阅读说明：本技术 一种乳酸链球菌素微胶囊及其制备方法 (Nisin microcapsule and preparation method thereof ) 是由 蒋悦 吴昕奕 董孝容 马灵 李惠 刘耀文 于 2021-08-19 设计创作，主要内容包括：本发明公开了一种乳酸链球菌素微胶囊及其制备方法,所述乳酸链球菌素微胶囊包括内水相层、油相层、外水相层三层,由油相层和外水相层组成的壁材包埋作为芯材的内水相层形成微胶囊,所述内水相层为乳酸链球菌素水溶液,所述油相层由司盘80和大豆油组成,所述外水相层由吐温80和明胶组成,本发明还包括了制备上述乳酸链球菌素微胶囊的方法。该乳酸链球菌素微胶囊无异味、稳定性强、抗菌时间长、不易失效、制备方法简单且成本低。(The invention discloses a nisin microcapsule and a preparation method thereof, wherein the nisin microcapsule comprises three layers, namely an inner aqueous phase layer, an oil phase layer and an outer aqueous phase layer, wherein a microcapsule is formed by embedding an inner aqueous phase layer serving as a core material in a wall material consisting of the oil phase layer and the outer aqueous phase layer, the inner aqueous phase layer is nisin aqueous solution, the oil phase layer consists of span 80 and soybean oil, and the outer aqueous phase layer consists of tween 80 and gelatin. The nisin microcapsule has no peculiar smell, strong stability, long antibacterial time, difficult invalidation, simple preparation method and low cost.)

1. A nisin microcapsule, characterized in that: comprises three layers of an inner water phase layer, an oil phase layer and an outer water phase layer; the outer water phase layer is the outermost layer, the inner water phase layer is the innermost layer, and the oil phase layer is the middle layer.

2. Nisin microcapsules according to claim 1, characterized in that:

the inner aqueous phase layer comprises a nisin aqueous solution;

the oil phase layer comprises vegetable oil and an emulsifier;

the outer aqueous phase layer includes a surfactant and a binder.

3. A method for preparing nisin microcapsules according to claim 2, comprising the steps of: dissolving the emulsifier in the vegetable oil, adding the nisin aqueous solution, stirring and mixing, adding the surfactant and the binder after stirring uniformly, stirring, adjusting the pH to 3-5 with hydrochloric acid after stirring uniformly again, and then sequentially carrying out ultrasonic treatment, centrifugation and freeze drying.

4. The production method according to claim 3, characterized in that: the vegetable oil comprises one of soybean oil, sunflower seed oil and olive oil.

5. The production method according to claim 3, characterized in that: the emulsifier comprises span 80, polyglycerol polyricinoleate and one of polyglycerol ester.

6. The production method according to claim 3, characterized in that: the surfactant comprises one of tween 80 and sucrose fatty acid ester.

7. The production method according to claim 3, characterized in that: the binder includes one of gelatin and chitosan.

8. The production method according to claim 3, characterized in that: the weight ratio of the emulsifier to the surfactant is 0.43-2.23: 1.

9. the production method according to claim 3, characterized in that: the ultrasonic treatment time is 1-5 min.

Technical Field

The invention relates to the field of food antibacterial preservation, in particular to a nisin microcapsule and a preparation method thereof.

Background

With the increasing living demands of people, the food safety problem becomes the key point of social attention. Food safety is closely related to food preservation, and people die every year due to eating microbial decayed food, but the food is inevitably decayed due to lipid oxidation or microbial activity during transportation and storage, so that the hot problem is how to effectively perform bacteriostatic treatment on the food, reduce the economic cost during transportation and storage of the food and protect the food safety.

In the prior art, antibacterial treatment is usually carried out by using an antibacterial agent, but the method has short antibacterial time, so that the shelf life of food is not prolonged obviously. The microcapsule technology is a novel technology, in which a core material is embedded by using a wall material, which is a special material artificially synthesized or natural, to form a microcapsule, and the core material can be slowly released into the environment by dissolution, diffusion, and the like. The technology prepares the natural antibacterial substance into a microcapsule product, has the function of enabling the natural antibacterial substance to avoid the interference of environmental factors, thereby achieving the protection effect, and the micro-nano capsule not only can effectively solve the problems that the natural antibacterial substance has smell and is easy to be influenced by the environment, but also can be slowly released, prolong the antibacterial aging, prolong the shelf life of fresh-keeping contents, and has profound significance for preserving the quality of food.

Nisin is a polypeptide substance produced by streptococcus lactis. Since nisin can inhibit most gram-positive bacteria, has strong inhibitory action on spores of bacillus, is a natural food preservative with high efficiency, no toxicity, safety and no side effect, and is often used as a food fresh-keeping material. Although it is non-toxic, harmless and has good antibacterial effect, it is volatile in common materials, so that the food fresh-keeping time is limited. In addition, the addition of nisin in common materials has the problems of peculiar smell, easy environmental influence and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a nisin microcapsule and a preparation method thereof, so as to at least solve the problems of peculiar smell, easy environmental influence, short antibacterial time and easy failure when nisin is used for food fresh-keeping materials.

The purpose of the invention is realized by the following technical scheme:

one of the objects of the present invention is: provides a nisin microcapsule, which is characterized in that: comprises three layers of an inner water phase layer, an oil phase layer and an outer water phase layer.

Further, the inner aqueous phase layer comprises a nisin aqueous solution; the oil phase layer comprises vegetable oil and an emulsifier; the outer aqueous phase layer includes a surfactant and a binder.

Wherein, the nisin has water solubility because the polar groups (-NH2, -COOH) contained in the nisin can form hydrogen bonds with water molecules, and the soybean oil is insoluble in water, so the oil and the water can be separated into an oil-water interface. However, the addition of lipophilic emulsifiers such as span 80 can link the groups between the oil phase and the aqueous phase, reduce the interfacial tension of the oil and water phases, form an interfacial film between the oil phase and the aqueous phase, form a water-in-oil (W/O) emulsion consisting of a solution of nisin encapsulated in soy oil, and prevent the single aggregation of oil and water. The resultant W/O emulsion was then dropped into a gelatin solution containing the hydrophilic emulsifier Tween 80. And simultaneously carrying out ultrasonic treatment on the mixture, and crushing the water phase and the oil phase into smaller liquid drops by utilizing cavitation energy and strong breaking force generated by ultrasonic waves. Tween 80 is adsorbed on the interface, and a lipophilic group is connected with an oil phase, and a hydrophilic group is connected with an aqueous phase to form a water-in-oil-in-water (W/O/W) emulsion which is stable in a suspension system for a long time.

Another object of the present invention is to: a preparation method of the nisin microcapsule is provided, which comprises the following steps:

dissolving an emulsifier in vegetable oil, adding nisin aqueous solution, stirring and mixing, adding a surfactant and a binder after stirring uniformly, adjusting the pH to 3-5 by using HCl after stirring uniformly, and then sequentially carrying out ultrasonic treatment, centrifugation and freeze drying.

Further, the emulsifier is one of span 80, polyglycerol polyricinoleate and polyglycerol ester.

Further, the surfactant is one of tween 80 and sucrose fatty acid ester.

Further, the binder is one of gelatin and chitosan.

Further, the weight ratio of the emulsifier to the surfactant is 0.43-2.23: 1.

further, the ultrasonic time is 1-5 min.

Wherein, ultrasonic agitation can lead the solution loaded with particles to have more uniform particle size, higher stability and lower polydispersity index. Compared with untreated nanofibers, the ultrasonic treatment improves the surface structure, light transmittance and mechanical properties of the nanofibers, microcapsules which are not subjected to ultrasonic treatment are easy to gather and sink in a solution, and the cavitation energy and breaking force of ultrasonic waves enable microcapsule particles to be uniformly dispersed, so that the particle size is reduced. Within 0-3min, the longer the ultrasonic treatment time, the larger the EN particle size, the smaller the PDI, the more uniform the suspended matter particle size and the higher the uniformity. At 3min optimum, excessive sonication may cause the microcapsules to degrade in uniformity or break.

The invention has the beneficial effects that:

1. the invention provides a nisin microcapsule, which solves the problems of peculiar smell, easy environmental influence, short antibacterial time and easy failure when nisin is used for food fresh-keeping materials.

2. The invention provides a preparation method of nisin microcapsules, which is environment-friendly, pollution-free, low in cost, simple in process and easy to operate.

Detailed Description

The technical solutions of the present invention are described in further detail below, but the scope of the present invention is not limited to the following.

Example 1

The nisin (nisin) microcapsule is prepared by the following specific preparation method:

0.2g of span 80 is weighed into 20mL of soybean oil (oil phase), 1g (0.1g mL-1) of nisin is weighed and dissolved in deionized water (internal water phase), and the oil phase and the internal water phase are stirred and mixed. The mixture was added to 0.15g of Tween 80 and 2.5g of 1% (w/v) gelatin (external aqueous phase) and stirred at 25 ℃ for 10 min. The pH of the mixed emulsions was then adjusted to 4.2 with HCl (1mM), respectively. Then, ultrasonic treatment was carried out at 43kHz and 200W for 3 min. Centrifuging at 4 ℃ and 6000rpm for 5min, and freeze-drying for 48h to obtain the nisin microcapsules.

8g of polyvinyl alcohol (PVA) and 10g of sodium Polyacrylate (PAAS) were weighed out and dissolved in 100mL of deionized water, respectively, and after sufficient stirring, the solutions were mixed, and different masses of the above nisin microcapsules (EN) were added. And (3) the mass ratio of the polymer to EN is 100: 0. 95: 5. 90: 10. 85: 15 and 80: the 20 samples are abbreviated as PVA/PAAS/EN-0%, PVA/PAAS/EN-5%, PVA/PAAS/EN-10%, PVA/PAAS/EN-15%, PVA/PAAS/EN-20%, and stirred for 2 h. Then sonicated at 40kHz and 50W for 15min, respectively.

The treated solution was charged into a metal capillary having a diameter of 0.5 mm. Setting the jet flow rate to 0.5mL h < -1 > by using a syringe pump, fixing the voltage to 20kV, and preparing the composite nanofiber by setting the distance between the tip of a capillary tube and a collector to 20 cm.

The experimental results are as follows: the average radius of the micro-nano capsule is 320nm, the micro-nano capsule is uniformly dispersed, the nano microcapsule is smooth and uniform in appearance, complete in network structure, excellent in mechanical property, good in nisin release and excellent in antibacterial activity. The nano-fiber prepared by adding the nisin microcapsule has good appearance, and good mechanical property, light transmittance and barrier property. In addition, the maximum release rate of nisin in the nanofibers within 16 days is (85.28 +/-2.38)%, so that the growth of escherichia coli and staphylococcus aureus can be effectively inhibited. The nanofiber can effectively inhibit the activity of food microorganisms and prolong the shelf life of food.

Example 2

The method is characterized in that nisin microcapsule nano-fiber and a commercially available PE preservative film (Liangterkitchen preservative film) are taken for carrying out a preservation experiment, and the specific method comprises the following steps: whole fresh slaughtered pork is placed on a surface-sterilized plate, then excess fat and connective tissue are trimmed with a surface-sterilized knife, and the meat is cut into small pieces of 100g in mass and drained into 4 groups of 5 parts each. Preparing nisin microcapsule nano fibers into a 15cm multiplied by 15cm bag, filling 100g pork sample into the bag and packaging; the second group made PVA/PAAS nanofiber into 15cm x 15cm bags, filled with 100g pork samples and packaged; the third group was wrapped with a commercial PE wrap, and pork pieces without any treatment were used as a control group, and stored at 4 ℃, and the following tests were performed on 5 groups of pork in the experiment on days 1, 3, 5, 9, 15, and 21, respectively.

1) Sensory testing: sensory evaluation is carried out on the pork sample in 3 aspects of color, smell and comprehensive evaluation, six fixed persons carry out sensory evaluation on the pork and score each time of measurement, the average value is taken as a final result, the experimental result is shown in table 1, and the sensory evaluation table is shown in table 2.

TABLE 1

TABLE 2

Score of	Color	Pork smell
			5	Bright red with luster	Has peculiar smell of fresh pork and no peculiar smell
4	Bright red and lustrous	Has normal smell of fresh pork and no peculiar smell
			3	Dull red without luster	Slightly putrefactive smell
2	Dull-red with no luster	Has putrefactive odor
			1	Dark brown with no luster	Has strong putrefactive odor and odor

2) Detection of thiobarbituric acid (TBARS) value: accurately weighing 10.00g of minced meat sample, adding 50mL of trichloroacetic acid (containing EDTA with mass fraction of 0.1%) with mass fraction of 7.5%, oscillating for 30min, and filtering for 2 times. Transferring 5mL of supernatant, adding 5mL of 0.02 mol/L2-thiobarbituric acid solution, keeping in a boiling water bath for 40min, cooling, centrifuging for 5min at a rotating speed of 2000r/min, taking out the supernatant, adding 5mL of chloroform, shaking up, standing for layering, taking out the supernatant, respectively measuring absorbance at 600nm and 532nm, wherein the TBARS value calculation formula is as follows: TBARS value (mg/100g) ═ a532-a600)/155 × 726. The results of the experiment are shown in table 3.

TABLE 3

3) Peroxide number (POV) test: weighing 5g of sample, putting the sample into an iodometric flask, adding 50mL of mixed solution of trichloroethane and glacial acetic acid (2:1), adding 1mL of saturated potassium iodide solution when grease is completely dissolved, covering a bottle stopper, uniformly mixing the solution for 1min, standing the solution for 5min in a dark place, adding 150mL of distilled water, shaking the solution uniformly, titrating the solution to be light yellow by using sodium thiosulfate as a standard solution, adding 0.5% of starch solution as an indicator, enabling the starch indicator to be blue, continuously dropwise adding a sodium thiosulfate reagent until the blue color disappears, and using water as a blankAnd (4) carrying out control experiments. The POV value is calculated by the following formula: POV (meq/kg) ═ V_l-V₂) Xc.times.1000/m. In the formula: v1 is the volume of sample consuming sodium thiosulfate standard solution (mL); v2 is the volume of sodium thiosulfate standard solution consumed (mL) in the blank test; c is the concentration (mol/L) of the standard solution of sodium thiosulfate; and m is the sample mass (g). The results of the experiment are shown in Table 4

TABLE 4

4) And (3) microorganism index determination: the determination was carried out according to GB4789.2-94 "determination of Total number of colonies for microbiological examination of food hygiene^[6]The results are expressed logarithmically. Log CFU/g (CFU, colony Forming Unit). Evaluation standard control meat quality hygiene index colony count generally suggests standard (fresh meat is less than l ten thousand/g, inferior fresh meat is 1-100 ten thousand/g, deteriorated meat is more than 100 ten thousand/g). The results of the experiment are shown in Table 5.

TABLE 5

In conclusion, from table 1, it can be seen that both the color and smell and the comprehensive evaluation show that the nisin microcapsule nanofiber prepared by the method has better preservation effect than the PE preservative film and the PVA/PAAS nanofiber. As can be seen from tables 3-5, in 21 days, the TBARS content, the peroxide value and the microorganism index value in four groups of pork are obviously increased, but the increment of the nisin microcapsule nanofiber group is the least, and the TBARS represents the fat oxidation degree of the meat sample, so that the refreshment effect nisin microcapsule nanofiber > PVA/PAAS nanofiber > PE preservative film is seen, and the nisin microcapsule prepared by the method has great application potential in the aspect of food refreshment.

The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.