CN105801896B - A kind of preparation method of bacteriocin preservative film - Google Patents
A kind of preparation method of bacteriocin preservative film Download PDFInfo
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- CN105801896B CN105801896B CN201610257606.2A CN201610257606A CN105801896B CN 105801896 B CN105801896 B CN 105801896B CN 201610257606 A CN201610257606 A CN 201610257606A CN 105801896 B CN105801896 B CN 105801896B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/04—Coating
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2327/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Derivatives of such polymers
- C08J2327/02—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Derivatives of such polymers not modified by chemical after-treatment
- C08J2327/04—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a halogen; Derivatives of such polymers not modified by chemical after-treatment containing chlorine atoms
- C08J2327/08—Homopolymers or copolymers of vinylidene chloride
Abstract
The present invention relates to a kind of preparation methods of bacteriocin biological preservation film, suitable for the fresh-keeping packaging field of low temperature food product, lactobacillus plantarum plant subspecies that the present invention utilizes(Lactobacillus plantarum subsp. plantarum)Zhang-LL CGMCC No.6936, is concentrated to get after activated, expansion culture, centrifuging and taking supernatant, ammonium sulfate precipitation, dialysis by Plantaricin by L. plantarum Zhang-LL solution；Coating feed liquid is prepared in Zhang-LL Plantaricin by L. plantarum solution and dehydrated alcohol mixing；Obtaining the bacteriostasis, preservation film that the bacteriocin food fresh keeping membrane present invention makes after coated testing machine (ZY-TB) coating, natural drying has good storage-stable, the bacteriocin preservative film that room temperature storage bacteriostatic activity at least keeps 6 months or more the present invention to make, simple process, it is low in cost, it has no toxic side effect, and there is good fungistatic effect, it is suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of preparation methods of bacteriocin preservative film, belong to packaging material for food technical field.
Background technique
Be used to prepare food fresh keeping membrane material be all using ethylene masterbatch as raw material, it is fresh-keeping according to the difference of ethylene masterbatch
Film can be divided into three categories: polyethylene (PE), polyvinyl chloride (PVC) and polyvinylidene chloride (PVDC) polyethylene (PE) can be divided into
Two major class: high density polyethylene (HDPE) and oligomerisation of ethylene (LDPE) but the use due to preservative film often will increase micro- life of food
Object pollution problem, the preparation of food antimicrobial preservative film is so that preservative film can increase guaranteeing the quality for food with certain biocidal property
Phase, patent CN103319735B disclosed a kind of preparation method of Konjac glucomannan antibacterial preservative film, by nano-titanium dioxide
It is mixed with konjak portuguese gansu polyose sugar juice, is adjusted to alkalinity after addition glycerol and obtains fresh-keeping liquid；Fresh-keeping liquid is uniformly coated on carrier table
Face, the drying and forming-film preservative film can be used for the packaging of fruits and vegetables at 65 DEG C, extend the freshness date of fruits and vegetables by south
Peaceful city Ju Xiang plastic products Co., Ltd Wei Chun Xiang and " a kind of antimicrobial preservative film " of Wei Decheng application (publication number:
CN105086305A a large amount of chemical reagent is used in), the antibacterial film is at high cost, dosage is limited, human body alimentary canal cannot digest
It absorbs, the " a kind of using polyvinyl alcohol as the Chinese medicine fruits and vegetables of carrier of tinkling of pieces of jade etc. thanks in Agricultural University Of Hunan general to the fresh-keeping effect of food
The method of report film carries out package fruit in preservative film " (publication number CN10496101A), can be by the fruit preservation under room temperature time
Extend to 7d.
Lactobacillus plantarum plant subspecies are the active microorganism beneficial to human body, lactobacillus plantarum as a kind of probiotics
Plant subspecies(Lactobacillus plantarum subsp. plantarum)Zhang-LL CGMCC No.6936 has
Inhibit pathogen Listeria Monocytogenes (hereinafter referred to as single increase listeria spp) effect (publication number:
CN104560799A) bacteriocin that is isolated and purified from the bacterial strain has no toxic side effect, and bacteriocin is to a variety of harmful bacterias
With stronger bacteriostatic activity, and it is preferable to heat and ph stability, it can be degraded by human protease, be a kind of natural safe
Its bacteriocin is used for the research of food fresh keeping membrane by biological preservative, is had to the shelf life and safety that extend food important
Meaning.
Summary of the invention
A kind of system for the Novel antibacterial biological preservation film that the object of the present invention is to provide preparation processes is simple, thermal stability is good
Standby technology.
The technical scheme is that realized by following steps:
1. lactobacillus plantarum plant subspecies(Lactobacillus plantarum subsp. plantarum)
Zhang-LL activation and fermentation liquid preparation:
(1) the lactobacillus plantarum lactic acid subspecies Zhang-LL of glycerol stocks at -80 DEG C is transferred and is arrived into 5mL MRS liquid
In 10ml centrifuge tube, 37 DEG C, 12h is cultivated on the shaking table of 180r/min；
(2) incubation time 12h in the MRS culture medium of culture Zhang-LL to 100ml is expanded with 2% bacterium amount that connects；
(3) culture lactobacillus plantarum incubation time 20h into the MRS culture medium of 1000ml is expanded with 2% bacterium amount that connects.
2. lactobacillus plantarum plant subspecies (Lactobacillus plantarum subsp. plantarum)Zhang-
The preparation of LL bacteriocin extracting solution:
(1) by the fermentation liquid of Zhang-LL in 4 DEG C, 8000 r/min, centrifugation 15min obtains fermented supernatant fluid；
(2) fermented supernatant fluid is used 4 DEG C of ammonium sulfate precipitation method, is placed in the stirring of magnetic stirring apparatus top, side slowly adds
Enter ammonium sulfate powder, makes the final saturation degree 75% of ammonium sulfate；
(3) for ammonium sulfate precipitation liquid after 4 DEG C of refrigerators precipitate 12h, 8000r/min is centrifuged 15min, and precipitating is suspended in volume
It is redissolved in deionized water for fermentating liquid volume 1/10；
(4) 4 DEG C, 12000r/min be centrifuged 15min remove redissolve liquid insoluble matter, obtain plant cream bar bacteriocin slightly mention it is molten
Liquid；
(5) it dialyses to the plant cream bar bacteriocin solution that (4) obtain, the molecular cut off of bag filter is 3500Da,
It is slowly stirred in 4 DEG C of deionised waters with magnetic stirring apparatus, dialysis is for 24 hours；
(6) further use molecular cut off for 4 DEG C of dialysis band of 7000Da dialysis for 24 hours, collect dialyzate, obtain
Zhang-LL Plantaricin by L. plantarum solution, then PEG8000 concentrate solution；
(7) coating feed liquid is prepared in Zhang-LL Plantaricin by L. plantarum solution (28600AU) and dehydrated alcohol mixing,
Ethyl alcohol final concentration of 75%.
3. coating machine coating film forming
(1) surface is smooth, non-wrinkled, unabroken PVDC film (22cm × 35cm) is laid in middle instrument coated test machine
(ZY-TB) on pedestal, clamp is used；
(2) suitable coating feed liquid is gone up before coating machine bar；
(3) coated test machine is coated with (28600AU) bacteriocin solution with 5c m/s speed, and coating thickness is 10 ~ 30 μm；
(4) coated film is taken out, spontaneously dries, obtains novel food antistaling film.
In the method, lactobacillus plantarum plant subspecies(Lactobacillus plantarum subsp. plantarum)It is common to be preserved in China Committee for Culture Collection of Microorganisms on December 04th, 2012 by Zhang-LL
Microorganism center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number
Are as follows: CGMCC No.6936. cultivates 2d in MRS culture medium, and bacterial strain Zhang-LL thallus is in rod-short, 0.5 × 1.33~2.0 μ
M in pairs or is in individually short catenation, spore of not sprouting, the Gram-positive bacterium fermented supernatant fluid is to enterococcus faecalis, single increasing Lee
The growth of the various bacterias such as this special Salmonella, lactobacillus curvatus, brown rot fungus has apparent inhibiting effect.
Bacteriocin is applied in food fresh keeping membrane by the present invention, has widened the application field of bacteriocin, has obtained having suppression
The novel food antistaling film of bacterium property provides new research direction and method for the research of packaging material for food.
In the method, using the lactobacillus plantarum plant subspecies of food source, and will be thin using coating machine coating method
Rhzomorph is applied to the surface of film, and bacteriocin bacteriostasis, preservation film is obtained after dry, and the use of the film can extend the shelf life of food
Plantaricin by L. plantarum solution manufacturing method is simple, at low cost, has no toxic side effect, and has good fungistatic effect this strain simultaneously
Plantaricin by L. plantarum also has characteristic resistant to high temperature, will not influence its fungistatic effect after high-temperature heating.
In the method, a kind of preparation method of novel food antistaling film belongs to the scope of the present invention.
Specific embodiment
The invention will be further described for following embodiments without limiting the scope of the invention.
Experimental method in following embodiments is unless otherwise instructed conventional method.
Percentage composition in following embodiments is unless otherwise instructed mass volume ratio concentration (W/V).
The preparation method of embodiment 1, bacteriocin preservative film
(1) the lactobacillus plantarum lactic acid subspecies Zhang-LL of glycerol stocks at -80 DEG C is transferred and is trained into 5mL MRS liquid
It supports in base, 37 DEG C, cultivates 12h. on the shaking table of 180r/min；
(2) incubation time 12h in the MRS culture medium of culture Zhang-LL to 100ml is expanded with 2% bacterium amount that connects；
(3) incubation time 20h in the MRS culture medium of culture Zhang-LL to 1000ml is expanded with 2% bacterium amount that connects；
(4) by the fermentation liquid of Zhang-LL in 4 DEG C, 8000 r/min, it is centrifuged 15min, obtains fermented supernatant fluid；
(5) fermented supernatant fluid is used 4 DEG C of ammonium sulfate precipitation method, is placed in the stirring of magnetic stirring apparatus top, side slowly adds
Enter ammonium sulfate powder, makes the final saturation degree 75% of ammonium sulfate；
(6) for ammonium sulfate precipitation liquid after 4 DEG C of refrigerators precipitate 12h, 8000r/min is centrifuged 15min, is redissolved in deionized water
To 100mL；
(7) 4 DEG C, 12000r/min is centrifuged 15min and removes the insoluble matter for redissolving liquid；
(8) it dialyses to the Plantaricin by L. plantarum solution that (4) obtain, the molecular cut off of bag filter is 3500Da, in 4
It is slowly stirred in DEG C deionised water with magnetic stirring apparatus, dialysis for 24 hours, obtains Plantaricin by L. plantarum extracting solution about fermentating liquid volume
1/10；
(9) further use molecular cut off for 4 DEG C of dialysis band of 7000Da dialysis for 24 hours, collect dialyzate, obtain plant
Lactobacillin Zhang-LL solution, then PEG8000 is concentrated into 100mL or so；
(10) Zhang-LL Plantaricin by L. plantarum solution (28600AU) 100mL and dehydrated alcohol 75mL mixing is prepared into
To coating feed liquid；
(11) surface is smooth, non-wrinkled, unabroken PVDC film 22cmx35cm is laid in middle instrument coated test machine (ZY-
TB) on pedestal, clamp is used；
(12) suitable coating feed liquid is gone up before coating machine bar；
(13) coated test machine is coated with (28600AU) bacteriocin solution with 5c m/s speed, and coating thickness is 10 ~ 30 μm；
(14) coated film is taken out, spontaneously dries, obtains novel food antistaling film.
The bacteriostatic experiment of embodiment 2, bacteriocin food fresh keeping membrane
(1) bacteriocin food fresh keeping membrane is prepared by embodiment 1；
(2) bacteriostatic experiment test strain: select some pathogens and common bacterium as test strain, including single increasing Lee
This special bacterium (Listeria monocytogenes) ATCC54003, escherichia coli (Escherichia coli )
CDC85933, shigella dysenteriae (Shigella dysenteriae) CMCC51105, enterococcus faecalis (Enterococcus facealis) AS 1.2984, Lactobacillus pentosus (Lactobacillus pentosus) ATCC 8041, Bacterium enteritidis
(Salmonella enteritidis) CMCC 50041, bacillus subtilis (Bacillus subtilis) and the newborn bar of bending
Bacterium (Lactobacillus curvatus) carry out bacteriostatic experiment；
(3) bacteriostatic experiment: bacteriostasis, preservation film is cut into the disk that diameter is 10mm, disk is laid in the plate of test bacterium
Fungistatic effect is detected on surface, cultivates 10h in 37 DEG C of incubator, observes and records the fungistatic effect of film；
(4) test result: as shown in table 1, Zhang-LL has Listeria monocytogenes, enterococcus faecalis, Lactobacillus pentosus
Obvious bacteriostatic activity has moderate fungistatic effect to shigella dysenteriae and lactobacillus curvatus, and to large intestine angstrom
Uncommon Salmonella, Bacterium enteritidis and bacillus subtilis have lesser fungistatic effect Zhang-LL in these test strains
To Listeria monocytogenes (Listeria monocytogenes) ATCC54003 fungistatic effect it is especially prominent.
Fungistatic effect of 1 Zhang-LL of table to several frequently seen bacterium
| Test strain | Condition of culture | Antibacterial circle diameter (mm) |
| Listeria monocytogenes (Listeria monocytogenes) ATCC54003 | TSB-YE, 37°C, 16 h | 20 |
| Escherichia coli (Escherichia coli )CDC85933 | LB, 37°C, 12 h | 5 |
| Shigella dysenteriae (Shigella dysenteriae) CMCC51105 | GN, 37°C, 24 h | 13 |
| Enterococcus faecalis (Enterococcus facealis) AS 1.2984 | MRS, 37°C, 16 h | 18 |
| Lactobacillus pentosus (Lactobacillus pentosus) ATCC 8041 | MRS, 37°C, 24 h | 16 |
| Bacterium enteritidis (Salmonella enteritidis) CMCC 50041 | BPY, 37°C, 16 h | 7 |
| Bacillus subtilis (Bacillus subtilis) | MRS, 37°C, 16 h | 8 |
| Lactobacillus curvatus (Lactobacillus curvatus) | MRS, 37°C, 24 h | 12 |
Embodiment 3, bacteriocin food fresh keeping membrane are to the fresh-keeping test of cold fresh meat
(1) by embodiment 1, bacteriocin food fresh keeping membrane is prepared；
(2) the cold fresh meat 25g bacteriocin food fresh keeping membrane of fresh, hygienic qualification and ordinary food preservative film are wrapped up,
Same processing package sample meat, 4 DEG C cold storing and fresh-keeping 0 day, 1 day, 3 days, 4 days, 6 days, 8 days 2 days；
(3) it is measured according to GB 4789.2-2010 national food safety standard food microbiological examination total plate count
Method is measured under different storage time respectively by the total plate count in the meat of two kinds of fresh-keeping dresses of film；
(4) experimental result: bacteriostasis, preservation film has the good effect for inhibiting bacterial growth compared with compareing common preservative film,
Concrete outcome is shown in Table 2, as the extension fungistatic effect of storage time is more prominent.
Fungistatic effect of the 2 bacteriostasis, preservation film of table to the cold fresh meat sample containing indicator bacteria
The cold fresh meat of common preservative film packaging has just exceeded state from the 4th day in 4 DEG C of cold storing and fresh-keepings as can be seen from Table 2
It marks 1 × 10 as defined in GB/T 9959.2-20086CFU/g, cold fresh meat can be with the fresh-keeping 3 days times, and bacteriocin preservative film packet
The cold fresh meat of dress exceeded national standard quantity at the 8th day, and cold fresh meat at least can be with the fresh-keeping 6 days time, that is to say, that bacteriocin
The relatively common preservative film of cold fresh meat of preservative film packaging at least extends one times of fresh keeping time thus bacteriocin is fresh-keeping and can use
It is packed in the cold storing and fresh-keeping of cold fresh meat, the freshness date of cold fresh meat can be extended.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose these descriptions of
It is not wishing to limit the invention to disclosed precise forms the purpose of selecting and describing the exemplary embodiment is that solution
Certain principles and practical application of the invention are released, so that those skilled in the art can be realized and utilize of the invention
A variety of different exemplary implementation schemes and various chooses and changes the scope of the present invention are intended to by claims
And its equivalent form is limited.
Claims (1)
1. a kind of preparation method of bacteriocin preservative film, which is characterized in that be prepared by the following steps to obtain:
(1) lactobacillus plantarum Zhang-LL is activated, 37 DEG C of expansions culture, 8000r/min are centrifuged 15min, take supernatant solution,
75% ammonium sulfate precipitation protein isolate, centrifuged deposit deionized water dissolving use molecular cut off for the dialysis of 3500Da
4 DEG C of dialysis of bag for 24 hours, further use molecular cut off to be 4 DEG C of dialysis band of 7000Da and dialyse for 24 hours, collection dialyzate,
Bacteriocin solution is concentrated in PEG8000, and the lactobacillus plantarum Zhang-LL is the plant cream that deposit number is CGMCC No.6936
Bacillus plant subspecies (Lactobacillus plantarum subsp.plantarum)；
(2) coating feed liquid is prepared in bacteriocin solution described in 28600AU and dehydrated alcohol mixing, ethyl alcohol is final concentration of
75%；
(3) PVDC film is laid in coated test machine, suitable coating feed liquid is gone up before coating machine bar；
(4) coating machine is coated with 28600AU bacteriocin solution with 5cm/s speed, and coating thickness is 10~30 μm；Certainly by the film coated
It is so dry, obtain bacteriocin preservative film.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769426A (en) * | 2005-10-08 | 2006-05-10 | 中国农业大学 | Pentose lactobacillus bacteriocin and its special production strain and uses |
| CN102061271A (en) * | 2010-09-09 | 2011-05-18 | 四川大学 | Lactobacillus casei bacteriocin and use thereof in feed |
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104629279A (en) * | 2015-02-03 | 2015-05-20 | 安徽民祯生物工程有限公司 | Bacteriocin bacteriostatic fresh-keeping biological composite film |
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2016
- 2016-04-21 CN CN201610257606.2A patent/CN105801896B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769426A (en) * | 2005-10-08 | 2006-05-10 | 中国农业大学 | Pentose lactobacillus bacteriocin and its special production strain and uses |
| CN102061271A (en) * | 2010-09-09 | 2011-05-18 | 四川大学 | Lactobacillus casei bacteriocin and use thereof in feed |
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104629279A (en) * | 2015-02-03 | 2015-05-20 | 安徽民祯生物工程有限公司 | Bacteriocin bacteriostatic fresh-keeping biological composite film |
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