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High-dose bacillus licheniformis viable bacterium composition as well as preparation method and application thereof

The technology of Bacillus licheniformis and the composition is applied in the field of microorganisms, which can solve the problems of reducing the fluidity of preparation products, difficult to achieve industrialized production, and low viable bacteria content, and achieves recovery of intestinal colonization resistance, fast onset, and viable bacteria content. high effect

Active Publication Date: 2013-05-15
NORTHEAST PHARMA GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] According to the data disclosed in invention patent 92113318.9 and 201010578718.0, the number of live bacteria in Bacillus licheniformis CMCC63516 (CGMCC No.0183) bacteria powder is relatively low, generally 25-53 billion / g. In addition, according to invention patent 92113318.9 and 201010578718.0 The disclosed bacterial powder prepared with anhydrous calcium carbonate as a diluent has poor fluidity (angle of repose: 45.0°), because of the limited addition amount in the existing 1 billion / g live Bacillus licheniformis preparation , has not affected the industrial production of the preparation, but if the addition amount is greater than 15% of the product, it will significantly reduce the overall fluidity of the preparation product, and it is difficult to realize industrial production. Therefore, in the past, this method used anhydrous calcium carbonate as a diluent to prepare lichen The method of bacillus bacterium powder can not be applicable to the high dose scope covered by the present invention
[0008] In addition, previous studies have paid too much attention to the output of each batch of fermentation liquid sludge, but this cannot solve the current situation that the content of viable bacteria in the unit mass of bacteria powder is not high, and it cannot satisfy the need to use a small amount of bacteria powder to prepare high-dose, high-activity bacteria. The goal of the finished product is how to increase the number of viable spores in the unit mass of sludge while ensuring the output of the sludge, which has become a key issue restricting the development of high-dose live Bacillus licheniformis preparations

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  • High-dose bacillus licheniformis viable bacterium composition as well as preparation method and application thereof
  • High-dose bacillus licheniformis viable bacterium composition as well as preparation method and application thereof
  • High-dose bacillus licheniformis viable bacterium composition as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. Materials and methods

[0019] 1.1 Materials

[0020] 1.1.1 Experimental animals

[0021] SPF-grade Kunming mice, age 6-8 weeks, weight 20±2g, male and female, half male and half male, provided by the animal room of Shenyang No. .

[0022] 1.1.2 Drugs

[0023] Ampicillin sodium for injection (Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., 0.5g / bottle), Bacillus licheniformis BL63516 bacterial powder (Northeast Pharmaceutical Group Shenyang First Pharmaceutical Factory, batch number: YJ12018, 29.4 billion / g)

[0024] 1.1.3 Main reagents

[0025]Bifidobacterium Selective Medium (BBL), Enterococcus Selective Medium (Bile-Escin-Sodium Azide Agar), Lactobacillus Selective Medium (MRS), Enterobacterial Medium (EMB), Gas Production Clostridium perfringens culture medium (TSC) was purchased from Beijing Land Bridge Technology Co., Ltd. Beef extract and peptone were purchased from Beijing Aoboxing Biotechnology Co., Ltd., and sodium chloride was purchased from Tianji...

Embodiment 2

[0068] 1 Materials and methods

[0069] 1.1 material is the same as embodiment one

[0070] 1.2 Method

[0071] 1.2.1 Bacillus licheniformis BL63516 bacterial suspension preparation

[0072] Dissolve 5g of bacterial powder in 600ml of normal saline to prepare a high-concentration bacterial solution of 8.33mg / ml. Take 500ml of high-concentration bacterial solution and add 250ml of normal saline to prepare a 5.56mg / ml bacterial solution. Carry out 2-fold serial dilution to 2.78mg / ml, 1.39mg / ml bacterial solution.

[0073] It needs to be prepared and fed before each gavage.

[0074] 1.2.2 Grouping of test animals

[0075] 72 healthy Balb / c mice were selected, weighing 20±2g. After feeding with mouse feed and clean water for 1 week, the mice were randomly divided into 6 groups, 12 in each group, half male and half male. Group 1 was the blank control group, and the other 5 model groups were made with ampicillin sodium to make the diarrhea model of intestinal flora imbalance, an...

Embodiment 3

[0099] MnSO 4 Concentrations were 0%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%.

[0100] Take a ring of Bacillus licheniformis BL63516 with a sterile hook, inoculate the nutrient broth medium, and cultivate it at 36-38°C for 9 hours to make a seed solution. Inoculate the seed liquid into the shake flask nutrient broth medium, the inoculum amount is 0.1%, the liquid volume is 200ml / 1000ml, cultivate at 37°C and 200r / min for 18-20h, then add different concentrations of manganese ions to the fermentation liquid, and cultivate to 22 -24h. The bacteria sludge was collected by centrifugation, and the vegetative body was killed by heating at 62.5°C for 15 minutes, and then 10-fold serial dilution was performed to measure the number of spores in the fermentation broth. The results are shown in Table 12.

[0101] Table 12 Manganese ion concentration test results (spore number unit: 10 7 CFU / mg)

[0102] Manganese ion concentration

[0103] The results showe...

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Abstract

The invention provides a high-dose bacillus licheniformis viable bacterium composition as well as a preparation method and an application thereof. Each gram of composition contains 1.5-20 billions of bacillus licheniformis viable bacteria; a diluent needs to be added into bacterial sludge when bacterium powder is prepared, wherein the ratio of the bacterial sludge to the diluent is 1: (3-5); the high-dose bacillus licheniformis viable bacterium composition can be applied to preparation of a drug used for preventing and treating alteration of intestinal flora as well as acute enteritis, chronic enteritis and diarrhea caused by the alteration of intestinal flora, wherein recommended dosage of the bacillus licheniformis viable bacterium composition is 0.025-0.34 billions of viable bacteria / Kg; and the preparation method of the high-dose bacillus licheniformis viable bacteria comprises the steps of: inoculating a seed solution into a sterilization culture medium of a fermentation tank, fermenting for 18-20 hours at the temperature of 36-38 DEG C, changing a culture temperature to 41-49 DEG C, adding Mn<2+> until final concentration of the culture medium is 0.005-0.05%, and culturing until fermentation is finished. The high-dose bacillus licheniformis viable bacterium composition provided by the invention has the advantages that the content of viable bacteria in unit mass of bacterium powder is high; retention rate of spore viable bacteria dried at high temperature is high; and liquidity of the bacterium powder is good, and obvious effects of taking effect quickly, realizing a short course treatment and quickly restoring intestinal tract field planting resistance can be achieved.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a high-dose live bacillus licheniformis composition and a preparation method and application thereof. Background technique [0002] Probiotics belong to a kind of microecological preparations. The Food and Agriculture Organization of the United Nations / World Health Organization defines probiotics as: "live microorganisms that, when ingested in sufficient quantities, will bring benefits to the health of the host." There are 3 key points in this definition: the first is the live bacteria, the second is the effective dose, and the last is the effect that can produce health benefits. The first two points belong to the category of quality standards, and the third point belongs to the category of health claims. All probiotic legislation and guidelines are refined around these three points. [0003] Activity is the key. Probiotics must be "alive" in order to play a health...

Claims

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Application Information

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IPC IPC(8): A61K35/74A61K47/38A61K47/26A61P1/00A61P1/12A61K35/742
CPCA61K9/1652A61K47/26A61K35/74A61K47/02A61K9/2018A61K47/38A61K9/2009A61K9/1623A61K9/2846A61K9/5026A61K9/2054A61K9/0095A61K9/1611A61K35/742A61P1/00A61P1/12C12N1/20
Inventor 李雪龙雷健陈伟吴楠彭勃
Owner NORTHEAST PHARMA GRP
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