Lactococcus lactis with high yield of nisin as well as preparation method and application of lactococcus lactis

Abstract

The invention discloses lactococcus lactis with tolerance and high yield of nisin as well as a preparation method and application of the lactococcus lactis. The strain is named as Lactococcus lactis in taxonomy. The obtained strain can normally grow under the condition of the nisin with the concentration of 2800IU / mL, the yield of the nisin is increased by 15%, and theoretical basis and practical experience can be provided for subsequent development of application of excellent strains in the field of fermented food bacteriostasis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a high-yield nisin Lactococcus lactis and a preparation method and application thereof. Background Art

[0002] Lactococcus lactis is a Gram-positive bacterium belonging to the Firmicutes, Bacillus, Lactobacilales, Streptococcus, and Lactococcus. Lactococcus lactis is spherical or oval in shape, is a facultative anaerobic bacterium, does not have a capsule or spore morphology, and has an optimal growth temperature of 30°C. Lactococcus lactis plays a key role in food acidification, flavor development, and the production of various dairy products such as cheese and fermented butter. It is an important microorganism in the dairy food fermentation industry. In addition, Lactococcus lactis has good genetic stability, and its fermentation function and probiotic function change little in continuous fermentation, making it an ideal strain for industrial production.

[0003] The most unique and most concerned function of Lactococcus lactis is its ability to synthesize nisin. Nisin is an efficient and safe bacteriocin that can be used in food antibacterial. However, the synthesis of nisin is subject to negative feedback regulation by Lactococcus lactis. Therefore, there is an urgent need in the art for a Lactococcus lactis mutant strain that is tolerant and can produce high levels of nisin. Summary of the invention

[0004] The present invention aims to solve the technical problem in the prior art that the growth of Lactococcus lactis is inhibited under high concentration nisin stress and the antibacterial efficiency of the strain is low. The present invention provides a Lactococcus lactis mutant strain that tolerates and produces high nisin and application thereof.

[0005] The first aspect of the present invention provides a high-yield nisin Lactococcus lactis, which is taxonomically named Lactococcus lactis and is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31426;

[0006] The second aspect of the present invention provides the use of the above-mentioned Lactococcus lactis in the fermentation environment containing high concentration of nisin for preparing nisin;

[0007] Furthermore, the yield of nisin produced by the fermentation of Lactococcus lactis is at least 15% higher than that of the starting strain Lactococcus lactis CICC 6242;

[0008] The third aspect of the present invention provides a method for preparing the above-mentioned Lactococcus lactis, comprising the following steps:

[0009] The starting strain Lactococcus lactis CICC 6242 is continuously cultured, the culture medium is continuously cultured, the concentration of nisin in the culture medium is continuously increased to a maximum concentration, and the culture is continuously cultured for at least 10 generations, thereby obtaining the above-mentioned Lactococcus lactis;

[0010] Further, the culture medium is MRS liquid culture medium;

[0011] Further, the maximum concentration of nisin in the culture medium is 2600-2800 IU / mL;

[0012] Beneficial Effects

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The present invention adopts continuous culture to continuously increase the concentration of nisin to carry out adaptive domestication of the starting strain for up to 116 generations, and the mutant strain obtained can grow in the MRS culture medium of 2800IU / mL nisin at most, and the yield of nisin is significantly improved. The present invention proves that adaptive domestication is an effective method to improve the tolerance of Lactococcus lactis and increase the yield of nisin, and by continuously culturing the strain under nisin stress, the strain is made to produce a large number of genetic diversity mutation sites and enriched to obtain a stable strain of tolerant and high-yield nisin. It is conducive to providing excellent strains and optimized targets for the development and optimization of subsequent cell factories, and provides theoretical basis and practical experience for the application of lactic acid bacteria in the field of antibacterial of fermented foods, with good commercial and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The growth curves of the starting strain and Lactococcus lactis in MRS medium containing 2800 IU / mL nisin

[0016] Figure 2 Electron microscope scanning images of Lactococcus lactis before and after domestication

[0017] Figure 3 The inhibition effects (A) and inhibition diameter (B) of nisin, wild type (WT) and domesticated Lactococcus lactis (outer circle) on Staphylococcus aureus. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings, but it should be understood that the examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used can be obtained through commercial channels.

[0019] Example 1: Obtaining mutant strains of Lactococcus lactis

[0020] Thaw the starting strain stored in a -80°C glycerol tube at room temperature, shake it evenly, pick up the bacterial liquid with a sterilized inoculation loop, make three-zone lines on an MRS solid culture medium, and incubate it upside down in an anaerobic incubator at 37°C for 48 hours to obtain a single colony.

[0021] Single colonies were picked from the plate and inoculated into MRS liquid medium at a ratio of 2% (v / v), cultured at 37°C under anaerobic conditions for 24 hours, and subcultured three times under the same culture conditions. The 80 IU / mL concentration of nisin was used as the starting point for domestication. When the growth condition of the domesticated strain was close to that of the wild-type strain grown in MRS medium without nisin, the concentration of nisin was increased.

[0022] The above steps were repeated until the strain reached the biological limit, that is, the strain could not grow normally under the concentration of 2900-3000 IU / mL of nisin. After 10 generations of culturing in the medium with the maximum acclimation concentration of 2800 IU / mL of nisin, the growth condition did not change, and the mutant strain 6N was obtained.

[0023] Example 2: Determination of the growth characteristic curve of Lactococcus lactis 6N

[0024] The wild-type (WT) and domesticated strains were cultured to the logarithmic phase, the old culture medium was washed away with PBS buffer, and the culture medium was resuspended in fresh MRS liquid medium. Nisin at a concentration of 2800 IU / mL was added, and the initial OD was adjusted to 600nm , so that it reaches 0.10±0.05. Then, 200μL of each culture was transferred to a sterile 96-well plate with a transparent cover and sealed with a sterile sealing film. The plate was then placed in a growth characteristic instrument and cultured at 37°C and 500rpm until it reached the stationary phase. The control group used the corresponding MRS medium. The OD of the culture was measured every 10min. 600nm .

[0025] The results are as follows Figure 1As shown in Figure 2, compared with wild-type Lactococcus lactis, the hysteresis phase of Lactococcus lactis 6N was significantly shortened under 2800 IU / mL nisin stress, but the growth rate and maximum OD value of the plateau phase were significantly reduced. 600nm The value was not much different from that of the wild type. The growth of the domesticated type was close to that of the wild type without nisin stress, and was significantly better than that of the wild type under stress conditions.

[0026] Example 3: Detection of cell morphology of Lactococcus lactis 6N

[0027] The wild-type (WT) and domesticated strains were cultured at a concentration of 100 IU / mL of nisin, and subcultured three times. The bacterial precipitate was collected after centrifugation at 3800 rpm for 10 minutes using a low-speed centrifuge. The bacterial precipitate was added to PBS buffer (phosphate buffered saline) and resuspended by oscillation. After centrifugation (3800 rpm, 5 minutes), the supernatant was discarded, and then PBS buffer was added. After repeating this step to wash the bacteria three times, the bacterial sludge was resuspended in PBS buffer again and stored at 4°C as a bacterial suspension for later use.

[0028] After adjusting the OD600nm to 0.5-0.8, pipette 1mL to 1.5mL of bacterial suspension into a centrifuge tube, centrifuge at 12000rpm and 4℃ for 15min, discard the supernatant, and collect the bacterial precipitate. Wash the bacterial precipitate twice with PBS and discard the supernatant. Then, slowly add 2.5% glutaraldehyde fixative precooled at 4℃ to the centrifuge tube and place it in a 4℃ refrigerator overnight to fix. After that, pour out the fixative and rinse the sample three times with 0.1M phosphate buffer at pH 7.0, each time for 15min. Then, use 1% osmium acid solution to fix the sample for 1-2h; remove the osmium acid and rinse the sample three times with 0.1M phosphate buffer at pH 7.0, each time for 15min. Next, the samples were dehydrated using five different concentration gradients of ethanol solutions (30 v / v%, 50 v / v%, 70 v / v%, 80 v / v%, 90 v / v% and 95 v / v%), with each concentration treated for 15 min. Finally, 100% ethanol was used for two treatments, each for 20 min. The sample treatment method was changed to a 1:1 volume ratio of ethanol and isoamyl acetate for 30 min, and then the sample was treated with pure isoamyl acetate for 1 h. The samples were critical point dried and then coated, and finally the treated samples were observed using a scanning electron microscope.

[0029] The results are as follows Figure 2 As shown, Figure 2 A shows the morphology of wild type (WT) Lactococcus lactis. Figure 2B is a domesticated strain. After being cultured under 100 IU / mL nisin stress conditions, the cell morphology of the wild-type strain changed, and the surface of some spherical or ellipsoidal cells showed irregular deformation and even depression, which indicated that the cell surface structure of the strain cells was damaged under the stress of nisin. Figure 2 As shown in Figure B, the cell structure of the domesticated strain is fuller and the surface is smooth and symmetrical, which indicates that the strain has improved tolerance to nisin. In addition, judging from the aggregation of the strain cells, the strain promotes cell aggregation to form a chain structure through the C-terminal amino acid of the NisI protein, thereby preventing nisin from contacting lipid II. This is one of the ways that Lactococcus lactis self-regulates to prevent nisin from damaging its own cells. The results show that adaptive domestication enhances the strain's tolerance to the nisin produced by itself, thereby potentially achieving a higher nisin production.

[0030] Example 4: Determination of the production of nisin by Lactococcus lactis 6N

[0031] Add 0.12% agar medium to the bottom of a sterile culture dish. After solidification, place the required number of Oxford cups on top and set aside. Autoclave the LB solid medium (add 0.12% agar) and inoculate 107CFU / mL Staphylococcus aureus at a constant temperature of 50°C, then shake well. Pour the medium into the prepared culture dish and wait for it to solidify. Then take out the Oxford cup, leaving a hole where it was pulled out. After culturing the wild-type strain and the domesticated strain in liquid MRS medium for 24 hours, adjust the pH value of the bacterial solution to 6.5-7.5 with 2mol / L NaOH to eliminate the interference of acid production by the strain. Then, take 200μL of supernatant and add it to the wells of the culture dish. The solution is centrifuged at 3800rpm for 10min. Add MRS medium to other wells as a blank control, and add 200μL of 4000IU / mL of MRS medium as a positive control. Each sample is tested in triplicate. The culture dish was then placed at 4°C for at least 4 hours to allow the sample to diffuse. It was then incubated at 37°C for 24 hours. The diameter of the inhibition zone produced around the sample well was measured using a vernier caliper to indicate the antibacterial ability of the strain.

[0032] The results are as follows Figure 3 As shown in A, MRS medium was added to the well as the blank control (CK), and no inhibition zone appeared around it. However, MRS medium added with 4000 IU / mL nisin as the positive control showed obvious inhibition zone, and the inhibition zone of Lactococcus lactis 6N in the outer circle was larger than that of the wild-type strain before domestication.

[0033] In order to quantify the production of nisin, a standard curve was prepared using the nisin concentration and the diameter of the inhibition zone, and the nisin production was obtained by converting the diameter of the inhibition zone with the standard curve. Figure 3 As shown in Figure B, the diameter of the inhibition zone of 4000 IU / mL nisin is 2.06±0.01 cm, the diameter of the inhibition zone of wild-type Lactococcus lactis is 1.48±0.02 cm, and the diameter of the inhibition zone of Lactococcus lactis 6N is 1.70±0.01 cm. It can be calculated that the yield of Lactococcus lactis 6N is about 3058 IU / mL, which is about 15% higher than the 2657 IU / mL of wild-type Lactococcus lactis.

[0034] Example 5: Transcriptomic analysis of Lactococcus lactis 6N

[0035] The starting strain and Lactococcus lactis 6N were subjected to RNA extraction, mRNA enrichment and fragmentation, reverse transcription into cDNA, adapter ligation, and Illumina sequencing.

[0036] After sequencing, the base distribution and quality fluctuation statistics of each cycle of the raw data were performed, and then the raw sequencing data was filtered to obtain high-quality sequencing data, namely clean data (reads). Subsequently, the Q20, Q30, GC content, etc. of the above clean reads were evaluated. The clean reads were aligned with the reference genome (NCBI accession number: JAVIXT000000000) using HiSat2 software to obtain mapped data (reads).

[0037] RSEM software was used to calculate the expression level of genes by the number of sequences (clean reads) located in the genomic region. After obtaining the read counts, the differentially expressed genes (DEGs) between samples were identified using DEGseq software, with the screening conditions being: |log2FC|>=1 and padjust<0.05. Functional enrichment analysis of DEGs was performed using the GO and KEGG databases, and gene sets were classified according to the biological processes they participated in, the components that constituted the cells, the molecular functions they achieved, and the pathways they participated in or the functions they performed.

[0038] The results showed that Lactococcus lactis 6N had 1410 DEGs compared with the starting strain, of which 708 DEGs were up-regulated and 702 DEGs were down-regulated. The genes of lantibiotic biosynthesis sensor histidine kinase, lantibiotic related protein and lantibiotic processing related protease involved in the two-component regulatory system and quorum effect were significantly up-regulated in this strain, and the genes of lantibiotic transport system permeabilin and lantibiotic biosynthesis response regulator Nis R / Spa R involved in ATP-binding cassette protein were significantly up-regulated, indicating that adaptive domestication changed the response mechanism of the strain in resisting nisin stress, and the production of nisin increased due to the feedback mechanism of Lactococcus lactis to produce nisin.

[0039] The results showed that the expression of genes involved in peptidoglycan metabolism and receptor activity of Lactococcus lactis 6N was upregulated under nisin stress, and adaptive domestication changed the strain's ability to resist nisin stress. The expression of genes involved in ATP biosynthesis and bacteriocin-related transport and synthesis proteins was upregulated, and adaptive domestication changed the synthetic metabolic mechanism of nisin in the strain.

[0040] The basic principles, main features and advantages of the present invention are shown and described above, but the present invention is not limited to the embodiments. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A high-yield nisin Lactococcus lactis, characterized in that: The taxonomic name of the Lactococcus lactis is Lactococcus lactis, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.31426.

2. Use of the Lactococcus lactis according to claim 1 in preparing nisin in a fermentation environment containing high concentration of nisin.

3. The application according to claim 2, characterized in that: The yield of nisin produced by the lactococcus lactis fermentation is at least 15% higher than that of the starting bacteria, the lactococcus lactis CICC 6242.

4. A method for preparing Lactococcus lactis according to claim 1, characterized in that: The following steps are involved: The starting strain Lactococcus lactis CICC 6242 is continuously cultured, the culture medium is continuously cultured, the concentration of nisin in the culture medium is continuously increased to a maximum concentration, and the culture is continuously cultured for at least 10 generations to obtain the above Lactococcus lactis.

5. The method for preparing Lactococcus lactis according to claim 4, characterized in that: The culture medium is MRS culture medium.

6. The method for preparing Lactococcus lactis according to claim 4, characterized in that: The maximum concentration of nisin in the culture medium is 2600-2800 IU / mL 。

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