Lactococcus lactis capable of generating stable heat and stabilizing bacteriocin pH, and screening method and application thereof
1. Lactococcus lactis producing heat-stable and pH-stable bacteriocins, characterized in that the lactococcus lactis sieve is selected from naturally fermented kimchi, named NCU 036018.
2. The lactococcus lactis thermogenic-stable and pH-stable bacteriocin according to claim 1, characterized in that the 16S rRNA sequence of lactococcus lactis is as set forth in SEQ ID: 1 is shown.
3. The lactococcus lactis strain producing heat-stable and pH-stable bacteriocins as claimed in claim 1, wherein said lactococcus lactis strain was deposited at the china general microbiological culture collection center on 26/3/2021 with the accession number of CGMCC No. 22077.
4. The screening method of lactococcus lactis producing heat-stable and pH-stable bacteriocins according to any one of claims 1 to 3, comprising the steps of:
(1) culturing: taking 1mL of pickle sample liquid to carry out gradient dilution by using normal saline, selecting a proper concentration gradient, coating the pickle sample liquid on an MRS solid culture medium, and carrying out inverted culture at 37 ℃ for 46-50 h;
(2) and (3) purification: selecting single colonies with different forms on the culture medium in the step (2) to carry out continuous streak purification culture;
(3) and (3) storage: selecting the single bacterial colony purified in the step (3) to incubate in 10mL of MRS liquid culture medium for 22-25h, respectively taking 500 mu L of culture solution and 50% of sterilized glycerol, mixing according to a ratio of 1:1(v/v), and storing in a refrigerator at-80 ℃;
(4) and (3) activation: inoculating the strain separated and stored in the step (3) into an MRS liquid culture medium, activating for 22-25h at 37 ℃, then inoculating into the MRS liquid culture medium by 2% of inoculation amount, and culturing for 22-25h at constant temperature of 37 ℃ to obtain lactobacillus fermentation liquor;
(5) separation: centrifuging the fermentation liquor obtained in the step (4) to collect supernatant, adjusting the pH of the supernatant to 6.5, filtering with a 0.22-micron microporous filter membrane, heating the filtrate at 121 ℃ for 30min by using an autoclave, and standing at 4 ℃ for later use;
(6) screening: adding the indicator bacteria cultured for 24 hours into an LB culture medium, uniformly mixing, pouring a flat plate, drilling 8mm holes after cooling and solidification, adding 200 mu L of the filtrate prepared in the step (5) into each hole, culturing for 14-16 hours at 37 ℃, determining the diameter of a bacteriostatic circle, and screening strains with excellent bacteriostatic performance according to the diameter of the bacteriostatic circle.
5. The method of claim 4The screening method of lactococcus lactis capable of generating stable heat and stabilizing bacteriocin pH is characterized in that the pickle samples in the step (1) are obtained from different types of naturally fermented pickles in the market, and the concentration gradient is set to be 10-3，10-4，10-5。
6. The method for screening lactococcus lactis capable of producing heat-stable and pH-stable bacteriocins as claimed in claim 4, wherein the centrifugation speed in step (5) is 6500-7000g and the separation time is 10-12 min.
7. The method of claim 4, wherein the indicator bacteria of step (6) are Staphylococcus aureus and Salmonella typhimurium added to LB medium at a concentration of 1.0X 107CFU/mL。
8. A bacteriocin produced by lactococcus lactis according to any one of claims 1 to 3, wherein the bacteriocin inhibits both Salmonella typhimurium and Staphylococcus aureus at a pH in the range of 2 to 10.
9. The bacteriocin according to claim 8, wherein the bacteriocin is produced for a period of 15 to 16 h.
10. Use of the bacteriocin according to claim 8 or 9 in food fermentation and preservation processes.
Background
During the preparation and preservation of the plant-based fermented product, various potential factors can cause bacteria contamination and food poisoning. Staphylococcus aureus and salmonella typhimurium are common food-borne pathogenic bacteria. The salmonella typhimurium is one of the main pathogenic bacteria causing acute gastroenteritis, and septicemia is caused if the salmonella typhimurium is not treated in time, namely, bacteria invade blood circulation and grow and reproduce in blood to generate toxin to cause acute systemic infection. The method comprises the steps of setting a limit standard of salmonella in national standards, specifying all 11 types of foods such as meat products, aquatic products and food products, collecting 5 samples in the same batch according to a secondary sampling scheme, wherein the salmonella is not allowed to be detected from any sample in the 5 detected samples; staphylococcus aureus can produce enterotoxin to cause food poisoning, the food poisoning caused by the Staphylococcus aureus accounts for about 25% of food-borne microbial food poisoning events, and the Staphylococcus aureus becomes a microbial pathogen in the third rank and causes symptoms such as vomiting and diarrhea due to damage to human intestinal tracts. The method comprises the steps of setting up a limit standard of staphylococcus aureus in national standards, setting 8 types of foods such as meat products, aquatic products, food products, instant bean products, instant fruit and vegetable products, beverages, frozen drinks, instant seasonings and the like, collecting 5 samples in the same batch according to a three-level sampling scheme, wherein the concentration of the staphylococcus aureus in each sample does not exceed 1000CFU/g (mL), and only allowing 1 sample to be between 100 and 1000CFU/g (mL).
At present, the plant-based fermentation products mainly rely on the traditional preservatives such as benzoic acid, sodium benzoate, potassium sorbate and Nisin (Nisin) to inhibit the food-borne pathogenic bacteria. However, the use of benzoic acid, sodium benzoate and potassium sorbate is limited due to their taste, low toxicity, and the intolerance of Nisin at neutral pH.
Disclosure of Invention
The invention aims to provide lactococcus lactis (L. lactis) capable of generating stable heat and stabilizing bacteriocin by pH, a screening method and application thereof, wherein the lactococcus lactis is NCU036018, the generated bacteriocin can inhibit salmonella typhimurium and staphylococcus aureus at the same time within the pH range of 2-10, and the bacteriostatic activity is still remarkable after high-temperature treatment under the neutral pH condition.
The invention provides a lactococcus lactis strain capable of generating stable heat and stabilizing bacteriocin pH, wherein the lactococcus lactis sieve is selected from naturally fermented pickle, the strain is NCU036018, and the 16S rRNA sequence of the lactococcus lactis strain is shown as SEQ ID: 1, the lactobacillus lactis strain is deposited in China general microbiological culture Collection center on 26.3.2021, and the address is: no. 3 of Xilu No. 1 of Beijing, Chaoyang, the institute for microbiology of Chinese academy of sciences, CGMCC No.22077, with the collection number of CGMCC, is named as Lactobacillus lactis.
The invention also provides a screening method of lactococcus lactis capable of generating stable heat and stabilizing bacteriocin pH, which comprises the following steps:
(1) culture, namely taking 1mL of pickle sample liquid to perform gradient dilution by using physiological saline, selecting a proper concentration gradient, coating the gradient on an MRS solid culture medium, and performing inversion culture at 37 ℃ for 46-50 h;
(2) and (3) purification: selecting single colonies with different forms on the culture medium in the step (2) to carry out continuous streak purification culture;
(3) and (3) storage: selecting the single bacterial colony purified in the step (3) to incubate in 10mL of MRS liquid culture medium for 22-25h, respectively taking 500 mu L of culture solution and 50% of sterilized glycerol, mixing according to a ratio of 1:1(v/v), and storing in a refrigerator at-80 ℃;
(4) and (3) activation: inoculating the strain separated and stored in the step (3) into an MRS liquid culture medium, activating for 22-25h at 37 ℃, then inoculating into the MRS liquid culture medium by 2% of inoculation amount, and culturing for 22-25h at constant temperature of 37 ℃ to obtain lactobacillus fermentation liquor;
(5) separation: centrifuging the fermentation liquor obtained in the step (4) to collect supernatant, adjusting the pH of the supernatant to 6.5, filtering with a 0.22-micron microporous filter membrane, heating the filtrate at 121 ℃ for 30min by using an autoclave, and standing at 4 ℃ for later use;
(6) screening: adding the indicator bacteria cultured for 24 hours into an LB culture medium, uniformly mixing, pouring a flat plate, drilling 8mm holes after cooling and solidification, adding 200 mu L of the filtrate prepared in the step (5) into each hole, culturing for 14-16 hours at 37 ℃, determining the diameter of a bacteriostatic circle, and screening strains with excellent bacteriostatic performance according to the diameter of the bacteriostatic circle.
In the technical scheme, the bacteriocin generated by the lactococcus lactis is subjected to high-temperature sterilization pretreatment, so that the lactococcus lactis producing heat-producing unstable bacteriocin is eliminated, and the lactococcus lactis producing heat-producing stable bacteriocin can be screened conveniently.
Further, in the technical scheme, the pickle samples in the step (1) are collected from different types of naturally fermented pickles in the market, and the concentration gradient is set to be 10-3，10-4，10-5。
Further, in the above technical scheme, the centrifugal rotation speed in step (5) is 6500-7000g, and the separation time is 10-12 min.
Further, in the above technical scheme, the indicator bacteria in step (6) are staphylococcus aureus and salmonella typhimurium, and the concentration of the indicator bacteria added into the LB culture medium is 1.0 × 107CFU/mL。
The present invention also provides a bacteriocin produced by lactococcus lactis according to the above, which is capable of inhibiting both salmonella typhimurium and staphylococcus aureus at a pH in the range of 2 to 10.
Further, the production time of the bacteriocin in the technical scheme is 15-16 h.
The invention also provides the use of the bacteriocin according to the above in food fermentation and preservation processes.
Compared with the prior art, the method has the beneficial effects that:
1. the lactococcus lactis NCU036018 sieve is selected from naturally fermented pickles, and is natural and safe;
2. the bacteriocin produced by the lactococcus lactis NCU03601 can inhibit salmonella typhimurium and staphylococcus aureus at the same time within the pH range of 2-10, has still significant bacteriostatic activity after high-temperature treatment under the neutral pH condition, has more advantages compared with the traditional chemical and biological preservatives, has no taste when being used as a preservative, does not influence the quality of food, and can be widely used for fermented products and preservation.
3. The invention provides a biological preservative measure which is safe, tasteless, biodegradable and heat stable under the condition of pH neutrality, and overcomes the limitations of low toxicity, flavor development and heat sensitivity under the condition of pH neutrality of the traditional preservative.
Drawings
FIG. 1 is a graph showing the growth inhibitory effect of lactococcus lactis NCU036018 of the present invention on Staphylococcus aureus (A) and Salmonella typhimurium (B);
FIG. 2 is a microscopic examination of a colony of lactococcus lactis NCU036018 according to the present invention;
FIG. 3 is a graphical representation of the metabolic kinetic growth of bacteriocin from lactococcus lactis NCU036018 of the present invention.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials in the following examples are all commercially available products and are commercially available, unless otherwise specified.
The above-mentioned technical features of the present invention and those described in detail below (e.g., in the embodiments) can be combined with each other to form a new or preferred embodiment.
The invention is described in further detail below with reference to the figures and examples:
1. screening of lactococcus lactis strains:
(1) culturing: several samples of different kinds of naturally fermented kimchi were collected from the market. Diluting 1mL of sauerkraut sample with physiological saline, and selecting appropriate concentration (10)-3，10-4，10-5) Gradient, coating on MRS solid culture medium, and culturing at 37 deg.C for 48 hr;
(2) and (3) purification: selecting single colonies with different forms on a culture medium to carry out continuous streak purification culture;
(3) and (3) storage: then, selecting the purified single colony to incubate in 10mL of MRS liquid culture medium for 24h, respectively taking 500 mu L of culture solution and 50% of sterilized glycerol, mixing according to a ratio of 1:1(v/v), and storing in a refrigerator at-80 ℃;
(4) and (3) activation: inoculating each isolate into an MRS liquid culture medium, activating at 37 ℃ for 24h, then inoculating into the MRS liquid culture medium by 2 percent of inoculation amount, and culturing at constant temperature of 37 ℃ for 24h to obtain lactobacillus fermentation liquor;
(5) separation: centrifuging the fermentation liquid (rotation speed of 6500g for 10min), collecting supernatant, adjusting pH of the supernatant to 6.5, filtering with 0.22 μm microporous membrane, heating the filtrate at 121 deg.C for 30min in autoclave, and standing at 4 deg.C for use.
(6) Screening: the indicator bacteria (Staphylococcus aureus, Salmonella typhimurium) after 24h culture were added to LB medium to a concentration of 1.0X 107And CFU/mL, uniformly mixing, pouring a flat plate, cooling, solidifying, punching (8mm), adding 200 mu L of prepared fermentation supernatant into each hole, culturing at 37 ℃ for 14-16h, measuring the diameter of an inhibition zone by using a vernier caliper, screening a strain with excellent antibacterial performance according to the diameter of the inhibition zone, and naming the strain as NCU 036018. Among them, as can be seen from fig. 1, the fermentation supernatant of the strain NCU036018 showed a significant inhibitory ability against the growth of staphylococcus aureus and salmonella typhimurium.
2. Lactococcus lactis colony morphology:
lactococcus lactis NCU036018 was applied to MRS containing bromocresol purple, and observed after culture, as can be seen from FIG. 2, the colony morphology of the strain NCU036018 on the plate was small, round convex, glossy, gram-positive, and streptococcal.
3. Catalase test:
a single colony of the target strain is picked up on a glass slide, and then a plurality of drops of 3% hydrogen peroxide are added dropwise and observed immediately, wherein the strain NCU036018 does not generate bubbles, which indicates that the strain is negative.
4. Sugar fermentation experiment:
performing physiological and biochemical tests such as hydrogen sulfide and sugar fermentation on the target strain by adopting a trace biochemical tube, and analyzing the physiological and biochemical characteristics of the strain according to a common bacteria system identification manual and a Bergey bacteria identification manual to determine the species of the target strain: respectively inoculating 24h culture solution of bacteria to be detected into NaCl (4%), hydrogen sulfide, glucose, arabinose, cellobiose, fructose, galactose, lactose, maltose, mannose, melezitose, melibiose, raffinose, rhamnose, ribose, saligenin, sorbose, sucrose, xylose and esculin micro fermentation tubes, placing the micro fermentation tubes with openings facing downwards, culturing in a sterilized culture dish at 37 ℃ for 24h, observing results and recording.
If the strain can ferment a certain sugar or alcohol to produce acid, the indicator bromocresol purple in the culture medium changes from purple (pH 7.0) to yellow (pH 5.4), and if the strain is not fermented, the purple is still kept. If gas is generated while fermentation is performed, bubbles may be generated in the inverted tube in the liquid medium.
As can be seen from the results of sugar fermentation of lactococcus lactis NCU036018 in Table 1, this strain was able to ferment glucose, galactose, maltose, fructose and mannose in liquid culture, and inulin was not available; experimental comparison also shows that the NaCl does not resist 4 percent.
TABLE 1 lactococcus lactis NCU036018 sugar fermentation results
5. Strain NCU036018 bacteriocin metabolism kinetics:
inoculating strain NCU036018 into MRS liquid culture medium, activating at 37 deg.C for 24 hr, inoculating 2% of strain into MRS liquid culture medium, continuously culturing at 37 deg.C for 36 hr, sampling every 2 hr, and detecting OD of fermentation liquid by ultraviolet spectrophotometer, pH meter and agar plate punching method600pH and bacteriostatic activity.
The growth curve of the strain NCU036018 in the MRS liquid culture medium is shown in FIG. 3, the logarithmic phase is started after about 4 hours of culture, the colony count begins to grow exponentially along with time, and the thallus begins to enter the stationary phase after about 8 hours; the pH of the fermentation supernatant liquid is rapidly reduced from about 6.53 initially until the pH gradually stabilizes at about 4.2 in the logarithmic phase; the bacteriostatic performance of the strain NCU036018 fermentation liquor is increased and then decreased with time, and the optimal production time of the bacteriocin is finally determined to be about 16 h.
6. Molecular biological identification:
(1) extraction of lactic acid bacteria genomic DNA was performed using a bacterial DNA extraction kit.
(2) The obtained genomic DNA was subjected to PCR amplification using bacterial universal primers. The PCR reaction system and reaction procedure are shown in tables 2 and 3. The PCR product was sent to Shanghai Biotech for sequencing, and the obtained sequences were subjected to homology comparison in Gene Bank database using BLAST tool, thereby determining the species of the isolate.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction procedure
And (3) identification result: the length of the obtained sequence fragment is 980bp, the obtained sequence is input into NCBI for BLAST sequence alignment, and the result shows that the homology of the obtained sequence fragment and Lactococcus latis is more than 97%.
Example 1: biodegradability of bacteriocin produced by strain NCU036018
Culturing the activated strain NCU036018 in an MRS culture medium at 37 ℃ for 24h, centrifuging at 4 ℃ for 10min at 6500g to obtain cell-free fermentation supernatant, dividing the cell-free fermentation supernatant into 6 groups, adding catalase, pepsin, trypsin, papain, proteinase K and alpha-chymotrypsin at 37 ℃ for treatment for 2h, wherein the final concentration of each enzyme is 1mg/mL, placing the mixed solution in a 100 ℃ water bath kettle for 5min to inactivate the enzymes, adjusting the pH to 6.5, respectively measuring the inhibitory activity of different treatment groups on the indicator bacterium staphylococcus aureus, and referring to Table 4 for the sensitivity of bacteriocin produced by the strain NCU036018 to different enzymes.
TABLE 4 susceptibility of bacteriocins produced by lactococcus lactis NCU036018 to different enzymes
The result shows that the bacteriocin produced by the bacterium is not sensitive to amylase and catalase, but sensitive to trypsin, papain, proteinase K and alpha-chymotrypsin, namely the bacteriocin can be biodegraded by a plurality of proteases.
Example 2: excellent pH stability of bacteriocin produced by strain NCU036018
The activated strain NCU036018 was cultured in MRS medium at 37 ℃ for 24h and centrifuged at 6500g for 10min at 4 ℃. Removing the precipitate to obtain cell-free fermented supernatant, dividing the cell-free fermented supernatant into 9 groups, adjusting the pH of the solution to 2, 3, 4, 5, 6, 7, 8, 9 and 10, treating for 30min, adjusting the solution to pH 6.5, and respectively measuring the inhibitory activity of different treatment groups on indicator bacteria staphylococcus aureus, wherein the pH stability of bacteriocin produced by the strain NCU036018 is shown in Table 5.
TABLE 5 pH stability of bacteriocins produced by lactococcus lactis NCU036018
The result shows that the bacteriocin produced by the lactococcus lactis NCU036018 shows significant bacteriostatic activity in the pH range of 2-10, which indicates that the bacteriocin has excellent pH stability.
Example 3: excellent thermal stability of bacteriocin produced by strain NCU036018
Inoculating strain NCU036018 into MRS culture medium, culturing overnight, centrifuging the fermentation liquid, removing thallus (6500g, 10min), adjusting pH of the obtained lactobacillus fermentation supernatant to 6.5, treating in water bath at 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C and high pressure steam at 121 deg.C for 30min, and using untreated fermentation supernatant as control. Staphylococcus aureus is used as an indicator bacterium, an agar diffusion method is adopted for determining the bacteriostatic activity, the influence of heat treatment on the bacteriostatic activity of the supernatant liquid of pH neutral fermentation is researched, and the thermal stability of bacteriocin produced by the strain NCU036018 is shown in Table 6.
TABLE 6 Heat stability of bacteriocins produced by lactococcus lactis NCU036018
The results show that the bacteriostatic activity is still remarkable after the pH neutral fermentation supernatant is treated at different temperatures for 30min, which indicates that the bacteriocin produced by the bacterium is high temperature resistant under the pH neutral condition.
In conclusion, the invention provides a biological preservative measure which is safe, tasteless, biodegradable and thermally stable under the condition of neutral pH, and overcomes the limitations of low toxicity, flavor development and heat sensitivity under the condition of neutral pH of the traditional preservative.
Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> university of Nanchang
<120> lactococcus lactis capable of generating stable heat and stabilizing bacteriocin pH, and screening method and application thereof
<130> 20210601
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 980
<212> DNA
<213> lactococcus lactis (L.lactis)
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