CN110734874A

CN110734874A - lactic acid bacteria zy-B with food preservation and fresh-keeping function and application thereof

Info

Publication number: CN110734874A
Application number: CN201910985561.4A
Authority: CN
Inventors: 刘颖; 张瀛; 徐春厚; 孙力军; 王雅玲; 房志家; 邓旗
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2019-10-17
Filing date: 2019-10-17
Publication date: 2020-01-31

Abstract

The invention belongs to the technical field of food preservation, and particularly relates to lactic acid bacteria zy-B with food preservation and preservation effects and application thereof, wherein lactic acid bacteria are separated from the intestinal tracts of noble sea fans collected from the east China wind market of Zhanjiang, 16S rRNA molecular biology identification shows that the lactic acid bacteria are highly similar to Pediococcus pentosaceus DSM 20336T and have the similarity of 99.53 percent, PCR identification, acidic substance interference elimination experiments, hydrogen peroxide interference elimination experiments and protease sensitivity experiments show that the bacterial strain is a type IIa bacteriocin producing bacterium, and bacteriostatic activity determination experiments show that the bacterium has bacteriostatic activity on Listeria monocytogenes, staphylococcus aureus, bacillus subtilis, escherichia coli and Vibrio parahaemolyticus, and the bacterium is lactic acid bacteria with strong bacteriostatic activity and antibacterial spectrum and can be applied to the field of food preservation.

Description

lactic acid bacteria zy-B with food preservation and fresh-keeping function and application thereof

Technical Field

The invention belongs to the technical field of food preservation, and particularly relates to lactic acid bacteria zy-B with food preservation and preservation effects and application thereof.

Background

In the process of processing and preserving food, the food is extremely easy to be polluted by microorganisms to cause spoilage, and the adoption of food preservatives to inhibit harmful microorganisms and delay the spoilage is which is an important technology of food preservation at present.

Lactic Acid Bacteria (LAB), a generic name for gram-positive bacteria capable of producing large amounts of lactic acid using carbohydrates, widely existing in nature and recognized as safe food-grade microorganisms, can produce a variety of metabolites with antibacterial activity during the metabolic process, and are mainly classified as (1) lactic acid bacteria can produce lactic acid, acetic acid, carbon dioxide, ethanol and other byproducts during the metabolic process, the production of lactic acid results in the reduction of the pH of the surrounding environment, and the growth of microorganisms is very pH-dependent, so that the growth of food spoilage or pathogenic bacteria that cannot tolerate a lower pH of the growing environment is inhibited, (2) the inhibition of hydrogen peroxide, wherein if oxygen exists in the growing environment of lactic acid bacteria, the lactic acid bacteria undergo aerobic fermentation, and the activity of flavoprotein oxidase therein causes the production of H₂O₂And the lactic acid bacteria do not contain catalase to cause H₂O₂The growth inhibitory effect on other microorganisms (e.g., Vibrio parahaemolyticus, Salmonella , etc.) (3) inhibitory effect of Lactobacilli, i.e., Lactobacillus bacteriocin is a peptide or precursor peptide having bacteriostatic activity of species synthesized by the ribosome of lactic acid bacteria during metabolism, wherein most of the Lactobacillus bacteriocin can endure high temperature,the lactobacillus species are different from the metabolic produced lactic acid bacteriocin species, and the bacterial inhibition spectra of different lactic acid bacteriocins are different, wherein the lactobacillus species producing the IIa type bacteriocin is type spectrum bacterial inhibition lactobacillus having strong inhibition effect on Listeria monocytogenes, and is which is the most promising natural food preservative.

Therefore, the discovery of lactic acid bacteria having a natural food preservative function is currently a focus of research.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides lactic acid bacteria zy-B with food preservation and fresh-keeping functions and application thereof, the bacterial strain is a type IIa bacteriocin producing strain, has the advantages of strong bacteriostatic activity and antimicrobial spectrum , and can be applied to the field of food preservation.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides lactic acid bacteria zy-B with food preservation and fresh-keeping effects, wherein the lactic acid bacteria zy-B is a type IIa bacteriocin producing bacterium, the lactic acid bacteria zy-B is preserved in eastern province microorganism strain preservation center in 8 and 19 months in 2019, and the preservation number is GDMCC No. 60743.

Preferably, the 16S rRNA of the lactobacillus zy-B has a nucleotide sequence shown as SEQ ID NO:1, and the 16S rRNA molecular biological identification shows that the lactobacillus (Pediococcus pentosaceus) is highly similar to lactobacillus pentosaceus DSM 20336T (JQBF01000022), and the similarity is 99.53%.

Preferably, the lactic acid bacteria zy-B are isolated from the intestine of the noble scallop.

, the above-mentioned Zy-B can be used for food preservation.

In order to screen potential lactic acid bacteria possibly applied to the field of food preservation, aiming at common spoilage bacteria causing food spoilage, Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli) and Vibrio parahaemolyticus (Vibrio parahaemolyticus) are selected as indicator bacteria. The antibacterial activity of the lactobacillus zy-B is measured by adopting a double-layer oxford cup method, and the potential lactobacillus with the natural food preservative function is screened according to the antibacterial activity, and the result shows that the lactobacillus zy-B has better antibacterial activity on the 5 indicator bacteria.

Through PCR identification, acidic substance interference elimination experiments, hydrogen peroxide interference elimination experiments and protease sensitivity experiments, the lactobacillus zy-B is type IIa bacteriocin producing bacteria.

The method comprises the following steps of extracting with ethyl acetate, purifying with a Sephadex LH-20 column chromatography system, purifying with an ion exchange chromatography and purifying with a semi-preparative reverse phase liquid chromatography to obtain bacteriocins PE-ZYB1 from lactic acid bacteria zy-B, and performing an antibacterial spectrum determination experiment on the bacteriocin PE-ZYB1, wherein the determination result shows that the bacteriocin PE-ZYB1 has an antibacterial spectrum of , has a strong inhibition effect on gram-positive bacteria such as Listeria monocytogenes and Staphylococcus aureus, has an inhibition effect on gram-negative bacteria such as vibrio parahaemolyticus, escherichia coli, enterobacter aerogenes, pseudomonas aeruginosa, proteus and hemolytic streptococcus, and has an inhibition effect on bacillus subtilis and bacillus cereus.

Preferably, the lactobacillus zy-B is fermented, supernatant is taken to prepare the bacteriostatic agent, and the lactobacillus zy-B is directly sprayed on the surface of solid food in the form of lactobacillus fermentation supernatant or is mixed in liquid food for inhibiting listeria monocytogenes, staphylococcus aureus, vibrio parahaemolyticus, escherichia coli, enterobacter aerogenes, pseudomonas aeruginosa, proteus vulgaris, streptococcus haemolyticus, bacillus subtilis and bacillus cereus in the food. The solid food comprises meat food (such as chicken feet, chicken wings, pig trotters, pigskin, pig ears, etc.) or vegetable food (such as bamboo shoots, radish, etc.), and the liquid food comprises beverage (such as fruit juice, etc.) and alcoholic beverage (such as beer, etc.).

More preferably, the lactobacillus zy-B is inoculated in an MRS liquid culture medium and cultured for 36-48h at 28 ℃ to obtain lactobacillus fermentation supernatant. The method specifically comprises the following steps: inoculating lactobacillus zy-B into MRS liquid culture medium, culturing in constant temperature shaking table at 28 deg.C and 150r/min, activating for 2 generations, inoculating into 1L MRS liquid culture medium according to 3% of inoculum size, culturing at 28 deg.C for 48h, collecting fermentation liquid, centrifuging at 12,000rpm and 4 deg.C for 10min, retaining fermentation supernatant, and filtering the fermentation supernatant with 0.22 μm filter membrane to obtain lactobacillus fermentation supernatant.

Compared with the prior art, the invention has the beneficial effects that:

the lactobacillus zy-B is characterized in that the lactobacillus zy-B is potential lactic acid bacteria with natural food preservative function, the lactobacillus zy-B is identified by PCR identification, acid substance interference elimination experiment, hydrogen peroxide interference elimination experiment and protease sensitivity experiment, the lactobacillus zy-B is IIa type bacteriocin producing bacteria, the four-step method of ethyl acetate extraction, Sephadex LH-20 system purification, ion exchange chromatography purification and semi-preparation type reversed phase liquid chromatography purification is adopted to separate and purify the lactobacillus zy-B to obtain listeria monocytogenes Listeriolysin PE-ZYB 5, the method is used for carrying out column chromatography purification on the listeria monocytogenes PE-ZYB, YB-389 PE, the bacterium ZYB-483, and the like, and the lactobacillus plantarum ZByByByByzb has strong bacteriostatic spectrum effect on pseudomonas aeruginosa, such as bacillus subtilis ZByByByByByByByByya, ZJ, the bacterium strain has strong bacteriostatic spectrum effect on Pseudomonas aeruginosa, the bacillus pyogenes, the bacillus subtilis ZYB-1, the bacillus pyogenes, the bacillus monocytogenesis also has strong bacteriostatic spectrum inhibitory effect on gram-lysobacter coli, the bacillus pyogenic bacteria, the bacteriostatic spectrum test can be applied to the determination, and the bacillus pyogenic bacteria such as bacillus subtilis Zymptococcus pyogenes.

Drawings

FIG. 1 shows the result of PCR amplification electrophoresis of specific primers identified by PCR;

FIG. 2 shows the inhibitory effect of crude bacteriocin extracts after fermentation of strain zy-B and ethyl acetate extraction on Listeria monocytogenes (in the figure, 1: supernatant of strain zy-B fermentation; 2: bacteriocin after ethyl acetate extraction; 3: ethyl acetate; CK: blank control);

FIG. 3 shows the inhibitory effect of 25-32 tube components on Listeria monocytogenes;

FIG. 4 elution profile of ion exchange chromatography purification of bacteriocins;

FIG. 5 is an elution profile of purified bacteriocin by semi-preparative reverse phase chromatography;

FIG. 6 is an RP-HPLC chromatogram identification of the purity of the H5 component;

FIG. 7 is a MS/MS plot of bacteriocin PE-ZYB 1.

Detailed Description

The present invention will be described in more detail with reference to the following , which is to be construed as an aid to understanding the present invention but not limiting the present invention.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Experimental materials: containing 0.3% CaCO₃The formula of the isolated culture medium and the MRS liquid culture medium is specifically referred to as follows: Delves-Broughton J.Nisin and its applications as a food preservative [ J.].International Journal ofDairy Technology,2010,43(3):73-76.

MightyAmp DNA polymerase, 2 XMightyAmp buffer, available from Bao bioengineering (Dalian) Ltd.

Tryptone soy yeast extract agar (TSA-YE medium), nutrient agar medium containing 3% sodium chloride, and agarose, purchased from Beijing land bridge technology, Inc.

Lactic acid, catalase, trypsin, pepsin, available from Shanghai Biotechnology Ltd; papain was purchased from Sigma, usa.

The indicator bacteria are commercially available and the details are shown in table 1:

table 1 indicates the species

EXAMPLE 1 isolation of the strains

Repeatedly washing noble sea fan clam (Mimachlamys nobilis) sample collected from the Zhanjiang Dongfeng market with sterile physiological saline, placing the sample on a super-clean workbench, taking out the intestinal tract of the sample through sterile operation, and cutting the sample into pieces with sterile scissors. Weighing 25g, transferring into 225mL sterile physiological saline homogenizing bag, repeatedly beating in homogenizer for 1-2min to thoroughly homogenize to obtain 10^-1The sample bacterial suspension of (4). Following the gradient dilution method, 10mL pipette is used^-1Sucking 1mL of the sample bacterial suspension, transferring the sample bacterial suspension into a test tube containing 9mL of sterile normal saline, repeatedly blowing and beating the sample bacterial suspension to fully mix the sample bacterial suspension and the sterile normal saline to obtain 10^-2Sample bacterial suspension is diluted into 10 in turn according to the same method^-3、10^-4、10^-5And (5) sample bacterial suspension for later use.

0.1mL of each sample solution was extracted from the sample solution after the gradient dilution and uniformly applied to 4 samples containing 0.3% CaCO₃The culture was carried out at 28 ℃ for 48 hours in 4 plates per dilution, and in 2 plates, layers of the same medium at a temperature of about 50 ℃ were poured over the coated upper layer and cultured at 28 ℃.

And (3) preliminarily determining according to the colony morphology in the calcium dissolving ring and the separation culture medium, and separating out 4 suspected lactobacillus strains from the sample.

Example 2 molecular biological identification of 16S rRNA of Strain

PCR amplification was carried out using 27F and 1492R as primers (synthesized by Shanghai Biotechnology Co., Ltd.), the nucleotide sequences of 27F and 1492R are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively, and the strains were subjected to colony PCR using MightyAmp DNA polymerase. The PCR reaction conditions are as follows: using a single colony of 24h of sterilized toothpick as a template, 2 XMightyAmpb buffer 15. mu.L, MightyAmp DNA polymerase 0.75. mu.L, 27F (10. mu. mol/. mu.L) 0.75. mu.L, 1492R (10. mu. mol/. mu.L) 0.75. mu.L, ddH₂O12.75. mu.L. Reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ 15s, extension at 68 ℃ for 1.5min, 40 cycles; storing at 10 deg.C for 10 min. The PCR product was sent to Biotechnology (Shanghai) Inc. for sequencing. The sequencing results were subjected to homology searches in the NCBI (GenBank) and ezBioCloud (ezTaxon) databases.

As shown in Table 2, these 4 strains were all belonging to lactic acid bacteria, and among them, strain zy-6 belongs to the genus Lactobacillus (Lactobacillus), strains zy-12 and zy-14 belong to the genus Enterococcus (Enterococcus), and strain zy-B belongs to the genus Pediococcus (Pediococcus).

TABLE 24 phylogenetic relationship between strains and their most phylogenetic canonical strains

Example 3 screening of strains by bacteriostatic Activity assay

In order to screen potential lactic acid bacteria which can be applied to the field of food preservation, aiming at common spoilage bacteria causing food spoilage, Listeria monocytogenes (Listeria monocytogenes), staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli) and Vibrio parahaemolyticus (Vibrio parahaemolyticus) are selected as indicator bacteria, and the indicator bacteria are shown in Table 1. And (3) performing antibacterial activity determination on the lactobacillus zy-B by adopting a double-layer oxford cup method.

Inoculating the 4 strains of lactic acid bacteria into an MRS liquid culture medium, placing the MRS liquid culture medium in a constant temperature shaking table for culture at 28 ℃ and 150r/min, activating for 2 generations, inoculating the activated MRS liquid culture medium into 1L according to the inoculation amount of 3%, culturing for 48h at 28 ℃, collecting fermentation liquor, centrifuging for 10min at 12,000rpm and 4 ℃, removing thalli, reserving fermentation supernatant, filtering the fermentation supernatant by using a 0.22 mu m filter membrane, and obtaining lactic acid bacteria fermentation supernatant for later use.

The specific operation method of the Oxford cup method refers to the antibacterial active lactic acid in the intestinal tracts of animals in the fields of Jojoba, Arvina, Zhao duck, et al, south China seaIsolation and diversity analysis of bacteria [ J]Food science, 2017(08):82-87. ". Listeriophytoenes is inoculated in a TSA-YE culture medium, Escherichia coli, Staphylococcus aureus and Bacillus subtilis are inoculated in a nutrient agar culture medium, Vibrio parahaemolyticus adopts a nutrient agar culture medium containing 3% of sodium chloride and is cultured at 37 ℃ for 24h, a thin layer of sterilized liquid containing only 2% of agar is added into a sterile culture dish, after the mixture is solidified, a sterilized Oxford cup is uniformly placed on solidified agar, and indicator bacteria cultured for 24h are adjusted to OD by using sterile physiological saline₆₀₀And (3) taking 2mL of the indicator bacterium suspension, adding the indicator bacterium suspension into 100mL of corresponding culture medium which is cooled to 50 ℃ for sterilization, shaking up, immediately pouring into a solidified culture dish only containing 2% agar, and slightly pulling up the Oxford cup by using a sterile forceps after solidification. And adding 200 mu L of lactobacillus fermentation supernatant into the hole of the Oxford cup, standing and diffusing at 4 ℃ for 4h, transferring to 37 ℃ for culturing for 24h, observing whether a bacteriostatic zone appears and measuring the diameter.

The results shown in Table 3 indicate that only the strain zy-B has antibacterial activity against 5 indicator bacteria simultaneously, and has the advantages of strong antibacterial activity and an antibacterial spectrum .

For this purpose, the strain zy-B was deposited.

The deposit information for strain zy-B is as follows:

preservation time: 8/19/2019;

the name of the preservation unit is eastern province microorganism strain preservation center;

the preservation number is: GDMCC No. 60743;

the address of the preservation unit is No. 59 building No. 5 building of No. 100 college of the Xieli Zhonglu city, ;

and (3) classification and naming: pediococcus pendosaceus zy-B.

TABLE 34 bacteriostatic action of lactic acid bacteria on different indicator bacteria

Note: the inner diameter of the oxford cup is 7.8mm, and the outer diameter is 8.0 mm; +++: the diameter of the bacteriostatic circle is more than 20 mm; ++: the diameter of the bacteriostatic zone: 14-20 mm; +: the diameter of the bacteriostatic zone: is less than 14 mm; -: has no antibacterial activity

Example 4 identification of whether Strain zy-B is a bacteriocin producing bacterium of class IIa

(1) And (3) PCR identification: colony PCR was performed by designing specific primers based on conserved sequences specific to class IIa bacteriocins, referring to the method of chenoting (preliminary study of PCR screening method for antibacterial peptides of class IIa of lactic acid bacteria [ D ]. harlbin industrial university, 2012).

The nucleotide sequences of the primer F and the primer R are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5 (synthesized by Shanghai Biotechnology Co., Ltd.), and the PCR reaction conditions are as follows: using a single colony cultured with a sterile toothpick for 24 hours as a template, 2 XMightyAmp buffer 5. mu.L, MightyAmp DNA polymerase 0.25. mu.L, primer F (10 pmol/. mu.L) 0.25. mu.L, primer R (10 pmol/. mu.L) 0.25. mu.L, ddH₂O5.25. mu.L. Reaction conditions are as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 53 ℃ for 15s, extension at 72 ℃ for 10s, and 35 cycles; storing at 10 deg.C for 10 min.

The PCR products were electrophoresed on 2% agarose Gel, and the electrophoresis results were visualized and photographed on a Gel Doc XR + Gel imaging system (BerleBIO-RAD, USA).

As shown in FIG. 1, amplified bands of about 150bp were bright and specific, and it was preliminarily determined that the strain zy-B was a group IIa bacteriocin-producing strain, and whether the antibacterial property was a bacteriocin or not was steps.

(2) Organic acid exclusion experiment: because the bacteriostatic effect of lactic acid bacteria is probably caused by organic acid and other substances generated by metabolism of lactic acid bacteria, in order to eliminate the interference of the organic acid, lactic acid is used for adjusting the pH value of a sterile MRS liquid culture medium (sterilized by high-pressure steam at 121 ℃ for 15min) to 3.0, 4.0, 4.5, 5.0, 6.0 and 7.0 respectively to carry out bacteriostatic experiments, and the maximum pH value without bacteriostatic effect on 5 indicator bacteria is selected as a control. And (3) regulating the supernatant of the lactobacillus fermentation liquor to be detected to the determined control pH value by using 1.0mol/L HCl and 1.0mol/L NaOH, and performing an acid discharge experiment by using a double-layer Oxford cup method. Observing and recording the strains with the inhibition zone after eliminating the acid action.

As shown in table 4, no inhibitory effect was observed for 5 indicator bacteria at pH 6.0, so pH 6.0 was selected as the control pH for acid exclusion.

Inoculating the strain zy-B into 50mL of MRS liquid culture medium, placing the strain zy-B in a constant temperature shaking table, culturing for 16h at 28 ℃ and 150r/min to obtain strain zy-B fermentation liquor, adjusting the pH value of the strain zy-B fermentation liquor to 6.0, using the fermentation liquor without pH adjustment as a reference, and performing an antibacterial experiment by using a double-layer Oxford cup method, wherein the result is shown in Table 5, when the pH value of the strain zy-B fermentation liquor is adjusted to 6.0, the strain zy-B still has antibacterial activity, which indicates that the inhibition effect of the strain zy-B on the indicator bacteria is not only caused by acid, but also can be the effect of other substances such as hydrogen peroxide and the like generated in the metabolism of lactic acid bacteria.

TABLE 4 inhibitory effect of different pH on indicator bacteria

TABLE 5 bacteriostatic effect after elimination of organic acids

(3) Exclusion experiments for hydrogen peroxide effect: absorbing 5mL of strain fermentation liquor which has the bacteriostatic effect on the indicator bacteria after eliminating the interference of the organic acid, adding 50mg of catalase until the catalase is completely dissolved, adjusting the pH to 7.0, placing the mixture in a water bath kettle at 37 ℃ for 2h, adjusting the pH to be the pH (6.0) when the interference effect of the organic acid is eliminated, and performing a bacteriostatic experiment by using a double-layer Oxford cup method by using the fermentation liquor which is not treated by the catalase and is subjected to the interference elimination of the acid as a control.

The lactobacillus can generate hydrogen peroxide through metabolism, hydrogen peroxide with a certain concentration has a sterilization effect, the bacterial strain after the interference of acidic substances is eliminated is subjected to a hydrogen peroxide enzymolysis test of steps to eliminate the interference, catalase is added into the fermentation supernatant of the bacterial strain zy-B to perform a water bath reaction, then the antibacterial effect of the fermentation liquid after the reaction is measured by using a double-layer oxford cup, and the antibacterial activity result is shown in table 6.

In conclusion, in addition to the presence of organic acids and hydrogen peroxide, other substances with bacteriostatic action are present in the fermentation supernatant of the strain zy-B.

TABLE 6 bacteriostatic effect after removal of Hydrogen peroxide

(3) Protease sensitivity assay: taking 3 parts of 2.0mL of strain fermentation liquor which still has bacteriostatic activity on the indicator bacteria after acid elimination and catalase treatment, respectively adding pepsin, papain and trypsin according to the standard that the pH values of the pepsin, the papain and the trypsin are 2.0, 6.0 and 7.4 respectively, enabling the final concentration of each protease to be 1.0mg/mL, carrying out water bath at 37 ℃ for 4 hours, then adjusting the original pH value (6.0), and determining the bacteriostatic activity before and after enzyme treatment.

The bacterial strain zy-B is still found to have bacteriostatic activity after eliminating the interference of acidic substances and the interference of hydrogen peroxide, and in order to finally determine whether the property of the bacteriostatic substances is protein or peptide, the zymohydrolysis test is carried out on the fermentation liquor of the bacterial strain zy-B which is subjected to the acid discharge and hydrogen peroxide discharge tests. In the test, after papain, trypsin and pepsin are respectively used for treating the strain fermentation supernatant, the change of the diameter of a bacteriostatic zone (taking Listeria monocytogenes as indicator bacteria) before and after treatment is measured. The results are shown in Table 7, and it is found that the activities of the supernatant of the fermentation broth of the strain zy-B are respectively maintained by 64.7% and 77.1% after the treatment of pepsin and papain; after trypsin treatment, the activity is only kept by 8.9%, and the bacteriostatic substances in the fermentation liquor of the strain zy-B are more sensitive to the treatment of the trypsin, so that the strain zy-B can be judged to be capable of generating bacteriostatic substances with protein or peptide properties.

The strain zy-B is a type IIa bacteriocin producing strain which can be obtained by combining PCR identification, acidic substance interference elimination experiments, hydrogen peroxide interference elimination experiments and protease sensitivity experiments.

TABLE 73 bacteriostatic effect after protease treatment

EXAMPLE 5 determination of the bacterial inhibition Profile of the Strain zy-B

The bacterial strains in the table 1 are taken as indicator bacteria, the bacterial strain zy-B is subjected to bacteriostatic spectrum determination by a double-layer Oxford cup method, and the diameter of a bacteriostatic circle is measured. Inoculating the strain zy-B into an MRS liquid culture medium, placing the strain zy-B into a constant temperature shaking table to be cultured at 28 ℃ and 150r/min, inoculating the strain into 1L of the MRS liquid culture medium according to the inoculation amount of 3 percent after 2 generations of activation, culturing for 48h at 28 ℃, collecting fermentation liquor, centrifuging for 10min at 12,000rpm and 4 ℃, removing thalli, reserving fermentation supernatant, filtering the fermentation supernatant by using a 0.22 mu m filter membrane, and obtaining lactic acid bacteria fermentation supernatant for later use.

The specific operation method of the Oxford cup method refers to the separation and diversity analysis of antibacterial active lactobacillus in intestinal tracts of animals in the areas of Jojoba, Arvina, Zhao duck Mei, et al south sea [ J ] food science, 2017(08):82-87 ]. Wherein Listeria monocytogenes is inoculated in a TSA-YE culture medium, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus cereus, Enterobacter aerogenes, Pseudomonas aeruginosa, Streptococcus thermophilus and Proteus Bacillus are inoculated in a nutrient agar culture medium, and Vibrio parahaemolyticus is inoculated in a nutrient agar culture medium containing 3% of sodium chloride and cultured for 24 hours at 37 ℃.

As can be seen from the determination results in Table 8, the strain zy-B not only has a strong inhibiting effect on gram-positive bacteria such as Listeria monocytogenes, Staphylococcus aureus and the like, but also has an inhibiting effect on gram-negative bacteria such as Vibrio parahaemolyticus, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus, Streptococcus hemolyticus, Bacillus subtilis and Bacillus cereus, and shows that the strain zy-B has an antibacterial spectrum wider than , and can be applied to the food industry as a potential natural antibacterial drug or biological preservative.

TABLE 8 determination of the bacterial inhibition spectra of the strain zy-B

Note: diameter of zone of inhibition (mm): 20, is used; ++: 14-20; +: < 14.

EXAMPLE 6 isolation and purification of bacteriocin PE-ZYB1

(1) Preparation of Zy-B fermentation supernatant of lactic acid bacteria and determination method of bacteriocin titer

Transferring lactobacillus zy-B stored on an inclined plane into an MRS liquid culture medium, culturing in a constant temperature shaking table at 28 ℃ and 150r/min, activating for 2 generations, inoculating into 1L of MRS liquid culture medium according to the inoculation amount of 3%, culturing at 28 ℃ for 48h, collecting fermentation liquor, centrifuging at 12,000rpm and 4 ℃ for 10min, removing thallus, retaining fermentation supernatant, filtering the fermentation supernatant by using a 0.22 mu m filter membrane, measuring the antibacterial activity of the fermentation supernatant by using listeria monocytogenes as indicator bacteria, and storing at 4 ℃ for later use. Wherein, the determination of the antibacterial activity adopts a double-layer agar oxford cup method, and the specific operation method of the oxford cup method refers to the separation and diversity analysis of antibacterial activity lactobacillus in animal intestines of the south sea area of Johnson winter, Arvina yew, Yamei, et al [ J ] food science, 2017(08):82-87 ].

The bacteriocin titer was determined by serial double dilution to quantitatively determine bacteriocin activity. The bacteriocin solution was serially diluted two-fold with sterile deionized water, and 100. mu.L of the dilution was added to the Oxford cup to determine the antibacterial activity (using Listeria monocytogenes as indicator). The calculation formula of the antibacterial potency (AU) is 2ⁿX 1000. mu.L/100. mu.L, where n is the dilution at which the zone of inhibition is evident (reference to specific procedures) "

A.C M,Carvalho M A R,Bemquerer M P,etal.Purification and partial characterization of a bacteriocin produced byEikenella corrodens[J].Journal of Applied Microbiology,2010,104(2):508-514.”)。

(2) Extraction of the crude bacteriocin extract (ethyl acetate extraction)

Taking 1L of the fermentation supernatant and ethyl acetate, and mixing the mixture according to the weight ratio of 1: 1, and placing the mixture on a shaking table at a rotating speed of 120r/min for overnight. The upper organic phase was collected and the lower fermentation supernatant was extracted twice more with equal volume of ethyl acetate. Mixing the organic phase, evaporating at 40 deg.C under vacuum using rotary evaporator, concentrating, and collecting the fraction containing residue to obtain bacteriocin crude extract. The crude bacteriocin extract was redissolved with 25mL PBS (phosphate buffered saline), and the bacteriostatic activity and potency of Listeria monocytogenes were determined using the double agar Oxford cup method.

The results of the bacteriostatic activity determination experiments in FIG. 2 show that the fermentation supernatant of lactic acid bacteria zy-B still has good bacteriostatic effect after being extracted by ethyl acetate, and the titer of the bacteriocin crude extract after being primarily purified is 6553.6AU/mL through titer determination, which indicates that ethyl acetate can be used for extracting bacteriocin from the fermentation supernatant for the next steps of purification.

(3) Purification of bacteriocin by Sephadex LH-20 column chromatography system

The Sephadex LH-20 column chromatography system uses Sephadex with the function of a molecular sieve to separate and purify components by utilizing different molecular sizes of different substances. The specific method refers to: separation and purification of bacteriocin produced by lactobacillus plantarum JLA-9 [ J ] in Zhao Shengming, Zhao rock, Ma Han Jun, food and fermentation industries, 2017,43(6):60-65.

Dissolving the crude bacteriocin extract obtained by ethyl acetate extraction by using PBS, concentrating the sample to the volume as small as possible, filtering a 0.22 mu m filter membrane to remove impurities, loading the sample by a wet method, eluting column volumes by using pure methanol to remove the impurities remained before, eluting column volumes by using an 80% methanol-water elution system, balancing the gel column, then directly adding the sample into a Sephadex LH-20 gel chromatographic column, eluting by using 80% methanol-water, setting the flow rate to be 1mL/5min, collecting 5 mL/tube by using a full-automatic collector, finally eluting by using pure methanol, and collecting the eluted sample of each tube for bacteriostatic determination.

Loading 25mL of ethyl acetate purified bacteriocin crude extract sample on an LH-20 gel column, eluting by using an elution system of methanol and water (8:2), collecting 40 tubes of eluted samples by using a computer automatic collector, measuring the activity of inhibiting the listeria monocytogenes of each tube, and determining the antibacterial activity of each tube to obtain the result shown in figure 3. No. 25, 26, 27, 28, 29, 30, 31 and 32 tubes have different antibacterial effects, combining freeze-drying and redissolving, measuring the titer to be 8192AU/mL, and performing cation exchange purification at the next step.

(4) Purification of bacteriocins by ion exchange chromatography

Most bacteriocins are positively charged when the pH is less than pI, so bacteriocins can be further purified steps using ion exchange chromatography using a bacteriocin solution at a suitable pH, and the bacteriocins can be isolated by adjusting the pH, loading, elution flow rate and NaCl concentration to obtain a suitable elution system, in particular by reference to "purification, characterization and molecular structure studies [ D ].2015 ] of novel Bifidobacterium bacteriocin bifidum A".

Loading 10mL of bacteriocin purified by an LH-20 gel column into an AKTA system, combining the bacteriocin with an SP FF cation column, washing unbound impurity protein by using a citric acid buffer solution (pH 6.0) of 20nmol/L, and washing until the balance of a baseline is achieved; and then, eluting the target protein by using a 1mol/L NaCl solution in a gradient manner, simultaneously detecting an absorption peak at 280nm, collecting samples by using an automatic collector, and respectively detecting the antibacterial activity of each tube.

The collected components are detected at 280nm, the elution curve is shown in figure 4, two absorption peaks S1 and S2 are detected, and the antibacterial activity determination shows that S2 has antibacterial activity and the titer is 4096 AU/mL.

(5) Purification of bacteriocins by semi-preparative reverse phase liquid chromatography:

the method comprises the steps of separating and purifying components collected through cation exchange chromatography by using an HP Plus 50D full-automatic medium-high pressure preparative liquid chromatograph, wherein C18 reverse phase column packing is SinoChromomODS-AP 15 mu m, the size is 20.0mm x 250mm, the column number is 2617138, a mobile phase is methanol-water, samples are filtered through a 0.22 mu m filter membrane before use and are subjected to ultrasonic degassing, the S2 sample is subjected to 0.22 mu m filter membrane cation exchange chromatography, is subjected to sample injection ring loading, is subjected to 0-4 min, is subjected to 10% methanol-water isocratic elution, is subjected to 4-13 min, is subjected to 10% methanol-water-25% methanol-water gradient elution, is subjected to 13-21 min, 25-30% methanol-water gradient elution 21-22 min, is subjected to 30-40% methanol gradient elution, and is subjected to 22-32 min by utilizing 100% methanol isocratic elution, so that components with inhibitory activity on Listeria monocytogenes are collected.

The elution curve is shown in figure 5, and the component H1 is obtained by 0-4 min and 10% methanol-water isocratic elution; performing gradient elution with 10% methanol-water and 25% methanol-water for 4-13 min to obtain a component H2; 13-21 min, and 25-30% of methanol-water to obtain a component H3 and a component H4; 21-22 min, 30-40% of methanol has no component; isocratic elution is carried out for 22-32 min by using 100% methanol to obtain a component H5. Antibacterial activity detection shows that only the peak H5 has antibacterial activity, and the potency is 5120 AU/mL.

The purity of the H5 peak was identified by RP-HPLC analysis, as shown in fig. 6, and it was found that peaks appeared only at t ═ 1.859min, indicating that the purified bacteriocin had reached a single fraction, thus obtaining purified bacteriocin PE-ZYB 1.

Example 7 molecular mass determination and amino acid analysis of bacteriocin PE-ZYB1

The purified bacteriocin PE-ZYB1 was sent to Betay park Biotechnology, Inc. of Beijing, and polypeptide fragments were analyzed by LC-MS/MS system (Shimadzu LC-20AD nano flow-LC and Thermo Fisher Scientific Q active mass spectrometer) -class mass spectrometry scan range was 350-1800(m/z), resolution was 70000, and secondary MS/MS resolution was 17500. the obtained amino acid sequences were compared with the sequences stored in the general protein database (UniProt), BLAST (http:/www.uniprot.org/BLAST /) and the anti-peptide database (APD database) by MaxQuant analysis software (http:/aps. unmamc. edu/ap/main. html.).

LC-MS/MS analysis shows that the molecular weight of the bacteriocin PE-ZYB1 is 2019.22Da, and the bacteriocin PE-ZYB1 has an amino acid sequence shown as SEQ ID NO:6 (FIG. 7). comparison with the general protein database (UniProt) shows that the bacteriocin PE-ZYB1 is similar to unidentified proteins in the database and has no homology with antibacterial peptides reported in other documents.

EXAMPLE 8 determination of the bacterial inhibition Profile of bacteriocin PE-ZYB1

The bacteriocin PE-ZYB1 solution (diluted to 50 mug/mL by PBS) adopts a double-layer agar oxford cup method, and indicator bacteria bacteriostasis spectrum experiments are carried out by indicator bacteria in Table 8, and the diameter of the bacteriostasis zone is measured. The specific operation method of the Oxford cup method refers to the separation and diversity analysis of antibacterial active lactobacillus in intestinal tracts of animals in the areas of Jojoba, Arvina, Zhao duck Mei, et al south sea [ J ] food science, 2017(08):82-87 ]. Wherein Listeria monocytogenes is inoculated in a TSA-YE culture medium, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus cereus, Enterobacter aerogenes, Pseudomonas aeruginosa, Streptococcus thermophilus and Proteus Bacillus are inoculated in a nutrient agar culture medium, and Vibrio parahaemolyticus is inoculated in a nutrient agar culture medium containing 3% of sodium chloride and cultured for 24 hours at 37 ℃.

The results of determination of bacteriocin PE-ZYB1 antibacterial spectrum on 10 indicator bacteria by Oxford cup method (Table 9) show that it not only has strong inhibiting effect on gram-positive bacteria such as Listeria monocytogenes, Staphylococcus aureus, etc., but also has inhibiting effect on gram-negative bacteria such as Vibrio parahaemolyticus, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus, Streptococcus haemolyticus, as well as Bacillus subtilis and Bacillus cereus.

TABLE 9 antibiogram of bacteriocin PE-ZYB1

Note: antibacterial diameter (mm): 20; 14-20 parts of ++; less than 14mm (8 mm diameter oxford cup); /: has no bacteriostatic effect.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> university of eastern ocean

<120> lactic acid bacteria zy-B with food preservation and fresh-keeping function and application thereof

<141>2019-10-17

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>1480

<212>DNA

<213> lactic acid bacterium zy-B (Pediococcus pentosaceus zy-B)

<400>1

gggcgttgcg gggtgctaat actgcagtcg acgaacttcc gttaattgat tatgacgtac 60

ttgtactgat tgagatttta acacgaagtg agtggcgaac gggtgagtaa cacgtgggta 120

acctgcccag aagtagggga taacacctgg aaacagatgc taataccgta taacagagaa 180

aaccgcatgg ttttctttta aaagatggct ctgctatcac ttctggatgg acccgcggcg 240

tattagctag ttggtgaggt aaaggctcac caaggcagtg atacgtagcc gacctgagag 300

ggtaatcggc cacattggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360

gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga agaagggttt 420

cggctcgtaa agctctgttg ttaaagaaga acgtgggtaa gagtaactgt ttacccagtg 480

acggtattta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 540

tggcaagcgt tatccggatt tattgggcgt aaagcgagcg caggcggtct tttaagtcta 600

atgtgaaagc cttcggctca accgaagaag tgcattggaa actgggagac ttgagtgcag 660

aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720

gtggcgaagg cggctgtctg gtctgcaact gacgctgagg ctcgaaagca tgggtagcga 780

acaggattag ataccctggt agtccatgcc gtaaacgatg attactaagt gttggagggt 840

ttccgccctt cagtgctgca gctaacgcat taagtaatcc gcctggggag tacgaccgca 900

aggttgaaac tcaaaagaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960

tcgaagctac gcgaagaacc ttaccaggtc ttgacatctt ctgacagtct aagagattag 1020

aggttccctt cggggacaga atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080

gatgttgggt taagtcccgc aacgagcgca acccttatta ctagttgcca gcattaagtt 1140

gggcactcta gtgagactgc cggtgacaaa ccggaggaag gtggggacga cgtcaaatca 1200

tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagtcgcga 1260

gaccgcgagg ttaagctaat ctcttaaaac cattctcagt tcggactgta ggctgcaact 1320

cgcctacacg aagtcggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1380

cccgggcctt gtacacaccg cccgtcacac catgagagtt tgtaacaccc aaagccggtg 1440

gggtaacctt taggagctag ccgtctaagt gacagaatga 1480

<210>2

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ggttaccttg ttacgactt 19

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

agagtttgat cctggctcag 20

<210>4

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atgaaaaaga aaktakt 17

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ccaatatacc caacattttt 20

<210>6

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Ala Leu Pro Ala Leu Pro Ala Ala Ser Met Gly Thr Gly Ala Gly Val

1 5 10 15

Gly

Claims

1, lactic acid bacteria zy-B with food preservation and fresh keeping functions, which is characterized in that the lactic acid bacteria zy-B is IIa type bacteriocin producing bacteria, the lactic acid bacteria zy-B is preserved in eastern province microorganism strain preservation center in 2019, 8 and 19 months, and the preservation number is GDMCC No. 60743.

2. The lactic acid bacteria zy-B with food preservation and fresh keeping effects of claim 1, wherein the 16S rRNA of said lactic acid bacteria zy-B has the nucleotide sequence shown in SEQ ID NO. 1.

3. The use of the lactic acid bacteria zy-B according to claim 1 for preserving food.

4. Use according to claim 3, characterized in that: fermenting the lactobacillus zy-B, and taking supernatant to prepare the bacteriostatic agent.

5. Use according to claim 4, characterized in that: inoculating the lactobacillus zy-B into an MRS liquid culture medium, and culturing at 28 ℃ for 36-48h to obtain lactobacillus fermentation supernatant.