CN101974468A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
- Publication number
- CN101974468A CN101974468A CN2010105271726A CN201010527172A CN101974468A CN 101974468 A CN101974468 A CN 101974468A CN 2010105271726 A CN2010105271726 A CN 2010105271726A CN 201010527172 A CN201010527172 A CN 201010527172A CN 101974468 A CN101974468 A CN 101974468A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- lactobacillus
- cgmcc
- application
- bacteriocin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a lactobacillus plantarum and application thereof. The lactobacillus plantarum is lactobacillus plantarum QZ-3, and the preservation number of the lactobacillus plantarum in China General Microbiological Culture Collection Center is CGMCC No.4199. The lactobacillus plantarum QZ-3 can produce bacteriocin having inhibition effect on various pathogenic bacteria and putrefying bacteria, and the bacteriocin has the advantages of good heat stability and acid stability, sensitivity to protease, wide antibacterial spectrum. The lactobacillus plantarum QZ-3 can be applied as a natural food preservative and a feed additive with wide application prospect.
Description
Technical field
The present invention relates to a lactobacillus plantarum and application thereof.
Background technology
Bacteriocin lab (Bacteriocins of LAB) is meant that milk-acid bacteria has the polypeptide or the protein substance of anti-microbial activity by rrna synthetic one class in metabolic process.They can suppress some common spoilage organism and pathogenic bacterium usually, as streptococcus aureus, micrococcus luteus, subtilis, monocyte hyperplasia listeria spp etc.And most of bacteriocin lab stability better, can heat use with food, and easily by the proteasome degradation in the human body, can not accumulate in vivo and cause untoward reaction, be considered to a kind of antiseptics for natural food and fodder additives with broad prospect of application.
Summary of the invention
The purpose of this invention is to provide plant lactobacillus and the application thereof of the bacteriocinogeny of a strain, the bacteriocin that this bacterial strain is produced suppresses common G
+And G
-Spoilage organism and pathogenic bacteria.
Plant lactobacillus provided by the invention is plant lactobacillus (Lactobacillus plantarum) QZ-3, and its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC № .4199.
Above-mentioned plant lactobacillus is with some modal G
+And G
-Spoilage organism and pathogenic bacteria are indicator, the lactobacterium plantarum strain of broad-spectrum bacteriocins is produced in screening, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 09 30th, 2010 and (be called for short CGMCC, the address is: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № .4199.
Application provided by the invention, be the application of plant lactobacillus (Lactobacillus plantarum) QZ-3CGMCC № .4199 in suppressing bacterium, and the application of plant lactobacillus (Lactobacillus plantarum) QZ-3CGMCC № .4199 in producing bacteriocin.
Described bacterium is above-mentioned described G
+And G
-Spoilage organism and pathogenic bacteria, specifically can be micrococcus luteus (Micrococcusluteus), streptococcus aureus (Staphylococcus aureus), subtilis (Bacillus subtilis), Salmonellas (Salmonella enterica), pseudomonas aeruginosa (Pseudomonas aeruginosa), intestinal bacteria (Escherichia coli) are preferably micrococcus luteus (Micrococcus luteus) 28001, streptococcus aureus (Staphylococcus aureus) 26003, subtilis (Bacillus subtilis) 63501, Salmonellas (Salmonella enterica) 50094, pseudomonas aeruginosa (Pseudomonas aeruginosa) 10104, intestinal bacteria (Escherichia coli) 30105.
Plant lactobacillus of the present invention (Lactobacillus plantarum) QZ-3 can produce multiple pathogenic bacteria and the inhibited bacteriocin of spoilage organism, this bacteriocin is to the sensitivity of proteolytic enzyme, has good thermostability, still keep active at 100 ℃ of effect 30min, this bacteriocin is handled 30min in the pH2.0-10.0 scope after, after adjusting back pH6.0, all keep the ability of very strong inhibition indicator micrococcus luteus (Micrococcus luteus) 28001.And this bacteriocin has the wide advantage of antimicrobial spectrum.Can be applied as antiseptics for natural food and fodder additives with broad prospect of application.
Description of drawings
Fig. 1 is the sensitivity Detection result of bacteriocin to enzyme;
Fig. 2 is that the bacteriocin extraction conditions is to its active influence;
Fig. 3 is the influence of plant lactobacillus (Lactobacillus plantarum) QZ-3 bacteriocinogeny to the micrococcus luteus growth curve
Embodiment
The acquisition of embodiment 1, plant lactobacillus (Lactobacillus plantarum) QZ-3
One, the acquisition of plant lactobacillus (Lactobacillus plantarum) QZ-3
Bacterial classification source: separate silage from Tibet.The present invention is with some modal G
+And G
-Spoilage organism and pathogenic bacteria are indicator, and the lactic bacterium strains of broad-spectrum bacteriocins is produced in screening, and concrete grammar is: under aseptic condition, get milk-product material 5g, join in the 45mL sterile distilled water, fully shake 5min, with 10 times of serial dilutions, from 10
-1～10
-7Gradient is selected suitable gradient, gets 100 μ L separate application in the culture dish that contains the MRS substratum, and 30 ℃ leave standstill cultivation and counting.Wherein, containing MRS culture medium culturing ware is that anaerobism is cultivated 48h, and anaerobism bag and anaerobism that the anaerobism culture device adopts strain formula chemistry society of Mitsubishi to produce are cultivated box.Picking list bacterium colony from the culture dish that contains the MRS substratum, line purifying 2 times, and carry out gramstaining test, microscopy.Utilize Oxford cup double-layer plate method: the indicator substratum that contains 1.5% agar is cooled to about 50 ℃, is poured in the sterile petri dish by every plate 15mL, cools off in the Bechtop; Preparation contains the indicator substratum of 0.8% agar, be cooled to about 50 ℃, inoculum size by 1% adds the indicator bacteria suspension as the upper strata substratum, toppling over the 5mL upper strata cultivates based on cooling off in the flat board that contains 1.5% nutrient agar, be positioned over the Oxford cup on the flat board gently with aseptic nipper then, to on Bechtop, spread 5h in the cup of cell free fermentation supernatant liquor adding Oxford, place the appearance of observing inhibition zone under the suitable culture condition behind the cultivation 24h, choosing the Oxford cup has the bacterial strain of obvious inhibition zone to do multiple sieve on every side.The result obtains the bacterial strain that a strain extensively suppresses indicator, called after QZ-3.
Two, the evaluation of plant lactobacillus (Lactobacillus plantarum) QZ-3
QZ-3 bacterial strain Physiology and biochemistry experimental result is as shown in table 1, and the carbon source through fermentation test-results is as shown in table 2; The 16SrRNA gene order is shown in sequence in the sequence table 1.According to cell microscopic morphology, Physiology and biochemistry data and 16S rRNA gene order data, with the QZ-3 identification of strains is plant lactobacillus (Lactobacillus plantarum), and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 09 30th, 2010 and (be called for short CGMCC, the address is: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № .4199.
Table 1, plant lactobacillus (Lactobacillus plantarum) QZ-3 physical and chemical experiment result
Annotate :+representative reaction is positive;-representative reaction is negative; W representative reaction is the weak positive
Table 2. carbon source through fermentation test-results
Annotate :+representative reaction is positive;-representative reaction is negative; W representative reaction is the weak positive
Find out that from embodiment 1 plant lactobacillus of the present invention (Lactobacillus plantarum) QZ-3 is to some modal G
+And G
-Therefore spoilage organism and pathogenic bacteria have biocidal property, infer that this bacterial strain can the secreting bacteria element, by the following method fungistatic effect and its excretory bacteriocin of plant lactobacillus (Lactobacillusplantarum) QZ-3 are identified.
One, eliminating factor is disturbed
1, gets rid of the interference of acid
The test method that suppresses pathogenic bacteria is an Oxford cup double-layer plate method: the indicator substratum NA substratum (being used for the cultivation of micrococcus luteus, streptococcus aureus, subtilis, pseudomonas aeruginosa, bacillus pumilus, Salmonellas) that contains 1.5% agar: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 15g, distilled water 1.0L, pH7.0～7.2.LB substratum (being used for colibacillary cultivation): peptone 10g, yeast extract 5g, sodium-chlor 10g, distilled water 1.0L, pH7.0.PDA substratum (being used for Candida albicans): potato is leached powder 4.0g, glucose 20g, agar 15g, distilled water 1.0L, pH5.6.Be cooled to about 50 ℃, be poured in the sterile petri dish, cool off in the Bechtop by every plate 15mL; Preparation contains the indicator substratum (the same) of 0.8% agar, be cooled to about 50 ℃, (be inoculated in the substratum that this bacterium is fit to transfering loop picking activatory indicator one ring, 37 ℃ leave standstill cultivation 24h to add indicator (shown in the table 2) bacteria suspension by the inoculum size of 1% (volumn concentration).And the adjustment bacterial concentration is 10
7Cfu/mL, 4 ℃ of refrigerators are preserved standby) mixing is as the upper strata substratum, topple in the flat board that contains 1.5% nutrient agar of 5mL upper strata cultivation based on above-mentioned preparation and cool off, be positioned over the Oxford cup on the flat board gently with aseptic nipper then, with on Bechtop, spreading 5h in the cup of above-mentioned liquid adding to be measured Oxford, place under the suitable culture condition and observe inhibition zone behind the cultivation 24h.
Transferring the pH value of distilled water and MRS liquid nutrient medium respectively with hydrochloric acid and lactic acid is pH3.0,4.0,5.0,6.0, for contrast pH value, utilizes Oxford cup double-layer plate method, and the distilled water and the MRS liquid nutrient medium of above-mentioned various pH values are done bacteriostatic test.With the pH value that lactic acid and hydrochloric acid are transferred distilled water and MRS liquid nutrient medium respectively, the result shows the fungistatic effect difference of different pH values to 6 kinds of indicators.Transfer distilled water pH value all not have fungistatic effect with lactic acid and hydrochloric acid 3.0～6.0 pairs of indicators; Lactic acid and hydrochloric acid transfer the MRS liquid nutrient medium that fungistatic effect is all arranged under pH3.0, pH4.0 is to intestinal bacteria (Escherichia coli) 30105, micrococcus luteus (Micrococcus luteus) 28001, pseudomonas aeruginosa (Pseudomonasaeruginosa) 10104 all has fungistatic effect, and to streptococcus aureus (Staphylococcus aureus) 26003, subtilis (Bacillus subtilis) 63501 and Salmonellas (Salmonella enterica) 50094 do not have fungistatic effect, pH5.0 is less to micrococcus luteus and pseudomonas aeruginosa restraining effect, to other four kinds of indicator unrestraint effects, and pH6.0 does not have fungistatic effect to six kinds of indicators, selects pH6.0 pH in contrast.
Cultivate 24 hours plant lactobacillus (Lactobacillus plantarum) QZ-3 on the picking MRS substratum, be inoculated in the MRS liquid nutrient medium, 30 ℃ leave standstill cultivation 24h, 12000rpm, behind 4 ℃ of centrifugal 15min, survey the pH value of supernatant liquor, and leave heart fermented supernatant fluid under order to above-mentioned definite contrast pH value 6.0, do bacteriostatic test according to above-mentioned Oxford cup double-layer plate method with 1mol/L NaOH (sterilizing) and 1mol/L HCl.
The MRS liquid nutrient medium: the MRS substratum can be available from Shanghai Hu Feng bio tech ltd, and article No. is HB0384, its prescription for every liter by peptone 10g; yeast extract paste 5g, extractum carnis 10g, glucose 20g; dipotassium hydrogen phosphate 2g, ammonium citrate 2g, sodium acetate 5g; sal epsom 0.58g; manganous sulfate 0.25g, tween 80 1mL, agar 15g; distilled water 1.0L forms, pH6.5.
The result is as shown in table 3, the result shows that plant lactobacillus (Lactobacillus plantarum) QZ-3 not only has the obvious suppression effect to micrococcus luteus (Micrococcus luteus) 28001, streptococcus aureus (Staphylococcus aureus) 26003, subtilis (Bacillus subtilis) 63501, Salmonellas (Salmonella enterica) 50094, pseudomonas aeruginosa (Pseudomonas aeruginosa) 10104, intestinal bacteria (Escherichia coli) 30105, and this restraining effect is not subjected to the influence of pH.Used indicator is as shown in table 3, and all indicators are all purchased in Henan Province's food and medicine check institute.
Table 3. is got rid of sour inhibiting test-results
Annotate: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm)
2, the eliminating of hydrogen peroxide effect
Milk-acid bacteria may produce the growth that hydrogen peroxide suppresses bacterium in metabolic process, therefore must get rid of the interference of hydrogen peroxide.Fermented supernatant fluid is handled with catalase, cell free fermentation supernatant liquor with the pH6.0 that handles without catalase is cooked contrast, with the micrococcus luteus is that indicator carries out bacteriostatic test, the fermented supernatant fluid of milk-acid bacteria is after the hydrogen peroxide enzyme is handled, antibacterial circle diameter is compared with the contrast antibacterial circle diameter, thereby main antibacterial substance is a hydrogen peroxide in the proof fermented liquid.Method: cultivate 24 hours plant lactobacillus (Lactobacillus plantarum) QZ-3 on the picking MRS substratum, be inoculated in the MRS liquid nutrient medium, 30 ℃ leave standstill cultivation 24h, and 12000rpm behind 4 ℃ of centrifugal 15min, obtains fermented supernatant fluid.Catalase is dissolved in the phosphoric acid buffer (pH 7.0) of 50mmol/L, according to the catalase final concentration is that 5.0mg/mL joins above-mentioned fermented supernatant fluid, 37 ℃ of water-bath 2h, detect catalase and handle the bacteriostatic activity of back cell free fermentation supernatant liquor, method is with the Oxford cup double-layer plate method of step 1.The result shows that catalase does not influence the restraining effect of this bacterium to micrococcus luteus (Micrococcus luteus) 28001, also has other antibacterial substance that indicator is played restraining effect.
3, proteolytic enzyme detects the protein properties of antibacterial substance
Earlier readjust the distribution the ferment supernatant liquor respectively to trypsin SIGMA company with 1mol/L NaOH (sterilizing) and 1mol/L HCI, article No.: C9322) the suitableeest action pH value 8.1 and Proteinase K (MERCK company, article No.: the suitableeest action pH value 8.0 WL558668.609), press 1.0mg/mL and add trypsinase and Proteinase K, 37 ℃ of water-bath 2h, again pH is recalled to contrast pH value 6.0, the Oxford agar diffusion method detects bacteriostatic activity, and with pH6.0 cell free fermentation supernatant liquor in contrast, detect the influence of trypsinase and Proteinase K to milk-acid bacteria cell free fermentation supernatant liquor bacteriostatic activity.Method is with the Oxford cup double-layer plate method of step 1.After the result showed trypsinase and Proteinase K effect, this bacterium did not have bacteriostatic action to micrococcus luteus (Micrococcus luteus) 28001.The material that this bacterium inhibition micrococcus luteus (Micrococcus luteus) 28001 is described be a protein substance for being decomposed by trypsinase and Proteinase K.
Bacteriostatic activity after table 4. catalase and the proteolytic enzyme effect
The experimental result explanation of above-mentioned steps 1-3, main antibacterial substance is not a hydrogen peroxide in plant lactobacillus (Lactobacillus plantarum) the QZ-3 fermented liquid, neither also have other antibacterial substance that indicator is played restraining effect because of the effect of acid.This antibacterial substance can be illustrated that they are protein substances by the enzymolysis of trypsinase and Proteinase K, is a bacterioid element.
Two, plant lactobacillus (Lactobacillus plantarum) QZ-3 Optimizing Conditions of Fermentation
Optimize plant lactobacillus (Lactobacillus plantarum) QZ-3 fermentation condition method: the glucose of measuring different concns (quality percentage composition) according to the Oxford agar diffusion method method among the embodiment 1, Tryptones, peptone, yeast extract paste, sal epsom and tween 80, and the inoculum size of different mass percentage composition, incubation time, plant lactobacillus (Lactobacillus plantarum) QZ-3 nutrient solution under temperature and the initial pH value condition obtains top condition to suppressing the influence of micrococcus luteus (Micrococcus luteus) 28001.
After optimization to conditions such as culture medium carbon source, nitrogenous source, incubation time, temperature, inoculum size, pH values, cultivate 24h for 30 ℃, cultivating initial pH value is 6.5, inoculum size quality percentage composition 2%, and substratum (quality percentage composition): QZ-3 is a glucose 3%, Tryptones 2%, peptone 2%, yeast extract paste 2%, sal epsom 0.058%, tween 80 0.2%, all the other are water.
The tire mensuration of standard equation
Tire and detect in the flat board, Nisin (the SIGMA company that adds 50IU/mL, 100IU/mL, 500IU/mL, 1000IU/mL, 1500IU/mL, 2000IU/mL, 5000IU/mL respectively, article No.: N5764) standardized solution, with micrococcus luteus (Micrococcus luteus) 28001 is indicator, cultivating 24h for 30 ℃, is X-coordinate with the logarithmic value of tiring, and antibacterial circle diameter is an ordinate zou, add the straight line asymptotic line, obtain the titration standard equation.Corresponding then typical curve calculates the relative potency of plant lactobacillus (Lactobacillus plantarum) QZ-3 bacteriocinogeny.
Cultivate 24 hours plant lactobacillus (Lactobacillus plantarum) QZ-3 before optimizing on the picking MRS substratum, be inoculated in the MRS liquid nutrient medium, 30 ℃ leave standstill cultivation 24h, initial pH value 6.0.Inoculum size is 10
6Cfu/ml.
The result is as shown in table 5, and the result shows that the bacteriostatic activity of plant lactobacillus (Lactobacillusplantarum) QZ-3 bacteriocinogeny obviously increases after the optimization of process culture condition.
Table 5 fermentation condition optimization result
Three, plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin biological Characteristics Study
1, bacteriocin is to the stability of heat
The single colony inoculation of picking activatory plant lactobacillus (Lactobacillus plantarum) QZ-3 is in the MRS liquid nutrient medium, sealing, 30 ℃ leave standstill cultivation 24h, fermented liquid is with 12000rpm, 4 ℃ of centrifugal 15min, collect supernatant liquor, supernatant liquor is removed thalline and other impurity with the aseptic membrane filtration in 0.22 μ m aperture.Obtain the cell free fermentation supernatant liquor of plant lactobacillus (Lactobacillusplantarum) QZ-3.
The pH6.0 cell free fermentation supernatant liquor of plant lactobacillus (Lactobacillus plantarum) QZ-3 is placed on 60 ℃, 80 ℃, 100 ℃ and 121 ℃ respectively handles 15min and 30min, according to the described Oxford of step 1 cup double-layer plate method, under 30 ℃ of conditions, do bacteriostatic test, determine after the treatment of different temperature bacteriocin to be suppressed micrococcus luteus (Micrococcus luteus) 28001 active influences.
The result is as shown in table 6, and the result shows that plant lactobacillus (Lactobacillus plantarum) QZ-3 is to the temperature-stable below 100 ℃.
Table 6. temperature is to the active influence of bacteriocin
2, plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin is to the stability of acid
It is 2.0～10.0 that plant lactobacillus (Lactobacillus plantarum) QZ-3 cell free fermentation supernatant liquor (method according to step 1 obtains) is transferred its pH value with 1mol/L HCL and 1mol/L NaOH respectively, 37 ℃ of following incubation 2h, adjust pH is 6.0, does bacteriostatic test according to the described Oxford of step 1 cup double-layer plate method then and surveys micrococcus luteus (Micrococcusluteus) 28001 bacteriostatic activities.Under the equal conditions, the liquid MRS medium pH value of adjusting the bacterium of going out with 1mol/LHCL and 1mol/LNaOH is 2.0～10.0,37 ℃ of following incubation 2h respectively, and adjusting the pH value is 6.0, does contrast according to the described Oxford of step 1 cup double-layer plate method equally.
Table 7. plant lactobacillus (Lactobacillus plantarum) QZ-3 bacteriocin is to the stability of acid
Annotate: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm), and on behalf of antibacterial circle diameter, "-" be lower than 8.0mm
3, plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin is to the susceptibility of enzyme
Get plant lactobacillus (Lactobacillus plantarum) the QZ-3 bacterium pH6.0 cell free fermentation supernatant liquor (method according to step 1 obtains) of equivalent, regulating pH with 1mol/L HCL and 1mol/L NaOH is the suitableeest action pH value of following each enzyme, add stomach en-(the suitableeest action pH value 3 respectively by final concentration 1mg/mL, Amresco company, article No.: 0685), the suitableeest action pH value 8.1 of trypsin, GLBCO company, 27250018), Proteinase K (the suitableeest action pH value 8.0, MERCK company, WL558668.609), papoid (the suitableeest action pH value 6.0, MERCK company, 107147) and Chymotrypsin (the suitableeest action pH value 8.5, SIGMA company, C8660), 37 ℃ of following incubation 2h, adjust pH and reach 6.0, do bacteriostatic experiment according to embodiment one described Oxford cup double-layer plate method, repeat 3 times, do contrast with the pH6.0 cell free fermentation supernatant liquor that does not pass through protease treatment simultaneously.
The result as shown in Figure 1,5 kinds of proteolytic enzyme can well decompose plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin, and micrococcus luteus (Micrococcus luteus) 28001 is not had restraining effect substantially.Among Fig. 1,1 is stomach en-; 2 is Proteinase K; 3 is trypsinase; 4 is papoid; 5 is Chymotrypsin; CK is not for passing through the pH6.0 cell free fermentation supernatant liquor of protease treatment.
4, the mensuration of the antimicrobial spectrum of plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin
Get plant lactobacillus (Lactobacillus plantarum) the QZ-3pH6.0 cell free fermentation supernatant liquor (method according to step 1 obtains) of equivalent, do bacteriostatic experiment according to the various indicators in the described Oxford cup double-layer plate method his-and-hers watches 8 among the embodiment 1, the liquid MRS substratum with pH6.0 is contrast simultaneously.The result is as shown in table 8, in selected indicator scope, plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin has restraining effect to micrococcus luteus 28001, streptococcus aureus 26003, subtilis 63501, bacillus pumilus 63202, bacillus megaterium 63201, pseudomonas aeruginosa 10104, Salmonellas 50094, intestinal bacteria 30105, and Candida albicans 98001, cereuisiae fermentum 98002, aspergillus niger 98003 and mould 98005 are not had restraining effect.
The antimicrobial spectrum of table 8. plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin
Annotate: antibacterial circle diameter comprises Oxford cup external diameter (7.8mm), and on behalf of antibacterial circle diameter, "-" be lower than 8.0mm.
Four, the extraction of plant lactobacillus (Lactobacillus plantarum) QZ-3 excretory bacteriocin
1, ammonium sulfate salting-out process is slightly carried bacteriocin
The 80 ℃ of following 10min of processing of cell free fermentation supernatant liquor (method according to step 1 in the step 3 obtains) that respectively get equivalent plant lactobacillus (Lactobacillus plantarum) QZ-3 prevent the bacteriocin degraded, the saturation ratio of adjusting ammonium sulfate respectively is 30%, 40%, 50%, 60%, 70%, 80%, 90%, after slowly stirring 1h, place 4 ℃ of refrigerator overnight.Centrifugal supernatant (the 10000rpm that abandons, 4 ℃, 30min), precipitate in the phosphate buffered saline buffer that is dissolved in 1mL (pH6.0), detect the bacteriostatic activity of each concentration throw out solution, indicator is a micrococcus luteus 28001, centrifugal (the 10000rpm of MRS liquid nutrient medium that while handled with same concentration, 4 ℃, abandon supernatant after 30min), be dissolved in that (pH6.0) does contrast in the phosphate buffered saline buffer of 1mL.
The result as shown in Figure 2.
2, bacteriocinogeny minimal inhibitory concentration (MIC)
Determine the minimal inhibitory concentration (MIC) of lactobacillin with the liquid coubling dilution.With common spoilage organism and pathogenic bacterium is indicator: micrococcus luteus, streptococcus aureus, subtilis, intestinal bacteria, pseudomonas aeruginosa and Salmonellas.Method is as follows:
(1) each bacteriocin lab crude extract is joined in each indicator nutrient solution, obtain various concentration, insert indicator, and to adjust bacterium dense is 10 with doubling dilution dilution
6Cfu/mL, the optimum temperuture overnight incubation.With the test tube that do not add the bacteriocin crude extract but connect bacterium as positive control, to add the bacteriocin crude extract but the test tube of not inoculating bacterium as negative control.
(2) the culture of respectively managing that will not see bacteria growing is successively drawn 100uL injection indicator plate culture medium respectively, cultivates 24h for 30 ℃, and the minimum concentration of the pairing bacteriocin crude extract of test group asepsis growth group is minimal inhibitory concentration (MIC).
The result is as shown in table 9, the result shows, the bacteriocin crude extract that QZ-3 produces is 302.88IU/mL to the minimal inhibitory concentration of subtilis, intestinal bacteria and Salmonellas three strain bacterium, and to micrococcus luteus, streptococcus aureus and pseudomonas aeruginosa is 151.44IU/M1.
Table 9.Lactobacillus plantarum QZ-3 bacteriocinogeny is to several indicator minimal inhibitory concentrations (MIC)
Annotate: "-" is lower than 8.0mm (the diameter 7.8mm of Oxford cup) to the inhibition loop diameter of indicator
"+" to the inhibition loop diameter of indicator greater than 8.0mm.
3, bacteriocinogeny is to the influence of indicator (micrococcus luteus) growth curve
Be inoculated in the NA liquid nutrient medium (extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 15g, distilled water 1.0L, pH7.0～7.2) with transfering loop picking micrococcus luteus 28001, get part bacterium liquid respectively, being diluted to viable count with liquid nutrient medium then is 10
6The bacteria suspension of cfu/mL continues to cultivate, and every the 2h sampling, surveys OD
600nmLight absorption value, METHOD FOR CONTINUOUS DETERMINATION 48h produces the contrast growth curve.
Get rest parts bacterium liquid,, make that viable count is 10 in the substratum to the Lactobacillus plantarum QZ-3 bacteriocinogeny crude extract that wherein adds MIC and 1/2MIC
6Cfu/mL continues to cultivate and take a sample every 2h, surveys OD
600nm, produce the growth curve of the micrococcus luteus under the bacteriocin effect.
The result as shown in Figure 3, the result shows with contrast and compares, the bacteriocin of MIC content suppresses fully to micrococcus luteus 28001, and the bacteriocin of 1/2MIC content has also suppressed the growth of micrococcus luteus 28001 greatly, the time lengthening that its logarithmic phase occurs, the maximum bacteria biomass of growth also reduces.
Claims (8)
1. plant lactobacillus (Lactobacillus plantarum) QZ-3, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC № .4199.
2. the application of plant lactobacillus (Lactobacillus plantarum) QZ-3 CGMCC № .4199 in suppressing bacterium.
3. application according to claim 2 is characterized in that: described bacterium is micrococcus luteus, streptococcus aureus, subtilis, bacillus pumilus, pseudomonas aeruginosa, Salmonellas or intestinal bacteria.
4. application according to claim 3 is characterized in that: described bacterium is micrococcus luteus 28001, streptococcus aureus 26003, subtilis 63501, bacillus pumilus 63202, pseudomonas aeruginosa 10104, Salmonellas 50094 and intestinal bacteria 30105.
5. the application of plant lactobacillus (Lactobacillus plantarum) QZ-3 CGMCC № .4199 in producing bacteriocin.
6. the application of plant lactobacillus (Lactobacillus plantarum) QZ-3 CGMCC № .4199 in preparation food or feed anticorrosion agent.
7. the substratum of culturing plants Bacterium lacticum (Lactobacillus plantarum) QZ-3 CGMCC № .4199, by glucose 3%, Tryptones 2%, peptone 2%, yeast extract paste 2%, sal epsom 0.058%, tween 80 0.2% and water are formed, and wherein percentage composition is the quality percentage composition.
8. the application of plant lactobacillus (Lactobacillus plantarum) QZ-3 CGMCC № .4199 in the preparation bacterial inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105271726A CN101974468B (en) | 2010-10-26 | 2010-10-26 | Lactobacillus plantarum and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105271726A CN101974468B (en) | 2010-10-26 | 2010-10-26 | Lactobacillus plantarum and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101974468A true CN101974468A (en) | 2011-02-16 |
| CN101974468B CN101974468B (en) | 2012-04-18 |
Family
ID=43574377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105271726A Expired - Fee Related CN101974468B (en) | 2010-10-26 | 2010-10-26 | Lactobacillus plantarum and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101974468B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643767A (en) * | 2012-04-19 | 2012-08-22 | 郑州大学 | Lactobacillus plantarum and application thereof in fermenting and ensiling sweet potato stem and leaf |
| CN102732447A (en) * | 2012-04-18 | 2012-10-17 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum for inhibiting mould and yeasts in bread, and application method thereof |
| CN102851233A (en) * | 2012-06-12 | 2013-01-02 | 华南农业大学 | Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof |
| CN103667093A (en) * | 2012-09-19 | 2014-03-26 | 成都与康科技有限公司 | Lactobacillus plantarum 929-2 strain having food preservative and fresh-keeping effect and application thereof |
| CN105816417A (en) * | 2016-04-29 | 2016-08-03 | 润盈生物工程（上海）有限公司 | Application experiment of lactobacillus rhamnosus extract in skin care product |
| JP2017108742A (en) * | 2015-12-15 | 2017-06-22 | ロッテ コンフェクショナリー カンパニー リミテッド | Low temperature resistant lactobacillus plantarum lrcc5309 and methods for producing ice cream using it |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
| CN110129235A (en) * | 2019-05-28 | 2019-08-16 | 青岛力力惠生物科技股份有限公司 | A kind of heat-resisting plant lactobacillus and its application in agricultural production |
| CN110205267A (en) * | 2019-05-31 | 2019-09-06 | 上海应用技术大学 | The lactobacillus plantarum of one plant of bacteriocinogeny and its application |
| CN113403227A (en) * | 2021-06-08 | 2021-09-17 | 昆明理工大学 | Lactobacillus plantarum and preparation method and application thereof |
| CN114058531A (en) * | 2021-03-16 | 2022-02-18 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN114276951A (en) * | 2021-12-01 | 2022-04-05 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
| CN116077534A (en) * | 2023-01-31 | 2023-05-09 | 大连工业大学 | Application of bacillus coagulans |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331104B (en) * | 2019-07-05 | 2020-12-18 | 四川大学 | Lactobacillus plantarum CV10D1 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888051A (en) * | 2005-06-28 | 2007-01-03 | 北京天佑达生物工程科技有限公司 | Plant lactobacillus strain and its application |
| CN101218935A (en) * | 2008-02-01 | 2008-07-16 | 中国农业科学院农业资源与农业区划研究所 | Application of plants lactobacillus in fresh-keeping of freezing meat |
| CN101812414A (en) * | 2009-08-06 | 2010-08-25 | 东北农业大学 | Lactobacillus plantarum and bacteriocins produced by lactobacillus plantarum and capable of inhibiting Gram negative bacteria |
-
2010
- 2010-10-26 CN CN2010105271726A patent/CN101974468B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888051A (en) * | 2005-06-28 | 2007-01-03 | 北京天佑达生物工程科技有限公司 | Plant lactobacillus strain and its application |
| CN101218935A (en) * | 2008-02-01 | 2008-07-16 | 中国农业科学院农业资源与农业区划研究所 | Application of plants lactobacillus in fresh-keeping of freezing meat |
| CN101812414A (en) * | 2009-08-06 | 2010-08-25 | 东北农业大学 | Lactobacillus plantarum and bacteriocins produced by lactobacillus plantarum and capable of inhibiting Gram negative bacteria |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732447A (en) * | 2012-04-18 | 2012-10-17 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum for inhibiting mould and yeasts in bread, and application method thereof |
| CN102732447B (en) * | 2012-04-18 | 2013-11-13 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum for inhibiting mould and yeasts in bread, and application method thereof |
| CN102643767A (en) * | 2012-04-19 | 2012-08-22 | 郑州大学 | Lactobacillus plantarum and application thereof in fermenting and ensiling sweet potato stem and leaf |
| CN102851233A (en) * | 2012-06-12 | 2013-01-02 | 华南农业大学 | Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof |
| CN102851233B (en) * | 2012-06-12 | 2014-05-28 | 华南农业大学 | Low-temperature-resistant lactobacillus strain lactobacillus plantarum CCZZ1 and application thereof |
| CN103667093A (en) * | 2012-09-19 | 2014-03-26 | 成都与康科技有限公司 | Lactobacillus plantarum 929-2 strain having food preservative and fresh-keeping effect and application thereof |
| CN103667093B (en) * | 2012-09-19 | 2015-11-11 | 成都与康科技有限公司 | A kind of plant lactobacillus 929-2 bacterial strain and application thereof with food antiseptic freshening effect |
| JP2017108742A (en) * | 2015-12-15 | 2017-06-22 | ロッテ コンフェクショナリー カンパニー リミテッド | Low temperature resistant lactobacillus plantarum lrcc5309 and methods for producing ice cream using it |
| CN105816417A (en) * | 2016-04-29 | 2016-08-03 | 润盈生物工程（上海）有限公司 | Application experiment of lactobacillus rhamnosus extract in skin care product |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
| CN108611299B (en) * | 2018-05-10 | 2022-03-08 | 南京师范大学 | Lactobacillus plantarum for producing antibacterial peptide and application thereof |
| CN110129235A (en) * | 2019-05-28 | 2019-08-16 | 青岛力力惠生物科技股份有限公司 | A kind of heat-resisting plant lactobacillus and its application in agricultural production |
| CN110205267A (en) * | 2019-05-31 | 2019-09-06 | 上海应用技术大学 | The lactobacillus plantarum of one plant of bacteriocinogeny and its application |
| CN114058531A (en) * | 2021-03-16 | 2022-02-18 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN114058531B (en) * | 2021-03-16 | 2023-05-30 | 中国农业大学 | Bacteriocin-producing lactobacillus plantarum and compound application thereof in silage |
| CN113403227A (en) * | 2021-06-08 | 2021-09-17 | 昆明理工大学 | Lactobacillus plantarum and preparation method and application thereof |
| CN114276951A (en) * | 2021-12-01 | 2022-04-05 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
| CN114276951B (en) * | 2021-12-01 | 2023-10-03 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
| CN116077534A (en) * | 2023-01-31 | 2023-05-09 | 大连工业大学 | Application of bacillus coagulans |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101974468B (en) | 2012-04-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974467B (en) | Lactobacillus plantarum and application thereof | |
| CN101974468B (en) | Lactobacillus plantarum and application thereof | |
| CN101974449B (en) | Lactobacillus plantarum and application thereof | |
| CN101974450B (en) | Leuconostoc mesenteroides and application thereof | |
| CN102120975B (en) | Bacillus subtilis strain with stronger bacteriostatic action and application thereof | |
| CN104818232B (en) | One strain has Lactobacillus plantarum AB-3 and the application thereof of bacteriostatic activity | |
| CN102676398B (en) | Separation and purification method of endophytic fungi from ginkgo biloba | |
| CN102640885B (en) | Applications of Lactobacillus plantarum in complete-strain Triticumspp silage | |
| CN101928678B (en) | Bacillus subtilis B2 microbial agent, compound microbial agent and application | |
| CN102061271B (en) | Lactobacillus casei bacteriocin and use thereof in feed | |
| CN102703513A (en) | Method for preparing red elemental selenium by utilizing super-selenium resistance microorganism | |
| CN102732468B (en) | Enterococcus faecalis and broad-spectrum antibacterial action thereof | |
| CN109161498B (en) | Bacillus subtilis M406 and application thereof in preparation of bacteriocin and cellulase | |
| CN102229902B (en) | Bacillus pumilus, feed additive thereof, premix, and feed mildewproof agent | |
| CN103409334A (en) | Lactobacillus for inhibiting pathogenic bacteria of vaginitis and application thereof | |
| CN103667106B (en) | A kind of plant lactobacillus, its bacteriocin and cultivation and separation purification method | |
| CN104611256A (en) | Microbial freeze-dried protective agent and preparation method and application thereof | |
| CN116836830A (en) | Bacteriocin-producing lactobacillus plantarum and application thereof | |
| CN106591174B (en) | The lactobacillus curvatus of one plant of bacteriocinogeny and its application | |
| CN105754886B (en) | One plant of nontoxic bacillus cereus strain pBC-1 and its application | |
| CN102174437B (en) | Lactobacillus delbrueckiisubsp.lactis and preparation method of bacteriocin thereof capable of resisting Listeria monocytogenes | |
| CN115161234B (en) | Bacillus bailii and application thereof in silage preservation | |
| CN116731908A (en) | Lactobacillus plantarum MT213 and application thereof | |
| CN104877986A (en) | Screening method and application of lactobacillus plantarum | |
| CN103146602A (en) | Lactic acid bacteria solid co-fermenting method, obtained bacterial agent and use of bacterial agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120418 Termination date: 20141026 |
|
| EXPY | Termination of patent right or utility model |