Probiotic bacteriocin as well as preparation method and application thereof in lactose-free liquid goat milk

Abstract

The invention discloses probiotic bacteriocin as well as a preparation method and application thereof in lactose-free liquid goat milk, and belongs to the technical field of bacteriocin. According to the invention, the bacteriocin is obtained by optimizing fermentation conditions of the rhamnosus lactobacillus KD5, separating and purifying, an amino acid sequence and an antibacterial spectrum of the bacteriocin are also identified, and a result shows that the bacteriocin can remarkably inhibit multi-drug-resistant pseudomonas aeruginosa and other various clinical common pathogenic bacteria, and has a good application prospect. The invention provides theoretical and technical references for the development of bacteriocin required for treating and preventing multi-drug-resistant pseudomonas aeruginosa infection. The bacteriocin is added into the lactose-free liquid goat milk for the first time, so that the growth of putrefying bacteria can be inhibited, and the shelf life is prolonged.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, in particular to a probiotic bacteriocin and a preparation method thereof and application thereof in lactose-free liquid goat milk. Background Art

[0002] Bacteria can produce a class of proteins and polypeptides called bacteriocins during metabolism, which have antibacterial activity. They can inhibit peptidoglycan synthesis by perforating target cells, inhibiting protein synthesis, interacting with ribosomes or tRNA, and directly degrading target cell DNA, thereby achieving an antibacterial effect. The protein antibacterial substances produced by lactic acid bacteria during their own metabolism are called lactic acid bacteriocins, which have a wide range of inhibitory effects on Gram-negative bacteria, especially on spoilage bacteria.

[0003] Bacteriocins are mainly prepared by liquid fermentation of lactic acid bacteria, followed by solid-liquid separation, extraction, ultrafiltration and freeze-drying. Many studies have focused on the screening of bacterial strains and the optimization of fermentation processes, but there are few literatures on the identification of bacteriocin amino acid sequences.

[0004] Pasteurized milk is popular in the market because of its low heating temperature and less damage to nutrients. However, pasteurization only kills harmful bacteria in milk and cannot completely kill spoilage bacteria that easily cause liquid milk to deteriorate. Therefore, the shelf life of pasteurized milk is short, resulting in a short sales time, which brings certain pressure on sales. Currently, there are few reports on the application of probiotic bacteriocins in pasteurized liquid milk. Summary of the invention

[0005] The purpose of the present invention is to provide a probiotic bacteriocin and a preparation method thereof and application in lactose-free liquid goat milk to solve the problems existing in the above-mentioned prior art. The bacteriocin separated and purified from the fermentation broth of Lactobacillus rhamnosus is added to lactose-free liquid goat milk to inhibit the growth of spoilage bacteria and prolong the shelf life of the lactose-free liquid goat milk product.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a probiotic bacteriocin, the amino acid sequence of the probiotic bacteriocin is shown as SEQ ID NO.1-22.

[0008] The present invention also provides a method for preparing probiotic bacteriocin, comprising the step of obtaining probiotic bacteriocin from the supernatant of probiotic fermentation liquid, wherein the probiotic is Lactobacillus rhamnosus KD5.

[0009] Preferably, the fermentation conditions of the probiotics are: the fermentation medium is MRS medium, the inoculation amount is 3%, the culture temperature is 37° C., and the culture time is 42 h.

[0010] Preferably, the fermentation broth supernatant is mixed with ethyl acetate for extraction, and the organic phase is collected and concentrated to obtain the probiotic bacteriocin.

[0011] Preferably, after the organic phase is concentrated, ultrafiltration is performed to obtain probiotic bacteriocins with molecular weights less than 3k, 3-5k, 5-10k and greater than 10k.

[0012] The present invention also provides the use of the probiotic bacteriocin in preparing a drug for inhibiting multi-drug resistant Pseudomonas aeruginosa.

[0013] The present invention also provides the use of the probiotic bacteriocin in the preparation of a drug for inhibiting Gram-positive bacteria and / or Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis and Acinetobacter baumannii.

[0014] The present invention also provides the use of the probiotic bacteriocin in the preparation of lactose-free liquid goat milk. The addition amount of the probiotic bacteriocin is 0.05%-0.10% w / v, which can extend the shelf life by 4 days.

[0015] The present invention also provides an antibacterial drug containing the probiotic bacteriocin, wherein the antibacterial effect includes inhibiting multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

[0016] The invention also provides a lactose-free liquid goat milk containing the probiotic bacteriocin.

[0017] The present invention discloses the following technical effects:

[0018] The invention optimizes the fermentation conditions of Lactobacillus rhamnosus KD, separates and purifies the bacteriocin from the fermentation supernatant, and determines the MIC value (0.78 mg / mL), MBC value (6.24 mg / mL) and amino acid sequence of the bacteriocin. The bacteriocin can significantly inhibit multi-drug resistant Pseudomonas aeruginosa and multiple clinical pathogens, and provides a theoretical basis for the development of multi-drug resistant Pseudomonas aeruginosa antibacterial agents.

[0019] The invention adds bacteriocin into lactose-free liquid goat milk, thereby inhibiting the growth of spoilage bacteria and extending the shelf life of the liquid goat milk by 4 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effect of culture time on the antibacterial activity of Lactobacillus rhamnosus KD5;

[0022] Figure 2 The effect of inoculum size on the antibacterial activity of Lactobacillus rhamnosus KD5;

[0023] Figure 3 The effect of culture temperature on the antibacterial activity of Lactobacillus rhamnosus KD5;

[0024] Figure 4 : The effect of lactic acid and acetic acid on the antibacterial agent; in the figure, KD5 and KD10 represent the fermentation supernatant of Lactobacillus rhamnosus KD5 and Lactobacillus paracasei KD10, respectively, and MRS represents MRS broth medium;

[0025] Figure 5 Effects of catalase on antimicrobial agents;

[0026] Figure 6 The effect of protease on the activity of antibacterial agent;

[0027] Figure 7 To ultrafilter the antibacterial activity of Lactobacillus rhamnosus KD5 in each component;

[0028] Figure 8 It is the liquid chromatogram of Lactobacillus rhamnosus KD5 bacteriocin;

[0029] Fig. 9 is the secondary mass spectrum of the peptide VISAVAQTNA;

[0030] Fig.10 This is the secondary mass spectrum of the peptide PQGPQGPRG;

[0031] Fig.11 It is the secondary mass spectrum of peptide GPQGPQGPR;

[0032] Fig.12 is the secondary mass spectrum of peptide KSVKFPVS;

[0033] Fig.13 is the concentration of bacteriocin produced by KD5 and its inhibitory effect on multidrug-resistant Pseudomonas aeruginosa;

[0034] Fig.14is the inhibition curve of KD5 bacteriocin concentration against multidrug-resistant Pseudomonas aeruginosa;

[0035] Fig.15 To investigate the effect of adding bacteriocins on the total colony count in pasteurized lactose-free liquid goat milk. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The experimental strains involved in the following examples: Lacticaseibacillus rhamnosus KD5 and Lacticaseibacillus paracasei are all stored in the 2065 laboratory of the Department of Biotechnology, School of Food Science and Engineering, multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Baumanii, Albicans Saccharomyces, Candida krusei and Bacillus cereus can all be purchased through conventional commercial channels and are all stored in the laboratory of Xianyang Central Hospital.

[0042] Culture medium: (1) Lactic acid bacteria activation and fermentation medium (MRS broth medium): 2% glucose, 0.8% beef extract, 1% peptone, 0.5% yeast powder, 0.2% diammonium citrate, 0.2% potassium dihydrogen phosphate, 0.5% anhydrous sodium acetate, 0.005% manganese sulfate, 0.01% magnesium sulfate, 0.1% Tween-80; pH 6.2±0.2; sterilize with high pressure steam at 121°C for 20 min.

[0043] (2) Antibacterial punch test medium (MRS broth agar medium): 1.5% agar powder was added to MRS broth, pH was 6.2-6.4, and sterilized at 121°C for 20 min.

[0044] Example 1 Optimization of fermentation conditions and analysis of antibacterial components of Lactobacillus rhamnosus KD5

[0045] 1. (1) Activation of lactic acid bacteria

[0046] The freeze-dried bacterial powder of Lactobacillus rhamnosus KD5 stored at low temperature (-18°C) was dissolved with 1 mL of sterile physiological saline, inoculated into MRS broth culture medium, and kept at 37°C for 24 hours to obtain the first generation of activated lactic acid bacteria; then, it was inoculated into MRS broth culture medium at a 5% inoculation rate, and cultured at 37°C for 24 hours, the activation was repeated 3 times, and the culture was refrigerated for later use.

[0047] 2. The activated Lactobacillus rhamnosus KD5 was inoculated into MRS broth medium at a rate of 5%, cultured at 37°C for 24h, centrifuged at a speed of 10000r / m and a temperature of 4°C for 20min, and the supernatant was filtered using a filter membrane with a pore size of 0.45μm to obtain the fermentation broth supernatant. At the same time, the precipitated bacterial mud was collected and placed in a 4°C refrigerator for standby use.

[0048] 3. Optimization of fermentation conditions

[0049] Using MRS broth as the fermentation medium, the effects of culture time, culture temperature and inoculation size on the production of antibacterial agents were studied through single-factor experiments to determine the appropriate range of each factor. The fermentation conditions were then optimized and verified through Box-Behnken Design experiments.

[0050] 4. Analysis of antibacterial components in fermentation supernatant

[0051] Lactobacillus rhamnosus KD5 will produce bacteriocins, lactic acid, acetic acid, hydrogen peroxide, protein and other substances during fermentation. In order to determine the effective antibacterial components, the agar diffusion method was designed to determine the effects of lactic acid, acetic acid, hydrogen peroxide and protein on the inhibition zone, so as to preliminarily determine the antibacterial substances.

[0052] 5. Results and Analysis

[0053] 5.1 Effect of fermentation conditions on the production of antibacterial agents by Lactobacillus rhamnosus KD5

[0054] (1) Cultivation time

[0055] Under the condition of ensuring that the inoculation volume is 5% and the culture temperature is constant at 37℃, the culture time is used as a variable to study the effect on the growth of Lactobacillus rhamnosus KD5. The pH value of the MRS broth medium was adjusted to 7.0, and KD5 was inoculated with 5% inoculation volume. The culture was carried out at 37℃ for 12h, 18h, 24h, 30h, 36h, 42h, and 48h, and the OD value of each culture time was measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the antibacterial test was performed. The results are shown in Figure 1 .

[0056] Depend on Figure 1 It can be seen that the OD of KD5 600 The pH value increased with the extension of the culture time, reaching a peak of 1.905 at 48h. The pH value decreased within 24h of culture, and stabilized after 24h, with a value of 3.94±0.08. The inhibition zone increased with the increase of culture time, reaching a maximum diameter of 19.12mm at 48h. (2) Inoculation amount

[0057] On the basis of the single factor study on culture time, the culture time was kept constant at 48 h and the culture temperature was kept at 37 °C. KD5 was inoculated into MRS broth medium at inoculation amounts of 1.5%, 3%, 4.5%, and 6% for 48 h. The OD of each inoculation amount was measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the inhibition zone test was performed. The results are shown in Figure 2 .

[0058] Depend on Figure 2 It can be seen that when the inoculation amount is 3%, KD5 OD 600 The maximum is 1.806; the pH value does not change much, both are 3.92±0.04; the diameter of the inhibition zone with an inoculation volume of 3% is the largest, which is 18.19mm

[0059] (3) Culture temperature

[0060] Keeping the culture time at 48h and the inoculum at 3%, KD5 was inoculated into MRS broth medium for culture at 33℃, 35℃, 37℃, 39℃, and 41℃, and the OD at each culture temperature was measured. 600 and pH, and then centrifuged at 8000r / min for 15min to obtain the supernatant, and the inhibition zone test was performed. The results are shown in Figure 3 .

[0061] Depend on Figure 3 It can be seen that through single factor experiment, under 37℃ culture conditions, KD5 OD 600 The maximum is 1.80; the maximum diameter of the inhibition zone is 18.58mm.

[0062] 5.2 Box-Behnken experimental design to optimize the fermentation conditions of Lactobacillus rhamnosus KD5 for antibacterial production

[0063] Under the condition that MRS broth medium was used as the basic medium, the culture conditions were further optimized by Box-Behnken experimental design according to the single-factor test results of culture time, inoculation amount and culture temperature. A three-factor four-level (N=17) experiment was set up, the initial pH value was set to 6.5, and three parallels were set for each group of experiments. 600 The values ​​of R1 and R2 were taken as response values, respectively. The factor level coding was shown in Table 1. The experimental design and results were shown in Table 1.

[0064] Table 1 Box-Behnken test design and results of fermentation conditions of Lactobacillus rhamnosus KD5 antibacterial agent

[0065]

[0066] 5.3 Analysis of antibacterial components in the fermentation supernatant of Lactobacillus rhamnosus KD5

[0067] (1) Effects of lactic acid and acetic acid on antibacterial agents

[0068] Lactic acid bacteria produce a large amount of lactic acid and acetic acid and other acidic substances during the fermentation process. In order to determine whether the antibacterial effect is caused by the action of MRS broth, lactic acid or acetic acid, an organic acid exclusion test was designed. After fermentation and cultivation of KD5, the supernatant was obtained by centrifugation and the pH was measured to be 3.5. At the same time, the pH of the 15% lactic acid and 20% acetic acid concentrations was adjusted to 3.5, which is equal to the pH value of the fermentation supernatant, and the antibacterial zone test was performed. Figure 4 It can be seen that MRS broth, lactic acid and acetic acid have no obvious antibacterial effect, so the antibacterial effect of lactic acid and acetic acid in the fermentation supernatant can be ruled out.

[0069] (2) Effect of hydrogen peroxide on antibacterial agents

[0070] Lactic acid bacteria produce hydrogen peroxide during metabolism. To eliminate the antibacterial effect of hydrogen peroxide, enzymatic hydrolysis was used to eliminate the antibacterial effect of hydrogen peroxide. 1 mL of KD5 fermentation supernatant was taken and thoroughly mixed with 1 mL of 10 mg / mL catalase. At the same time, the untreated fermentation supernatant was used as a control. Figure 5 It can be seen that there is no significant difference between the experimental group and the control group (P>0.05), among which the diameters of the inhibition zones treated with KD5 and untreated are 20.79±0.17mm and 19.98±0.22mm respectively. From the experimental data, it can be seen that the diameters of the inhibition zones treated with KD5 and untreated are similar, which shows that hydrogen peroxide has almost no antibacterial effect, thus excluding the antibacterial effect of hydrogen peroxide.

[0071] (3) Effect of proteases on antibacterial agents

[0072] On the basis of excluding the antibacterial effects of acetic acid, lactic acid and hydrogen peroxide, pepsin, trypsin and papain were added to the supernatant of KD5 fermentation broth for treatment, and the optimum pH of each protease was adjusted by 1mol / L HCl and 2mol / LNaOH, among which the pH of pepsin, trypsin and papain was adjusted to 2.0, 7.0 and 5.7 respectively. 1mL / L of the three proteases were added to the antibacterial agent and mixed, and then treated at 37℃ water bath for 2h, and then treated at 80℃ for 10min to eliminate the activity of the enzyme, and then the pH was adjusted to be consistent with the original supernatant, and the antibacterial effect was determined by punching test with multidrug-resistant Pseudomonas aeruginosa as indicator bacteria.

[0073] Depend on Figure 6It can be seen that there is no significant difference between the experimental group and the control group (P>0.05). After adding enzyme substances, the diameter of the inhibition zone decreased to varying degrees. The results show that KD5 produces antibacterial substances with protein characteristics that can be decomposed by proteases. Based on the combined exclusion of lactic acid, acetic acid and hydrogen peroxide tests, the antibacterial substance was determined to be bacteriocin.

[0074] Example 2 Isolation, purification, identification and property determination of rhamnosus lactis KD5 bacteriocin

[0075] 1. Experimental methods

[0076] 1.1 Bacteriocin extraction

[0077] The fermentation liquid obtained under the optimal conditions of Lactobacillus rhamnosus KD5 was centrifuged at a rotation speed of 10000 r / m and a temperature of 4° C. for 20 minutes to obtain a cell-free fermentation supernatant (CFS).

[0078] Alcohol precipitation extraction: CFS was concentrated 10 times using a vacuum rotary evaporator, 95% anhydrous ethanol was added, and the final anhydrous ethanol concentration was adjusted to 75% after thorough mixing. The mixture was precipitated at 4°C for 12 hours, and centrifuged (10,000 r / m, 20 min) to obtain the precipitate and aqueous phase, respectively, and their antibacterial effects were determined. The protein concentration of the supernatant and precipitate was determined using a Roche biochemical analyzer, and the specific activity, total activity, and recovery rate were calculated using the relationship (1), (2), and (3).

[0079] Organic solvent extraction: Based on the principle of like dissolves like, CFS was concentrated 4 times using a vacuum rotary evaporator, fully mixed with ethyl acetate at a ratio of 1:3, placed in a shaker at a speed of 120 r / min for 12 hours, then left to rest for 2 hours, and the extraction was repeated 3 times to obtain an organic phase and an aqueous phase, respectively. The organic phase was then concentrated using a rotary evaporator at a temperature of 60°C and a speed of 90 r / min until the ethyl acetate was completely evaporated, and then redissolved in pure water to determine its antibacterial effect. The protein concentrations of the organic and aqueous phases were determined using a Roche biochemical analyzer, and the specific activity, total activity, and recovery rate were calculated using equations (1), (2), and (3).

[0080] Ammonium sulfate precipitation method: add 60%, 70%, 80%, 90%, 100% saturation ammonium sulfate solution to CFS, stir for 2 hours using a magnetic stirrer, leave for 12 hours, centrifuge (10000r / m, 20min) to obtain precipitate and supernatant, and measure their antibacterial effect. Use Roche biochemical analyzer to measure the protein concentration of precipitate and water, and calculate the specific activity, total activity and recovery rate by equations (1), (2), and (3).

[0081] Specific activity (AU / mg) = titer (AU / mL) / protein concentration (mg / mL) (1);

[0082] Total activity (AU) = specific activity (AU / mg) × volume of protein-containing solution (mL) (2);

[0083] Recovery rate (%) = total activity (AU) / total activity of supernatant (AU) (3).

[0084] 1.2 Ultrafiltration purification of bacteriocin KD5 and activity determination of each component

[0085] Ultrafiltration technology is used to pressurize the sample through the filter membranes of different pore sizes to purify, separate and concentrate the stock solution. The filter membranes with cut-off molecular weights of less than 3k, 3-5k, 5-10k and greater than 10k are selected for filtration respectively. The ultrafiltration purification multiple is obtained by calculating the volume of the stock solution and the volume of the liquid after ultrafiltration. The antibacterial test is carried out on the different components obtained by ultrafiltration to determine their activity.

[0086] 1.3 Preparation of bacteriocin

[0087] First, the fermentation broth prepared by Lactobacillus rhamnosus KD5 under optimal fermentation conditions was centrifuged to obtain the fermentation supernatant, and then ethyl acetate was added to extract the obtained organic phase, which was then concentrated at a temperature of 60°C and a rotation speed of 90r / min until the ethyl acetate was completely evaporated, and then it was redissolved in pure water, and finally ultrafiltration was used to obtain the component with the best antibacterial effect. After freezing at -80°C for 24 hours, it was freeze-dried in a vacuum freeze dryer for 48 hours to obtain bacteriocin freeze-dried powder, which was stored at -20°C for future use.

[0088] 1.4 Determination of bacteriocin inhibition spectrum

[0089] Seven common clinical pathogens were selected as indicator bacteria, including Gram-positive cocci represented by Staphylococcus aureus, Enterobacteriaceae represented by Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, non-fermenting bacteria represented by Acinetobacter baumannii, and yeast-like fungi represented by Candida albicans and Candida krusei, for antibacterial tests.

[0090] 1.5 LC-MS / MS identification and structure prediction of bacteriocins

[0091] The bacteriocin lyophilized powder was prepared into a solution with a concentration of 0.1 g / mL, and then separated, purified and identified by LC-MS / MS. The identified peptides were compared in UniProtKB, and the three-dimensional structure of the bacteriocin was predicted using Swiss-Mode.

[0092] 1.6 Determination of minimum inhibitory concentration and minimum bactericidal concentration of KD5 bacteriocin

[0093] Determine the minimum inhibitory concentration (MIC) of bacteriocin freeze-dried powder, take the indicator bacteria in the logarithmic phase, and prepare a bacterial suspension (the bacterial suspension concentration is 10 6 CFU / ml), weigh 100 mg of KD5 bacteriocin freeze-dried powder, dilute with 4 mL of nutrient broth and add to a 96-well microplate, add 300 μL of the diluted stock solution to the first well, add 150 μL of nutrient broth to the 2nd to 11th wells respectively, aspirate 150 μL from the first well and add to the second well and mix evenly, aspirate 150 μL into the third well and so on, aspirate 150 μL from the 10th well and discard, the 11th well is the positive control. The bacterial suspension and nutrient broth medium without adding bacteriocin are the negative control group. Culture at 35°C for 24 hours for the determination of MIC value. Determine the OD of the test group and the control group 600 The difference is taken as the mass concentration of bacteriocin freeze-dried powder, OD 600 The difference is used as the vertical axis to draw a curve, the inflection point is MIC, and the concentration corresponding to the dilution with the minimum inhibitory effect is the minimum inhibitory concentration MIC.

[0094] Based on the MIC determination, the concentrations of bacteriocin were set to MIC, 2MIC, 4MIC, and 8MIC, respectively. 100 μl was mixed with the indicator bacteria, spread on broth agar medium, and cultured at 37°C for 24 hours. The number of colonies on the plate was observed. If the number of colonies was less than 5, the inhibitory concentration was the minimum bactericidal concentration (MBC).

[0095] 2. Results and Analysis

[0096] 2.1 Effect of extraction method on antibacterial effect and recovery rate of bacteriocin

[0097] Under the optimal fermentation conditions of Example 1, the supernatant of the fermentation broth of Lactobacillus rhamnosus KD5 was obtained, and the multidrug-resistant Pseudomonas aeruginosa was used as the indicator bacteria. The bacteriocins in the supernatant of the fermentation broth of Lactobacillus rhamnosus KD5 were extracted according to the following three extraction methods. The results are shown in Table 5.

[0098] As shown in Table 2, when the alcohol precipitation method is used for crude extraction, relatively more bacteriocin remains in the aqueous phase, and the activity of bacteriocin is lost to a certain extent during the ethanol extraction process; when the ammonium sulfate precipitation method is used, the effect is relatively good when the concentration is 70% and 80%, but there is also a certain antibacterial effect in the supernatant, indicating that the extraction of bacteriocin is incomplete; when ethyl acetate is used for extraction, only a small amount of bacteriocin is contained in the aqueous phase, and the antibacterial effect is the best. As shown in Table 3, the total activity and recovery rate of ethyl acetate extraction are the highest, and the effects of ammonium sulfate precipitation and alcohol precipitation are equivalent. In summary, ethyl acetate is used to extract bacteriocin in the subsequent process.

[0099] Table 2 Effect of extraction method on the antibacterial activity of bacteriocin of Lactobacillus rhamnosus KD5

[0100]

[0101]

[0102] Table 3 Effect of extraction method on the recovery rate of Lactobacillus rhamnosus KD5 bacteriocin

[0103]

[0104] 2.2 Effect of ultrafiltration on the activity and recovery of bacteriocin components

[0105] Lactobacillus rhamnosus KD5 was extracted with ethyl acetate to obtain fractions less than 3k, 3-5k, 5-10k, and greater than 10k. The fermentation supernatant was used as a control to determine the antibacterial activity against multidrug-resistant Pseudomonas aeruginosa. Figure 7 As shown, the diameters of the inhibition zones of the supernatant solution, less than 3k, 3-5k, 5-10k, and greater than 10k of KD5 were 26.59±0.12mm, 26.74±0.24mm, 14.37±0.17mm, 16.39±0.19mm, and 7.44±0.32mm, respectively. It can be seen that there is no obvious change in the antibacterial effect of the supernatant and the supernatant solution after treatment with an ultrafiltration membrane less than 3k, while the antibacterial effects of 3-5k, 5-10k, and greater than 10k are all reduced.

[0106] 2.3 LC-MS / MS purification and identification of bacteriocins

[0107] The component with the strongest antibacterial effect on multidrug-resistant Pseudomonas aeruginosa obtained by ultrafiltration was purified and its amino acid sequence was identified by LC-MS / MS. The liquid chromatogram is shown in Figure 8 As shown, the amino acid series of bacteriocins are shown in Table 4. The peptide sequences corresponding to the bacteriocins No. 1-22 in Table 4 are numbered as SEQ ID NO: 1-SEQ ID NO: 22, respectively.

[0108] Table 4 Amino acid sequences of bacteriocins

[0109]

[0110]

[0111] Twenty-two peptides were isolated and identified from the fraction with the strongest inhibitory activity using LC-MS / MS, as shown in Table 4.

[0112] In the antibacterial test of each component of ultrafiltration, it was found that bacteriocins with a molecular weight less than 3k had the best antibacterial effect. The main reason is that low molecular weight peptides have good structural stability. Therefore, the molecular weight of the peptide will be used as an important reference for the screening of bacteriocin segments; studies have shown that bacteriocins have hydrophilic and hydrophobic groups that can form an amphiphilic structure. When entering the hydrophobic area of ​​the bacterial cell membrane, they can destroy the bacterial cell membrane and cause the bacteria to die. Therefore, hydrophobicity is also an important reference for screening peptides; the positive charge of bacteriocins plays a core role in the electrostatic interaction between bacterial cell membranes. It can change the charge of bacterial cell membranes, causing cell membrane damage and death. Most bacteriocins have positive charges ranging from +2 to +9. Therefore, the number of charges is also an important reference for screening. In summary, considering the molecular weight, hydrophobicity, and charge number at the same time, peptides with 8-10 amino acids were selected. The peptides of the KD5 strain are VISAVAQTNA, PQGPQGPRG, GPQGPQGPR, and KSVKFPVS. The secondary mass spectra of the selected peptides are shown in Figure 9-12 shown.

[0113] 2.4 Antibacterial spectrum of bacteriocin produced by Lactobacillus rhamnosus KD5

[0114] The antibacterial results of the bacteriocin produced by Lactobacillus rhamnosus KD5 (concentration of 0.1 g / mL) against 7 common clinical pathogens (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Candida albicans, and Candida krusei) are shown in Table 5.

[0115] The results showed that the bacteriocin produced by KD5 was effective against Staphylococcus aureus (G + ), Klebsiella pneumoniae (G - ), Escherichia coli (G - ), Proteus mirabilis (G - ), Acinetobacter baumannii (G - ) all had significant antibacterial effects, with the diameter of the inhibition zone ranging from 20 to 29 mm, among which Acinetobacter baumannii had the best antibacterial effect; it had no antibacterial effect on yeasts such as Candida albicans and Candida krusei, with the diameter of the inhibition zone ranging from 7.2 to 7.3 mm, which was close to the diameter of the Oxford cup ( 7mm), in summary, this shows that the bacteriocin produced by KD5 has a certain broad-spectrum antibacterial effect against both Gram-positive and Gram-negative bacteria.

[0116] Table 5 Antibacterial spectrum of KD5 bacteriocin

[0117]

[0118] 2.5 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of bacteriocins against multidrug-resistant Pseudomonas aeruginosa

[0119] The MIC and MBC of the antibacterial agent produced by Lactobacillus rhamnosus KD5 against multidrug-resistant Pseudomonas aeruginosa were determined by the two-fold dilution method. Figure 13 to Figure 14 shown.

[0120] from Figure 13 to Figure 14 It can be seen that the bacteriocin produced by KD5 still has a significant antibacterial effect after 6 consecutive dilutions, but after the 7th dilution, the antibacterial effect is significantly weakened, and its MIC is 0.78 mg / mL. Therefore, the concentration greater than the MIC within 24 hours of OD 600 The value remained at 0; in the 5 groups of experiments where the bacteriocin concentration was less than the MIC, the OD 600 Mainly kept at 0, the main reason is that the bacteria are in the delayed period, 6-14h OD 600 Logarithmic growth trend, 14-24h OD 600 Maintain high trend.

[0121] Based on the MIC determination, the two-fold dilution method was used to dilute the bactericidal concentrations of different concentrations. The results showed that the MIC of KD5 bacteriocin was 0.78 mg / mL and the MBC was 6.24 mg / mL.

[0122] Example 3 Application of Lactobacillus rhamnosus KD5 bacteriocin in lactose-free liquid goat milk

[0123] 1‰ lactase was added to goat milk, and enzymolysis was performed at 55℃ for 2h to obtain lactose-free liquid goat milk. Then 0.05% (w / v) and 0.10% (w / v) bacteriocin of Lactobacillus rhamnosus KD5 were added respectively. The lactose-free goat milk without bacteriocin was used as the control. The milk was pasteurized and then refrigerated at 4℃ for 12d. The total number of colonies was measured every 4d. The results are shown in Table 1. Fig.15 .

[0124] Depend on Fig.15 It can be seen that the total number of colonies in the control group without adding bacteriocin increased rapidly, and at 12:00 its total number of colonies was higher than the national standard requirement of 10 5 CFUmL, while the total colony counts of pasteurized lactose-free liquid goat milk with 0.05% and 0.10% bacteriocin added were all less than 10 5 CFUmL, it can be seen that adding bacteriocin can extend the shelf life of lactose-free liquid goat milk by 4 days.

[0125] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A probiotic bacteriocin, characterized in that The amino acid sequence of the probiotic bacteriocin is shown in SEQ ID NO.1-22.

2. A method for preparing a probiotic bacteriocin, characterized in that: The method comprises the step of obtaining probiotic bacteriocin from the supernatant of probiotic fermentation liquid, wherein the probiotic is Lactobacillus rhamnosus KD5, and the preservation number is CCTCC NO: M20231641.

3. The preparation method according to claim 2, characterized in that: The probiotics were fermented under the following conditions: the fermentation medium was MRS medium, the inoculation amount was 3%, the culture temperature was 37° C., and the culture time was 42 h.

4. The preparation method according to claim 2, characterized in that: The fermentation broth supernatant is mixed with ethyl acetate for extraction, and the organic phase is collected and concentrated to obtain the probiotic bacteriocin.

5. The preparation method according to claim 3, characterized in that: After the organic phase is concentrated, it is ultrafiltered to obtain probiotic bacteriocins with molecular weights less than 3k, 3-5k, 5-10k and greater than 10k.

6. Use of the probiotic bacteriocin according to claim 1 in the preparation of a medicament for inhibiting multidrug-resistant Pseudomonas aeruginosa.

7. Use of the probiotic bacteriocin according to claim 1 in the preparation of a medicament for inhibiting Gram-positive bacteria and / or Gram-negative bacteria, characterized in that: The Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis and Acinetobacter baumannii.

8. The use of the probiotic bacteriocin according to claim 1 in preparing lactose-free liquid goat milk, characterized in that: The added amount of the probiotic bacteriocin is 0.05%-0.10% w / v.

9. An antibacterial drug, characterized in that: The probiotic bacteriocin according to claim 1 is contained, wherein the antibacterial activity includes inhibiting multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis and / or Acinetobacter baumannii.

10. A lactose-free liquid goat milk, characterized in that: Containing the probiotic bacteriocin according to claim 1.

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