CN110240720A - A kind of methods and applications preparing polysaccharide composite film using thermostabilization recombinant bacteria laccase - Google Patents
A kind of methods and applications preparing polysaccharide composite film using thermostabilization recombinant bacteria laccase Download PDFInfo
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- A—HUMAN NECESSITIES
- A22—BUTCHERING; MEAT TREATMENT; PROCESSING POULTRY OR FISH
- A22C—PROCESSING MEAT, POULTRY, OR FISH
- A22C13/00—Sausage casings
- A22C13/0013—Chemical composition of synthetic sausage casings
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
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Abstract
The invention discloses a kind of methods and applications that polysaccharide composite film is prepared using thermostabilization recombinant bacteria laccase.The composite membrane is to be adjusted to 3-5 by the way that carboxymethyl chitosan solution, bacteria cellulose, phenolic acid and plasticizer, surfactant, mixed liquor pH value in edible polysaccharide Curdlan suspension, is added；Thermostabilization recombinant bacteria laccase is added in ultrasonic degassing, obtains into film liquid；Poured into polyfluortetraethylene plate at film liquid, 90-120 DEG C insulation reaction 8-10 minutes, be formed and sterilization processing, ultraviolet treatment with irradiation 5-10 minutes, 90-120 DEG C of hot air circulation is dried 30-60 minutes, is taken off film after 4 DEG C of cooling control humidity, temperature are dry, is obtained natural complex fresh-keeping film.The composite membrane that the present invention prepares, tensile strength is high, and extensibility is strong, has preferable anti-oxidant and antibacterial activity, and can effectively extend the shelf life of fruits and vegetables and meat products.
Description
Technical field
The invention belongs to technical field of food industry biology, it is excellent to be related to a kind of good mechanical performance, vapor barrier properties
Good, degradable polysaccharide composite membrane with antibacterial and anti-oxidation function and preparation method thereof.
Background technique
Polysaccharide degradable film is using animals and plants polysaccharide and microbial polysaccharide as film made of primary raw material, it has good
Good mechanical performance and transparency, is had a good application prospect using the film that polysaccharide material is prepared as matrix, but single polysaccharide
The film mechanical performance of preparation, block-water performance and stability are to be improved.
Curdlan, Chinese curdlan can also known as obtain right polysaccharide, be a kind of microbial source polyglucose, by list
One D glucose molecule is in the position C1 and C3, by β -1, branchiess linear molecule, the degree of polymerization made of 3 glucosides key connections
It is not soluble in water between 135-455.Curdlan has the peculiar property for forming gel in a heated condition, is 55 in temperature
Curdlan is capable of forming a kind of reversible low-intensity gel within the scope of DEG C -60 DEG C, and the general when temperature rises to 80 DEG C or more
Form a kind of irreversible high elastic and strength gel.
Curdlan is because of its extensive concern of the special physicochemical property by field of food, now using as coagulator, water holding
Agent, stabilizer, film forming agent etc. are applied in cereal preparation and frozen food.Curdlan has outstanding film forming, can obtain so
It is poured into mold after polysaccharide solution heating, drying can form a film, but pure Curdlan film mechanical performance is poor, so both at home and abroad
It is actually rare to the research that can obtain right polysaccharide membrane.There is document report Curdlan/ chitosan (Preparation and
characterization of novel curdlan/chitosan blending membranes for
Antibacterial applications, Carbohydrate Polymers, 2011,84:952-959) and konjac glucomannan/
Curdlan(Structural characterization and properties of konjac glucomannan/
Curdlan blend films, Carbohydrate Polymers, 2012,89:497-503) blend film.Both films or
Tensile strength (TS) is not high or extension property (EB) is not strong, and these problems limit the application of Curdlan correlation membrane product.
Laccase (Laccase, EC1.10.3.2, Lac) is a kind of generally existing copper-containing metal enzyme of nature, can urge
Change various phenolic substancess such as catechin, tannin, flavones etc. and is oxidized to quinone.Largely have drawn from fungi about the research work of laccase,
The fungi of secretion laccase focuses primarily upon Basidiomycota (Basidiomycota), Ascomycota (Ascomycota) and Fungi Imperfecti
The fungies such as class (Imperfect fungi), wherein the white-rot fungi most importantly in Basidiomycota.Fungal laccase is maximum to ask
Topic is that its thermal stability is poor, and pH stability difference is not suitable for actual production and processing application.Bacterial origin laccase has good heat
Stability can play synergistic effect with heating physical denaturation in catalytic reaction process, promote the progress of reaction.
Currently, right polysaccharide (Curdlan), carboxymethyl chitosan (CMCH), bacteria cellulose, phenolic acid can be obtained as matrix,
It is had not been reported using the research that thermostabilization recombinant bacteria laccase prepares composite membrane as crosslinking agent.
Summary of the invention
Right polysaccharide/carboxymethyl is obtained in view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of function admirable
Chitosan/bacteria cellulose/phenolic acid composite membrane.
It is a further object of the present invention to provide the preparation methods of the degradable natural complex fresh-keeping film.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of degradable polysaccharide composite membrane, in film liquid contain following raw material:
The film forming agent of 3%-8%: Curdlan polysaccharide；
The antibacterial material of 0.5%-2.0%: carboxymethyl chitosan (CMCH)；
0.1-1% film mechanical performance hardening agent: bacteria cellulose；
0.1-1% antioxidant: it preferably is selected from ferulic acid in phenolic acid, caffeic acid, chlorogenic acid, gallic acid, do not have
Gallate-based, catechin, tannic acid, any one in tea polyphenols；
The plasticizer of 0.1-0.5%: glycerol, sorbierite, any one in polyethylene glycol preferably are selected from；
The surfactant of 0.1-0.5%: Tween 80, sapn, alkyl glucoside, appointing in fatty glyceride preferably are selected from
It anticipates one kind；The above % indicates mass percentage.
The polysaccharide composite film is preferably also containing 10-20U/100ml at the crosslinking agent of film liquid: thermostabilization recombinant bacteria paint
Enzyme (Lac).
The polysaccharide composite film is preferably prepared by the following method to obtain:
(1) it takes Curdlan powder, in molten pure water, after Curdlan is sufficiently swollen, under high-speed shearing machine effect, prepares
The Curdlan suspension of 10-20g/100mL；
(2) chitosan phenol powder, the bacteria cellulose, phenol of formula ratio are added in the Curdlan suspension in step (1)
Acid prepares suspension A under the action of magnetic stirring apparatus；
(3) plasticizer, surfactant are added into the suspension A of step (2), under the action of magnetic stirring apparatus, system
Standby suspension B out；
(4) pH is adjusted with one of lactic acid, hydrochloric acid, acetic acid, phosphoric acid or citric acid of 1-3mol/L or a variety of acid solutions
To 3-5, homogeneous, ultrasonic degassing are added thermostabilization recombinant bacteria laccase, obtain into film liquid；Wherein, the thermostabilization recombinant bacteria
Laccase additional amount is 10-20U/100ml；
(5) polyfluortetraethylene plate is poured at film liquid；55-65 DEG C enzymic catalytic reaction 30-60 minutes；
(6) ultraviolet light irradiation-sterilize is handled 5-10 minutes；
(7) 90-120 DEG C of hot air circulation is dried 30-60 minutes；
After (8) 4 DEG C of coolings are handled 10-20 minutes, dry 12-18h is taken off under the conditions of RH30%-50%, 20-30 DEG C of control
Film.
The dead paddy bacillus laccase of the preferably heat-staple recombination of the recombinant bacteria laccase, the recombinant bacteria laccase are logical
Cross following method preparation:
(1) the dead paddy bacillus fmb-103 genomic DNA for being CGMCC No.6198 using deposit number is template, sequence
The downstream primer PCR amplification that upstream primer and sequence for SEQ ID NO.3 are SEQ ID NO.4 contains SacI and XhoI digestion
The laccase gene sequence in site；
(2) the previous step PCR product of purifying is inserted into the SacI/XhoI of carrier pET-23a after SacI, XhoI double digestion
Between restriction enzyme site, the expression plasmid pET-23a-fmb-L103 containing laccase gene is obtained；
(3) expression plasmid pET-23a-fmb-L103 is converted into Bacillus coli expression host strain BL21 (DE3) pLysS,
In 37 DEG C of picking petites after culture 10-11 hours, the 50ml LB liquid medium containing ampicillin, 70- are accessed
30 DEG C of overnight incubations of 90rpm take seed liquor to be added to the 100ml LB liquid containing ampicillin according to the volume ratio of 1:40
Culture medium, 35 DEG C of 180rpm, which vibrate, adds IPTG (100 μ g/ml of final concentration) induction for 2-3 hour when to OD600 being about 0.6, and 5 hours
Thalline were collected by centrifugation afterwards；
(4) thallus for being collected into inducing expression, with phosphate buffer be resuspended thallus, ultrasonic disruption thalline, 4 DEG C,
10000 × g is centrifuged 10min, collects supernatant as crude enzyme liquid, the crude enzyme liquid that processing is obtained is according to Ni-NTA His Tag
Kit specification is purified.
The preparation method of Compound Edible Coating for Preservation of the present invention comprising the steps of:
(1) Curdlan powder is weighed, is dissolved in pure water, after Curdlan is sufficiently swollen, under high-speed shearing machine effect, system
The Curdlan suspension of standby 10-20g/100mL out；
(2) chitosan phenol powder, the bacteria cellulose, phenol of formula ratio are added in the Curdlan suspension in step (1)
Acid prepares suspension A under the action of magnetic stirring apparatus；
(3) plasticizer, surfactant are added into the suspension A of step (2), under the action of magnetic stirring apparatus, system
Standby suspension B out；
(4) acid adjust suspension B pH to 3-5, homogeneous, ultrasonic degassing, be added thermostabilization recombinant bacteria laccase obtain at
Film liquid；Wherein, the thermostabilization recombinant bacteria laccase additional amount is 10-20U/100ml；
(5) polyfluortetraethylene plate will be poured at film liquid；55-65 DEG C enzymic catalytic reaction 30-60 minutes；
(6) ultraviolet light irradiation-sterilize is handled 5-10 minutes；
(7) 90-120 DEG C of hot air circulation is dried 30-60 minutes；
After (8) 4 DEG C of coolings are handled 10-20 minutes, dry 12-18h takes off film under the conditions of RH30%-50%, 20-30 DEG C of control
Obtain the Compound Edible Coating for Preservation.
Wherein, the acid preferably is selected from one of lactic acid, hydrochloric acid, acetic acid, phosphoric acid or citric acid of 1-3mol/L or more
Kind acid solution.
Application of the degradable composite membrane of the present invention in fruits and vegetables, food preservation.
Application of the degradable composite membrane of the present invention in terms of food processing, answering preferably in terms of preparing casing
With.
Application of the degradable composite membrane liquid in fruits and vegetables, food preservation.
It is preferred that degradable composite fresh-keeping is directly applied to fruits and vegetables, meat products surface at film liquid.
Further preferably edibility composite fresh-keeping is directly applied to behind fruits and vegetables, meat products surface at ultraviolet light irradiation at film liquid
Reason 5-10 minutes.
The utility model has the advantages that
Currently, the progress of degradable membrane is rapid both at home and abroad.However, compared with chemical synthesis film, degradable composite membrane
Bad mechanical strength, block-water performance is poor, problem of stability difference still need to it is urgently to be resolved.
Curdlan is a kind of thermal gels, has excellent filming performance, thermal stability and water-insoluble；Carboxymethyl chitosan
Sugared (abbreviation CMCH) has preferable film forming and antibacterial activity；Bacteria cellulose can enhance the mechanical performance of film；Phenolic acid class
Substance has excellent antioxygenic property；Laccase can be catalyzed the crosslinking of carboxymethyl chitosan and phenolic acid, and catalysis phenolic acid is formed
The stronger dimeric structure of antioxygenic property, thus the catalytic action of laccase can be enhanced film mechanical performance and anti-oxidant function
Energy.Therefore, Curdlan, CMCH of special ratios, bacteria cellulose, phenolic acid are film forming matrix, and with heat-staple recombinant bacteria
Laccase is cross-linking catalyst, the machinery mutually coordinated, interaction organism can effectively improve protein film as one
Performance, thermal stability, and assign film anti-microbial property and antioxygenic property.The present invention selects above-mentioned from existing filmogen
Specific degradable polysaccharide component, antimicrobial component, antioxidant content, surfactant, plasticizer and crosslinking agent, and pass through this
The compounding of the specific amount ranges of a little ingredients is aided with effective compounding method and realizes various saccharides substance and phenolic acid and recombinant bacteria paint
The blending of enzyme, by controlling film preparation condition, the polysaccharide composite film of acquisition in mechanical performance, thermal stability, inoxidizability and resists
Good performance is shown in terms of bacterium property, therefore, there is important application value.
Specific embodiment
The preparation of recombinant bacteria laccase and isolation and purification method are detailed in 109111584 A embodiment 1-4 of CN in the present invention.
Embodiment 1: composite membrane preparation process
(1) 8g Curdlan polysaccharide powder is weighed respectively, in molten pure water, after Curdlan is sufficiently swollen, high-speed shearing machine
Under effect, the Curdlan suspension of 8g/100mL is prepared；
(2) in 50mL Curdlan suspension, it is separately added into 1g carboxymethyl chitosan, 0.2g bacteria cellulose, 0.1g is green
Ortho acid prepares suspension A under the action of magnetic stirring apparatus
(3) 0.3g glycerol is added into the suspension A of step (2), 0.3g Tween 80 is settled to 100mL with pure water, in magnetic
Under the action of power blender, suspension B is prepared；
(4) with the salt acid for adjusting pH of 2mol/L to 4, homogenizer homogenization, ultrasonic degassing, addition 10U thermostabilization recombination
Bacterial laccase (Lac) is obtained into film liquid；
(5) polyfluortetraethylene plate is poured at film liquid；60 DEG C enzymic catalytic reaction 40 minutes；
(6) ultraviolet light irradiation-sterilize is handled 10 minutes；
(7) 90 DEG C of hot air circulations are dried 60 minutes；
After (8) 4 DEG C of coolings are handled 15 minutes, under the conditions of RH50%, 30 DEG C, dry 18h takes off film.
Embodiment 2: composite membrane preparation process
(1) 12g Curdlan polysaccharide powder is weighed respectively, in molten pure water, after Curdlan is sufficiently swollen, high speed shear
Under machine effect, the Curdlan suspension of 12g/100mL is prepared；
(2) in 50mL Curdlan suspension, 1.5g carboxymethyl chitosan, 0.4g bacteria cellulose, 0.1g are separately added into
Catechin prepares suspension A under the action of magnetic stirring apparatus；
(3) 0.5g glycerol is added into the suspension A of step (2), 0.5g Tween 80 is settled to 100mL with pure water, in magnetic
Under the action of power blender, suspension B is prepared；
(4) with the salt acid for adjusting pH of 2mol/L to 4, homogenizer homogenization, ultrasonic degassing, addition 15U thermostabilization recombination
Bacterial laccase (Lac) is obtained into film liquid；
(5) polyfluortetraethylene plate is poured into；60 DEG C enzymic catalytic reaction 40 minutes；
(6) ultraviolet light irradiation-sterilize is handled 10 minutes；
(7) 90 DEG C of hot air circulations are dried 60 minutes；
After (8) 4 DEG C of coolings are handled 15 minutes, under the conditions of RH50%, 30 DEG C, dry 18h takes off film.
Embodiment 3: composite membrane preparation process
(1) 16g Curdlan polysaccharide powder is weighed respectively, in molten pure water, after Curdlan is sufficiently swollen, high speed shear
Under machine effect, the Curdlan suspension of 16g/100mL is prepared；
(2) in 50mL Curdlan suspension, it is separately added into 2g carboxymethyl chitosan, 0.6g bacteria cellulose, 0.1g does not have
Gallate-based prepares suspension A under the action of magnetic stirring apparatus；
(3) 0.3g sorbierite is added into the suspension A of step (2), 0.3g sapn is settled to 100mL with pure water, in magnetic
Under the action of power blender, suspension B is prepared；
(4) with the salt acid for adjusting pH of 2mol/L to 4, homogenizer homogenization, ultrasonic degassing, addition 10U thermostabilization recombination
Bacterial laccase (Lac) is obtained into film liquid；
(5) polyfluortetraethylene plate is poured into；60 DEG C enzymic catalytic reaction 40 minutes；
(6) ultraviolet light irradiation-sterilize is handled 10 minutes；
(7) 90 DEG C of hot air circulations are dried 60 minutes；
After (8) 4 DEG C of coolings are handled 15 minutes, under the conditions of RH50%, 30 DEG C, dry 18h takes off film.
Embodiment 4: natural complex fresh-keeping film performance measurement
(1) mechanical performance
Film is cut into the strip of 8cm*2cm specification, and is fixed on physical property instrument and measures.Two fixture of physical property instrument is set
Initial distance is 80mm, speed 0.8mm/sec.After measurement, two folders when recording film breaks the power (g) and film fracture of moment
The distance L of tool.Each sample takes five parallel sample measurements.
Tensile strength TS (g) is calculated as follows:
TS=Fm/d*W
In formula: TS-tensile strength, (MPa)；
The maximum tension born when Fm-sample fracture, (N)；
D-film thickness, (m)；
W-film is wide, (m).
Elongation at break E (%) is calculated as follows:
E=(L1- L0)/L0
In formula: E-film elongation at break (%)；
L1The distance (mm) of two folder fixtures when-film is broken；
L0Distance (mm) when-two fixtures are initial.
(2) block-water performance
Moisture-vapor transmission
The open plastic cup of circle for taking diameter 3cm, depth 4cm, is added the dry CaC1 of 3g2, the film of diameter 7cm will be cut into
Sample covers rim of a cup, and the interface between film and cup is sealed with paraffin.Each cup is then placed in the distilled water that 1000mL is added in bottom
Drier in (21 DEG C, relative humidity 75%).Every 12h is weighed 1 time, for 1 week.Vapor is determined by the incrementss of cup weight
Transit dose.Moisture-vapor transmission (WVP) is calculated by ASTM method (E96-93,1993).
(3) natural complex fresh-keeping film thermal stability
The polysaccharide gel composite membrane prepared is boiled 30 minutes in boiling water, 30 DEG C of dry 12h are measured according to the method described above
Tensile strength, elongation percentage, moisture-vapor transmission and the water imbibition of film.
According to the method described above, the performance indicator for the composite membrane that different components and condition are prepared such as table 1.It can be with from table 1
Find out after thermostabilization recombinant bacteria Laccase Catalyzed, tensile property, elongation percentage and the vapor barrier properties of natural complex fresh-keeping film refer to
Mark is improved.
The different component edible freshness-keeping thin coat mechanical performances of table 1
Wherein, film 1 is Curdlan/ carboxymethyl chitosan/bacteria cellulose component film, and preparation process is referring to embodiment
1, chlorogenic acid and thermostabilization recombinant bacteria laccase component is not added only；
Film 2 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 1；
Film 3 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid/thermostabilization recombination prepared by embodiment 1 is thin
Bacterium laccase component film.
Film 4 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 2；
Film 5 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin/thermostabilization recombination prepared by embodiment 2 is thin
Bacterium laccase component film；
Film 6 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid component film, and preparation process is referring to reality
Example 3 is applied, thermostabilization recombinant bacteria laccase component is not added only；
Film 7 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid/thermostabilization recombination prepared by embodiment 3
Bacterial laccase component film.
5 natural complex fresh-keeping film antioxygenic property of embodiment
The oxidation resistance of edible composite film preservative film is indicated by measurement DPPH Scavenging activity.Composite membrane is cut into
20mm × 20mm is put into beaker, and 100mL distilled water is added, and in stirring on magnetic stirring apparatus, revolving speed 150r/min, temperature is
25±0.5℃.Timing sampling 1mL is into test tube, and using 1mL distilled water as blank control, 4mLDPPH methanol solution is added
(150umol/L) is mixed, and is protected from light standing 30min and is sufficiently reacted, measures absorbance at 516nm with ultraviolet specrophotometer.
DPPH free radical scavenging ability calculates following formula:
DPPH free radical scavenging ability=(1-Asample) × 100%/Acontrol
AsampleFor sample absorbance value, AcontrolFor the absorbance value of blank.
The oxidation resistance for the preservative film that different components and condition are prepared, as shown in table 2.
As can be seen from Table 2 after thermostabilization recombinant bacteria Laccase Catalyzed, the antioxygenic property index of natural complex fresh-keeping film
It is improved.
The different component edible freshness-keeping thin coat antioxygenic properties of table 2
Wherein, film 1 is Curdlan/ carboxymethyl chitosan/bacteria cellulose component film, and preparation process is referring to embodiment
1, chlorogenic acid and thermostabilization recombinant bacteria laccase component is not added only；
Film 2 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 1；
Film 3 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid/thermostabilization recombination prepared by embodiment 1 is thin
Bacterium laccase component film.
Film 4 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 2；
Film 5 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin/thermostabilization recombination prepared by embodiment 2 is thin
Bacterium laccase component film；
Film 6 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid component film, and preparation process is referring to reality
Example 3 is applied, thermostabilization recombinant bacteria laccase component is not added only；
Film 7 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid/thermostabilization recombination prepared by embodiment 3
Bacterial laccase component film.
6 natural complex fresh-keeping film anti-microbial property of embodiment
Composite membrane is surveyed to the bacteriostasis of Escherichia coli, staphylococcus aureus, bud pole bacterium and saccharomycete.It will be added
0.1mL indicator bacteria (bacterial concentration 105~106CFU/mL culture medium (bacterium: beef-protein medium)；Saccharomycete:
PDA culture medium) inverted plate, natural cooling 30min.Natural complex fresh-keeping film is cut into the disk of diameter 1cm, is placed in plate culture
Primary surface.Plate is placed in incubator and cultivates (Escherichia coli, staphylococcus aureus, 37 DEG C of bacillus subtilis for 24 hours；
28 DEG C of saccharomycete), measure antibacterial circle diameter.
The edible freshness-keeping thin coat that different components and condition are prepared is to Escherichia coli, staphylococcus aureus, withered grass gemma
The bacteriostatic activity of bacillus and saccharomycete is as shown in table 3.
As can be seen from Table 3 after thermostabilization recombinant bacteria Laccase Catalyzed, the antibacterial energy index of natural complex fresh-keeping film is obtained
Improvement.
The bacteriostasis property of 3 edible freshness-keeping thin coat of table
Wherein, film 1 is Curdlan/ carboxymethyl chitosan/bacteria cellulose component film, and preparation process is referring to embodiment
1, chlorogenic acid and thermostabilization recombinant bacteria laccase component is not added only；
Film 2 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 1；
Film 3 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid/thermostabilization recombination prepared by embodiment 1 is thin
Bacterium laccase component film.
Film 4 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 2；
Film 5 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin/thermostabilization recombination prepared by embodiment 2 is thin
Bacterium laccase component film；
Film 6 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid component film, and preparation process is referring to reality
Example 3 is applied, thermostabilization recombinant bacteria laccase component is not added only；
Film 7 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid/thermostabilization recombination prepared by embodiment 3
Bacterial laccase component film.
Fresh-keeping application of the 7 composite membrane preservative film of embodiment to fruits and vegetables
It is illustrated so that edible freshness-keeping thin coat is to the fresh-keeping effect of strawberry as an example.
Strawberry processing
Select in the same size, solid colour, shape is uniform, and the strawberry of intact defect and disease is fitted into crisper (each
Fresh-keeping box-packed 10), control group is sealed with Curdlan one-component film, and processing group is sealed with natural complex fresh-keeping film.Then it is put into
RH50%, 6 days in 21 DEG C of climatic chamber, the physiological and biochemical index of survey in every two days.
Strawberry physiological index determining
Strawberry fruit weight uses electronic balance weighing.The measurement of SOD enzyme activity is carried out using kit (WST-1 method)；
Conductivity uses conductivity gauge measuring method；Vitamin C content measurement uses 2,6- dichloropheno-lindophenol method；Organic acid uses acid base titration
Method；The measurement of strawberry fruit hardness uses GY-1 type hand-held hardometer, and unit is ox (N；kg/cm2)
Strawberry preservation effect
The fresh-keeping effect that the preservative film that different components and condition are prepared stores 6 days to strawberry is as shown in table 4.
6 days strawberry items physiological and biochemical indexs are stored from table 4, it can be seen that Curdlan/ carboxymethyl chitosan/thin
Fungin/chlorogenic acid/laccase natural complex fresh-keeping film, it is best to strawberry keeping-freshness storage effect.
The fresh-keeping effect to strawberry of 4 edible freshness-keeping thin coat of table
Wherein, film 1 is Curdlan/ carboxymethyl chitosan/bacteria cellulose component film, and preparation process is referring to embodiment
1, chlorogenic acid and thermostabilization recombinant bacteria laccase component is not added only；
Film 2 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 1；
Film 3 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid/thermostabilization recombination prepared by embodiment 1 is thin
Bacterium laccase component film.
Film 4 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 2；
Film 5 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin/thermostabilization recombination prepared by embodiment 2 is thin
Bacterium laccase component film；
Film 6 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid component film, and preparation process is referring to reality
Example 3 is applied, thermostabilization recombinant bacteria laccase component is not added only；
Film 7 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid/thermostabilization recombination prepared by embodiment 3
Bacterial laccase component film.
Fresh-keeping application of the 8 composite membrane preservative film of embodiment to meat products
It is illustrated so that edible freshness-keeping thin coat is to the fresh-keeping effect of chilled meat as an example.
Chilled meat processing
Cold fresh meat is accurately weighed according to the standard according to measurement, is randomly divided into six groups, wherein five groups are answered with different
It closes film and carries out package processing, control group is wrapped up with Curdlan one-component film, and processing group is wrapped up with natural complex fresh-keeping film, then put
It sets in refrigerator and is stored, storage temperature is 4 ± 0.5 DEG C.Index is measured by sampling in every 2d.
Chilled meat total plate count
Side referring to GB 4789.2-2010 national food safety standard, in the measurement of food microbiological examination total plate count
Method measurement.
Volatile Base Nitrogen (TVB-N)
Referring to the semi-micro nitrogen method measurement in GB/T 5009.44-2003.
Thiobarbituric acid reaction object (TBARS)
Weigh 5g sample be added 15mL A liquid (weigh 75.0g trichloroacetic acid, 1.0gEDTA and 1.0g propylgallate,
With ddH2O is settled to 1L), it filters after homogeneous.5.0mL filtrate is drawn in plug test tube, is mixed after 5mL B liquid is added
It is even, 40min is reacted in 100 DEG C of boiling water, measures absorbance.It compares malonaldehyde (MDA) standard curve and calculates MDA content, TBARS
Value is expressed as mg MDA/kg.
The measurement of pH value
It is measured referring to GB/T 9695.5-2008 " meat is measured with meat products pH ".
Chilled meat fresh-keeping effect
The fresh-keeping effect that the edible freshness-keeping thin coat that different components and condition are prepared stores 6 days to chilled meat is as shown in table 5.
Every index of quality of 6 days chilled meats is stored from table 5, it can be seen that Curdlan/ carboxymethyl chitosan/thin
Fungin/chlorogenic acid/laccase natural complex fresh-keeping film, it is best to chilled meat keeping-freshness storage effect.
Fresh-keeping effect of 5 edible freshness-keeping thin coat of table to chilled meat
Wherein, film 1 is Curdlan/ carboxymethyl chitosan/bacteria cellulose component film, and preparation process is referring to embodiment
1, chlorogenic acid and thermostabilization recombinant bacteria laccase component is not added only；
Film 2 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 1；
Film 3 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/chlorogenic acid/thermostabilization recombination prepared by embodiment 1 is thin
Bacterium laccase component film.
Film 4 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin component film, and preparation process is referring to implementation
Thermostabilization recombinant bacteria laccase component is not added only in example 2；
Film 5 is that Curdlan/ carboxymethyl chitosan/bacteria cellulose/catechin/thermostabilization recombination prepared by embodiment 2 is thin
Bacterium laccase component film；
Film 6 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid component film, and preparation process is referring to reality
Example 3 is applied, thermostabilization recombinant bacteria laccase component is not added only；
Film 7 is Curdlan/ carboxymethyl chitosan/bacteria cellulose/gallic acid/thermostabilization recombination prepared by embodiment 3
Bacterial laccase component film.
Claims (10)
1. a kind of polysaccharide composite film, it is characterised in that in film liquid contain following raw material:
(a) Curdlan: content 3%-8%；
(b) carboxymethyl chitosan: content 0.5-2%；
(c) bacteria cellulose: content 0.1%-1%；
(d) phenolic acid, ferulic acid, caffeic acid, chlorogenic acid, gallic acid, gallic acid, catechin, tannic acid, tea polyphenols
Deng one of or it is a variety of: content 0.1%-1%；
(e) plasticizer, one of glycerol, sorbierite, polyethylene glycol or a variety of: content 0.1-0.5%；
(f) surfactant, Tween 80, sapn, alkyl glucoside, fatty glyceride it is one or more: 0.1-0.5%；
The above % indicates mass volume ratio g/100ml.
2. Compound Edible Coating for Preservation according to claim 1 or 2, it is characterised in that be prepared by the following method to obtain:
(1) it takes Curdlan powder, in molten pure water, after Curdlan is sufficiently swollen, under high-speed shearing machine effect, prepares 10-
The Curdlan suspension of 20g/100mL；
(2) chitosan phenol powder, the bacteria cellulose, phenolic acid class of formula ratio are added in the Curdlan suspension in step (1)
Substance prepares suspension A under the action of magnetic stirring apparatus；
(3) plasticizer, surfactant are added into the suspension A of step (2) to prepare under the action of magnetic stirring apparatus
Suspension B；
(4) pH to 3- is adjusted with one of lactic acid, hydrochloric acid, acetic acid, phosphoric acid or citric acid of 1-3mol/L or a variety of acid solutions
5, homogeneous, ultrasonic degassing are added thermostabilization recombinant bacteria laccase, obtain into film liquid；Wherein, the thermostabilization recombinant bacteria laccase
Additional amount is 10-20U/100ml；
(5) polyfluortetraethylene plate is poured at film liquid；55-65 DEG C enzymic catalytic reaction 30-60 minutes；
(6) ultraviolet light irradiation-sterilize is handled 5-10 minutes；
(7) 90-120 DEG C of hot air circulation is dried 30-60 minutes；
After (8) 4 DEG C of coolings are handled 10-20 minutes, dry 12-18h takes off film under the conditions of RH30%-50%, 20-30 DEG C of control.
3. the preparation method of Compound Edible Coating for Preservation described in claim 1, it is characterised in that comprise the steps of:
(1) Curdlan powder is weighed, is dissolved in pure water, after Curdlan is sufficiently swollen, under high-speed shearing machine effect, is prepared
The Curdlan suspension of 10-20g/100mL；
(2) chitosan phenol powder, the bacteria cellulose, phenolic acid class of formula ratio are added in the Curdlan suspension in step (1)
Substance prepares suspension A under the action of magnetic stirring apparatus；
(3) plasticizer, surfactant are added into the suspension A of step (2) to prepare under the action of magnetic stirring apparatus
Suspension B；
(4) acid adjusts the pH to 3-5 of suspension B, and homogeneous, ultrasonic degassing are added thermostabilization recombinant bacteria laccase and obtain into film liquid；
Wherein, the thermostabilization recombinant bacteria laccase additional amount is 10-20U/100ml；
(5) polyfluortetraethylene plate will be poured at film liquid；55-65 DEG C enzymic catalytic reaction 30-60 minutes；
(6) ultraviolet light irradiation-sterilize is handled 5-10 minutes；
(7) 90-120 DEG C of hot air circulation is dried 30-60 minutes；
After (8) 4 DEG C of coolings are handled 10-20 minutes, dry 12-18h takes off film and obtains institute under the conditions of RH30%-50%, 20-30 DEG C of control
The Compound Edible Coating for Preservation stated.
4. preparation method according to claim 3, it is characterised in that the acid be selected from the lactic acid of 1-3mol/L, hydrochloric acid,
One of acetic acid, phosphoric acid or citric acid or a variety of acid solutions.
5. application of the polysaccharide composite film of any of claims 1 or 2 in fruits and vegetables, food preservation.
6. application of the polysaccharide composite film of any of claims 1 or 2 in terms of food processing.
7. application according to claim 5, it is characterised in that polysaccharide composite of any of claims 1 or 2 is protected film and prepared
Application in terms of casing.
8. the fresh-keeping application at film liquid in fruits and vegetables, food preservation of polysaccharide composite as stated in claim 2.
9. application according to claim 8, it is characterised in that polysaccharide composite film is directly applied to fruits and vegetables, meat products at film liquid
Surface.
10. application according to claim 9, it is characterised in that polysaccharide composite film is directly applied to fruits and vegetables, meat system at film liquid
Ultraviolet treatment with irradiation 5-10 minutes behind product surface.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628089A (en) * | 2019-10-11 | 2019-12-31 | 浙江理工大学 | Preparation method of poly-gallic acid/chitosan antibacterial material |
| CN115160609A (en) * | 2022-06-20 | 2022-10-11 | 湖南省农产品加工研究所 | Pullulan/bacterial cellulose/ferulic acid active composite membrane and preparation method and application thereof |
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| JPS60153998A (en) * | 1984-01-20 | 1985-08-13 | Hitachi Plant Eng & Constr Co Ltd | Waste water treating agent and its preparation |
| CN108586828A (en) * | 2018-04-10 | 2018-09-28 | 南京农业大学 | A kind of Compound Edible Coating for Preservation and its preparation method and application |
| CN109111584A (en) * | 2018-06-28 | 2019-01-01 | 南京农业大学 | It is a kind of to prepare albumen/polysaccharide composite film method and products thereof and application using thermostabilization recombinant bacteria laccase |
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2019
- 2019-06-06 CN CN201910491106.9A patent/CN110240720A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60153998A (en) * | 1984-01-20 | 1985-08-13 | Hitachi Plant Eng & Constr Co Ltd | Waste water treating agent and its preparation |
| CN108586828A (en) * | 2018-04-10 | 2018-09-28 | 南京农业大学 | A kind of Compound Edible Coating for Preservation and its preparation method and application |
| CN109111584A (en) * | 2018-06-28 | 2019-01-01 | 南京农业大学 | It is a kind of to prepare albumen/polysaccharide composite film method and products thereof and application using thermostabilization recombinant bacteria laccase |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628089A (en) * | 2019-10-11 | 2019-12-31 | 浙江理工大学 | Preparation method of poly-gallic acid/chitosan antibacterial material |
| CN115160609A (en) * | 2022-06-20 | 2022-10-11 | 湖南省农产品加工研究所 | Pullulan/bacterial cellulose/ferulic acid active composite membrane and preparation method and application thereof |
| CN115160609B (en) * | 2022-06-20 | 2024-01-26 | 湖南省农产品加工研究所 | Pullulan/bacterial cellulose/ferulic acid active composite membrane, and preparation method and application thereof |
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