A biological method for preparing a zein film

Abstract

The application discloses a biological preparation method of a zein film and belongs to the technical field of natural high polymer material functional modification. In the application, a reducing agent L-cysteine is used to reduce disulfide bonds in zein, so that the molecular structure is loose, and more glutamine residues are exposed. On the basis, a transglutaminase catalytic acyl transfer reaction is used to introduce epsilon-polylysine rich in primary amino groups into the protein structure, so that the protein film with high physical and mechanical properties, water-soluble stability and high antibacterial activity is prepared, and the application effect of the protein film as food preservation material is improved.

Description

TECHNICAL FIELD

[0001] The application relates to a biological preparation method of a zein film and belongs to the technical field of functional modification of natural high-molecular materials. BACKGROUND

[0002] Zein is a natural plant protein, has good biodegradability, biocompatibility and non-toxicity, can be processed into various forms of regenerated materials, and has wide application prospects in food packaging materials, tissue engineering materials and the textile field.

[0003] Zein is rich in hydrophobic amino acids, and is connected by disulfide bonds and hydrophobic bonds between molecules, so that the zein is easy to form a film. However, the film material directly formed by the zein is brittle, has low physical and mechanical properties, and is easy to swell in water, which seriously affects the actual application performance. Generally, the mechanical strength and water-soluble stability of the zein film can be improved through a chemical crosslinking method or blending with other functional polymers. However, the use of a chemical crosslinking agent has potential threats to the environment and human health, and physical mixing makes the interface compatibility between the modified material and the protein matrix poor, which affects the enhancement effect of the macro mechanical properties and stability of the film. SUMMARY

[0004] [TECHNICAL PROBLEM]

[0005] The film material formed by the zein in the prior art is brittle, has low physical and mechanical properties, and is easy to swell in water; and the film formed through a chemical crosslinking method or blending with other functional polymers has poor interface compatibility, which seriously affects the macro mechanical properties and stability of the film.

[0006] [TECHNICAL SCHEME]

[0007] In view of the defects and deficiencies in the prior art, the application provides a biological preparation method of a zein film, and the zein film prepared through the method has higher physical and mechanical properties, higher water-soluble swelling stability, antibacterial activity and regeneration performance.

[0008] The principle involved in the application is that the zein seriously lacks lysine and is rich in glutamine groups, can be catalyzed by a glutamine transaminase to have a crosslinking grafting reaction with a functional compound; the zein contains many sulfur-containing amino acids, and these amino acids can form very strong intramolecular and intermolecular disulfide bonds. Therefore, the application uses a reducing agent to open the disulfide bonds in the protein molecules, so that the molecular structure is loose, which is beneficial to the exposure of the reaction groups in the protein structure; and then the catalytic action of the glutamine transaminase is used to make the zein crosslink with a functional polymer epsilon-polylysine through an acyl transfer reaction, so that the zein grafted and modified by the polylysine is obtained.

[0009] The technical scheme adopted is:

[0010] The application provides a biological preparation method of a zein film, and the preparation method specifically comprises the following steps:

[0011] (1) reduction pretreatment of zein

[0012] The zein is dissolved in an ethanol aqueous solution to prepare a zein solution, then water and a buffer are added to adjust the pH value of the zein solution, and then L-cysteine is added to react, so that a reduction pretreated zein solution is obtained;

[0013] (2) enzyme-promoted graft modification of zein by ε-polylysine

[0014] The pH value of the reduction pretreated zein solution obtained in step (1) is adjusted to 6-7 by acetic acid, and then ε-polylysine and glutamine transaminase are added to react, the enzyme is inactivated, dialysis and freeze-drying are performed, and a graft modified zein powder is obtained;

[0015] (3) preparation of a zein film

[0016] The graft modified zein powder obtained in step (2) is weighed and dissolved in an acetic acid solution, and glycerol is added, after being fully stirred and dissolved, bubbles are removed, a film forming solution is formed, the film forming solution is poured into a mold, and drying is performed until the solvent is completely volatilized, so that a zein film is obtained.

[0017] In an embodiment of the application, the ethanol aqueous solution in step (1) is an ethanol aqueous solution with a volume fraction of 60-80%.

[0018] In an embodiment of the application, the concentration of the zein solution in step (1) is 3-5 g / 100 ml.

[0019] In an embodiment of the application, the buffer in step (1) is one or more of borax-sodium hydroxide buffer, sodium carbonate-sodium bicarbonate buffer and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0020] In an embodiment of the application, the pH value in step (1) is 9-10.

[0021] In an embodiment of the application, the addition amount of L-cysteine in step (1) is 0.1-0.2 moL / 100 mL (relative to the zein solution after the pH value is adjusted).

[0022] In an embodiment of the application, the reaction condition in step (1) is constant temperature reaction at 40-50 DEG C for 10-15 h.

[0023] In one embodiment of the present application, the ε-polylysine is added in an amount of 0.1-0.3 g / L, relative to the total amount of the zein solution adjusted with acetic acid; and the transglutaminase is added in an amount of 20-30 U / g of zein.

[0024] In one embodiment of the present application, the reaction in step (2) is carried out at 50°C for 5-10 hours.

[0025] In one embodiment of the present application, the dialysis in step (2) is carried out in deionized water for 60-72 hours.

[0026] In one embodiment of the present application, the freeze-drying in step (2) is carried out at -50 to -80°C.

[0027] In one embodiment of the present application, the acetic acid solution in step (3) is 70-80% (m / v, g / ml).

[0028] In one embodiment of the present application, the glycerol is added in an amount of 1-2% (m / v, g / ml) in step (3).

[0029] In one embodiment of the present application, after the solution is sufficiently stirred and dissolved in step (3), the bubbles are removed by ultrasonic degassing under room temperature.

[0030] In one embodiment of the present application, the mold in step (3) is a polytetrafluoroethylene mold.

[0031] Another object of the present application is to provide a zein film prepared by the method described above.

[0032] A third object of the present application is to provide the use of the zein film described above in food preservation.

[0033] A fourth object of the present application is to provide a food preservation method, which comprises immersing a food to be preserved in the zein film-forming solution described above, and then storing the food at room temperature after the film-forming solution forms a film on the surface of the food.

[0034] Advantages:

[0035] The present application utilizes the structural characteristics of high content of sulfur-containing amino acids and glutamine groups in zein, the reducing property of L-cysteine and the catalytic property of transglutaminase in catalyzing acyl transfer reaction, first reduces the disulfide bonds in zein by using reducing agent L-cysteine to loosen the molecular structure and expose more glutamine residues, and then introduces epsilon-polylysine into the protein structure by using transglutaminase to catalyze acyl transfer, so as to prepare a protein film with high physical and mechanical properties, high water-soluble stability and high antibacterial activity.

[0036] Compared with the traditional chemical modification method, the preparation method of the present application has mild reaction conditions, no environmental problems and toxic side effects caused by chemical reagents, and has strong practicability. When the modified zein obtained by the method of the present application is used for fruit preservation treatment, the shelf life is significantly improved. DETAILED DESCRIPTION

[0037] The present application will be further described by way of examples, but the present application is not limited in the scope of the examples.

[0038] The test method involved in the present application is as follows:

[0039] 1. Mechanical property test of zein film

[0040] The average thickness of the film is randomly read by using a micrometer, and then the film is cut into a size of 50mm*10mm for testing the breaking strength (Ts) and elongation at break (E) of the film. Three strips of each film are taken as parallel samples, and the running speed of the clamp is 5mm / min.

[0041] 2. Water-soluble stability test of zein film

[0042] The concentration of the protein film dissolved in water over time is determined by using ultraviolet spectrophotometry. 80mg of the protein film is weighed and placed in 40ml of deionized water and 70% ethanol solution, respectively, and is placed in a constant temperature water bath at 25℃. After 2 hours, the supernatant is taken after centrifugation, and the absorbance at 280nm is determined to calculate the degree of protein dissolution in the film. The standard curve is prepared by using bovine serum albumin as the standard protein.

[0043] 3. Antibacterial rate test of zein film

[0044] According to GB / T 20944.3-2008, the antibacterial experiment is carried out by using the shaking method, and the antibacterial circle method and colony counting method are used to evaluate the antibacterial activity of the protein film on gram-negative Escherichia coli strain (E.coli, ATCC-25922) and gram-positive Staphylococcus aureus strain (S.aureus, ATCC-6538). The antibacterial rate is calculated by using the colony counting method.

[0045] 4. Fresh-keeping effect test of zein film on fruits

[0046] Select the strawberries with similar color, uniform size and no obvious surface damage, wash them with deionized water, and then randomly divide them into four groups. Select the untreated strawberries as the control group, and the remaining three groups are immersed in zein solution. After the solution forms a film on the surface of the strawberries, store them at room temperature. Record the appearance changes of the strawberries every day.

[0047] The raw materials involved in the embodiments of the present application are as follows:

[0048] Zein, with a protein content of 99%;

[0049] Epsilon-polylysine: degree of polymerization 3000-5000;

[0050] Glutamine transaminase: enzyme activity 100 U / g;

[0051] L-cysteine: analytical pure.

[0052] Example 1

[0053] A method for preparing a zein film by a biological method, the method comprising the following steps:

[0054] (1) Reducing pretreatment of zein

[0055] Weigh a certain amount of zein powder, dissolve it in an 80% volume fraction ethanol aqueous solution, ultrasonicate for 2 min, and then magnetically stir at room temperature for 5 min to obtain a clear yellow zein solution with a concentration of 3 g / 100 mL. Then, add 15.0 mL of deionized water, 10.0 mL of borax-sodium hydroxide buffer (to adjust the pH of the zein solution to 10), and 0.2 moL / 100 mL of L-cysteine, respectively, to obtain a reaction solution. Then, place the reaction solution in a constant-temperature oven at 50°C for 15 h to obtain a reduced pretreated zein solution.

[0056] (2) Enzymatic graft modification of zein by epsilon-polylysine

[0057] Adjust the pH of the pretreated zein solution obtained in step (1) to 7 with acetic acid, and then add 0.3 g / L of epsilon-polylysine and 30 U / g of zein glutamine transaminase, respectively, and react at 40°C for 6 hours. Then, terminate the reaction by water-bathing the protein solution in boiling water for 5 min, dialyze it in deionized water for 72 hours, and freeze-dry it at -50°C to obtain a grafted and modified zein powder.

[0058] (3) Preparation of zein film

[0059] Take 0.5 g of the grafted and modified zein powder of step (2) and dissolve it in 10 ml of 80% (m / v, g / ml) acetic acid solution, add 1% (m / v, g / ml) glycerol, stir at room temperature until completely dissolved, then ultrasonic for 5 min to remove bubbles, then slowly pour into a 5x7 cm polytetrafluoroethylene mold, and place in a 40°C oven until the solvent completely evaporates, to obtain a zein film.

[0060] Example 2

[0061] A method for preparing a zein film by a biological method, the method comprising the following steps:

[0062] (1) Reductive pretreatment of zein

[0063] Take a certain amount of zein powder and dissolve it in an 80% (v / v) ethanol aqueous solution, ultrasonic for 2 min, then magnetically stir at room temperature for 5 min to obtain a clear yellow zein solution with a concentration of 3 g / 100 ml, then sequentially add 15.0 ml of deionized water, 10.0 ml of borax-sodium hydroxide buffer (to adjust the pH of the zein solution to 10), and 0.1 mol / L of L-cysteine per 100 ml, to obtain a reaction solution; then place the reaction solution in a constant temperature oven at 40°C for 10 h to obtain a reductively pretreated zein solution;

[0064] (2) Enzymatic grafting modification of zein by ε-polylysine

[0065] Adjust the pH of the pretreated zein solution obtained in step (1) to 6 with acetic acid, then add 0.1 g / L ε-polylysine and 20 U / g of zein of transglutaminase, and react at 40°C for 4 h; terminate the reaction by water bath in boiling water for 5 min, dialyze in deionized water for 72 h, and freeze-dry at -50°C to obtain grafted and modified zein powder;

[0066] (3) Preparation of a zein film

[0067] Take 0.5 g of the grafted and modified zein powder of step (2) and dissolve it in 10 ml of 80% (m / v, g / ml) acetic acid solution, add 1% (m / v, g / ml) glycerol, stir at room temperature until completely dissolved, then ultrasonic for 5 min to remove bubbles, then slowly pour into a 5x7 cm polytetrafluoroethylene mold, and place in a 40°C oven until the solvent completely evaporates, to obtain a zein film.

[0068] Comparative Example 1 Zein without any treatment

[0069] 0.5 g of zein powder was weighed into 10 ml of 80% (m / v, g / ml) acetic acid solution, 1% (m / v, g / ml) glycerol was added, and the mixture was stirred at room temperature until completely dissolved, then ultrasonic treatment was performed for 5 min to remove air bubbles, and then the mixture was slowly poured into a 5x7 cm polytetrafluoroethylene mold, and the mold was placed in a 40°C oven until the solvent completely evaporated, thereby obtaining a zein film.

[0070] Comparative Example 2 without L-cysteine reduction pretreatment

[0071] The difference between Example 1 and this example is that the reduction pretreatment of zein in step (1) is omitted, and the other parameters and conditions are the same as in Example 1.

[0072] Comparative Example 3 with sodium sulfite reduction pretreatment

[0073] The difference between Example 1 and this example is that 1% (m / v, g / ml) sodium sulfite is used instead of L-cysteine for the reduction pretreatment of zein, and the other parameters and conditions are the same as in Example 1.

[0074] Comparative Example 4

[0075] The difference between Example 1 and this example is that 0.9 g / L α-pentapolylysine is used instead of ε-polylysine for the modification of zein, and the other parameters and conditions are the same as in Example 1.

[0076] Result analysis

[0077] The zein films prepared in Examples 1-2 and Comparative Examples 1-4 were equilibrated at 20°C and 65% constant temperature and humidity, and then their physical and mechanical properties, water-soluble stability, and antibacterial rate were measured.

[0078] Strawberries with similar color, uniform size, and no obvious surface damage were selected, washed with deionized water, and then immersed in the film-forming solution of zein prepared in Examples 1-2 and Comparative Examples 1-4, respectively. After the solution formed a film on the surface of the strawberries, they were stored at room temperature, and the appearance of the strawberries was recorded every day. The results are shown in Table 1.

[0079] Table 1. Performance index measurement

[0080]

[0081] The examples provided above are not intended to limit the scope of the present application, and the described steps are not intended to limit the order of their execution. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for food preservation, characterized in that, The method involves immersing the food to be preserved in a zein film-forming solution, and then storing the food at room temperature after the film-forming solution forms a film on the food surface. The preparation of the zein film-forming solution includes the following steps: (1) Pretreatment of zein reduction Zeatin was dissolved in an aqueous ethanol solution to prepare a zeatin solution. Then, water and buffer were added to adjust the pH of the zeatin solution. L-cysteine ​​was then added to carry out the reaction. After the reaction, a zeatin solution that had been pretreated by reduction was obtained. The ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 60-80%; The concentration of the zein solution is 3-5 g / 100 ml; The pH value is 9-10; the amount of L-cysteine ​​added is 0.1-0.2 mol / 100 mL; (2) ε-Polylysine promotes grafting modification of zeinin-promoted protease The pH of the reduced pretreated zein solution obtained in step (1) was adjusted to 6-7 with acetic acid. ε-polylysine and transglutaminase were added to react, and the enzymes were inactivated, dialyzed, and freeze-dried to obtain grafted modified zein powder. The amount of ε-polylysine added is 0.1~0.3 g / L, which is relative to the total amount of zein solution after acetic acid adjustment; the amount of transglutaminase is 20~30 U / g zein. (3) Preparation of zein membrane Weigh the grafted modified zein powder obtained in step (2), dissolve it in acetic acid solution, add glycerol, stir thoroughly to dissolve, remove air bubbles, and form zein film-forming solution.

2. The method according to claim 1, characterized in that, The reaction conditions in step (1) are a constant temperature reaction at 40~50℃ for 10~15 h.

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