Preparation method and application of antibacterial gel film based on fish scale gelatin compounded with probiotics

Abstract

The invention belongs to the technical field of biological gel films, and particularly relates to a preparation method and application of an antibacterial gel film based on fish scale gelatin compounded with probiotics. The fish scale gelatin and the probiotics are creatively compounded, and the prepared gel film has good performance; meanwhile, the alcohol-soluble protein, the trehalose and the amino acid are added, so that the fish scale gelatin can form a special space structure, pores are uniform, the structure is compact, and the physicochemical properties of the fish scale gelatin are further improved; the prepared gel film also has a good bacteriostatic effect, and can be applied to storage and preservation of perishable food; the method not only realizes waste utilization of fish scales, but also solves the problem that the prepared product is easy to be infected by microorganisms and spoilage, and achieves a remarkable technical effect. In addition, the invention has the advantages of safety, environmental protection, simple preparation method, easy popularization and application, and very wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biogel membranes, and specifically to a preparation method and application of an antibacterial gel membrane based on fish scale gelatin compounded with probiotics. Background Art

[0002] my country is the world's largest aquatic country. Fish scales are by-products of fish processing. As a deformed product of the fish dermis, the total mass accounts for about 5% of the fish body mass, of which the total protein content is 50% to 70%. It is rich in protein, iron, zinc, calcium and a variety of trace elements necessary for the human body. It not only has good nutritional value, but also can form gel by itself after hot water extraction and cooling, without adding additional chemical gelling agents, and is safe and environmentally friendly. However, as a by-product of fish processing, fish scales have not been well utilized and are often thrown away as waste, which not only leads to a waste of resources, but also the irrational use of fish scales will cause certain pollution to the environment. If the recycling of fish scale waste can be achieved, it can not only turn waste into treasure and protect the environment, but also achieve good social and economic benefits.

[0003] On the other hand, because fish scales are rich in collagen, they are widely used in food, such as preparing gel products or polypeptide products; but at the same time, the food prepared based on them is also very easy to be infected by microorganisms and deteriorate, which not only brings food safety problems, but also inevitably causes economic losses. In addition, the current preservation of food is mainly controlled by preservatives, so how to better utilize fish scales and solve food safety problems has become a current research hotspot. It has been reported in the literature that probiotics can inhibit the growth of spoilage microorganisms by producing antibacterial substances and nutrient competition, and are safe and environmentally friendly; however, probiotics are particularly sensitive to the external environment, such as temperature, ultraviolet rays, and pH value; these factors will cause a large number of probiotics to die, seriously affecting their effects, and therefore restricting the application of probiotics. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention creatively compounds fish scale gelatin with probiotics, and the prepared gel film has good performance; at the same time, the addition of alcohol-soluble protein, trehalose and amino acids is beneficial to the formation of a special spatial structure of fish scale gelatin with uniform pores and dense structure, further improving its physical and chemical properties; and the prepared gel film also has good antibacterial effect, and can be used for the storage and preservation of perishable foods; not only does it realize the utilization of waste fish scales, but it also solves the problem of the prepared product being easily infected by microorganisms and spoilage, and achieves significant technical effects.

[0005] In order to achieve the above technical objectives, the present invention adopts the following scheme:

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

[0007] Step 1:

[0008] The probiotics are inoculated into MRS liquid culture medium, cultured to a stable period, then centrifuged to collect the bacteria, and then resuspended with sterile physiological saline to obtain a probiotic suspension;

[0009] Step 2: Preparation of fish scale gelatin solution:

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

[0011] (2) adding the pretreated fish scale particles into distilled water for segmented heating treatment; wherein the segmented heating treatment is divided into three stages: the first stage is microwave treatment, with the microwave power set to 150-200 W and the treatment time being 4-6 min; the second stage is water bath treatment, with the microwave power set to 60-80° C. for 10-15 min; the third stage is microwave treatment, with the microwave power set to 350-500 W and the treatment time being 5-10 min; after the treatment, the supernatant is centrifuged to obtain fish scale gelatin solution for later use;

[0012] Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, and stir to obtain an aqueous phase; then mix the vegetable oil and Tween-80 to obtain an oil phase; finally, drop the aqueous phase into the oil phase, stir to mix evenly, and after mixing evenly, perform shear emulsification and ultrasonic treatment to obtain a probiotic nanoemulsion;

[0013] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3, stir evenly, add to a mold and place under a certain temperature to solidify to form an antibacterial gel film.

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

[0015] Preferably, in step 1, 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 step 2 (1), the concentration of the HCl solution is 0.2 mol / L; and the number of cleaning times is 3-5 times.

[0017] Preferably, in step 2 (2), the centrifugation conditions are: 4000-5000 rpm, 3-5 min.

[0018] Preferably, in step 3, 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 dosage relationship of the zein, trehalose and probiotic suspension is 10-15g:3-5g:20-40mL.

[0019] Preferably, in step 3, the volume ratio of the vegetable oil to Tween-80 is 1:1, the vegetable oil is soybean oil; the volume ratio of the water phase to the oil phase is 1:1-2;

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

[0021] Preferably, in step 4, 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°C.

[0022] Beneficial effects of the present invention:

[0023] (1) The materials used in the present invention, such as trehalose, lysine and zein, are all safe and non-toxic materials with food safety. At the same time, the probiotics used are plant lactobacillus, which has a good health care effect, can regulate immunity, inhibit pathogens, reduce serum cholesterol, prevent cardiovascular and cerebrovascular diseases, etc.; and the probiotics can also maintain the balance of intestinal flora, promote nutrient absorption, and alleviate acute and chronic diarrhea, constipation, indigestion, and abdominal distension caused by intestinal flora imbalance. In addition, the present invention 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 waste recycling.

[0024] (2) The use of the probiotics of the present invention adopts an embedding form, which is conducive to prolonging the survival rate of the probiotics; at the same time, trehalose and lysine are added to the mixture, wherein trehalose is used as a protective agent to form a unique protective film on the cell surface, effectively protecting the biological molecular structure from being destroyed, thereby further improving the survival rate of the probiotics. In addition, the lysine added by the present invention can improve the stability of the water-in-oil emulsion, and at the same time, it can also supplement the deficiency of lysine missing from zein, thereby improving the nutritional value of the gel film. However, it should be noted that if the amount of lysine is too high, it will have an adverse effect on the emulsion system, so it is generally not suitable to add too much.

[0025] (3) The present invention utilizes microwave heating combined with water bath treatment to achieve sufficient extraction of fish scale gelatin; the segmented heating treatment is divided into three stages, the first stage: microwave treatment, setting the microwave power to 150-200W, and treating for 4-6min; the second stage: water bath treatment, heating treatment in a constant temperature water bath of 60-80°C for 10-15min; the third stage: microwave treatment, setting the microwave power to 350-500W, and treating for 5-10min; the present invention performs segmented heating under specific conditions, which can more advantageously and fully extract fish scale gelatin.

[0026] (4) The fish scales used in the present invention are safe and environmentally friendly, and belong to waste recycling. In the process of composite gel formation, fish scale gelatin liquid and probiotic nanoemulsion have a synergistic effect, and the ratio of the two needs to be controlled within an appropriate range. The ratio of fish scale gelatin liquid to probiotic nanoemulsion is 1:2-4. By utilizing the adhesion and stability of fish scale gelatin, it is easier to form a gel in this range and has good gel strength. On the other hand, the combination of probiotic nanoemulsion and fish scale gelatin, supplemented by the use of zein, trehalose and lysine, can form a more ordered and dense spatial structure, which is beneficial to the improvement of the mechanical properties of the gel film, and has achieved outstanding technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The following are scanning electron microscope images of the gel membrane, wherein (A) is comparative example 6 and (B) is example 2.

[0028] Figure 2 It is the antibacterial rate of the gel film of Example 1-3 and Comparative Example 1-2.

[0029] Figure 3 It is the survival rate of the gel membrane of Examples 1-3 and Comparative Examples 5-6. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

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

[0032] Embodiment 1:

[0033] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0034] Step 2: Preparation of fish scale gelatin solution:

[0035] (1) firstly, clean the fish scales, dry them and crush them 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0036] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0037] Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, and stir evenly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the dosage relationship of zein, trehalose and probiotic suspension is 10 g:5 g:20 mL; then mix vegetable oil and Tween-80 in a volume ratio of 1:1 to obtain an oil phase;

[0038] Finally, the aqueous phase was added dropwise to the oil phase at a volume ratio of 1:2, and the mixture was stirred with a magnetic stirrer. After being mixed evenly, the mixture was subjected to shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) to obtain a probiotic nanoemulsion;

[0039] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3 at a volume ratio of 2:1, stir evenly, add into a mold and place at 4°C to form an antibacterial gel film.

[0040] Embodiment 2:

[0041] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0042] Step 2: Preparation of fish scale gelatin solution:

[0043] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0044] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0045] Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, stir evenly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the dosage relationship of zein, trehalose and probiotic suspension is 10 g:5 g:20 mL; then mix the vegetable oil and Tween-80 in a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase dropwise to the oil phase in a volume ratio of 1:2, and stir the mixture with a magnetic stirrer, mix evenly, and then obtain a probiotic nanoemulsion after shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min);

[0046] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3 at a volume ratio of 3:1, stir evenly, add to the mold and place at 4°C to form an antibacterial gel film.

[0047] Embodiment 3:

[0048] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0049] Step 2: Preparation of fish scale gelatin solution:

[0050] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0051] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

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

[0053] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3 at a volume ratio of 4:1, stir evenly, add to the mold and place at 4°C to form an antibacterial gel film.

[0054] Comparative Example 1:

[0055] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0056] (2) The pretreated fish scale particles are then added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150 W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400 W, and treating for 5 minutes; after treatment, centrifuge (5000 rpm, 5 minutes) to obtain the supernatant, which is the fish scale gelatin solution; add the solution into a mold and place it at 4°C to form a gel film.

[0057] Comparative Example 2:

[0058] Step 1: Preparation of fish scale gelatin solution:

[0059] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0060] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0061] Step 2: Zein, trehalose, lysine and MRS liquid culture medium are mixed and stirred to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the dosage relationship of zein, trehalose and MRS liquid culture medium is 10 g:5 g:20 mL; then the vegetable oil and Tween-80 are mixed in a volume ratio of 1:1 to obtain an oil phase; finally, the aqueous phase is added dropwise to the oil phase in a volume ratio of 1:2, and the mixture is stirred with a magnetic stirrer, and after mixing evenly, shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min) are performed to obtain a nanoemulsion;

[0062] Step 3: Add the fish scale gelatin solution prepared in step 2 to the nanoemulsion in step 3 at a volume ratio of 3:1, stir evenly, add to a mold and place at 4°C to form a gel film.

[0063] Comparative Example 3:

[0064] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0065] Step 2: Preparation of fish scale gelatin solution:

[0066] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0067] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0068] Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, stir evenly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the dosage relationship of zein, trehalose and probiotic suspension is 10 g:5 g:20 mL; then mix the vegetable oil and Tween-80 in a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase dropwise to the oil phase in a volume ratio of 1:2, and stir the mixture with a magnetic stirrer, mix evenly, and then obtain a probiotic nanoemulsion after shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min);

[0069] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3 at a volume ratio of 1:1, stir evenly, add to the mold and place at 4°C. A good gel cannot be formed and there is local solid-liquid mixing.

[0070] Comparative Example 4:

[0071] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0072] Step 2: Preparation of fish scale gelatin solution:

[0073] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0074] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0075] Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, stir evenly to obtain an aqueous phase, wherein the final concentration of lysine in the aqueous phase is 40 mM, and the dosage relationship of zein, trehalose and probiotic suspension is 10 g:5 g:20 mL; then mix the vegetable oil and Tween-80 in a volume ratio of 1:1 to obtain an oil phase; finally, add the aqueous phase dropwise to the oil phase in a volume ratio of 1:2, and stir the mixture with a magnetic stirrer, mix evenly, and then obtain a probiotic nanoemulsion after shear emulsification (12000 rpm, 3 min) and ultrasonic treatment (150 W, 5 min);

[0076] Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3 at a volume ratio of 5:1, stir evenly, add to the mold and place at 4°C to form an antibacterial gel film.

[0077] Comparative Example 5:

[0078] Step 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile 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 1: Inoculate Lactobacillus plantarum at a rate of 1% (1 g: 100 mL) into MRS liquid culture medium, culture at 35°C until stable period (24 h), then centrifuge, collect the cells, resuspend the cells with sterile saline to obtain a probiotic suspension, and adjust the concentration of the probiotic suspension to 10 9 cfu / mL;

[0081] Step 2: Preparation of fish scale gelatin solution:

[0082] (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles into a 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, wash them with distilled water for 3 times, and dry them naturally to obtain pretreated fish scale particles;

[0083] (2) Then the pretreated fish scale particles are added to distilled water for segmented heating treatment; the segmented heating treatment is divided into three stages: the first stage: microwave treatment, setting the microwave power to 150W, and treating for 5 minutes; the second stage: water bath treatment, heating treatment in a constant temperature water bath at 80°C for 10 minutes; the third stage: microwave treatment, setting the microwave power to 400W, and treating for 5 minutes; after treatment, centrifugation (5000rpm, 5min) is performed to obtain the supernatant, which is the fish scale gelatin solution, for standby use;

[0084] Step 3: Add the fish scale gelatin solution prepared in step 2 to the probiotic suspension in step 1 at a volume ratio of 3:1, stir evenly, add to a mold and place at 4°C to form a gel film.

[0085] 1. Texture performance determination: Gel strength: The instrument used is TA.XT.Plus food property analyzer produced by Stable Micro Systems, UK. A p / 0.5 cylindrical probe is selected for pressure test. The preparatory 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 gel strength is used as the standard for judging the gel texture performance. After measurement and calculation, the gel strength of the sample in Example 1 is 73.2 g·mm; the gel strength of the sample in Example 2 is 81.7 g·mm; the gel strength of the sample in Example 3 is 76.5 g·mm; the gel strength of the sample in Comparative Example 1 is 47.3 g·mm; the gel strength of the sample in Comparative Example 2 is 72.5 g·mm. The gel strength of the sample in Comparative Example 4 is 80.5 g·mm; and the gel strength of the sample in Comparative Example 6 is 50.1 g·mm.

[0087] The gel strength of the sample prepared by the present invention 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 invention is improved by 42.1% compared with that of comparative example 1, and by 38.7% compared with that of comparative example 6; while realizing the recycling of waste, an unexpected significant effect is achieved; and increasing the dosage of fish scale gelatin solution cannot significantly improve the gel strength, so the dosage of fish scale gelatin solution should not be too large.

[0088] 2. Test the tensile strength and elongation at break of the membrane.

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

[0090] Table 1 Tensile strength and elongation at break results

[0091]

[0092]

[0093] Note: The values ​​in the table are expressed as mean ± standard deviation; different lowercase letters indicate significant differences (P<0.05)

[0094] The results show that the tensile strength and elongation at break of the gel films prepared in Examples 1-3 are significantly improved compared to Comparative Example 1. Figure 1) It can be seen that the gel film of Comparative Example 6 is Figure A, which has a disordered structure and uneven pores; while the gel film of Example 2 (Figure B) has uniform pores and a dense structure, indicating that the addition of alcohol-soluble protein, trehalose and amino acids is more conducive to the formation of a special spatial structure of fish scale gelatin and the improvement of its physical and chemical properties. On the other hand, it can be seen from Example 3 and Comparative Example 4 that continuing to increase the volume ratio of fish scale gelatin solution to probiotic suspension has no significant effect on promoting membrane performance, so the optimal volume ratio is 3:1.

[0095] 3. Determination of antibacterial activity and survival rate of membrane:

[0096] (1) Antimicrobial activity;

[0097] The selected pathogens were Escherichia coli, Salmonella and Staphylococcus aureus; 20 mL of nutrient agar solution was added to each plate and allowed to solidify, and then the cultured pathogens (10 6 CFU / mL, 100μL) was applied to sterile culture medium; the probiotic film was cut into small discs with a diameter of 6mm and placed on the inoculated agar medium, cultured at 35℃ for 24h. The antibacterial ability of the film was evaluated by measuring the diameter of the inhibition zone of different probiotic films on the tested microorganisms, and the experiment was set up in three parallel groups.

[0098] The antibacterial efficiency was calculated according to the following formula:

[0099] Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%.

[0100] (2) Survival rate determination;

[0101] The culture medium was stored at 20 ± 2 °C and 25% relative humidity for 30 days and colony forming units were determined by using the plate count method in triplicate.

[0102] Figure 2 The antibacterial rates of the gel films of Examples 1-3 and Comparative Examples 1-2 are shown in the results. The gel films prepared in Examples 1-3 have good antibacterial effects compared to Comparative Examples 1-2. The antibacterial rates against Escherichia coli, Salmonella and Staphylococcus aureus are all above 80%; the best inhibition effect against Escherichia coli is above 85%. Fish scale gelatin itself also has certain antibacterial properties, but the effect of using it alone is not ideal; after compounding with probiotics according to the present invention, significant technical effects are achieved, indicating that the added Lactobacillus plantarum can be used as an effective antibacterial agent to enhance the antibacterial activity of the gel film.

[0103] Figure 3 is the survival rate of the gel membranes of Examples 1-3 and Comparative Examples 5-6; combined with Figure 3It can also be seen that the high antibacterial activity of the present invention is also due to the good survival rate of probiotics; after storage for 30 days, the survival rate of probiotics in the gel film prepared by the present invention can also be maintained at more than 70%; the survival period of plant lactobacillus in sterile physiological saline is relatively short, and it can be seen from comparative examples 5-6 that preserving plant lactobacillus in the form of gel film is conducive to improving its survival rate. In addition, the present invention can effectively protect probiotic cells in the form of nanoemulsion, and trehalose is used as a protective agent to form a unique protective film on the cell surface, effectively protecting the biomolecular structure from being destroyed; at the same time, the present invention does not need to dry, dry and other operations, thereby further improving the survival rate of probiotics; maintaining the effective number of probiotic cells as an antibacterial agent, thereby effectively improving the antibacterial performance of the gel film.

[0104] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics, characterized in that: The following steps are involved: Step 1: The probiotics are inoculated into MRS liquid culture medium, cultured to a stable phase, then centrifuged to collect the bacterial cells, and then resuspended with sterile physiological saline to obtain a probiotic suspension; Step 2: Preparation of fish scale gelatin solution: (1) firstly, clean the fish scales, dry them and crush them to obtain fish scale particles; add the fish scale particles to an HCl solution at a solid-liquid ratio of 1:10-15, stir for 40-90 minutes, then collect the fish scale particles by filtration, wash them with distilled water several times, and dry them naturally to obtain pretreated fish scale particles; (2) then adding the pretreated fish scale particles into distilled water and performing a stepwise heating treatment; The segmented heating treatment is divided into three stages: the first stage is microwave treatment, with the microwave power set at 150-200W and the treatment time at 4-6min; the second stage is water bath treatment, with the constant temperature water bath at 60-80℃ for 10-15min; the third stage is microwave treatment, with the microwave power set at 350-500W and the treatment time at 5-10min; after the treatment, the supernatant is centrifuged to obtain the fish scale gelatin solution for later use; Step 3: Mix zein, trehalose, lysine and the probiotic suspension obtained in step 1, and stir to obtain an aqueous phase; then mix the vegetable oil and Tween-80 to obtain an oil phase; finally, drop the aqueous phase into the oil phase, stir to mix evenly, and after mixing evenly, perform shear emulsification and ultrasonic treatment to obtain a probiotic nanoemulsion; Step 4: Add the fish scale gelatin solution prepared in step 2 to the probiotic nanoemulsion in step 3, stir evenly, add to a mold and place under a certain temperature to solidify to form an antibacterial gel film.

2. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 1, the dosage of the bacterial powder and the MRS liquid culture medium is 1-2 g: 100 mL; the culture time is 24-48 h, and the temperature is 35-36° C.

3. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 1, 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.

4. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 2 (1), the concentration of the HCl solution is 0.2 mol / L; and the number of cleaning times is 3-5 times.

5. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 2 (2), the centrifugation conditions are: 4000-5000 rpm, 3-5 min.

6. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 3, 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 dosage relationship of the zein, trehalose and probiotic suspension is 10-15g:3-5g:20-40mL.

7. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step three, the volume ratio of the vegetable oil to Tween-80 is 1:1, and the vegetable oil is soybean oil; the volume ratio of the aqueous phase to the oil phase is 1:1-2; the shear emulsification conditions are: 10000-20000rpm, time 3-5min; the ultrasonic power is 100W-150W, and the ultrasonic time is 5-10min.

8. The method for preparing an antibacterial gel film based on fish scale gelatin compounded with probiotics according to claim 1, characterized in that: In step 4, 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°C.

9. The antibacterial gel film prepared according to any one of the methods of claims 1 to 8 is used for antibacterial and fresh-keeping purposes.

10. The use according to claim 9, characterized in that The antibacterial agents include Escherichia coli, Salmonella and Staphylococcus aureus.

Images