Bifidobacterium pseudominutus freeze-drying protective agent and application thereof

Abstract

The invention discloses a bifidobacterium pseudocatenum freeze-drying protective additive and application, and the bifidobacterium pseudocatenum freeze-drying protective additive is prepared from the following raw materials in percentage by weight: 2%-10% of dried skim milk, 5%-20% of cane sugar, 1%-6% of fructo-oligosaccharide, 0.5%-3% of sodium glutamate, 0.5%-2% of sodium ascorbate, 3%-15% of betaine and the balance of distilled water. The freeze-drying protective agent is simple in component composition, the survival rate of the bifidobacterium pseudocatenum in the freeze-drying process can be increased by using the freeze-drying protective agent, the freeze-drying survival rate of the bifidobacterium pseudocatenum reaches 76.3% or above, the stability of the freeze-drying preparation is guaranteed, and meanwhile the obtained freeze-drying preparation has the advantages of being easy to operate and the like, and has good application prospects. The preparation method of the freeze-dried preparation is simple and feasible, mild in condition, good in reproducibility, strong in controllability, suitable for large-scale production, and more convenient for clinical use and storage.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically relates to a freeze-drying protective agent for Bifidobacterium pseudocatenulatum and an application thereof. Technical Background

[0002] Bifidobacterium pseudocatenulatum is a species of Bifidobacterium. It is a Gram-positive, anaerobic, non-spore-forming probiotic. It is commonly found in the intestines of humans and animals and is an important component of the intestinal microbiota. Bifidobacterium pseudocatenulatum competitively inhibits the growth of harmful microorganisms and promotes the health of the intestinal mucosa, reduces the chance of invasion by pathogens, and maintains the balance of the intestinal environment. In recent years, Bifidobacterium pseudocatenulatum has been shown to improve host lipid metabolism, inflammatory response, and the nervous system. In addition, like other bifidobacteria, Bifidobacterium pseudocatenulatum may affect mental health through the gut-brain axis. Studies have suggested that it may help alleviate anxiety and depression symptoms. Therefore, Bifidobacterium pseudocatenulatum has extremely high research value and broad application prospects.

[0003] Probiotics are dormant when their water activity is low, which is more conducive to preservation. Therefore, the bacteria are usually dried to remove water for long-term preservation and subsequent bacterial preparation development. Vacuum freeze-drying technology is usually used in industry to dehydrate bacteria so that bacterial preparations can maintain high activity and stability during long-term storage. However, during the vacuum freeze-drying process, bacteria will inevitably suffer physiological damage, mainly including mechanical damage, solute damage, cell membrane damage, protein denaturation and inactivation, DNA damage, etc.

[0004] Adding appropriate protective agents is one of the best strategies to reduce damage, increase the freeze-dried survival rate of probiotics, and improve the stability of bacterial powder during storage.

[0005] However, the freeze-drying protective agent and application of Bifidobacterium pseudocatenulatum have not been reported yet. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a freeze-drying protective agent for Bifidobacterium pseudocatenulatum.

[0007] The second object of the present invention is to provide the use of the above-mentioned Bifidobacterium pseudocatenulatum freeze-drying protective agent.

[0008] The technical solution of the present invention is summarized as follows:

[0009] A freeze-dried protective agent for Bifidobacterium pseudomicrocystis is prepared from the following raw materials by weight percentage: 2%-10% skim milk powder, 5%-20% sucrose, 1%-6% oligofructose, 0.5%-3% sodium glutamate, 0.5%-2% sodium ascorbate, 3%-15% betaine, and the balance is distilled water.

[0010] Skimmed milk powder is skimmed cow's milk powder or skimmed goat's milk powder.

[0011] The above-mentioned freeze-drying protective agent for Bifidobacterium pseudocatenulatum is used in freeze-drying of Bifidobacterium pseudocatenulatum bacterial mud.

[0012] The above application includes the following steps:

[0013] 1) inoculating Bifidobacterium pseudocatenulatum into MRS medium and performing anaerobic fermentation to obtain activated seed solution;

[0014] 2) inoculating the activated seed liquid into the MRS medium, culturing at 37°C for 24-36 hours, centrifuging at 7000-9000 rpm for 8-12 minutes to obtain bacterial sludge 1; resuspending with PBS, centrifuging at 7000-9000 rpm for 8-12 minutes to obtain bacterial sludge 2;

[0015] 3) adding the freeze-drying protective agent of Bifidobacterium pseudocatenulatum to the bacterial mud 2 obtained in step 2), wherein the weight ratio of the bacterial mud 2 to the freeze-drying protective agent of Bifidobacterium pseudocatenulatum is 1:2-4, and stirring evenly to obtain a bacterial mud-protective agent mixed solution;

[0016] 4) The bacterial sludge-protectant mixture is pre-frozen at -40°C for 10 hours, then sublimated and dried at a vacuum degree of 5 Pa at -15°C to -25°C for 6 hours to 12 hours, and then analytically dried at a vacuum degree of ultimate vacuum at 25°C for 5 hours.

[0017] Advantages of the present invention:

[0018] The freeze-drying protective agent of Bifidobacterium pseudocatenulatum of the present invention has simple composition. The freeze-drying protective agent of the present invention can improve the survival rate of Bifidobacterium pseudocatenulatum in the freeze-drying process, so that the freeze-drying survival rate of Bifidobacterium pseudocatenulatum reaches 83.6%, and the stability of the freeze-dried preparation is guaranteed. At the same time, the obtained freeze-dried preparation has the advantages of simple operation, etc. The preparation method of the freeze-dried preparation is simple and feasible, the conditions are mild, the reproducibility is good, the controllability is strong, it is suitable for large-scale production, and it is more convenient for clinical use and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the number of live bacteria of Bifidobacterium pseudocatenulatum before and after freeze-drying using the freeze-drying protectant of the present invention. Specific implementation methods

[0021] The Bifidobacterium pseudocatenulatum used in each embodiment of the present invention is MF-009588 (Xinyang Mufan Biotechnology Co., Ltd.) as an example, so that those skilled in the art can better understand the present invention. Bifidobacterium pseudocatenulatum sold by other companies can also be used in the present invention.

[0022] The present invention will be further described below in conjunction with specific embodiments.

[0023] The components of MRS medium are: peptone 10g, beef extract powder 8.6g, yeast extract powder 5g, K2HPO4 2g, triammonium citrate 2g, sodium acetate 5g, glucose 20g, L-cysteine ​​hydrochloride 0.5g, Tween-80 1mL, MgSO4·7H2O 0.58g, MnSO4·4H2O 0.25g, distilled water to 1000mL; sterilize at 115℃ for 30min.

[0024] The anaerobic conditions in Examples 4 to 6 were: 85% nitrogen, 5% hydrogen, and 10% carbon dioxide.

[0025] Example 1

[0026] A freeze-dried protective agent for Bifidobacterium pseudocatenulatum, which is prepared from the following raw materials by weight percentage:

[0027] 2% skim milk powder, 20% sucrose, 1% fructooligosaccharide, 3% sodium glutamate, 0.5% sodium ascorbate, 15% betaine, and the balance is distilled water.

[0028] Example 2

[0029] A freeze-dried protective agent for Bifidobacterium pseudocatenulatum, which is prepared from the following raw materials by weight percentage:

[0030] 5% skim milk powder, 10% sucrose, 3% fructooligosaccharide, 1.5% sodium glutamate, 1% sodium ascorbate, 10% betaine, and the balance is distilled water.

[0031] Example 3

[0032] A freeze-dried protective agent for Bifidobacterium pseudocatenulatum, which is prepared from the following raw materials by weight percentage:

[0033] 10% skim goat milk powder, 5% sucrose, 6% fructooligosaccharide, 0.5% sodium glutamate, 2% sodium ascorbate, 3% betaine, and the balance is distilled water.

[0034] The preparation method of a freeze-dried protective agent of Bifidobacterium pseudocatenulatum in Example 1 is as follows: weigh the raw materials according to Example 1: skimmed (cow) milk powder, sucrose, oligofructose, sodium glutamate, sodium ascorbate, betaine and distilled water, mix them evenly, and obtain the freeze-dried protective agent.

[0035] The preparation methods of the freeze-dried protective agent of Bifidobacterium pseudocatenulatum in Examples 2 and 3 are the same as the steps of the freeze-dried protective agent of Bifidobacterium pseudocatenulatum in Example 1.

[0036] Example 4

[0037] The invention discloses an application of a freeze-drying protective agent of Bifidobacterium pseudocatenulatum in freeze-drying of Bifidobacterium pseudocatenulatum bacterial mud, comprising the following steps:

[0038] 1) Inoculate Bifidobacterium pseudocatenulatum MF-009588 into MRS medium and perform anaerobically fermentation for 24 hours to obtain activated seed solution;

[0039] 2) The activated seed solution was inoculated into MRS medium at an inoculum rate of 5% (v / v), and cultured anaerobically at 37°C for 30 hours, and centrifuged at 7000 rpm for 12 minutes to obtain bacterial sludge 1; the culture was resuspended in PBS, and centrifuged at 7000 rpm for 12 minutes to obtain bacterial sludge 2;

[0040] 3) adding a freeze-drying protective agent of Bifidobacterium pseudocatenulatum (prepared in Example 1) to the bacterial mud 2 obtained in step 2), wherein the weight ratio of the bacterial mud 2 to the freeze-drying protective agent of Bifidobacterium pseudocatenulatum is 1:2, and stirring the mixture to obtain a bacterial mud-protectant mixture;

[0041] 4) The bacterial sludge-protectant mixture was pre-frozen at -40°C for 10 hours, then sublimated and dried at a vacuum degree of 5 Pa at -15°C for 12 hours, and then analytically dried at a vacuum degree of ultimate vacuum at 25°C for 5 hours to obtain a freeze-dried powder.

[0042] Example 5

[0043] The invention discloses an application of a freeze-drying protective agent of Bifidobacterium pseudocatenulatum in freeze-drying of Bifidobacterium pseudocatenulatum bacterial mud, comprising the following steps:

[0044] 1) Inoculate Bifidobacterium pseudocatenulatum MF-009588 into MRS medium and perform anaerobically fermentation for 24 hours to obtain activated seed solution;

[0045] 2) The activated seed solution was inoculated into MRS medium at an inoculum rate of 5% (v / v), and cultured anaerobically at 37°C for 24 hours, and centrifuged at 8000 rpm for 10 min to obtain bacterial sludge 1; the culture was resuspended in PBS, and centrifuged at 8000 rpm for 10 min to obtain bacterial sludge 2;

[0046] 3) adding a freeze-drying protective agent of Bifidobacterium pseudocatenulatum (prepared in Example 2) to the bacterial mud 2 obtained in step 2), wherein the weight ratio of the bacterial mud 2 to the freeze-drying protective agent of Bifidobacterium pseudocatenulatum is 1:3, and stirring the mixture to obtain a bacterial mud-protectant mixture;

[0047] 4) The bacterial sludge-protectant mixture was pre-frozen at -40°C for 10 hours, then sublimated and dried at -20°C at a vacuum degree of 5 Pa for 8 hours, and then analytically dried at a vacuum degree of ultimate vacuum at 25°C for 5 hours to obtain a freeze-dried powder.

[0048] Example 6

[0049] The invention discloses an application of a freeze-drying protective agent of Bifidobacterium pseudocatenulatum in freeze-drying of Bifidobacterium pseudocatenulatum bacterial mud, comprising the following steps:

[0050] 1) Inoculate Bifidobacterium pseudocatenulatum MF-009588 into MRS medium and perform anaerobically fermentation for 24 hours to obtain activated seed solution;

[0051] 2) Then the activated seed solution was inoculated into MRS medium at an inoculum rate of 5% (v / v), and cultured anaerobically at 37°C for 36 hours, and centrifuged at 9000 rpm for 8 minutes to obtain bacterial sludge 1; resuspended with PBS, and centrifuged at 9000 rpm for 8 minutes to obtain bacterial sludge 2;

[0052] 3) adding a freeze-drying protective agent of Bifidobacterium pseudocatenulatum (prepared in Example 3) to the bacterial mud 2 obtained in step 2), wherein the weight ratio of the bacterial mud 2 to the freeze-drying protective agent of Bifidobacterium pseudocatenulatum is 1:4, and stirring evenly to obtain a bacterial mud-protective agent mixed solution;

[0053] 4) The bacterial sludge-protectant mixture was pre-frozen at -40°C for 10 hours, then sublimated and dried at a vacuum degree of 5 Pa at -25°C for 6 hours, and then analytically dried at a vacuum degree of ultimate vacuum at 25°C for 5 hours to obtain a freeze-dried powder.

[0054] Example 7

[0055] The survival rate of Bifidobacterium pseudocatenulatum after vacuum freeze-drying was calculated as follows:

[0056] 1) Take 1 mL of the bacterial sludge-protectant mixed solution obtained in step 3) of Example 4, and determine the initial viable bacterial count, which is A0;

[0057] 2) Take 1 mL of the bacterial sludge-protectant mixture obtained in step 3) of Example 4, and perform the operation in step 4) of Example 4 to obtain a lyophilized powder. Add physiological saline under a sterile environment and restore to the original volume of 1 mL. Measure the number of viable bacteria after freeze-drying, which is A1, and calculate the freeze-drying survival rate according to the following formula:

[0058]

[0059] I: freeze-drying survival rate; A0: initial viable bacterial count; A1: viable bacterial count after freeze-drying.

[0060] The results showed that the freeze-dried survival rate of Bifidobacterium pseudocatenulatum in Example 4 was 83.6% (see Figure 1 ).

[0061] According to the above method, the freeze-dried survival rate of Bifidobacterium pseudocatenulatum in Example 5 was 76.3%, and the freeze-dried survival rate of Bifidobacterium pseudocatenulatum in Example 6 was 80.5%.

Claims

1. A freeze-drying protective agent for Bifidobacterium pseudosmall chain, characterized in that The invention is prepared from the following raw materials by weight percentage: 2%-10% skim milk powder, 5%-20% sucrose, 1%-6% oligofructose, 0.5%-3% sodium glutamate, 0.5%-2% sodium ascorbate, 3%-15% betaine, and the balance is distilled water.

2. The freeze-dried protective agent of Bifidobacterium pseudocatenulatum according to claim 1, characterized in that The skimmed milk powder is skimmed cow's milk powder or skimmed goat's milk powder.

3. Use of a Bifidobacterium pseudocatenulatum freeze-drying protectant according to claim 1 or 2 in freeze-drying of Bifidobacterium pseudocatenulatum bacterial sludge.

4. The use according to claim 3, characterized in that it comprises the following steps: 1) inoculating Bifidobacterium pseudocatenulatum into MRS medium and performing anaerobic fermentation to obtain activated seed solution; 2) inoculating the activated seed liquid into the MRS medium, culturing at 37°C for 24-36 hours, centrifuging at 7000-9000 rpm for 8-12 minutes to obtain bacterial sludge 1; resuspending with PBS, centrifuging at 7000-9000 rpm for 8-12 minutes to obtain bacterial sludge 2; 3) adding the freeze-drying protective agent of Bifidobacterium pseudocatenulatum to the bacterial mud 2 obtained in step 2), wherein the weight ratio of the bacterial mud 2 to the freeze-drying protective agent of Bifidobacterium pseudocatenulatum is 1:2-4, and stirring evenly to obtain a bacterial mud-protective agent mixed solution; 4) The bacterial sludge-protectant mixture is pre-frozen at -40°C for 10 hours, then sublimated and dried at a vacuum degree of 5 Pa at -15°C to -25°C for 6 hours to 12 hours, and then analytically dried at a vacuum degree of ultimate vacuum at 25°C for 5 hours.

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