Method for extracting bile salt hydrolase by fermenting lactobacillus salivarius

A technology of Lactobacillus salivarius and bile salt hydrolase, which is applied in the field of bile salt hydrolase, can solve problems such as reduction, and achieve the effects of simple extraction process, convenient source of raw materials and short fermentation period

Inactive Publication Date: 2014-06-18
青岛博之源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a large number of clinical experiments at home and abroad have confirmed that taking probiotics and related products can reduce the serum cholesterol content of the human body and reduce the incidence of cardiovascular diseases, which may be closely related to the bile salt hydrolyzing enzyme BSH produced by probiotics

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Medium components (w / v) for cultivating Lactobacillus salivarius high-yield bile salt hydrolase: glucose 1%, casein 1%, manganese sulfate heptahydrate 0.02%, beef extract 1%, yeast extract 0.5 %, Tween 80 0.1%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, diamine citrate 0.2%, magnesium sulfate heptahydrate 0.058%, calcium carbonate 2%, distilled water 1000 ml, sterilized at 0.07 MPa for 20 min. The fermentation process conditions for high production of bile salt hydrolase: fermentation temperature 37 °C, fermentation time 20 h, initial pH value of medium 6.5, inoculum size 2%.

[0020] Under these fermentation conditions, the bile salt hydrolase activity of Lactobacillus salivarius was 2.25 times that before optimization.

Embodiment 2

[0022] Lactobacillus salivarius fermented liquid is used to extract bile salt hydrolyzing enzyme, and the purpose of separating and purifying bile salt hydrolyzing enzyme is to extract the target intracellular enzyme and make it reach the required purity through purification. The key technology of extraction is to choose an effective method for breaking cells. Centrifuge the lactic acid bacteria fermentation broth obtained under the above fermentation conditions at 8 000 r / min, 4 °C for 8 min, discard the supernatant, and centrifuge and wash the bacteria twice with sterile physiological saline (conditions are the same as above), discard the supernatant , adding 0.02 mol / L phosphate buffer (pH 6.0) to the original volume of the fermentation broth, and ultrasonically crushing the cell suspension in an ice bath for 15 min (power 400-450 w, working for 1 s, intermittent for 2 s), and Centrifuge at 10 000 r / min at 4 °C for 10 min to remove cell debris and collect the supernatant co...

Embodiment 3

[0024] The method for measuring the enzyme activity of the bile salt hydrolyzing enzyme in the fermented liquid and the extracted liquid is improved, and it is used for micro-determination. Take 20 μl of a solution containing bile salt hydrolyzing enzyme, add an equal volume of pig bile salt with a concentration of 1%, shake and mix on a vortex mixer for 1 min, and shake the mixture at 37 °C for 30 min for enzymatic hydrolysis. After the enzyme reaction is completed, add 40 μl of 15% trichloroacetic acid immediately, mix well, stop the enzyme reaction for 1 min, and then centrifuge (the conditions are the same as above), and the supernatant is the enzyme reaction solution. For the control group, 20 μl of a solution containing bile salt hydrolyzing enzyme was shaken at 37 °C for 30 minutes, 40 μl of 15% trichloroacetic acid was added, and 20 μl of 1% porcine bile salt was added immediately, and the subsequent operations were treated with the same sample. The activity of the bil...

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PUM

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Abstract

The invention relates to an extraction method of BSH (Bile Salt Hydrolase) contained in lactobacillus salivarius. The extraction method is suitable for developing and producing a novel functional food by taking BSH as a functional factor in functional milk products. The extraction method comprises the following step of: acquiring lactobacillus bile salt hydrolase through optimized fermentation condition and extraction method by utilizing the Lactobacillus salivarius LMG14476 capable of producing the BSH. By adopting suitable ultrasonic cell disruption, the extraction method disclosed by the invention solves the problem that the intracellular bile salt hydrolase is difficult to extract and enhances the extraction rate of the BSH. The BSH prepared through the extraction method has the advantages of simple fermentation process and extraction method, shorter fermentation period, low requirement on equipment, lower cost and suitability for industrialized production.

Description

technical field [0001] The invention discloses a method for extracting bile salt hydrolyzing enzyme from Lactobacillus salivarius by ultrasonic wave for cell wall breaking combined with ammonium sulfate precipitation. The obtained BSH product is suitable for use as a functional factor in functional dairy products to develop and produce novel functional food. Background technique [0002] High cholesterol content in serum is an important factor in inducing cardiovascular diseases such as hypertension and coronary heart disease. Studies have found that different types of probiotics have the effect of lowering cholesterol, and this effect is closely related to the bile salt hydrolase (Bile salt hydrolase, BSH, EC 3·5·1·24) produced by them. [0003] BSH enzymes are ubiquitously found in gut microbes and are rarely found in microbes from other environments. At present, many BSHs enzymes have been purified and identified from various microorganisms. BSH activity is found in lac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12R1/225
CPCC12N9/80C12Y305/01024
Inventor 齐宏涛
Owner 青岛博之源生物技术有限公司
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