milk powder contains a large amount of protein that can effectively form a protective layer on the surface of lactic acid bacteria cells, helping to maintain the stability of the lactic acid bacteria structure, maintain the morphology of lactic acid bacteria cells, help lactic acid bacteria cells resist mechanical damage caused by freeze drying, and also avoid free radical damage caused by freeze drying, reducing the crystallization of lactic acid bacteria cells during freeze drying. Figure 8 As shown, the optimal skim milk powder concentration for freeze-drying lactic acid bacteria is 16%. At lower skim milk powder concentrations, the viscosity is low, resulting in rapid ice crystal formation during freeze-drying and ineffective protection of the lactic acid bacteria. At higher skim milk powder concentrations, the excessive viscosity hinders uniform ice crystal formation and sublimation, prolonging drying time. Furthermore, excessive skim milk powder concentrations form an overly thick protective film, hindering the exchange of substances within the lactic acid bacteria. Therefore, a skim milk powder concentration of 8% to 16% in the protective agent results in a high survival rate for strain MRS1026.4.

[0088] 2.5 Preliminary study on the effect of inulin concentration on survival rate

[0089] Prepare a certain concentration of trehalose, skim milk powder and inulin solution, mix thoroughly to obtain a protective agent solution, and set aside.

[0090] Protective agent 6: The mass concentration of trehalose in the protective agent 6 solution is 10%, the mass concentration of inulin is 4%, and the mass concentration of skim milk powder is 10%.

[0091] Protective agent 7: The mass concentration of trehalose in the protective agent 7 solution is 10%, the mass concentration of inulin is 8%, and the mass concentration of skim milk powder is 10%.

[0092] Protective agent 8: The mass concentration of trehalose in the protective agent 8 solution is 10%, the mass concentration of inulin is 12%, and the mass concentration of skim milk powder is 10%.

[0093] Protective agent 9: The mass concentration of trehalose in the protective agent 9 solution is 10%, the mass concentration of inulin is 16%, and the mass concentration of skim milk powder is 10%.

[0094] Protective agent 10: The mass concentration of trehalose in the protective agent 10 solution is 10%, the mass concentration of inulin is 20%, and the mass concentration of skim milk powder is 10%.

[0095] Protective agents 6 to 10 were used to prepare freeze-dried bacterial powder according to the method in 2.2, and the survival rate of strain MRS1026.4 in the freeze-dried bacterial powder was analyzed.

[0096] Inulin alone as a freeze-drying protective agent cannot significantly improve the survival rate of lactic acid bacteria during freeze-drying. It is usually cross-linked with other freeze-drying protective agents, such as trehalose, to better protect lactic acid bacteria. Inulin can reduce relative humidity, reduce water loss, maintain glassy state, form hydrogen bonds with proteins, etc., and enhance the stability of lactic acid bacteria. Figure 9 As shown in the figure, the optimal concentration of inulin for freeze-drying is 16%. At lower concentrations, the inulin binds weakly to proteins, resulting in weaker glassy mechanical strength and inability to effectively protect lactic acid bacteria. At higher concentrations, inulin crystallizes, destroying the lactic acid bacteria cell membrane, damaging the cell structure, and reducing cell viability.

[0097] 2.6 Preliminary study on the effect of trehalose concentration on survival rate

[0098] Prepare a certain concentration of trehalose, skim milk powder and inulin solution, mix thoroughly to obtain a protective agent solution, and set aside.

[0099] Protective agent 11: The mass concentration of trehalose in the protective agent 11 solution is 4%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 10%.

[0100] Protective agent 12: The mass concentration of trehalose in the protective agent 12 solution is 8%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 10%.

[0101] Protective agent 13: The mass concentration of trehalose in the protective agent 13 solution is 12%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 10%.

[0102] Protective agent 14: The mass concentration of trehalose in the protective agent 14 solution is 16%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 10%.

[0103] Protective agent 15: The mass concentration of trehalose in the protective agent 15 solution is 20%, the mass concentration of inulin is 10%, and the mass concentration of skim milk powder is 10%.

[0104] Protective agents 11 to 15 were used to prepare freeze-dried bacterial powder according to the method in 2.2, and the survival rate of strain MRS1026.4 in the freeze-dried bacterial powder was analyzed.

[0105] Trehalose can combine with phospholipids and proteins to form a hydration film. Under the action of hydrogen bonds, it can inhibit phospholipid phase transition, protein denaturation and ice crystal formation, which helps to maintain the stability of the cell membrane. At the same time, trehalose can form a glassy shell on the outside during freeze-drying of lactic acid bacteria, reducing mechanical damage and further protecting lactic acid bacteria. Figure 10 As shown in the figure, the optimal concentration for freeze-drying lactic acid bacteria is 12%. When the trehalose concentration is low, the binding of trehalose to phospholipids and proteins is weak, and it cannot effectively inhibit protein denaturation, phospholipid phase transition, and ice crystal formation. When the trehalose concentration is high, the glassy shell formed by trehalose on the outside of the lactic acid bacteria is too strong, hindering the growth and metabolism of the lactic acid bacteria.

[0106] 2.7 Optimization of freeze-drying of lactic acid bacteria by response surface methodology

[0107] Taking the survival rate of lactic acid bacteria as an indicator, the concentration of skim milk powder in the freeze-dried protective agent screened in the above experiment is 12% to 20%, the mass concentration of inulin is 12% to 20%, and the mass concentration of trehalose is 8% to 16%.

[0108] A three-factor, three-level experiment was designed using Box-Behnken software. Based on the preliminary findings above, high and low concentrations were input. The experimental factors and levels are coded as shown in Table 1.

[0109] Table 1 Box-Behnken experimental factor and level coding table

[0110] level Skim milk powder (%) Inulin (%) Trehalose (%) +1 20 20 16 0 16 16 12 -1 12 12 8

[0111] The Box-behnken software designed the experiment based on high-level and low-level concentrations as shown in Table 2 below.

[0112] Table 2 Box-Behnken experimental design

[0113] Serial number skim milk powder Inulin Trehalose 1 0 0 0 2 0 -1 -1 3 -1 1 0 4 0 0 0 5 1 -1 0 6 -1 0 1 7 -1 -1 0 8 0 0 0 9 1 0 1 10 0 0 0 11 0 0 0 12 0 1 -1 13 0 1 1 14 0 -1 1 15 -1 0 -1 16 1 1 0 17 1 0 -1

[0114] The Box-Behnken experiment results of Table 2 are shown in Table 3. The same serial numbers in Table 2 and Table 3 represent the same experiment.

[0115] Table 3 Box-behnken experiment results

[0116]

[0117]

[0118] Note: The protectant concentrations of No. 1, 4, 8, 10 and 11 were the same, but the survival rates were different because these were repeated experiments in the response surface experiment.

[0119] 2.8 Data Analysis of Lactic Acid Bacteria Freeze-Drying Model

[0120] According to the results of the Box-Behnken experimental design, a difference analysis of the regression model of lactic acid bacteria immobilization was performed, and the results are shown in Table 4 below.

[0121] Table 4 Difference analysis results of regression model for immobilization of lactic acid bacteria

[0122]

[0123] Note: A is skim milk powder; B is trehalose; C is inulin.

[0124] The regression equation obtained by fitting the results of the Box-Behnken experimental design is: survival rate (%) = -565.24 + 21.88A + 32.24B + 38.68C + 0.70AB - 0.53AC - 0.52BC - 0.90A 2 -1.21B 2 -0.89C 2 Among them, the model difference P value is <0.0001, the difference is extremely significant, indicating that the model is highly credible; the lack of fit P value is 0.2702>0.05, the difference is not significant, indicating that the regression equation of the model has fewer abnormal errors; the square error of the model regression equation: R 2 =0.9808, indicating that the regression equation fits well, and 98.08% of the experimental results can be explained by the model regression equation. The adjusted variance is: Adjusted R 2 =0.9562, indicating that the model regression equation can explain 95.62% of the variation in the experimental results; the difference between the corrected variance and the predicted variance (Predicted R 2) difference is less than 0.2, indicating high reliability of the regression equation. Among the linear terms, A has a P value < 0.001, indicating a highly significant impact, and has the largest F value. B has a P value < 0.00145, indicating a significant impact. A comparison of F values: F value (A) > F value (B) > F value (C) indicates that the effects of freeze-drying on lactic acid bacteria follow the order: A > B > C, i.e., skim milk powder > trehalose > inulin.

[0125] 2.9 Response surface interaction analysis

[0126] If the contour plot of the response surface analysis is a distinct ellipse, it indicates a significant interaction between the two factors; if the contour plot is approximately circular, it indicates a weak or no interaction between the two factors. The response surface plot generated based on the Box-Behnken experimental design results in Table 3 shows an inverted bowl-shaped pattern, indicating the presence of a peak, indicating the optimal ratio of skim milk powder, inulin, and trehalose.

[0127] The contour map of the interaction between skim milk powder and trehalose is elliptical (see Figure 11 ), skimmed milk powder (kimmedmilk) and trehalose (trehalose) response surface interaction results are shown in the figure Figure 12 , indicating that there is a significant interaction between skim milk powder and trehalose during freeze-drying of lactic acid bacteria; from the response surface diagram of skim milk powder and trehalose, when the concentration of skim milk powder is between 10.9% and 14.9% and the concentration of trehalose is between 9.0% and 13.0%, the survival rate of lactic acid bacteria also increases accordingly.

[0128] The interaction contour map of skimmed milk and inulin is elliptical. Figure 13 The interactive results of skim milk powder and inulin response surface are shown in Figure 14 , indicating that there is a significant interaction between skim milk powder and inulin during freeze-drying of lactic acid bacteria; from the response surface diagram of skim milk powder and inulin, when the concentration of skim milk powder is 10.2%-14.2% and the concentration of inulin is 11.1%-15.1%, the survival rate of lactic acid bacteria also increases accordingly.

[0129] The contour map of the interaction between trehalose and inulin is elliptical. Figure 15 The results of the interaction between trehalose and inulin are shown in the figure Figure 16 , indicating that the interaction between trehalose and inulin is significant during freeze-drying of lactic acid bacteria; from the response surface diagram of trehalose and inulin, when the trehalose concentration is 10.2%-14.2% and the inulin concentration is 11.1%-15.1%, the survival rate of lactic acid bacteria also increases accordingly.

[0130] 2.10 Response surface model validation experiment

[0131] According to the response surface model analysis, the optimal ratio of the lactic acid bacteria immobilization process was obtained as follows: the mass concentration of skim milk powder in the freeze-drying protectant was 13.422%, the mass concentration of trehalose was 14.155%, and the mass concentration of inulin was 12.949%. The volume ratio of the freeze-drying protectant to the bacterial suspension (prepared by the same method as in Example 1) was 1:1. The optimal ratio of the freeze-drying protectant was verified by the test, and the survival rate of the immobilized lactic acid bacteria under these conditions was 79.64%. Compared with the immobilized lactic acid bacteria survival rate of 76.82% predicted by the regression equation simulation of DX13 software for 2.8, the relative error was small, and the model credibility was high.

[0132] 2.11 Continuous simulation experiment of gastrointestinal fluid with freeze-dried lactic acid bacteria powder

[0133] 2.10 The freeze-dried lactic acid bacteria powder prepared was used in the following experiments:

[0134] Treatment 1: Determination of the number of viable bacteria after gastric juice treatment: Weigh 2 g of freeze-dried lactic acid bacteria powder, add 18 mL of simulated gastric juice (purchased from Shanghai Maokang Biotechnology Co., Ltd.), and shake in a constant temperature shaker at 37°C, 180 rpm, for 2 h. The resulting culture solution was centrifuged at 10,000 rpm, washed with PBS buffer solution, and the bacterial sludge was obtained for plate counting.

[0135] Control 1: 2 mL of MRS1026.4 bacterial suspension (prepared as in Example 1) was added to 18 mL of simulated gastric fluid (purchased from Shanghai Maokang Biotechnology Co., Ltd.) and incubated in a constant temperature shaker at 37°C, 180 rpm, for 2 hours. The resulting culture was centrifuged at 10,000 rpm, washed with PBS buffer, and the resulting bacterial slurry was counted for plate counts. The survival rate was 64.56%.

[0136] Treatment 2: Determination of viable bacterial counts after continuous gastrointestinal fluid treatment: Weigh 2 g of freeze-dried lactic acid bacteria powder and add it to 18 mL of simulated gastric fluid. Incubate the mixture in a shaker at 37°C, 180 rpm, for 2 hours. Centrifuge the resulting culture at 10,000 rpm and wash with PBS buffer to obtain a slurry for plate count. Simultaneously, transfer the resulting slurry to 20 mL of simulated intestinal fluid (purchased from Shanghai Maokang Biotechnology Co., Ltd.) and incubate the mixture in a shaker at 37°C, 180 rpm, for 4 hours. Count the plate counts.

[0137] Control 2: 2 mL of MRS1026.4 bacterial suspension (prepared as in Example 1) was added to 18 mL of simulated gastric fluid and incubated in a constant temperature shaker at 37°C, 180 rpm, for 2 hours. The resulting culture was centrifuged at 10,000 rpm and washed with PBS buffer to obtain a bacterial slurry for plate count. Simultaneously, the resulting bacterial slurry was transferred to 20 mL of simulated intestinal fluid (purchased from Shanghai Maokang Biotechnology Co., Ltd.) and incubated in a constant temperature shaker at 37°C, 180 rpm, for 4 hours. Plate counts were then performed. The survival rate was 34.67%.

[0138] The results are shown in Table 5. After 2 hours of treatment in simulated gastric fluid, the survival rate of the probiotics encapsulated by freeze-drying was 86.30%. After 2 hours of treatment in simulated gastric fluid and 4 hours of treatment in simulated intestinal fluid, the survival rate was 80.14%. This indicates that the survival rate of unfreeze-dried MRS1026.4 in gastric fluid was 64.56% after 2 hours of treatment, while the survival rate of MRS1026.4 after 2 hours of treatment in simulated gastric fluid and 4 hours of treatment in simulated intestinal fluid was 34.67%. This indicates that freeze-drying significantly enhances the activity of lactic acid bacteria MRS1026.4 in gastrointestinal fluid.

[0139] Table 5 Results of continuous simulation experiment on gastrointestinal fluid of freeze-dried lactic acid bacteria powder

[0140]

[0141] In summary, the present invention provides a lyoprotectant, which is used to lyophilize lactic acid bacteria, thereby increasing the survival rate of lactic acid bacteria and improving the activity of lactic acid bacteria in the gastrointestinal tract.

[0142] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A freeze-drying protective agent for lactic acid bacteria, characterized in that: Calculated by weight, the invention comprises 12 to 20 parts of skim milk powder, 12 to 20 parts of inulin and 8 to 16 parts of trehalose.

2. The freeze-drying protective agent according to claim 1, characterized in that Calculated by weight, the mixture further comprises 44 to 68 parts of water.

3. The method for preparing the lyoprotectant according to claim 1 or 2, characterized in that: include: The skimmed milk powder, inulin and trehalose are mixed to obtain a freeze-drying protective agent.

4. Use of the lyoprotectant according to claim 1 or 2 or the lyoprotectant prepared by the preparation method according to claim 3 in the following 1) and / or 2), 1) Improve the freeze-dried survival rate of lactic acid bacteria; 2) Increase the number of viable lactic acid bacteria in the gastrointestinal tract.

5. The application according to claim 4, characterized in that: The lactic acid bacteria include Lactobacillus plantarum.

6. The application according to claim 4, characterized in that: Increasing the number of viable lactic acid bacteria in the gastrointestinal tract includes improving the gastrointestinal tolerance of lactic acid bacteria, and improving the gastrointestinal tolerance of lactic acid bacteria includes improving the survival rate of lactic acid bacteria in gastric juice and / or improving the survival rate of lactic acid bacteria in intestinal juice.

7. A method for preparing freeze-dried bacterial powder, characterized in that: The freeze-drying protectant is mixed with lactic acid bacteria and then dried.

8. The method according to claim 7, characterized in that The application form of the lactic acid bacteria includes bacterial suspension.

9. The method according to claim 8, characterized in that The volume ratio of the freeze-drying protective agent to the bacterial suspension is 1:

1.

10. The method according to claim 7, characterized in that The drying method includes sequentially performing pre-freezing and vacuum freeze drying.

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