CN115399481A - Functional edible film loaded with probiotics and preparation method thereof - Google Patents
Functional edible film loaded with probiotics and preparation method thereof Download PDFInfo
- Publication number
- CN115399481A CN115399481A CN202210859329.8A CN202210859329A CN115399481A CN 115399481 A CN115399481 A CN 115399481A CN 202210859329 A CN202210859329 A CN 202210859329A CN 115399481 A CN115399481 A CN 115399481A
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- CN
- China
- Prior art keywords
- liposome
- protamine
- probiotic
- probiotics
- lipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W90/00—Enabling technologies or technologies with a potential or indirect contribution to greenhouse gas [GHG] emissions mitigation
- Y02W90/10—Bio-packaging, e.g. packing containers made from renewable resources or bio-plastics
Abstract
The invention discloses a functional edible film carrying probiotics and a preparation method thereof. The active probiotics are wrapped by the liposome to endow the slow release function of the active probiotics. The surface of the liposome is modified by beta-cholesterol-D-glucoside (beta-CG), and the liposome can cooperate with zwitterion lipid to induce protamine to generate spherical fiber aggregates around the liposome through hydrogen bond action, wherein the diameter of the protamine fiber aggregates is 10-30um. The protamine has high heat stability, and the fiber aggregate formed by the protamine forms a stable carrier protection effect around the liposome, so that the heat stability of the probiotic active substance is improved. Can overcome the defect that the edible film can be produced only by a wet film forming method due to the heat instability of the probiotics.
Description
Technical Field
The invention relates to the field of food processing, in particular to a functional edible film loaded with probiotics and a preparation method thereof.
Background
The edible film is an edible film with specific functions, which is formed by interaction between film-forming agent molecules through a certain processing technology by using an edible biopolymer and a food additive. The prepared film can be directly applied to food as a packaging or covering material to prolong the shelf life of the food. With the improvement of the requirements of people on food quality and the enhancement of environmental awareness, the functional edible film is more and more widely applied in daily life of people. For example, a variety of active compounds such as antioxidants, antimicrobials, micronutrients, natural pigments, flavors, bioactive peptides, bacterial extracellular secondary metabolites, synbiotics, probiotics, and metazoans may be added to the food packaging film. The food preservative can not only help active ingredients to exert effects, but also prolong the shelf life of food.
The probiotics are microorganisms which can maintain the micro-ecological balance of a host organism and are beneficial to the health of the organism. The types of probiotics currently applied to human beings mainly comprise lactobacillus, bifidobacterium, enterococcus, lactococcus, streptococcus thermophilus and the like. However, the probiotic bacteria are susceptible to pH, oxygen concentration and temperature changes during production, storage and passage through the gastrointestinal tract, which may reduce their viability. In particular, edible films containing probiotics can only be produced by a low temperature wet film forming process due to the heat labile nature of the probiotic cells. It is therefore important how to maintain high activity and stability of the probiotic.
Disclosure of Invention
Aiming at the instability problems of easy inactivation and the like of probiotics, the invention provides a functional edible film carrying the probiotics and a preparation method thereof, which can improve the stability, delivery and absorption efficiency and application value of active ingredients.
The invention also provides a protamine aggregate protected probiotic liposome, which is a nano particle with good stability, high intracellular delivery efficiency and certain antibacterial capacity.
The specific technical scheme is as follows:
a functional edible film loaded with probiotics comprises the following raw materials in percentage by mass: 25-30% of alcohol soluble protein, 15-20% of algal polysaccharide, 1-3% of plasticizer, 0.1-0.5% of protamine aggregate protected probiotic liposome and the balance of solvent.
The prolamin comprises at least one of zein, gliadin, etc.;
the seaweed polysaccharide is one or more of agar, sodium alginate and chitosan;
the plasticizer is at least one of glycerol, propylene glycol, sorbitol, xylitol, mannitol, ethylene glycol, glucose, fructose and the like;
the protamine aggregate protected probiotic liposome is a spherical aggregate protected probiotic liposome formed by protamine on the surface of the liposome. Adding probiotic bacteria into a lipid film formed by lipoid, beta-cholesterol-D-glucoside and antioxidant to obtain probiotic bacteria liposome, and forming spherical aggregates on the surface of the liposome by protamine to protect the probiotic bacteria liposome.
beta-cholesterol-D-glucoside (beta-CG) modification is adopted, the synergistic zwitterionic lipid induces protamine fibers to form spherical aggregates on the surface of the liposome, and the liposome and the protamine are mainly connected through a hydrogen bond network.
The functional edible film carrying the probiotics is prepared by a tape casting method and comprises the following steps:
s1, adding prolamin into an ethanol solution, and stirring in a hot water bath until the prolamin is dissolved;
s2, adding algal polysaccharide into glacial acetic acid, and stirring in a hot water bath until the algal polysaccharide is dissolved;
s3, uniformly mixing the solution, adding a plasticizer and a protamine aggregate protected probiotic liposome, fully stirring, carrying out cross-linking reaction in a hot water bath for a period of time, and carrying out vacuum degassing and defoaming;
and S4, pouring the solution on a clean and dry polytetrafluoroethylene film forming plate, and performing heat treatment to fix the film to obtain the functional edible film loaded with the probiotics.
In the S1, the mass concentration of the prolamin-ethanol solution is 9-13%; the temperature of the hot water bath is 40-50 ℃.
In the S2, the mass concentration of the algal polysaccharide-acetic acid solution is 3% -5%; the temperature of the hot water bath is 40-50 ℃.
In the S3, the crosslinking reaction condition is 50-55 ℃, and the reaction is carried out in a water bath for 1-2h;
in the S4, the heat treatment condition is 90-130 ℃, and the treatment time is 10-20min.
The invention also provides a protamine aggregate protected probiotic liposome, wherein the probiotic is added into a lipid film formed by a lipoid, beta-cholesterol-D-glucoside and an antioxidant to obtain the probiotic liposome, and protamine forms a spherical aggregate on the surface of the liposome to protect the probiotic liposome.
Wherein: the lipoid is zwitterionic lipid, and comprises one or more of soybean lecithin, yolk lecithin, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and dimyristoyl phosphatidylcholine (DMPC). The antioxidant is any one of VE acetate and propyl gallate. The molar ratio of the lipid to the beta-CG is 3.5-4:1, the mass ratio of the lipid to the probiotics is 6-15, and the molar ratio of the lipid to the protamine is 1.3-2:1.
The particle size of the protamine aggregate protected probiotic liposome is 10-30um, and the distribution is uniform.
The preparation method of the protamine aggregate protected probiotic liposome comprises the following steps:
(1) Weighing lipoid, beta-cholesterol-D-glucoside (beta-CG) and antioxidant, dissolving in organic solvent, evaporating in rotary evaporator to form lipid film, and drying under vacuum overnight;
(2) Suspending the probiotic culture in a phosphate buffer solution to obtain a bacterial solution;
(3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing under high pressure to obtain a probiotic liposome suspension;
(4) Dissolving protamine powder in deionized water, then uniformly mixing the protamine powder and the liposome suspension, and stirring and incubating for a period of time to prepare the protamine aggregate protected probiotic liposome emulsion.
The lipoid in the step (1) is zwitterionic lipid, and comprises one or more of soybean lecithin, egg yolk lecithin, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dimyristoyl phosphatidylcholine (DMPC). The solvent preferably comprises at least one of chloroform, methanol and ethanol; the antioxidant is any one of VE acetate and propyl gallate; the addition amount of the antioxidant is 0.5 to 1 percent of the mass of the liposome; the molar ratio of lipid to beta-CG is 3.5-4:1.
Preferably, in the step (1), the temperature of the reduced pressure rotary evaporation is 40-50 ℃, the time of the reduced pressure rotary evaporation is 30-60 min, and the rotating speed of the reduced pressure rotary evaporation is 50-150 rpm.
Preferably, the concentration of the probiotics in the phosphate buffer solution in the step (2) is 0.01-0.03 mg/mL; the probiotic bacteria are selected from one or more strains of Lactobacillus, bifidobacterium, enterococcus, escherichia, bacillus, clostridium butyricum and Saccharomyces;
the phosphate buffer pH was 7.5.
In the step (3), the mass ratio of the lipoid to the probiotics is 6-15; the parameter conditions of the high-pressure homogenization treatment are as follows: homogenizing for 1-10 times under the pressure of 300-800 bar.
The condition of stirring incubation in the step (4) is incubation for 16-24h at room temperature.
The concentration of the protamine aqueous solution in the step (4) is 0.2-0.5 mg/ml, and the molar ratio of the lipoid to the protamine is 1.3-2:1.
The prepared protamine aggregates generated around the probiotic liposome have uniform particle size distribution, only 10-30um, which is far smaller than other protein aggregate microcapsules (1-10 mm).
In the invention, protamine is an alkaline protein, has high nutrition and functionality, can reduce blood pressure, promote digestion and the like, has strong bacteriostatic ability and high thermal stability, still has activity after being heated for 1 hour at 210 ℃, and by considering the heat-resistant stability of the protamine, the heat-resistant performance and the survival rate of the prepared probiotic liposome are greatly improved after being protected by protamine fiber aggregates, and can meet the requirements of various film-forming processing methods.
Advantageous effects
The invention integrates the probiotic liposome in the edible film, and combines the preparation process of the liposome and the preparation process of the edible film after improving the heat resistance of the probiotic liposome, thereby completely meeting the requirements of various film-forming processing methods. The prepared edible film has good antibacterial and preservative effects, and can be conveniently clamped in hamburgers, biscuits, cakes and the like, so that the edible film is greatly convenient for matching probiotics with other foods to eat.
The invention provides a liposome which is modified by beta-cholesterol-D-glucoside (beta-CG) and can cooperate with zwitter-ion lipid to induce the formation of protamine spherical fiber aggregates, and at the moment, the liposome and the protamine are mainly under the action of a hydrogen bond network. After the probiotic is encapsulated by the method, the survival rate and the heat stability of the live bacteria are obviously improved.
Drawings
FIG. 1 is a schematic diagram of the mechanism of protamine forming spherical fiber aggregates on the surface of probiotic liposome;
fig. 2 is a total internal reflection fluorescence microscopy image of protamine aggregate protected probiotic liposomes;
fig. 3 is a graph of the particle size distribution of the probiotic liposomes of example 1.
FIG. 4 shows a comparison of the number of active bacteria.
Fig. 5 is a comparison of probiotic survival rates.
Detailed Description
Example 1
The functional edible film material loaded with bifidobacterium of the embodiment comprises the following components in percentage by mass: 25% of zein, 15% of chitosan, 1% of glycerol, 0.1% of bifidobacterium liposome protected by protamine aggregates and the balance of solvent.
The functional edible film loaded with the bifidobacteria is prepared by a tape casting method and comprises the following steps:
step 2, adding 6g of chitosan into 114g of glacial acetic acid, stirring in a hot water bath at 50 ℃ until the chitosan is dissolved, and preparing a zein-ethanol solution with the mass concentration of 5%;
step 3, uniformly mixing the zein-ethanol solution and the chitosan-glacial acetic acid solution, adding 0.4g of glycerol and 0.04g of bifidobacterium liposome protected by protamine aggregates, fully stirring at 300r/min, carrying out crosslinking reaction in a hot water bath at 55 ℃ for 1h, and carrying out vacuum degassing and defoaming;
and 4, pouring the solution on a clean and dry polytetrafluoroethylene film-forming plate, and performing heat treatment at 120 ℃ for 10min to fix the film to prepare the functional edible film loaded with the bifidobacterium.
The synthesis method of the bifidobacterium liposome protected by the protamine aggregate comprises the following steps:
(1) Weighing 15.16mg of soybean lecithin, 13.56mg of DMPC, 3.87mg of beta-CG and 0.36mg of VE acetate, dissolving in chloroform, evaporating at 150rpm for 30min in a 40 ℃ water bath rotary evaporator to remove the solvent, forming a lipid film, and then drying under vacuum overnight;
(2) Suspending 3.59mg of Bifidobacterium culture in 140mL of phosphate buffer solution with pH of 7.5 to obtain 0.026mg/mL Bifidobacterium solution;
(3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing at 800bar under high pressure for 5 times to obtain a bifidobacterium liposome suspension;
(4) Dissolving 6.62mg of protamine powder in 20mL of deionized water to prepare a mixed solution with the concentration of 0.33mg/mL, uniformly mixing the mixed solution with the liposome suspension, and stirring and incubating for 20 hours to prepare the bifidobacterium liposome emulsion with the concentration of 0.27mg/mL of protamine aggregate protection.
Example 2
The functional edible film material loaded with escherichia coli comprises the following raw materials in percentage by mass: 28% of zein, 17% of chitosan, 2% of mannitol, 0.4% of escherichia coli liposome protected by protamine aggregates and the balance of solvent.
The functional edible film loaded with the escherichia coli is prepared by a tape casting method and comprises the following steps:
step 2, adding 1.27g of chitosan into 24.13g of glacial acetic acid, stirring in a hot water bath at 50 ℃ until the chitosan is dissolved, and preparing a chitosan-glacial acetic acid solution with the mass concentration of 5%;
step 3, uniformly mixing the solution, adding 0.15g of mannitol and 0.03g of protamine aggregate protected escherichia coli liposome, fully stirring at 300r/min, carrying out crosslinking reaction in a hot water bath at 50 ℃ for 1.5h, and carrying out vacuum degassing and defoaming;
and 4, pouring the solution on a clean and dry polytetrafluoroethylene film-forming plate, and performing heat treatment at 100 ℃ for 20min to fix the film to prepare the functional edible film loaded with the bifidobacterium.
The liposome is an escherichia coli liposome protected by protamine aggregates, and the synthesis method comprises the following steps:
(1) Weighing yolk lecithin 4.71mg, POPC 19mg, beta-CG 3.87mg and VE acetate 0.15mg, dissolving in chloroform, evaporating the solvent in a water bath rotary evaporator at 50 deg.C and 100rpm for 40min to form lipid film, and drying under vacuum overnight;
(2) Suspending 2.37mg of E.coli culture in 100mL of phosphate buffer pH 7.5 to give a Bifidobacterium solution at a concentration of 0.024 mg/mL;
(3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing at 800bar under high pressure for 5 times to obtain an escherichia coli liposome suspension;
(4) 8.28mg of protamine powder is dissolved in 20mL of deionized water to prepare a mixed solution with the concentration of 0.41mg/mL, and then the mixed solution is uniformly mixed with the liposome suspension, and stirred and incubated for 24 hours to prepare the escherichia coli liposome emulsion with the concentration of 0.32mg/mL protamine aggregate protection.
Comparative example 1
The difference from example 1 is that: the bifidobacterium liposome is not added with beta-CG.
The specific synthesis method of the bifidobacterium liposome comprises the following steps:
(1) 15.16mg of soybean lecithin, 13.56mg of DMPC and 0.36mg of VE acetate were weighed out and dissolved in chloroform, after which the solvent was removed by evaporation in a 40 ℃ water bath rotary evaporator at 150rpm for 30min to form a lipid film, which was subsequently dried under vacuum overnight.
(2) Suspending 3.59mg of Bifidobacterium culture in 140mL of phosphate buffer solution with pH of 7.5 to obtain 0.026mg/mL Bifidobacterium solution;
(3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing at 800bar under high pressure for 5 times to obtain a bifidobacterium liposome suspension;
(4) Dissolving 6.62mg protamine powder in 20mL deionized water to prepare a mixed solution with the concentration of 0.33mg/mL, then uniformly mixing the mixed solution with the liposome suspension, and stirring and incubating for 20h to prepare the bifidobacterium liposome emulsion with the concentration of 0.27 mg/mL.
Comparative example 2
The difference from example 1 is that: the bifidobacterium liposome is not protected by protamine aggregates.
The specific synthesis method of the bifidobacterium liposome comprises the following steps:
(1) 15.16mg of soybean lecithin, 13.56mg of DMPC, 3.87mg of beta-CG and 0.36mg of VE acetate were weighed out and dissolved in chloroform, and then the solvent was removed by evaporation in a 40 ℃ water bath rotary evaporator at 150rpm for 30min to form a lipid film, followed by drying under vacuum overnight.
(2) Suspending 3.59mg of Bifidobacterium culture in 140mL of phosphate buffer solution with pH of 7.5 to obtain 0.026mg/mL Bifidobacterium solution;
(3) Adding the solution in the step (2) into the lipid film in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing for 5 times under high pressure of 800 bar.
The rest is the same. Preparing bifidobacterium liposome emulsion.
Comparative example 3
The difference from example 1 is that: the bifidobacterium liposome is not protected by protamine aggregates, and the film-fixing heat treatment temperature is 50 ℃.
The functional edible film material loaded with bifidobacterium comprises the following components in percentage by mass: 25% of zein, 15% of chitosan, 1% of glycerol, 0.1% of bifidobacterium liposome and the balance of solvent.
The functional edible film loaded with the bifidobacteria is prepared by a tape casting method and comprises the following steps:
step 2, adding 6g of chitosan into 114g of glacial acetic acid, stirring in a hot water bath at 50 ℃ until the chitosan is dissolved, and preparing a zein-ethanol solution with the mass concentration of 5%;
step 3, uniformly mixing the zein-ethanol solution and the chitosan-glacial acetic acid solution, adding 0.4g of glycerol and 0.04g of bifidobacterium liposome protected by protamine aggregates, fully stirring at 300r/min, carrying out crosslinking reaction in a hot water bath at 55 ℃ for 1h, and carrying out vacuum degassing and defoaming;
and 4, pouring the solution on a clean and dry polytetrafluoroethylene film-forming plate, and performing heat treatment at 50 ℃ for 10min to fix the film to prepare the functional edible film loaded with the bifidobacterium.
The invention provides a bifidobacterium liposome, and the synthesis method comprises the following steps:
(1) 15.16mg of soybean lecithin, 13.56mg of DMPC, 3.87mg of beta-CG and 0.36mg of VE acetate were weighed out and dissolved in chloroform, and then the solvent was removed by evaporation in a 40 ℃ water bath rotary evaporator at 150rpm for 30min to form a lipid film, followed by drying under vacuum overnight.
(2) Suspending 3.59mg of Bifidobacterium culture in 140mL of phosphate buffer solution with pH of 7.5 to obtain 0.026mg/mL Bifidobacterium solution;
(3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing at 800bar under high pressure for 5 times to obtain the bifidobacterium liposome emulsion.
Test procedure
1. Total internal reflection fluorescence microscopy Test (TIRFM)
To observe the behavior of protamine fibers forming spherical aggregates on the surface of liposomes, the sample emulsion prepared in example 1 was mixed with an equal amount of 5uM thioflavin T phosphate buffer solution, which specifically binds to the fiber aggregates, and 14uL of the mixed sample was aspirated and used with a total internal reflection fluorescence microscope to observe the fluorescence image of the aggregates
As a result, as shown in FIG. 2, it was observed that linear fibers were stained with thioflavin T and then exhibited a fluorescent characteristic, and protamine fibers were aggregated around liposomes to form spherical particles having a size of about 20 μm.
2. Particle size and zeta potential testing
The probiotic liposome emulsions of example 1, example 2 and comparative example 1 were diluted to 10 times with deionized water and measured with a nano-particle size analyzer and a zeta potential analyzer.
The measured average particle size and zeta potential of the probiotic liposome are shown in the following table 1, the average particle size of the probiotic liposome prepared in the examples and the comparative examples is about 100-130nm, the particle size distribution curve of the probiotic liposome prepared in the example 1 is shown in fig. 3, and the absolute value of the zeta potential of the examples is more than 20mV, which is obviously higher than that of the comparative example 1. This shows that the addition of β -CG can suitably increase the absolute value of zeta potential on the liposome surface, thereby further avoiding aggregation between liposomes.
TABLE 1
2. Probiotic viability test
Samples of the functionalized edible films prepared in example 1, comparative example 1 and comparative example 2 were subjected to ultrasonic demulsification with a methanol solvent, and then microbial cells were released, and then were subjected to anaerobic culture at 39 ℃ for 48 to 72 hours by a viable count plate method, and the number of colonies on the plate was counted.
As shown in FIG. 4, the viable count of the probiotic bacteria in example 1 is 6.4X 10 8 cfu/mL, whereas the number of viable bacteria in the control 1 without β -CG added to the liposomes and in the control 2 without protamine aggregates added to the probiotic liposomes was essentially absent. The method proves that after the probiotics liposome is protected by the protamine aggregate, the damage effect of high-temperature film-fixing heat treatment on the probiotics can be avoided. In contrast, in comparative examples 1 and 2, since β -CG and protamine were not added, protamine aggregates could not be formed on the surface of the probiotic liposome, and the activity of the probiotic was greatly affected by the heat treatment of high-temperature membrane fixation.
3. Simulated gastric fluid digestion test
The prepared functional edible film carrying probiotics is subjected to in vitro simulation. The detection method of the viable count of the probiotics comprises the following steps: the national standard GB/T4789.35-2016 food safety national standard food microbiology detection lactic acid bacteria is adopted.
Simulated gastric fluid preparation: to 1L of deionized water were added 2g of sodium chloride and 0.26g of pepsin, and the pH of the solution was adjusted to 1-2 with 1M hydrochloric acid to prepare a simulated gastric fluid. 1g of the sample to be tested prepared in the example 1 and the comparative example 3 is added into simulated gastric fluid, and meanwhile, a probiotic edible membrane without being wrapped by liposome is used as a blank sample control. Oscillating and incubating in a water bath shaker at 37 ℃ and 180rpm at constant temperature, taking out 1mL of simulated gastric juice at 60min, 120min and 180min respectively, and measuring the survival rate of the probiotics by using a plate colony counting method after gradient dilution.
As shown in fig. 5, after the probiotics are embedded in the liposome and protected by protamine fiber aggregation, the survival rate of the probiotics in the gastric environment is greatly improved. At the end of gastric digestion (180 min), the probiotic of example 1 showed a survival rate of 96%, the liposomal probiotic of control 3, unprotected by protamine fiber aggregation, showed a survival rate of 32%, and the probiotic, unprotected by both liposome encapsulation and protamine fiber aggregation, showed a survival rate of only 5%. This indicates that, after the liposome is used to encapsulate the probiotics, the survival rate of the probiotics in the gastric juice environment can be improved, but the survival rate of the probiotics in the probiotics liposome protected by protamine fiber aggregation prepared by the invention is further greatly improved, the sample prepared in example 1 is subjected to high-temperature heat treatment and has no damage to the probiotics, and the protamine fiber aggregation provides a certain protection effect on the probiotics liposome.
Claims (10)
1. A protamine aggregate protected probiotic liposome is characterized in that probiotics are added into a lipid film formed by lipoid, beta-cholesterol-D-glucoside and an antioxidant to obtain the probiotic liposome, and protamine forms spherical aggregate protected probiotic liposome on the surface of the liposome.
2. A protamine aggregate protected probiotic liposome according to claim 1, characterized in that said lipid is a zwitterionic lipid; the antioxidant is any one of VE acetate and propyl gallate.
3. The protamine aggregate protected probiotic liposome of claim 1, wherein the amount of antioxidant is 0.5% -1% of the liposome mass; the molar ratio of the lipid to the beta-CG is 3.5-4:1, the mass ratio of the lipid to the probiotics is 6-15, and the molar ratio of the lipid to the protamine is 1.3-2:1.
4. The method for preparing protamine aggregate protected probiotic liposomes according to claim 1, comprising the steps of:
1) Weighing lipoid, beta-cholesterol-D-glucoside and antioxidant, dissolving in organic solvent, evaporating to remove solvent to form lipid film, and vacuum drying overnight;
2) Suspending the probiotic culture in a phosphate buffer solution to obtain a bacterial solution;
3) Adding the solution obtained in the step (2) into the lipid film obtained in the step (1), hydrating and vortexing to form a liposome suspension, and homogenizing under high pressure to obtain a probiotic liposome suspension;
4) And (3) dissolving protamine powder in deionized water, then uniformly mixing the protamine powder with the probiotic liposome suspension obtained in the step (3), stirring and incubating, and preparing the protamine aggregate protected probiotic liposome.
5. The method for preparing protamine aggregate protected probiotic liposomes according to claim 1, wherein the conditions of the parameters of the high pressure homogenization treatment in step 3) are: homogenizing for 1-10 times under the pressure of 300-800 bar.
6. A functional edible film loaded with probiotics is characterized by comprising the following raw materials in percentage by mass: 25-30% of alcohol soluble protein, 15-20% of algal polysaccharide, 1-3% of plasticizer, 0.1-0.5% of protamine aggregate protected probiotic liposome and the balance of solvent.
7. The probiotic-loaded, functionalized edible film according to claim 6, wherein the prolamin is at least one of zein and wheat gliadin; the algal polysaccharide is at least one of agar, sodium alginate and chitosan.
8. The method of claim 6, wherein the method comprises the steps of:
s1, adding prolamin into an ethanol solution, and stirring in a hot water bath until the prolamin is dissolved;
s2, adding algal polysaccharide into glacial acetic acid, and stirring in a hot water bath until the algal polysaccharide is dissolved;
s3, uniformly mixing the solution, adding a plasticizer and a protamine aggregate protected probiotic liposome, fully stirring, carrying out cross-linking reaction in a hot water bath for a period of time, and carrying out vacuum degassing and defoaming;
and S4, pouring the solution on a clean and dry polytetrafluoroethylene film forming plate, and performing heat treatment to fix the film to obtain the functional edible film loaded with the probiotics.
9. The method of claim 6, wherein the prolamin-ethanol solution in S1 has a mass concentration of 9% to 13%; the mass concentration of the algal polysaccharide-acetic acid solution in the S2 is 3% -5%; the temperature of the hot water bath is 40-50 ℃; in the S3, the crosslinking reaction condition is 50-55 ℃, and the reaction is carried out in a water bath for 1-2h; in the S4, the heat treatment condition is 90-130 ℃, and the treatment time is 10-20min.
10. Use of protamine aggregate protected probiotic liposomes in the preparation of an edible film.
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