Freeze-drying production method of plant lactobacillus powder with high viable count

Abstract

The invention relates to a freeze-drying production method of high-viable-count plant lactobacillus bacterial powder, which comprises the following steps: step 1, uniformly mixing plant lactobacillus bacterial sludge with a protective solution to obtain a bacterial suspension; step 2, dropwise adding the bacterial suspension into liquid nitrogen, and performing cryogenic granulation to obtain cryogenic granules; and step 3, filling the cryogenic granules into a tray of a freeze dryer, then placing the tray filled with the cryogenic granules between adjacent heating plate layers of the freeze dryer, and heating the cryogenic granules in a 360-degree heat radiation mode under a vacuum condition to sublimate moisture, thereby obtaining the plant lactobacillus powder with high viable count. According to the method, the freeze-drying efficiency is greatly improved, the freeze-drying time is shortened, the damage to thalli in the freeze-drying process is reduced, and the survival rate of strains is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial freeze-drying, and in particular relates to a freeze-drying production method of Lactobacillus plantarum powder with high viable count. Background Art

[0002] Lactobacillus plantarum is widely used in various probiotic products because it can improve gastrointestinal flora, relieve diarrhea, constipation, indigestion, promote nutrient absorption, and inhibit pathogenic bacteria such as Helicobacter pylori.

[0003] Plant lactobacillus is usually obtained by liquid fermentation. Before it is prepared into a probiotic product, it must be freeze-dried. However, due to the poor low-temperature resistance of plant lactobacillus, the current freeze-drying method usually involves placing a bacterial suspension in a vacuum freeze-drying tray for vacuum low-temperature drying. During the vacuum freeze-drying process, the temperature of the bacterial suspension is controlled by plate-layer heat-conducting oil, which has a low cooling efficiency, causes greater damage to the bacteria, and results in a low freeze-drying survival rate, which affects the number of viable bacteria in the product. On the other hand, its production efficiency is low and the freeze-drying time is long.

[0004] Therefore, it is very important to develop a freeze-drying method that can improve the freeze-drying efficiency of Lactobacillus plantarum, reduce freeze-drying damage, and increase the viable bacterial rate of the strain for use in Lactobacillus plantarum powder with high viable bacterial count. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a freeze-drying production method for Lactobacillus plantarum powder with a high viable count, which greatly improves the freeze-drying efficiency, reduces the freeze-drying time, reduces the damage of the bacteria during the freeze-drying process, and improves the survival rate of the strain.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A freeze-drying production method for high-viable count Lactobacillus plantarum powder comprises the following steps:

[0008] Step 1, mixing the plant lactobacillus slurry with the protective solution to obtain a bacterial suspension;

[0009] Step 2, dropping the bacterial suspension into liquid nitrogen for cryogenic granulation to obtain cryogenic pellets;

[0010] Step 3: Load the cryogenic pellets into a tray of a freeze dryer, and then place the tray containing the cryogenic pellets between adjacent heating plate layers of the freeze dryer. Under vacuum conditions, heat the cryogenic pellets using 360-degree heat radiation to sublime the water, thereby obtaining Lactobacillus plantarum powder with a high viable count.

[0011] As a further technical solution, the protective solution includes the following raw materials in percentage by weight:

[0012] Trehalose 8-10%; skim milk powder 8-10%; sodium glutamate 1-2%; sucrose 2-4%; water balance; pH adjusted to 6.9-7.1;

[0013] As a further technical solution, the protective solution is sterilized at 115° C. for 20 minutes before being mixed with the Lactobacillus plantarum cells.

[0014] As a further technical solution, in step 1, the Lactobacillus plantarum bacterial slurry and the protective solution are evenly mixed in a ratio of 1:0.8-1 based on the dry weight of the Lactobacillus plantarum cells.

[0015] As a further technical solution, in step 2, the inner diameter of the dropper used for dropping the bacterial suspension into liquid nitrogen is 2 mm, and the particle size of the obtained cryogenic particles is 2-4 mm.

[0016] As a further technical solution, in step 3, the cryogenic pellets are loaded into a tray of a freeze dryer with a tray thickness of 0.8-1.4 cm.

[0017] As a further technical solution, in step 3, the heating treatment procedure is: under a vacuum degree of 30-40 Pa, the temperature is raised to 20°C and maintained for 10-12 hours, then, under a vacuum degree of 30-40 Pa, the temperature is raised to 30°C and maintained for 6-8 hours, and then the vacuum degree is controlled at 10-20 Pa and maintained at 30°C for 4-6 hours.

[0018] As a further technical solution, the heating plate layer is at least 2 layers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention uses trehalose, skim milk powder, sodium glutamate and sucrose as freeze-drying protective agents for strains. The macromolecular materials are combined with small molecular materials and can form a stable suspended structure after mixing with bacterial mud. The structure is evenly dispersed on the surface of the bacteria, which can reduce the damage of ice crystals to the bacteria during the pre-freezing process and prevent the collapse of deep-cold particles during the heating process.

[0021] 2) In the traditional freeze-drying process, the pre-freezing speed of the bacterial suspension is slow and the ice crystals formed are large. However, the present invention performs deep freezing granulation before placing the bacterial suspension in the freeze-drying equipment, which greatly reduces the pre-freezing time in the freeze-drying process. The ice crystals formed by the bacterial suspension can reduce the mechanical damage during the pre-freezing process.

[0022] 3) The present invention is simple to operate, can increase the number of live bacteria of Lactobacillus plantarum powder to more than 1 trillion, enhances the market competitiveness of local strains, solves the problem of low production efficiency of Lactobacillus plantarum, and reduces production costs and energy loss in the freeze-drying process.

[0023] Instruction Manual

[0024] Figure 1 A positional relationship diagram of adjacent heating plate layers and trays of a freeze dryer;

[0025] exist Figure 1 1. Heating plate layer, 2. Tray with cryogenic pellets.

[0026] Figure 2 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0029] In the present invention,

[0030] Lactobacilus plantarum YMC 1005 (commercial number LP45), deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number CGMCC NO.8072, is a published strain;

[0031] Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0032] The following is further explained with specific examples.

[0033] Embodiment 1:

[0034] A freeze-drying production method for high-viable count Lactobacillus plantarum powder comprises the following steps:

[0035] Step 1: Prepare protective solution:

[0036] The protective solution comprises the following raw materials in percentage by weight: trehalose 8%; skim milk powder 8%; sodium glutamate 1%; sucrose 2%; the balance is water; the pH is adjusted to 6.9-7.1, and sterilized at 115°C for 20 minutes;

[0037] Step 2, preparation of bacterial suspension: collecting Lactobacillus plantarum bacterial mud, and mixing the Lactobacillus plantarum bacterial mud and protective solution in a ratio of 1:1 based on the dry weight of Lactobacillus plantarum cells to obtain a bacterial suspension;

[0038] Step 3, cryogenic granulation: dropping the bacterial suspension into liquid nitrogen for cryogenic granulation to obtain cryogenic pellets;

[0039] The inner diameter of the dropper used to drop the bacterial suspension into liquid nitrogen was 2 mm, and the particle size of the cryogenic pellets obtained was 2-4 mm;

[0040] Step 4, freeze drying: Place the cryogenic pellets into the freeze dryer tray with a thickness of 0.8-1.4 cm, and then Figure 1 As shown, a tray 2 containing cryogenic particles is placed between adjacent heating plate layers 1 of a freeze dryer, and the temperature is raised to 20°C for 4 hours and maintained for 6 hours under a vacuum degree of 40 Pa, and then, the temperature is raised to 30°C for 3 hours under a vacuum degree of 40 Pa, and then, the vacuum degree is controlled at 20 Pa and maintained at 30°C for 4 hours, so that a plant lactobacillus powder with a high viable count can be obtained.

[0041] Example 2

[0042] A freeze-drying production method for plant lactobacillus powder with high viable count comprises the following steps: the same as Example 1, except that, in step 4, the thickness of the plate is 1.2 cm, and the heating procedure is: under the condition of vacuum degree of 30 Pa, the temperature of the heating layer is increased to 20° C. for 4 hours and maintained for 8 hours, under the condition of 40 Pa, the temperature is increased to 30° C. for 3 hours and maintained for 5 hours, and then the vacuum degree is controlled at 10-20 Pa, and the temperature is maintained at 30° C. for 6 hours.

[0043] Comparative Example 1

[0044] A freeze-drying production method for Lactobacillus plantarum powder comprises the following steps:

[0045] Step 1: Prepare protective solution:

[0046] The protective solution comprises the following raw materials in percentage by weight: trehalose 8%; skim milk powder 8%; sodium glutamate 1%; sucrose 2%; the balance is water; the pH is adjusted to 6.9-7.1, and sterilized at 115°C for 20 minutes;

[0047] Step 2, preparation of bacterial suspension: collecting Lactobacillus plantarum bacterial mud, and mixing the Lactobacillus plantarum bacterial mud and protective solution in a ratio of 1:1 based on the dry weight of Lactobacillus plantarum cells to obtain a bacterial suspension;

[0048] Step 3, pre-freezing of bacterial suspension: pour the prepared bacterial suspension into the freeze dryer tray, and place it on the tray according to the thickness of 0.8 cm. The pre-freezing procedure is to reduce the temperature of the bacterial suspension to -45°C for 2 hours, and keep it for 1.5 hours after the bacterial suspension drops to -45°C;

[0049] Step 4, freeze-drying: under the condition of vacuum degree at 40Pa, the heating layer is heated to 0°C for 8h and maintained for 10h, then, under the condition of 40Pa, the temperature is raised to 10°C for 6h and maintained for 10h, then, under the condition of 40Pa, the temperature is raised to 20°C for 6h and maintained for 6h, the vacuum degree is controlled at 20Pa, and maintained at 30°C for 4h to obtain Lactobacillus plantarum powder.

[0050] Comparative Example 2

[0051] A freeze-drying production method for Lactobacillus plantarum powder comprises the following steps:

[0052] Step 1: Prepare protective solution:

[0053] The protective solution comprises the following raw materials in percentage by weight: trehalose 8%; skim milk powder 8%; sodium glutamate 1%; sucrose 2%; the balance is water; the pH is adjusted to 6.9-7.1, and sterilized at 115°C for 20 minutes;

[0054] Step 2, preparation of bacterial suspension: collecting Lactobacillus plantarum bacterial mud, and mixing the Lactobacillus plantarum bacterial mud and protective solution in a ratio of 1:1 based on the dry weight of Lactobacillus plantarum cells to obtain a bacterial suspension;

[0055] Step 3, pre-freezing of bacterial suspension: pour the prepared bacterial suspension into the freeze dryer tray, and place it on the tray according to the thickness of 0.8 cm. The pre-freezing procedure is to reduce the temperature of the bacterial suspension to -45°C for 2 hours, and keep it for 1.5 hours after the bacterial suspension drops to -45°C;

[0056] Step 4, freeze-drying: under the condition of vacuum degree at 40Pa, the heating layer is heated to 0°C for 10 hours, maintained for 10 hours, then, under the condition of 40Pa, the temperature is raised to 10°C for 8 hours, maintained for 10 hours, then, under the condition of 40Pa, the temperature is raised to 20°C for 6 hours, maintained for 6 hours, the vacuum degree is controlled at 20Pa, and maintained at 30°C for 4 hours to obtain Lactobacillus plantarum powder.

[0057] Effect example:

[0058] The Lactobacillus plantarum powder prepared in each embodiment and comparative example was weighed, and viable bacteria detection was performed to calculate the number of viable bacteria and the freeze-drying survival rate. The results are shown in Table 1;

[0059] Table 1

[0060]

[0061]

[0062] From the results in Table 1, we can see that:

[0063] 1) Under the same tray thickness, the freeze-drying time of Example 1 and Example 2 is shorter than that of the control group. Compared with the conventional freeze-drying technology, the overall freeze-drying time of liquid nitrogen cryogenic granulation and 360-degree thermal radiation vacuum freeze-drying is shortened by more than 40%, indicating that the liquid nitrogen cryogenic granulation and 360-degree thermal radiation vacuum freeze-drying technology can significantly shorten the freeze-drying time and increase production efficiency.

[0064] 2) Example 1-2 uses liquid nitrogen cryogenic granulation and 360-degree thermal radiation vacuum freeze-drying, and its freeze-drying survival rate can reach more than 80%, while using conventional freeze-drying technology, the freeze-drying survival rate is only about 60%. This shows that the use of cryogenic granulation and 360-degree thermal radiation vacuum freeze-drying technology can greatly improve the freeze-drying survival rate of the bacteria and improve the quality of the product.

[0065] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A freeze-drying production method for high viable count Lactobacillus plantarum powder, characterized in that: The steps include: Step 1, mixing the plant lactobacillus slurry with the protective solution to obtain a bacterial suspension; Step 2, dropping the bacterial suspension into liquid nitrogen for cryogenic granulation to obtain cryogenic pellets; Step 3: Load the cryogenic pellets into a tray of a freeze dryer, and then place the tray containing the cryogenic pellets between adjacent heating plate layers of the freeze dryer. Under vacuum conditions, heat the cryogenic pellets using 360-degree heat radiation to sublime the water, thereby obtaining Lactobacillus plantarum powder with a high viable count.

2. The freeze-drying production method of a high viable count plant lactobacillus powder according to claim 1, characterized in that: The protective solution includes the following raw materials in percentage by weight: Trehalose 8-10%; skim milk powder 8-10%; sodium glutamate 1-2%; sucrose 2-4%; water as the balance; pH adjusted to 6.9-7.

1.

3. The freeze-drying production method of a high viable count plant lactobacillus powder according to claim 1, characterized in that: The protective solution is sterilized at 115° C. for 20 minutes before being mixed with Lactobacillus plantarum cells.

4. The freeze-drying production method of a high viable count plant lactobacillus powder according to claim 1, characterized in that: In step 1, the Lactobacillus plantarum bacterial sludge and the protective solution are evenly mixed in a ratio of 1:0.8-1 based on the dry weight of the Lactobacillus plantarum cells.

5. The freeze-drying production method of a high viable count plant lactobacillus powder according to claim 1, characterized in that: In step 2, the inner diameter of the dropper used to drop the bacterial suspension into liquid nitrogen is 2 mm, and the particle size of the obtained cryogenic particles is 2-4 mm.

6. The freeze-drying production method of a high viable count Lactobacillus plantarum powder according to claim 1, characterized in that: In step 3, the cryogenic pellets are loaded into the tray of the freeze dryer with a tray thickness of 0.8-1.4 cm.

7. The freeze-drying production method of a high viable count Lactobacillus plantarum powder according to claim 1, characterized in that: In step 3, the heating treatment procedure is: under the condition of vacuum degree of 30-40Pa, the temperature is raised to 20℃ and maintained for 10-12h, then, under the condition of 30-40Pa, the temperature is raised to 30℃ and maintained for 6-8h, and then the vacuum degree is controlled at 10-20Pa and maintained at 30℃ for 4-6h.

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