CN110205266B

CN110205266B - Lactobacillus paracasei capable of producing bacteriocin and application thereof

Info

Publication number: CN110205266B
Application number: CN201910466468.2A
Authority: CN
Inventors: 邵丽; 臧雪梅; 马霞; 张业萃; 何艳; 蒋愫婧
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2022-06-10
Anticipated expiration: 2039-05-31
Also published as: CN110205266A

Abstract

The invention provides a bacteriocin-producing lactobacillus paracasei and application thereof. The Lactobacillus paracasei (Lactobacillus paracasei) S20 strain collection number related by the invention is as follows: CGMCC NO.17120, which is separated from traditional fermented food "milk lump" in Tibet. And extracting a crude bacteriocin extract from the MRS fermentation supernatant of the strain by adopting ammonium sulfate fractional precipitation. Experiments prove that the bacteriocin crude extract produced by the strain has strong inhibiting effect on staphylococcus aureus S.aureus and escherichia coli E.coli, and the minimum inhibitory concentrations measured by a double dilution method are 312 and 625 mu g/mL respectively. The bacteriocin produced by the strain can be hydrolyzed by protease such as neutral protease and trypsin, and can not be accumulated in vivo. The lactobacillus paracasei and the bacteriocin thereof screened by the invention have wide application prospects in fermented foods.

Description

Lactobacillus paracasei capable of producing bacteriocin and application thereof

Technical Field

The invention relates to the technical field of food microorganisms, in particular to a lactobacillus paracasei strain for producing bacteriocin and application thereof.

Background

Bacteriocins (Bacteriocins) are a class of small peptides or proteins secreted extracellularly by the ribosome of certain bacteria to inhibit or kill closely related microorganisms. Bacteriocins are widely available and can be produced by both gram-positive and gram-negative bacteria, where the bacteriocins produced by lactic acid bacteria are known as lactobacilli. Lactic acid bacteria are well-known safe microorganisms in food, and bacteriocins produced by the lactic acid bacteria have the advantages of safety, no toxicity, easiness in degradation by protease, no accumulation in the body and the like, so that the lactic acid bacteria are widely concerned.

Since the british food preservative committee and the world health organization in 1969 combined with the expert committee of food additives confirmed that Nisin (Nisin) was a food preservative, Nisin was sequentially accepted by many countries due to its gene production stability, good bacteriostatic effect, and safe eating, as the first Nisin to be applied to foods. Hundreds of lactobacillin have been discovered so far, but few lactobacillin are actually used for commercialization, and one of the main reasons is that the yield of the lactobacillin is low, the separation and purification are complicated, the cost is too high, and the industrial production is difficult to realize.

With the improvement of living standard of people, the requirements of food and cosmetic additives tend to be natural, healthy, safe, nutritional and multifunctional, and the development of the lactein as a natural preservative has wide market prospect. However, the low yield of bacteriocin from lactic acid bacteria is one of the bottlenecks limiting the mass production of the bacteriocin from lactic acid bacteria, and the current ways for improving the yield of the bacteriocin from lactic acid bacteria are as follows: screening high-yield strains, regulating fermentation conditions, modifying strains through genetic engineering, inducing regulation, stress stimulation response and the like. Currently, the optimization of fermentation conditions is still the most common method for improving the bacteriocin of the lactic acid bacteria, but has the defects of time consumption, complex operation, large workload, small increase amplitude of yield, multiple uncertain factors and the like; constructing a genetic engineering strain, wherein the expression product has the problems of low activity and safety; induction regulation and stress stimulation response are regulation on transcription level, and are still at the initial stage at present, and further researches on induction mechanisms and regulation methods thereof are needed (octopamine, ruilingya, Yihuaxi, and the like. high-efficiency expression method of lactobacillus bacteriocin research [ J ]. brewing in China, 2014,33(7): 29-33.). Therefore, screening of a high-producing strain of lactein is still one of effective ways to improve lactein.

China is wide in territory and rich in lactobacillus resources, the traditional fermented foods (yoghourt, pickled vegetables, milk fans and the like) have a history of thousands of years in preparation and eating, and some lactobacillus with excellent fermentation characteristics in the traditional fermented foods are reserved after thousands of years of natural domestication. Meanwhile, the natural environments and climates of different regions, and the differences of raw materials, processes and the like adopted by traditional fermented foods prepared in different regions of China make the biological diversity and the gene diversity of the abundant lactobacillus resources in China unavailable for any other countries. Therefore, the method is an effective method for obtaining the novel lactobacilli by digging lactobacillus resources in traditional fermented food in China and screening high-efficiency broad-spectrum lactobacilli producing bacteria from the lactobacillus resources.

Disclosure of Invention

The invention aims to provide a bacteriocin-producing lactobacillus paracasei and application thereof.

The invention screens and provides a strain of lactobacillus paracasei for producing bacteriocin from Chinese traditional fermented food, provides a new strain resource for the development of novel bacteriocin, and lays a foundation for further researching the property and structure of the bacteriocin produced by the strain and developing novel, efficient and broad-spectrum bacteriocin.

The technical scheme of the invention is as follows:

the invention provides a bacteriocin-producing Lactobacillus paracasei strain which is classified and named as Lactobacillus paracasei (Lactobacillus paracasei) S20, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is addressed to the institute of microbiology, Zhongkoyage institute of Zhongkoyami No.1 North Chen West Lu of the Korean district in Beijing, and the strain preservation numbers are as follows: CGMCC NO.17120, with the preservation time of 1 month and 7 days in 2019.

The lactobacillus paracasei S20 strain provided by the invention is separated from a Tibetan traditional fermented food, namely a milk lump, is of a facultative anaerobic type, and has a round colony, a neat edge and a milky white color on an MRS solid plate, and the surface is moist, smooth, raised and easy to pick up; gram staining is positive, and the thallus is rod-shaped; the strain was identified as lactobacillus paracasei (lactobacillus. paracasei) by 16S rDNA sequence analysis. In MRS liquid culture medium, 14h reaches logarithmic phase, and 24h reaches stationary phase.

The lactobacillus paracasei S20 strain provided by the invention is a strain with good bacteriostatic effect screened by adopting a double-layer agar diffusion method from the centesian strain lactobacillus preserved in a laboratory and taking escherichia coli and staphylococcus aureus as indicator bacteria to eliminate the interference of organic acid and hydrogen peroxide.

The invention also provides a bacteriocin, which is characterized in that the bacteriocin is prepared by fermenting the lactobacillus paracasei.

The invention also provides a preparation method of the bacteriocin, which is characterized by comprising the following steps: culturing the bacteriocin-producing lactobacillus paracasei in an MRS liquid culture medium at 28-37 ℃ for 28-48 h, centrifuging, carrying out fractional precipitation on the obtained fermentation supernatant through ammonium sulfate, collecting precipitation components with 20-80% of saturation degree, dialyzing, and freeze-drying to obtain the bacteriocin-producing lactobacillus paracasei.

The crude extract of the bacteriocin produced by the lactobacillus paracasei S20 provided by the invention is further determined to be the bacteriocin by analyzing the protein content, the ultraviolet spectrum scanning analysis and the protease sensitivity, and the purification is more than 85%.

The bacteriocin crude extract produced by the lactobacillus paracasei S20 provided by the invention has strong inhibiting effect on staphylococcus aureus S.aureus and escherichia coli E.coli, and the minimum inhibitory concentrations measured by a double dilution method are 312 and 625 mu g/mL respectively.

The lactobacillus paracasei S20 provided by the invention is derived from traditional fermented food, belongs to Generally Recognized As Safe (GRAS) strains, and can be used in lactobacillus food. Therefore, the invention also relates to the application of the lactobacillus paracasei S20 in fermented food. Wherein the food product containing lactobacillus paracasei S20 is a fermented dairy product.

The invention also provides application of the lactobacillus paracasei in preparing fermented food.

The invention also provides a fermented food which is characterized in that the raw material of the fermented food contains the lactobacillus paracasei.

Preferably, the fermented food is a fermented dairy product.

The invention also provides application of the bacteriocin in preparation of a bacterial inhibitor, a food additive or a biological preservative.

A bacteria inhibitor, food additive or biological preservative, characterized by containing the above bacteriocin.

In recent years, there are many reports on bacteriocin production by lactic acid bacteria, and the research contents include screening of strains, physicochemical properties, structural analysis, bacteriostatic spectrum, application development and the like of novel bacteriocins. It can be seen that different bacterial strains (such as lactobacillus paracasei) produce bacteriocins with different amino acid compositions, peptide chain structures, molecular weights and physicochemical properties, and the produced bacteriocins have different bacteriostasis (bacteriostasis spectra) and different application ranges. These differences are caused by differences in the bacteriocin-producing strains, depending on the strain itself and the growth environment. Therefore, the method is an effective way for obtaining the novel, high-efficiency and broad-spectrum bacteriocin by screening and obtaining the bacterial strains for producing the bacteriocin from the separation samples of different sources, and lays a foundation for the development of the novel bacteriocin.

Therefore, the lactobacillus paracasei separated from the traditional fermented food (the milk lumps in Tibet) provides a new strain resource for the development of novel bacteriocin, lays a solid foundation for further researching the property and the structure of the produced bacteriocin, developing novel, efficient and broad-spectrum bacteriocin and lays a theoretical foundation for the industrialization of the lactobacillus bacteriocin. Experiments prove that the bacteriocin crude extract produced by the strain has strong inhibiting effect on staphylococcus aureus S.aureus and escherichia coli E.coli, and the minimum inhibitory concentrations measured by a double dilution method are 312 and 625 mu g/mL respectively. The bacteriocin produced by the strain can be hydrolyzed by protease such as neutral protease and trypsin, and can not be accumulated in vivo. The lactobacillus paracasei and the bacteriocin thereof screened by the invention have wide application prospects in fermented foods.

Preservation information

The Lactobacillus paracasei (Lactobacillus paracasei) S20 provided by the invention is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the address: the microorganism research institute of Zhongkou institute of Xilu No.1 Hospital, Beijing, Chaoyang, with the accession number: CGMCC No.17120, with preservation time of 2019, 1 month and 7 days.

Description of the drawings:

fig. 1 is a graph showing the bacteriostatic effect of fermentation liquor of lactobacillus paracasei S20 (wherein 1, 2, and 3 sequentially represent MRS fermentation supernatant, acid-inhibited fermentation supernatant, and hydrogen peroxide-inhibited fermentation supernatant of S20, with S. aureus as indicator bacteria);

FIG. 2 colony morphology of Lactobacillus paracasei S20 (MRS plate);

FIG. 3 shows the form (. times.1000) of Lactobacillus paracasei S20;

FIG. 4 shows that the ammonium sulfate of Lactobacillus paracasei S20 is graded to salt out the bacteriostatic activity of each section (0%, 40%, 53%, 65%, 80% salting-out saturation time zone, S.aureus is used as indicator bacteria);

FIG. 5 is a UV scanning scan of crude bacteriocin produced by Lactobacillus paracasei S20;

FIG. 6 shows the proteolytic activity of S20 crude and crude rhzomorph (1, 2, 3 represent, in order, aqueous solution of S20, hydrolyzed solution of S20+ neutral protease, hydrolyzed solution of S20+ trypsin, and E.coli as indicator bacteria);

FIG. 7 growth curve of Lactobacillus paracasei S20.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

EXAMPLE 1 screening and identification of bacteriocin-producing lactic acid bacteria

(1) Activation of bacterial strains

(a) Activation of bacteriocin-producing lactic acid bacteria: in the early stage, hundreds of lactobacillus strains are separated from traditional fermented foods collected from various places, such as samples of farmer self-made yoghourt, milk tofu, pickles, milk pimples, milk fans, sweet fermented grains and the like, by adopting MRS and M17 culture media, and after streaking and purifying, freeze-drying pipes are stored in a refrigerator.

Streaking an MRS plate (purchased from Haibo organisms) of the lactobacillus strain, placing the MRS plate at 37 ℃ for anaerobic culture for 48h, picking a single colony of the plate, transferring an MRS liquid culture medium to activate for 24h at 37 ℃, and repeating the activation step for 2 times for later use.

(b) Activating indicator bacteria: escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) are used as indicator bacteria, the E.coli and S.aureus strains stored in a glycerol tube are respectively marked on LB plates (purchased from Haibo organisms), activated for 16-18h at 37 ℃, and then the activation step is repeated for one time for standby.

(c) Preparing an indicator bacterium suspension:

choose a loop of single colony on LB plates of E.coli and S.aureus and dissolve in an EP tube containing 1mL of sterile water, indicating the concentration of bacteria to be controlled at 10⁶CFU/mL, spare.

(2) Screening of bacteriocin-producing lactic acid bacteria

(a) The preparation of the fermentation supernatant comprises respectively inoculating lactobacillus strains in MRS liquid culture medium (purchased from Haibobo), standing at 37 deg.C for 28-48 h to obtain fermentation liquid, and centrifuging at 4 deg.C and 8000r/min for 10min to obtain the fermentation supernatant.

(b) Experiment for inhibiting bacteria

The bacteriocin-producing strains were screened by the classical agar diffusion method. A first layer of sterilized 1.2% LB agar medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 12g/L, water 1000mL) was poured into a sterile plate, and after the agar was cooled, 1mL of an indicator suspension containing about one ring of single colony was added (the concentration of indicator was controlled at 10%⁶CFU/mL), added to 100mL of 0.8% LB agar medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 8g/L, water 1000mL) at 50 ℃, mixed well and poured into a plate as a second layer medium. After the culture medium solidified for 1h, the Oxford cup was taken out with a sterile forceps and gently placed on the agar surface (taking care not to make the Oxford cup sink into the second layer of agar), 0.2mL of the solution to be measured was added, the culture was carried out at 37 ℃ for 12-18 h, multiple repetitions were set, MRS liquid culture medium was used as a control, and the diameter of the zone of inhibition was measured with a vernier caliper.

(c) Eliminating inhibition of acids

Adjusting the pH of the fermentation supernatant to 6.0 by using 2mol/L NaOH solution to perform a bacteriostasis test; the initial pH was recorded before adjusting the pH, and lactic acid and acetic acid at the same pH were used as controls to exclude interference from organic acids.

(d) Inhibition by scavenging hydrogen peroxide

And (3) preserving the temperature of the fermentation supernatant at 80 ℃ for 10min to remove the interference of hydrogen peroxide, and detecting the antibacterial activity by adopting an agar diffusion method.

For hundreds of strains of lactic acid bacteria preserved in a laboratory, a double-layer agar oxford cup method is adopted, and E. The result shows that nearly 60 plants have obvious bacteriostatic zones and show stronger bacteriostatic effects. Because the metabolism of lactic acid bacteria can produce a plurality of substances with bacteriostatic effects, such as organic acid, hydrogen peroxide, diacetyl, bacteriocin and the like, the growth of other bacteria can be inhibited. Therefore, the selection of bacteriocin-producing strains requires exclusion of interference from these factors, where the effects of organic acids and hydrogen peroxide are greater. Then 60 strains of bacteria are subjected to acid inhibition and hydrogen peroxide inhibition interference elimination experiments.

The experimental result shows that 9 strains of the 60 strains of test strains still have strong bacteriostatic effects on E.coli and S.aureus (table 1), which indicates that bacteriocins may exist in the fermentation broth. The bacterial strain S20 shows good bacteriostatic activity by comprehensively eliminating bacteriostatic activity after acid inhibition and hydrogen peroxide inhibition, the growth state of the bacterial strain and the culture time (the result is shown in figure 1).

TABLE 1 bacterial strain rescreening table

(3) Identification of bacteriocin-producing strains

(a) And (3) observing the morphology of bacterial colonies: MRS solid plate is streaked by S20, anaerobic culture is carried out for 48h at 37 ℃, the colony is round, the edge is neat, the colony is milky white, the surface is wet, smooth and raised, and the colony is easy to pick up (figure 2); gram staining positive; the bacterial cells were rod-shaped (FIG. 3).

(b)16S rDNA sequence analysis: inoculating the S20 strain into MRS liquid culture medium, culturing to logarithmic phase, and centrifuging to collect thallus. Extracted with a genomic DNA extraction kit (TIAN GEN Co.). Two synthetic universal primers (16s 27F: GAGAGTTTGATCCTGGCTCAG; 16s 1492R: CGGCTACCTTGTTACGACTT) were used for PCR amplification, and the PCR product was recovered using a gel cutting recovery kit (BioFlux), purified and sequenced by Shanghai Producer sequencing company. The 16S rDNA nucleotide sequence of the obtained strain S20 is 1519bp (SEQ ID NO: 1), and Blast analysis is carried out on the GenBank NCBI website. The strain S20 with the highest homology is Lactobacillus paracasei KL1(GenBank accession number: CP013921.1), and the homology is 100%. Species having a G + C (mol%) of the DNA of 10 to 12% or less and a sequence homology of 16S rDNA of 95% or more as described by Goodfellow and O' Donnell are classified as a genus, and Embley and Stackelbungdt consider that a species having a sequence homology of 16S rDNA of 97% or more is a species. From this it can be concluded that: strain S20 and lactobacillus paraasei KL1 belong to the same species. Strain S20 was identified as Lactobacillus paracasei.

Through colony, thallus morphology observation and 16S rDNA sequence analysis, the S20 strain is identified as lactobacillus paracasei (Lactobacillus. paracasei), which is preserved in the China general microbiological culture Collection center in 2019, 1 month and 7 days, and the preservation number is CGMCC No. 17120.

EXAMPLE 2 preparation of crude bacteriocin extract produced by Strain S20

1) Fractional precipitation for determining salting-out saturation

Inoculating lactobacillus paracasei S20 in MRS liquid culture medium, standing and culturing at 37 ℃ for 28-48 h to obtain fermentation liquor, and centrifuging at 4 ℃ and 8000r/min for 10min to obtain fermentation supernatant.

Fractional precipitation with ammonium sulfate was used to prepare the crude bacteriocin extract. Firstly, determining salting-out saturation of fractional precipitation, grinding ammonium sulfate into powder, slowly stirring and adding into fermentation supernatant, when turbidity appears and the fermentation supernatant is not clear within 2min, calculating the salting-out saturation according to a salting-out table, standing overnight in a refrigerator at 4 ℃, centrifuging (4 ℃, 8000r/min, 20min), and respectively collecting supernatant and precipitate. And (3) taking the saturation as the initial salting-out degree of the supernatant, continuing salting-out, when the secondary turbidity occurs, operating by the same method and recording the data, after salting-out is carried out to 100%, redissolving several gradient salting-out precipitates by water, carrying out bacteriostasis test, selecting the salting-out saturation with the best bacteriostasis effect, and determining the final saturation of the crude bacteriocin extract.

Comparing with the analysis of ammonium sulfate salting-out saturation table, S20 precipitates at 20%, 40%, 53%, 65% and 80% saturation respectively, meanwhile, precipitates centrifugally collected by fermentation liquor at each saturation are redissolved by equal amount of sterile water and then are subjected to bacteriostasis test by a double-layer agar Oxford cup method, and E.coli and S.aureus are taken as indicator bacteria, and three experiments are carried out in parallel, and the results are shown in Table 2.

Experimental results show that when the salting-out saturation reaches 80%, the component antibacterial effect is the best, antibacterial substances extracted when the salting-out saturation of the MRS fermentation supernatant of S20 is 65% -80% are preliminarily determined to have strong antibacterial effect, and salting-out components with the saturation of 65% -80% are selected in subsequent experiments to prepare the bacteriocin crude extract.

TABLE 2 results of bacteriostatic activity of each fraction precipitated by ammonium sulfate

2) Crude bacteriocin extract

Collecting a crude extract corresponding to 65-80% saturation, redissolving in water, putting into a 3500Da dialysis bag for desalination, dialyzing with deionized water overnight, replacing the dialysate for 3 times, collecting the dialysate, freeze-drying to obtain a powdery crude bacteriocin product, culturing in an MRS culture medium by weighing and calculating S20, and obtaining about 0.8 +/-0.14 g of the crude bacteriocin product per liter of the culture medium.

3) Determination of the bacteriostatic Activity of bacteriocin crude extracts

And (3) determining the bacteriostatic activity of the crude extract by taking E.coli and S.aureus as indicator bacteria and taking the original fermentation supernatant as a reference. The crude lyophilized bacteriocin was reconstituted in sterile distilled water to a concentration of 12mg/mL and the zone of inhibition was measured according to the previous method, the results are shown in Table 3. The result shows that the crude bacteriocin product after separation and purification has bacteriostatic activity, and compared with the original supernatant, the bacteriostatic activity is obviously enhanced. Indicating that the purified sample is likely to be the desired bacteriocin and further experiments are required to verify.

TABLE 3 comparison of the bacteriostatic effect of bacteriocins with the stock solutions

Example 3 determination of the Properties of crude bacteriocin extract produced by S20

(1) Crude extract protein content determination

The protein content of the crude bacteriocin extract prepared in example 2 was measured by the coomassie brilliant blue method, a bovine serum albumin solution at a concentration of 1mg/mL was used as a standard protein solution, the standard protein content was used as the abscissa, and the absorbance at 595nm was used as the ordinate, and a standard curve was plotted to obtain a linear regression equation of y ═ 1.021x +0.017(R ═ 1.021x + (R) (²0.999). The crude extract was reconstituted with water to a concentration of 2.5mg/mL and diluted appropriately to determine protein content. The calculation shows that the protein content in the crude bacteriocin product is 2.12mg/2.5mg, the protein content reaches 85 percent, which indicates that the crude bacteriocin product is mainly protein substances, and also indicates that the bacteriocin obtained by the fractional ammonium sulfate precipitation method has higher purity.

(2) Ultraviolet spectral analysis of bacteriostatic substances

0.625mg/mL of the bacteriocin crude extract solution prepared in example 2 was prepared from sterile distilled water, and subjected to UV scanning analysis in a wavelength range of 190-600 nm. The result is shown in fig. 5, the bacteriostatic substance produced by S20 has a strong absorption peak near 220nm, which is the characteristic absorption characteristic of peptide bond in far ultraviolet region, and is consistent with the literature report, and the bacteriostatic substance produced is further proved to be a polypeptide substance.

(3) Protease sensitivity test of antibacterial substance

A freeze-dried crude bacteriocin extract sample prepared in the embodiment 2 is re-dissolved in sterile distilled water (12mg/mL) to be used as a bacteriocin solution, neutral protease and trypsin are respectively prepared into 5mg/mL solutions, 0.1mL of each solution is put into a centrifuge tube, 0.4mL of bacteriocin solution (12mg/mL) is respectively added, the pH of a protease mixed solution is adjusted to 6-7 by using 0.1mol/L hydrochloric acid, 0.4mL of bacteriocin solution is simultaneously added into 0.1mL of distilled water to be used as a control, and the mixture is subjected to enzymolysis at 37 ℃ for 4 hours to perform a bacteriostatic experiment. As shown in Table 4, the results show that the bacteriostatic activity disappeared after the hydrolysis with neutral protease and trypsin, while the bacteriocin aqueous solution had bacteriostatic activity, and it was presumed that the bacteriostatic active substance was a protein (FIG. 6), which is consistent with the report that bacteriocin is sensitive to protease and can be decomposed by protease, and it was confirmed that the extracted crude extract was bacteriocin.

TABLE 4 results of the antimicrobial protease sensitivity experiments

EXAMPLE 4 double dilution method for determining bacteriocin minimum inhibitory concentration

The bacteriocin crude extract prepared in the embodiment 2 is dissolved in deionized water, diluted by a double dilution method, and prepared into 10.0, 5.0, 2.5, 1.25, 0.625, 0.312 and 0.156mg/mL solutions respectively, and the bacteriostatic activity of the bacteriocin crude extract is measured by taking sterile water as a reference and taking E.coli and S.aureus as indicator bacteria. As is clear from Table 5, the bacteriostatic activity against the indicator bacteria decreased as the bacteriocin concentration decreased. The minimum inhibitory concentration to golden yellow wine bacteria is 0.312mg/mL, and the minimum inhibitory concentration to colibacillus is 0.625 mg/mL.

TABLE 5 minimum inhibitory concentration (mg/mL) of bacteriocin produced by S20

Note: no bacteria-inhibiting ring

EXAMPLE 5 plotting of growth curves of the strains

Inoculating activated Lactobacillus paracasei S20 into MRS liquid culture medium at 1% (v/v), culturing at 37 deg.C for 32 hr, sampling every 2 hr, and determining OD of the culture solution₆₀₀And a pH value. By OD₆₀₀And the pH value is plotted against the cultivation time, and the strain S2 is plotted0 growth curve in MRS. The results are shown in fig. 7, strain S20 grows faster in MRS liquid medium, 14h enters logarithmic phase, and 24h enters stationary phase. With the prolonging of the culture time, the bacterial strain grows to produce acid, the pH value is continuously reduced, and the pH value reduction trend is gradually slowed after the stable period. When the culture is finished, the pH value of the culture solution is 3.89, and the concentration of viable bacteria in the culture solution can reach more than 2.5.

SEQUENCE LISTING

<110> Shanghai applied technology university

<120> lactobacillus paracasei for producing bacteriocin and application thereof

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Claims

1. The lactobacillus paracasei for producing bacteriocin is characterized in that the strain preservation number is as follows: CGMCC NO. 17120.

2. A bacteriocin produced by fermentation of the Lactobacillus paracasei strain of claim 1.

3. The method of producing a bacteriocin according to claim 2, which comprises: the bacteriocin-producing lactobacillus paracasei of claim 1 is cultured in MRS liquid medium at 28-37 ℃ for 28-48 h, centrifuged, the obtained fermentation supernatant is subjected to fractional precipitation by ammonium sulfate, and the precipitated components with 20-80% saturation degree are collected, dialyzed and freeze-dried to obtain the bacteriocin-producing lactobacillus paracasei.

4. Use of lactobacillus paracasei according to claim 1 for the preparation of fermented food products.

5. A fermented food characterized in that its raw material contains the Lactobacillus paracasei according to claim 1.

6. The fermented food according to claim 5, wherein the fermented food is a fermented dairy product.

7. Use of the bacteriocin of claim 2 for the preparation of a bacterial inhibitor, a food additive or a biological preservative.

8. A bacteriostatic, food additive or biological preservative comprising the bacteriocin of claim 2.