CN103351471B - Whey protein film and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of food industrial biotechnology, and relates to a whey protein film and a preparation method thereof. The whey protein film is prepared by adding whey protein and glycerol into purified water to prepare film-forming liquid, heating the film-forming liquid in 45-50 DEG C water bath for 0.5-2h, cooling to room temperature, adding recombinant lipoxygenase and linoleic acid, ultrasonically degassing, pouring into a glass culture dish covered with a plastic film, reacting for 2-3h at room temperature (20-25 DEG C), drying at 50-55 DEG C for 36-48h, and uncovering the film. With the whey protein as a based material and the glycerol as a plasticizer, the whey protein film prepared by adding the recombinant anabaena lipoxygenase as a crosslinking agent is green, edible and good in tensile strength, transparency, water absorption and plasticity.

Description

A kind of lactalbumin membrane and preparation method thereof

Technical field

The invention belongs to technical field of food industry biology, relate to a kind of lactalbumin membrane and preparation method thereof.

Background technology

Plastics since generation to society bring huge simultaneously easily, it also brings great threat to environment for human survival.The packaging that this polymer chemistry polymeric material is made not only is difficult to degraded, also can produce dusty gas because of burning.Along with people are to the enhancing of environmental consciousness, natural green material is adopted to prepare the focus that edible packing film becomes many investigators.Edible packing material is with edible natural material, and if protein, polysaccharide (starch, Mierocrystalline cellulose) etc. are raw material, the structure with holey formed by the intermolecular effect of being cross-linked with each other, edible, easily to be degraded, and have certain packaging protecting function.

But edible packaging film application is at present not extensive, the use of plastics cannot be replaced, trace it to its cause, the edible film performance mainly prepared cannot be compared with chemical polymer plastic, and such as tensile strength, light transmission, absorption of water, plasticity-, the how hard product (packing box) of soft product (packing film) are less; Cost Problems is also a factor of its development of restriction in addition.Cause the reason of the problem of above-mentioned two aspects just one of the key problem of edible packing material be search out the good linking agent playing the intermolecular effect of being cross-linked with each other not yet.At present early stage in the research about linking agent, softening agent based on inorganic reagent, as ammoniacal liquor, CMC-Na, polyalcohols epoxy chloropropane, di-carboxylic acid, NaH ₂pO ₄deng.Above-mentioned result of study, although the performance that improve packaging material for food to a certain extent, still can not match in excellence or beauty with chemical material.In addition, the inorganic reagent linking agent adopted has coverd with shade also to " green ".Zymin, as a kind of pure natural biological product, has the essence that catalytic efficiency is high, itself is exactly protein, and after food-processing, sex change is nontoxic food component, and zymin is the green food additive of standard.Therefore, utilize the crosslinked action of enzyme catalysis albumen, polysaccharide, using zymin as the linking agent of edible packing material will be inexorable trend.In recent years, because LOX (Lipoxygenase, EC1.13.11.12, LOX) can the strong congregation of catalysis soybean protein, and the extensive concern of Chinese scholars is caused.

LOX belongs to oxydo-reductase, extensively be present in plant materials, containing nonheme iron in its structure, can single-minded catalysis contain suitable, the polyunsaturated fatty acid of cis-Isosorbide-5-Nitrae-pentadiene and ester, by molecule hydrogenation, form the hydroperoxidation derivative with conjugated double bond, these hydroperoxidation properties are active, are important chemical reaction intermediates.LOX has specific requirement for the substrate that it acts on, and needs containing suitable, the straight chain fatty acid of cis-Isosorbide-5-Nitrae-pentadiene, fatty acid ester and alcohol etc.Such as, LOX by with its natural substrate linolic acid effect, the Fe that the Fe of the reduction-state be in the enzyme of non-active state becomes oxidation state, thus make enzyme activition, the mechanism that the enzyme of activated state is assembled by catalysis linolic acid inducible protein mainly comprises the following aspects: one is the polymerization that intermediate product free radical causes protein molecule, namely linoleic acid peroxidation Primary product mainly hydroperoxide cracking produce lipid free radical, lipid free radical makes protein molecule become free radical as initiator by taking out hydrogen, the latter's initiated polymerization formula chain reaction, cause the polymerization of protein molecule, two is that the secondary metabolite etc. of Linoleic Acid Oxidation causes the crosslinked of protein molecule, the aldehyde compound namely in linoleic acid peroxidation secondary metabolite can with the side-chain radical such as the amino of protein molecule have an effect cause polypeptide chain chain in crosslinked and interchain linkage, three is re-assemblying of protein molecule, namely under the aldehyde product attack of lipid free radical and lipid peroxidation, participate in the protein reacted, further by the effect of hydrophobic interaction, electrostatic interaction and hydrogen bond etc., form high molecular protein aggregate.More than illustrate that restructuring ana-LOX has the potentiality preparing packing film as linking agent, and the research of this respect also there is not report at present.

Summary of the invention

What the object of the invention is for prior art is above-mentioned, provides a kind of preparation method of lactalbumin membrane.

Object of the present invention realizes by following technical scheme:

A kind of lactalbumin membrane, it is characterized in that this lactalbumin membrane be by adding whey-protein in pure water, glycerine obtains film forming liquid, film forming liquid is in 45-50 DEG C of heating in water bath 0.5-2h, adjust ph, after being cooled to room temperature, adds ana-LOX and linolic acid, after ultrasonic degas, pour into and be covered with in the glass culture dish of plastic packaging film, room temperature (20-25 DEG C) reaction 2-3h, 50-55 DEG C of dry 36-48h takes off film.

Wherein, in described film forming liquid, the mass percentage of whey-protein is 8%-12%, glycerine and whey-protein quality ratio are 1:4-1:5, restructuring ana-LOX enzyme amount is 90U/g-110U/g film forming liquid, linoleic acid con is 0.8 μm ol/g-1.2 μm ol/g film forming liquid, and film forming liquid pH is 5.5-6.5, and temperature of reaction is 20-25 DEG C, reaction times is that 2-3h, 50-55 DEG C of dry 36-48h takes off film.

Described LOX is restructuring anabena ana-LOX, and this restructuring anabena ana-LOX is prepared by the following method:

(1) with anabena genomic dna for template, the ana-LOX-R of sequence to be the ana-LOX-F of SEQ ID NO.3 and sequence be SEQ ID NO.4 is the LOX gene order of primer synthesis containing SacI and XhoI restriction enzyme site;

(2) by between the SacI/XhoI restriction enzyme site of the PCR primer of previous step purifying insertion vector pET-23a after SacI, XhoI double digestion, the expression plasmid pET-23a-ana-LOX containing ana-LOX gene is obtained;

(3) will containing ana-LOX expression plasmid pET-23a-ana-LOX transformation of E. coli expressive host bacterial strain BL21 (DE3) pLysS, picking small colonies after cultivating 10-11 hour at 37 DEG C, the 50ml LB liquid nutrient medium of access containing penbritin, 70-90rpm30 DEG C of overnight incubation, get seed liquor according to the volume ratio of 1:40 and join 100mlLB liquid nutrient medium containing penbritin, 35 DEG C of 180rpm vibrate, and 2-3 is little adds IPTG (final concentration 100 μ g/ml) induction, collected by centrifugation thalline after 5 hours when OD600 is about 0.6;

(4) thalline abduction delivering collected, with the resuspended thalline of phosphoric acid buffer, ultrasonic disruption thalline, 4 DEG C, the centrifugal 10min of 10 000 × g, collects supernatant liquor as crude enzyme liquid, carries out purifying by processing the crude enzyme liquid obtained according to Ni-NTA His Tag Kit specification sheets.

The mass percentage 10% of whey-protein in described film forming liquid, glycerine and whey protein proportions be preferred 1:4 further, restructuring ana-LOX enzyme amount preferred 100U/g film forming liquid further, linoleic acid con preferred 1 μm of ol/g film forming liquid further, film forming liquid pH further preferably 6.

The described method preparing lactalbumin membrane is optimized for further: take lactalbumin powder and be dissolved in ultrapure water and be mixed with mass percentage 10%, glycerine and whey protein proportions further preferably 1:4 slowly add in whipping process and obtain film forming liquid, pH to 6 is regulated with NaOH or HCl solution after magnetic agitation makes it fully dissolve, film forming liquid is heated 1h as in 45 DEG C of constant temperature waters, after liquid cooling but to be filmed, add restructuring ana-LOX, enzyme amount preferred 100U/g film forming liquid, linoleic acid con is 1 μm of ol/g film forming liquid preferably, ultrasonic degas process, pour into and be covered with in the glass culture dish of plastic packaging film, room temperature 20 DEG C reaction 2h, 55 DEG C of dry 36h take off film.

Beneficial effect:

The present invention take whey-protein as base-material, take glycerine as softening agent, by adding restructuring anabena lipoxygenase as the lactalbumin membrane prepared by linking agent, green edible, tensile strength, light transmission, absorption of water, plasticity-all show good character, performance is better than the control group utilizing commodity soybean lipoxygenase and do not use ferment treatment, has important using value.

Accompanying drawing explanation

Fig. 1 does not add linolic acid, does not add the whey-protein film-formation result of enzyme

Fig. 2 adds the whey-protein film-formation result of restructuring restructuring ana-LOX,

Fig. 3 adds linoleic whey-protein film-formation result

Fig. 4 adds restructuring ana-LOX, linoleic whey-protein film-formation result.

Embodiment

Embodiment 1: anabena (Anabaena sp.PCC 7120) LOX gene clone

Collected by centrifugation anabena (Anabaena sp.PCC 7120) thalline, extracts the genomic dna of anabena with the raw work genome DNA extracting reagent kit in Shanghai.

Two primers are designed according to the genome sequence (NC_003267) that NCBI announces:

Upstream primer: 5 '-GGAGTGTCTGGTGCC-3 ' (SEQ ID NO.3)

Downstream primer: 5 '-CTAAATGTTGATACTCATCAT-3 ' (SEQ ID NO.4)

In 50 μ l systems, primer final concentration is respectively 1 μM, and dNTPs final concentration is 0.2mM, anabena genomic dna 10ng, 2U Pfu archaeal dna polymerase.Amplification program is 94 DEG C of 3min; 30 × (94 DEG C of 30s, 59 DEG C of 50s, 72 DEG C of 40s); 72 DEG C of 10min.Agarose gel electrophoresis, cut glue, adopt the raw work test kit in Shanghai to reclaim, the PCR primer reclaimed is connected with TaKaRa pMD19-T vector, Transformed E .coli DH5 α, the LB be applied to containing IPTG, X-gal, penbritin is dull and stereotyped, cultivate 13-14 hour, choose white colony, shaking culture for 37 DEG C, extract plasmid, after determining successful connection, deliver to the raw work order-checking in Shanghai.Analyze sequencing result with computer software DNAMAN, obtain the ORF of a 1368bp, i.e. anabena fat oxygenase gene ana-Lox, sequence is SEQ ID NO.1, and the protein be made up of 455 amino acid of encoding, sequence is SEQ ID NO.2.

Embodiment 2: the structure of anabena (Anabaena sp.PCC 7120) ana-LOX expression vector

According to the LOX gene order obtained, design two primers, upstream primer adds SacI recognition sequence, and downstream primer adds XhoI recognition sequence (underscore part is restriction enzyme recognition sequence):

Upstream primer ana-LOX-F:5 '-CGC gAGCTCgGAGTGTCTGGTGCC-3 ' (SEQ ID NO.5)

Downstream primer ana-LOX-R:5 '-CCG cTCGAGcTAAATGTTGATACTCATCAT-3 ' (SEQ ID NO.6)

Each composition is added, amplification LOX gene according to following PCR system:

PCR program is 94 DEG C of 2min; 30 × (94 DEG C of 45s; 58 DEG C of 50s; 72 DEG C of 4min); 72 DEG C of 10min.

By Shanghai raw work PCR primer Purification Kit PCR primer, add SacI, XhoI double digestion, deactivation, alcohol settling, ddH ₂o is heavy molten, connects, transformation of E. coli DH5 α with appropriate with the carrier pET-23a that identical restriction enzyme digests.The several bacterium colony of random picking from transformation plate, access LB liquid nutrient medium, shaking culture, extracts plasmid, electrophoresis in a small amount, carries out PCR checking with the plasmid that electrophoresis is delayed for template, delivers to the raw work order-checking in Shanghai after determining successful connection.

Embodiment 3: the expression of anabena (Anabaena sp.PCC 7120) LOX gene in intestinal bacteria

Will containing ana-LOX expression plasmid pET-23a-ana-LOX transformation of E. coli expressive host bacterial strain BL21 (DE3) pLysS(purchased from Novagen company), picking small colonies after cultivating 10-11 hour at 37 DEG C, the 50ml LB liquid nutrient medium of access containing penbritin, 70-90rpm30 DEG C of overnight incubation, get seed liquor according to the volume ratio of 1:40 and join 100ml LB liquid nutrient medium containing penbritin, 35 DEG C of 180rpm vibrate, and 2-3 is little adds IPTG (final concentration 100 μ g/ml) induction when OD600 is about 0.6.Collected by centrifugation thalline after 5 hours.

Embodiment 4: the separation and purification of restructuring ana-LOX

The thalline that abduction delivering is collected, with phosphoric acid buffer (50mmol/L PBS+0.3mol/L NaCl+0.5% TritonX-100) resuspended thalline, ultrasonic disruption thalline (400W, ultrasonic 5s, interval 10s, altogether ultrasonic 15min), 4 DEG C, the centrifugal 10min of 10000 × g, collects supernatant liquor as crude enzyme liquid.The elution Ni-NTA post containing different concns imidazoles (50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L) is used successively according to Ni-NTA His Tag Kit specification sheets by processing the crude enzyme liquid obtained, collect elution peak, measure restructuring ana-LOX active.

Embodiment 5: restructuring ana-LOX vitality test

Recombinate ana-LOX activation analysis with linolic acid sodium as substrate.Contain in enzyme reaction system: the phosphoric acid buffer 2.79mL of pH6.0, enzyme liquid 10 μ L, linolic acid sodium 200 μ L, puts into 35 DEG C of water-baths and starts timing after mixing, measures absorbancy after reaction 3min.Enzyme unit definition alive: increase by 0.001 as an enzyme activity unit U using 3mL reaction system in 1min in the absorbancy of 234nm under these conditions.Enzyme work through Ni-NTA column separating purification is: 13.72U × 10 ³/ mg.

Embodiment 6: the preparation flow of whey-protein packing film

Take 10g lactalbumin powder (WPC, protein content 80%) be dissolved in the ultrapure water of 100ml, and slowly stir add be equivalent to whey-protein quality 1/5 glycerine (glycerine, analytical pure), magnetic agitation makes NaOH or the HCl solution that material fully dissolves rear 2mol/L regulate pH, film forming liquid is placed in 45 DEG C of thermostat water baths and heats 1h, after liquid to be filmed is cooled to room temperature, add restructuring ana-LOX and linolic acid, the restructuring ana-LOX enzyme amount added is 100U/g film forming liquid, linoleic acid con is 1 μm of ol/g film forming liquid, ultrasonic degas process, pour into and be covered with in the glass culture dish of plastic packaging film, 20 DEG C of reaction 2h, 55 DEG C of dry 36h take off film.

Embodiment 7: the mensuration of whey-protein film properties

The mensuration of film Oranoleptic indicator: the color of subjective appreciation film, whether the stickiness of surface flatness and film, easily take off film.

The mensuration of film thickness (mm): random on tested film (membrane removal most edge) gets five points, measures its thickness, get its mean value and record with digimatic calipers.

The mensuration of film folding line (FM): by film doubling, judges the folding line of film, with 0-4 as index according to folding line degree, 0 indicates without folding line, and 1 represents that folding line is slight, and 2 represent that film folding line is obvious, 3 represent the fracture of film doubling rear section, the part complete rupture of 4 expression film doublings.

The mensuration of transmittance: rectangle sample being cut into certain specification (0.8mm*4.5cm), is pasted into the interior shiny surface of cuvette, measures its transmittance and transmittance under the wavelength of 500nm, with empty cuvette in contrast.Each sample do 2 parallel.

Film water dissolubility measures: film sample (2cm × 2cm) is dried to constant weight under 105 DEG C of conditions, moisture content is calculated according to difference that is dry, weight in wet base after weighing. the film having measured moisture content is put into the beaker filling 30mL water, at room temperature dissolve 24h, again film is dried to constant weight under 105 DEG C of conditions, weigh, the difference according to twice dry weight calculates water-soluble.

Water vapor transmission rate (WVTR) measures: the uncovered plastic cup of circle of cut-off footpath 3cm, dark 4cm, adds the CaC1 of 3g drying ₂, by cutting into the membrane sample of diameter 7cm, cover rim of a cup, the interface paraffin between film and cup seals.Then each cup is placed in the moisture eliminator (25 DEG C, relative humidity 100%) that bottom adds the distilled water of 1000mL.Every 12h weighs 1 time, continues 1 week.The transit dose of the increasing amount determination water vapour weighed by cup.Water vapour transfer rate (WVTR) and water vapor transmission rate (WVTR) (WVP) is calculated by ASTM method (E96-93,1993).

For Measuring Mechanical Properties: film is cut into the rectangular of 4cm*2cm specification, and be fixed on physical property instrument and measure.The initial distance arranging physical property instrument two fixture is 20mm, and speed is 1mm/sec.After mensuration terminates, recording film breaks the power (g) of moment and the distance L of two fixtures during film fracture.Each sample is got three parallel sample and is measured.

Tensile strength TS (g): when breaking with film, the power of moment represents tensile strength.

Elongation at break E (%) is calculated as follows:

E＝(L1－L0)/L0

In formula: the elongation at break (%) of E-film;

The distance (mm) of two fixtures during L1-film fracture;

Distance (mm) when L0-two fixture is initial.

According to the method described above, the lactalbumin membrane performance index prepared restructuring ana-LOX, soybean lipoxygenase (preparation method is with embodiment 7 for sigma Products, 100U/g film forming liquid), not enzyme-added contrast three kinds of methods are to such as table 1:

Whey-protein film properties prepared by table 1 different treatment

Claims (1)

1. the preparation method of a lactalbumin membrane, it is characterized in that by adding whey-protein in pure water, glycerine obtains film forming liquid, film forming liquid is in 45-50 DEG C of heating in water bath 0.5-2h, after being cooled to 20-25 DEG C, add restructuring lipoxygenase and linolic acid, after ultrasonic degas, pour into and be covered with in the glass culture dish of plastic packaging film, 20-25 DEG C of reaction 2-3h, 50-55 DEG C of dry 36-48h takes off film; Described restructuring lipoxygenase is prepared by the following method:

(1) with anabena genomic dna for template, the ana-Lox-R of sequence to be the ana-Lox-F of SEQ ID NO.5 and sequence be SEQ ID NO.6 is that primer synthesis contains sacIwith xhoIthe fat oxygenase gene sequence of restriction enzyme site;

(2) by the PCR primer warp of previous step purifying sacI, xhoIinsertion vector pET-23a after double digestion sacI/ xhoIbetween restriction enzyme site, obtain the expression plasmid pET-23a-ana-Lox containing ana-Lox gene;

(3) will containing ana-Lox expression plasmid pET-23a-ana-Lox transformation of E. coli expressive host bacterial strain BL21 (DE3) pLysS, picking small colonies after cultivating 10-11 hour at 37 DEG C, the 50ml LB liquid nutrient medium of access containing penbritin, 70-90rpm 30 DEG C of overnight incubation, get seed liquor according to the volume ratio of 1:40 and join 100ml LB liquid nutrient medium containing penbritin, 35 DEG C of 180rpm 2-3 that vibrate are little of OD ₆₀₀add IPTG 100 μ g/ml when being 0.6 to induce, collected by centrifugation thalline after 5 hours;

(4) thalline abduction delivering collected, with the resuspended thalline of phosphoric acid buffer, ultrasonic disruption thalline, 4 DEG C, centrifugal 10 min of 10 000 × g, collect supernatant liquor as crude enzyme liquid, carry out purifying by processing the crude enzyme liquid obtained according to Ni-NTA His Tag Kit specification sheets.

2. the preparation method of lactalbumin membrane according to claim 1, it is characterized in that the mass percentage of whey-protein in film forming liquid is 8%-12%, glycerine and whey-protein quality ratio are 1:4-1:5, restructuring lipoxygenase addition is 90U/g-110 U/g film forming liquid, linoleic acid con is 0.8 μm ol/g-1.2 μm ol/g film forming liquid, and film forming liquid pH is 5.5-6.5.

3. the preparation method of lactalbumin membrane according to claim 2, it is characterized in that described restructuring lipoxygenase is for restructuring anabena lipoxygenase, this restructuring anabena lipoxygenase is prepared by the following method:

(1) with anabena genomic dna for template, the ana-Lox-R of sequence to be the ana-Lox-F of SEQ ID NO.5 and sequence be SEQ ID NO.6 is that primer synthesis contains sacIwith xhoIthe fat oxygenase gene sequence of restriction enzyme site;

(2) by the PCR primer warp of previous step purifying sacI, xhoIinsertion vector pET-23a after double digestion sacI/ xhoIbetween restriction enzyme site, obtain the expression plasmid pET-23a-ana-Lox containing ana-Lox gene;

(3) will containing ana-Lox expression plasmid pET-23a-ana-Lox transformation of E. coli expressive host bacterial strain BL21 (DE3) pLysS, picking small colonies after cultivating 10-11 hour at 37 DEG C, the 50ml LB liquid nutrient medium of access containing penbritin, 70-90rpm 30 DEG C of overnight incubation, get seed liquor according to the volume ratio of 1:40 and join 100ml LB liquid nutrient medium containing penbritin, 35 DEG C of 180rpm 2-3 that vibrate are little of OD ₆₀₀add IPTG 100 μ g/ml when being 0.6 to induce, collected by centrifugation thalline after 5 hours;

(4) thalline abduction delivering collected, with the resuspended thalline of phosphoric acid buffer, ultrasonic disruption thalline, 4 DEG C, centrifugal 10 min of 10 000 × g, collect supernatant liquor as crude enzyme liquid, carry out purifying by processing the crude enzyme liquid obtained according to Ni-NTA His Tag Kit specification sheets.

4. lactalbumin membrane prepared by the preparation method according to any one of claim 1 ~ 3.