Preparation method for antibacterial gel film based on fish scale gelatin compounded with,probiotics and use thereof

Abstract

Provided are a preparation method for an antibacterial gel film based on fish scale gelatin compounded with probiotics, and the use thereof. Fish scale gelatin is compounded with probiotics to prepare a gel film with excellent properties. Meanwhile, prolamin, trehalose and an amino acid are supplemented to facilitate the formation of a unique spatial structure in the fish scale gelatin, with uniform pores and a compact structure, thereby further enhancing the physicochemical properties thereof. The prepared gel film exhibits a good antibacterial effect and can be used for the storage and preservation of perishable foods. Therefore, the present invention not only achieves the utilization of fish scale waste, but also solves the problems of spoilage and deterioration caused by the microbial contamination of the prepared product, thereby achieving a significant technical effect.

Description

Preparation method and application of fish scale gelatin and probiotic compound bacteriostatic gel film TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological gel film, and particularly relates to a preparation method and application of fish scale gelatin and probiotic compound bacteriostatic gel film. BACKGROUND

[0002] China is the world's largest aquatic products producer, and fish scales are by-products of fish processing. As a deformation of fish corium, the total mass of fish scales accounts for about 5% of the mass of fish, and the total protein content is 50%-70%, which contains rich protein, iron, zinc, calcium and various trace elements essential for human body. Fish scales not only have good nutritional value, but also can form gel by hot water extraction and cooling without adding additional chemical gelling agents, which is safe and environmentally friendly. However, fish scales have not been well utilized as by-products of fish processing, and are often discarded as waste, which not only leads to resource waste, but also causes environmental pollution due to unreasonable utilization of fish scales. If fish scale waste can be recycled, it not only turns waste into treasure and protects the environment, but also achieves good social and economic benefits.

[0003] On the other hand, because of the high content of collagen in fish scales, fish scales are widely used in food, such as preparation of gel products or polypeptide products. However, the food prepared from fish scales is prone to microbial infection and deterioration, which not only causes food safety problems, but also inevitably causes economic losses. In addition, the current food preservation is mainly controlled by preservatives, so how to better utilize fish scales and solve the food safety problem has become a research hotspot. Some documents report that probiotics can inhibit the growth of spoilage microorganisms by producing antibacterial substances and nutritional competition, and are safe and environmentally friendly. However, probiotics are particularly sensitive to external environment such as temperature, ultraviolet light and pH value. These factors can cause a large number of probiotics to die, which seriously affects the effect of probiotics and restricts the application of probiotics. SUMMARY

[0004] In view of the deficiencies of the prior art, the fish scale gelatin and probiotics are compounded in the present application, and the prepared gel film has good performance. The addition of prolamin, trehalose and amino acids is beneficial to the formation of a special spatial structure of fish scale gelatin, and the pore is uniform and the structure is dense, which further improves the physicochemical properties of the gel film. Moreover, the prepared gel film has good bacteriostatic effect and can be applied to the storage and preservation of perishable food. The present application not only realizes the utilization of fish scale waste, but also solves the problem of spoilage and deterioration of the prepared products due to microbial infection, and achieves remarkable technical effects.

[0005] In order to achieve the above technical purposes, the present application adopts the following scheme.

[0006] A preparation method of an antibacterial gel film based on fish scale gelatin and probiotics, the steps are as follows:

[0007] Step one:

[0008] Inoculate the probiotics into the MRS liquid medium, culture to the stable phase, then centrifuge, collect the bacterial bodies, resuspend the bacterial bodies with sterile normal saline to obtain a probiotic suspension;

[0009] Step two: preparation of fish scale gelatin solution:

[0010] (1) First, wash the fish scales, dry and crush after washing, to obtain fish scale particles; add the fish scale particles to the HCl solution according to the solid-liquid ratio of 1:10-15 (g / mL), stir for 40-90 min, then collect the fish scale particles by filtration and wash several times with distilled water, and dry naturally to obtain pretreated fish scale particles;

[0011] (2) Then add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages: the first stage is microwave treatment, set the microwave power to 150-200 W and treat for 4-6 min; the second stage is water bath treatment, heat treatment at 60-80℃ constant temperature water bath for 10-15 min; the third stage is microwave treatment, set the microwave power to 350-500 W and treat for 5-10 min; after treatment, centrifuge to obtain the supernatant, which is the fish scale gelatin solution, ready for use;

[0012] Step three: mix the zein, trehalose, lysine and the probiotic suspension obtained in step one, stir uniformly to obtain an aqueous phase; then mix the vegetable oil and Tween-80 to obtain an oil phase; finally, add the aqueous phase to the oil phase, stir and mix uniformly, then perform shear emulsification and ultrasonic treatment to obtain a probiotic nanoemulsion;

[0013] Step four: add the fish scale gelatin solution prepared in step two to the probiotic nanoemulsion in step three, stir uniformly, then add to a mold and solidify under certain temperature conditions to form an antibacterial gel film.

[0014] Preferably, in step one, the amount of bacterial powder and MRS liquid medium is 1-2 g: 100 mL; the culture time is 24-48 h, and the temperature is 35-36℃.

[0015] Preferably, in step one, the probiotics are one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brevis or Lactobacillus acidophilus; the concentration of the probiotic suspension is 10 8 -10 10 cfu / mL.

[0016] Preferably, in (1) of step two, the concentration of the HCl solution is 0.2 mol / L; and the number of cleaning times is 3-5 times.

[0017] Preferably, in (2) of step two, the centrifugation condition is 4000-5000 rpm for 3-5 min.

[0018] Preferably, in step three, the final concentration of lysine in the aqueous phase is 40-60 mM, the concentration of probiotics in the aqueous phase is not less than 10 6 cfu / mL; and the amount ratio of zein, trehalose and probiotic suspension is 10-15 g:3-5 g:20-40 mL.

[0019] Preferably, in step three, the volume ratio of the plant oil to Tween-80 is 1:1, and the plant oil is soybean oil; and the volume ratio of the aqueous phase to the oil phase is 1:1-2.

[0020] Preferably, in step three, the shearing emulsification condition is 10000-20000 rpm for 3-5 min; and the ultrasonic power is 100W-150W, and the ultrasonic time is 5-10 min.

[0021] Preferably, in step four, the volume ratio of the fish scale gelatin solution to the probiotic nanoemulsion is 2-4:1; and the certain temperature condition is 0-4℃.

[0022] The beneficial effects of the present application are:

[0023] (1) The materials used in the present application, such as trehalose, lysine and corn alcohol-soluble protein, are safe and non-toxic materials with food safety. At the same time, the probiotics used are plant lactobacillus, which has good health care effect, can regulate immunity, inhibit pathogenic bacteria, reduce serum cholesterol, prevent cardiovascular and cerebrovascular diseases, etc.; and the probiotics can also maintain the balance of intestinal flora, promote nutrient absorption, and relieve acute and chronic diarrhea, constipation, indigestion and abdominal distension caused by intestinal flora imbalance. In addition, the present application also uses fish scales as materials, and the prepared fish scale gelatin has good biocompatibility and no biological toxicity, fully utilizes the value of fish scales, and realizes the recycling of waste.

[0024] (2) The use of the probiotics of the present application adopts an embedding form, which is beneficial to prolong the survival rate of the probiotics; meanwhile, trehalose and lysine are added in the mixing, wherein the trehalose is used as a protective agent, can form a unique protective film on the cell surface, effectively protect the biological molecular structure from being destroyed, so as to further improve the survival rate of the probiotics. In addition, the lysine added in the present application can improve the stability of the oil-in-water emulsion, and also can make up for the lack of lysine in the zein, and improve the nutritional value of the gel film. However, it should be noted that if the amount of lysine is too high, it will bring adverse effects to the emulsion system, so it is generally not appropriate to add too much.

[0025] (3) The present application realizes the sufficient extraction of fish scale gelatin by using microwave heating combined with water bath treatment; the segmented heating treatment is divided into three stages, the first stage: microwave treatment, the microwave power is set to 150-200W, and the treatment time is 4-6min; the second stage: water bath treatment, constant temperature water bath heating treatment is carried out at 60-80℃ for 10-15min; the third stage: microwave treatment, the microwave power is set to 350-500W, and the treatment time is 5-10min; the present application carries out segmented heating under specific conditions, which can more favorably and more sufficiently extract fish scale gelatin.

[0026] (4) The fish scale used in the present application is safe and environmentally friendly, and belongs to waste recycling. In the process of forming the composite gel, there is a synergistic effect between the fish scale gelatin liquid and the probiotic nanoemulsion, and the ratio of the fish scale gelatin liquid and the probiotic nanoemulsion needs to be controlled within a proper range, the ratio of the fish scale gelatin liquid and the probiotic nanoemulsion is 1:2-4; the adhesion and stability of the fish scale gelatin are utilized, and it is easier to form a gel and has good gel strength in this interval; on the other hand, the probiotic nanoemulsion is combined with the fish scale gelatin, supplemented with the use of zein, trehalose and lysine, which can form a more ordered and dense spatial structure, which is beneficial to the improvement of the mechanical properties of the gel film, and outstanding technical effects are achieved. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a scanning electron microscope image of the gel film, wherein (A) is the comparative example 6, and (B) is the example 2.

[0028] Figure 2 is the antibacterial rate of the gel films of examples 1-3 and comparative examples 1-2.

[0029] Figure 3 is the survival rate of the gel films of examples 1-3 and comparative examples 5-6. DETAILED DESCRIPTION

[0030] The present application is described in detail below in conjunction with the drawings, but the present application is not limited to these embodiments. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0031] The probiotic bacteria of this embodiment is Lactobacillus plantarum, which is purchased from China General Microbiological Culture Collection Center, and the strain number is CGMCC NO. 1.568.

[0032] Example 1:

[0033] Step one: inoculate Lactobacillus plantarum into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultivate at 35°C until the stable phase (24 h), then centrifuge, collect the bacterial bodies, resuspend the bacterial bodies with sterile normal saline to obtain a probiotic bacteria suspension, and adjust the concentration of the probiotic bacteria suspension to 10 9 cfu / mL;

[0034] Step two: preparation of fish scale gelatin solution:

[0035] (1) First, wash the fish scales, dry and crush after washing to obtain fish scale particles; add the fish scale particles to a 0.2 mol / L HCl solution at a solid-liquid ratio (g / mL) of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0036] (2) Then, add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages, the first stage: microwave treatment, set the microwave power to 150 W and treat for 5 min; the second stage: water bath treatment, heat at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) to take the supernatant, which is the fish scale gelatin solution, ready for use;

[0037] Step three: mix the zein, trehalose, lysine and the probiotic bacteria suspension obtained in step one, stir uniformly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the amount relationship of zein, trehalose and probiotic bacteria suspension is 10 g:5 g:20 mL; then mix the vegetable oil and Tween-80 at a volume ratio of 1:1 to obtain an oil phase;

[0038] Finally, add the aqueous phase to the oil phase at a volume ratio of 1:2, and stir the mixture using a magnetic stirrer, mix uniformly, then perform shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic bacteria nanoemulsion;

[0039] Step four: add the fish scale gelatin solution prepared in step two to the probiotic bacteria nanoemulsion in step three at a volume ratio of 2:1, stir uniformly, then add to a mold and place at a temperature of 4°C to form an antibacterial gel film.

[0040] Example 2:

[0041] Step one: Lactobacillus plantarum was inoculated into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultured at 35°C until the stable phase (24 h), then centrifuged, the bacterial cells were collected, resuspended with sterile normal saline to obtain a probiotic bacterial suspension, and the concentration of the probiotic bacterial suspension was adjusted to 10 9 cfu / mL;

[0042] Step two: preparation of fish scale gelatin solution:

[0043] (1) First, wash the fish scales, dry and crush after washing to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution at a solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0044] (2) Then add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages, the first stage: microwave treatment, set the microwave power to 150 W and treat for 5 min; the second stage: water bath treatment, heat treatment at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) to take the supernatant, which is the fish scale gelatin solution, ready for use;

[0045] Step three: mix the zein, trehalose, lysine and probiotic bacterial suspension obtained in step one, stir uniformly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the amount relationship of zein, trehalose and probiotic bacterial suspension is 10 g: 5 g: 20 mL; then mix the vegetable oil and Tween-80 at a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase to the oil phase at a volume ratio of 1:2, and stir the mixture using a magnetic stirrer, mix uniformly, then emulsify by shearing (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic bacterial nanoemulsion;

[0046] Step four: add the fish scale gelatin solution prepared in step two to the probiotic bacterial nanoemulsion in step three at a volume ratio of 3:1, stir uniformly, then add to a mold and place at a temperature of 4°C to form an antibacterial gel film.

[0047] Example 3:

[0048] Step one: inoculate Lactobacillus plantarum into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultivate at 35°C until the stable phase (24 h), then centrifuge, collect the bacterial cells, resuspend the bacterial cells with sterile normal saline to obtain a probiotic bacterial suspension, and adjust the concentration of the probiotic bacterial suspension to 10 9 cfu / mL;

[0049] Step two: preparation of fish scale gelatin solution

[0050] (1) First, wash the fish scales, dry and crush them after washing to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution at a solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0051] (2) Then add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages, the first stage: microwave treatment, set the microwave power to 150 W and treat for 5 min; the second stage: water bath treatment, heat at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) and take the supernatant, which is the fish scale gelatin solution, for standby use;

[0052] Step three: mix the zein, trehalose, lysine and the probiotic bacterial suspension obtained in step one, stir uniformly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the amount relationship of zein, trehalose and probiotic bacterial suspension is 10 g: 5 g: 20 mL; then mix the vegetable oil and Tween-80 at a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase to the oil phase at a volume ratio of 1:2, and stir the mixture using a magnetic stirrer, mix uniformly, then perform shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic bacterial nanoemulsion;

[0053] Step four: add the fish scale gelatin solution prepared in step two to the probiotic bacterial nanoemulsion in step three at a volume ratio of 4:1, stir uniformly, then add a mold and place it at a temperature of 4°C to form an antibacterial gel film.

[0054] Comparative Example 1

[0055] (1) First, wash the fish scales, dry and crush them after washing to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution at a solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0056] (2) Then the pretreated fish scale particles were added into distilled water for stepwise heating treatment; the stepwise heating treatment was divided into three stages, the first stage: microwave treatment, the microwave power was set to 150 W, and the treatment time was 5 min; the second stage: water bath treatment, the heating treatment was carried out in a constant temperature water bath at 80℃ for 10 min; the third stage: microwave treatment, the microwave power was set to 400 W, and the treatment time was 5 min; after the treatment, centrifugation (5000 rpm, 5 min) was carried out to take the supernatant, which was the fish scale gelatin liquid; the fish scale gelatin liquid was added into a mold and placed in a temperature condition of 4℃ to form a gel film.

[0057] Comparative Example 2:

[0058] Step one: preparation of fish scale gelatin liquid:

[0059] (1) First, the fish scales were washed, dried and crushed to obtain fish scale particles; the fish scale particles were added into 0.2 mol / L HCl solution according to a solid-liquid ratio of 1:10, stirred for 50 min, then the fish scale particles were collected by filtration and washed with distilled water for 3 times, and naturally dried to obtain pretreated fish scale particles;

[0060] (2) Then the pretreated fish scale particles were added into distilled water for stepwise heating treatment; the stepwise heating treatment was divided into three stages, the first stage: microwave treatment, the microwave power was set to 150 W, and the treatment time was 5 min; the second stage: water bath treatment, the heating treatment was carried out in a constant temperature water bath at 80℃ for 10 min; the third stage: microwave treatment, the microwave power was set to 400 W, and the treatment time was 5 min; after the treatment, centrifugation (5000 rpm, 5 min) was carried out to take the supernatant, which was the fish scale gelatin liquid, for standby;

[0061] Step two: corn alcohol soluble protein, trehalose, lysine and MRS liquid medium were mixed to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase was 40 mM, and the amount of corn alcohol soluble protein, trehalose and MRS liquid medium was in the ratio of 10 g:5 g:20 mL; then plant oil and Tween-80 were mixed according to a volume ratio of 1:1 to obtain an oil phase; finally, the aqueous phase was added dropwise into the oil phase according to a volume ratio of 1:2, and a magnetic stirrer was used to stir the mixture; after mixing, the mixture was subjected to shearing emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a nanoemulsion;

[0062] Step three: the fish scale gelatin liquid prepared in step two was added into the nanoemulsion in step three according to a volume ratio of 3:1, and the mixture was stirred uniformly; then the mixture was added into a mold and placed in a temperature condition of 4℃ to form a gel film.

[0063] Comparative Example 3:

[0064] Step one: Lactobacillus plantarum was inoculated into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultured at 35°C until the stationary phase (24 h), then centrifuged, the bacterial cells were collected, resuspended with sterile normal saline, and a probiotic bacterial suspension was obtained. The concentration of the probiotic bacterial suspension was adjusted to 10 9 cfu / mL;

[0065] Step two: preparation of fish scale gelatin solution:

[0066] (1) First, wash the fish scales, dry and crush them after washing to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution at a solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0067] (2) Then, the pretreated fish scale particles are added to distilled water and subjected to stepwise heating treatment; the stepwise heating treatment is divided into three stages: the first stage: microwave treatment, set the microwave power to 150 W, and treat for 5 min; the second stage: water bath treatment, heat at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W, and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) to take the supernatant, which is the fish scale gelatin solution, ready for use;

[0068] Step three: mix the zein, trehalose, lysine and probiotic bacterial suspension obtained in step one, stir uniformly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the amount of zein, trehalose and probiotic bacterial suspension is 10 g: 5 g: 20 mL; then mix the vegetable oil and Tween-80 at a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase to the oil phase at a volume ratio of 1:2, and stir the mixture using a magnetic stirrer, mix uniformly, then emulsify by shearing (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic bacterial nanoemulsion;

[0069] Step four: add the fish scale gelatin solution prepared in step two to the probiotic bacterial nanoemulsion in step three at a volume ratio of 1:1, stir uniformly, then add to a mold and place at a temperature of 4°C, which cannot form a good gel, and there is a local solid-liquid mixing phenomenon.

[0070] Comparative Example 4:

[0071] Step one: Lactobacillus plantarum was inoculated into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultured at 35°C until the stationary phase (24 h), then centrifuged, the bacterial cells were collected, resuspended with sterile normal saline, and a probiotic bacterial suspension was obtained. The concentration of the probiotic bacterial suspension was adjusted to 109 cfu / mL;

[0072] Step two: preparation of fish scale gelatin solution:

[0073] (1) First, wash the fish scales, after washing, dry and crush to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution according to the solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0074] (2) Then add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages, the first stage: microwave treatment, set the microwave power to 150 W and treat for 5 min; the second stage: water bath treatment, heat at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) and take the supernatant, which is the fish scale gelatin solution, for standby use;

[0075] Step three: mix the zein, trehalose, lysine and probiotic suspension obtained in step one to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the amount of zein, trehalose and probiotic suspension is 10 g:5 g:20 mL; then mix the vegetable oil and Tween-80 according to the volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase to the oil phase according to the volume ratio of 1:2, and stir the mixture using a magnetic stirrer, mix uniformly, then emulsify by shearing (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic nanoemulsion;

[0076] Step four: add the fish scale gelatin solution prepared in step two to the probiotic nanoemulsion in step three according to the volume ratio of 5:1, stir uniformly, then add to a mold and place it at a temperature of 4°C to form an antibacterial gel film.

[0077] Comparative example 5:

[0078] Step one: inoculate Lactobacillus plantarum into MRS liquid medium according to the inoculation amount of 1% (1 g:100 mL), culture at 35°C until the stable phase (24 h), then centrifuge, collect the bacterial cells, resuspend the bacterial cells with sterile normal saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL.

[0079] Comparative example 6:

[0080] Step one: inoculate Lactobacillus plantarum into MRS liquid medium at an inoculation amount of 1% (1 g: 100 mL), cultivate at 35°C until the stable phase (24 h), then centrifuge, collect the bacterial cells, resuspend the bacterial cells with sterile normal saline to obtain a probiotic bacterial suspension, and adjust the concentration of the probiotic bacterial suspension to 10 9 cfu / mL;

[0081] Step two: preparation of fish scale gelatin solution:

[0082] (1) First, wash the fish scales, dry and crush them after washing to obtain fish scale particles; add the fish scale particles to 0.2 mol / L HCl solution at a solid-liquid ratio of 1:10, stir for 50 min, then collect the fish scale particles by filtration and wash them with distilled water for 3 times, and dry naturally to obtain pretreated fish scale particles;

[0083] (2) Then, add the pretreated fish scale particles to distilled water and perform stepwise heating treatment; the stepwise heating treatment is divided into three stages, the first stage: microwave treatment, set the microwave power to 150 W and treat for 5 min; the second stage: water bath treatment, heat at 80°C constant temperature water bath for 10 min; the third stage: microwave treatment, set the microwave power to 400 W and treat for 5 min; after treatment, centrifuge (5000 rpm, 5 min) to take the supernatant, which is the fish scale gelatin solution, ready for use;

[0084] Step three: add the fish scale gelatin solution prepared in step two to the probiotic bacterial suspension in step one at a volume ratio of 3:1, stir uniformly, add to a mold and place at 4°C to form a gel film.

[0085] I. Texture property determination: gel strength: the instrument used is TA.XT.Plus food texture analyzer produced by Stable Micro Systems Company in the United Kingdom, a p / 0.5 cylindrical probe is selected, a pressure test is performed, the preliminary speed is set to 1.0 mm / s; the test speed is 1.0 mm / s; the post-test speed is 2.0 mm / s; the distance is 5 mm; the trigger force is 3 g, and the breaking force (g) after compression is recorded;

[0086] The size of the gel strength is used as the standard for judging the gel texture performance, and the calculation by determination shows that the gel strength of the sample of Example 1 is 73.2 g·mm; the gel strength of the sample of Example 2 is 81.7 g·mm; the gel strength of the sample of Example 3 is 76.5 g·mm; the gel strength of the sample of Comparative Example 1 is 47.3 g·mm; the gel strength of the sample of Comparative Example 2 is 72.5 g·mm; the gel strength of the sample of Comparative Example 4 is 80.5 g·mm; and the gel strength of the sample of Comparative Example 6 is 50.1 g·mm.

[0087] The gel strength of the sample prepared by the present application can reach 81.7 g·mm, which is significantly higher than the gel strength of the product in Comparative Example 1, wherein the gel strength of Comparative Example 1 is only 47.3 g·mm, the gel strength of the present application is increased by 42.1% compared with Comparative Example 1, and the gel strength is increased by 38.7% compared with Comparative Example 6; while realizing waste recycling, unexpected significant effects are achieved; and increasing the amount of fish scale gelatin liquid cannot significantly improve the gel strength, so the amount of fish scale gelatin liquid should not be too large.

[0088] II. Test the tensile strength and elongation at break of the film.

[0089] Before testing, all films were cut into 5 cm long, 3 cm wide, 0.2 cm thick strips. During testing, the film samples were fixed at both ends of the tensile grips, the initial grip distance was 40 mm, and the stretching speed was 0.1 mm s -1 Each film sample was tested 3 times, and the results are shown in Table 1.

[0090] Table 1 Tensile Strength and Elongation at Break Results

[0091] Note: The values in the table represent the mean ± standard deviation; different lowercase letters indicate significant differences (P < 0.05)

[0092] The results show that the tensile strength and elongation at break of the gel film prepared in Examples 1-3 are significantly improved compared to Comparative Example 1. At the same time, combining the scanning electron microscope images of Example 2 and Comparative Example 6 (Figure 1), it can be seen that the gel film of Comparative Example 6 is A, which has a disordered structure and uneven pores; while the gel film of Example 2 (B) has uniform pores and a dense structure, indicating that the addition of prolamin, trehalose and amino acids is more conducive to the formation of a special spatial structure of fish scale gelatin and improves its physicochemical properties. On the other hand, combining Example 3 and Comparative Example 4, it can be seen that continuing to increase the volume ratio of fish scale gelatin liquid to probiotic suspension has no significant promoting effect on the film performance, so the optimal volume ratio is selected as 3:1.

[0093] III. Determination of antibacterial activity and survival rate of the film:

[0094] (1) Antibacterial activity;

[0095] The selected pathogenic bacteria are Escherichia coli, Salmonella and Staphylococcus aureus; 20 mL of nutrient agar solution is added to each plate and allowed to solidify, then the cultured pathogenic bacteria (10 6CFU / mL, 100 μL) was spread into sterile medium; the probiotic film was cut into small discs of 6 mm in diameter and placed on the inoculated agar medium, 35 °C, incubated for 24 h. The antibacterial ability of the film was evaluated by measuring the diameter of the inhibition zone of the tested microorganism against different probiotic films, and three parallel groups were set up.

[0096] The antibacterial efficiency was calculated according to the following formula:

[0097] The inhibition rate = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group x 100%.

[0098] (2) Survival rate determination;

[0099] Stored at 20 ± 2 °C and 25% relative humidity for 30 days, and the colony forming units were determined by using plate counting method, and three parallel groups were set up.

[0100] Figure 2 is the inhibition rate of the gel films of Examples 1-3 and Comparative Examples 1-2. The results show that the gel films prepared in Examples 1-3 have good antibacterial effect, and the inhibition rates against Escherichia coli, Salmonella and Staphylococcus aureus are all above 80%; among them, the inhibition effect against Escherichia coli is the best, reaching more than 85%. Fish scale gelatin also has certain antibacterial property, but the effect of single use is not ideal; after being compounded with probiotics according to the present application, significant technical effects are achieved, which shows that the added Lactobacillus plantarum can be used as an effective antibacterial agent to enhance the antibacterial activity of the gel film.

[0101] Figure 3 is the survival rate of the gel films of Examples 1-3 and Comparative Examples 5-6; it can also be seen from Figure 3 that the high antibacterial activity of the present application is also due to the good survival rate of the probiotics; after being stored for 30 days, the survival rate of the probiotics in the gel films prepared in the present application can still be maintained at more than 70%; the survival period of Lactobacillus plantarum in sterile normal saline is short, and it can be seen from Comparative Examples 5-6 that storing Lactobacillus plantarum in the form of gel film is beneficial to improving its survival rate. Moreover, the present application can effectively protect the probiotic cells in the form of nanoemulsion, and with trehalose as a protective agent, a unique protective film can be formed on the cell surface, effectively protecting the biological molecular structure from being destroyed; at the same time, the present application does not need to be dried, baked and the like, thereby further improving the survival rate of the probiotics; maintaining the effective number of probiotic cells as antibacterial agents, thereby effectively improving the antibacterial performance of the gel film.

[0102] Description: The above examples are only used to illustrate the technical solutions described in the present application and do not limit the present application; therefore, although the present application has been described in detail with reference to the above various embodiments, those of ordinary skill in the art should understand that the present application can still be modified or equivalently replaced; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for preparing an antibacterial gel film based on fish scale gelatin and probiotics, characterized in that, The method comprises the following steps: Step one: Inoculate the probiotic bacteria into MRS liquid medium, culture to the stable phase, then centrifuge, collect the bacteria, resuspend the bacteria with sterile normal saline to obtain a probiotic bacteria suspension; Step two: preparation of fish scale gelatin solution: (1) First, wash the fish scales, dry and crush after washing, to obtain fish scale particles; add the fish scale particles into HCl solution according to a solid-liquid ratio of 1:10-15, stir for 40-90 min, then collect the fish scale particles by filtration and wash several times with distilled water, and dry naturally to obtain pretreated fish scale particles; (2) Then add the pretreated fish scale particles into distilled water and perform stepwise heating treatment; The stepwise heating treatment comprises three stages: the first stage is microwave treatment, set the microwave power to 150-200 W and treat for 4-6 min; the second stage is water bath treatment, heat at 60-80℃ in a constant temperature water bath for 10-15 min; the third stage is microwave treatment, set the microwave power to 350-500 W and treat for 5-10 min; after treatment, centrifuge to obtain the supernatant, which is the fish scale gelatin solution and is ready for use; Step three: mix the zein, trehalose, lysine and the probiotic bacteria suspension obtained in step one to obtain an aqueous phase; then mix the vegetable oil and Tween-80 to obtain an oil phase; finally, add the aqueous phase into the oil phase, stir and mix uniformly, then perform shear emulsification and ultrasonic treatment to obtain a probiotic bacteria nanoemulsion; Step four: add the fish scale gelatin solution prepared in step two into the probiotic bacteria nanoemulsion in step three, stir uniformly, then pour into a mold and solidify under a certain temperature condition to form an antibacterial gel film.

2. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step one, the amount of the bacteria powder and the MRS liquid medium is 1-2 g:100 mL; the culture time is 24-48 h and the temperature is 35-36℃.

3. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step one, the probiotic bacteria are one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brevis, or Lactobacillus acidophilus; the concentration of the probiotic bacteria suspension is 10 8 -10 10 cfu / mL.

4. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step two (1), the concentration of the HCl solution is 0.2 mol / L; the number of washing times is 3-5 times.

5. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step two (2), the centrifugation condition is 4000-5000 rpm for 3-5 min.

6. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step three, the final concentration of lysine in the aqueous phase is 40-60 mM, and the concentration of probiotics in the aqueous phase is not less than 10 6 cfu / mL; the use amount relationship of the zein, trehalose and probiotic suspension is 10-15 g: 3-5 g: 20-40 mL.

7. The method of claim 1, wherein the fish scale gelatin and probiotics are mixed in a ratio of 1:1 to 1:

3. In step three, the volume ratio of the vegetable oil and Tween-80 is 1:1, and the vegetable oil is soybean oil; the volume ratio of the aqueous phase and the oil phase is 1:1-2; the shear emulsification condition is 10000-20000 rpm for 3-5 min; the ultrasonic power is 100 W-150 W and the ultrasonic time is 5-10 min. ​ 8. The method for preparing the bacteriostatic gel film based on fish scale gelatin and probiotics according to claim 1, characterized in that, In step four, the volume ratio of the fish scale gelatin solution and the probiotic bacteria nanoemulsion is 2-4:1; the certain temperature condition is 0-4℃.

9. The antibacterial gel film prepared by the method of any one of claims 1-8 is applied to the use of antibacterial and fresh-keeping.

10. Use according to claim 9, characterized in that, The bacteria include Escherichia coli, Salmonella and Staphylococcus aureus.

Images