Lactobacillus paracasei producing bacteriocin and application thereof

Abstract

The invention provides a lactobacillus paracasei producing bacteriocin and an application thereof. The present invention relates to a Lactobacillus paracasei S20 strain with a preservation number of CGMCC NO. 17120, The strain is isolated from a Tibetan traditional fermented food-milk block. A crude bacteriocin extract is obtained by extracting from a MRS fermentation supernatant of the strain byusing ammonium sulfate grading deposition. The experiment proves that the bacterial extract produced by the strain has a strong inhibitory effect against Staphylococcus aureus and Escherichia coli, and the minimum inhibitory concentration measured by a double dilution method is 312 and 625 [mu]g / mL, respectively. The bacteriocin produced by the strain can be hydrolyzed by proteases such as neutralprotease and trypsin, and does not accumulate in the body. The Lactobacillus paracasei and its bacteriocin screened by the invention have broad application prospects in fermented foods.

Description

technical field

[0001] The invention relates to the technical field of food microorganisms, in particular to a bacteriocin-producing Lactobacillus paracasei and its application. Background technique

[0002] Bacteriocins are a class of small molecule peptides or proteins that are synthesized and secreted by the ribosomes of some bacteria and have the effect of inhibiting or killing related microorganisms. Bacteriocins come from a wide range of sources and can be produced by both Gram-positive and Gram-negative bacteria. Among them, the bacteriocins produced by lactic acid bacteria are called lactobacillus. Lactic acid bacteria are recognized as safe-grade microorganisms in food. The bacteriocins produced by them have attracted widespread attention due to their safety, non-toxicity, easy degradation by proteases, and no accumulation in the body.

[0003] Nisin, as the first lactic acid bacterium used in food, has been recognized as a food preservative since the British Commi...

Examples

Embodiment 1

[0035] Implementation Example 1 Screening and identification of bacteriocin-producing lactic acid bacteria

[0036] (1) Strain activation

[0037] (a) Activation of bacteriocin-producing lactic acid bacteria: samples of traditional fermented foods collected from various places in the early stage, such as homemade yogurt, milk tofu, kimchi, milk bumps, milk fans, sweet fermented grains, etc., were isolated from more than 100 strains using MRS and M17 medium Lactic acid bacteria strains were purified by streaking, and the freeze-dried tubes were stored in the refrigerator.

[0038]Streak the lactic acid bacteria strain on the MRS plate (purchased from Haibo Biology), place it in 37°C for anaerobic culture for 48 hours, pick a single colony on the plate, and then transfer it to MRS liquid medium for activation at 37°C for 24 hours, repeat the activation step 2 times, and set aside.

[0039] (b) Activation of indicator bacteria: with Escherichia coli (E.coli) and Staphylococcus a...

Embodiment 2

[0058] Implementation Example 2 The preparation of the crude extract of bacteriocin produced by bacterial strain S20

[0059] 1) Determination of salting-out saturation by fractional precipitation

[0060] Lactobacillus paracasei S20 was inoculated in MRS liquid medium, placed at 37°C for static culture for 28-48 hours to obtain a fermentation liquid, and centrifuged at 4°C and 8000r / min for 10 minutes to obtain a fermentation supernatant.

[0061] The crude extract of bacteriocin was prepared by fractional precipitation with ammonium sulfate. First determine the salting-out saturation of the graded precipitation, grind the ammonium sulfate into powder, slowly stir and add it to the fermentation supernatant, when it appears turbid and does not clarify for 2 minutes, calculate the salting-out saturation at this time according to the salting-out table, and store it in a 4°C refrigerator Stand overnight, centrifuge (4°C, 8000r / min, 20min), and collect supernatant and precipitate...

Embodiment 3

[0072] Determination of the properties of the crude extract of bacteriocins produced by implementation example 3 S20

[0073] (1) Determination of protein content in crude extract

[0074] Adopt Coomassie Brilliant Blue method to measure the protein content of the bacteriocin crude extract prepared in Example 2, the bovine serum albumin solution with a concentration of 1mg / mL is used as the standard protein solution, the standard protein content is the abscissa, and the absorbance at 595nm Is the ordinate, draws the standard curve, obtains the linear regression equation to be y=1.021x+0.017 (R 2 = 0.999). After the crude extract was redissolved in water, the concentration of 2.5mg / mL was prepared, and after appropriate dilution, the protein content was determined. It can be obtained by calculation that the protein content in the crude bacteriocin is 2.12mg / 2.5mg, and the protein content reaches 85%, which shows that the crude bacteriocin is mainly a protein substance, and at...

technical field

[0001] The invention relates to the technical field of food microorganisms, in particular to a bacteriocin-producing Lactobacillus paracasei and its application. Background technique

[0002] Bacteriocins are a class of small molecule peptides or proteins that are synthesized and secreted by the ribosomes of some bacteria and have the effect of inhibiting or killing related microorganisms. Bacteriocins come from a wide range of sources and can be produced by both Gram-positive and Gram-negative bacteria. Among them, the bacteriocins produced by lactic acid bacteria are called lactobacillus. Lactic acid bacteria are recognized as safe-grade microorganisms in food. The bacteriocins produced by them have attracted widespread attention due to their safety, non-toxicity, easy degradation by proteases, and no accumulation in the body.

[0003] Nisin, as the first lactic acid bacterium used in food, has been recognized as a food preservative since the British Commi...

Examples

Embodiment 1

[0035] Implementation Example 1 Screening and identification of bacteriocin-producing lactic acid bacteria

[0036] (1) Strain activation

[0037] (a) Activation of bacteriocin-producing lactic acid bacteria: samples of traditional fermented foods collected from various places in the early stage, such as homemade yogurt, milk tofu, kimchi, milk bumps, milk fans, sweet fermented grains, etc., were isolated from more than 100 strains using MRS and M17 medium Lactic acid bacteria strains were purified by streaking, and the freeze-dried tubes were stored in the refrigerator.

[0038]Streak the lactic acid bacteria strain on the MRS plate (purchased from Haibo Biology), place it in 37°C for anaerobic culture for 48 hours, pick a single colony on the plate, and then transfer it to MRS liquid medium for activation at 37°C for 24 hours, repeat the activation step 2 times, and set aside.

[0039] (b) Activation of indicator bacteria: with Escherichia coli (E.coli) and Staphylococcus a...

Embodiment 2

[0058] Implementation Example 2 The preparation of the crude extract of bacteriocin produced by bacterial strain S20

[0059] 1) Determination of salting-out saturation by fractional precipitation

[0060] Lactobacillus paracasei S20 was inoculated in MRS liquid medium, placed at 37°C for static culture for 28-48 hours to obtain a fermentation liquid, and centrifuged at 4°C and 8000r / min for 10 minutes to obtain a fermentation supernatant.

[0061] The crude extract of bacteriocin was prepared by fractional precipitation with ammonium sulfate. First determine the salting-out saturation of the graded precipitation, grind the ammonium sulfate into powder, slowly stir and add it to the fermentation supernatant, when it appears turbid and does not clarify for 2 minutes, calculate the salting-out saturation at this time according to the salting-out table, and store it in a 4°C refrigerator Stand overnight, centrifuge (4°C, 8000r / min, 20min), and collect supernatant and precipitate...

Embodiment 3

[0072] Determination of the properties of the crude extract of bacteriocins produced by implementation example 3 S20

[0073] (1) Determination of protein content in crude extract

[0074] Adopt Coomassie Brilliant Blue method to measure the protein content of the bacteriocin crude extract prepared in Example 2, the bovine serum albumin solution with a concentration of 1mg / mL is used as the standard protein solution, the standard protein content is the abscissa, and the absorbance at 595nm Is the ordinate, draws the standard curve, obtains the linear regression equation to be y=1.021x+0.017 (R 2 = 0.999). After the crude extract was redissolved in water, the concentration of 2.5mg / mL was prepared, and after appropriate dilution, the protein content was determined. It can be obtained by calculation that the protein content in the crude bacteriocin is 2.12mg / 2.5mg, and the protein content reaches 85%, which shows that the crude bacteriocin is mainly a protein substance, and at...

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