1. pH and NH₃The preparation method of the high-response food secondary fresh color sensitive indicating film is characterized by comprising the following steps of: stirring and mixing the gelatinized starch solution and the chitosan solution to obtain a starch-chitosan mixed solution; adding the crude extract solution of lonicera edulis anthocyanin into the starch-chitosan mixed solution, and stirring to obtain a starch-chitosan-lonicera edulis anthocyanin mixed solution; adding a certain amount of glycerol into the starch-chitosan-loniceraedulis anthocyanin mixed solution, adjusting the pH of the solution to 2-3, and continuously stirring for a period of time to obtain a film forming solution; drying the film-forming solution to form a film, thus obtaining the product.

2. The method of claim 1, wherein: the preparation method of the gelatinized starch solution comprises the following steps: gradually heating the starch solution with the concentration of 50-70 mg/mL to 60-65 ℃ while stirring, so that the starch is in a just gelatinized state, wherein the stirring speed is 800-1000 r/min, and the stirring time is 20-40 min.

3. The method of claim 1, wherein: the chitosan solution is 1-1.5% (v: v) acetic acid water solution of chitosan, the concentration of the chitosan solution is 6-10 mg/mL, and the mass ratio of starch to chitosan is 5-15: 1.

4. The method of claim 1, wherein: the crude extract solution of the loniceraedulis anthocyanin is an acidic ethanol solution of the loniceraedulis anthocyanin crude extract, the purity of the loniceraedulis anthocyanin crude extract is more than 20%, and the concentration of the loniceraedulis anthocyanin crude extract is 2-6 mg/mL; the acidic ethanol solution is composed of 1-1.5 mol/L acetic acid and absolute ethyl alcohol according to a volume ratio of 1: 4-6.

5. The method of claim 1, wherein: the mass ratio of the loniceraedulis anthocyanin to the chitosan is 20-40: 1.

6. The method of claim 1, wherein: after the pH value of the solution is adjusted, stirring at the speed of 800-1000 r/min for 20-40 min; the addition amount of the glycerol is 1-3% of the total volume of the film forming solution.

7. The method of claim 1, wherein: and casting the obtained film forming liquid into a film forming plate, placing the film forming plate in an air blowing drying oven, and drying to form a film, wherein the drying temperature is 40-45 ℃, and the drying time is 12-24 hours.

8. The method of claim 1, wherein: adjusting the pH value of the starch-chitosan-loniceraedulis anthocyanin mixed solution to 2-3 by using 0.1-1.0 mol/L HCl solution or 0.1-1.0 mol/L NaOH solution.

9. The pH and NH prepared by the method of any one of claims 1 to 8₃High response type food inferior freshness level color sensitive indicating film.

10. pH and NH₃The application of the high-response food secondary freshness color-sensitive indicating film in monitoring the shelf life of fresh products which generate volatile nitrogen-containing compounds in the process of spoilage.

Background

Since a large amount of volatile nitrogen and biogenic amine generated by microbial decay of high-fat and high-protein food are easy to occur in the storage process, the method brings great challenges to the food packaging industry and also becomes a hotspot of current research. In order not to influence the appearance and the integrity of the food monitoring, the pH color-sensitive indicating film is widely applied to intelligent packaging of fresh food as a convenient and quick nondestructive testing mode. Among them, the use of chemical dyes as an indicator dye in freshness indicating packages is not desirable because they have high toxicity and have harmful effects on human health and the environment. Because the natural pigment has the advantages of safety, no toxicity, convenient preparation, biodegradability and the like, researchers take the natural pigment extracted from plants as an indicating substance of the pH color-sensitive indicating membrane. Anthocyanin is edible natural water-soluble pigment, and anthocyanin exists in different chemical forms according to different pH values of solutions, has obvious color change under different structures, and can be used as an ideal selection of pH sensitive dye.

A large number of researches prove that anthocyanin from different sources can be used as a bio-based pH color-sensitive indicating dye to be applied to food freshness monitoring. Among them, loniceraedulis anthocyanin is used as an indicator dye in the present invention because it is poor in fresh food and shows a significant color change under a wide range of pH conditions. The existing report has good effect on distinguishing the freshness and the spoilage of the indicated food in the application of the indicating film, but the identification of the level of the next freshness of the monitored food in the food storage process is still not clear enough. Some existing anthocyanin indicating films improve the color responsiveness of the indicating films by adjusting and controlling the content of anthocyanin or compounding pigments and the like, but researches on improving the response sensitivity and stability of the anthocyanin indicating films by adjusting and controlling the pH value of a film forming solution are rarely reported.

Disclosure of Invention

The object of the present invention is to provide a pH and NH₃The invention relates to a high-response food secondary fresh color-sensitive indicating film, a preparation method and application thereof₃The color sensitive indicating film with good responsiveness improves the sensitivity of the indicating film and enhances the indicating effect. The color of the indicator film changes with the freshness of the food product, and can quickly indicate the quality state (freshness grade, secondary freshness grade and spoilage) of the product in the package. The indicating film can be used for monitoring the freshness, the secondary freshness and the spoilage grade of fresh shrimps in the storage process, visually monitoring and early warning the food spoilage of manufacturers and consumers in real time, and providing reference for the natural anthocyanin indicating film applied to the shelf life monitoring of high-protein high-fat products.

The invention provides a pH and NH₃A preparation method of a high-response food secondary fresh grade color sensitive indicating film comprises the steps of stirring and mixing a gelatinized starch solution and a chitosan solution to obtain a starch-chitosan mixed solution; adding the crude extract solution of lonicera edulis anthocyanin into the starch-chitosan mixed solution, and stirring to obtain a starch-chitosan-lonicera edulis anthocyanin mixed solution; adding a certain amount of glycerol into the starch-chitosan-loniceraedulis anthocyanin mixed solution, adjusting the pH of the solution to 2-3, and continuously stirring for a period of time to obtain a film forming solution; drying the film-forming solution to form a film, thus obtaining the product.

In the technical scheme, the mass ratio of the starch to the chitosan is preferably 5-15: 1.

In the technical scheme, the concentration of the chitosan solution is preferably 6-10 mg/mL.

Further, the chitosan solution is 1-1.5% (v: v) acetic acid aqueous solution of chitosan.

In the above technical solution, the starch is preferably potato starch.

Further, the preparation method of the gelatinized starch solution comprises the following steps: gradually heating the starch solution with the concentration of 50-70 mg/mL to 60-65 ℃ while stirring, so that the starch is in a just gelatinized state, wherein the stirring speed is 800-1000 r/min, and the stirring time is 20-40 min.

In the technical scheme, the crude extract solution of the loniceraedulis anthocyanin is preferably an acidic ethanol solution of the loniceraedulis anthocyanin crude extract, and the concentration of the loniceraedulis anthocyanin crude extract is 2-6 mg/mL.

Further, the acidic ethanol solution is composed of 1-1.5 mol/L acetic acid and anhydrous ethanol according to the volume ratio of 1: 4-6.

In the technical scheme, the mass ratio of the loniceraedulis anthocyanin to the chitosan is preferably 20-40: 1.

Further, the stirring speed is 800-1000 r/min, and the stirring time is 20-40 min.

In the technical scheme, a certain amount of glycerol is preferably added into the obtained starch-chitosan-loniceraedulis anthocyanin mixed solution, the pH value of the solution is adjusted to be 2-3, then the solution is continuously stirred for 20-40 min by magnetic force, and the stirring speed is 800-1000 r/min.

Further, the addition amount of the glycerol is 1-3% of the total volume of the deposition solution. The glycerol is mainly used as a plasticizer, so that the physical properties of the film are improved, and the film is prevented from being dried and cracked.

Further, the pH value of the starch-chitosan-loniceraedulis anthocyanin mixed solution is adjusted to 2-3 by using 0.1-1.0 mol/L HCl solution or 0.1-1.0 mol/L NaOH solution.

In the technical scheme, the obtained film-forming solution is preferably cast into a film-forming plate, placed in an air-blowing drying oven, and dried to form a film, wherein the drying temperature is 40-45 ℃, and the drying time is 12-24 hours.

The invention also provides the pH and NH prepared by the method₃High response type food inferior freshness level color sensitive indicating film.

The invention also provides a pH and NH₃The application of the high-response food secondary freshness color-sensitive indicating film in monitoring the shelf life of fresh products which generate volatile nitrogen-containing compounds in the process of spoilage.

A preferred technical scheme of the invention is as follows:

pH and NH₃The preparation method of the high-response food secondary fresh color sensitive indicating film comprises the following steps:

(1) gelatinizing starch with a certain concentration to obtain a gelatinized starch solution;

(2) adding the gelatinized starch solution obtained in the step (1) into a chitosan solution with a certain concentration, and magnetically stirring for 10-30 min;

(3) slowly adding an acidic ethanol solution of a lonicera edulis anthocyanin extract with a certain concentration into the starch-chitosan mixed solution obtained in the step (2), and magnetically stirring for 10-30 min;

(4) dropwise adding glycerol with a certain volume fraction into the starch-chitosan-lonicera edulis anthocyanin solution obtained in the step (3), adjusting the final pH to 2-3, and continuously stirring for 20-40 min by magnetic force;

(5) and (4) casting the solution obtained in the step (4) into a film forming plate, placing the film forming plate in an air blowing drying box, drying the film forming plate to form a film, balancing the film forming plate in a dryer for a period of time, and then removing the film.

Preferably, in the step (1), the starch is potato starch, the starch is dissolved in distilled water to form a starch solution with the concentration of 50-70 mg/mL, the starch solution is gradually heated to a starch just gelatinized state under magnetic stirring, the stirring speed is 800-1000 r/min, and the stirring time is 20-40 min.

Preferably, in the step (2), the concentration of the chitosan solution is 6-10 mg/mL, the chitosan solution is a 1% acetic acid aqueous solution of chitosan, and the stirring speed is 600-800 r/min; the mass ratio of the starch to the chitosan is 5-15: 1.

Preferably, in the step (3), the concentration of the loniceraedulis anthocyanin extract is 1-4 mg/mL, and the acidic ethanol solution is prepared by mixing 1-1.5 mol/L acetic acid and absolute ethyl alcohol in a ratio of 1: 4-6; the mass ratio of the lonicera edulis anthocyanin to the chitosan is 20-40: 1, and the stirring speed is 600-800 r/min.

Preferably, in the step (4), the final volume fraction of the glycerol is 1-3%, the final pH is adjusted to 2-4, and the stirring speed is 800-1000 r/min.

Preferably, in the step (5), the drying temperature is 40-45 ℃ and the drying time is 12-24 h.

The invention has the beneficial effects that: the indicating film is prepared by compounding two film forming materials with good physical properties, namely starch and chitosan, taking lonicera edulis anthocyanin as an indicator, and regulating and controlling the pH value of a film forming solution to prepare the food freshness indicating film with excellent physical properties and color response properties. The indicating film prepared by the method is applied to the field of intelligent packaging of food, aims to monitor and identify the secondary freshness level of the food, and provides whole-process real-time monitoring for consumers to purchase fresh food so as to accurately identify the freshness, secondary freshness and putrefaction levels of the packaged food.

Drawings

FIG. 1(a) is a graph showing the color change of a loniceraedulis anthocyanin solution used in the present invention at a pH of 2-12. The solution is red at the pH value of 2-3, the red degree of the solution is gradually reduced to be light pink and the color intensity is gradually reduced with the increase of the pH value to 4-6, the solution is dark brown at the pH value of 7, the solution is light gray at the pH value of 8-9 and the color intensity is gradually increased, the solution is purple at the pH value of 10, and the solution is brown and the color intensity is gradually increased at the pH value of 11-12; FIG. 1(b) is a UV-Vis spectrum of a loniceraedulis anthocyanin solution at a pH of 2-12. The maximum absorption peak of the anthocyanin solution under the acidic condition is near the wavelength of 520nm, and the maximum absorption wavelength of the anthocyanin moves from 520nm to 580nm along with the increase of pH, so that red shift is generated.

Fig. 2 is a result of measuring mechanical properties of the indicating films prepared in example 1, example 2 and comparative example 1 according to the present invention. Different lower case letters represent significant differences (p < 0.05).

Fig. 3 is a water solubility measurement result of the indicating films prepared in example 1, example 2 and comparative example 1 of the present invention. Different lower case letters represent significant differences (p < 0.05).

Fig. 4 is an ammonia response measurement result (a) and a color change chart (b) before and after the response of the indicating films prepared in example 1, example 2 and comparative example 1 of the present invention.

FIG. 5 is a photograph indicating that membranes of example 1(PS-CS-LCA pH2.5) of the present invention are respectively at pH 2-12. When the pH value is 2-4, the PS-CS-LCA of the indicating membrane is pink, and the redness is reduced along with the increase of the pH value; when the pH is 5 and 6, the PS-CS-LCA of the indicating membrane has a pH of 2.5 and is light purple and gray pink respectively; the a value is significantly reduced in the process of the change of the pH2-6 (3.14 → 0.74); when the pH is neutral, i.e., 7, the indicator membrane PS-CS-LCA pH2.5 appears bluish gray; when the pH value is increased to 8-9, the PS-CS-LCA pH2.5 of the indicating film respectively presents cyan and water green; when the pH value is 10, the indicating films PS-CS-LCA respectively show grayish blue color at pH2.5; when the pH value reaches the range of 11-12, the PS-CS-LCA of the indicating film with pH2.5 gradually appears blue, and the intensity of the blue color is decreased gradually.

FIG. 6(a) is the result of pH, total number of colonies measurement of an indicator film in deterioration monitoring of example 1 of the present invention; FIG. 6(b) is the result of measuring the content of volatile basic nitrogen and the Δ E value of the indicator film of example 1 of the present invention in the monitoring of deterioration. Different lower case letters represent significant differences (p < 0.05).

FIG. 7 is a graph showing the color change of the indicating film in the monitoring of the deterioration of fresh shrimps in accordance with example 1 of the present invention: red (0h) → rose red (12h) → grey pink (24h) → grey (30h) → blue grey (36 h).

FIG. 8 is the result of principal component analysis of color parameters of the indicating film of example 1 of the present invention in a fresh shrimp spoilage monitoring application.

FIG. 9 is a graph showing the color change of an indicating film of comparative example 1 of the present invention in the monitoring of the deterioration of fresh shrimps: the color nature has no obvious difference, and the gray intensity is reduced within 0-36 h.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1

A method for preparing a starch-chitosan-lonicera edulis anthocyanin food secondary fresh level color sensitive indicating film with a pH value of 2.5 comprises the following steps:

gradually heating the potato starch water solution with the concentration of 60mg/mL to 62 ℃ under magnetic stirring to enable the starch to reach a just gelatinized state, wherein the magnetic stirring speed is 800r/min, and the stirring time is 30min, so as to obtain the potato starch solution subjected to gelatinization. The chitosan is dissolved in 1% acetic acid solution to prepare 8mg/mL chitosan solution. Mixing the gelatinized potato starch solution and the chitosan solution according to the volume ratio of 1:1 to obtain a starch-chitosan mixed solution.

The loniceraedulis anthocyanin is dissolved in acidic ethanol solution (the acidic ethanol solution is composed of 1mol/L of acetic acid and absolute ethyl alcohol according to the volume ratio of 3: 17) to prepare 6mg/mL loniceraedulis anthocyanin crude extract solution.

Adding 2mL of crude extract solution of lonicera edulis anthocyanin into 80mL of starch-chitosan mixed solution to obtain starch-chitosan-lonicera edulis anthocyanin mixed solution. Adding glycerol into the starch-chitosan-loniceraedulis anthocyanin mixed solution according to the addition amount of 2.5 percent of volume fraction, adjusting the final pH of the solution to be 2.5, magnetically stirring for 30min at the stirring speed of 800r/min to obtain a film-forming solution, and preparing the final film-forming solution with the volume of about 80 mL. Pouring the film-forming solution into a film-forming plate, drying in an oven at 45 deg.C for 12 hr to form a film, balancing in a dryer for 10 hr, and removing the film to obtain starch-chitosan-loniceraedulis anthocyanin pH and NH₃The thickness of the high-response food secondary fresh grade anthocyanin indicating film is 1.5-2.0 mm, and the high-response food secondary fresh grade anthocyanin indicating film is named as PS-CS-LCA pH2.5 indicating film.

Example 2

A preparation method of a starch-chitosan-lonicera edulis anthocyanin food secondary fresh level color sensitive indicating film with a pH value of 3.0 comprises the following steps:

the final pH of the adjusted solution in example 1 was changed to pH3.0 instead of 2.5, and the remaining preparation conditions were unchanged. This was designated as PS-CS-LCA pH3.0 indicating membrane.

Comparative example 1

A preparation method of a starch-chitosan-lonicera edulis anthocyanin food secondary fresh level color sensitive indicating film with a pH value of 3.5 comprises the following steps:

the final pH of the adjusted solution in example 1 was changed to pH3.5 instead of pH2.5, and the remaining preparation conditions were unchanged. This was designated as PS-CS-LCA pH3.5 indicator membrane.

The color change and the ultraviolet-visible spectrum analysis of the loniceraedulis anthocyanin under different pH conditions are as follows:

measuring the ultraviolet-visible spectrum of the loniceraedulis anthocyanin extract solution under the condition of pH2-12 by using an ultraviolet spectrophotometer, scanning the spectrum at the wavelength of 380-780nm, drawing an ultraviolet-visible spectrum diagram, and recording the color change by using a camera.

The color change of the loniceraedulis anthocyanin solution under the condition of pH2-12 is shown in figure 1 (a). The solution is red at 2-3, gradually decreases to light pink and gradually decreases in color intensity as the pH value increases to 4-6, and is brown at pH 7, light gray at pH 8-9 and gradually increases in color intensity, purple at pH 10, and brown at pH 11-12 and gradually increases in color intensity. The UV-Vis spectrum of the loniceraedulis anthocyanin solution at pH2-12 is shown in FIG. 1 (b). The maximum absorption peak of the anthocyanin solution under the acidic condition is near the wavelength of 520nm, and the maximum absorption wavelength of the anthocyanin moves from 520nm to 580nm along with the increase of pH, so that red shift is generated. The results show that the loniceraedulis anthocyanin solution shows a remarkable color change under the condition of pH 2-12.

The mechanical properties, water solubility, apparent color, ammonia response, pH response of the indicated films of example 1 and example 2, comparative example 1 were tested and the results were as follows.

Firstly, measuring the mechanical property of the indicating film: the mechanical properties were measured by a method described in GB/T1040.3-2006 "measurement of Plastic Tensile Property", wherein a film was cut into a 100mm × 15mm long strip, and the Tensile Strength (Tensil Strength, TS) and Elongation at Break (EBA) of the film were measured by fixing the film to an electronic Tensile tester. The initial nip was 80mm, the stretching rate was 250mm/s, and the measurement was repeated 10 times for each set of films. The tensile strength and the elongation at break are calculated according to the formulas [1] and [2], respectively.

TS＝F/W×d [1]

Wherein TS is tensile strength, MPa;

f is the maximum tensile force borne by the sample when the sample is broken, and N;

w is the width of the film, mm;

d is the thickness of the film, mm.

EBA＝L₁/L₀×100 [2]

Wherein EBA is elongation at break,%;

L₁the stretched length at break of the film, mm;

L₀is the initial grip distance, mm.

Determination of water solubility of the indicator film: the indicating film is put in an oven at 105 ℃ and dried to constant mass W₁(g) Then soaking in 50mL of distilled water for 24h, pouring out the soak solution, putting into a 105 ℃ oven again to be dried to constant mass, and weighing the mass W₂(g) The assay was repeated 3 times per group. According to the formula [3]Calculating the water solubility:

water solubility/100% (W)₁-W₂)/W₁×100 [3]

The mechanical property results of the indicator film are shown in fig. 2, indicating that the tensile strength of the film increases slightly (5.98MPa → 6.43MPa) with increasing pH of the film forming solution from 2.5 to 3.0, but there is no significant difference; the tensile strength (4.74MPa) decreased significantly when the film-forming solution pH increased to 3.5. The elongation at break of the indicating film has no significant difference in the change process of the pH value of the film forming solution being 2.5-3.5. The water solubility results of the indicator film are shown in fig. 3, and the water solubility of the indicator film is 33.11% and 33.20% at pH values of the film forming solution of 2.5 and 3.0, respectively, with no significant difference; when the pH of the film forming solution was 3.5, the water solubility of the indicator film was 36.46%, which is higher than those of examples 1 and 2. In conclusion, the pH of the deposition solution had a small effect on the physical properties of the indicator film, but all showed good physical properties.

Secondly, measuring the apparent color and parameters of the indicating film: color parameters (L, a, b) were measured for the composite films of examples 1, 2 and 1 using a colorimeter, while recording the color of each composite film using a cameraColor photograph. With a standard white board (L)₀＝97.02,a₀＝-0.92,b₀2.06) as a colour difference reference according to equation [4]]And calculating the total color difference delta E value of each composite film.

TABLE 1 results of color parameter measurement of example 1, example 2 and comparative example 1

Note: different lower case letters in the same column represent significant differences, p < 0.05.

Table 1 shows color parameters (L, a, b), Δ E values and indicating film images of example 1 and example 2, comparative example 1. As the pH of the film-forming solution increased from 2.5 to 3.5, both the a value (4.72 → 2.66 → -0.59) and the b value (-0.23 → -0.54 → -0.98) of the indicator film decreased significantly. Correspondingly, the color of the indicating film of the example 1, the example 2 and the comparative example 1 is red, pink and gray. The color difference value of the indicator film (example 1) of the film-forming solution ph2.5 was the greatest (57.79) while it exhibited the greatest color intensity from the image. In conclusion, the reduction of the pH value of the film forming solution has obvious influence on the color change of the final indicating film, the color intensity of the indicating film is the largest at a low pH value, the redness is the strongest, the visual presenting effect of the indicating film is stronger, the color state of the indicating film is easy to observe, and a good basis is provided for subsequent color development application.

Thirdly, ammonia response determination: a100 mL Erlenmeyer flask containing 10mL of ammonia (20%, v/v) was covered with an indicator membrane, its colorimetric response to ammonia was determined, the color change was captured by a camera, the color parameters (L, a, b) were measured by a colorimeter, the Δ E value was calculated according to equation [4] with reference to the initial membrane color, at 1min intervals, and monitoring was continued for 20 min.

To evaluate the sensitivity of the indicator film to alkaline gases, the study performed a color-response test on volatile ammonia. Exposing the indicating film above the ammonia solution, and observing the indication through ammonia volatilizationThe color change of the film, which acts by the mechanism of ammonia gas combining with water in the indicator film to form NH₃·H₂O, then hydrolyzed to form NH⁴⁺And OH^-，OH^-The anthocyanin color can be changed, and the color of the indicating film is further changed. The ammonia response results are shown in fig. 4(a), indicating that the L, a value of the membrane varies significantly with increasing response time, gradually decreases first, and then remains substantially unchanged. Accordingly, PS-CS-LCA pH2.5, PS-CS-LCA pH3.0, and PS-CS-LCA pH3.5 indicate that the total color difference is 9.18, 8.13, and 5.86 respectively when the response time of the membrane reaches 15, 11, and 10min, and all remain stable and have no significant change thereafter. As can be seen visually in fig. 4(b), the three indicator films exhibited a distinct color change after 20min of response: PS-CS-LCA pH2.5 changes from red to water green; PS-CS-LCA pH3.0 changes from pink to blue-gray; PS-CS-LCA pH3.5 changes from red to dark cyan. The PS-CS-LCA pH2.5 (example 1) showed the highest color difference value among all the indicator films, with the best ammonia response.

In summary, comparing example 1 with example 2 and comparative example 1, example 1 shows superiority in mechanical properties, water solubility, apparent color, ammonia response, and the like. Therefore, example 1(PS-CS-LCA pH2.5 indicator membrane) was characterized for the application study.

The pH responsiveness of PS-CS-LCA pH2.5 membranes was evaluated by soaking the membranes in buffer solutions of different pH values (pH 2-12). The color parameters (L, a, b) and Δ E and the color change image are shown in table 2.

TABLE 2 color parameters and color change measurements for example 1(PS-CS-LCA pH2.5 indicator film)

Note: different letters in the same column represent significant differences, p < 0.05.

As can be observed from table 2 and fig. 5, the indicator membrane PS-CS-LCA pH2.5 is pink when pH is 2-4, and the redness decreases with increasing pH; when the pH is 5 and 6, the PS-CS-LCA of the indicating membrane has a pH of 2.5 and is light purple and gray pink respectively; the a value is significantly reduced in the process of the change of the pH2-6 (3.14 → 0.74); when the pH is neutral, i.e., 7, the indicator membrane PS-CS-LCA pH2.5 appears bluish gray; when the pH value is increased to 8-9, the PS-CS-LCA pH2.5 of the indicating film respectively presents cyan and water green; when the pH value is 10, the indicating membrane PS-CS-LCA is gray blue at the pH value of 2.5; when the pH value reaches the range of 11-12, the PS-CS-LCA of the indicating film with pH2.5 gradually appears blue, and the intensity of the blue color is decreased gradually. The indicator membrane PS-CS-LCA pH2.5 exhibits a clear, distinct color change over a pH range of 2-12. However, the pH range of product deterioration caused by microbial spoilage of most animal product proteins is approximately between 5 and 8, and the color change of the indication film PS-CS-LCA pH2.5 in the pH range is obvious and can be easily distinguished by naked eyes, so that powerful support is provided for subsequent next freshness indication.

pH and NH₃The application of the high-response food secondary fresh color sensitive indicating film comprises the following steps:

the prepared indicating film is applied to freshness monitoring of packaged shrimps, the fresh shrimps are stored for 36 hours at 4 ℃, the pH, the content of volatile basic nitrogen (TVB-N) and the change of total number of bacterial colonies (TVC) indexes of the shrimps are measured according to the operation requirements of GB 5009.237-2016 (food pH value measurement), GB 5009.228-2016 (volatile basic nitrogen measurement) and GB 4789.2-2016 (food microbiology test total number measurement), the color change of the indicating film is recorded by photographing, and the delta E value is calculated.

Measurement results (fig. 6) and color change of indicator film (fig. 7) results: when the storage time reaches 24h and 30h, the TVB-N value of the fresh shrimps is increased from the initial 9.80mg/100g to 22.87mg/100g and 29.40mg/100g respectively, the pH value of the shrimps (6.78 → 7.43 → 7.50) and the TVC value (3.17 → 4.05 → 5.35(lg (CFU/g)) are all changed remarkably, the corresponding indicator film color is changed from the initial red color to the grey pink color (24h) and the grey color (30h), the delta E value is respectively 8.01 and 8.95, and the TVB-N in the fresh seawater fish and shrimps is regulated to be less than 30mg/100g according to the national standard GB 2733-2015, so that the fresh shrimps are judged to be of a secondary freshness grade during the storage period from 24h to 30h and are the critical point of spoilage, and then the merchants and consumers are warned that the food spoilage is about to happen. When the storage time reaches 36h, the TVB-N value of the fresh shrimps is 38.50mg/100g, which indicates that the shrimps are completely rotten, and the corresponding pH value is 7.68, the TVC value is 6.37(lg (CFU/g)), and then the indicating film presents blue gray, the delta E value is 10.95, and the color change of the indicating film can be visually perceived by naked eyes.

And (3) main component analysis: each group of fresh shrimp samples are parallelly detected for 10 times, 5 multiplied by 10 multiplied by 3 dimensional data (5 represents days 0h, 12h, 24h, 30h and 36h, 10 represents the parallel test times of each day, and 3 represents R, G, B variable) are obtained after the image of the indicating membrane is subjected to feature extraction, statistical analysis is carried out on the experimental data through PCA, and a three-dimensional graph is drawn.

Characteristic signals indicative of the membrane are extracted under R, G, B color space model, and the characteristic signals are recombined into three principal components. As shown in fig. 8, the contribution rate of the first principal component is 79.69%; the contribution rate of the second principal component is 19.88%; the contribution ratio of the third main component was 0.43%. The fresh shrimp samples of 0 and 12h were judged to be fresh grade, the fresh shrimp samples of 24 and 30h were sub-fresh grade, and the fresh shrimp samples of 36h were putrefactive according to the TVB-N measurement results. The three dimensions of the main components are shown in FIG. 8, and under the storage environment of 4 ℃, the shrimp samples with different storage times have obvious clustering tendency and can be well divided into three grades of freshness grade, secondary freshness grade and putrefaction grade. The PCA results demonstrate that the prepared PS-CS-LCA indicator film with pH2.5 can be used as a secondary freshness indicator of shrimps.

Similarly, when the indicator film of comparative example 1(PS-CS-LCA ph3.5) is applied to the deterioration monitoring of fresh shrimps, the color change of the indicator film in the whole monitoring process cannot be clearly distinguished by naked eyes (fig. 9), so that the state of the monitored product is not defined, and the consumer cannot be warned that the product is about to decay.

In conclusion, the PS-CS-LCA pH2.5 optimized by regulating the pH value of the film-forming solution indicates that the film has good physical performance, ammonia response performance and pH response performance. In the application experiment result, the indicating film can obviously distinguish the fresh, sub-fresh and putrefactive grades of the fresh shrimps. Therefore, the indicating film provided by the invention can visually monitor and warn manufacturers and consumers of food spoilage in real time through color change, so as to provide reference for the shelf life monitoring of fresh products which generate volatile nitrogen-containing compounds in the spoilage process of the natural anthocyanin indicating film.