CN115305653A

CN115305653A - Preparation method and application of food antibacterial nanofiber membrane

Info

Publication number: CN115305653A
Application number: CN202211060791.8A
Authority: CN
Inventors: 黄晓玮; 都柳孜; 李志华; 邹小波; 石吉勇; 杨志坤; 张俊俊; 张宁; 孙伟
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2022-08-31
Filing date: 2022-08-31
Publication date: 2022-11-08
Anticipated expiration: 2042-08-31
Also published as: CN115305653B

Abstract

The invention relates to a preparation method and application of an antibacterial food nanofiber membrane, and belongs to the technical field of food intelligent packaging membranes. The method comprises the following steps: dissolving gum arabic, polyvinyl alcohol and polyoxyethylene in deionized water to obtain a solution A; dissolving pullulan in deionized water to obtain a solution B; inoculating probiotic strains into the MRS broth culture medium, and culturing to obtain a probiotic solution; then mixing and stirring the solution A, the solution B and the probiotic solution to obtain a spinning solution; and (2) adopting an electrostatic spinning technology to carry out spinning, injecting the spinning solution into an injector, setting corresponding parameters to carry out spinning, taking the tin foil paper as a receiving base material and collecting the tin foil paper by a rotary drum to finally obtain the nanofiber membrane, namely the edible antibacterial nanofiber membrane. The method can effectively improve the effective contact area between the antibacterial component in the film and the food; the obtained material is used for food preservation and antibiosis, has obvious effect, and is safe and environment-friendly.

Description

Preparation method and application of food antibacterial nanofiber membrane

Technical Field

The invention belongs to the technical field of food intelligent packaging films, and particularly relates to a preparation method of a food antibacterial nanofiber film by using probiotic metabolites as a bacteriostatic agent.

Background

The processing, storage and transportation of food products in various countries of the world are currently in constant progress, and various food products are distributed around the world, which requires that the food products not only have a long shelf life, but also remain fresh during storage and transportation. Therefore, various bacteriostatic films are gradually emerging, and although the appearance of the bacteriostatic films can prolong the shelf life of food, the abuse of bacteriostatic agents also causes certain attack to the food industry. For example, the preservative is directly added into the food, so that a certain fresh-keeping effect is achieved, but the taste of the food is influenced by excessive use; metal ions with antibacterial function in the inorganic compound are used as antibacterial substances, but the metal ions are difficult to digest and absorb by human bodies; utilizes natural extracts such as chitosan, horseradish and the like to inhibit bacteria, but has low sterilization rate and small quantity and can not be used for broad spectrum and long effect.

The probiotics can produce a plurality of antibiotics to achieve the bacteriostatic action, and can be digested and absorbed in intestinal tracts to achieve the edible action, so that the probiotics becomes a hot point of research. However, probiotics are extremely demanding on the storage conditions, and it is a research hotspot to improve the survival rate of probiotics by embedding the probiotics in a suitable material, among the existing probiotic encapsulating materials, gao et al (Impact of encapsulation of probiotics in oil-in-water high-internal phase emulsions on the same thermal and structural stability, volume 126, may 2022, 107478) encapsulates probiotics in oil-in-water high-internal emulsion, and improves the activity of probiotics by the difference of the added pectin content; ajallouean et al (Multi-layer PLGA-pullulan-PLGA electrophoresis nanoparticles for biological delivery, food Hydrocolloids, volume 123, february 2022, 107112) encapsulate probiotics in a Multi-layer polymer matrix, which can significantly increase their encapsulation efficiency but decrease their release efficiency. Although the above-described methods reduce the attack of probiotics by harsh environments to some extent, the extreme temperatures, oxidative stress, and the use of organic solvents and multistep processes all result in significant cell death. Therefore, it is very important to select a material which has a strong bacteriostatic action and is edible to prepare the film.

Disclosure of Invention

In view of the deficiencies of the prior art, the present invention aims to solve one of the problems; provides a preparation method of an edible nano antibacterial fiber membrane. The purpose of bacteriostasis is achieved by mainly utilizing probiotics to generate a plurality of bacteriostatics, and then the edible bacteriostatic nanofiber membrane prepared by the electrostatic spinning device has large specific surface area, porosity and easy absorption, and the existence of the probiotics enables the membrane to have bacteriostatic action and be edible.

In order to achieve the above purpose, the method comprises the following steps:

a preparation method of an edible bacteriostatic nanofiber membrane is specifically implemented by an electrostatic spinning technology and comprises the following steps:

1. preparation of the spinning solution

(1) Dissolving Gum Arabic (GA), polyvinyl alcohol 1750 +/-50 (PVA) and polyethylene oxide (PEO) in deionized water, stirring to form a uniform solution, and recording as a mixed solution A;

(2) Dissolving Pullulan (PUL) in deionized water, and stirring to form a uniform solution, thus obtaining a pullulan solution which is marked as solution B;

(3) Inoculating probiotic strains into the MRS broth culture medium, and culturing for a certain time to obtain a probiotic solution;

(4) Mixing the mixed solution A obtained in the step (1), the solution B obtained in the step (2) and the probiotic solution obtained in the step (3) according to a certain proportion, and uniformly stirring to obtain a spinning solution;

2. preparation of antibacterial nanofiber membrane

And (3) spinning the spinning solution prepared in the step one by adopting an electrostatic spinning technology, firstly injecting the spinning solution into an injector, secondly setting corresponding parameters for spinning, and finally obtaining the nanofiber membrane, namely the edible antibacterial nanofiber membrane by taking tin foil paper as a receiving base material and collecting the nanofiber membrane by a rotary drum.

Preferably, the Gum Arabic (GA), the polyvinyl alcohol (PVA), the polyethylene oxide (PEO), and the deionized water are used in an amount ranging from 3.0 to 5.0g: 0.5-1.0 g:0.5 to 1.0g: 15-20 ml; the polyvinyl alcohol is specifically 1750 +/-50 of polyvinyl alcohol.

Preferably, the mass fraction of the solution B in the step (2) is 10-15%.

Preferably, the probiotic bacteria in step (3) are any one of lactobacillus acidophilus, lactobacillus casei, bifidobacterium thermophilum or lactobacillus bulgaricus.

Preferably, in step (3), the probiotic strains are inoculated into the MRS broth, and the inoculation dosage is 1-2g:100ml.

Preferably, the culture temperature of the probiotics in the step (3) is 37 ℃, and the culture time is 12h; the concentration of the probiotic solution is 10 ⁹ ～10 ¹⁰ lg(CFU/g)；

Preferably, the mixed solution a, the pullulan solution B and the probiotic solution in the step (4) are mixed according to a mass ratio of 1:3-4:1-2.5, and mixing.

Preferably, the electrostatic spinning technology in the second step has the condition parameters that the voltage applied by a high-voltage power supply is 15-20 KV, the receiving distance is 10-20 cm, the solution advancing speed is 0.1-0.4 ml/h, the spinning temperature is 25-35 ℃, and the relative humidity is 30-50%.

The invention has the advantages of

Compared with the traditional film, the edible bacteriostatic nanofiber membrane designed by the electrostatic spinning technology has larger specific surface area, so that bacteriostatic substances can be uniformly dispersed in the fiber membrane, and the effective contact area of antibacterial ingredients in the membrane and food is increased;

the addition of the Gum Arabic (GA) and the Pullulan (PUL) provides nutrient substances for the survival of probiotics to prolong the bacteriostatic time and effect of the probiotics; on the other hand, the unique thickening property, film forming property and solubility of the substances play a great role in film forming; in addition, the different weight ratios when the two are mixed are extremely important for the encapsulation of the probiotics, when the weight ratio of the two is 1:4, the solution has excellent conductivity, surface tension and viscosity; furthermore, different weight ratios will result in different nanofiber diameters when 1:4 its nanofiber diameter is more suitable for encapsulation of probiotics.

The probiotic is added, so that on one hand, the probiotic can generate lactic acid, acetic acid and various antibiotics to have a certain inhibiting effect on bacteria such as escherichia coli and staphylococcus aureus; on the other hand, the edible antibacterial film can be prepared by utilizing the properties of being capable of adjusting the balance among floras in intestinal tracts, improving the immunity of human bodies, being harmless to the human bodies and the like;

the film forming material is safe and nontoxic, meets the food safety, is used for food preservation and antibiosis, and has an obvious effect.

Drawings

FIG. 1 is a schematic view of an electrospinning apparatus used in the method of the present invention.

FIG. 2 is a scanning electron microscope image of the nanofiber membrane with antibacterial properties in the method of the present invention.

FIG. 3 shows the measurement of pH (A), total number of colonies (B) and TVB-N (C) of meat with antibacterial film prepared under different bacteria liquid ratio conditions in the method of the present invention.

FIG. 4 shows the survival rate of the antibacterial film prepared under the conditions of different ratios of bacteria liquid in the method of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, which are used in conjunction with the following embodiments.

Example 1:

a preparation method of a nanofiber membrane with antibacterial property adopts an electrostatic spinning technology and comprises the following steps;

1. preparing a spinning solution:

(1) Dissolving 3g of GA, 1g of polyvinyl alcohol 1750 +/-50 and 1g of PEO in 20ml of deionized water, stirring to form a uniform solution, and marking as a mixed solution A;

(2) Dissolving 5g PUL in 20ml deionized water, and stirring to obtain a uniform solution, namely a pullulan solution and marking as a solution B;

(3) Adding 1g of bacteria powder of the bulgaria lactobacillus into 50ml of MRS broth, culturing for 12h at 37 ℃, and then centrifuging and resuspending the bacteria liquid to 1ml to obtain a bulgaria lactobacillus solution;

(4) Uniformly mixing the solution A, the solution B and the Bulgaria lactobacillus solution to obtain a spinning solution; the mixing ratio is 1 ⁹ lg(CFU/g)；

2. Preparing a nanofiber membrane:

and (2) injecting the spinning solution into an injector by adopting an electrostatic spinning technology, wherein the corresponding condition parameters are as follows: the voltage applied by a high-voltage power supply is 16KV, the receiving distance is 10cm, the solution advancing speed is 0.4ml/h, the spinning temperature is 25 ℃, and the relative humidity is 50%; collecting with tin foil paper as receiving base material by a rotary drum to obtain nanometer fiber film, i.e. edible antibacterial film, as shown in figure 2, electron microscope structure diagram of electrostatic spinning film;

fig. 2 is a scanning electron microscope image of the nanofiber membrane prepared in example 1, from which a plurality of small balls which are scattered can be seen, and a plurality of probiotics are coated in the small balls to play a role in inhibiting bacteria.

Table 1 shows the effect of the mixed solution A and the PUL solution on the pH value, viscosity, conductivity and surface tension of the solutions under different mass ratios;

mass ratio of	pH	Conductivity	Surface tension	Viscosity at 0.1s ^-1
					0：10	4.79±0.02 ^a	0.76±0.04 ⁱ	41.22±0.65 ^a	1272.64±6.88 ^a
2：8	4.47±0.01 ^b	2.59±0.16 ^h	39.23±1.29 ^b	708.93±7.21 ^b
					3：7	4.41±0.01 ^c	4.28±0.19 ^g	35.19±2.04 ^c	526.66±6.32 ^c
4：6	4.35±0.03 ^d	5.87±0.08 ^f	34.16±2.15 ^c	386.85±8.98 ^d
					5：5	4.31±0.00 ^e	7.48±0.07 ^e	34.81±1.82 ^c	309.5±3.27 ^e
6：4	4.28±0.02 ^f	9.30±0.15 ^d	33.92±1.53 ^c	249.54±4.04 ^f
					7：3	4.25±0.03 ^fg	11.04±0.06 ^c	34.55±1.38 ^c	201.12±8.49 ^g
8：2	4.22±0.02 ^g	13.30±0.22 ^b	34.08±2.18 ^c	171.08±4.53 ^h
					10：0	4.17±0.02 ^h	17.26±0.21 ^a	34.38±0.37 ^c	206.18±6.93 ^g

During the spinning process, the higher the conductivity of the solution is, the more easily a large electrostatic repulsion force is generated under high electric field strength, so that the high tensile strain rate is caused, and the thinner fiber can be prepared; the splitting capacity of the droplets after the dope leaves the nozzle during spinning decreases with the increase of the surface tension of the dope; too low a viscosity of the solution during spinning results in string fibers, while too high a viscosity does not allow the solution to be drawn into fibers due to insufficient charge. In summary, when the mass ratio of the mixed solution a to the PUL solution is 1:4, the pH value, viscosity, conductivity and surface tension of the probiotic bacteria are in proper states and the probiotic bacteria are conveniently encapsulated.

The optimal ratio of the mixed solution A to the PUL solution can be further determined to be 1:4, the film prepared under the condition of the proportion is the optimal nanofiber film;

the nanofiber membrane is used for pork preservation experiments:

under the aseptic environment, after removing excessive fat, selecting pork with uniform appearance and good texture, uniformly dividing the pork into small blocks of about 40g, and then selecting 40g of fresh pork from the cut pork and filling the fresh pork into food-grade Polyethylene (PE) self-sealing bags to serve as blank groups. The film made of 1.0g is taken to wrap 40g of pork and put into a PE self-sealing bag to isolate the influence of the external environment. And then putting the processed pork into a refrigerator with the temperature of 4 ℃ for refrigeration, taking out every other day from 0 to 10 days, and respectively measuring the pH value, the total number of bacteria, TVB-N and other physical and chemical indexes of the pork sample.

FIG. 3 is the measurement of pH (A), total number of colonies (B) and TVB-N (C) of meat with antibacterial films prepared under different bacteria-liquid ratio conditions; wherein GA-PUL represents a fiber membrane prepared by mixing a gum arabic mixed solution and a pullulan solution in a mass ratio of 1; the weight ratio of the GA-PUL-LB (a), the GA-PUL-LB (b), the GA-PUL-LB (c) and the GA-PUL-LB (d) to the gum arabic mixed solution, the pullulan solution and the bulgaria lactobacillus solution is 1 ⁹ lg(CFU/g)。

As can be seen from FIG. 3 (A), the initial pH of pork was 5.86. + -. 0.12, after one week of refrigerator storage at 4 ℃ the pH of uncoated hymexazol was 8.25. + -. 0.21, while that of GA-PUL-LB (d) was 6.54. + -. 0.15; it can be found from FIG. 3 (B) that the initial colony count of pork was 5.55. + -. 0.14, the colony count of uncoated bacteriostatic coating was 8.52. + -. 0.16 and the colony count of GA-PUL-LB (d) bacteriostatic coating was 7.50. + -. 0.18 after storage in a refrigerator at 4 ℃ for one week; it can be seen from FIG. 3 (C) that the initial TVB-N value of pork after one week of refrigerator storage at 4 ℃ was 7.30. + -. 0.14, the TVB-N value of uncoated hymexazol was 18.70. + -. 0.11 on the fourth day, while the TVB-N value of GA-PUL-LB (d) hymexazol was 15.32. + -. 0.15 on the seventh day (it is known from national standards that when the TVB-N value of a meat product reached 15mg/100g, it is indicated that the meat had spoiled); as is apparent from FIG. 3, as the ratio of the added bacteria solution increases, the pH value is slightly lower than that of the control group; the total number of colonies is far less than that of the control group, which shows that the inhibition of the meat spoilage bacteria is improved by adding the probiotics; moreover, the results in the figures show that the shelf life of meat is significantly improved upon the addition of probiotics.

In summary, the weight ratio of the GA-PUL-LB (d) bacteriostatic coating, i.e., the gum arabic mixed solution, the pullulan solution and the lactobacillus bulgaricus solution, was 1:4:2.5 the antibacterial film prepared under the condition is more beneficial to the fresh-keeping of food.

In order to better characterize the bacteriostatic ability of the package, one characterization can be performed by testing the survival rate of the probiotics in the nanofiber membrane;

determination of probiotic survival rate in fibrous membranes:

dissolving 0.2g of nanofiber membrane in 9.8ml of 0.85% physiological saline, shaking uniformly at 37 ℃ for 1h, performing corresponding gradient dilution, coating 200 microliters of bacterial liquid on an MRS plate, culturing at 37 ℃ for 72h, and then characterizing the survival rate of the MRS plate in a plate counting mode.

FIG. 4 is a graph showing the survival rate of the antibacterial film under different ratios of bacteria liquid in the method of the present invention, wherein GA-PUL-LB refers to a mixed solution of gum arabic and pullulan containing probiotics; wherein the mass ratio of GA-PUL-LB (a), GA-PUL-LB (b), GA-PUL-LB (c) and GA-PUL-LB (d) to the gum arabic solution, the pullulan solution and the bacteria liquid is 1;

as can be seen from FIG. 4, the probiotic count of GA-PUL-LB (a) in the initial case is 8.03. + -. 0.12[ lg (CFU/g) ]; the probiotic count of GA-PUL-LB (b) is 8.54. + -. 0.13[ lg (CFU/g) ]; the probiotic count of GA-PUL-LB (c) is 9.72. + -. 0.15[ lg (CFU/g) ]; the probiotic count of GA-PUL-LB (d) is 10.15. + -. 0.17 lg (CFU/g) ];

the number of probiotics of GA-PUL-LB (a) after 28 days of storage at room temperature is 4.52. + -. 0.16[ lg (CFU/g) ]; the probiotic number of GA-PUL-LB (b) is 4.85. + -. 0.14[ CFU/g ]; the probiotic number of GA-PUL-LB (c) is 5.98. + -. 0.18[ 2 ], [ CFU/g) ]; the probiotic number of GA-PUL-LB (d) is 6.45. + -. 0.14[ CFU/g ]; the survival rate of GA-PUL-LB (a) was 56.29% as compared with the initial colony count; the survival rate of GA-PUL-LB (b) was 56.79%; the survival rate of GA-PUL-LB (c) is 61.52%; the survival rate of GA-PUL-LB (d) was 63.55%.

In conclusion, the method of electrostatic spinning for encapsulating probiotics greatly improves the survival rate of the probiotics, and is more beneficial to food preservation.

Description of the invention: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.

Claims

1. The preparation method of the food antibacterial nanofiber membrane is characterized by comprising the following steps:

1. preparing a spinning solution;

(1) Dissolving gum arabic, polyvinyl alcohol and polyoxyethylene in deionized water, and stirring to form a uniform solution, which is marked as a mixed solution A;

(2) Dissolving pullulan in deionized water, and stirring to form a uniform solution, namely obtaining a pullulan solution and marking as a solution B;

2. preparing an antibacterial nanofiber membrane;

2. The method for preparing antibacterial nanofiber membrane for food as claimed in claim 1, wherein the amount of said gum arabic, polyvinyl alcohol, polyethylene oxide and deionized water in step (1) is in the range of 3.0-5.0 g:0.5 to 1.0g: 0.5-1.0 g: 15-20 ml; the polyvinyl alcohol is specifically 1750 +/-50 of polyvinyl alcohol.

3. The preparation method of the food antibacterial nanofiber membrane as claimed in claim 1, wherein the mass fraction of the solution B in the step (2) is 10-15%.

4. The method for preparing the antibacterial food nanofiber membrane according to claim 1, wherein the probiotic bacteria in step (3) are any one of lactobacillus acidophilus, lactobacillus casei, bifidobacterium thermophilum, or lactobacillus bulgaricus.

5. The method for preparing the food antibacterial nanofiber membrane according to claim 1, wherein probiotic strains are inoculated in the MRS broth culture medium in the step (3) in an amount of 1-2g:100ml.

6. The preparation method of the food antibacterial nanofiber membrane as claimed in claim 1, wherein the culture temperature of the probiotics in the step (3) is 37 ℃, and the culture time is 12 hours; the concentration of the probiotic solution is 10 ⁹ ～10 ¹⁰ lg(CFU/g)。

7. The preparation method of the food antibacterial nanofiber membrane as claimed in claim 1, wherein the mixed solution A, the pullulan solution B and the probiotic solution in the step (4) are mixed according to a mass ratio of 1:3-4: mixing at a ratio of 1-2.5.

8. The preparation method of the food antibacterial nanofiber membrane according to claim 7, wherein in the step (4), the mass ratio of the mixed solution A to the pullulan solution B to the probiotic solution is 1.

9. The preparation method of the food antibacterial nanofiber membrane as claimed in claim 1, wherein the electrostatic spinning technology in the second step has the condition parameters that the voltage applied by a high-voltage power supply is 15-20 KV, the receiving distance is 10-20 cm, the solution advancing rate is 0.1-0.4 ml/h, the spinning temperature is 25-35 ℃, and the relative humidity is 30-50%.

10. The use of the food antibacterial nanofiber membrane prepared according to any one of claims 1-9 in food antibacterial and fresh-keeping applications.