CN105385633A - Lactobacillus plantarum with function of inhibiting mould activity and application of lactobacillus plantarum - Google Patents
Lactobacillus plantarum with function of inhibiting mould activity and application of lactobacillus plantarum Download PDFInfo
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- CN105385633A CN105385633A CN201510868539.3A CN201510868539A CN105385633A CN 105385633 A CN105385633 A CN 105385633A CN 201510868539 A CN201510868539 A CN 201510868539A CN 105385633 A CN105385633 A CN 105385633A
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- lactobacillus plantarum
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Abstract
The invention discloses lactobacillus plantarum with a function of inhibiting mould activity and application of the lactobacillus plantarum. The lactobacillus plantarum is preserved in China Center for Type Culture Collection (CCTCC for short) with the preservation number of CCTCC M 2015615 and the preservation date of October 19th, 2015. According to the lactobacillus plantarum with the function of inhibiting mould activity, disclosed by the invention, an inhibition action for inhibiting common decay mould in pickled vegetables can be realized, the inherit quality of the pickled vegetables can be maintained, the shelf life of pickled vegetable foods with low salt and low preservative addition can be prolonged, and certain guiding and promoting action for the development of the pickled vegetable industry is realized; meanwhile, a biological preservative prepared from the lactobacillus plantarum disclosed by the invention is simple and convenient in operation and is low in cost.
Description
Technical field
The present invention relates to a kind of plant lactobacillus and application thereof, be specifically related to a kind of plant lactobacillus and the application thereof with mould fungus inhibition function.
Background technology
Chinese like eating pickles, but existing pickles usually adopt salt marsh for extending the pickles quality guaranteed period and add sanitas, make pickles salinity large, sodium content is high, and containing a large amount of sanitass, easily damage human body, people also lose interest to pickles gradually.Therefore, need a kind of quality that pickles can be kept intrinsic badly, can also reach extend less salt, the biological preservative of pickles product preservation term that low sanitas adds.
Summary of the invention
Technical problem to be solved by this invention is, provides a kind of cultivation with low cost, easy, practical has to mould the pickles biological preservative and application thereof that the lactobacterium plantarum strain of good inhibition, less salt and low sanitas add.
The technical scheme that the present invention solves the employing of its technical problem is, a kind of plant lactobacillus with mould fungus inhibition function, called after plant lactobacillus JCM1149 (LactobacillusplantarumJCM1149), described bacterial strain JCM1149 is preserved in China typical culture collection center, it is referred to as CCTCC, deposit number is CCTCCM2015615, preservation date is on October 19th, 2015, preservation address: Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan University's preservation centers, postcode 430072.
The technical scheme that the present invention solves the employing of its technical problem is further that a kind of biological preservative, activeconstituents is described plant lactobacillus.
The technical scheme that the present invention solves the employing of its technical problem is further, a kind of preparation method of biological preservative, plant lactobacillus after activation is inoculated in fresh MRS liquid nutrient medium with 1 ~ 3% inoculum size, at 35 ~ 37 DEG C of constant incubator quiescent culture 24 ~ 26h, then by bacteria suspension in refrigerated centrifuge with the centrifugal 10 ~ 15min of the rotating speed of 2000 ~ 4000r/min, get its supernatant liquor, with the filtering with microporous membrane removing thalline of 0.22 μm, be placed in-60 ~ 80 DEG C of refrigerator and cooled and freeze 12 ~ 15h, through concentrate drying, then dissolve with sterile distilled water and prepare tenfold concentrated solution, .
Further, described MRS liquid nutrient medium is by peptone 10.0g, extractum carnis 5.0g, yeast leaching powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, Triammonium citrate 2.0g, potassium acetate 5.0g, bitter salt 0.2g, manganous sulfate 0.05g, tween 80 1.0mL, distilled water 1000mL, 121 DEG C of sterilizing 15min make.
The technical scheme that the present invention solves the employing of its technical problem is further, a kind of application of plant lactobacillus in pickles processing with mould fungus inhibition function, containing 6 ~ 7 × 10 in every kilogram of pickles
8plant lactobacillus described in cfu.
The technical scheme that the present invention solves the employing of its technical problem is further, the application of a kind of biological preservative in pickles processing, adds biological preservative described in 6 ~ 10g in every kilogram of pickles.
The lactobacterium plantarum strain with mould fungus inhibition function of the present invention can play good restraining effect to Sapromyces common in pickles food, the quality that pickles are intrinsic can be kept, also can reach extend less salt, the preservation term of pickles product that low sanitas adds, certain guidance and the promotion effect is played to the development of pickles industry; Meanwhile, it is easy and simple to handle that the present invention prepares biological preservative, with low cost.
Embodiment
Below in conjunction with embodiment, the present invention is illustrated further.
Embodiment 1: the separation of lactobacterium plantarum strain, purifying and qualification
1, the separation of lactobacterium plantarum strain:
The aseptic technique of 6 kinds of pickles samples is respectively taken Fresh Yuxincao 10g, is cut into the little fourth of 5mm, add 90mL stroke-physiological saline solution vortex concussion 3min, then get suspension and carry out gradient dilution to 10
-7, then respectively from 10
-4-10
-7extent of dilution is drawn 1mL and is added in sterile petri dish, is cooled to room temperature, is positioned in 37 DEG C of constant incubators and cultivates 24-48h and occur to there being molten calcium circle bacterium colony after the MRS solid medium 15-20mL containing calcium carbonate pouring 50 DEG C into mixes.
2, the purifying of lactobacterium plantarum strain:
Picking has the bacterium colony of molten calcium circle to continue to carry out separation and purification on MRS solid medium.Gramstaining and catalase test is carried out, stained positive and catalase negative strain is tentatively decided to be milk-acid bacteria by producing the bacterium colony of molten calcium circle.Freezing Glycerine preservation.
3, strain morphology feature and Physiology and biochemistry qualification:
3.1 strain morphology
This bacterium bacterium colony is circular, and color is creamy white, and bacterium colony performance is smooth, and neat in edge is homogeneous.
3.2 Physiology and biochemistry qualifications, the results are shown in Table 1
The Physiology and biochemistry qualification result of table 1 bacterial strain LY-4
| Index | Result | Index | Result | Index | Result |
| Pectinose | － | Gluconate | － | Melibiose | + |
| Cellobiose | + | Lactose | + | Raffinose | + |
| Polychrom | + | Maltose | + | Rhamnosyl | － |
| Fructose | + | N.F,USP MANNITOL | + | Sorbyl alcohol | － |
| Semi-lactosi | + | Seminose | + | Sucrose | + |
| Glucose | + | Melizitose | + | Wood sugar | + |
Note: "+" represents positive; "-" represents negative.
As shown in Table 1, bacterial strain is positive through gramstaining, and thalline is shaft-like.Catalase test is negative, and gelatin hydrolysis is negative, does not produce H
2s, and most of carbohydrate that can ferment, but negative in fermentation to pectinose, rhamnosyl, sorbose etc.
4, the molecular biology identification of active bacterial strain:
The extraction of 4.1DNA
CTAB method is adopted to extract the genomic dna of milk-acid bacteria.Concrete steps are:
(1) bacterium is collected: get 2mL, and 37 DEG C of shaking tables cultivate the MRS bacterium liquid after 16-18h in 2mL sterile centrifugation tube, and the centrifugal 5min of 12000rpm, removes substratum as much as possible, in order to avoid residual substratum affects subsequent experimental result.
(2) suspension thalline: add the TEbuffer solution of 560 μ L in centrifuge tube with liquid-transfering gun, blow and beat gently, suspensoid, thalline and solution are fully mixed.
(3) the breaking of cell walls: the lysozyme soln (20mg/mL) adding 2 μ L-4 μ L in bacteria suspension, blow and beat 2 ~ 3 times gently with liquid-transfering gun fully to contact with thalline to make N,O-Diacetylmuramidase, under 37 DEG C of water-baths, react 40min somatic cells wall is broken completely.After add Proteinase K Solution (10mg/mL) solubilising protein of 6 μ L, the concentration adding 30 μ L is again that the SDS solution precipitation protein of 10% is (if precipitation appears in SDS solution, should first under 37 DEG C of water-baths, make it dissolve, otherwise the concentration of SDS solution can be affected).By above solution liquid-transfering gun piping and druming mixing, under 37 DEG C of water bath condition, react 45min-60min (transparent to solution).
(4) Polysaccharide removing composition: the NaCI solution (5mol/L) adding 100 μ L in above-mentioned clear solution, shakes mixing gently, puts into 65 DEG C of constant temperature water tank reaction 2min.Add the CTAB/NaCI solution (using front 65 DEG C of preheatings) of 80 μ L, several lower 65 DEG C of water-bath 10min of rifle head piping and druming.
(5) extracting: in the centrifuge tube of last action, chloroform/the isoamyl alcohol (24:1) adding equal-volume (about 800 μ L) comes sex change and precipitating proteins, upper transparent liquid is transferred in the 2mL centrifuge tube of another cleaning after the centrifugal 5min of 12000rpm.
(6) extracting again: the phenol/chloroform/primary isoamyl alcohol mixed solution (25:24:1) adding equal-volume (about 800 μ L) in upper transparent liquid is with precipitating proteins again, and upper transparent liquid is transferred in the 2mL centrifuge tube of another cleaning by the centrifugal 5min of 12000rpm.
(7) three extractings: the chloroform/primary isoamyl alcohol mixed solution (24:1) adding equal-volume (about 600 μ L) in supernatant liquor 2, so that the impurity such as protein are eliminated, the centrifugal 5min of 12000rpm, is transferred to supernatant liquor (containing DNA) in the 1.5mL centrifuge tube of another cleaning.
(8) precipitate: the Virahol adding 0.7 times of volume (about 400 μ L) in supernatant liquor 3, put upside down centrifuge tube gently for several times.Room temperature places 20min to precipitate DNA.The centrifugal 15min of 12000rpm, outwells supernatant liquor, and extraction effect can see that adularescent precipitation occurs at the bottom of pipe preferably.
(9) wash: with 70% ethanol (dissolved impurity, not dissolving DNA) washing precipitation, add-on is 500 μ L, with rifle piping and druming, precipitation is disperseed, the centrifugal 15min of 12000rpm after abundant washing, removes upper strata ethanol, and back-off centrifuge tube is to remove the moisture of tube wall attachment.
(10) dissolve: add 100 μ L distilled water dissolving DNA precipitations.
(11) with agarose gel electrophoresis detect extract the effect of DNA, agarose percentage composition 0.7%, DNA solution point sample amount is 5 μ L, with the mark of λ-EcoT14 I digestDNAMarker as molecular size, point sample amount is 3 μ L, and after electrophoresis completes, whether gel imaging to can observe carried DNA complete.Global DNA solution is put in-20 DEG C of refrigerators in what and saves backup.
The pcr amplification of 4.2 plant lactobacillus 16SrDNA and order-checking
With lactic bacterium strains genomic dna for template, utilize universal primer 27f (5 '-AGAGTTTGATCCTGGCTCAG) and the 1495r (5 '-CTACGGCTACCTTGTTACGA) of amplification bacterial 16 S rDNA, pcr amplification is carried out to the 16SrDNA of milk-acid bacteria.Pcr amplification program is: 94 DEG C of denaturation 4min; Then 94 DEG C of sex change 45s, 56 DEG C of annealing 1min, 72 DEG C of extension 1min, 30 circulations; 72 DEG C extend 10min.Target stripe, after agarose gel electrophoresis detection validation, is delivered to the handsome biotechnology company limited in Beijing and is checked order by PCR primer size.
The structure of 4.3 homology analysis and phylogenetic tree
Adopt nucleic acid BLAST technology, the GeneBank database in the sequence information of order-checking acquisition and NCBI website is contrasted, finds the bacterial classification of the known classification position the highest with surveyed sequence homology.Then from GenBank/EMBL/DDBJ database, obtain the 16SrDNA gene order of known bacterial strain, together with the 16SrDNA sequence of surveyed bacterial strain, adopt MEGA6.0 to compare, drawing system grows tree, and determine strain classification status, result is as shown in table 2.
The comparison of table 2 strain sequence and qualification result
According to the homology analysis of 16SrDNA gene order, determine that this bacterial strain is plant lactobacillus (Lb.plantarum).
Embodiment 2: the analysis of the mould fungus inhibition activity of lactobacterium plantarum strain
1, the preparation of aseptic concentrated plant lactobacillus fermented liquid: the plant lactobacillus after activation is inoculated in fresh MRS liquid nutrient medium (MRS liquid culture based formulas: peptone 10.0g with 1% inoculum size, extractum carnis 5.0g, yeast leaching powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, Triammonium citrate 2.0g, potassium acetate 5.0g, bitter salt 0.2g, manganous sulfate 0.05g, tween 80 1.0mL, distilled water 1000mL, 121 DEG C of sterilizing 15min), at 37 DEG C of constant incubator quiescent culture 24h, then by bacteria suspension in refrigerated centrifuge with the centrifugal 12min of the rotating speed of 3000r/min, get its supernatant liquor, with the filtering with microporous membrane removing thalline of 0.22 μm, be placed in-80 DEG C of refrigerator and cooled and freeze 12h, through vacuum freeze drier concentrate drying, then dissolve with sterile distilled water and prepare tenfold concentrated solution.
2, the preparation of mould spores liquid: choose the mould test bacterium detected as plant lactobacillus restraining effect of a spore, to treat that examination inoculation is on PDA inclined-plane, 28 DEG C of about constant temperature culture 7d produce to a large amount of spore, add 5mL sterile saline in slant tube, vortex concussion 5min, adopt 400 order sterile gauzes to cross and filter vegetative mycelium, the spore suspension of collection uses blood counting chamber counting, for subsequent use.
3, plant lactobacillus Fermented Condensed liquid is to the mensuration of mould inhibit activities: adopt Odontothrips loti to measure and press down mold activity.Be 5 × 10 with aseptic spreader by 0.4mL spore concentration
4individual/mL spore suspension is uniformly coated on the fresh PDA solid plate substratum for preparing, and be as the criterion without visible water droplet with flat board after coating, flat board now carries out bacteriostatic test immediately.With aseptic nipper, aseptic Oxford cup is put into above-mentioned culture dish gently, in the plate of horizontal positioned, place 3 Oxford cups equably.60 μm of germ-free plant lactobacillus ferment concentrated solutions are added, with ten times of concentrated MRS liquid nutrient mediums for contrast in every Oxford cup.Be placed in 28 DEG C of incubator quiescent culture 72h, measure its antibacterial circle diameter to the such as Sapromyces such as mould, Aspergillus ochraceus, that detects concentrated solution presses down mold activity.
4, the analysis of effective component of plant lactobacillus mould fungus inhibition
(1) protease treatment is on the impact of concentrated solution mould fungus inhibition activity: in plant lactobacillus Fermented Condensed liquid, add the stomach en-and Proteinase K that final concentration is 1mg/mL, take out after 37 DEG C of water-bath 2h, 80 DEG C of water-bath 2min make enzyme deactivation.Adopting Odontothrips loti to detect the mould inhibit activities of concentrated solution after protease treatment, is that experimental result is in table 3 in contrast with the stomach en-of the concentrated solution of unused protein ferment treatment and final concentration 1mg/mL and Proteinase K buffered soln respectively.
Table 3 protease treatment presses down the impact of mold activity to concentrated solution
Note: "-", without inhibition zone, "+" antibacterial circle diameter is > 6mm and≤12mm, and " ++ " antibacterial circle diameter is > 12mm and≤18mm, " +++ " antibacterial circle diameter > 18mm.
As shown in Table 3, in the test of protease treatment plant lactobacillus Fermented Condensed liquid, plant lactobacillus fermented liquid its bacteriostatic activity after pepsin does not almost change, and it has certain susceptibility to Proteinase K.
(2) organic acid is on the impact of concentrated solution mould fungus inhibition activity: record concentrated solution original pH, concentrated solution pH value to 4.0,5.0,6.0 is regulated to adopt Odontothrips loti to measure the concentrated solution after process to the restraining effect of mould respectively by the NaOH solution of lmol/L, with the concentrated solution of non-adjusted to ph and NaOH solution in contrast, experimental result is in table 4.
Table 4 organic acid presses down mold activity impact to concentrated solution
Note: "+" antibacterial circle diameter is > 6mm and≤12mm, " ++ " antibacterial circle diameter is > 12mm and≤18mm, " +++ " antibacterial circle diameter > 18mm and≤24mm, " ++++" antibacterial circle diameter > 24mm.
As shown in Table 4, plant lactobacillus concentrated broth presses down the impact that mold activity is subject to pH value adjustment.When fermented liquid pH value is adjusted to 4.0, fermented liquid press down mold activity decline to some extent, pH value be 5.0 or continue to be increased to neutrality time, the bacteriostatic activity of fermented liquid remains unchanged.
(3) thermal treatment is on the impact of concentrated solution mould fungus inhibition activity: plant lactobacillus Fermented Condensed liquid is placed in 60,80,100 DEG C of water-baths respectively and processes 5min, with Odontothrips loti measure process after concentrated solution press down mold activity, detect antibacterial substance thermostability, with without heat treated concentrated solution for contrast, experimental result is in table 5.
Table 5 thermal treatment is on the impact of mould fungus inhibition activity
Note: in table, data are the data obtained mean value of 4 revision tests; In table, after vertical data, same letter person represents that difference is not remarkable, and alphabetical different person represents significant difference, is p<5% horizontal check.
As shown in Table 5, plant lactobacillus Fermented Condensed liquid is after 60,80,100 DEG C of process, and its bacteriostatic activity and control group there are no significant difference, illustrates in plant lactobacillus fermented liquid but the thermostability of mold activity material is higher.
Embodiment 3: the application of plant lactobacillus (biological preservative) in pickles processing
1, apply
Plant lactobacillus of the present invention has good bacteriostatic activity to the contaminated mold in pickles and common pathogen, adds containing 6.5 × 10 according in every kilogram of pickles
8cfu plant lactobacillus, also its bacteria suspension and Sodium Benzoate can be made an addition to pickles sample, namely 10g biological preservative is added in every kilogram of pickles, the two all has good bacteriostatic action in the process suppressing spoilage organism growth, when adopting biological preservative, then there is good synergy, make fungistatic effect stronger.
2, the impact on pickles quality is evaluated
Use Sodium Benzoate, plant lactobacillus and Sodium Benzoate and plant lactobacillus combined treatment pickles sample respectively, do blank, through 20 DEG C, the storage of 90d, investigate different treatment group in the impact of different steps on pickles quality, experimental result is as table 6.
The impact of table 6 different treatment method on pickles organoleptic quality is compared
As shown in Table 6, swollen bag phenomenon is there is during preservation 30d in blank group under the condition of 20 DEG C, there is fat bag when 90d in the Sodium Benzoate treatment group of interpolation standard limitation, and fat bag phenomenon does not appear in the treatment group of adding the compound preservative of L544 and plant lactobacillus and Sodium Benzoate in preservation term.In addition, compared with blank group, use Sodium Benzoate, plant lactobacillus and compound preservative to have obvious effect to maintenance pickles quality, and use the change of the pickles sample quality of compound preservative less.Therefore, contrasted from preservation effect, use Sodium Benzoate and plant lactobacillus can keep pickles quality to a greater extent as compound preservative.
Claims (6)
1. have a plant lactobacillus for mould fungus inhibition function, it is characterized in that, described plant lactobacillus is preserved in China typical culture collection center, and it is referred to as plant lactobacillus, and deposit number is CCTCCM2015615, and preservation date is on October 19th, 2015.
2. a biological preservative, is characterized in that, activeconstituents is plant lactobacillus described in claim 1.
3. the preparation method as biological preservative according to claim 2, it is characterized in that, plant lactobacillus after activation is inoculated in fresh MRS liquid nutrient medium with 1 ~ 3% inoculum size, at 35 ~ 37 DEG C of constant incubator quiescent culture 24 ~ 26h, then by bacteria suspension in refrigerated centrifuge with the centrifugal 10 ~ 15min of the rotating speed of 2000 ~ 4000r/min, get its supernatant liquor, with the filtering with microporous membrane removing thalline of 0.22 μm, be placed in-60 ~ 80 DEG C of refrigerator and cooled and freeze 12 ~ 15h, through concentrate drying, then dissolve with sterile distilled water and prepare tenfold concentrated solution, .
4. the preparation method of biological preservative according to claim 3, is characterized in that, described MRS liquid nutrient medium is by peptone 10.0g, extractum carnis 5.0g, yeast leaching powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, Triammonium citrate 2.0g, potassium acetate 5.0g, bitter salt 0.2g, manganous sulfate 0.05g, tween 80 1.0mL, distilled water 1000mL, 121 DEG C of sterilizing 15min make.
5. the application of plant lactobacillus in pickles processing with mould fungus inhibition function according to claim 1, is characterized in that, containing 6 ~ 7 × 10 in every kilogram of pickles
8plant lactobacillus described in cfu.
6. the application of biological preservative according to claim 2 in pickles processing, adds biological preservative described in 6 ~ 10g in every kilogram of pickles.
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Cited By (2)
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| CN107183484A (en) * | 2017-06-01 | 2017-09-22 | 烟台东和饲料科技有限公司 | It is a kind of for the composition of feedstuff mildew, preparation method and its application method |
| CN109679880A (en) * | 2019-01-28 | 2019-04-26 | 合肥工业大学 | A kind of lactobacillus plantarum antistaling agent for less salt meat products |
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| WO2005045053A1 (en) * | 2003-11-07 | 2005-05-19 | Ajinomoto Co., Inc. | Process for producing lactic acid bacerium culture containing bacteriocin and method of storing food using the same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107183484A (en) * | 2017-06-01 | 2017-09-22 | 烟台东和饲料科技有限公司 | It is a kind of for the composition of feedstuff mildew, preparation method and its application method |
| CN109679880A (en) * | 2019-01-28 | 2019-04-26 | 合肥工业大学 | A kind of lactobacillus plantarum antistaling agent for less salt meat products |
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