CN115769832A - Preparation method and application of nisin nano composite coacervate - Google Patents
Preparation method and application of nisin nano composite coacervate Download PDFInfo
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
The invention discloses a preparation method and application of nisin nano complex coacervate, wherein the method comprises the following steps: (1) Weighing rhamnolipid, dissolving in water, and stirring at room temperature to obtain a rhamnolipid solution; (2) Weighing nisin, dissolving in water, and stirring at room temperature to obtain nisin solution; (3) Proportionally mixing the rhamnolipid solution and the nisin solution, adjusting the pH value with a NaOH solution, and stirring at room temperature to obtain a mixed system A; (4) Weighing chitosan, dissolving the chitosan in an acetic acid solution, and stirring at room temperature to obtain a mixed system B; (5) And mixing the mixed system A and the mixed system B, adjusting the pH value with NaOH solution, and stirring to obtain the nisin nano composite coacervate. The preparation method disclosed by the invention is simple in preparation process, environment-friendly and pollution-free, and the nano composite coacervate prepared by the method disclosed by the invention solves the problem of narrow antibacterial spectrum of nisin, and can be applied to beef antibacterial preservation.
Description
Technical Field
The invention relates to the field of food antibacterial preservation, and particularly relates to a preparation method and application of nisin nano composite coacervate.
Background
In recent years, with the improvement of living standards and the enhancement of food safety awareness, the quality assurance, shelf life extension, and prevention of food spoilage caused by microorganisms have become urgent problems in the field of food science. Food safety problems caused by improper or excessive addition of chemical preservatives have prompted a focus on the development of natural preservatives. As a natural and efficient food preservative, the bacteriocin has broad-spectrum antibacterial activity, good safety, mass production by a microbial fermentation method and low cost. The bacteriocin can be degraded into natural nutrient components in the digestive tract of a human body, does not influence the flora in the digestive tract, does not lose the using effect of the bacteriostatic agent, has a certain nutritive value, is one of hot spots of food science research, meets the development requirement of future preservatives and meets the pursuit of people on health food.
Nisin (Nisin) is a cationic polypeptide which is generated by streptococcus lactis (lactococcus lactis subsp.lactis) and consists of 34 amino acid residues, has good antibacterial activity, has an inhibiting effect on the growth and reproduction of most gram-positive bacteria and spores, is the only publicly recognized efficient and nontoxic bacteriocin the prior art, and can be widely applied to the preservation and fresh-keeping of dairy products, meat products and alcoholic beverages as a natural food preservative. Meanwhile, nisin can also reduce energy consumption in the food processing process and obviously improve the nutrition, appearance, flavor and texture of food. However, nisin often has the problems of poor stability, low antibacterial ability, narrow antibacterial spectrum and the like, and can react with components in food, thereby seriously restricting the practical application of Nisin in food preservation and fresh-keeping. The Nisin is subjected to stable treatment by adopting a special microencapsulation technology, and the dissolubility, the antibacterial effect and the biocompatibility of the Nisin are improved at the same time, so that the method is one of the most effective means for solving the problems. Complex coacervation is a self-aggregation phenomenon based on electrostatic interactions that occurs between two or more charged macromolecules (e.g., polysaccharide-protein, polysaccharide-polysaccharide, protein-protein). The complex coacervation preparation condition is mild, the yield is high, the efficiency is high, the prepared microcapsule shows excellent performance in the aspects of environmental tolerance, controllable release and the like, the embedding, transportation and controlled release of sensitive or active substances can be realized, and the complex coacervation preparation method becomes a research hotspot in the technical field of microencapsulation at home and abroad.
Beef as a high-grade food is popular with consumers due to the characteristics of rich nutritional value, good sensory characteristics, delicious taste and the like, but fresh beef is easily polluted by microorganisms and decayed. Generally, the beef is preserved by adopting the technologies of irradiation, vacuum packaging, preservative addition and the like, so that the shelf life of the beef can be effectively prolonged, but the defects of high cost, possibility of causing food safety problems and the like exist. At present, beef is packaged and sold by plastic preservative films commonly used, the mode has the advantages of low cost, convenience, practicability and the like, but the shelf life of the beef is usually short, and the traditional preservative films are not degradable and can even generate toxic and harmful substances.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a preparation method and application of nisin nanocomposite coacervate.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, a method for preparing a nisin nanocomposite coacervate comprises the steps of:
(1) Weighing rhamnolipid, dissolving in water, and stirring at room temperature to obtain a rhamnolipid solution;
(2) Weighing nisin, dissolving the nisin in water, and stirring at room temperature to obtain nisin solution;
(3) Proportionally mixing the rhamnolipid solution and the nisin solution, adjusting the pH value with a NaOH solution, and stirring at room temperature to obtain a mixed system A;
(4) Weighing chitosan, dissolving the chitosan in an acetic acid solution, and stirring at room temperature to obtain a mixed system B;
(5) And mixing the mixed system A and the mixed system B, adjusting the pH value with NaOH solution, and stirring to obtain the nisin nano composite coacervate.
Preferably, in the step (1), the water is deionized water, the stirring is magnetic stirring, and the stirring time is 1-3 hours.
More preferably, in step (1), the stirring time is 2h.
Preferably, in the step (1), the rhamnolipid solution has a mass concentration of 2.0mg/mL.
Preferably, in the step (2), the water is deionized water, the stirring is magnetic stirring, and the stirring time is 1-3 hours.
More preferably, in step (1), the stirring time is 2h.
Preferably, in the step (2), the mass concentration of the nisin solution is 4.0mg/mL.
Preferably, in the step (3), the ratio is 1.
More preferably, in the step (3), the ratio is 1.
Preferably, in the step (3), the concentration of the NaOH solution is 0.1mol/L.
Preferably, in step (3), the pH is adjusted to 7 to 8 with NaOH solution.
More preferably, in step (3), the pH is adjusted to 7.5 with NaOH solution.
Preferably, in the step (3), the stirring is magnetic stirring, and the stirring time is 40min.
Preferably, in the step (4), the volume concentration of the acetic acid solution is 1%, and the mass concentration of the chitosan in the mixed system B is 1.0-4.0 mg/mL.
More preferably, in the step (4), the mass concentration of the chitosan in the mixed system B is 2.0mg/mL.
Preferably, in the step (4), the stirring is magnetic stirring, and the stirring time is 12h.
Preferably, in the step (5), the concentration of the NaOH solution is 0.1mol/L.
Preferably, in step (5), the pH is adjusted to 7 to 8 with NaOH solution.
More preferably, in step (5), the pH is adjusted to 7.5 with NaOH solution.
Preferably, in the step (5), the stirring is magnetic stirring, and the stirring time is 30min.
In a second aspect, the invention also provides a nisin nanocomposite aggregate prepared by the method.
In a third aspect, the invention also provides an application of the nisin nanocomposite aggregate in beef antibiosis and freshness preservation. The application comprises the following steps: and (3) soaking beef in the nisin nano complex coacervate prepared by the method to obtain beef blocks treated by the nisin nano complex coacervate. Preferably, the soaking time is 10min.
The application can be used for antibacterial preservation and applied to the field of environmental remediation.
Compared with the prior art, the invention has the following beneficial effects:
(1) The preparation method of the nisin nano composite condensate has the advantages of simple preparation process, low cost, easy realization of industrialization, environmental protection and no pollution, and provides technical support for the research and development of food preservation and fresh-keeping;
(2) The nisin nano complex condensate takes rhamnolipid and chitosan as complex condensate reaction matrixes, the used materials have good biocompatibility, safety and no toxicity, and good antibacterial performance, and the prepared nano complex condensate solves the problem of narrow nisin antibacterial spectrum;
(3) The nisin nano composite aggregate is used for treating beef in a coating manner, so that the color, texture and sensory quality of the beef are effectively maintained, the growth of pathogenic microorganisms can be effectively inhibited, and the shelf life of the beef is prolonged.
Drawings
FIG. 1 is a diagram showing the inhibition zone effect of different solutions in Experimental example 1 on Escherichia coli, wherein in FIG. 1, (a) is a diagram showing the inhibition zone effect of blank groups (No. 1), 2.0mg/mL chitosan solution (No. 2) and 2.0mg/mL rhamnolipid solution (No. 3) on Escherichia coli, and (b) is a diagram showing the inhibition zone effect of 4.0mg/mL nisin solution (No. 4), the mixed system A (No. 5) prepared in example 5 and the nisin nanocomposite aggregate (No. 6) prepared in example 5 on Escherichia coli;
FIG. 2 is a diagram of the inhibition zone effect of different solutions in Experimental example 2 on Staphylococcus aureus, in FIG. 2, (a) is a diagram of the inhibition zone effect of blank groups (number 1), 2.0mg/mL chitosan solution (number 2), 2.0mg/mL rhamnolipid solution (number 3) on Staphylococcus aureus, and (b) is a diagram of the inhibition zone effect of 4.0mg/mL nisin solution (number 4), the mixed system A (number 5) prepared in example 5, and the nisin nanocomposite aggregate (number 6) prepared in example 5 on Staphylococcus aureus;
FIG. 3 shows the change of pH values of Nisin nanocomposite condensate-treated beef pieces obtained in example 6 and untreated beef pieces obtained in comparative example 1 when stored at different temperatures, respectively, wherein the Nisin nanocomposite condensate-treated beef pieces obtained in example 6 were used as an experimental group, the untreated beef pieces obtained in comparative example 1 were used as a control group, and FIG. 3 shows (a) the change of pH values of the experimental group (Nisin/RL/CS) and the control group stored at 4 ℃ and (b) the change of pH values of the experimental group (Nisin/RL/CS) and the control group stored at 25 ℃;
FIG. 4 is a photograph showing changes in the appearance of beef treated with nisin nanocomposite condensate obtained in example 6 and untreated beef obtained in comparative example 1 when stored at 4 ℃ in the same manner, wherein the untreated beef obtained in example 6 was used as an experimental group and the untreated beef obtained in comparative example 1 was used as a control group, and wherein (a) is a photograph showing the appearance of the control group when stored at 4 ℃ for 0 day, (b) is a photograph showing the appearance of the experimental group when stored at 4 ℃ for 0 day, (c) is a photograph showing the appearance of the control group when stored at 4 ℃ for 3 days, (d) is a photograph showing the appearance of the experimental group when stored at 4 ℃ for 3 days, (e) is a photograph showing the appearance of the control group when stored at 4 ℃ for 7 days, (f) is a photograph showing the appearance of the experimental group when stored at 4 ℃ for 7 days;
FIG. 5 is a photograph showing changes in the appearance of beef treated with nisin nanocomposite condensate obtained in example 6 and untreated beef obtained in comparative example 1 when stored at 25 ℃, wherein the untreated beef obtained in example 6 was used as an experimental group, and the untreated beef obtained in comparative example 1 was used as a control group, and wherein (a) is a photograph showing the appearance of the control group when stored at 25 ℃ for 0 day, (b) is a photograph showing the appearance of the experimental group when stored at 25 ℃ for 0 day, (c) is a photograph showing the appearance of the control group when stored at 25 ℃ for 3 days, (d) is a photograph showing the appearance of the experimental group when stored at 25 ℃ for 3 days, (e) is a photograph showing the appearance of the control group when stored at 25 ℃ for 7 days, (f) is a photograph showing the appearance of the experimental group when stored at 25 ℃ for 7 days;
FIG. 6 shows the change of the surface bacteria count of the beef extract treated with Nisin nanocomposite aggregate obtained in example 6 and the untreated beef extract obtained in comparative example 1 when stored at different temperatures, and in FIG. 6, (a) shows the change of the surface bacteria count of the experimental group (Nisin/RL/CS) and the control group stored at 4 ℃ and (b) shows the change of the surface bacteria count of the experimental group (Nisin/RL/CS) and the control group stored at 25 ℃;
FIG. 7 shows changes in TBARS values when the beef pieces treated with Nisin nanocomposite aggregate obtained in example 6 and the untreated beef pieces obtained in comparative example 1 were stored at different temperatures, respectively, wherein the beef pieces treated with Nisin nanocomposite aggregate obtained in example 6 were used as an experimental group, the beef pieces untreated beef pieces obtained in comparative example 1 were used as a control group, and (a) in FIG. 7 are changes in TBARS values when the experimental group (Nisin/RL/CS) and the control group were stored at 4 ℃ and (b) are changes in TBARS values when the experimental group (Nisin/RL/CS) and the control group were stored at 25 ℃.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, and is not intended to limit the present invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
A method for preparing a nisin nanocomposite coacervate, the method comprising the steps of:
(1) Weighing rhamnolipid, dissolving the rhamnolipid in deionized water, and magnetically stirring at room temperature for 1h to obtain a rhamnolipid solution with the mass concentration of 2.0 mg/mL;
(2) Weighing nisin, dissolving nisin in deionized water, and magnetically stirring at room temperature for 1h to obtain nisin solution with the mass concentration of 4.0 mg/mL;
(3) Mixing the rhamnolipid solution and the nisin solution according to a mass ratio of 1;
(4) Weighing chitosan, dissolving the chitosan in an acetic acid solution with the volume concentration of 1%, and magnetically stirring for 12 hours at room temperature to obtain a mixed system B with the mass concentration of the chitosan of 1.0 mg/mL;
(5) And (3) mixing the mixed system A and the mixed system B in equal volume, adjusting the pH value to 7.0 by using 0.1mol/L NaOH solution, and magnetically stirring at room temperature for 30min to obtain the nisin nano composite coacervate.
Example 2
A method for preparing a nisin nanocomposite coacervate, the method comprising the steps of:
(1) Weighing rhamnolipid, dissolving the rhamnolipid in deionized water, and magnetically stirring at room temperature for 3h to obtain a rhamnolipid solution with the mass concentration of 2.0 mg/mL;
(2) Weighing nisin, dissolving nisin in deionized water, and magnetically stirring at room temperature for 3h to obtain nisin solution with the mass concentration of 4.0 mg/mL;
(3) Mixing a rhamnolipid solution and a nisin solution according to a mass ratio of 1;
(4) Weighing chitosan, dissolving the chitosan in an acetic acid solution with the volume concentration of 1%, and magnetically stirring for 12 hours at room temperature to obtain a mixed system B with the mass concentration of the chitosan of 4.0 mg/mL;
(5) And (3) mixing the mixed system A and the mixed system B in equal volume, adjusting the pH value to 7.0 by using 0.1mol/L NaOH solution, and magnetically stirring at room temperature for 30min to obtain the nisin nano composite coacervate.
Example 3
A method for preparing a nisin nanocomposite coacervate, the method comprising the steps of:
(1) Weighing rhamnolipid, dissolving the rhamnolipid in deionized water, and magnetically stirring at room temperature for 2 hours to obtain a rhamnolipid solution with the mass concentration of 2.0 mg/mL;
(2) Weighing nisin, dissolving nisin in deionized water, and magnetically stirring at room temperature for 2h to obtain nisin solution with mass concentration of 4.0 mg/mL;
(3) Mixing a rhamnolipid solution and a nisin solution according to a mass ratio of 1;
(4) Weighing chitosan, dissolving the chitosan in an acetic acid solution with the volume concentration of 1%, and magnetically stirring for 12 hours at room temperature to obtain a mixed system B with the mass concentration of the chitosan of 2.0 mg/mL;
(5) And (3) mixing the mixed system A and the mixed system B in equal volume, adjusting the pH value to 7.0 by using 0.1mol/L NaOH solution, and magnetically stirring at room temperature for 30min to obtain the nisin nano complex coacervate.
Example 4
Example 4 is essentially the same as example 3, except that: in example 4, the rhamnolipid solution and the nisin solution were mixed at a ratio of 1.
Example 5
Example 5 is essentially the same as example 3, except that: in example 5, the rhamnolipid solution and the nisin solution were mixed at a ratio of 1.
The nisin nanocomposite aggregates obtained in examples 3 to 5 were subjected to laser particle size analyzer (Litesizer) TM 500, austria-east pascal corporation), the particle diameter, the polydispersity index (PDI), and the Zeta potential of the nisin nanocomposite aggregate obtained in inventive example 3 were found to be 684.9nm, PDI 0.275, and potential +33.1mV, the particle diameter of the nisin nanocomposite aggregate obtained in inventive example 4 was 683.4nm, PDI 0.281, and potential +32.6mV, and the particle diameter of the nisin nanocomposite aggregate obtained in inventive example 5 was 682.5nm, PDI 0.286, and potential +31.0mV. As is clear from the potentials, particle diameters, and PDI of examples 3 to 5, the production method described in example 5 is a preferred production method among the production methods of nisin nanocomposite aggregates of the present invention.
Experimental example 1
In this experimental example, the effect of the nisin nanocomposite aggregate prepared in example 5 on the inhibition zone of escherichia coli was mainly examined, which included the following steps:
(1) Setting a blank group and a control group, wherein 1 is the blank group, 2 is a 2.0mg/mL chitosan solution, 3 is a 2.0mg/mL rhamnolipid solution, 4 is a 4.0mg/mL nisin solution, 5 is the mixed system A prepared in the steps (1) to (3) of the example 5, and 6 is the nisin nanocomposite condensate prepared in the example 5;
(2) Culturing a bacterial suspension of Escherichia coli at 37 ℃ to logarithmic phase;
(3) The bacterial suspension of the Escherichia coli is diluted to 10 degrees in a gradient way 6 CFU/mL；
(4) Sucking 200 mu L of the bacterial suspension, adding the bacterial suspension into 20mL of LB solid culture medium, pouring the plate to obtain 2 LB solid culture media added with the bacterial suspension;
(5) Standing for 20min, pricking 3 holes with diameter of 5mm on 2 LB solid culture media added with bacterial suspension, sequentially adding 20 μ L blank group and control group solutions, standing in a super clean bench for 5min, and culturing for 24h.
The inhibition zone effects of different solutions on escherichia coli are shown in fig. 1, wherein (a) in fig. 1 is a diagram of inhibition zone effects of a blank group (number 1), a 2.0mg/mL chitosan solution (number 2), and a 2.0mg/mL rhamnolipid solution (number 3) on escherichia coli, and (b) is a diagram of inhibition zone effects of a 4.0mg/mL nisin solution (number 4), the mixed system a (number 5) prepared in example 5, and the nisin nanocomposite condensate (number 6) prepared in example 5 on escherichia coli; as can be seen from fig. 1 (a) and (b), the rhamnolipid, nisin and mixed system a have no zone of inhibition on escherichia coli, the chitosan and nisin nanocomposite condensate prepared in example 5 have a zone of inhibition on escherichia coli, and the nisin nanocomposite condensate prepared in example 5 has the largest zone of inhibition diameter (13 mm), which indicates that the nisin nanocomposite condensate prepared in example 5 has the best effect on escherichia coli inhibition.
Experimental example 2
In this experimental example, the inhibition zone effect of the nisin nanocomposite condensate prepared in example 5 on staphylococcus aureus was mainly considered, the process was substantially the same as that of example 1, except that the bacterial suspension of example 2 was a suspension of staphylococcus aureus, and the results are shown in fig. 2, where (a) in fig. 2 is a graph showing the inhibition zone effect on staphylococcus aureus of blank groups (No. 1), 2.0mg/mL chitosan solution (No. 2), and 2.0mg/mL rhamnolipid solution (No. 3), and (b) is a graph showing the inhibition zone effect on staphylococcus aureus of 4.0mg/mL nisin solution (No. 4), the mixed system a (No. 5) prepared in example 5, and the nisin nanocomposite condensate (No. 6) prepared in example 5; as can be seen from fig. 2 (a) and (b), the rhamnolipid and chitosan have no inhibition zone against staphylococcus aureus, nisin, the mixed system a and the nisin nanocomposite aggregate prepared in example 5 have an inhibition zone against staphylococcus aureus, and the nisin nanocomposite aggregate prepared in example 5 has the largest diameter (18 mm) of the inhibition zone, which indicates that the nisin nanocomposite aggregate prepared in example 5 has the best inhibition effect against staphylococcus aureus.
Experimental example 3
In this experimental example, the nisin nanocomposite coacervate prepared in example 5 was examined mainly for the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) against escherichia coli and staphylococcus aureus, comprising the following steps:
(1) Weighing 4.5mL of LB liquid medium, sterilizing, adding 5mL of nisin nanocomposite aggregate prepared in example 5 into the medium, and adding 0.5mL of Escherichia coli to obtain a mixed system C;
(2) Vortexing and uniformly mixing the mixed system C to respectively obtain mixed liquid culture media with final concentrations of nisin nano composite coacervate of 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0mg/mL, carrying out shaking culture at the temperature of 37 ℃ for 4 hours at the speed of 150r/min, observing the mixed liquid culture media by naked eyes, and defining the minimum concentration with clear color as the Minimum Inhibitory Concentration (MIC);
(3) Selecting MIC, 2-time MIC and 4-time MIC, respectively sucking 200 μ L of bacterial liquid with corresponding concentration to an LB solid culture medium plate, uniformly coating with a coating rod, culturing at 37 ℃ for 24h, observing the plate, and taking the minimum sample concentration for completely inhibiting the bacterial growth as the Minimum Bactericidal Concentration (MBC).
From the results, it was found that the MIC value of nisin nanocomposite aggregate obtained in example 5 for E.coli was 0.5mg/mL, and the MBC value was 1.0mg/mL, respectively.
Experimental example 4
In this experimental example, the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) against Staphylococcus aureus of the nisin nanocomposite aggregate prepared in example 5 were mainly examined, and in experimental example 4, which is substantially the same as in experimental example 3, except that Staphylococcus aureus was added to LB liquid medium in experimental example 4, it was found that the MIC and the MBC of the nisin nanocomposite aggregate prepared in example 5 were 0.25mg/mL and 0.50mg/mL, respectively, against Staphylococcus aureus.
Example 6
A method for the antimicrobial and freshness-retaining treatment of beef by the nisin nanocomposite coacervate prepared in example 5, comprising the steps of:
(1) Cleaning fresh beef in sterile water in a sterile environment, cutting into cubes of 30 multiplied by 25 multiplied by 5mm to obtain beef blocks, subpackaging for later use, inoculating staphylococcus aureus into an LB liquid culture medium, and culturing for 12-24 hours at 37 ℃ to obtain a logarithmic phase staphylococcus aureus liquid for later use;
(2) Sterilizing the Nisin nanocomposite aggregate (Nisin/RL/CS) prepared in example 5 by irradiating the aggregate under an ultraviolet lamp for 1 h;
(3) In an aseptic environment, placing the beef blocks in Staphylococcus aureus liquid, soaking for 2min, and taking out to make the initial bacteria concentration on the surface about 10 5 ～10 6 CFU/mL to obtain beef blocks inoculated with staphylococcus aureus;
(4) After being left for 30min, the beef blocks inoculated with staphylococcus aureus were placed in the nisin nanocomposite aggregate prepared in example 5 to be soaked for 10min, so as to obtain beef blocks treated by nisin nanocomposite aggregate.
Comparative example 1
An unprocessed beef piece comprising the steps of:
(1) Cleaning fresh beef in sterile water in a sterile environment, cutting into cubes of 30 multiplied by 25 multiplied by 5mm to obtain beef blocks, subpackaging for later use, inoculating staphylococcus aureus into an LB liquid culture medium, and culturing for 12-24 hours at 37 ℃ to obtain a logarithmic phase staphylococcus aureus liquid for later use;
(2) Placing the phosphate buffer under an ultraviolet lamp for irradiation for 1h for sterilization;
(3) In an aseptic environment, placing the beef blocks in Staphylococcus aureus liquid, soaking for 2min, and taking out to make the initial bacteria concentration on the surface about 10 5 ～10 6 CFU/mL to obtain beef blocks inoculated with staphylococcus aureus;
(4) And (3) placing the beef blocks inoculated with the staphylococcus aureus for 30min, and then placing the beef blocks in a phosphate buffer solution for soaking for 10min to obtain untreated beef blocks.
pH value test
The beef pH value was measured by the standard method of "national food safety Standard for determination of food pH value" (GB 5009.237-2016), and the evaluation criteria are shown in Table 1.
TABLE 1 evaluation criteria
The beef pieces treated with nisin nanocomposite aggregate obtained in example 6 were used as an experimental group, the untreated beef pieces obtained in comparative example 1 were used as a control group, the experimental group and the control group were stored at 4 ℃ and 25 ℃, respectively, and the change of beef pH at different temperatures was measured, and the results are shown in fig. 3. In FIG. 3, (a) shows the change in pH at 4 ℃ in the experimental group (Nisin/RL/CS) and the control group, and (b) shows the change in pH at 25 ℃ in the experimental group (Nisin/RL/CS) and the control group. As can be seen from fig. 3 (a), the pH of the beef decreases when the experimental group and the control group are stored at 4 ℃ for 1 day, and then gradually increases with the increase of the storage time, the pH of the experimental group is lower than that of the control group during the whole storage period, and the pH of the experimental group and the control group is lower than 6.3 (first-grade freshness pH), which indicates the quality of the beef which is still fresh meat after being stored for 7 days in the environment of 4 ℃. As can be seen from FIG. 3 (b), the pH trends of the beef in the experimental group and the control group were similar to those in the control group at 25 ℃ when stored at 25 ℃, but the beef in the control group became deteriorated meat when stored at 25 ℃ for more than 2 days, whereas the beef in the experimental group became deteriorated meat only on the 4 th day. It is thus understood that the nisin nanocomposite aggregate prepared in example 5 effectively suppresses the growth and propagation of bacteria and delays the deterioration of beef.
Appearance change condition
Beef pieces treated with nisin nanocomposite aggregate obtained in example 6 were used as an experimental group, untreated beef pieces obtained in comparative example 1 were used as a control group, and the experimental group and the control group were stored at 4 ℃ and 25 ℃, respectively, and changes in appearance of beef during storage were photographed with a digital camera on days 0, 3, and 7, respectively, and the results are shown in fig. 4 and 5. FIG. 4 is a graph showing changes in the appearance of the experimental group and the control group when they are stored at 4 ℃ respectively, in which (a) is an image showing the appearance of the control group when it is stored at 4 ℃ for 0 day, (b) is an image showing the appearance of the experimental group when it is stored at 4 ℃ for 0 day, (c) is an image showing the appearance of the control group when it is stored at 4 ℃ for 3 days, (d) is an image showing the appearance of the experimental group when it is stored at 4 ℃ for 3 days, (e) is an image showing the appearance of the control group when it is stored at 4 ℃ for 7 days, and (f) is an image showing the appearance of the experimental group when it is stored at 4 ℃ for 7 days; FIG. 5 shows the change of the appearance of the experimental group and the control group when they are stored at 25 ℃, respectively, in FIG. 5, (a) is an image of the appearance of the control group when it is stored at 25 ℃ for 0 day, (b) is an image of the appearance of the experimental group when it is stored at 25 ℃ for 0 day, (c) is an image of the appearance of the control group when it is stored at 25 ℃ for 3 days, (d) is an image of the appearance of the experimental group when it is stored at 25 ℃ for 3 days, (e) is an image of the appearance of the control group when it is stored at 25 ℃ for 7 days, and (f) is an image of the appearance of the experimental group when it is stored at 25 ℃ for 7 days. As can be seen from the figure 4 (a), the fresh beef has bright and uniform surface color, slightly moist surface, no mucus and good elasticity, when the experimental group is stored for 7 days at 4 ℃, the beef surface loses the original color, the texture begins to gradually become fuzzy, and a small amount of water slowly begins to appear on the beef surface, and the gray part at the lower side of the beef block in the figure 4 (f) is water. The grey part around the beef piece in fig. 5 (c) is water, and as can be seen by comparing fig. 5 (c) with fig. 5 (d), a small amount of water appears already when the control group is stored at 25 ℃ for 3 days, which indicates that the beef is rotten, while the experimental group is still in a good meat state when the control group is stored at 25 ℃ for 3 days, which indicates that the nisin nanocomposite aggregate prepared in example 5 has a good fresh-keeping effect on beef when stored at 25 ℃ and a better fresh-keeping effect on beef when stored at 4 ℃.
Color test
Beef pieces treated with Nisin nanocomposite condensate obtained in example 6 were used as an experimental group, and untreated beef pieces obtained in comparative example 1 were used as a control group, and the experimental group (Nisin/RL/CS) and the control group were stored at 4 ℃ and 25 ℃ respectively, and a spectrophotometer was used(Color Quest XE, hunter Lab, USA) measures the change in the Color of beef during storage. Setting the aperture of the spectrocolorimeter to be 19.0mm, the light-emitting source to be a xenon lamp and the angle of a standard observer to be 8 degrees, and measuring the L of the sample by using the spectrocolorimeter * (luminance) a * (degree of redness) and b * (yellowness) values, using a standard white board calibrated spectrocolorimeter, the colorimetric parameters were determined at day 0, 3 and 7 for the experimental and control groups at 4 ℃ and 25 ℃ respectively. The results are shown in tables 2 and 3, in which table 2 shows the change in color of nisin nanocomposite aggregates treated beef during storage at 4 ℃ and table 3 shows the change in color of nisin nanocomposite aggregates treated beef during storage at 25 ℃ under dark conditions.
TABLE 2 color change of beef of experimental group and control group during storage at 4 deg.C
Note: different letters indicate significant differences, P <0.05.
TABLE 3 color change of beef of the experimental group and the control group during storage at 25 deg.C
Note: different letters indicate significant differences, P <0.05.
As can be seen from tables 2 and 3, L in the experimental group * The value is higher than that of a control group, which shows that the Nisin nano-composite aggregate (Nisin/RL/CS) prepared in example 5 can well inhibit the loss of beef water and delay L * The value is decreased. a is * Value b and * the change of the value is the result of a series of combined actions of the intramuscular fat of the beef, the oxidation of the myoglobin and the like, and the a of the beef in the control group and the experimental group is controlled at 4 ℃ and 25 DEG C * Value b and * the values all tended to decrease with increasing storage time. It can be seen that storage at 4 ℃ and 25 ℃ is carried outUnder the condition, the whole chromatic value of the beef is inversely proportional to the storage time, and the change is more obvious under the condition of 25 ℃; in addition, compared with a control group, the change range of the beef chroma value of the experimental group is small, which shows that the nisin nanocomposite aggregate prepared in example 5 has a certain effect of maintaining the beef color.
Texture testing
The beef pieces treated with Nisin nanocomposite aggregate obtained in example 6 were used as an experimental group, the untreated beef pieces obtained in comparative example 1 were used as a control group, the experimental group (Nisin/RL/CS) and the control group were stored at 4 ℃ and 25 ℃, respectively, and the change in texture parameters of the beef samples during storage was measured using a food property tester (ta.xt Plus, stable Micro Systems, uk), and the results are shown in table 4, which are changes in texture of Nisin nanocomposite aggregate treated beef during storage under different temperature conditions.
TABLE 4 texture change of beef in the experimental and control groups during storage at different temperatures
Note: different letters indicate significant differences, P <0.05.
As can be seen from table 4, at 4 ℃ and 25 ℃, the texture parameters of the beef both significantly decrease with the increase of the storage time, and the higher the temperature, the larger the decrease, and the change range of the texture parameters of the experimental group is smaller than that of the control group, which indicates that the nisin nanocomposite aggregate prepared in example 5 can well maintain the original texture characteristics of the beef.
Determination of the number of surface microbial residues
The beef pieces treated with the Nisin nanocomposite aggregate obtained in example 6 were used as an experimental group, the untreated beef pieces obtained in comparative example 1 were used as a control group, the experimental group and the control group were stored at 4 ℃ and 25 ℃ respectively, the number of surface bacteria on the beef samples was analyzed by the plate colony counting method, samples were taken on days 0 to 7 respectively, the beef pieces were homogenized in a sterile homogenizer bag for 5min using a homogenizer 12,000r/min, the number of surface microorganisms remaining on the beef samples was measured according to the determination of the total number of colonies for food safety national standard food microbiology examination (GB 4789.2-2016), and the results are shown in fig. 6, in which (a) in fig. 6 are the changes in the number of surface bacteria stored at 4 ℃ in the experimental group (Nisin/RL/CS) and the control group, and (b) in the changes in the number of surface bacteria stored at 25 ℃ in the experimental group (Nisin/RL/CS) and the control group. As can be seen from FIG. 6, staphylococcus aureus rapidly grows and breeds on the beef surface in the experimental groups at 4 ℃ and 25 ℃, but the growth rate of the colony count is reduced in the later period of storage; the viable count of staphylococcus aureus in the experimental group is gradually reduced along with the prolonging of the storage time, and the viable count of the beef surface treated by Nisin nano composite aggregate (Nisin/RL/CS) is respectively reduced to 4.13Log CFU/g and 4.81Log CFU/g after the beef is stored for 7 days, which shows that the Nisin nano composite aggregate prepared in the example 5 has an inhibiting effect on staphylococcus aureus on the beef surface at 4 ℃ and 25 ℃, and can play a fresh-keeping effect on beef.
Determination of Thiobabarbituric acid reactant content (TBARS)
The beef pieces treated with Nisin nanocomposite condensate obtained in example 6 were used as an experimental group, the untreated beef pieces obtained in comparative example 1 were used as a control group, the experimental group and the control group were stored at 4 ℃ and 25 ℃, respectively, and the change of TBARS during beef storage was measured by spectrophotometry, and the results are shown in fig. 7, in which (a) is the change of TBARS stored at 4 ℃ in the experimental group (Nisin/RL/CS) and the control group, and (b) is the change of TBARS stored at 25 ℃ in the experimental group (Nisin/RL/CS) and the control group. The TBARS value is an important index reflecting the beef lipid oxidation degree. Generally, the meat is considered to be good quality meat when the TBARS value is between 0.2 and 0.66 mg/kg; if the content exceeds 1mg/kg, the meat is deteriorated.
Referring to the determination of malondialdehyde in national food safety standards (GB 5009.181-2016), it can be seen from FIG. 7 that TBARS values of the experimental group and the control group both increased with the increase of storage time at 4 ℃ and 25 ℃. The TBARS value of the beef stored for 2 days at 4 ℃ is not obvious; after 2 days, the TBARS value increases significantly with the increase of the storage time; on day 7, the TBARS value of the control group reached 0.51mg/kg; the TBARS value of the experimental group has a similar change trend to that of the control group, but the change amplitude is relatively small, and the TBARS value is only increased by 0.21mg/kg. The variation trend of the TBARS values in the control group and the experimental group is consistent with that at the temperature of 4 ℃ at 25 ℃, but the TBARS value of the control group reaches the index value of the deteriorated meat on the 2 nd day; the TBARS value of the beef in the experimental group changes slowly and reaches the range value of deteriorated beef only after being stored for 6 days, which shows that the nisin nanocomposite aggregate prepared in example 5 has good fresh-keeping effect on the beef at different temperatures.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (10)
1. A method for preparing a nisin nanocomposite coacervate, comprising the steps of:
(1) Weighing rhamnolipid, dissolving in water, and stirring at room temperature to obtain a rhamnolipid solution;
(2) Weighing nisin, dissolving the nisin in water, and stirring at room temperature to obtain nisin solution;
(3) Proportionally mixing the rhamnolipid solution and the nisin solution, adjusting the pH value with a NaOH solution, and stirring at room temperature to obtain a mixed system A;
(4) Weighing chitosan, dissolving the chitosan in an acetic acid solution, and stirring at room temperature to obtain a mixed system B;
(5) And mixing the mixed system A and the mixed system B, adjusting the pH value with NaOH solution, and stirring to obtain the nisin nano composite coacervate.
2. The method for preparing nisin nanocomposite condensate according to claim 1, wherein in the step (1), the water is deionized water, the stirring is magnetic stirring, and the stirring time is 1 to 3 hours.
3. The method for preparing a nisin nanocomposite condensate according to claim 1, wherein in step (1), the rhamnolipid solution has a mass concentration of 2.0mg/mL.
4. The preparation method of nisin nanocomposite condensate according to claim 1, wherein in step (2), the water is deionized water, the stirring is magnetic stirring, the stirring time is 1 to 3 hours, and the nisin solution has a mass concentration of 4.0mg/mL.
5. The method for producing a nisin nanocomposite aggregate according to claim 1, wherein in the step (3), the ratio is 1.
6. The method for preparing nisin nanocomposite aggregates according to claim 1, wherein in step (4), the volume concentration of the acetic acid solution is 1%, the mass concentration of chitosan in the mixed system B is 1.0 to 4.0mg/mL, and the stirring is performed by magnetic stirring for 12 hours.
7. The method for producing a nisin nanocomposite aggregate according to claim 1, wherein in the step (5), the concentration of the NaOH solution is 0.1mol/L, the pH value is adjusted to 7 to 8 by the NaOH solution, and the stirring is magnetic stirring for 30min.
8. A nisin nanocomposite coacervate prepared according to the process of any one of claims 1 to 7.
9. Use of the nisin nanocomposite coacervate of claim 8 for antimicrobial preservation of beef.
10. The use of nisin nanocomposite coacervate according to claim 9 for antimicrobial preservation of beef, wherein the use comprises the following steps:
and (3) putting the beef into the nisin nano complex coacervate prepared by the method, and soaking for 10min to obtain beef blocks treated by the nisin nano complex coacervate.
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| Title |
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| 唐春红: "《天然防腐剂与抗氧化剂》", vol. 1, 31 May 2010, 中国轻工业出版社, pages: 35 * |
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