CN105380067A - Coating inhibiting listeria monocytogenes and preparation method and application - Google Patents
Coating inhibiting listeria monocytogenes and preparation method and application Download PDFInfo
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Abstract
The invention provides a coating inhibiting listeria monocytogenes and a preparation method and application and relates to the field of food safety. The coating inhibiting listeria monocytogenes contains, by mass, 95-105 parts of polylactic acid and 20-65 parts of bacteriocin capable of inhibiting listeria monocytogenes. The bacteriocin comes from an enterococcus durans NJ strain. The preparation method of the coating includes the steps that the polylactic acid and the bacteriocin capable of inhibiting listeria monocytogenes are added into a solvent and stirred till the polylactic acid is dissolved and the bacteriocin are evenly dispersed in the solvent, and a coating solution is obtained; the coating solution is poured into a container or the surface of an object is coated with the coating solution, the solvent is removed, and the coating is dried. The coating can efficiently inhibit growth of listeria monocytogenes, prevents the situation that broad-spectrum bactericidal bacteriocin is used and inhibits other beneficial bacteria in food, has no toxic and side effects, can be used on glass, plastic, ceramic and iron products, and is safe and simple in preparation method, low in cost and suitable for being widely popularized in the food industry.
Description
Technical field
The present invention relates to field of food safety, be specifically related to the listerial coating of a kind of suppression, its preparation method and application.
Technical background
The harm of Listeria monocytogenes (Listeria monocytogenes) is long-standing, and this bacterium is ubiquitous in the environment.Nineteen eighty-three, break out together by the case of at least 14 people's death of polluting listerial pasteurize whole milk and cause containing the milk of 2% fat at Massachusetts, United States.1985, again break out at California, USA the case that people that the soft cheese of Mexico's type that polluted by Listeria monocytogenes causes infects listeriosis.Have the confirmed cases more than 100 examples in this outburst, and it is dead to cause at least 40 people.The investigation display that U.S. food and drug administration carried out in 1986, in the Milk Processing Plant of 357, the whole America, has the factory of 2.5% in its product containing Listeria.The people such as Lwasor isolated Listeria monocytogenes in 1989 from egg pulp, subsequently to all having found the existence of Listeria monocytogenes in the detection of meat, vegetables etc.Have also appeared the listeriosis outburst case caused by the bean sprouts of polluting, cabbage and muskmelon in recent years.Listeria is a kind of Gram-positive bacillus, can 4 DEG C to 45 DEG C growths, independent refrigeration measure is not enough to the breeding preventing it in food, the people such as Doyle find that in the investigation of 1987 Listeria monocytogenes can be survived under pasteurize condition, so it inevitably exists in raw material and food processing engineering, therefore re-pasteurization process is very necessary.
In prior art, antibacterial, the bactericide that add chemical composition is needed in the food storage processes such as milk, because chemical composition is antibacterial, the most of employing chemical method synthesis of bactericide, there is more potential safety hazard, do not meet consumer for safety, health and natural requirement.
The bacteriocin that Enterococcus durans NJ strain produces can suppress, to bacterial strain unrestraint effects such as Lactobacillus plantarum, Lactobacillus casei, lactobacillus fermenti, saliva chain coccus thermophilous subspecies and Escherichia coli listeria (especially Listeria monocytogenes), Enterococcus durans, enterococcus faecalis and VREF.
Summary of the invention
The object of this invention is to provide the listerial coating of a kind of suppression, can efficiently suppress listerial growth, the broad-spectrum sterilization bacteriocin can effectively avoiding present stage to use during application food fresh keeping is to the suppression of other beneficial bacteriums in food, have no side effect, may be used on glass, plastics, pottery and ironwork.
Another object of the present invention is to provide the preparation method of described coating, and the method safely, simple, cost is low, is adapted at food industry and promotes widely.
Another object of the present invention is to provide the application of described coating in field of food safety.
The present invention seeks to be realized by following scheme:
The listerial coating of a kind of suppression, containing 95-105 mass parts PLA, 20-65 mass parts can suppress listerial bacteriocin; Described bacteriocin derives from Enterococcus durans (Enterococcusdurans) NJ strain.
In the present invention, describedly listerial bacteriocin is suppressed to be tired as 7*10
5-9*10
5aU/g.
In the present invention, the thickness of described coating is 0.1-0.3 millimeter.
In the present invention, described suppress listerial bacteriocin adopt with the following method prepare: fermented and cultured Enterococcus durans (Enterococcusdurans) NJ strain, get zymotic fluid, centrifugation supernatant and bacterial sediment; Adopt macroporous absorbent resin to be separated described supernatant, collect the eluent A containing described bacteriocin; Adopt the bacteriocin on isopropanol water solution washing bacterial sediment surface, obtain eluent B; Merge eluent A and B, be separated with the bag filter that molecular cut off is 1000-3000Da, get trapped fluid, described in obtaining, listerial bacteriocin can be suppressed.
The present invention also provides a kind of method preparing described suppression Listeria coating, comprise the steps: PLA and listerial bacteriocin can be suppressed to add in solvent, be stirred to that PLA dissolves, described bacteriocin is dispersed in solvent, obtain coating solution; Described coating solution is poured in container or is coated on body surface, except desolventizing, drying.
In preferred technical scheme, described solvent is carrene.
In preferred technical scheme, the PLA added in every 15ml solvent is 0.5-1.5g.
In preferred technical scheme, the temperature of described drying is 25-90 DEG C, and drying time is 2-20 hour.
In the present invention, the material of described container or object is plastics, pottery, metal or glass.
The present invention also provides the application of described suppression Listeria coating in food security.
The present invention suppresses listerial coating, can efficiently suppress listerial growth, the broad-spectrum sterilization bacteriocin can effectively avoiding present stage to use during application food fresh keeping storage art is to the suppression of other beneficial bacteriums in food, have no side effect, may be used on glass, plastics, pottery and ironwork, with low cost.The preparation method of coating of the present invention, safely, simple, cost is low, is adapted at food industry and promotes widely.
Accompanying drawing illustrates:
Fig. 1 shows the change of vial before and after coating.Be respectively from left to right without coated glass bottle (not applying any material), control coatings vial, coated glass bottle (this vial inwall is coated with the coating containing bacteriocin NJ) containing bacteriocin NJ.
After Fig. 2 cultivates different time under showing 4 DEG C of conditions, the clump count of Listeria monocytogenes ScottA in milk sample in each vial, wherein "None" represents in vial without coating, PLA represents control coatings vial, NJ represents that inwall is coated with the vial of the coating containing bacteriocin NJ, and the content of bacteriocin NJ in the numeral coating of NJ front.
After Fig. 3 cultivates different time under showing 25 DEG C of conditions, the clump count of Listeria monocytogenes ScottA in milk sample in each vial, wherein "None" represents in vial without coating, " PLA " represents control coatings vial, " NJ " represents that inwall is coated with the vial of the coating containing bacteriocin NJ, and the content of bacteriocin NJ in the numeral coating of NJ front.
Fig. 4 shows milk and preserve state after 42 days in vial, under 4 DEG C of conditions, and the coating wherein scheming vial in A is interior containing 250mg bacteriocin NJ, and in figure B, the coating of vial is interior containing 500mg bacteriocin NJ.
Fig. 5 be in coating bacteriocin NJ and streptococcus lactis bacteriocin Nisin addition to the comparison of growing state suppressing Listeria monocytogenes ScottA.Wherein "None" represents in vial without coating, " PLA " represents control coatings vial, " NJ " represents that inwall is coated with the vial of the coating containing bacteriocin NJ, " NISN " represents that inwall is coated with the vial of the coating containing streptococcus lactis bacteriocin Nisin, the content of bacteriocin in the numeral coating of NJ or NISN front.
Fig. 6 contrasts before and after container made of iron coating, and wherein (A) is without coating, and (B) has coating.
Detailed description of the invention
The preparation of embodiment 1 bacteriocin NJ
Fermentative medium formula: tryptone (OXOID) 30g/L, dusty yeast (OXOID) 10g/L, beef extract (OXOID) 10g/L, dipotassium hydrogen phosphate 5g/L, lactose 5g/L, use phosphoric acid adjust ph 6.5, solvent is water.
Enterococcus durans (Enterococcusdurans) the NJ strain (being disclosed in ZL201310265229.3 patent of invention) being kept at-70 DEG C is inoculated in fermentation medium, 37 DEG C, under anaerobic condition incubated overnight in order to activate NJ strain.And then with the inoculum concentration of 1% (v/v), the NJ strain bacterium liquid of activation is transferred in fermentation medium, 37 DEG C, quiescent culture 18h under anaerobic condition in water isolation type constant incubator.
Enterococcus durans NJ strain institute bacteriocinogeny called after bacteriocin NJ.The purge process of bacteriocin NJ is as follows: collect the Enterococcus durans NJ strain zymotic fluid after cultivating 18h, 4 DEG C, the centrifugal 20min of 12000r/min, separation of supernatant and bacterial sediment, wherein supernatant is as bacteriocin NJ crude extract.Macroreticular resin (XAD-16 resin) is carried out pretreatment: employing pH2.0, concentration are the isopropanol water solution rinsing of 70% (v/v), then adopt ultra-pure water balance.Bacteriocin NJ crude extract adopts pretreated macroporous resin adsorption, and pH2.0, concentration are the isopropanol water solution wash-out of 70% (v/v), collects eluent A.In addition, be the isopropanol water solution Eddy diffusion bacterial sediment of 70% (v/v) by pH2.0, concentration, wash the bacteriocin that sticks on enterococcus, 4 DEG C, centrifugal 20min gets supernatant to 12000r/min, as eluent B.Merge eluent A, B, at 75 DEG C of constant temperature rotary evaporations to 25mL, adopt molecular cut off to be the bag filter dialysed overnight of 1000Da, get trapped fluid again 75 DEG C of constant temperature rotary evaporations to 25mL, after freeze drying, obtain bacteriocin NJ freeze-dried powder.
The cost calculation process of bacteriocin NJ is as follows: 1L culture medium can obtain bacteriocin freeze-dried powder 5g through above preparation method, and cost is 454.11 yuan, and concrete analysis is as table one.Therefore, the cost of bacteriocin NJ is 90.8 yuan/gram.
Table one: the cost of bacteriocin NJ
The mensuration that bacteriocin NJ tires is using Listeria monocytogenes as indicator bacteria, concrete grammar is as follows: 1g/mL bacteriocin solution is carried out 2 times of doubling dilutions and (in EP pipe, adds the ultra-pure water of 100 μ L, then getting 100 μ L enterococcins adds in ultra-pure water, dilutes downwards step by step after mixing).Get the bacteriocin solution 5 μ L diluted, be added drop-wise to inoculation to have on the BHI solid medium (Beijing overpass technical concern Co., Ltd) of the Listeria monocytogenes that 0.5% (v/v) is fresh, detected the fungistatic effect of this gradient bacteriocin dilution by agar diffusion method.Tiring of bacteriocin calculates with following formula:
X＝2
n×(1000/A)
In formula: n-bacteriocin has the maximum dilution number of times of fungistatic effect to Listeria monocytogenes; The volume (μ L) of the bacteriocin solution that A-drips; Tire (AU/mL or AU/g) of X-bacteriocin.
Through detecting, the bacteriocin NJ prepared in the present embodiment tires as 819200AU/g.
Embodiment 2 suppresses listerial coating in the coating of vial inwall
1. the solvent preparing coating is selected
In order to select safety, efficiently solvent, investigate the solubility property of a large amount of solvent to PLA (PLA), wherein the solubility property of partial solvent lists in table two.PLA, purchased from NatureWorksLLC, model is 4060D, proportion 1.24, and solution density is 1.08 230 DEG C time, and glass transition temperature is 55-60 DEG C, and relative viscosity is 3.4.
The dissolving situation (room temperature) of table two: PLA
| Solvent | Dissolubility | Dissolution time | Quantity of solvent needed for 1gPLA |
| Chloroform | Dissolve | Dissolve immediately | 12mL |
| Carrene | Dissolve | Dissolve immediately | 15mL |
| Ethanol | Do not dissolve | — | — |
| Acetone | Dissolve | 30min | 25mL |
| Methyl alcohol | Do not dissolve | — | — |
| Benzinum | Do not dissolve | — | — |
As can be seen from Table II, PLA has good dissolubility in carrene and chloroform, room temperature condition and solubilized, and dissolubility is relatively slightly poor in acetone, and because the toxicity of chloroform is comparatively large, therefore this experiment chooses carrene as solvent.
2. in the coating of vial inwall coating containing bacteriocin NJ
Accurately take one gram of PLA and 80mg bacteriocin NJ.PLA, bacteriocin NJ are added in 15mL carrene, stirs this mixture by non magnetic rod, until PLA dissolves completely, bacteriocin NJ is distributed in solvent equably, obtain coating solution.Poured into by coating solution in the vial (100mL another name for Sichuan Province ox silk mouth bottle) of uncovered, at room temperature (22 DEG C-26 DEG C) carry out rotary evaporation removing carrene.Vertically be placed in vacuum drying oven by vial after removing carrene, drying 12 hours at 90 DEG C, then cover lid is for subsequent use.
3. coating is containing the coating of different amount bacteriocin
According to method in the present embodiment title 2 at vial inwall applying coating, only change the consumption of bacteriocin NJ into 125,250 and 500mg, other are constant, to investigate in coating bacteriocin NJ consumption to Listeria inhibition.
The preparation of control coatings vial: according to method in the present embodiment title 2 at vial inwall applying coating, difference is only not add bacteriocin NJ in coating solution.
Average coating thickness containing bacteriocin NJ is 0.15 millimeter, with the PLA coating layer thickness almost indifference of control coatings vial inwall.As seen from Figure 1, the coating containing bacteriocin NJ can effectively be coated on glass medium, and adding of bacteriocin NJ affects to some extent on PLA coated optical performance, makes vial become translucent from transparent.
4. coating is to listerial inhibition
For Listeria monocytogenes (Listermonocytogenes) ScottA (being abbreviated as Listeria monocytogenes ScottA), detect coating to listerial inhibition.
Milk add 100mL sterilizing in the cated each vial of inwall after, adds wherein by the Listeria monocytogenes ScottA of incubated overnight, makes Listeria monocytogenes ScottA cell density in milk be about 1 × 10
4individual cell/mL, cover lid, cultivates under 200rpm condition, investigates 4 DEG C and 25 DEG C of impacts on Listeria monocytogenes ScottA breeding situation respectively.Three parallel tests are carried out in each experiment.In 28 days that cultivate, sampling (1mL) detects the clump count in milk.Sample uses phosphate buffer continuous gradient dilution (pH7.2), then 100ul dilution is got in the upper coating of PALCAM solid medium (purchased from Beijing overpass technical concern Co., Ltd), cultivate 24 hours at 37 DEG C, calculate the clump count in each sample.With uncoated vial, control coatings vial in contrast, same procedure is adopted to cultivate Listeria monocytogenes ScottA.In coating, bacteriocin NJ content is on suppressing the impact of Listeria monocytogenes ScottA effect as table 1, table 2, Fig. 2 and Fig. 3.After 17d, in control coatings vial in milk Listeria monocytogenes ScottA cell more than 10
7cFU/mL.Under 4 DEG C and 25 DEG C of conditions, the coating of control coatings vial not containing bacteriocin NJ, therefore to Listeria monocytogenes ScottA without any inhibitory action.Along with the increase of bacteriocin NJ concentration in coating, inhibitory action increases gradually, but add 80mg bacteriocin NJ coating and contrast between there is no significant difference.
Analyze fungistatic effect (table 1 and Fig. 2) under 4 DEG C of conditions.Coating containing 250 milligrams of bacteriocin NJ, in one day, Listeria monocytogenes ScottA cell concentration significantly reduces, lower than 10
2can't detect Listeria monocytogenes ScottA in sample after CFU/mL, 3d, and all do not detect Listeria monocytogenes ScottA in the storage period of 3-28 days.Coating containing 500 milligrams of bacteriocin NJ, can't detect Listeria monocytogenes ScottA in sample, in 1-28 days, all can't detect Listeria monocytogenes ScottA in sample; Even, after cultivation 42d, still Listeria monocytogenes ScottA do not detected containing the sample in 250 milligrams, 500 milligrams coated glass bottles, and sample is unchanged on sense organ, without lamination, intact (as Fig. 4).In addition, inventor directly adds 500mg bacteriocin NJ in uncoated vial, finds, after cultivation 1d, to can't detect Listeria monocytogenes ScottA.Can only ensure that the system being added with bacteriocin has bactericidal effect owing to directly adding bacteriocin in food, and coating is owing to being solidificated on container, can returnable container, reduce the use cost of bacteriocin.
After different time cultivated by table 14 DEG C, the list in each vial in milk increases Listeria monocytogenes ScottA clump count (logcfu/ml)
Note: data are all repeat experimental result mean+SD 3 times.Without significant difference (P>0.05) between the data that often in row there is same letter."None" represents in sample and can't detect Listeria monocytogenes ScottA (<1CFU/mL).
Analyze fungistatic effect (as table 2, Fig. 3) under 25 DEG C of conditions.Just 10 are reached after Listeria monocytogenes ScottA3d in milk in control coatings vial
7cFU/mL.Coating containing 250mg bacteriocin NJ can not suppress the growth of Listeria monocytogenes ScottA completely; Coating containing 500 milligrams of bacteriocin NJ, after 1d, just can't detect Listeria monocytogenes ScottA in sample; After 42d, in sample, still Listeria monocytogenes ScottA do not detected, and unchanged on sense organ of sample, without lamination, intact.
After showing 2:25 DEG C of cultivation different time, single clump count (logcfu/ml) increasing Listeria monocytogenes ScottA in milk in each vial
Note: data are all repeat experimental result mean+SD 3 times.Without significant difference (P>0.05) between the data that often in row there is same letter."None" represents in sample and can't detect Listeria monocytogenes ScottA (<1CFU/ milliliter).
Applicant carried out great many of experiments, find that this coating can not only suppress other bacterial strains in Listeria monocytogenes kind, comprise ListermonocytogenesV37, ListermonocytogenesF3VJG, ListermonocytogenesH2NG and ListermonocytogenesNR30 (wherein ListermonocytogenesNR30 is known bacterial strain nisin Nisin being had to resistance), and can listera innocua be suppressed.
5. contrast test
Adopt method in the present embodiment title 3, only changing bacteriocin NJ in coating is 250mg and 500mg streptococcus lactis bacteriocin Nisin (available from Sigma), then with carry out comparing of fungistatic effect under 4 DEG C of conditions containing 250mg bacteriocin NJ coating, result is as Fig. 5.Can see, under 4 DEG C of conditions, coating containing 250mg streptococcus lactis bacteriocin Nisin can not suppress the growth of Listeria monocytogenes ScottA completely, coating containing 500mg streptococcus lactis bacteriocin Nisin could suppress the growth of Listeria monocytogenes ScottA completely, but the coating only containing 250mg bacteriocin NJ just can suppress the growth of Listeria monocytogenes ScottA completely.Cost due to streptococcus lactis bacteriocin Nisin is 1065 yuan/gram, the cost of bacteriocin NJ of the present invention is 90.8 yuan/gram, therefore, the coating cost of the suppression Listeria monocytogenes ScottA adopting the inventive method to prepare is significantly lower than employing streptococcus lactis bacteriocin Nisin.
Embodiment 3 applies and suppresses listerial coating on iron ware
Accurately take 1gPLA and 300mg bacteriocin NJ.PLA, bacteriocin are added in 15mL carrene, stirs this mixture by non magnetic rod, until PLA dissolves completely, bacteriocin is distributed in solvent equably, obtain coating solution.Coating solution is poured in container made of iron, carry out evaporating to remove carrene in room temperature.Container made of iron after removing carrene is placed in vacuum drying oven, drying 14 hours at 80 DEG C.Contrast as shown in Figure 6 before and after the coating of container made of iron, can see that the coating containing bacteriocin NJ can be coated to the surface of irony medium.
Claims (10)
1. suppress a listerial coating, it is characterized in that: containing 95-105 mass parts PLA, 20-65 mass parts can suppress listerial bacteriocin; Described bacteriocin derive from Enterococcus durans (
enterococcusdurans) NJ strain.
2. suppress listerial coating according to claim 1, it is characterized in that: describedly suppress listerial bacteriocin to be tired as 7*10
5-9*10
5aU/g.
3. suppress listerial coating according to claim 2, it is characterized in that: the thickness of described coating is 0.1-0.3 millimeter.
4. according to claim 1,2 or 3, suppress listerial coating, listerial bacteriocin can be suppressed described in it is characterized in that to adopt and prepare with the following method: fermented and cultured Enterococcus durans (
enterococcusdurans) NJ strain, get zymotic fluid, centrifugation supernatant and bacterial sediment; Adopt macroporous absorbent resin to be separated described supernatant, collect the eluent A containing described bacteriocin; Adopt the bacteriocin on isopropanol water solution washing bacterial sediment surface, obtain eluent B; Merge eluent A and B, be separated with the bag filter that molecular cut off is 1000-3000Da, get trapped fluid, described in obtaining, listerial bacteriocin can be suppressed.
5. prepare the method suppressing Listeria coating described in claim 1 for one kind, it is characterized in that comprising the steps: PLA and listerial bacteriocin can being suppressed to add in solvent, be stirred to that PLA dissolves, described bacteriocin is dispersed in solvent, obtain coating solution; Described coating solution is poured in container or is coated on body surface, except desolventizing, drying.
6. prepare the method suppressing Listeria coating according to claim 5, it is characterized in that described solvent is carrene.
7. according to claim 5 or 6, prepare the method suppressing Listeria coating, it is characterized in that the PLA added in every 15ml solvent is 0.5-1.5g.
8. prepare the method suppressing Listeria coating according to claim 7, it is characterized in that the temperature of described drying is 25-90 DEG C, drying time is 2-20 hour.
9. prepare the method suppressing Listeria coating according to claim 8, it is characterized in that the material of described container or object is plastics, pottery, metal or glass.
10. the application of one of claim 1-4 described suppression Listeria coating in food security.
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| CN108872573A (en) * | 2018-07-04 | 2018-11-23 | 中国水产科学研究院东海水产研究所 | The Listeria monocytogenes rapid detection method of group is swashed based on nanometer magnetic bead-fluorescence |
| CN115160613A (en) * | 2022-08-17 | 2022-10-11 | 江苏省农业科学院 | Preparation process of fresh-keeping hydrogel film |
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