CN102286393B - 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
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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 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 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 Ensuring Food Safety, thereby the technology of preparing of screening new bacteriocin producing bacterial strain and antibacterial peptide has great importance to the development of national economy.
Milk-acid bacteria is just widely used in the anti-corrosive fresh-keeping of food since ancient times, and the numerous antibacterial substance small molecular peptide matters bacteriocins that produce milk-acid bacteria, owing to can, by the enzyme liberating of human secretory, more having highlighted its security and the using value in food fresh keeping.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 7g/kg body weight), in saliva, in 10min, can be completely degraded, can not cause anaphylaxis, thereby be widely used in the anti-corrosive fresh-keeping of milk-product, meat product, acid canned food and leavened food.But because Nisin can only be soluble status under acidic conditions, under neutrallty condition, solvability is low, poor stability, and easily by meat proteins, adsorbed, these characteristics have greatly affected the application of Nisin in food preservation, are therefore necessary to develop new polypeptide antiseptic-germicide.
Summary of the invention
The object 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's common micro-organisms center (CGMCC), deposit number is:
cGMCC No.4584.
Lactococcus lactis in the present invention (Lactococcus lactis subsp.lactis) LLC518 is that the pregnant solution of preparing from commercially available cucumber, screening obtains, concrete screening method is for first screening acid-producing bacteria strain with the MRS selective medium containing 0.01% purpurum bromocresolis, again by screening bacterium dibbling at MRS solid culture primary surface, after the obvious 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, by the bacterial strain of generation inhibition zone further by fermentation, obtain fermented supernatant fluid, and by getting rid of organic acid in fermented liquid, the bacteriostatic action of hydrogen peroxide, thereby further by Chymotrypsin and papoid, can eliminate in the fermented liquid of bacteriostatic activity proof bacterial strain and contain protein-based antibacterial substance.By morphological feature, Physiology and biochemistry, learn feature and 16SrDNA sequential analysis, according to < < uncle Jie Shi division bacteria handbook > > and the classification of < < lactic-acid-bacterium, identify and experimental technique > >, show that this bacterial strain belongs to Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), is numbered LLC518.
Second object of the present invention is to provide 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 obtaining 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: the Lactococcus lactis that first picking has activated from MRS slant medium (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 is inoculated in MRS seed culture medium, 28 ℃ of shaking tables are cultivated 12 hours, seed liquor is inoculated in MRS fermention medium by 2～6% inoculum size, 28 ℃ of standing cultivation 24h can obtain fermented liquid again;
Again that the fermented liquid obtaining is centrifugal; Get supernatant liquor, 0.45 μ m membrane filtration, carries out ammonium sulfate precipitation to filtered solution; Then centrifugal, get the protein precipitation that precipitation obtains; Protein precipitation is dissolved in water, adopts the dialysis tubing dialysis treatment of molecular weight cut-off 2000Da, vacuum lyophilization dialyzate obtains rough dry powder; Again rough dry powder is dissolved in the water, and through Sephadex G-50 gel chromatography, collects Peak Activity and carry out reversed-phase HPLC processing, to obtain antibacterial peptide sterling.
The actual conditions of the above-mentioned purification step of antibacterial peptide is as follows: by 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, after 12h, 4 ℃, 8000r/min is centrifugal, and 45min is precipitated albumen, the 4h that dialyses in the dialysis tubing of molecular weight cut-off 2000Da after getting protein precipitation and being dissolved in water, makes described rough dry powder with freeze drier after dialyzate being put into-20 ℃ of refrigerator freezings; Rough dry powder is dissolved in water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect Peak Activity, RP-HPLC prepares antibacterial peptide sterling;
Described gel chromatography adopts Sephadex G-50 column packing (26 * 100cm), and 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 Peak Activity is collected retention time;
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 is ultrapure water, B is 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 Peak Activity is collected retention time.
Second approach: the Lactococcus lactis that first picking has activated from MRS slant medium (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 is inoculated in MRS seed culture medium, 28 ℃ of shaking tables are cultivated 12 hours, by 2～6% inoculum size, be inoculated in MRS fermention medium, 28 ℃ of standing cultivation 24h can obtain fermented liquid again;
Again by fermented liquid after ceramic membrane filter degerming, spraying is dry obtains pulverous antibacterial peptide finished product.
Spraying drying step actual conditions as follows: by fermented liquid under the condition of 25 ℃ of temperature, by working pressure, be 0.02Mpa, membrane pore size is after the ceramic membrane filter of 0.2 μ m, to obtain removing the filtered liquid of thalline, in this filtered liquid, add solid salt, the volume ratio that the quality of solid salt accounts for filtered liquid is 5～20%, after dissolving completely, solid salt carries out spray dried dry, the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, obtains pulverous antibacterial peptide finished product.
The antibacterial peptide being made by above-mentioned any approach also all belongs to protection scope of the present invention.
The 3rd object of the present invention is to provide a kind of above-mentioned antibacterial peptide purposes in food preservation.
Antibacterial peptide in the present invention is particularly useful for pork to carry out fresh-keeping, and actual conditions is in every 100g pork, to add 12ml antibacterial peptide solution, 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 from existing antibacterial peptide Nisin, and Nisin can be degraded by alpha-chymotrypsin, insensitive to pronase e, trypsinase and stomach en-.And this antibacterial peptide is except can being degraded by alpha-chymotrypsin, also can be degraded by papoid, pronase e and Proteinase K.
The antimicrobial spectrum of the antibacterial peptide that 2, this Lactococcus lactis produces is different from existing antibacterial peptide Nisin, the micrococcus luteus unrestraint effect to Nisin sensitivity.
3, the antibacterial peptide that this Lactococcus lactis produces has activity within the scope of wider pH, particularly under alkaline condition, still has bacteriostatic action.
4, the mode of action of this antibacterial peptide is killed bacterium, and under storage conditions, has significant germicidal action 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.
Accompanying drawing explanation
Fig. 1 be rough dry powder water-soluble and after membrane filtration by the elution curve of Sephadex G-50;
Fig. 2 is the bacteriostatic action schematic diagram of Sephadex-G-50 detached peaks;
Fig. 3 is antibacterial peptide high performance liquid chromatography elution curve;
Fig. 4 is antibacterial peptide mass spectrum;
Fig. 5 A is the mode of action of antibacterial peptide to LSX801 at 37 ℃;
Fig. 5 B is the mode of action of antibacterial peptide to LSX801 at 4 ℃;
Fig. 5 C is the mode of action of antibacterial peptide to listeria monocytogenes at 37 ℃;
Fig. 5 D is the mode of action of antibacterial peptide to listeria monocytogenes at 4 ℃;
The restraining effect of Fig. 6 antibacterial peptide to bacterium in pork;
The restraining effect of Fig. 7 sterling antibacterial peptide to indicator;
The morphological specificity of Fig. 8 Lactococcus lactis LLC518 is observed figure.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is quality percentage composition.
Lactococcus lactis LLC518 of the present invention obtains in the following ways:
1, the new fresh cucumbers of purchasing from market, Hefei ,Anhui is cleaned and is cut into small pieces, fill the sterile test tube of 50ml, compacting, with plastic skin and kraft paper sealing, 30 ℃, after enrichment culture 48h, the MRS that sampling is coated containing 0.01% purpurum bromocresolis selects substratum.
2, picking makes to select containing the MRS of 0.01% purpurum bromocresolis the acid-producing bacteria strain of substratum flavescence, further separation and purification;
2, take Bacterium lacticum (Lactobacillus sp.) LSX801 is indicator, obtains the bacterial strain with bacteriostasis by dull and stereotyped dibbling method;
3, by fermentation culture, obtain fermented liquid, 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.By in the water-soluble solution of dry powder, be added in ox Feng cup, adopt Agar diffusion test screening to have the bacterial strain of bacteriostasis;
4. by Chymotrypsin, papoid, pronase e, Proteinase K, stomach en-, trypsinase, lipase treatment crude protein dry powder, 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:
On MRS plate culture medium, bacterium colony is rounded, central uplift, smooth surface, neat in edge, oyster white, colony diameter 0.5～1.0mm.Sterile film in MRS liquid nutrient medium, bacterium liquid is muddy, has precipitation to produce.Gram-positive microorganism, spherical, thalline diameter 0.5～0.8um, without gemma, atrichia, single or existence in pairs.Under anaerobic growing way, than good under aerobic condition, increases about 0.5mm under the relative aerobic condition of colony diameter under anaerobic condition, and under aerobic conditions, growing state is also good, belongs to facultative anaerobe.
Can between 20～40 ℃, grow, not grow for 45 ℃, wherein optimum growth temperature is 30 ℃.Under 0-7% sodium chloride concentration, all can grow.
Catalase is negative; Can decomposition glucose produce acid, but aerogenesis not also do not produce hydrogen sulfide, but hydrolyzable arginine produces ammonia; Can decompose Vitamin C2, but not starch-splitting and gelatin; Litmus milk experiment is positive, but does not produce proteolytic enzyme, can not make milk peptonize; Voges-Proskauer test is negative, shows that the unfermentable glucose of this bacterium generates acetyl methyl carbinol.
Glucose fermentation produces acid, but aerogenesis not, can xylose-fermenting, semi-lactosi, lactose, sucrose, pectinose, lactose, maltose and N.F,USP MANNITOL produces acid, and utilize raffinose and inositol but do 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: the Lactococcus lactis that picking has activated from MRS slant medium (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 is inoculated in MRS seed culture medium, 28 ℃ of shaking tables are cultivated 12 hours, seed liquor is inoculated in MRS fermention medium by 4% inoculum size, 28 ℃ of standing cultivation 24h can obtain fermented liquid again;
The isolation and purification of B, antibacterial peptide: by 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, after 12h, 4 ℃, 8000r/min is centrifugal, and 45min is precipitated albumen, and the 4h that dialyses in the dialysis tubing of molecular weight cut-off 2000Da after getting protein precipitation and being dissolved in water makes described rough dry powder with freeze drier after dialyzate being put into-20 ℃ of refrigerator freezings; Rough dry powder is dissolved in water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect Peak Activity, RP-HPLC prepares antibacterial peptide sterling;
Described gel chromatography adopts Sephadex G-50 column packing (26 * 100cm), and 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 Peak Activity is collected retention time;
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 is ultrapure water, B is 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 Peak Activity is collected retention time.
Rough dry powder water-soluble and after membrane filtration the elution curve by Sephadex G-50 as shown in Figure 1, and the bacteriostatic action of Sephadex-G-50 detached peaks as shown in Figure 2, in Fig. 2,1 is Sephadex-G-50 first peak, 2 is Sephadex-G-50 the 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: the Lactococcus lactis that picking has activated from MRS slant medium (Lactococcus lactis subsp.lactis) LLC518CGMCC No.4584 is inoculated in MRS seed culture medium, 28 ℃ of shaking tables are cultivated 12 hours, seed liquor is inoculated in MRS fermention medium by 4% inoculum size, 28 ℃ of standing cultivation 24h can obtain fermented liquid again;
B, by fermented liquid under the condition of 25 ℃ of temperature, by working pressure, be 0.02Mpa, membrane pore size is to obtain filtered liquid after 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%, carries out spray dried dry after solid salt dissolves completely, and the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, obtains pulverous antibacterial peptide finished product.
The impact of the mass ratio of solid salt on antibacterial peptide vigor: add respectively 5%, 10%, 15%, 20% salt in the fermented supernatant fluid of 150ml Lactococcus lactis LLC518, inlet temperature is made as 140 ℃, air outlet temperature is made as 120 ℃, after spray drying treatment, get 0.1 gram, sample under each condition in 5ml water, measure its vigor, the vigor of gained sample is as shown in table 1, can obtain: when adding 15% salt, total activity is maximum simultaneously.
The impact of table 1 auxiliary material comparison product vigor
The impact of different inlet temperature on product vigor: Lactococcus lactis LLC518 fermented liquid is added after film system is filtered to 15% salt, carry out spray dried after dissolving completely dry.The hot blast inlet temperature of spray-drier is made as respectively 130 ℃, 135 ℃, 140 ℃, 145 ℃, 150 ℃, air outlet temperature is made as 120 ℃, collect powdery substance, get 0.1 gram, sample under each condition 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 ℃, total activity is maximum.
The impact of table 2 inlet temperature on product vigor
The impact of different air outlet temperatures on total activity: Lactococcus lactis LLC518 fermented liquid is added after film system is filtered to 15% salt, carry out spray dried after dissolving completely dry.The hot blast air outlet temperature of spray-drier is made as respectively 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃, inlet temperature is made as 150 ℃, collect powdery substance, get 0.1 gram, sample under each condition 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 ℃, total activity is maximum.
The impact of table 3 air outlet temperature on 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 are had to stronger restraining effect.Within the scope of wider pH, bacteriostatic activity is stable, can be by multiple protein enzyme liberating, because of but the higher antibacterial peptide of a kind of security.
Antibacterial peptide is also that the feature of Lacticin LLC518 is as follows:
1, the antibacterial substance that this bacterial strain produces has protein properties, with Chymotrypsin, papoid, pronase E and Proteinase K, processes, and anti-microbial activity disappears, as shown in table 4.Molecular weight through this bacteriocin of mass spectroscopy is 3344.6Da;
The susceptibility of table 4Lacticin LLC518 to enzyme
Lacticin LLC518 is responsive to Chymotrypsin, papoid, pronase E, Proteinase K as can be seen from the above table, 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 to restraining effect, to Lactococcus lactis, micrococcus luteus unrestraint effect.
Antibacterial peptide is also 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, the susceptibility to temperature: under acidic conditions, can tolerate 100 ℃ of high temperature, but under alkaline condition, 100 ℃ of processing vigor are down to zero;
Antibacterial peptide is as shown in table 6 to hot stability:
Table 6lacticin LLC518 is to hot stability
| 100℃ | Quality (mg) | (AU) tires | Specific activity |
| 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, the susceptibility to pH: this antibacterial peptide is stable in pH2～10, and vigor remains unchanged, shown in table 7.
The stability of table 7Lacticin LLC518 to pH
| pH | Quality (mg) | (AU) tires | Specific activity |
| 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 at 4 ℃ and 37 ℃ to indicator.
The mode of action of antibacterial peptide: this antibacterial peptide all has germicidal action at 4 ℃ and 37 ℃ to indicator (Bacterium lacticum LSX801, listeria monocytogenes).Experimental result as shown in Fig. 5 A, Fig. 5 B, Fig. 5 C, Fig. 5 D, in Fig. 5 A～D ▲ be CK, ■ is antibacterial peptide.
By 25mg/ml antibacterial peptide solution, MRS, LB nutrient solution join respectively OD for contrasting
600be, in 0.2 bacteria suspension, to be placed in 4 ℃ of preservations and 37 ℃ of cultivations, regularly coat in MRS, 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 viable count all reduces.Viable count 3 orders of magnitude that decline 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 process 2h with bacteriocin, 4h, 6h, 8h, the listeria monocytogenes of 10h is coated with respectively to cultivate and observes, and viable count first reduces rear rising.In treatment time 10h, rise and only have an order of magnitude, than contrast, have obvious bacteriostatic action.
A lot of food needs 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 at random sample meat 20g with aseptic scissors, random 3 are divided into one group.Adopt respectively preservative film and vacuum packaging, 4 ℃ of preservations, regularly survey total plate count.
The mensuration of total plate count: add sterilized water 180ml in above-mentioned sample, with the centrifugal 1min of 8000r/min, dilute by decimal dilution method after mixing.Respectively from 10
-2～10
-7in the diluent of gradient, take out 0.1ml and coat on LB substratum, 37 ℃, cultivate 48h, carry out enumeration, calculate contained total plate count in every gram of primary sample.
The mensuration of antibacterial peptide to the bacteriostatic action of Bacterium lacticum LSX801 in pork: by sterilizing pork in the bacterium liquid (OD of Bacterium lacticum LSX0801
600=0.2) in, soak 5min and drain after 5min, the antibacterial peptide that is 3.1% by the concentration of mass percent is applied in pork surface, vacuumizes 4 ℃ of Refrigerator stores, regularly detects lactic acid bacteria number and pH value, adds sterilized water in pork as a control group.
Test result as shown in Figure 6, Fig. 6 shows that the initial germicidal action of antibacterial peptide is remarkable, adds antibacterial peptide and processes rear the 1st day total plate count at 1.46～1.62log (cfug
-1) between, control group is 2.3～2.4log (cfug
-1).In lay up period antibacterial peptide treatment group total plate count increasing degree, be less than control group, so after changing matrix environment, this antibacterial peptide still has stable bacteriostatic action, and it is of practical significance in the fresh-keeping application of meat-based food simultaneously.
The anti-microbial effect of the composite preservative that antibacterial peptide and conventional fungistat proportioning form in fresh-keeping meat.
Use 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.
In composite preservative, the concentration range of each preservation agent is: 0.18%～0.6%EDTA, 0.05%～0.24% potassium sorbate, 0.63%～3.1% antibacterial peptide Lacticin LLC518.
Above-mentioned composite preservative is processed to pork sample by the mode in embodiment 5, coat pork surface, vacuumize 4 ℃ of Refrigerator stores, regular bacterial detection content, it is control group that the sterilized water of take substitutes antibacterial peptide solution.Experimental result is carried out to statistical analysis.
Research finds that the concentration as EDTA is 0.33%, the concentration of potassium sorbate is 0.12%, the concentration of antibacterial peptide is while being 1.44%, the total plate count of pork sample is minimum, and wherein antibacterial peptide and EDTA exist significant interaction (P=0.003 < 0.01).
Claims (6)
1. a preparation method for antibacterial peptide, its by Lactococcus lactis (
lactococcus lactissubsp.
lactis) LLC518 CGMCC No.4584 ferments to obtain antibacterial peptide, described CGMCC No.4584 be Lactococcus lactis (
lactococcus lactissubsp.
lactis) LLC518 is at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described antibacterial peptide is purifying in accordance with the following methods: the fermented liquid obtaining is centrifugal; Get supernatant liquor, 0.45 μ m membrane filtration, carries out ammonium sulfate precipitation to filtered solution; Then centrifugal, get protein precipitation; Protein precipitation is dissolved in water, adopts the dialysis tubing dialysis treatment of molecular weight cut-off 2000Da, dialyzate vacuum lyophilization is obtained to rough dry powder; Again rough dry powder is dissolved in the water, and through Sephadex G-50 gel chromatography, collects Peak Activity and carry out reversed-phase HPLC processing, to obtain antibacterial peptide sterling.
2. the preparation method of antibacterial peptide according to claim 1, it comprises following preparation process: the Lactococcus lactis that picking has activated from MRS slant medium (
lactococcus lactissubsp.
lactis) LLC518 CGMCC No.4584 is inoculated in MRS seed culture medium, 28 ℃ of shaking tables are cultivated and within 12 hours, are reached stationary phase, then are inoculated in MRS fermention medium by 2～6% inoculum size, and 28 ℃ of standing cultivation 24h reach stationary phase, can obtain fermented liquid.
3. the preparation method of antibacterial peptide according to claim 1, it is characterized in that: by 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, after 12h, 4 ℃, 8000r/min is centrifugal, and 45min is precipitated albumen, the 4h that dialyses in the dialysis tubing of molecular weight cut-off 2000Da after getting protein precipitation and being dissolved in water, makes described rough dry powder with freeze drier after dialyzate being put into-20 ℃ of refrigerator freezings; Rough dry powder is dissolved in water and the membrane filtration of via hole diameter 0.45 μ m after, through Sephadex G-50 gel chromatography, collect Peak Activity, RP-HPLC prepares antibacterial peptide sterling;
Described gel chromatography adopts Sephadex G-50 column packing, the column dimension of Sephadex G-50 is 26 * 100cm, and applied sample amount is 5ml, and flow velocity is 1.5 ml/min, elutriant is through 0.22 μ m membrane filtration secondary water, and it is 70-85min that Peak Activity is collected retention time;
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 is ultrapure water, B is 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 Peak Activity is collected retention time.
4. a preparation method for antibacterial peptide, its by Lactococcus lactis (
lactococcus lactissubsp.
lactis) LLC518 CGMCC No.4584 ferments to obtain antibacterial peptide, described CGMCC No.4584 be Lactococcus lactis (
lactococcus lactissubsp.
lactis) LLC518 is at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center; By fermented liquid under the condition of 25 ℃ of temperature, by working pressure, be 0.02Mpa, membrane pore size is after the ceramic membrane filter of 0.2 μ m, to obtain removing the filtered liquid of thalline, in filtered liquid, add solid salt, the volume percent that the quality of solid salt accounts for filtered liquid is 5～20%, carries out spray dried dry after solid salt dissolves completely, and the inlet temperature of spray-drier is 130～150 ℃, air outlet temperature is 110～130 ℃, obtains pulverous antibacterial peptide finished product.
5. the application of the antibacterial peptide being prepared by method described in any one in claim 1～4 in food preservation.
6. the application of antibacterial peptide according to claim 5, is characterized in that: antibacterial peptide is fresh-keeping for pork, and actual conditions is in every 100g pork, to add 12ml antibacterial peptide solution, and the concentration of the mass percent of antibacterial peptide is 3.1%.
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| 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 |
| 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 |
| JPWO2015087919A1 (en) * | 2013-12-10 | 2017-03-16 | 株式会社明治 | Antibacterial peptide inducer |
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| CN104855492A (en) * | 2015-04-02 | 2015-08-26 | 曹庸 | A natural preservative for meat products and a preparation and use method thereof |
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| 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 |
| CN114958663B (en) * | 2022-05-13 | 2023-06-02 | 齐鲁工业大学 | Lactococcus lactis subspecies lactate A32 strain, derivative product and application thereof |
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