CN104593294A - Enterococcus faecalis with high bacteriocin yield and application of enterococcus faecalis - Google Patents

Abstract

The invention discloses a high-yielding bacteriocin enterococcus faecalis (Enterococcus faecalis) and its application. The present invention relates to a bacteriocin-producing Enterococcus faecalis with a preservation number of CGMCC NO.9966. The strain is isolated from traditional dairy products, and the bacteriocin produced by it is obtained through ammonium sulfate salting out and a series of purifications by gel chromatography. . Experiments have proved that the bacteriocin produced by the strain can be degraded by protease, has a strong inhibitory effect on Listeria monocytogenes (listeria monolytogenes), and has good heat and acid resistance. Adding the bacteriocin to fresh cheese can effectively inhibit the growth of Listeria monocytogenes. Therefore, a high-yield bacteriocin-producing Enterococcus faecalis of the present invention has the potential to be applied to fresh cheese as a natural food additive.

Description

A kind of enterococcus faecalis producing bacteriocin and its application

technical field

The invention relates to a high-production bacteriocin enterococcus faecalis and its application, belonging to the field of biotechnology.

Background technique

With the continuous improvement and improvement of people's living standards, the consumption of dairy products is also increasing, especially cheese, which has not been accepted by people, has gradually become a part of people's daily diet. Fresh cheese refers to raw milk, cream, whey Or their mixture is fermented by starter, rennet curds the milk, and excludes the products formed by whey. Fresh cheese omits the step of fermentation and maturation in the production process, which makes its flavor soft and light, with a water content greater than 67%, a texture between yogurt and cheese, and a good taste. It has the highest recognition among domestic consumers. However, cheese is rich in nutrients and is an ideal environment for the growth and reproduction of most microorganisms, especially pathogenic bacteria and spoilage bacteria. Frequent manual operations in cheese production can also cause post-processing pollution. Fresh cheese is particularly sensitive to the colonization of Listeria (L.Monocytogenes) and is particularly susceptible to contamination by Listeria (L.Monocytogenes), causing microbial food poisoning , Seriously affect people's quality of life, so it is particularly important to ensure the safety of cheese.

At present, people mainly add chemical preservatives to control the growth of microorganisms in food and prolong the shelf life of food. However, long-term consumption of these chemical additives has very bad effects on human heart, blood pressure and kidneys, and even some chemical additives have adverse effects of carcinogenesis, mutagenesis and teratogenicity. In the production and processing of food, heat sterilization is often used to control the growth of microorganisms, but heat sterilization also has some disadvantages, which will affect the quality of food and the nutritional content will be destroyed. Although physical sterilization methods such as ultraviolet disinfection and filter sterilization can avoid damaging the quality of food, the package is easily infected by bacteria after opening. Therefore, it has become an urgent hope to develop efficient, stable and safe natural biological preservatives.

Lactic acid bacteria are widely used in the food industry, and are already a type of microorganisms that people often consume. Some lactic acid bacteria will produce bacteriocins when they grow to a certain stage. Bacteriocins are a kind of polypeptide, protein or protein complex substances with antibacterial activity. It can effectively inhibit the growth of most Gram-positive bacteria and pathogenic bacteria, has good acid and heat resistance, and can be hydrolyzed and digested by protease in the body. It is a very good type of biological preservative. Applying lactic acid bacteria bacteriocin to fresh cheese processing will not affect the taste and taste of cheese, and can also effectively inhibit the growth of Listeria (L.Monocytogenes).

Bacteriocins, as a kind of biological preservatives, have great potential for application in cheese. At present, commercial bacteriocin products are still very limited, and it has become people's expectation to develop new types of bacteriocins. With the continuous improvement of technology, bacteriocin will eventually become a safe natural biological preservative widely used in cheese.

Contents of the invention

The first object of the present invention is to provide a high-yield bacteriocin-producing Enterococcus faecalis (Enterococcus faecalis), named after KLDS6001, classified as Enterococcus faecalis (Enterococcus faecalis), preserved in the General Microorganism Center of China Microorganism Culture Preservation Management Committee , located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

The Enterococcus faecalis provided by the present invention is obtained by separating from traditional dairy products. On the MRS solid medium, the colony form is round and protruding, and the color is milky white and opaque. Gram staining is positive, and the thalli form is Arranged in circles and short chains, it was identified as Enterococcus faecalis by 16SrDNA.

The second object of the present invention is to provide an Enterococcus faecalis bacteriocin and a method for producing the Enterococcus faecalis bacteriocin.

The bacteriocin provided by the invention is obtained by fermentation of Enterococcus faecalis of the invention. Specifically, the following steps are included:

(1) Preparation of Enterococcus faecalis fermentation broth

Inoculate the Enterococcus faecalis (Enterococcus faecalis) described in claim 1 in the MRS medium, obtain the Enterococcus faecalis fermentation liquid after cultivating for 12-16h at 37°C;

(2) Preliminary purification of Enterococcus faecalis bacteriocin

Centrifuge the fermented liquid obtained in step (1), absorb the supernatant, add ammonium sulfate to the supernatant for salting out, centrifuge, collect the precipitate, and redissolve it in a sodium acetate buffer solution to obtain the bacteriocin crude extract;

(3) Secondary purification of Enterococcus faecalis bacteriocin

The crude extract of the bacteriocin is further separated and purified by a Sephadex G-25 gel chromatography column to obtain the purified bacteriocin.

In the method of the present invention, preferably, step (1) is used to cultivate the MRS medium composition of faecalis (Enterococcus faecalis) as follows: peptone 10.0g, beef extract 10.0g, yeast powder 5g, glucose 20.0g, Tween 801.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, diamine citrate 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g and distilled water 1000mL.

In the method of the present invention, preferably, the culture temperature in step (1) is 37° C., and the culture time is 14 hours.

In the method of the present invention, preferably, preliminary purification is carried out in step (2) according to the following method: the fermentation broth obtained in step (1) is centrifuged, and the supernatant is drawn, and the supernatant is added Salt out ammonium sulfate with a saturation of 70%, centrifuge at 10,000×g at 4°C for 30 min, collect the precipitate, and redissolve in 0.02 mol/L sodium acetate buffer solution with pH 6.0 to obtain the crude extract of bacteriocin.

In the method of the present invention, preferably, the chromatographic conditions used for separation and purification using Sephadex G-25 gel chromatography column in step (3) are: use 50% (w/w) sodium chloride and 50% (w/w) sodium acetate buffer for elution, the flow rate is 1mL/min, the sample loading volume is 400μL, and the protein peak product with a retention time of 14-16min is collected as the purified bacteriocin.

Studies have shown that adding the bacteriocin obtained by the method and the Enterococcus faecalis fermentation broth containing the bacteriocin to fresh cheese containing 1% Listeria at 2% can effectively inhibit the growth of Listeria within 5 days of storage. growth, and the moisture and protein content of fresh cheese did not change significantly.

In addition, the bacteriocin prepared according to the method of the present invention and the Enterococcus faecalis fermentation broth containing the bacteriocin have good heat resistance, and they still maintain antibacterial activity after being treated at 121°C for 30 minutes, and are stable to acids. Maintain stable antibacterial activity in the range of -7.5. Pepsin, proteinase K, α-amylase can partially inactivate bacteriocin.

Therefore, the third object of the present invention is to provide the application of the Enterococcus faecalis bacteriocin or the Enterococcus faecalis fermentation liquid containing the bacteriocin in inhibiting the growth of Listeria monocytogenes (L.Monocytogenes). as well as

The application of the enterococcus faecalis bacteriocin or the enterococcus faecalis fermentation broth containing the bacteriocin in the preparation of fresh cheese starter.

Description of drawings

Fig. 1 is the inhibitory situation of bacteriocin to Listeria in fresh cheese;

Fig. 2 is the making process of fresh cheese.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, the examples are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Isolation and identification of Enterococcus faecalis

The Enterococcus faecalis provided by the present invention is obtained by separating from traditional dairy products. On the MRS solid medium, the colony form is round and protruding, and the color is milky white and opaque. Gram staining is positive, and the thalli form is Arranged in circles and short chains. It was identified as Enterococcus faecalis by 16SrDNA.

Described Enterococcus faecalis (Enterococcus faecalis), named as KLDS6001, classified named as Enterococcus faecalis (Enterococcus faecalis), preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms, the address is No. 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is: CGMCC No.9966, and the preservation time is November 13, 2014.

The preparation of embodiment 2 enterococcus faecalis bacteriocin

(1) Preparation of Enterococcus faecalis fermentation broth

Enterococcus faecalis KLDS6001 was inoculated in MRS liquid medium at an inoculum size of 3%, and its ingredients were: peptone 10.0g, beef extract 10.0g, yeast powder 5g, glucose 20.0g, Tween 801.1g, dipotassium hydrogen phosphate 2.0 g, 3.0 g of sodium acetate, 2.0 g of diamine citrate, 0.6 g of magnesium sulfate, 0.25 g of manganese sulfate, and 1000 mL of distilled water. Cultivate at 37°C for 16h, centrifuge at 12000×g at 4°C for 15min to obtain the fermentation supernatant;

(2) Preliminary purification of Enterococcus faecalis bacteriocin

Centrifuge the fermented liquid obtained in step (1), absorb the supernatant, add ammonium sulfate with a saturation of 70% to the supernatant for salting out, centrifuge at 10000 × g for 30 min at 4°C, collect the precipitate, Redissolve in 0.02mol/L sodium acetate buffer solution with pH 6.0 to obtain the crude extract of bacteriocin.

(3) Secondary purification of Enterococcus faecalis bacteriocin

The crude extract of bacteriocin is further separated and purified by Sephadex G-25 gel chromatography column, and the chromatographic conditions used are: use 50% (w/w) sodium chloride and 50% (w/w) The sodium acetate buffer was used for elution, the flow rate was 1 mL/min, the sample loading volume was 400 μL, and the protein peak product with a retention time of 14-16 min was collected as the purified bacteriocin.

Antibacterial activity analysis of embodiment 3 faecium enterococcus (Enterococcus faecalis)

1. Analysis method of antibacterial activity

Pour 15mL of Listeria (L.Monocytogenes) solid medium into each plate, take 100μL of indicator bacteria, and spread it evenly on the plate. After the bacteria solution is completely absorbed, put 3 Oxford cups with a diameter of 6mm into each plate. , put 50 μL of fermentation supernatant into each Oxford cup, place it at room temperature for 1 hour, put it in a constant temperature incubator at 37°C for 24 hours, observe and measure the diameter of the inhibition zone.

Enterococcus faecalis was inoculated in MRS liquid medium at an inoculum size of 3%, and its components were: 10.0 g of peptone, 10.0 g of beef extract, 5 g of yeast powder, 20.0 g of glucose, 801.1 g of Tween, and 2.0 g of dipotassium hydrogen phosphate , 3.0 g of sodium acetate, 2.0 g of diamine citrate, 0.6 g of magnesium sulfate, 0.25 g of manganese sulfate, and 1000 mL of distilled water. Cultivate at 37°C for 16 hours, centrifuge at 12000×g at 4°C for 15 minutes to obtain the fermentation supernatant, which is used for the following experiments.

(1) Eliminate the influence of organic acids: adjust the pH of the fermentation supernatant to neutral, and then measure the antibacterial activity of the fermentation supernatant.

(2) Eliminate the influence of hydrogen peroxide: dissolve catalase in phosphate buffer solution with pH 7.0 to make mother liquor, add it to the fermentation supernatant to make the final concentration of catalase 5.0mg/mL, and put it in a water bath at 37°C Take it out after 2 hours, and detect the antibacterial activity of the fermentation supernatant after catalase treatment.

(3) Determination of protein antibacterial substances: proteinase K was made into mother liquor with sterile MilliQ water, added to the supernatant of cell-free fermentation, so that the final concentration of proteinase K was 1.0mg/mL, mixed well and then in a water bath at 37°C Take it out after 2 hours, and detect the antibacterial activity of the fermented liquid after treatment.

After the treatment of (1) and (2), the antibacterial activity of the fermentation supernatant did not disappear, but after the treatment of (3), the antibacterial activity of the fermentation supernatant was lost. Preliminarily, it can be determined that the antibacterial substances in the fermentation supernatant are bacteriocins.

2. Antibacterial effect of bacteriocin

The fermentation supernatant has a strong antibacterial effect on Listeria (L.Monocytogenes), and the antibacterial diameter can reach 16.11±0.11mm.

3. Research on the biological characteristics of bacteriocin

(1) Sensitivity of bacteriocins to temperature:

Take 8 equal amounts of 1mL fermentation supernatant, keep them at 60°C, 80°C, 100°C and 121°C for 10min and 30min respectively, and do the antibacterial experiment after cooling with ice. The results are shown in Table 1: Strong heat resistance, after being treated at 121°C for 30 minutes, it still maintains antibacterial activity.

The influence of table 1 temperature on the antibacterial activity of bacteriocin

(2) Sensitivity of bacteriocins to pH: Take 8 parts of the same amount of fermentation supernatant, adjust the pH value to 2-10 with 3mol/L HCl or NaOH, incubate at 37°C for 2 hours, and do the antibacterial test, the results are shown in Table 2 Shown: bacteriocins are active under the condition of pH 2.5-6.5, and the activity weakens with the increase of pH value, and when the pH is above 7.5, the bacteriocins lose their antibacterial activity.

The influence of table 2pH on the antibacterial activity of bacteriocin

(3) Sensitivity of bacteriocins to enzymes: Take an equal amount of fermentation supernatant and adjust the pH to the following optimum pH values for each enzyme with HCl and NaOH. Add pepsin, trypsin, proteinase K, α-chymotrypsin, papain and α-amylase at a final concentration of 1 mg/mL, and incubate at 37°C for 2 hours. The pH was adjusted back to the optimum pH value of the bacteriocin, and the antibacterial experiment was carried out. The results are shown in Table 3: pepsin, proteinase K, and α-amylase can partially inactivate the bacteriocin.

The impact of table 3 protease on bacteriocin antibacterial activity

Example 4 Bacteriocin and the application of Enterococcus faecalis fermentation broth containing bacteriocin in fresh cheese

The production process of fresh cheese is shown in Figure 2.

After milk pasteurization, add fermentation agent while adding indicator bacterium Listeria (L.Monocytogenes) (1%), the Enterococcus faecalis fermented liquid (2%) containing bacteriocin prepared in Example 2 and bacteriocin ( 2%), get rid of the original pathogenic bacteria in the milk, use the sample that only adds the indicator bacteria as a control, and count the Listeria (L. (L.Monocytogenes) inhibition. The results show that: within 5 days of storage, the addition of bacteriocin and the Enterococcus faecalis fermentation broth containing bacteriocin can effectively inhibit the growth of Listeria in fresh cheese, and the results are shown in Figure 1.

Claims (10)

1. A strain of Enterococcus faecalis with high bacteriocin production, named KLDS6001, is preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms, and its strain preservation number is: CGMCC No.9966. 2. A method for producing Enterococcus faecalis bacteriocin, characterized in that it obtains by fermenting Enterococcus faecalis (Enterococcus faecalis) described in claim 1. 3. The method according to claim 2, characterized in that it comprises the following steps: (1) Preparation of Enterococcus faecalis fermentation broth Inoculate the Enterococcus faecalis (Enterococcus faecalis) described in claim 1 in the MRS medium, obtain the Enterococcus faecalis fermentation liquid after cultivating for 12-16h at 37°C; (2) Preliminary purification of Enterococcus faecalis bacteriocin Centrifuge the fermented liquid obtained in step (1), absorb the supernatant, add ammonium sulfate to the supernatant for salting out, centrifuge, collect the precipitate, and redissolve it in a sodium acetate buffer solution to obtain the bacteriocin crude extract; (3) Secondary purification of Enterococcus faecalis bacteriocin The crude extract of the bacteriocin is further separated and purified by a Sephadex G-25 gel chromatography column to obtain the purified bacteriocin. 4. The method according to claim 2, characterized in that: step (1) is used to cultivate the MRS medium composition of Enterococcus faecalis (Enterococcus faecalis) as follows: peptone 10.0g, beef extract 10.0g, yeast powder 5g, glucose 20.0g, Tween 801.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, diamine citrate 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g and distilled water 1000mL. 5. The method according to claim 2, characterized in that: the cultivation temperature in step (1) is 37°C, and the cultivation time is 14h. 6. The method according to claim 2, characterized in that: in the step (2), preliminary purification is carried out according to the following method: the fermented liquid obtained by the step (1) is centrifuged, and the supernatant is drawn, and the supernatant is added to the supernatant. Add ammonium sulfate with a saturation of 70% to the solution for salting out, centrifuge at 10000×g for 30 min at 4°C, collect the precipitate, and redissolve it in 0.02mol/L sodium acetate buffer solution with pH 6.0 to obtain the crude bacteriocin Extract. 7. The method according to claim 2, characterized in that: adopt Sephadex G-25 gel chromatography column in step (3) to carry out separation and purification, and the chromatographic conditions used are: use 50% (w/w) Sodium chloride and 50% (w/w) sodium acetate buffer solution for elution, the flow rate is 1mL/min, the sample loading volume is 400μL, and the protein peak product with a retention time within 14-16min is collected as the purified bacteriocin. 8. The Enterococcus faecalis bacteriocin prepared by the method according to any one of claims 2 to 7 or the Enterococcus faecalis fermentation broth containing the bacteriocin. 9. The application of the Enterococcus faecalis bacteriocin according to claim 8 or the Enterococcus faecalis fermentation broth containing the bacteriocin in inhibiting the growth of Listeria (L.Monocytogenes). 10. the application of the Enterococcus faecalis bacteriocin described in claim 8 or the Enterococcus faecalis fermentation broth containing the bacteriocin in the preparation of fresh cheese starter.