CN103404939A - Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution - Google Patents
Antibacterial peptide mixed solution and method for preserving freshness of foods by antibacterial peptide mixed solution Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial resource utilization, in particular relates to a method for preparing an antibacterial peptide mixed solution generated by lactococcus lactis subsp lactis and lactobacillus sake, and relates to an application of the antibacterial peptide mixed solution in beancurd preservation. The method comprises the following steps of: preserving freshness of beancurd by adopting 10wt% antibacterial peptide mixed solution, and uniformly spraying the antibacterial peptide mixed solution on the surface of a beancurd block in a ratio of adding 2ml of antibacterial peptide mixed solution in every 10g of beancurd. According to the antibacterial peptide mixed solution, the sense score of the beancurd can be effectively prevented from being reduced, the growing speed of spoilage microorganisms can be inhibited, and the growth of total number of bacteria in the beancurd in the storing period can be suspended. Therefore, the antibacterial peptide mixed solution provided by the invention plays an active effect in beancurd preservation, and has better practical value.
Description
Technical field
The invention belongs to microbial resources and utilize technical field, be specifically related to a kind of antibacterial peptide mixed liquor and for the method for food fresh keeping.
Background technology
Bean curd is owing to containing the compositions such as protein, fat, carbohydrate and moisture, and the utmost point is beneficial to microbial growth.But the physics such as high temperature sterilization, microwave keeping method easily causes tofu texture to change and mouthfeel descends, chemical preservative can produce adverse influence to health and ecological environment again.Therefore being necessary to study the natural biological preservation liquid with good bacteriostasis extends effective period of food quality.Existingly studies have shown that the natural frond extracts such as natural colouring matter, allicin, extract solution of bamboo leaves and Chinese herbal medicine have certain anti-corrosive fresh-keeping effect to bean curd.But the most of natural frond extract used at present is all semifinished product, and its effective component content often changes with season and geographical environment, and concrete action component is also very unclear, is difficult to isolate sterling and carries out toxicological evaluation.In addition, the researchs such as the mechanism of action of various anticorrisive agents, antimicrobial spectrum and range of application is also deep not.Therefore be necessary to develop more efficient safer biological preservative.
Lactic acid bacteria just is widely used in the anti-corrosive fresh-keeping of food since ancient times, and the small-molecule peptide material that the lactic acid bacteria secretion produces can be degraded by the enzyme of human secretory, and its security has shown the using value in food fresh keeping.The bacteriocin Nisin that Lactococcus lactis subsp. lactis produces is unique bacteriocin that can be used for food fresh keeping of FAO approval, it can suppress or kill some gram-positive pathogens and food spoilage bacterium, and have efficient, nontoxic, noresidue, without advantages such as the resistances to the action of a drug, be widely used in the anti-corrosive fresh-keeping of milk, cheese, canned food, fish product, beer, beverage etc.Because Nisin can only be soluble status under acid condition, dissolubility is low under neutrallty condition, and poor stability has greatly affected the application of Nisin in bean product are fresh-keeping.
Summary of the invention
One of purpose of the present invention is to provide a kind of antibacterial peptide mixed liquor that can be used for food fresh keeping.
For achieving the above object, the present invention has adopted following technical scheme: a kind of antibacterial peptide mixed liquor, and this antibacterial peptide mixed liquor is mixed by antibacterial peptide prepared by two kinds of bacterial strains, wherein:
A kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518, its
The deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.4584;
A kind of Lactobacillus saki (Lactobacillus sakei) LSJ618, its
Chinese microorganism strain is protected The deposit number of hiding administration committee's common micro-organisms center is: CGMCC No.7017;
By the antibacterial peptide that the described Lactococcus lactis subsp. lactis lactic acid subspecies that ferments (Lactococcus lactis subsp.lactis) LLC518 obtains, called after Lacticin LLC518; By the antibacterial peptide that the described Lactobacillus saki that ferments (Lactobacillus sakei) LSJ618 obtains, called after Sakacin LSJ618;
Antibacterial peptide Lacticin LLC518 and Sakacin LSJ618 be take to mass ratio and be mixed to get mixture as the ratio of 1:1, the more described mixture that will obtain be take mass ratio and mixed mutually and can obtain this antibacterial peptide mixed liquor as the ratio of 1:10 with water.
The preparation method of antibacterial peptide is by following approach:
At first the Lactococcus lactis subsp. lactis lactic acid subspecies activated from picking on MRS solid slant medium (Lactococcus lactis subsp.lactis) LLC518 and Lactobacillus saki (Lactobacillus sakei) LSJ618, and be inoculated in respectively in the MRS liquid seed culture medium, 37 ℃ of shaking tables were cultivated 12 hours, by 4% inoculum concentration, be inoculated in respectively in the MRS fermentation medium again, 37 ℃ of standing cultivation 24h, can obtain Lactococcus lactis subsp. lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid: by two kinds of zymotic fluids respectively after the ceramic membrane filter degerming, spray-drying can obtain respectively pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product again.
The actual conditions of spray-drying step is as follows: by Lactococcus lactis subsp. lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid respectively under the condition of 25 ℃ of temperature, by operating pressure, be 0.02Mpa, membrane aperture is after the ceramic membrane filter of 0.2 μ m, to obtain removing the supernatant of thalline, in two kinds of supernatants, add solid salt respectively, the ratio that the quality of the solid salt added accounts for described supernatant volume is 1:10, after dissolving fully, solid salt carries out spray-drying by spray dryer, the EAT of described spray dryer is 150 ℃, leaving air temp is 130 ℃, after spray-drying, namely obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
Two of purpose of the present invention is to provide the method for a kind of above-mentioned antibacterial peptide mixed liquor for food fresh keeping, and its concrete scheme is as follows: by the even sprinkle of antibacterial peptide mixed liquor in food surface.
Further, the every 10g bean curd of take adds 2mL antibacterial peptide mixed liquor and is ratio, by antibacterial peptide mixed liquor sprinkle in block bean curd surface.
Beneficial effect of the present invention is as follows:
1), the Powdered antibacterial peptide finished product that obtains by spray-drying has water-soluble and stability preferably, and has higher protein active.
2), isolate three strain different strains in corrupt bean curd, be initially identified as bacillus, pseudomonad and streptococcus, evidence, the antibacterial peptide aqueous solution all has inhibitory action well to above three strain bacterium.
3), this antibacterial peptide mixed liquor is mixed by Lacticin LLC518 and two kinds of antibacterial peptides of Sakacin LSJ618, wherein the fresh-keeping effect of Lacticin LLC518 obviously is better than the fresh-keeping effect of Sakacin LSJ618, yet the output of Lacticin LLC518 is lower.Although the fresh-keeping effect of Sakacin LSJ618 is relatively poor, but when Lacticin LLC518 and Sakacin LSJ618 mix according to the method in the present invention, two kinds of antibacterial peptides are worked in coordination while using suitable complementation are arranged, therefore obtain the antibacterial peptide mixed liquor of fresh-keeping effect excellence, thereby effectively promoted the ability to food fresh keeping.
4), this antibacterial peptide mixed liquor has stably bacteriostasis, the obvious shelf-life that extends bean curd under 4 ℃ of storage requirements in the bean curd storage process.
The accompanying drawing explanation
Fig. 1 is the growth curve chart of Lactobacillus saki.
Fig. 2 is the gel electrophoresis figure of Lactobacillus saki genomic DNA.
Fig. 3 is the pcr amplification electrophoretogram of Lactobacillus saki 16S rRNA gene.
Fig. 4 is the systematic evolution tree of Lactobacillus saki 16S rDNA sequence.
Fig. 5 is the Gram’s staining figure of bean curd spoilage organisms dfl-1.
Fig. 6 is the Gram’s staining figure of bean curd spoilage organisms dfl-2.
Fig. 7 is the Gram’s staining figure of bean curd spoilage organisms dfl-3.
Fig. 8 is the inhibitory action schematic diagram of the antibacterial peptide aqueous solution to dfl-1.
Fig. 9 is the inhibitory action schematic diagram of the antibacterial peptide aqueous solution to dfl-2.
Figure 10 is the inhibitory action schematic diagram of the antibacterial peptide aqueous solution to dfl-3.
Figure 11 is the affect schematic diagrames of different antibacterial peptides on total number of bacteria in the bean curd storage process.
The specific embodiment
Experimental technique in following embodiment, if no special instructions, be conventional method.
Percentage composition in following embodiment, if no special instructions, be the quality percentage composition.
Lactococcus lactis subsp. lactis involved in the present invention (Lactococcus lactis subsp.lactis) LLC518, in
On January 26th, 2011Be preserved in
China Committee for Culture Collection of Microorganisms is common Microorganism center (CGMCC), deposit number is:
CGMCC No.4584.
The biological property of described Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518 can referring to announced, publication number is the Chinese patent literature of CN102286393A.
Lactobacillus saki involved in the present invention (Lactobacillus sakei) LSJ618, in
2012 On December 18, inBe preserved in
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is: CGMCC No.7017.
The evaluation of Lactobacillus saki LSJ618
With reference to " uncle's outstanding Bacteria Identification handbook the 8th edition, " common bacteria system identification handbook, " lactic acid bacteria-Basic of Biology and application ", " the lactic acid bacteria classification is identified and experimental technique ", " lactic acid bacteria-basis, technology and application " and 16S rRNA sequence homology analysis carry out the bacterial classification evaluation to the detection bacterium.
A, individual morphology feature
By after Gram’s staining, the morphological features of observing the detection bacterium is as follows by the detection strain culture of growth 12h.
Table 1 morphological features
| Dyeing property | Cell size (μ m) | The thalline shape | Arrangement mode | The bacterial classification source |
| G + | （0.46-0.64）×（1.82-1.94） | Tubbiness is shaft-like | Single or gathering exists | The Taiyuan, Shanxi pickled radish |
B, cultural character
1) colony characteristics: will detect bacterium and be inoculated in the MRS solid medium, and cultivate 24h for 30 ℃, and observe colony characteristics, result is as shown in table 2.
Table 2 colony morphology characteristic
2) Liquid Culture proterties: a little detection bacterial classification of picking is inoculated in the MRS fluid nutrient medium, cultivates 24h for 30 ℃, after bacterial growth, observes the liquid culture feature, and result is as shown in table 3.
Table 3 liquid culture feature
| Sterile film is arranged | Turbidity | Have or not precipitation | Growth type |
| Nothing | Muddy | Have | Homogeneous |
3) lawn feature: will detect bacterium and on the MRS slant medium, draw the straight line inoculation, and cultivate 24h for 30 ℃, and observe the lawn growth characteristics, result is as shown in table 4.
Table 4 lawn feature
| Abundant degree | Edge | Surface configuration | Color | Viscosity | Transparency |
| Abundant | Smooth | Kick | Milky | Thickness | Opaque |
4) observation of puncture cultural characteristic: with the transfer needle percutaneous puncture-inoculation, detect bacterium at the MRS semisolid culturemedium in vitro, cultivate 24h for 30 ℃, the growth type of observing detection bacterium as can be known is the fine hair shape.
The physiological characteristic analysis of C, bacterial strain
1) to the utilization of oxygen: will detect bacterium and line dull and stereotyped above, put to anaerobic jar, 30 ℃ of cultivations, the aerobic cultivation of take simultaneously is contrast, after 24h, observes the growing state of bacterium colony.Result shows that detecting bacterium all can grow under the anaerobic and aerobic condition, and upgrowth situation is also better under aerobic conditions, belongs to facultative anaerobe
2) growth temperature and heat resistance: the liquid culture that will cultivate 24h proceeds in the MRS fluid nutrient medium, is placed in 4 ℃, and 15 ℃, 20 ℃, 30 ℃, 37 ℃, 41 ℃, the different temperatures such as 45 ℃ are cultivated, and after 24h, observe growing state.As shown in Table 5, detect bacterium and can in the temperature range of 4～45 ℃, grow, optimum growth temperature is 30 ℃, and result is as shown in table 5.
The growth temperature of table 5 bacterial strain and heat resistance
| 4℃ | 15℃ | 20℃ | 30℃ | 37℃ | 41℃ | 45 |
65℃ |
| + | + | ++ | +++ | ++ | + | + | - |
3) salt tolerance and need salt: get 6～12h children strain liquid in age and be inoculated in the MRS fluid nutrient medium, add respectively 1%, 2%, 5%, 7%, 10% variable concentrations NaCl, cultivate 24h for 30 ℃, detect bacterium and can in 1%～10% salinity, all can grow, the suitableeest salinity is 2%.
4) tolerance of pH: by eugonic detection bacterium be inoculated in respectively pH value be 4.5 and pH value be in 9.5 MRS fluid nutrient medium, 30 ℃ cultivate 24h after, the detection bacterium grows vigorous in the pH value is 4.5 MRS nutrient solution; In being 9.5 nutrient solution, the pH value still can grow.
The biochemical character analysis of D, bacterial strain
1) biochemical test of bacterial strain: according to catalase test, arginine produces ammonia test, and glucose produces sour aerogenesis test, gluconic acid sodium salt produces sour aerogenesis test, Starch Hydrolysis test, aesculin hydrolysis experiment, the litmus milk test, gelatin liquefaction test, V-P test, hydrogen sulfide produces test, arginine hydrolysis experiment, glucan test, the determination test of urase, and the inspection of motility, to detecting bacteria strain, carry out Analysis of Biochemical Characteristics, result is as shown in table 6.
The biochemical character of table 6 bacterial strain
| Biochemical test | Detect bacterial strain |
| Catalase | - |
| Arginine produces ammonia | + |
| Glucose produces acid | + |
| The glucose aerogenesis | + |
| Gluconic acid sodium salt produces acid | + |
| The gluconic acid sodium salt aerogenesis | + |
| Starch Hydrolysis | - |
| The aesculin hydrolysis | + |
| Litmus milk | Reduction |
| Gelatin liquefaction | - |
| The V-P test | + |
| Hydrogen sulfide produces | + |
| The arginine hydrolysis | + |
| Glucan produces | + |
| The mensuration of urase | - |
| Motility | - |
Annotate: "+" means positive, and "-" means negative.
Detecting as can be seen from Table 6 bacteria strain is the catalase feminine gender, can not hydrolyzed starch, and the hydrolyzable aesculin, can produce glucan, reducible litmus milk, gelatin liquefaction and urase are measured all negative, and V-P test, glucose produce sour aerogenesis test and gluconic acid sodium salt product acid test is all positive; It is positive that hydrogen sulfide produces test; It is all positive that arginine produces ammonia test, gluconic acid sodium salt aerogenesis and arginine hydrolysis.
2) carbohydrate fermentation and acid experiment: get a little nutrient solution in colorimetric disc, get simultaneously do not add carbohydrate the PY nutrient solution in contrast, drip BTB-MR reagent, relatively the variation of color.According to change color, but preliminary judgement produces the power of bacterium hydrolysis carbohydrate, and experimental result is as shown in table 7.
The carbohydrate fermentation character of table 7 bacterial strain
| The carbohydrate fermentation and acid | Detect bacterial strain |
| Arabinose | + |
| Cellobiose | + |
| Aesculin | + |
| D-Fructose | + |
| The D-galactolipin | + |
| Glucose | + |
| Lactose | + |
| D (+) maltose | - |
| D (+) mannose | + |
| PEARLITOL 25C | - |
| Melezitose | - |
| Melibiose | + |
| D (+) raffinose | - |
| Rhamnose | - |
| D-(-) ribose | + |
| Salicin | + |
| D-glucitol | - |
| Sucrose | + |
| Trehalose | + |
| Wood sugar | - |
| Gluconic acid sodium salt | + |
| The DL-erythrite | - |
| Glycerine | - |
| Inositol | - |
| The L-sorbose | - |
| Soluble starch | - |
Annotate: "+" means positive, and "-" means negative.
As can be seen from the above table, detect bacterium and can utilize aesculin, D-Fructose, D-galactolipin, glucose, D (+) mannose, D-(-) ribose, sucrose, trehalose, gluconic acid sodium salt, Arabinose, cellobiose, lactose, melibiose, salicin to ferment, can not be hydrolyzed PEARLITOL 25C, melezitose, D (+) raffinose, D (+) maltose, D-glucitol, wood sugar, DL-erythrite, glycerine, inositol, L-sorbose, rhamnose and soluble starch.
3) Analysis of Biochemical Characteristics of bacterial strain: according to catalase test, arginine produces ammonia test, glucose produces sour aerogenesis test, gluconic acid sodium salt produces sour aerogenesis test, the Starch Hydrolysis test, the aesculin hydrolysis experiment, the litmus milk test, gelatin liquefaction test, the V-P test, hydrogen sulfide produces test, the arginine hydrolysis experiment, the glucan test, the determination test of urase, the inspection of motility and carbohydrate produce the acid test, to detecting bacteria strain, carry out Analysis of Biochemical Characteristics, with reference to " the outstanding Bacteria Identification handbook of uncle the 8th edition, " the lactic acid bacteria classification is identified and experimental technique ", " lactic acid bacteria-Basic of Biology and application " and " lactic acid bacteria-basis, technology and application ", by comparing the lactobacillus of detection bacterial strain as can be known from lactic acid bacteria, and the lactobacillus Lactobacillus saki has higher similitude, therefore preliminary definite bacterial strain is the lactobacillus Lactobacillus saki.
The structure of E, 16S rRNA gene sequencing and systematic evolution tree
1) drafting of strain growth curve: for extracting bacterial genomes DNA, carried out the mensuration of growth curve to detecting bacterium, to obtain the growth characteristics of different bacterium, determined exponential phase.Inoculating strain in the 10mLMRS fluid nutrient medium, 30 ℃, 160rpm shaken cultivation 12h.Inoculum concentration with 7% is inoculated in the conical flask that fills 100mL MRS.Mix and survey its OD
600Value.30 ℃ afterwards, the 160rpm shaken cultivation, surveyed OD every one hour
600Value.The incubation time of take is abscissa, OD value OD
600For ordinate is drawn the growth curve (see figure 1), in Fig. 1, bacterial strain 14-1 detects bacterial strain for experiment.By Fig. 1, can determine that the bacterium liquid of the logarithmic phase of cultivating 6h is the experiment material of extracting genomic DNA.
2) extraction of bacterial genomes DNA: reference reagent box specification carries out the extraction of bacterial genomes DNA to detecting bacterium, and concrete operations are as follows:
By appropriate exponential phase bacterium, 10000rpm, centrifugal 1min, thoroughly abandon clean culture medium.Add 200 μ L to contain the TE of 400 μ g/mL lysozymes.Mix, under room temperature, enzymolysis is 3～5 minutes.In 200 μ LTE suspended sample, add 400 μ L digestion buffer solutions, after mixing, add 3 μ L Proteinase K, mix 55 ℃ of insulation 5min.
Add 260 μ L absolute ethyl alcohols to mix, with the 1-mLTip head, sample is all transferred to cover and be put in the UNIQ-10 post in the 2-mL collecting pipe.The centrifugal 1min of 8000rpm room temperature, discard the waste liquid in collecting pipe.Add 500 μ L washing lotions, the centrifugal 1min of 10000rpm room temperature, discard the whole waste liquids in collecting pipe again.Post is put back in collecting pipe, 10000rpm, centrifugal 1 minute of room temperature, remove residual washing lotion.
Post is put back in new clean 1.5mL centrifuge tube, added 50 μ L elution buffers in post central authorities, room temperature or 37 ℃ of placement 2min.10000rpm, the centrifugal 1min of room temperature.Liquid in centrifuge tube is genomic DNA.
3) gel electrophoresis of genomic DNA: by genomic DNA electrophoresis under 0.7% Ago-Gel that extraction obtains, concrete operations are as follows:
Accurately take the 0.14g agarose, be poured in little erlenmeyer flask, add 20mL1 * TAE.On bottleneck, cover preservative film, and prick a little apertures, then heating for dissolving agar pulverized sugar in micro-wave oven on film.When the agarose solution melted is cooled to 60 ℃ of left and right, add EB, making its final concentration is 0.5 μ g/mL, fully mixes glue.Take Marker(λ DNA/Hind III) be contrast.The 60v constant voltage, under sample moves to apart from offset plate during along about 1cm place, stop electrophoresis.Under uviol lamp, observe, and adopt gel imaging system to take pictures, as shown in Figure 2, in figure, M is λ DNA/Hind III to result; Swimming lane 4 is for detecting bacterial strain.Detect as seen from the figure the size of genome band of bacteria strain in about 20kb, meet the size of lactic acid bacteria genomic DNA.
4) pcr amplification of 16S rRNA gene: be template with the total DNA extracted, take prokaryotes universal primer 8f and 1509r and be upstream and downstream primer amplification 16S rRNA gene.
Primer fP1(5 '-AGAGTTTGATCCTGGCTCAG-3 ') and primer rP2(5 '-TACGGTTACCTTGTTACGACTT-3 ') respectively 8～27 and 1531～1509 nucleotides of target Escherichia coli 16S rRNA.25 μ L PCR reaction systems are as follows:
Table 8 25 μ L PCR reaction systems
| Reagent dosage (μ L) |
| 25mmol/LMgCl 2 | 1.5 | 1.5mmol/L |
| Taq enzyme (5U/ μ L) | 0.1 | 0.5 |
| Template DNA | ||
| 1 | ? | |
| ddH 2O | 14.8 | ? |
Amplification condition is:
The genomic DNA of bacterial strain is after the pcr amplification of 16S rRNA gene, amplified production is electrophoresis under 1.0% Ago-Gel, usings Marker F as standard control, and 5 μ L directly add in the point sample hole, the PCR product of 4 μ L mixes with 6 * loading buffer of 1 μ L, 70V electrophoresis 1h.As shown in Figure 3, wherein M is Marker, and swimming lane 4 is for detecting bacterial strain.Stripe size, in the 1500bp left and right, meets the size of 16SrDNA as seen from the figure.
5) sequencing result: pcr amplification product is sent to the mensuration that Shanghai Sangon company carries out sequence.The sequence that detects bacteria strain 14-1 is as follows.
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCACTCTCGTTTAGATTGAAGGAGCTTGCTCCTGATTGATAAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAACCTAACACCGCATGGTGTAGGGTTGAAAGATGGTTTCGGCTATCACTTTAGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCACCAAGACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGGAGAAGAATGTATCTAATAGTAACTGATCAGGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCCTTCGGGGAGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAACC
6) sequence homology analysis: related microorganisms sequence similarity known in the 16S rRNA gene order of detection bacteria strain and GenBank shows, with other Lactobacillus sakis that search, very high similitude is arranged.According to Search Results, to detecting bacterial strain called after LSJ618, its similitude with Lactobacillus saki has reached 99%, and this result has confirmed that equally Physiology and biochemistry tests the conclusion of gained.
7) structure of systematic evolution tree: in order to show affiliation and the system status thereof between bacterial strain and similar bacterial classification, sequencing result carries out homology relatively by the BLAST on NCBI and the sequence in GenBank.Use the adjacent method (Neighbour-joining) in bioinformatics software Clustal X and MEGA4.0 to carry out sequence homology comparison and constructing system chadogram.As shown in Figure 4, systematic evolution tree has reflected the affiliation between each bacterial strain and similar bacterium to result, and as can be seen from the figure, detecting bacterial strain LSJ618 and Lactobacillus saki (Lactobacillus sakei) has nearer affiliation, and similitude has reached 99%.This means that detecting bacterial strain 14-1 belongs to lactobacillus on taxonomy, be Lactobacillus saki.In addition, detect bacterial strain LSJ618 and in GenBank, log in, the number of logging in is HQ992696.
The preparation process of antibacterial peptide mixed liquor is as follows:
A, antibacterial peptide produces the fermentation of bacterium: Lactococcus lactis subsp. lactis (the Lactococcus lactis subsp.lactis) LLC518 and Lactobacillus saki (Lactobacillus sakei) LSJ618 that from picking on the MRS slant medium, have activated are inoculated in respectively the MRS seed culture medium, 37 ℃ of shaking tables were cultivated 12 hours, respectively seed liquor is inoculated in the MRS fermentation medium by 4% inoculum concentration again, 37 ℃ of standing cultivation 24h can obtain Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid,
B, the preparation of antibacterial peptide finished product: by Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid under the condition of 25 ℃ of temperature, by operating pressure, be 0.02Mpa, membrane aperture is to obtain filtered fluid after the ceramic membrane filter of 0.2 μ m, in this filtered fluid, add solid salt, the percent by volume that the quality of solid salt accounts for filtered fluid is 10%, after dissolving fully, solid salt carries out spray-drying, the EAT of spray dryer is 150 ℃, leaving air temp is 130 ℃, obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
The preparation of C, antibacterial peptide mixed liquor: antibacterial peptide Lacticin LLC518 and Sakacin LSJ618 be take to mass ratio and be mixed to get mixture as the ratio of 1:1, the more described mixture that will obtain be take mass ratio and mixed mutually and can obtain this antibacterial peptide mixed liquor as the ratio of 1:10 with water.
The inhibitory action of the antibacterial peptide aqueous solution to the bean curd spoilage organisms
The separation of A, bean curd spoilage organisms: under under gnotobasis, will be cut into approximately 4 * 3 * 0.5cm of size behind commercially available new fresh bean curd removal surface
3, heavily about 10g fritter, be placed in sterile petri dish in 37 ℃ of cultivations.After the bean curd corruption, get in the sterilized water that about 4g joins 60ml, vibration mixes rear 4 layers of filtered through gauze, and the separation and purification of filtrate dilution coated plate obtains three kinds of different bacterial strains.
The Preliminary Identification of B, three strain spoilage organisms: observe the colony morphology characteristic of bacterial strain, as shown in Fig. 5～7, result is as follows:
Dfl-1 bacterium colony dark yellow is opaque, diameter 3-4mm; Microprotrusion in the middle of circular, edge is irregular;
The dfl-2 bacterium colony is creamy white spherical, diameter 1-2mm, transparent glossy;
The dfl-3 bacterium colony is spherical, the diameter 1-2mm of white, and smooth surface is opaque, neat in edge.
By Physiology and biochemistry experiment Preliminary Identification dfl-1 such as Gram’s staining, sugar fermentation, indoles experiment, VP experiment, MR experiment, gelatin liquefaction, Starch Hydrolysis, nitrate reduction and citrate utilizations, be bacillus; Dfl-2 is pseudomonas, and dfl-3 is streptococcus.
Three strain bacterial strains are made to bacteria suspension and be sprayed on respectively new fresh bean curd surface, cultivate 48h for 37 ℃, result demonstration three strain bacterial strains can obviously be accelerated the corruption of bean curd, and three strain bacterial strains are the main spoilage organisms that causes the bean curd corruption as can be seen here.
C, the antibacterial peptide bacteriostasis to spoilage organisms: using three strain bean curd spoilage organisms as indicator bacteria, adopt Oxford cup double-layer plate method to carry out bacteriostatic test, in the cup of Oxford, add respectively each 200 μ L of the Lacticin LLC518 aqueous solution and the Sakacin LSJ618 aqueous solution, in the antibacterial peptide aqueous solution, the mass ratio of antibacterial peptide and sterilized water is that 1:10(is that the mass percent concentration of the antibacterial peptide aqueous solution is 10%).Plate 37 ℃ of cultivations after 4 ℃ of diffusion 5h, every kind of indicator bacteria do two parallel.Experimental result such as Fig. 8～shown in Figure 10, in figure, 1 is that mass percent concentration is 10% the Lacticin LLC518 aqueous solution; The 2nd, the Sakacin LSJ618 aqueous solution of same concentration.The Lacticin LLC518 aqueous solution that is 10% by Fig. 8～10 visual quality percent concentrations all has obvious bacteriostasis to three strain spoilage organisms, wherein bacterial strain dfl-3 is had to maximum inhibitory action, and antibacterial circle diameter can reach 24mm.The Sakacin LSJ618 aqueous solution of same concentration has different inhibitory action to three strain spoilage organisms, better to the inhibitory action of bacterial strain dfl-3, and bacterial strain dfl-2 is not had to bacteriostasis fully.
The antibacterial peptide mixed liquor is fresh-keeping for bean curd
A: bean curd organoleptic indicator's evaluation: the fritter that commercially available new fresh bean curd is cut into to heavy 10g after removing surface under gnotobasis is placed in respectively different aseptic triangular flasks, respectively by the 2mL Lacticin LLC518 aqueous solution, the Sakacin LSJ618 aqueous solution and the even sprinkle of antibacterial peptide mixed liquor in the bean curd surface, control group adds the 2mL sterilized water.Wherein the aqueous solution of Lacticin LLC518 and Sakacin LSJ618 is 1:10 according to the mass ratio of antibacterial peptide and sterilized water the ratio preparation obtains; The antibacterial peptide mixed liquor first be take mass ratio by Lacticin LLC518 and Sakacin LSJ618 and is mixed as the ratio of 1:1, then the mixture that will obtain and the sterilized water ratio that is 1:10 according to mass ratio is prepared and obtained.Each triangular flask sealing is placed on to 4 ℃ of storages, every 48h, draws the variation of bean curd sense organ score value in storage process according to the evaluation criterion of table 9, result is as shown in table 10.
The evaluation criterion of table 9 bean curd sense organ score value
The impact of table 10 different disposal on bean curd sense organ score value
As seen from Table 10, after different antibacterial peptides were processed, the sensory evaluation score value of bean curd was all high than control group, and each antibacterial peptide of general description all helps the fresh-keeping of bean curd to guarantee the quality, but score value all is downward trend gradually along with the prolongation of storage time.Control group is after 4 ℃ of storage 6d, and the bean curd surface starts to be clamminess, and slight tart flavour is arranged, and after 10d, the institutional framework heavy damage of bean curd, have strong acid smell.The variation of the bean curd sense organ score value that the Lacticin LLC518 aqueous solution is processed is slow, during to 10d, slight tart flavour just occurs, and corruption appears in 4d in evening than control group.The bean curd that the Sakacin LSJ618 aqueous solution is processed, storing the dehydration of 8d rear surface, be clamminess, and corruption appears in 2d in evening than control group.While with the antibacterial peptide mixed liquor, bean curd being carried out to Preservation Treatment, bean curd tart flavour just occurs after 4 ℃ of storage 10d, slight hair powder phenomenon is arranged, and corruption appears in 4d in evening than control group, and the fresh-keeping effect that its fresh-keeping effect and the Lacticin LLC518 aqueous solution are processed is very approaching.
Result shows that three kinds of processing modes can both be in the decline that delays in varying degrees bean curd sensory evaluation score value.Mass percent concentration is that the preservation of 10% the Lacticin LLC518 aqueous solution obviously is better than the Sakacin LSJ618 aqueous solution.When adopting the antibacterial peptide mixed liquor to process bean curd, in the antibacterial peptide mixed liquor, contain two kinds of antibacterial peptides, and the mass ratio of two kinds of antibacterial peptides is 1:1, be also half of the consumption of each antibacterial peptide consumption when antibacterial peptide is used separately for this reason, but the fresh-keeping effect of the fresh-keeping effect of antibacterial peptide mixed liquor and the Lacticin LLC518 aqueous solution is very approaching, so Lacticin LLC518 and Sakacin LSJ618 the two having certain complementation when fresh-keeping, effectively promoted the ability to beancurd fresh keeping.
B: the mensuration of spoilage organisms sum in storage period bean curd
The processing of bean curd sample: commercially available new fresh bean curd is removed behind surface the fritter that is cut into heavy 10g and is placed in respectively different aseptic triangular flasks under gnotobasis, according to described method before, add respectively the 2mL mass percent concentration to be 10% the Lacticin LLC518 aqueous solution, the Sakacin LSJ618 aqueous solution and antibacterial peptide mixed liquor, control group adds the 2mL sterilized water.After each triangular flask sealing, in 4 ℃ of storages, measure the spoilage organisms sum every 48h.
The mensuration of spoilage organisms sum: get the triangular flask that one group of different modes was processed, add wherein the 100ml sterilized water, dilute by decimal dilution method after vibration mixes.From the dilution of different gradients, taking out 0.1mL, evenly coat on the LB solid medium respectively, carry out colony counting after 37 ℃ of cultivation 24h, calculate contained spoilage organisms sum in every gram bean curd sample.Experimental result is shown in Figure 11, and in figure, " " represents control group; " ■ " representation quality percent concentration is 10% Lacticin LLC518 aqueous solution processed group; "●" representation quality percent concentration is 10% Sakacin LSJ618 aqueous solution processed group; " ▲ " representation quality percent concentration is 10% antibacterial peptide mixed liquor processed group.
As can be seen from Figure 11, after different antibacterial peptide processed group were processed, the spoilage organisms number in bean curd all was starkly lower than control group, and each antibacterial peptide processed group of general description all can effectively suppress the growth of bean curd spoilage organisms.According to national standard (GB2711.1998), the total number of bacteria of bean curd in bulk answers≤1 * 10
5CFU/g, control group bean curd spoilage organisms sum after 4 ℃ of storage 2d has surpassed national standard, has reached 1.2 * 10
5CFU/g, thus the shelf-life of control group bean curd be less than 2d.Through the processing of the Lacticin LLC518 aqueous solution and after 4 ℃ of storage 4d, in bean curd, the spoilage organisms sum reaches 4.8 * 10
4CFU/g, so in LacticinLLC518 aqueous solution processed group, the shelf-life of bean curd is at least 4d.Bean curd total number of bacteria after 2d in Sakacin LSJ618 aqueous solution processed group is 1.3 * 10
4CFU/g, than the low order of magnitude of control group.The antibacterial peptide mixed liquor carries out Preservation Treatment to bean curd, the total number of bacteria 7.1 * 10 after 4d
4CFU/g, reach 6.9 * 10 after 6d
5CFU/g, the shelf-life is at least 4d, and the fresh-keeping effect of this and Lacticin LLC518 aqueous solution processed group is very approaching.When the independent antibacterial peptide aqueous solution is identical with the antibacterial peptide mixed liquid concentration, the consumption of two kinds of antibacterial peptides in the antibacterial peptide mixed liquor is respectively its half of consumption while using separately, hence one can see that, and two kinds of antibacterial peptides mix while using, obvious complementation is arranged, can effectively improve the ability to food fresh keeping.
Prolongation along with period of storage, after 6-14d, although in each processed group bean curd, the spoilage organisms sum has surpassed set quota, but still be starkly lower than control group, illustrate that the antibacterial peptide mixed liquor in this invention is comparatively stable to the inhibitory action of bean curd spoilage organisms, be not subjected to the impact of bean-curd product composition and environment, thereby have using value preferably, further can be generalized in other food fresh-keeping.
Claims (5)
1. antibacterial peptide mixed liquor, this antibacterial peptide mixed liquor is mixed by antibacterial peptide prepared by two kinds of bacterial strains, wherein:
A kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518, its
The deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.4584;
A kind of Lactobacillus saki (Lactobacillus sakei) LSJ618, its
Chinese microorganism strain is protected The deposit number of hiding administration committee's common micro-organisms center is: CGMCC No.7017;
By the antibacterial peptide that the described Lactococcus lactis subsp. lactis that ferments (Lactococcus lactis subsp.lactis) LLC518 obtains, called after Lacticin LLC518; By the antibacterial peptide that the described Lactobacillus saki that ferments (Lactobacillus sakei) LSJ618 obtains, called after Sakacin LSJ618;
Antibacterial peptide Lacticin LLC518 and Sakacin LSJ618 be take to mass ratio and be mixed to get mixture as the ratio of 1:1, the more described mixture that will obtain be take mass ratio and mixed mutually and can obtain this antibacterial peptide mixed liquor as the ratio of 1:10 with water.
2. antibacterial peptide mixed liquor according to claim 1, it is characterized in that: Lactococcus lactis subsp. lactis (the Lactococcus lactis subsp.lactis) LLC518 and Lactobacillus saki (Lactobacillus sakei) LSJ618 that have activated from picking on the MRS slant medium, and be inoculated in respectively in the MRS seed culture medium, 37 ℃ of shaking tables were cultivated 12 hours, by 4% inoculum concentration, be inoculated in respectively in the MRS fermentation medium again, 37 ℃ of standing cultivation 24h, can obtain Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid: by two kinds of zymotic fluids respectively after the ceramic membrane filter degerming, spray-drying can obtain respectively pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product again.
3. antibacterial peptide mixed liquor according to claim 1, it is characterized in that: by Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) LLC518 zymotic fluid and Lactobacillus saki (Lactobacillus sakei) LSJ618 zymotic fluid respectively under the condition of 25 ℃ of temperature, by operating pressure, be 0.02Mpa, membrane aperture is after the ceramic membrane filter of 0.2 μ m, to obtain removing the supernatant of thalline, in two kinds of supernatants, add solid salt respectively, the quality of the solid salt added and the mass ratio of described supernatant are 1:10, after dissolving fully, solid salt carries out spray-drying by spray dryer, the EAT of described spray dryer is 150 ℃, leaving air temp is 130 ℃, after spray-drying, namely obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
4. the method for food fresh keeping of an antibacterial peptide mixed liquor as claimed in claim 1 is characterized in that: by the even sprinkle of antibacterial peptide mixed liquor in food surface.
5. the method for food fresh keeping of antibacterial peptide mixed liquor according to claim 4 is characterized in that: every 10g bean curd of take adds 2mL antibacterial peptide mixed liquor and is ratio, by the even sprinkle of antibacterial peptide mixed liquor in block bean curd surface.
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