CN102286393A - Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide - Google Patents
Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide Download PDFInfo
- Publication number
- CN102286393A CN102286393A CN2011100489122A CN201110048912A CN102286393A CN 102286393 A CN102286393 A CN 102286393A CN 2011100489122 A CN2011100489122 A CN 2011100489122A CN 201110048912 A CN201110048912 A CN 201110048912A CN 102286393 A CN102286393 A CN 102286393A
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- lactis
- lactococcus lactis
- lactococcus
- antibacterial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and the application of antibacterial peptide. In the invention, the collection number of the lactococcus lactis subsp.lactis LLC518 in the General Microorganism Center of China Microorganism Culture Collection Management Committee is CGMCC No. 4584. In the invention, the antibacterial peptide produced by the lactococcus lactis subsp.lactis can restrain micrococcus tetragenus, Bacillus thuringiensis, lactococcus lactis and listeria innocua, particularly, has strong restraint function to L. monocytogenes which can cause serious food poisoning. Thus, the lactococcus lactis subsp.lactis and the antibacterial peptide thereof have active function in food fresh keeping.
Description
Technical field
The invention belongs to Microbial resources and utilize the field, be specifically related to a kind of Lactococcus lactis and the antibacterial peptide of this Lactococcus lactis generation and the purposes of this antibacterial peptide.
Background technology
Foodborne bacterial pathogens and food spoilage bacterium not only have a strong impact on human health, and cause a large amount of wastes of grain.Some bacteriogenic bacteriocins are antibacterial peptides of class protein character, generally believe that at present utilizing aliment security level bacteriocin lab instead of chemical antisepsis antistaling agent is the key that improves food safety and ensure grain security, thereby screen the technology of preparing that new bacteriocin produces bacterial strain and antibacterial peptide development and national economy is had great importance.
Milk-acid bacteria just is widely used in the anti-corrosive fresh-keeping of food since ancient times, and small-molecule peptide material bacteriocin in numerous antibacterial substances that milk-acid bacteria produces is owing to can more have been highlighted its security and the using value in food fresh keeping by the enzyme liberating of human secretory.The bacteriocin Nisin that Lactococcus lactis produces is unique bacteriocin that can be used for food fresh keeping of FAO approval, the toxicity of Nisin extremely low (LD50 is about the 7g/kg body weight), in saliva, can be degraded fully in the 10min, can not cause anaphylaxis, thereby be widely used in the anti-corrosive fresh-keeping of milk-product, meat product, acid tinned pre-and leavened food.But because Nisin can only be soluble status under acidic conditions, solvability is low under neutrallty condition, poor stability, and easily adsorbed by meat proteins, these characteristics have greatly influenced the application of Nisin in meat product is fresh-keeping, therefore are necessary to develop new polypeptide antiseptic-germicide.
Summary of the invention
The purpose of this invention is to provide a kind of Lactococcus lactis.
For achieving the above object, the present invention has adopted following technical scheme: a kind of Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518, in
On January 26th, 2011Be preserved in
China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), deposit number is:
CGMCC No.4584
Lactococcus lactis among the present invention (Lactococcus lactis subsp.lactis) LLC518 is that screening obtains from the pregnant solution of commercially available cucumber preparation, concrete screening method is for screening the acid-producing bacteria strain earlier with the MRS selective medium that contains 0.01% purpurum bromocresolis, again by screening the bacterium dibbling at MRS solid culture primary surface, after the tangible bacterial plaque of microscler one-tenth to be generated, the one deck that falls again above contains the upper strata substratum of indicator Bacterium lacticum (Lactobacillus sp.) LSX801,28 ℃ of cultivations, with the bacterial strain that produces inhibition zone further by fermentation, obtain fermented supernatant fluid, and by getting rid of organic acid in the fermented liquid, the bacteriostatic action of hydrogen peroxide contains protein-based antibacterial substance thereby further can eliminate in the fermented liquid of bacteriostatic activity proof bacterial strain by Chymotrypsin and papoid.Learn feature and 16SrDNA sequential analysis by morphological feature, Physiology and biochemistry, according to " uncle Jie Shi division bacteria handbook and " the lactic-acid-bacterium classification is identified and experimental technique ", show that this bacterial strain belongs to Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), is numbered LLC518.
Second purpose of the present invention provides a kind of antibacterial peptide of above-mentioned Lactococcus lactis generation and the production method of antibacterial peptide.
Antibacterial peptide of the present invention is the antibacterial peptide that obtains by lactic acid-fermenting galactococcus (Lactococcus lactissubsp.lactis) LLC518CGMCC No.4584, called after LacticinLLC518.
The preparation method of antibacterial peptide can pass through two kinds of approach:
First approach: at first from the MRS slant medium picking activatory Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 be inoculated in the MRS seed culture medium, 28 ℃ of shaking tables were cultivated 12 hours, seed liquor is inoculated in the MRS fermention medium by 2～6% inoculum size, 28 ℃ leave standstill and cultivate 24h and can obtain fermented liquid again;
Again that the fermented liquid that obtains is centrifugal; Get supernatant liquor, 0.45 μ m membrane filtration carries out ammonium sulfate precipitation to filtered solution; Centrifugal then, get the protein precipitation that precipitation obtains; Protein precipitation is dissolved in the water, adopts the dialysis tubing dialysis treatment of molecular weight cut-off 2000Da, the vacuum lyophilization dialyzate obtains rough dry powder; Again rough dry powder is dissolved in the water, and, collects active peak and carry out the reversed-phase HPLC processing, to obtain the pure product of antibacterial peptide through Sephadex G-50 gel chromatography.
The actual conditions of the above-mentioned purification step of antibacterial peptide is as follows: with described fermented liquid through 4 ℃, the centrifugal 25min of 8000r/min, get the membrane filtration of supernatant liquor via hole diameter 0.45 μ m, gained filtrate is carried out ammonium sulfate precipitation with 30～50% saturation ratio, behind the 12h, 4 ℃, 8000r/min is centrifugal, and 45min obtains protein precipitation, get the 4h that in the dialysis tubing of molecular weight cut-off 2000Da, dialyses after protein precipitation is dissolved in the water, dialyzate is put into-20 ℃ of freezing backs of refrigerators and made described rough dry powder with freeze drier; Rough dry powder is dissolved in the water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect active peak, RP-HPLC prepares the pure product of antibacterial peptide;
Described gel chromatography adopts Sephadex G-50 column packing, and (26 * 100cm), applied sample amount is 5ml, and flow velocity is 1.5ml/min, and elutriant is through 0.22 μ m membrane filtration secondary water, and it is 70-85min that retention time is collected at active peak;
The RP-HPLC chromatography condition is: chromatographic column: C18 post SymmetryShield
TM, specification: 19 * 150mm, applied sample amount: 970 μ l, flow velocity: 2.5ml/min, temperature: 25 ℃, wavelength: 215nm, elutriant A are ultrapure water, and B is a trifluoroacetic acid aqueous solution.Program: 0-6min:0-5%B, 6-66min:5-100%B, 66-80min:100%B; It is 32.2-32.5min that retention time is collected at active peak.
Second approach: at first from the MRS slant medium picking activatory Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 be inoculated in the MRS seed culture medium, 28 ℃ of shaking tables were cultivated 12 hours, be inoculated in the MRS fermention medium by 2～6% inoculum size, 28 ℃ leave standstill and cultivate 24h and can obtain fermented liquid again;
Again with fermented liquid after the ceramic membrane filter degerming, spraying drying obtains pulverous antibacterial peptide finished product.
The actual conditions of spraying drying step is as follows: with fermented liquid under the condition of 25 ℃ of temperature, by working pressure is 0.02Mpa, membrane pore size is the filtered liquid that obtains removing thalline behind the ceramic membrane filter of 0.2 μ m, in this filtered liquid, add solid salt, the volume ratio that the quality of solid salt accounts for filtered liquid is 5～20%, treat to carry out spraying drying after solid salt dissolves fully, the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, promptly gets pulverous antibacterial peptide finished product.
The antibacterial peptide that is made by above-mentioned any approach also all belongs to protection scope of the present invention.
The 3rd purpose of the present invention provides a kind of above-mentioned antibacterial peptide purposes in meat product is fresh-keeping.
Antibacterial peptide among the present invention is particularly useful for pork is carried out fresh-keeping, and actual conditions is to add 12ml antibacterial peptide solution in every 100g pork, and the concentration of the mass percent of antibacterial peptide is 3.1%.
Beneficial effect of the present invention is as follows:
1, this antibacterial peptide Lacticin LLC518 is different with existing antibacterial peptide Nisin, and Nisin can be degraded by alpha-chymotrypsin, and is insensitive to pronase e, trypsinase and stomach en-.And this antibacterial peptide is except that can also being degraded by papoid, pronase e and Proteinase K by the alpha-chymotrypsin degraded.
2, the antimicrobial spectrum of the antibacterial peptide of this Lactococcus lactis generation is different from existing antibacterial peptide Nisin, to the micrococcus luteus unrestraint effect of Nisin sensitivity.
3, the antibacterial peptide of this Lactococcus lactis generation has activity in wider pH scope, particularly still has bacteriostatic action under alkaline condition.
4, the mode of action killing bacteria of this antibacterial peptide, and have significant germicidal action under the storage conditions under 4 ℃ of conditions.
5, this antibacterial peptide has stable bacteriostatic action in meat product, when with EDTA, potassium sorbate combined action, can make bacteriostatic action reach maximum value.
Description of drawings
Fig. 1 be rough dry powder water-soluble and behind membrane filtration the elution curve by Sephadex G-50;
Fig. 2 is the bacteriostatic action synoptic diagram of Sephadex-G-50 detached peaks;
Fig. 3 is an antibacterial peptide high performance liquid chromatography elution curve;
Fig. 4 is the antibacterial peptide mass spectrum;
Fig. 5 A is the modes of action of 37 ℃ of following antibacterial peptides to LSX801;
Fig. 5 B is the modes of action of 4 ℃ of following antibacterial peptides to LSX801;
Fig. 5 C is the modes of action of 37 ℃ of following antibacterial peptides to listeria monocytogenes;
Fig. 5 D is the modes of action of 4 ℃ of following antibacterial peptides to listeria monocytogenes;
Fig. 6 antibacterial peptide is to the restraining effect of bacterium in the pork;
The pure product antibacterial peptide of Fig. 7 is to the restraining effect of indicator;
The morphological specificity of Fig. 8 Lactococcus lactis LLC518 is observed figure.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Lactococcus lactis LLC518 of the present invention obtains in the following ways:
1, the fresh cucumber that will purchase from market, Hefei ,Anhui is cleaned and is cut into small pieces, and fills the sterile test tube of 50ml, compacting, and with plastic skin and kraft paper sealing, 30 ℃, behind the enrichment culture 48h, sampling is coated the MRS that contains 0.01% purpurum bromocresolis and is selected substratum.
2, picking makes the acid-producing bacteria strain of the MRS selection substratum flavescence that contains 0.01% purpurum bromocresolis, further separation and purification;
2, be indicator with Bacterium lacticum (Lactobacillus sp.) LSX801, obtain to have the bacterial strain of bacteriostasis by dull and stereotyped dibbling method;
3, obtain fermented liquid by fermentation culture, centrifugal treating obtains supernatant liquor, and removes thalline by 0.45 μ m membrane filtration, adds 40% ammonium sulfate processing filtrate, and makes crude protein dry powder through dialysis, lyophilize.In the water-soluble solution of dry powder, be added in the ox Feng cup, adopt dull and stereotyped diffusion process screening that the bacterial strain of bacteriostasis is arranged;
4. handle crude protein dry powder by Chymotrypsin, papoid, pronase e, Proteinase K, stomach en-, trypsinase, lipase, determine that antibacterial substance has protein properties;
5. by the compare of analysis of morphology and physiological and biochemical property and 16SrDNA sequence signature, determine that Lactococcus lactis LLC518 is Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis).
The biological property of Lactococcus lactis LLC518 is as follows:
Bacterium colony is rounded on the MRS plate culture medium, central uplift, smooth surface, neat in edge, oyster white, colony diameter 0.5～1.0mm.Do not have mycoderm in the MRS liquid nutrient medium, bacterium liquid muddiness has precipitation to produce.Gram-positive microorganism, sphere, the about 0.5～0.8um of thalline diameter, no gemma, atrichia, single or paired existence.Under anaerobic growing way is than good under the aerobic condition, and the relative aerobic condition of colony diameter increases about 0.5mm down under the anaerobic condition, and growing state is also good under aerobic conditions, belongs to facultative anaerobe.
Can grow between 20～40 ℃, not grow for 45 ℃, wherein optimum growth temperature is 30 ℃.Under the 0-7% sodium chloride concentration, all can grow.
The catalase feminine gender; But decomposition glucose produces acid, but aerogenesis not also do not produce hydrogen sulfide, but the hydrolyzable arginine produces ammonia; Can decompose Vitamin C2, but not starch-splitting and gelatin; The litmus milk experiment is positive, but does not produce proteolytic enzyme, and milk is peptonized; The voges-Proskauer test feminine gender shows that the unfermentable glucose of this bacterium generates acetyl methyl carbinol.
Glucose fermentation produces acid, but aerogenesis not, but xylose-fermenting, semi-lactosi, lactose, sucrose, pectinose, lactose, maltose and N.F,USP MANNITOL produce acid, utilizes raffinose and inositol but does not produce acid.
The sequence of the 16SrDNA of Lactococcus lactis LLC518 is:
GCAGGCACGCTGGCGGCGTGCTATACATGCAAGTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGGGTGAGTAACGCGTGGGGAATCTGCCTTTGAGCGGGGGACAACATTTGGAAACGAATGCTAATACCGCATAAAAACTTTAAACACAAGTTTTAAGTTTGAAAGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACGTTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGTATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATTC CTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTCCTAAGTAAGACCGAGCTTC
The preparation process of antibacterial peptide is as follows:
The fermentation of A, antibacterial peptide: from the MRS slant medium picking activatory Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 be inoculated in the MRS seed culture medium, 28 ℃ of shaking tables were cultivated 12 hours, seed liquor is inoculated in the MRS fermention medium by 4% inoculum size, 28 ℃ leave standstill and cultivate 24h and can obtain fermented liquid again;
The isolation and purification of B, antibacterial peptide: with described fermented liquid through 4 ℃, the centrifugal 25min of 8000r/min, get the membrane filtration of supernatant liquor via hole diameter 0.45 μ m, gained filtrate is carried out ammonium sulfate precipitation with 30～50% saturation ratio, behind the 12h, 4 ℃, 8000r/min is centrifugal, and 45min obtains protein precipitation, gets the 4h that dialyses in the dialysis tubing of molecular weight cut-off 2000Da after protein precipitation is dissolved in the water, dialyzate is put into-20 ℃ of freezing backs of refrigerators and is made described rough dry powder with freeze drier; Rough dry powder is dissolved in the water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect active peak, RP-HPLC prepares the pure product of antibacterial peptide;
Described gel chromatography adopts Sephadex G-50 column packing, and (26 * 100cm), applied sample amount is 5ml, and flow velocity is 1.5ml/min, and elutriant is through 0.22 μ m membrane filtration secondary water, and it is 70-85min that retention time is collected at active peak;
The RP-HPLC chromatography condition is: chromatographic column: C18 post SymmetryShield
TM, specification: 19 * 150mm, applied sample amount: 970 μ l, flow velocity: 2.5ml/min, temperature: 25 ℃, wavelength: 215nm, elutriant A are ultrapure water, and B is a trifluoroacetic acid aqueous solution.Program: 0-6min:0-5%B, 6-66min:5-100%B, 66-80min:100%B; It is 32.2-32.5min that retention time is collected at active peak.
Rough dry powder water-soluble and behind membrane filtration the elution curve by Sephadex G-50 as shown in Figure 1, and the bacteriostatic action of Sephadex-G-50 detached peaks is as shown in Figure 2,1 is the Sephadex-G-50 first peak among Fig. 2,2 is Sephadex-G-50 second peak.
The elution curve of RP-HPLC as shown in Figure 3.
The preparation process of antibacterial peptide is as follows:
The fermentation of A, antibacterial peptide: from the MRS slant medium picking activatory Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 be inoculated in the MRS seed culture medium, 28 ℃ of shaking tables were cultivated 12 hours, seed liquor is inoculated in the MRS fermention medium by 4% inoculum size, 28 ℃ leave standstill and cultivate 24h and can obtain fermented liquid again;
B, with fermented liquid under the condition of 25 ℃ of temperature, by working pressure is 0.02Mpa, membrane pore size is to obtain filtered liquid behind the ceramic membrane filter of 0.2 μ m, in this filtered liquid, add solid salt, the volume percent that the quality of solid salt accounts for filtered liquid is 5～20%, treats to carry out spraying drying after solid salt dissolves fully, and the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, promptly gets pulverous antibacterial peptide finished product.
The mass ratio of solid salt is to the influence of antibacterial peptide vigor: add 5%, 10%, 15%, 20% salt in the fermented supernatant fluid of 150ml Lactococcus lactis LLC518 respectively, inlet temperature is made as 140 ℃, air outlet temperature is made as 120 ℃, after the spray drying treatment, the sample of getting under each condition 0.1 restrains in 5ml water, measures its vigor, and the vigor of gained sample is as shown in table 1, can obtain simultaneously: when adding 15% salt, the total activity maximum.
The influence of table 1 auxiliary material comparison product vigor
Different inlet temperature are to the influence of product vigor: Lactococcus lactis LLC518 fermented liquid is added 15% salt behind the film system filtration, carry out spraying drying after dissolving fully.The hot blast inlet temperature of spray-drier is made as 130 ℃, 135 ℃, 140 ℃, 145 ℃, 150 ℃ respectively, air outlet temperature is made as 120 ℃, collect powdery substance, the sample of getting under each condition 0.1 restrains in 5ml water, survey its vigor, the vigor of gained sample is as shown in table 2, can obtain simultaneously: when inlet temperature is 150 ℃, and the total activity maximum.
Table 2 inlet temperature is to the influence of product vigor
Different air outlet temperatures are to the influence of total activity: Lactococcus lactis LLC518 fermented liquid is added 15% salt behind the film system filtration, carry out spraying drying after dissolving fully.The hot blast air outlet temperature of spray-drier is made as 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃ respectively, inlet temperature is made as 150 ℃, collect powdery substance, the sample of getting under each condition 0.1 restrains in 5ml water, survey its vigor, the vigor of gained sample is as shown in table 3, can obtain simultaneously: when air outlet temperature is 130 ℃, and the total activity maximum.
Table 3 air outlet temperature is to the influence of antibacterial peptide vigor
The physico-chemical property of antibacterial peptide
The antibacterial peptide that Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 produces is different from present widely used lactobacillin Nisin, and multiple food-borne pathogens and food spoilage bacterium are had stronger restraining effect.Bacteriostatic activity is stable in wider pH scope, can be by the multiple protein enzyme liberating, thereby be the higher antibacterial peptide of a kind of security.
Antibacterial peptide also is that the feature of Lacticin LLC518 is as follows:
1, the antibacterial substance of this bacterial strain generation has protein properties, handles with Chymotrypsin, papoid, pronase E and Proteinase K, and anti-microbial activity disappears, and is as shown in table 4.Molecular weight through this bacteriocin of mass spectroscopy is 3344.6Da;
Table 4Lacticin LLC518 is to the susceptibility of enzyme
Lacticin LLC518 is to Chymotrypsin, papoid, pronase E, Proteinase K sensitivity as can be seen from the above table, and is insensitive to stomach en-, trypsinase, lipase.
2, the antimicrobial spectrum of antibacterial peptide: tetrads (Micrococcus tetragenus), Tribactur (Bacillus thuringiensis), Bacterium lacticum (Lactobacilluas sp.) LSX0801, Ying Nuoke listeria bacteria (Lister innocua) GIM1.230, Listeria monocytogenes (Lister monocytogenes) are had restraining effect, to Lactococcus lactis, micrococcus luteus unrestraint effect.
Antibacterial peptide also is that the antimicrobial spectrum of lacticin LLC518 is as shown in table 5.
The antimicrobial spectrum of table 5Lacticin LLC 518
3, the character of antibacterial peptide:
A, to the susceptibility of temperature: under acidic conditions, can tolerate 100 ℃ of high temperature, but under alkaline condition, 100 ℃ of processing vigor reduce to zero;
Antibacterial peptide is stable as shown in table 6 to heat:
Table 6lacticin LLC518 is to the stability of heat
| 100℃ | Quality (mg) | (AU) tires | Than living |
| pH2.0 | 2 | ||
| |
2 4 | 8 | |
| |
2 4 | 8 | |
| |
2 3 | 4 | |
| pH4.0 | 2 | ||
| |
2 3 | 4 | |
| |
2 2 | 2 | |
| |
2 1 | 1 | |
| pH7.0 | 2 | ||
| |
2 1 | 1 | |
| |
2 1 | 1 | |
| |
0 | 0 | |
| pH8.0 | 2 | ||
| |
0 | 0 | |
| |
0 | 0 | |
| |
0 | 0 |
B, to the susceptibility of pH: this antibacterial peptide is stable in pH2～10, and vigor remains unchanged, shown in the table 7.
Table 7Lacticin LLC518 is to the stability of pH
| pH | Quality (mg) | (AU) tires | Than living |
| 2.0 | 2 | 2 4 | 8 |
| 3.0 | 2 | 2 4 | 8 |
| 4.0 | 2 | 2 4 | 8 |
| 5.0 | 2 | 2 4 | 8 |
| 6.0 | 2 | 2 4 | 8 |
| 7.0 | 2 | 2 4 | 8 |
| 8.0 | 2 | 2 4 | 8 |
| 10.0 | 2 | 2 4 | 8 |
4, the mode of action of antibacterial peptide: this antibacterial peptide all has germicidal action 4 ℃ and 37 ℃ to indicator.
The mode of action of antibacterial peptide: this antibacterial peptide all has germicidal action 4 ℃ and 37 ℃ to indicator (Bacterium lacticum LSX801, listeria monocytogenes).Experimental result shown in Fig. 5 A, Fig. 5 B, Fig. 5 C, Fig. 5 D, among Fig. 5 A～D ▲ be CK, ■ is an antibacterial peptide.
With 25mg/ml antibacterial peptide solution, MRS, LB nutrient solution join OD respectively for contrast
600Be in 0.2 the bacteria suspension, to place 4 ℃ of preservations and 37 ℃ of cultivations, regularly coat in MRS, the LB solid medium and carry out live bacterial count.
In the time of 37 ℃, antibacterial peptide solution is sterilization for the mode of action of Bacterium lacticum LSX801.Untreated initial viable count is 1 * 10
7, will be with antibacterial peptide solution-treated 2h, 4h, 6h, 8h, the LSX801 bacterium of 10h is coated with respectively to cultivate and observes, and the viable bacteria number average reduces.Viable count 3 orders of magnitude that descend altogether in treatment time 8h, germicidal action is stronger.Antibacterial peptide solution is antibacterial for the mode of action of listeria monocytogenes.Untreated initial viable count is 3 * 10
7, will handle 2h with bacteriocin, 4h, 6h, 8h, the listeria monocytogenes of 10h are coated with respectively to cultivate and observe, and viable count reduces the back earlier and slowly rises.Rising in treatment time 10h only has an order of magnitude, than contrast tangible bacteriostatic action is arranged.
A lot of food need cryopreservation, but some bacteriocin can not be brought into play its effect well under frozen state.And in the time of 4 ℃, this antibacterial peptide solution still has stronger germicidal action for Bacterium lacticum LSX801, listeria monocytogenes.
Antibacterial peptide has certain anti-microbial effect in fresh-keeping meat.
The mensuration of fresh pork microbial count
The processing of fresh pork sample: get sample meat 20g at random with aseptic scissors, 3 are divided into one group at random.Adopt preservative film and vacuum packaging respectively, total plate count is regularly surveyed in 4 ℃ of preservations.
The mensuration of total plate count: in above-mentioned sample, add sterilized water 180ml,, press the decimal dilution method dilution behind the mixing with the centrifugal 1min of 8000r/min.Respectively from 10
-2～10
-7Take out 0.1ml in the diluent of gradient and coat on the LB substratum, 37 ℃, cultivate 48h, carry out enumeration, calculate contained total plate count in every gram primary sample.
Antibacterial peptide is to the mensuration of the bacteriostatic action of Bacterium lacticum LSX801 in the pork: the pork of will sterilizing is in the bacterium liquid (OD of Bacterium lacticum LSX0801
600=0.2) after soaking 5min in and draining 5min, is that 3.1% antibacterial peptide is applied in the pork surface, vacuumizes 4 ℃ of refrigerators and preserve that regularly detect lactic acid bacteria number and pH value, the interpolation sterilized water is organized in contrast in pork with the concentration of mass percent.
Test the result as shown in Figure 6, Fig. 6 shows that the initial germicidal action of antibacterial peptide is remarkable, adds antibacterial peptide and handles the 1st day total plate count in back at 1.46～1.62log (cfug
-1) between, control group is 2.3～2.4log (cfug
-1).Simultaneously in lay up period antibacterial peptide treatment group total plate count increasing degree less than control group, therefore change matrix environment after this antibacterial peptide still have stable bacteriostatic action, it is of practical significance in the fresh-keeping application of meat-based food.
The anti-microbial effect of composite preservative in fresh-keeping meat that antibacterial peptide and fungistat proportioning commonly used form.
Use the fence effect principle, investigate the fresh-keeping effect of EDTA, potassium sorbate and Lacticin LLC518 compound prescription, analyze the effect of antibacterial peptide in the fresh-keeping application of reality.
The concentration range of each preservation agent is in the composite preservative: 0.18%～0.6%EDTA, 0.05%～0.24% potassium sorbate, 0.63%～3.1% antibacterial peptide Lacticin LLC518.
Above-mentioned composite preservative is handled the pork sample by the mode among the embodiment 5, coat the pork surface, vacuumize 4 ℃ of refrigerators and preserve, regularly bacterial detection content is control group with the alternative antibacterial peptide solution of sterilized water.Experimental result is carried out statistical analysis.
Discover that concentration as EDTA is 0.33%, the concentration of potassium sorbate is 0.12%, when the concentration of antibacterial peptide is 1.44%, the total plate count minimum of pork sample, wherein there are significant interaction (P=0.003＜0.01) in antibacterial peptide and EDTA.
Claims (10)
1. a Lactococcus lactis (Lactococcus lactis subsp.lactis) LLC518, its
The deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No.4584.
2. the preparation method of an antibacterial peptide, its by Lactococcus lactis (Lactococcus lactissubsp.lactis) LLC518CGMCC No.4584 fermentation to obtain antibacterial peptide.
3. the preparation method of antibacterial peptide according to claim 2, it comprises following preparation process: from the MRS slant medium picking activatory Lactococcus lactis (Lactococcus lactissubsp.lactis) LLC518CGMCC No.4584 be inoculated in the MRS seed culture medium, 28 ℃ of shaking tables are cultivated and were reached stationary phase in 12 hours, be inoculated in the MRS fermention medium by 2～6% inoculum size again, 28 ℃ leave standstill and cultivate 24h and reach stationary phase, can obtain fermented liquid.
4. according to the preparation method of claim 2 or 3 described antibacterial peptides, it is characterized in that the antibacterial peptide that to obtain purifying in accordance with the following methods: the fermented liquid that obtains is centrifugal; Get supernatant liquor, 0.45 μ m membrane filtration carries out ammonium sulfate precipitation to filtered solution; Centrifugal then, get protein precipitation; Protein precipitation is dissolved in the water, adopts the dialysis tubing dialysis treatment of molecular weight cut-off 2000Da, the dialyzate vacuum lyophilization is obtained rough dry powder; Again rough dry powder is dissolved in the water, and, collects active peak and carry out the reversed-phase HPLC processing, to obtain the pure product of antibacterial peptide through Sephadex G-50 gel chromatography.
5. the preparation method of antibacterial peptide according to claim 4, it is characterized in that: with described fermented liquid through 4 ℃, the centrifugal 25min of 8000r/min, get the membrane filtration of supernatant liquor via hole diameter 0.45 μ m, gained filtrate is carried out ammonium sulfate precipitation with 30～50% saturation ratio, behind the 12h, 4 ℃, 8000r/min is centrifugal, and 45min obtains protein precipitation, get the 4h that in the dialysis tubing of molecular weight cut-off 2000Da, dialyses after protein precipitation is dissolved in the water, dialyzate is put into-20 ℃ of freezing backs of refrigerators and made described rough dry powder with freeze drier; Rough dry powder is dissolved in the water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect active peak, RP-HPLC prepares the pure product of antibacterial peptide;
Described gel chromatography adopts Sephadex G-50 column packing, and (26 * 100cm), applied sample amount is 5ml, and flow velocity is 1.5ml/min, and elutriant is through 0.22 μ m membrane filtration secondary water, and it is 70-85min that retention time is collected at active peak;
The RP-HPLC chromatography condition is: chromatographic column: C18 post SymmetryShield
TM, specification: 19 * 150mm, applied sample amount: 970 μ l, flow velocity: 2.5ml/min, temperature: 25 ℃, wavelength: 215nm, elutriant A are ultrapure water, and B is a trifluoroacetic acid aqueous solution.Program: 0-6min:0-5%B, 6-66min:5-100%B, 66-80min:100%B; It is 32.2-32.5min that retention time is collected at active peak.
6. according to the preparation method of claim 2 or 3 described antibacterial peptides, it is characterized in that: after the ceramic membrane filter degerming, spraying drying obtains pulverous antibacterial peptide finished product with fermented liquid.
7. the preparation method of antibacterial peptide according to claim 6, it is characterized in that: with fermented liquid under the condition of 25 ℃ of temperature, by working pressure is 0.02Mpa, membrane pore size is the filtered liquid that obtains removing thalline behind the ceramic membrane filter of 0.2 μ m, in filtered liquid, add solid salt, the volume percent that the quality of solid salt accounts for filtered liquid is 5～20%, treat to carry out spraying drying after solid salt dissolves fully, the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, promptly gets pulverous antibacterial peptide finished product.
8. the antibacterial peptide that obtains by each described antibacterial peptide preparation method in the claim 2～7.
9. the purposes of the described antibacterial peptide of claim 8 in meat product is fresh-keeping.
10. the purposes of antibacterial peptide according to claim 9, it is characterized in that: antibacterial peptide is used for the fresh-keeping of pork, and actual conditions is to add 12ml antibacterial peptide solution in every 100g pork, and the concentration of the mass percent of antibacterial peptide is 3.1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110048912.2A CN102286393B (en) | 2011-03-01 | 2011-03-01 | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110048912.2A CN102286393B (en) | 2011-03-01 | 2011-03-01 | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102286393A true CN102286393A (en) | 2011-12-21 |
| CN102286393B CN102286393B (en) | 2014-04-30 |
Family
ID=45333155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110048912.2A Expired - Fee Related CN102286393B (en) | 2011-03-01 | 2011-03-01 | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102286393B (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173372A (en) * | 2012-07-12 | 2013-06-26 | 北京伟嘉人生物技术有限公司 | Lactococcus lactis with high bacteriostatic activity and application thereof |
| CN103190470A (en) * | 2013-03-24 | 2013-07-10 | 新疆农垦科学院 | Composite biological fresh-keeping agent for meat and preparation method of composite biological fresh-keeping agent |
| CN103404939A (en) * | 2013-03-15 | 2013-11-27 | 安徽农业大学 | Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution |
| CN103989190A (en) * | 2014-06-06 | 2014-08-20 | 海南椰国食品有限公司 | Method for preparing low-temperature meat paste product |
| CN104046585A (en) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | Bifidobacterium animal bacteriocin, production method thereof and specific production strain |
| CN104388503A (en) * | 2014-11-24 | 2015-03-04 | 天津商业大学 | Extraction method and application of lactobacillus extracellular protein |
| CN104855492A (en) * | 2015-04-02 | 2015-08-26 | 曹庸 | A natural preservative for meat products and a preparation and use method thereof |
| CN105002240A (en) * | 2015-07-07 | 2015-10-28 | 汕头大学 | Antibiotic, and preparation method and application thereof |
| CN105960244A (en) * | 2013-12-10 | 2016-09-21 | 株式会社明治 | Antibacterial peptide-inducing agent |
| CN106191179A (en) * | 2016-08-30 | 2016-12-07 | 林州中农颖泰生物肽有限公司 | A kind of production technology of feedstuff antibacterial peptide |
| CN108753875A (en) * | 2018-05-23 | 2018-11-06 | 浙江工商大学 | The method that spray drying prepares lactobacillus plantarum ZJ316 bacteriocins |
| CN109112079A (en) * | 2018-03-31 | 2019-01-01 | 华中农业大学 | There is the thuringiensis and its bacteriocin of bacteriostatic activity to several pathogenetic bacterias |
| CN110590918A (en) * | 2019-09-12 | 2019-12-20 | 渤海大学 | Inhibitory peptide for Vibrio harveyi quorum sensing and preparation method and application thereof |
| CN111011482A (en) * | 2019-12-24 | 2020-04-17 | 四川东坡中国泡菜产业技术研究院 | Preparation method of fresh-keeping liquid, fresh-keeping liquid and fresh-keeping method |
| CN114958663A (en) * | 2022-05-13 | 2022-08-30 | 齐鲁工业大学 | Lactococcus lactis subsp lactis A32 strain and derivative product and application thereof |
| CN115386523A (en) * | 2022-09-30 | 2022-11-25 | 北京诺安百汇医药科技有限公司 | Lactococcus lactis and application thereof in resisting helicobacter pylori infection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005080550A1 (en) * | 2004-02-23 | 2005-09-01 | Ajinomoto Co., Inc. | Lactic acid with high nisin-productivity and method of selecting the same |
| CN1687110A (en) * | 2005-04-26 | 2005-10-26 | 浙江银象生物工程有限公司 | Method for separating lacti-streptocin from fermentation liquid of lacti-galactococcus |
-
2011
- 2011-03-01 CN CN201110048912.2A patent/CN102286393B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005080550A1 (en) * | 2004-02-23 | 2005-09-01 | Ajinomoto Co., Inc. | Lactic acid with high nisin-productivity and method of selecting the same |
| CN1687110A (en) * | 2005-04-26 | 2005-10-26 | 浙江银象生物工程有限公司 | Method for separating lacti-streptocin from fermentation liquid of lacti-galactococcus |
Non-Patent Citations (4)
| Title |
|---|
| FROSETH B R等: "Molecular characterization of the Nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3", 《APPL ENVIRON MICROBIOL》 * |
| Y.CAI等: "Isolation and characterization of nisin-producing Lactococcus lactis subsp.lactis from bean-sprouts", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 吴琼等: "乳酸乳球菌发酵液中乳链菌肽的分离纯化", 《食品科学》 * |
| 周绪霞等: "乳链菌肽产生菌的筛选及其鉴定", 《浙江大学学报(农业与生命科学版)》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173372A (en) * | 2012-07-12 | 2013-06-26 | 北京伟嘉人生物技术有限公司 | Lactococcus lactis with high bacteriostatic activity and application thereof |
| CN103173372B (en) * | 2012-07-12 | 2015-05-20 | 北京伟嘉人生物技术有限公司 | Lactococcus lactis with high bacteriostatic activity and application thereof |
| CN103404939A (en) * | 2013-03-15 | 2013-11-27 | 安徽农业大学 | Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution |
| CN103404939B (en) * | 2013-03-15 | 2014-11-05 | 安徽农业大学 | Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution |
| CN103190470A (en) * | 2013-03-24 | 2013-07-10 | 新疆农垦科学院 | Composite biological fresh-keeping agent for meat and preparation method of composite biological fresh-keeping agent |
| CN103190470B (en) * | 2013-03-24 | 2015-09-09 | 新疆农垦科学院 | A kind of meat complex biological preservative and preparation method thereof |
| CN105960244A (en) * | 2013-12-10 | 2016-09-21 | 株式会社明治 | Antibacterial peptide-inducing agent |
| CN103989190B (en) * | 2014-06-06 | 2016-03-23 | 海南椰国食品有限公司 | A kind of preparation method of low temperature meat emulsion product |
| CN103989190A (en) * | 2014-06-06 | 2014-08-20 | 海南椰国食品有限公司 | Method for preparing low-temperature meat paste product |
| CN104046585A (en) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | Bifidobacterium animal bacteriocin, production method thereof and specific production strain |
| CN104388503A (en) * | 2014-11-24 | 2015-03-04 | 天津商业大学 | Extraction method and application of lactobacillus extracellular protein |
| CN104855492A (en) * | 2015-04-02 | 2015-08-26 | 曹庸 | A natural preservative for meat products and a preparation and use method thereof |
| CN105002240B (en) * | 2015-07-07 | 2018-09-04 | 汕头大学 | A kind of antibiotic and the preparation method and application thereof |
| CN105002240A (en) * | 2015-07-07 | 2015-10-28 | 汕头大学 | Antibiotic, and preparation method and application thereof |
| CN106191179A (en) * | 2016-08-30 | 2016-12-07 | 林州中农颖泰生物肽有限公司 | A kind of production technology of feedstuff antibacterial peptide |
| CN109112079B (en) * | 2018-03-31 | 2020-11-10 | 华中农业大学 | Bacillus thuringiensis with bacteriostatic activity on several pathogenic bacteria and bacteriocin thereof |
| CN109112079A (en) * | 2018-03-31 | 2019-01-01 | 华中农业大学 | There is the thuringiensis and its bacteriocin of bacteriostatic activity to several pathogenetic bacterias |
| CN108753875A (en) * | 2018-05-23 | 2018-11-06 | 浙江工商大学 | The method that spray drying prepares lactobacillus plantarum ZJ316 bacteriocins |
| CN110590918A (en) * | 2019-09-12 | 2019-12-20 | 渤海大学 | Inhibitory peptide for Vibrio harveyi quorum sensing and preparation method and application thereof |
| CN110590918B (en) * | 2019-09-12 | 2022-09-30 | 渤海大学 | Inhibitory peptide for Vibrio harveyi quorum sensing and preparation method and application thereof |
| CN111011482A (en) * | 2019-12-24 | 2020-04-17 | 四川东坡中国泡菜产业技术研究院 | Preparation method of fresh-keeping liquid, fresh-keeping liquid and fresh-keeping method |
| CN114958663A (en) * | 2022-05-13 | 2022-08-30 | 齐鲁工业大学 | Lactococcus lactis subsp lactis A32 strain and derivative product and application thereof |
| CN115386523A (en) * | 2022-09-30 | 2022-11-25 | 北京诺安百汇医药科技有限公司 | Lactococcus lactis and application thereof in resisting helicobacter pylori infection |
| CN115386523B (en) * | 2022-09-30 | 2024-01-30 | 北京诺安百汇医药科技有限公司 | Lactococcus lactis and application thereof in resisting helicobacter pylori infection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102286393B (en) | 2014-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102286393B (en) | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide | |
| CN105432667B (en) | A kind of preparation method of bacillus subtilis wettable powder | |
| CN108148794B (en) | Bacillus subtilis DYr3.3 with broad-spectrum antibacterial activity, and preparation method and application thereof | |
| CN104830713B (en) | The Bacillus strain of a variety of antibacterial lipopeptids of one plant of coproduction and γ polyglutamic acids | |
| CN104904751B (en) | A kind of wheat scab liquid biocontrol agent and preparation method thereof | |
| CN102524518B (en) | Method for producing antibacterial peptide by using brevibacillus laterosporu | |
| CN104531562A (en) | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin | |
| CN107022355A (en) | A kind of biologic ferment soil conditioner and preparation method thereof | |
| CN101691540A (en) | Muscodor endophytic fungi ZJLQ070 and application thereof and fungicide | |
| CN103333819A (en) | Bacillus subtilis capable of producing broad spectrum and efficient antibacterial peptide and application of Bacillus subtilis | |
| CN103421704A (en) | Lactobacillus plantarum for freshwater fish fermentation product and application thereof | |
| CN104970086B (en) | Bio-preservative and its preparation method and application | |
| CN104087524A (en) | Lactobacillus paracasei subsp. tolerans strain, screening method and use | |
| CN103667106B (en) | A kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method | |
| CN107475147A (en) | A kind of Lactobacillus plantarum LP1 and its application in feed addictive is prepared | |
| CN103404939B (en) | Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution | |
| CN105638744B (en) | A kind of preparation method of Brevibacillus brevis wettable powder | |
| CN104450551A (en) | Bacillus subtilis DPPG-26 for preventing and treating damping off and application thereof | |
| CN104311645B (en) | Spirulina polypeptide P1 and its application with bacteriostatic activity | |
| CN102250806B (en) | Method for screening lactic acid bacteria capable of producing bacteriocin from plant source materials | |
| CN106591174B (en) | The lactobacillus curvatus of one plant of bacteriocinogeny and its application | |
| CN101691542B (en) | Muscodor endophytic fungi and application thereof and fungicide | |
| CN106342900B (en) | A kind of Streptomyces jingyangensis wettable powder and preparation method thereof | |
| CN103911321A (en) | Cypermethrin- and/or deltamethrin- degrading bacterium in seawater environment, and separation, purification and applications thereof | |
| CN103224902B (en) | Biocontrol bacterium Bacillus subtilis sbz-106, and wettable pulvis and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140430 Termination date: 20180301 |