CN103404939B - 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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- CN103404939B CN103404939B CN201310082580.9A CN201310082580A CN103404939B CN 103404939 B CN103404939 B CN 103404939B CN 201310082580 A CN201310082580 A CN 201310082580A CN 103404939 B CN103404939 B CN 103404939B
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- antibacterial peptide
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- peptide mixed
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 solution and the method for food fresh keeping thereof.
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 radiation keeping method easily causes tofu texture to change and mouthfeel declines, Chemical Preservative can produce adverse influence to HUMAN HEALTH and ecotope again.Therefore be necessary that the natural biological preservation liquid that research has a good bacteriostatic action extends effective period of food quality.Existingly studies have shown that the natural frond extracts such as natural pigment, garlicin, extract solution of bamboo leaves and herbal medicine have certain anti-corrosive fresh-keeping effect to bean curd.But the most of natural frond extract using is at present all raw 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.What in addition, the mechanism of action of various sanitass, antimicrobial spectrum and range of application etc. were studied is also deep not.Therefore be necessary the more efficient safer biological preservative of exploitation.
Milk-acid bacteria is just widely used in the anti-corrosive fresh-keeping of food since ancient times, and the small-molecule peptide material that milk-acid bacteria secretion produces can be by the enzyme liberating 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 pathogenic bacterias and food spoilage bacterium, and there is efficient, nontoxic, noresidue, without advantages such as resistance, be widely used in the anti-corrosive fresh-keeping of milk, cheese, tinned food, fish product, beer, beverage etc.Because Nisin can only be soluble status under acidic conditions, under neutrallty condition, solvability is low, and poor stability has greatly affected the application of Nisin in bean product are fresh-keeping.
Summary of the invention
One of object of the present invention is to provide a kind of antibacterial peptide mixed solution 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 solution, and the antibacterial peptide that this antibacterial peptide mixed solution is prepared by two kinds of bacterial strains mixes, 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 sake (Lactobacillus sakei) LSJ618, its
chinese microorganism strain is protected the deposit number of hiding management committee's common micro-organisms center is: CGMCC No.7017;
The antibacterial peptide obtaining by the described Lactococcus lactis subsp.lactis lactic acid subspecies that ferments (Lactococcus lactis subsp.lactis) LLC518, called after Lacticin LLC518; The antibacterial peptide obtaining by the described lactobacillus sake that ferments (Lactobacillus sakei) LSJ618, called after Sakacin LSJ618;
By antibacterial peptide Lacticin LLC518 and Sakacin LSJ618, the ratio taking mass ratio as 1:1 is mixed to get mixture, the more described mixture obtaining is mixed mutually and can obtain this antibacterial peptide mixed solution with the ratio of water taking mass ratio as 1:10.
The preparation method of antibacterial peptide is by following approach:
First Lactococcus lactis subsp.lactis lactic acid subspecies (the Lactococcus lactis subsp.lactis) LLC518 that picking has activated from MRS solid slant culture base and lactobacillus sake (Lactobacillus sakei) LSJ618, and be inoculated in respectively in MRS liquid seed culture medium, 37 DEG C of shaking tables are cultivated 12 hours, be inoculated in respectively in MRS fermention medium by 4% inoculum size again, 37 DEG C leave standstill cultivation 24h, can obtain Lactococcus lactis subsp.lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid: by two kinds of fermented liquids respectively after ceramic membrane filter degerming, spraying is dry again can obtain respectively pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
The actual conditions of spraying drying step is as follows: by Lactococcus lactis subsp.lactis lactic acid subspecies (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid respectively under the condition of 25 DEG C of temperature, be 0.02Mpa by working pressure, membrane pore size is the supernatant liquor that obtains removing thalline after the ceramic membrane filter of 0.2 μ m, in two kinds of supernatant liquors, add solid salt respectively, the ratio that the quality of the solid salt adding accounts for described supernatant liquor volume is 1:10, after dissolving completely, solid salt sprays by spray-drier dry, the inlet temperature of described spray-drier is 150 DEG C, air outlet temperature is 130 DEG C, after spraying is dry, obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
Two of object of the present invention is to provide the method for a kind of above-mentioned antibacterial peptide mixed solution for food fresh keeping, and its concrete scheme is as follows: by even antibacterial peptide mixed solution sprinkle in food surfaces.
Further, add 2mL antibacterial peptide mixed solution as ratio taking every 10g bean curd, by antibacterial peptide mixed solution sprinkle in block bean curd surface.
Beneficial effect of the present invention is as follows:
1), have water-soluble and stability preferably by the dry Powdered antibacterial peptide finished product obtaining of spraying, and there is higher protein-active.
2), isolate three strain different strains in corrupt bean curd, be initially identified as genus bacillus, pseudomonas and suis, evidence, the antibacterial peptide aqueous solution all has restraining effect well to above three strain bacterium.
3), this antibacterial peptide mixed solution is mixed by Lacticin LLC518 and two kinds of antibacterial peptides of Sakacin LSJ618, wherein the fresh-keeping effect of Lacticin LLC518 is obviously better than the fresh-keeping effect of Sakacin LSJ618, but the output of Lacticin LLC518 is lower.Although the fresh-keeping effect of Sakacin LSJ618 is relatively poor, but in the time that Lacticin LLC518 and Sakacin LSJ618 mix according to the method in the present invention, two kinds of antibacterial peptides have suitable complementary action while working in coordination use, therefore obtain the antibacterial peptide mixed solution of fresh-keeping effect excellence, thereby effectively promoted the ability to food fresh keeping.
4), this antibacterial peptide mixed solution has stably bacteriostatic action, the obvious quality guaranteed period that extends bean curd under 4 DEG C of storage requirements in bean curd storage process.
Brief description of the drawings
Fig. 1 is the growth curve chart of lactobacillus sake.
Fig. 2 is the gel electrophoresis figure of lactobacillus sake genomic dna.
Fig. 3 is the pcr amplification electrophorogram of lactobacillus sake 16S rRNA gene.
Fig. 4 is the systematic evolution tree of lactobacillus sake 16S rDNA sequence.
Fig. 5 is the gramstaining figure of bean curd spoilage organism dfl-1.
Fig. 6 is the gramstaining figure of bean curd spoilage organism dfl-2.
Fig. 7 is the gramstaining figure of bean curd spoilage organism dfl-3.
Fig. 8 is the restraining effect schematic diagram of the antibacterial peptide aqueous solution to dfl-1.
Fig. 9 is the restraining effect schematic diagram of the antibacterial peptide aqueous solution to dfl-2.
Figure 10 is the restraining effect schematic diagram of the antibacterial peptide aqueous solution to dfl-3.
Figure 11 is the affect schematic diagram of different antibacterial peptides on total plate count in bean curd storage process.
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 subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 involved in the present invention, in
on January 26th, 2011be preserved in
in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City china Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of academy of sciences of state (CGMCC), deposit number is:
cGMCC No.4584.
The biological property of described Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 can be referring to the Chinese patent literature of having announced, publication number is CN102286393A.
Lactobacillus sake (Lactobacillus sakei) LSJ618 involved in the present invention, in
2012 on December 18, inbe preserved in
the China of Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica is micro- biological inoculum preservation management committee's common micro-organisms center (CGMCC), deposit number is: CGMCC?
no.7017.
The qualification of lactobacillus sake LSJ618
Carry out strain identification with reference to " the outstanding Bacteria Identification handbook of uncle " the 8th edition, " common bacteria system identification handbook ", " milk-acid bacteria-Basic of Biology and application ", " lactic-acid-bacterium classification qualification and experimental technique ", " lactic-acid-bacterium-basis, technology and application " and 16S rRNA sequence homology analysis to detecting bacterium.
A, individual morphology feature
By the detection strain culture of growth 12h, by after gramstaining, the morphological features of observing detection bacterium is as follows.
Table 1 morphological features
| Dyeing property | (μ m) for cell size | Thalline shape | Arrangement mode | Bacterial classification source |
| G + | （0.46-0.64）×（1.82-1.94） | Tubbiness is shaft-like | Single or gathering exists | Taiyuan, Shanxi pickled radish |
B, cultural characters
1) colony characteristics: detection bacterium is inoculated in to MRS solid medium, cultivates 24h for 30 DEG C, 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 MRS liquid nutrient medium, cultivates 24h for 30 DEG C, observes liquid culture feature after bacterial growth, and result is as shown in table 3.
Table 3 liquid culture feature
| There is sterile film | Opacity | Have or not precipitation | Growth form |
| Nothing | Muddy | Have | Homogeneous |
3) lawn feature: will detect bacterium and draw straight line inoculation on MRS slant medium, and cultivate 24h for 30 DEG C, and observe lawn growth characteristics, result is as shown in table 4.
Table 4 lawn feature
| Abundant degree | Edge | Surface shape | Color | Viscosity | Transparency |
| Abundant | Smooth | Pimple | Oyster white | Thickness | Opaque |
4) observation of stab culture feature: use inoculating needle percutaneous puncture-inoculation to detect bacterium at MRS semisolid medium in vitro, cultivate 24h for 30 DEG C, the growth form of observing known detection bacterium is fine hair shape.
The physiological characteristic analysis of C, bacterial strain
1) utilization to oxygen: detection bacterium is lined to flat board upper, put to anaerobic jar, 30 DEG C of cultivations, simultaneously taking aerobic cultivation as contrast, observe the growing state of bacterium colony after 24h.Result shows that detect bacterium all can grow under anaerobic and aerobic condition, and under aerobic conditions, upgrowth situation is also better, belongs to facultative anaerobe
2) growth temperature and thermotolerance: proceed in MRS liquid nutrient medium cultivating the liquid culture of 24h, be placed in 4 DEG C, 15 DEG C, 20 DEG C, 30 DEG C, 37 DEG C, 41 DEG C, the differing tempss such as 45 DEG C are cultivated, and observe growing state after 24h.As shown in Table 5, detect bacterium and can in the temperature range of 4～45 DEG C, grow, optimum growth temperature is 30 DEG C, and result is as shown in table 5.
The growth temperature of table 5 bacterial strain and thermotolerance
| 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 MRS liquid nutrient medium, add respectively 1%, 2%, 5%, 7%, 10% different concns NaCl, cultivate 24h for 30 DEG C, detect bacterium and can in 1%～10% salinity, all can grow, the suitableeest salt concn is 2%.
4) tolerance of pH: by eugonic detection bacterium be inoculated in respectively pH value be 4.5 and the pH value MRS liquid nutrient medium that is 9.5 in, after 30 DEG C of cultivation 24h, in the MRS nutrient solution that detection bacterium is 4.5 in pH value, grow vigorous; In the nutrient solution that is 9.5 in 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, glucose produces sour aerogenesis test, Sunmorl N 60S produces sour aerogenesis test, Starch Hydrolysis test, Vitamin C2 hydrolysis experiment, litmus milk test, gelatin liquification test, V-P test, hydrogen sulfide produces test, arginine hydrolysis experiment, dextran test, the determination test of urase, and the inspection of mobility, carry out Analysis of Biochemical Characteristics to detecting bacteria strain, 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 | + |
| Glucose aerogenesis | + |
| Sunmorl N 60S produces acid | + |
| Sunmorl N 60S aerogenesis | + |
| Starch Hydrolysis | - |
| Vitamin C2 hydrolysis | + |
| Litmus milk | Reduction |
| Gelatine liquefication | - |
| V-P test | + |
| Hydrogen sulfide produces | + |
| Arginine hydrolysis | + |
| Dextran produces | + |
| The mensuration of urase | - |
| Mobility | - |
Note: "+" represents positive, "-" represents negative.
Detecting as can be seen from Table 6 bacteria strain is catalase feminine gender, can not hydrolyzed starch, and hydrolyzable Vitamin C2, can produce dextran, reducible litmus milk, gelatine liquefication and urase are measured all negative, and V-P test, glucose produce sour aerogenesis test and Sunmorl N 60S product acid test is all positive; It is positive that hydrogen sulfide produces test; It is all positive that arginine produces ammonia test, Sunmorl N 60S aerogenesis and arginine hydrolysis.
2) carbohydrate fermentation and acid experiment: get a little nutrient solution in colorimetric disc, get simultaneously do not add carbohydrate PY nutrient solution in contrast, drip BTB-MR reagent, relatively the variation of color.According to colour-change, can preliminary judgement produce the power of bacterium hydrolysis carbohydrate, experimental result is as shown in table 7.
The carbohydrate fermentation character of table 7 bacterial strain
| Carbohydrate fermentation and acid | Detect bacterial strain |
| L-arabinose | + |
| Cellobiose | + |
| Vitamin C2 | + |
| D-Fructose | + |
| D-semi-lactosi | + |
| Glucose | + |
| Lactose | + |
| D (+) maltose | - |
| D (+) seminose | + |
| PEARLITOL 25C | - |
| Melizitose | - |
| Melibiose | + |
| D (+) raffinose | - |
| Rhamnosyl | - |
| D-(-) ribose | + |
| Saligenin | + |
| D-glucitol | - |
| Sucrose | + |
| Trehalose | + |
| Wood sugar | - |
| Sunmorl N 60S | + |
| DL-tetrahydroxybutane | - |
| Glycerine | - |
| Inositol | - |
| L-sorbose | - |
| Zulkovsky starch | - |
Note: "+" represents positive, "-" represents negative.
As can be seen from the above table, detect bacterium and can utilize Vitamin C2, D-Fructose, D-semi-lactosi, glucose, D (+) seminose, D-(-) ribose, sucrose, trehalose, Sunmorl N 60S, L-arabinose, cellobiose, lactose, melibiose, saligenin to ferment, can not be hydrolyzed PEARLITOL 25C, melizitose, D (+) raffinose, D (+) maltose, D-glucitol, wood sugar, DL-tetrahydroxybutane, glycerine, inositol, L-sorbose, rhamnosyl and Zulkovsky starch.
3) Analysis of Biochemical Characteristics of bacterial strain: according to catalase test, arginine produces ammonia test, glucose produces sour aerogenesis test, Sunmorl N 60S produces sour aerogenesis test, Starch Hydrolysis test, Vitamin C2 hydrolysis experiment, litmus milk test, gelatin liquification test, V-P test, hydrogen sulfide produces test, arginine hydrolysis experiment, dextran test, the determination test of urase, the inspection of mobility and carbohydrate produce acid test, carry out Analysis of Biochemical Characteristics to detecting bacteria strain, with reference to " the outstanding Bacteria Identification handbook of uncle " the 8th edition, " lactic-acid-bacterium classification qualification and experimental technique ", " milk-acid bacteria-Basic of Biology and application " and " lactic-acid-bacterium-basis, technology and application ", by comparing the lactobacillus of known detection bacterial strain from milk-acid bacteria, and lactobacillus lactobacillus sake has higher similarity, therefore tentatively determine that bacterial strain is lactobacillus lactobacillus sake.
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 logarithmic phase.Inoculating strain in 10mL MRS liquid nutrient medium, 30 DEG C, 160rpm shaking culture 12h.Be inoculated in the Erlenmeyer flask that fills 100mL MRS with 7% inoculum size.Mix and survey its OD
600value.30 DEG C afterwards, 160rpm shaking culture, surveyed OD every one hour
600value.Taking incubation time as X-coordinate, optical density value OD
600for ordinate zou is drawn growth curve (see figure 1), in Fig. 1, bacterial strain 14-1 is experiment detection bacterial strain.Can determine that by Fig. 1 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 sheets carries out the extraction of bacterial genomes DNA to detecting bacterium, and concrete operations are as follows:
By appropriate logarithmic phase bacterium, 10000rpm, centrifugal 1min, thoroughly abandons clean substratum.Add the TE of 200 μ L containing 400 μ g/mL N,O-Diacetylmuramidases.Mix enzymolysis 3～5 minutes under room temperature.In 200 μ LTE suspended sample, add 400 μ L digestion damping fluids, after mixing, add 3 μ L Proteinase K, mix 55 DEG C of insulation 5min.
Add 260 μ L dehydrated alcohols to mix, with 1-mLTip head, sample is all transferred to cover and be put in the UNIQ-10 post in 2-mL collection tube.The centrifugal 1min of 8000rpm room temperature, discards the waste liquid in collection tube.Add 500 μ L washing lotions, the centrifugal 1min of 10000rpm room temperature, discards the whole waste liquids in collection tube again.Post is put back in collection tube, 10000rpm, centrifugal 1 minute of room temperature, removes 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 DEG C of placement 2min.10000rpm, the centrifugal 1min of room temperature.Liquid in centrifuge tube is genomic dna.
3) gel electrophoresis of genomic dna: the genomic dna that extraction is obtained is electrophoresis under 0.7% sepharose, and concrete operations are as follows:
Accurately take 0.14g agarose, be poured in little erlenmeyer flask, add 20mL1 × TAE.On bottleneck, cover preservative film, and on film, prick a little apertures, then heating for dissolving agar pulverized sugar in microwave oven.In the time that the agarose solution melting is cooled to 60 DEG C of left and right, add EB, making its final concentration is 0.5 μ g/mL, fully mixes glue.Taking Marker(λ DNA/Hind III) be contrast.60v constant voltage during along about 1cm place, stops electrophoresis under sample moves to apart from offset plate.Under ultraviolet 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 milk-acid bacteria genomic dna.
4) pcr amplification of 16S rRNA gene: with the total DNA extracting be template, taking prokaryotic organism universal primer 8f and 1509r as 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 Nucleotide of target intestinal bacteria 16S rRNA.25 μ L PCR reaction systems are as follows:
Table 8 25 μ L PCR reaction systems
| Reagent | Consumption (μ L) | Final concentration |
| 10×PCR?buffer | 2.5 | 1× |
| 2mmol/L?dNTP | 2.5 | Every kind of 0.2mmol/L |
| Upstream primer (10 μ M) | 1.30 | 5μmol/L |
| Downstream primer (10 μ M) | 1.30 | 5μmol/L |
| 25mmol/LMgCl 2 | 1.5 | 1.5mmol/L |
| Taq enzyme (5U/ μ L) | 0.1 | 0.5U |
| 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% sepharose, and using Marker F as standard control, 5 μ L directly add in 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 1500bp left and right, meets the size of 16SrDNA as seen from the figure.
5) sequencing result: the mensuration that pcr amplification product is sent to Shanghai Sangon company and 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 there is very high similarity with other lactobacillus sakes that search.According to Search Results, to detecting bacterial strain called after LSJ618, it has reached 99% with the similarity of lactobacillus sake, and this result has confirmed that Physiology and biochemistry tests the conclusion of gained equally.
7) structure of systematic evolution tree: in order to show sibship and the system status thereof between bacterial strain and similar bacterial classification, sequencing result carries out homology comparison by the sequence in BLAST and GenBank on NCBI.Use the adjacent method (Neighbour-joining) in bioinformatics software Clustal X and MEGA4.0 to carry out sequence homology comparison and constructing system evolutionary tree.As shown in Figure 4, systematic evolution tree has reflected the sibship between each bacterial strain and similar bacterium to result, and as can be seen from the figure, detecting bacterial strain LSJ618 and lactobacillus sake (Lactobacillus sakei) has nearer sibship, and similarity has reached 99%.This means that detect bacterial strain 14-1 belongs to lactobacillus on taxonomy, is lactobacillus sake.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 solution is as follows:
A, antibacterial peptide produces the fermentation of bacterium: Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 and lactobacillus sake (Lactobacillus sakei) LSJ618 that from MRS slant medium, picking has activated are inoculated in respectively in MRS seed culture medium, 37 DEG C of shaking tables are cultivated 12 hours, respectively seed liquor is inoculated in MRS fermention medium by 4% inoculum size again, 37 DEG C leave standstill cultivation 24h and can obtain Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid,
B, the preparation of antibacterial peptide finished product: by Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid under the condition of 25 DEG C of temperature, be 0.02Mpa by working pressure, 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 10%, after dissolving completely, solid salt carries out spray dried dry, the inlet temperature of spray-drier is 150 DEG C, air outlet temperature is 130 DEG C, obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
The preparation of C, antibacterial peptide mixed solution: the ratio taking mass ratio as 1:1 is mixed to get mixture by antibacterial peptide Lacticin LLC518 and Sakacin LSJ618, the more described mixture obtaining is mixed mutually and can obtain this antibacterial peptide mixed solution with the ratio of water taking mass ratio as 1:10.
The restraining effect of the antibacterial peptide aqueous solution to bean curd spoilage organism
The separation of A, bean curd spoilage organism: 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 DEG C of cultivations.After 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 evaluation of B, three strain spoilage organism: 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;
Dfl-2 bacterium colony is creamy white spherical, diameter 1-2mm, transparent glossy;
Dfl-3 bacterium colony is spherical, the diameter 1-2mm of white, and smooth surface is opaque, neat in edge.
Be bacillus by Physiology and biochemistry experiment preliminary evaluation dfl-1 such as gramstaining, sugar-fermenting, indoles experiment, VP experiment, MR experiment, gelatine liquefication, Starch Hydrolysis, nitrate reduction and Citrate trianion utilizations; Dfl-2 is Rhodopseudomonas, 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 DEG C, result shows that three strain bacterial strains can obviously accelerate the corruption of bean curd, and three strain bacterial strains are the main spoilage organism that causes bean curd corruption as can be seen here.
C, the bacteriostatic action of antibacterial peptide to spoilage organism: using three strain bean curd spoilage organism as indicator, adopt Oxford cup double-layer plate method to carry out bacteriostatic test, in the cup of Oxford, add respectively the 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 DEG C of cultivations after 4 DEG C of diffusion 5h, every kind of indicator do two parallel.Experimental result is as shown in Fig. 8～Figure 10, and 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 percentage concentration all has obvious bacteriostatic action to three strain spoilage organism, wherein bacterial strain dfl-3 is had to maximum restraining effect, and antibacterial circle diameter can reach 24mm.The Sakacin LSJ618 aqueous solution of same concentration has different restraining effect to three strain spoilage organism, better to the restraining effect of bacterial strain dfl-3, and bacterial strain dfl-2 is not had to bacteriostatic action completely.
Antibacterial peptide mixed solution is fresh-keeping for bean curd
A: bean curd Oranoleptic indicator's evaluation: the fritter that is cut into heavy 10g after commercially available new fresh bean curd is removed to 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 solution in bean curd surface, control group adds 2mL sterilized water.The ratio preparation that wherein aqueous solution of Lacticin LLC518 and Sakacin LSJ618 is 1:10 according to the mass ratio of antibacterial peptide and sterilized water obtains; Antibacterial peptide mixed solution by Lacticin LLC518 and Sakacin LSJ618 first the ratio taking mass ratio as 1:1 mix, then the ratio that is 1:10 according to mass ratio by the mixture obtaining and sterilized water is prepared and is obtained.Each triangular flask sealing is placed on to 4 DEG C of storages, the variation that draws bean curd sense organ score value in storage process every 48h according to the judgement criteria of table 9, result is as shown in table 10.
The judgement criteria of table 9 bean curd sense organ score value
The impact of table 10 different treatment on bean curd sense organ score value
As seen from Table 10, after different antibacterial peptides are processed, the sensory evaluation score value of bean curd is all high compared with control group, and the each antibacterial peptide of general description all contributes to the fresh-keeping of bean curd to guarantee the quality, but score value is along with the prolongation of storage time is all downward trend gradually.Control group is after 4 DEG C of storage 6d, and bean curd surface starts to be clamminess, and has slight tart flavour, and after 10d, the weave construction havoc of bean curd, has strong acid smell.The variation of the bean curd sense organ score value of Lacticin LLC518 aqueous solution processing is slow, during to 10d, just occurs slight tart flavour, and compared with control group, corruption appears in 4d in evening.The bean curd of Sakacin LSJ618 aqueous solution processing, in storage 8d rear surface dehydration, is clamminess, and compared with control group, corruption appears in 2d in evening.While bean curd being carried out to Preservation Treatment with antibacterial peptide mixed solution, after 4 DEG C of storage 10d, just there is tart flavour in bean curd, have slight hair powder phenomenon, compared with control group there is corruption in 4d in evening, and the fresh-keeping effect of its fresh-keeping effect and the processing of the Lacticin LLC518 aqueous solution 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 freshening effect of 10% the Lacticin LLC518 aqueous solution is obviously better than the Sakacin LSJ618 aqueous solution.In the time adopting antibacterial peptide mixed solution to process bean curd, in antibacterial peptide mixed solution, contain two kinds of antibacterial peptides, and the mass ratio of two kinds of antibacterial peptides is 1:1, also be the half of the consumption of each antibacterial peptide consumption when antibacterial peptide uses separately for this reason, but the fresh-keeping effect of the fresh-keeping effect of antibacterial peptide mixed solution and the Lacticin LLC518 aqueous solution is very approaching, so Lacticin LLC518 and Sakacin LSJ618 the two thering is certain complementary action when fresh-keeping, effectively promoted the ability to beancurd fresh keeping.
B: the mensuration of spoilage organism sum in storage period bean curd
The processing of bean curd sample: commercially available new fresh bean curd is cut into heavy 10g fritter remove surface under gnotobasis after is placed in respectively different aseptic triangular flasks, add respectively 2mL mass percent concentration to be 10% the Lacticin LLC518 aqueous solution, the Sakacin LSJ618 aqueous solution and antibacterial peptide mixed solution according to described before method, control group adds 2mL sterilized water.After each triangular flask sealing, in 4 DEG C of storages, measure spoilage organism sum every 48h.
The mensuration of spoilage organism sum: get the triangular flask that one group of different modes was processed, add wherein 100ml sterilized water, dilute by decimal dilution method after vibration mixes.From the diluent of different gradients, take out respectively 0.1mL and evenly coat on LB solid medium, after 37 DEG C of cultivation 24h, carry out enumeration, calculate spoilage organism sum contained in every gram of bean curd sample.Experimental result is shown in Figure 11, and in figure, " " represents control group; The Lacticin LLC518 aqueous solution treatment group that " ■ " representation quality percentage concentration is 10%; The Sakacin LSJ618 aqueous solution treatment group that "●" representation quality percentage concentration is 10%; The antibacterial peptide mixed solution treatment group that " ▲ " representation quality percentage concentration is 10%.
As can be seen from Figure 11, after different antibacterial peptide treatment group are processed, the spoilage organism number in bean curd is all starkly lower than control group, and the each antibacterial peptide treatment group of general description all can effectively suppress the growth of bean curd spoilage organism.According to national standard (GB2711.1998), the total plate count of bean curd in bulk answers≤1 × 10
5cFU/g, control group bean curd spoilage organism sum after 4 DEG C of storage 2d has exceeded national standard, has reached 1.2 × 10
5cFU/g, thus the quality guaranteed period of control group bean curd be less than 2d.Process and store after 4d at 4 DEG C through the Lacticin LLC518 aqueous solution, in bean curd, spoilage organism sum reaches 4.8 × 10
4cFU/g, so the quality guaranteed period of bean curd is at least 4d in Lacticin LLC518 aqueous solution treatment group.Bean curd total plate count after 2d in Sakacin LSJ618 aqueous solution treatment group is 1.3 × 10
4cFU/g, compared with the low order of magnitude of control group.Antibacterial peptide mixed solution carries out Preservation Treatment to bean curd, the total plate count 7.1 × 10 after 4d
4cFU/g, reaches 6.9 × 10 after 6d
5cFU/g, the quality guaranteed period is at least 4d, and the fresh-keeping effect of this and Lacticin LLC518 aqueous solution treatment group is very approaching.In the time that the independent antibacterial peptide aqueous solution is identical with antibacterial peptide mixed liquid concentration, the half of consumption when the consumption of two kinds of antibacterial peptides in antibacterial peptide mixed solution is respectively its independent use, hence one can see that, and two kinds of antibacterial peptides mix while use, there is obvious complementary action, can effectively improve the ability to food fresh keeping.
Along with the prolongation of period of storage, after 6-14d, although spoilage organism sum has exceeded set quota in each treatment group bean curd, but still be starkly lower than control group, illustrate that the antibacterial peptide mixed solution in this invention is comparatively stable to the restraining effect of bean curd spoilage organism, be not subject to the impact of bean-curd product composition and environment, thereby there is good using value, further can be generalized in other food fresh-keeping.
Claims (4)
1. an antibacterial peptide mixed solution, this antibacterial peptide mixed solution is that the antibacterial peptide of being prepared by two kinds of bacterial strains mixes, 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 sake (Lactobacillus sakei) LSJ618, its
chinese microorganism strain is protected the deposit number of hiding management committee's common micro-organisms center is: CGMCC No.7017;
The antibacterial peptide obtaining by the described Lactococcus lactis subsp.lactis that ferments (Lactococcus lactis subsp.lactis) LLC518, called after Lacticin LLC518; The antibacterial peptide obtaining by the described lactobacillus sake that ferments (Lactobacillus sakei) LSJ618, called after Sakacin LSJ618;
By antibacterial peptide Lacticin LLC518 and Sakacin LSJ618, the ratio taking mass ratio as 1:1 is mixed to get mixture, the more described mixture obtaining is mixed mutually and can obtain this antibacterial peptide mixed solution with the ratio of water taking mass ratio as 1:10;
Lactococcus lactis subsp.lactis (the Lactococcus lactis subsp.lactis) LLC518 that picking has activated from MRS slant medium and lactobacillus sake (Lactobacillus sakei) LSJ618, and be inoculated in respectively in MRS seed culture medium, 37 DEG C of shaking tables are cultivated 12 hours, be inoculated in respectively in MRS fermention medium by 4% inoculum size again, 37 DEG C leave standstill cultivation 24h, can obtain Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid: by two kinds of fermented liquids respectively after ceramic membrane filter degerming, spraying is dry again can obtain respectively pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
2. antibacterial peptide mixed solution according to claim 1, it is characterized in that: by Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) LLC518 fermented liquid and lactobacillus sake (Lactobacillus sakei) LSJ618 fermented liquid respectively under the condition of 25 DEG C of temperature, be 0.02Mpa by working pressure, membrane pore size is the supernatant liquor that obtains removing thalline after the ceramic membrane filter of 0.2 μ m, in two kinds of supernatant liquors, add solid salt respectively, the quality of solid salt adding and the mass ratio of described supernatant liquor are 1:10, after dissolving completely, solid salt sprays by spray-drier dry, the inlet temperature of described spray-drier is 150 DEG C, air outlet temperature is 130 DEG C, after spraying is dry, obtain pulverous Lacticin LLC518 finished product and Sakacin LSJ618 finished product.
3. the method for food fresh keeping of antibacterial peptide mixed solution as claimed in claim 1, is characterized in that: by even antibacterial peptide mixed solution sprinkle in food surfaces.
4. the method for food fresh keeping of antibacterial peptide mixed solution according to claim 3, is characterized in that: add 2mL antibacterial peptide mixed solution as ratio taking every 10g bean curd, by even antibacterial peptide mixed solution sprinkle in block bean curd surface.
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| CN105961623A (en) * | 2016-05-05 | 2016-09-28 | 杭州正熵科技咨询有限公司 | Bacteriostat production method based on fermentation of yellow soybean curd liquid |
| CN107960584A (en) * | 2017-11-08 | 2018-04-27 | 常州武城服饰有限公司 | A kind of dry beancurd fresh keeping method |
| CN108753875A (en) * | 2018-05-23 | 2018-11-06 | 浙江工商大学 | The method that spray drying prepares lactobacillus plantarum ZJ316 bacteriocins |
| CN111374317B (en) * | 2019-12-18 | 2022-08-30 | 苏州大学 | Application of natural antibacterial peptide Hc-CATH in food preservation and fresh-keeping |
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