Bacterial cellulose-aloe-emodin-vanillin composite membrane, preparation method and application

Abstract

The invention belongs to the field of biotechnology and biological materials, and particularly relates to a bacterial cellulose-aloe-emodin-vanillin composite membrane, a preparation method and application. The invention provides a bacterial cellulose-aloe-emodin-vanillin composite membrane. The bacterial cellulose-aloe-emodin is formed by suspending bacterial cellulose and aloe-emodin through xanthan gum; the vanillin is cross-linked to the bacterial cellulose-aloe-rheum officinale to form a bacterial cellulose-aloe-rheum emodin-vanillin composite membrane. The composite film prepared by the invention has antibacterial activity, free radical scavenging capacity, excellent mechanical property, biocompatibility and biodegradability, can prolong the shelf life of food, and has pH responsiveness to indicate the freshness of the food.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and biomaterials, and particularly relates to a composite film of bacterial cellulose-aloe-emodin-vanillin, a preparation method and an application thereof. Background Art

[0002] Ideal food packaging is safe, biodegradable, and pollution-free to the environment. At the same time, it should have excellent barrier properties to effectively prevent the penetration of oxygen, water vapor, and light to protect the quality of food. It should also have good mechanical properties, such as high strength and flexibility, for easy processing and transportation. Heat resistance, cold resistance, and moisture stability enable it to adapt to various storage conditions. In addition, packaging materials should focus on sustainability, preferably using biodegradable or recyclable materials and preferably sourced from renewable resources. Packaging materials with intelligent functions such as time-temperature indication or pH-active packaging can further enhance food preservation effects.

[0003] Most traditional plastic polymer packaging has good mechanical strength, but most of its monomers are high molecular weight organic compounds that cannot be completely degraded in a short time. Moreover, due to low recovery rates, it is easy to cause environmental pollution and does not have intelligent functions.

[0004] Protein-rich foods are susceptible to enzymatic and microbial metabolism during storage and are extremely perishable, which not only poses a threat to human health but also causes significant economic losses. The internal environment of the packaging changes due to food spoilage, and this change can be easily detected by an intelligent colorimetric film. Therefore, intelligent colorimetric films have attracted increasing attention from researchers in food packaging. Intelligent colorimetric films consist of a solid matrix loaded with pH-sensitive dyes. However, due to their toxicity, they are not suitable for use in the development of intelligent food packaging.

[0005] In recent years, researchers have increasingly focused on natural pigments obtained from various plant sources as pH-sensitive dyes. However, among them, aloe-emodin (AE) has poor water solubility and is almost insoluble in water. The water-insolubility to a certain extent limits its wide application in fields such as food packaging. Bacterial cellulose (BC) has several significant advantages as a food packaging material, but it does not have antibacterial activity itself. Therefore, it needs to be modified to enhance its antibacterial, free radical scavenging, and other functions. Although existing BC modification methods have achieved certain results, they mostly rely on chemical modification, increasing complexity and cost, and the application effects of in-situ modification methods are not good.

[0006] In summary, the food packaging materials applied in the prior art face various challenges such as environmental protection and sustainability, and there is an urgent need to develop more efficient, environmentally friendly, and functional packaging materials. Summary of the Invention

[0007] In view of this, the present invention provides a bacterial cellulose-aloe-emodin-vanillin composite film, which can be used as an intelligent packaging material, has antibacterial activity, free radical scavenging ability and pH responsiveness, and is easily degradable, solving the technical problems of non-environmental protection and poor sustainability of packaging materials in the prior art.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention provides a composite film of bacterial cellulose-aloe-emodin-vanillin, comprising bacterial cellulose-aloe-emodin and vanillin; the vanillin is crosslinked to the bacterial cellulose-aloe-emodin; the bacterial cellulose-aloe-emodin is formed by suspending bacterial cellulose with xanthan gum and aloe-emodin.

[0010] Preferably, the preparation method of the bacterial cellulose-aloe-emodin includes: culturing Enterobacter sp. FY-07 to obtain bacterial cellulose-aloe-emodin; the components of the culture medium include N-acetylglucosamine, xanthan gum and aloe-emodin.

[0011] Preferably, using distilled water as a solvent, the composition of the culture medium includes: 0.01-5 g / L of aloe-emodin, 0.1-1.0 g / L of xanthan gum, 0.1-7.5 g / L of yeast powder, 0.1-10 g / L of peptone, 0.1-10 g / L of Na 2 HPO 4 , 0.1-1.0 g / L of KNO 3 and 0.1-25 g / L of N-acetylglucosamine.

[0012] Preferably, the temperature of the culture is 28-35 °C; the time of the culture is 22-26 h.

[0013] The present invention provides a preparation method of the composite film described in the above technical solution, comprising the following steps:

[0014] Crosslink the bacterial cellulose-aloe-emodin and the vanillin solution to obtain a bacterial cellulose-aloe-emodin-vanillin composite film.

[0015] Preferably, the volume concentration of the vanillin solution is 0.1% - 1% (w / v).

[0016] Preferably, the temperature of the crosslinking is 30-110 °C; the time of the crosslinking is 4-8 h; the rotation speed of the crosslinking is 80-120 rpm.

[0017] The present invention provides the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution for use in one or more of the following 1)-5);

[0018] 1) Food preservation;

[0019] 2) Food antioxidant;

[0020] 3) As an intelligent packaging;

[0021] 4) Bacteriostasis;

[0022] 5) As a food freshness detection reagent.

[0023] The present invention provides an application method of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution, including: using the composite film to package food.

[0024] The effective effect of the present invention: The present invention provides a composite film of bacterial cellulose-aloe emodin-vanillin, including bacterial cellulose-aloe emodin and vanillin; the vanillin is crosslinked to bacterial cellulose-aloe emodin; the bacterial cellulose-aloe emodin is formed by suspending bacterial cellulose with xanthan gum and aloe emodin.

[0025] Since aloe emodin and vanillin have bacteriostatic activity, the prepared bacterial cellulose-aloe emodin-vanillin composite film has bacteriostatic activity. Since aloe emodin is affected by the pH value and presents different colors at different pH values, the composite film can indicate the freshness of food according to pH responsiveness. In addition, like aloe emodin, vanillin has an aromatic ring with a conjugated π-electron system. This structure can disperse the energy of free radicals through resonance effects, stabilize free radicals, and reduce their oxidative damage to cells or biomolecules. In addition, the phenolic hydroxyl group (-OH) in the vanillin molecule can provide hydrogen atoms to reduce free radical molecules to relatively stable molecules, while itself is transformed into a stable phenoxy free radical, avoiding the expansion of the free radical chain reaction, so that the composite film has the ability to scavenge free radicals. In addition, the composite film prepared by the present invention also has excellent mechanical properties, biocompatibility, biodegradability and can extend the shelf life of food, which provides an important reference for the preparation of food preservation composite films in the future. Description of the Drawings

[0026] Figure 1 Detection diagram of the free radical scavenging ability of the composite film prepared in Example 1;

[0027] Figure 2 BC, BCA and BCA@Van composite films prepared in Example 1 n Diagram of the bacteriostatic effect and biofilm scavenging ability of the composite film;

[0028] Figure 3 pH responsiveness result diagram of aloe emodin solution and the composite film;

[0029] Figure 4 Application effect diagram of the film prepared in Example 1 for the preservation of salmon;

[0030] Figure 5 Biocompatibility result diagram of the film prepared in Example 1;

[0031] Figure 6 FTIR, TGA, DTG, XRD and WCA curve graphs of the film prepared in Example 1;

[0032] Figure 7 For degradable starch film, BC, BCA@Van 3 Soil natural degradation diagram of the composite film;

[0033] Figure 8 SEM surface morphology diagram of the composite film. Detailed implementation manners

[0034] The present invention provides a bacterial cellulose-aloe emodin-vanillin intelligent packaging composite film, comprising bacterial cellulose-aloe emodin and vanillin; the vanillin is crosslinked to the bacterial cellulose-aloe emodin; the bacterial cellulose-aloe emodin is formed by suspending aloe emodin in bacterial cellulose through xanthan gum.

[0035] The bacterial cellulose-aloe emodin of the present invention is formed by suspending aloe emodin in bacterial cellulose through xanthan gum. As an alternative implementation manner, the bacterial cellulose-aloe emodin of the present invention utilizes the suspending effect of xanthan gum to effectively suspend aloe emodin (AE) in the bacterial cellulose membrane (BC), so that aloe emodin (AE) is tightly bound to the cellulose network by the bacterial cellulose, and a bacterial cellulose-aloe emodin composite film is prepared. The vanillin of the present invention is crosslinked to the bacterial cellulose-aloe emodin and is formed by the crosslinking reaction of the amide bond of the bacterial cellulose and the aldehyde group of the vanillin through the Schiff base reaction.

[0036] As an alternative embodiment, the preparation method of the bacterial cellulose-aloe emodin preferably includes: culturing Enterobacter FY-07 to obtain bacterial cellulose-aloe emodin; the components of the culture medium include N-acetylglucosamine, xanthan gum, and aloe emodin. The preservation number of Enterobacter sp. FY-07 of the present invention is CGMCC No. 6103. Enterobacter sp. FY-07 of the present invention can in-situ utilize N-acetylglucosamine to ferment to obtain a bacterial cellulose membrane (BC) with amide bonds. Meanwhile, by using the suspension effect of xanthan gum, aloe emodin (AE) can be effectively suspended in BC, and then a BC-AE composite membrane is prepared by a one-step fermentation method. As an alternative embodiment, before inoculating the Enterobacter into the culture medium for culturing, a seed solution is prepared first in the present invention; the preparation method of the seed solution includes inoculating Enterobacter FY-07 on an LB-Congo red solid medium plate for activation culture to obtain single colonies; as an alternative embodiment, the temperature of the activation culture in the present invention is 28-32 °C, preferably 30 °C; the time of the activation culture is 22-26 h, more preferably 24 h. The present invention has no special limitation on the inoculation method, and a conventional method can be used. In the examples of the present invention, the inoculation method is preferably the dense streaking method.

[0037] As an alternative embodiment, in the present invention, the single colonies are inoculated on an LB solid medium for the first slant culture to obtain a first bacterial cellulose membrane; as an alternative embodiment, the temperature of the first slant culture in the present invention is 28-32 °C, preferably 30 °C; the time of the first slant culture is preferably 22-26 h, more preferably 24 h. The present invention has no special limitation on the inoculation method, and a conventional method can be used. In the examples of the present invention, the inoculation method is preferably the dense streaking method. As an alternative embodiment, in the present invention, the obtained first bacterial cellulose membrane is inoculated on an LB solid medium for the second slant culture to obtain a seed slant; the seed slant is washed and mixed with water to obtain an Enterobacter FY-07 seed solution. As an alternative embodiment, the temperature of the second slant culture in the present invention is 28-32 °C, preferably 30 °C; the time of the second slant culture in the present invention is preferably 22-26 h, more preferably 24 h. The present invention has no special limitation on the inoculation method, and a conventional method can be used. In the examples of the present invention, the inoculation method is preferably the dense streaking method. The viable count of the seed solution in the present invention is preferably 10 5 ~10 7CFU / mL. The present invention does not particularly limit the manner of rinsing and mixing, and conventional methods can be adopted. As an alternative embodiment, the composition of the LB solid medium in the present invention is: using distilled water as a solvent, including 1-5 g / L of yeast powder, 5-10 g / L of peptone, 5-10 g / L of NaCl, and 15-20 g / L of agar; more preferably: using distilled water as a solvent, including 5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of NaCl, and 20 g / L of agar, with a pH value of 7.4-7.6.

[0038] As an alternative embodiment, the present invention inoculates the Enterobacter FY-07 seed solution into a culture medium for culturing to obtain bacterial cellulose-aloe emodin. The inoculation amount of the seed solution in the present invention is preferably 1%-3% of the volume of the culture medium, more preferably 1%. As an alternative embodiment, the components of the culture medium in the present invention preferably include N-acetylglucosamine, xanthan gum, and aloe emodin. The components of the culture medium in the present invention include: 0.1-0.5 g / L of aloe emodin, 0.1-1.0 g / L of xanthan gum, 0.1-7.5 g / L of yeast powder, 0.1-10 g / L of peptone, 0.1-10 g / L of Na 2 HPO 4 , 0.1-1.0 g / L of KNO 3 and 0.1-25 g / L of N-acetylglucosamine; more preferably including: 0.1 g / L of aloe emodin, 1.0 g / L of xanthan gum, 7.5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of Na 2 HPO 4 , 1.0 g / L of KNO 3And 25 g / L N-acetylglucosamine. The culture medium of the present invention is obtained by improving on the basis of the HS-XGK culture medium. The glucose in the HS-XGK culture medium is replaced with N-acetylglucosamine. Enterobacter FY-07 uses N-acetylglucosamine as a carbon source. By means of a sustainable in-situ modification method, the amide bond in N-acetylglucosamine is introduced into BC. An amide bond is added in-situ to the 2nd carbon of bacterial cellulose, obtaining bacterial cellulose (BC) containing an amide bond. At the same time, by utilizing the suspension effect of xanthan gum, aloe-emodin is suspended in the bacterial cellulose containing an amide bond, obtaining a BC-aloe-emodin composite film with an amide bond. The sustainable in-situ modification of bacterial cellulose in the present invention means that during the synthesis process of bacterial cellulose, N-acetylglucosamine is directly added to the fermentation system, thereby obtaining a bacterial cellulose material with specific properties. Using N-acetylglucosamine (GlcNAc) as a carbon source, there are some components of the phosphotransferase system (PTS) in the bacterial cellulose-producing bacteria. Besides glucose, glucosamine can also be transported as a fermentation substrate through PTS. N-acetylglucosamine is sequentially converted into GIcNAc-1-P (N-acetylglucosamine-1-phosphate) and UDP-GlcNAc (uridine diphosphate-N-acetylglucosamine) in the bacteria. UDP-GlcNAc is continuously transferred to the newly formed polysaccharide chain under the action of cellulose synthase, forming a β-(1→4)-D-glucan chain and being secreted extracellularly through the outer membrane, forming a cellulose membrane with an amide bond at the 2nd carbon. This amide bond is derived from the carbon source N-acetylglucosamine. As an alternative embodiment, the temperature of the culture in the present invention is 28-35 °C, preferably 30 °C; the culture time is 22-26 h, preferably 24 h.

[0039] As an alternative embodiment, the present invention takes out the cultured film-like substance, and after sequentially rinsing, lysing, and washing the cultured film-like substance, a bacterial cellulose-aloe-emodin wet film is obtained. In the present invention, the rinsing is preferably completed with tap water, and the rinsing removes most of the bacteria and impurities; the bacterial cellulose-aloe-emodin wet film after rinsing in the present invention is lysed. The lysis is preferably completed with 0.2% (w / v) lysozyme. After mixing the bacterial cellulose-aloe-emodin wet film with the lysozyme solution, it is soaked; the temperature of the lysis is 30 °C, and the lysis time is preferably 2 h. After the soaking is completed, the lysozyme solution is removed, and then the bacterial cellulose-aloe-emodin wet film is washed. The washing is preferably completed with deionized water; the function of the washing is to make the bacterial cellulose-aloe-emodin wet film neutral and improve the accuracy of pH response.

[0040] Aloe-emodin (AE) is an anthraquinone compound with the chemical formula C 15 H 10 O 5, mainly derived from the dried roots and rhizomes of Rheum palmatum, Rheum tanguticum or Rheum officinale, which are plants of the Polygonaceae family. The anthraquinone structure of AE undergoes mutual isomerization when the pH value changes. When the environment is alkaline, AE shows a red anthraquinone structure. Under acidic conditions, AE has a macroscopic yellow anthranol structure. The ratio of the anthraquinone structure to the anthranol structure is affected by the pH value. Therefore, it exhibits different colors macroscopically. And AE has broad-spectrum antibacterial activity. Its function is 1,8-dihydroxy-3-hydroxymethyl anthraquinone, which is coplanar with three rings. AE can easily enter bacteria and slow down their metabolism. The hydroxyanthraquinone structure can cause apoptosis of bacterial cells. However, due to the poor water solubility of AE itself, it is almost insoluble in water. If only physically mixed, phase separation may occur and a stable structure may not be formed. The water insolubility limits its wide application in the fields of food packaging to a certain extent.

[0041] Bacterial cellulose (BC) has several significant advantages as a food packaging material. First of all, BC is synthesized from natural sources. It is an extracellular polysaccharide nanofiber polymer produced by certain bacteria through enzymatic reactions. It has high strength and crystallinity, excellent biocompatibility and safety, does not contain chemical additives, and poses no threat to food safety. Secondly, it has excellent mechanical properties, high strength and good toughness, and can maintain the structural integrity under dry and wet conditions. In addition, BC has good gas and liquid barrier properties, effectively delaying food oxidation and moisture absorption, and extending the shelf life. BC also has high customizability and can adjust the thickness, porosity and mechanical properties according to needs. And BC is also biodegradable, meeting the requirements of environmental protection and sustainable development. BC films also have high transparency and good appearance, which can enhance the visual attractiveness of packaging. Generally speaking, bacterial cellulose is an ideal food packaging material with strong functionality, high safety, environmental protection and sustainability. However, BC itself does not have antibacterial activity and needs to be modified to further enhance its antibacterial, free radical scavenging and food shelf life extension properties. Although the existing BC modification methods have achieved certain results, they mostly rely on chemical modification, increasing complexity and cost, and the application effect of in-situ modification methods is not good.

[0042] The present invention prepares a bacterial cellulose-aloe-emodin-vanillin composite film. By in-situ suspending aloe-emodin in the culture medium and obtaining the bacterial cellulose-aloe-emodin composite film through a one-step fermentation method, the in-situ modification method of bacterial cellulose is simple and low-cost. At the same time, it solves the problem that aloe-emodin is poorly water-soluble and difficult to directly utilize. Additionally, the cross-linking combined with vanillin solves the problem that the antibacterial effect of single aloe-emodin is not good and the antioxidant ability is poor, resulting in an unsatisfactory food preservation effect, and the application effect is good. In the bacterial cellulose-aloe-emodin-vanillin composite film prepared by the present invention, vanillin and aloe-emodin synergistically have strong antibacterial activity and free radical scavenging ability; it can be used for food preservation, and combined with the pH responsiveness of aloe-emodin, it can be used to intelligently indicate the spoilage of food; the preservation period is effectively extended. Moreover, the bacterial cellulose-aloe-emodin-vanillin composite film has good biocompatibility and can be degraded under natural conditions, being safe and non-toxic.

[0043] The present invention provides a preparation method of the composite film described in the above technical solution, including the following steps:

[0044] Cross-link the bacterial cellulose-aloe-emodin and vanillin solutions to obtain a composite film of bacterial cellulose-aloe-emodin and vanillin.

[0045] The preparation method of the bacterial cellulose-aloe-emodin described in the present invention has been elaborated above and will not be repeated here.

[0046] As an alternative embodiment, the present invention cross-links the bacterial cellulose-aloe-emodin and vanillin solutions to obtain a bacterial cellulose-aloe-emodin film; more preferably, cross-links the wet bacterial cellulose-aloe-emodin film and the vanillin solution to obtain a bacterial cellulose-aloe-emodin-vanillin composite film. As an alternative embodiment, during the cross-linking process, the vanillin solution can just submerge the bacterial cellulose-aloe-emodin film. The submerging can achieve the complete cross-linking of vanillin and bacterial cellulose. As an alternative embodiment, the volume concentration of the vanillin solution in the present invention is 0.1% - 0.3% (w / v), more preferably 0.3% (w / v).

[0047] The present invention cross-links vanillin molecules to the wet bacterial cellulose-aloe-emodin film by Schiff base reaction, and cross-links through the spontaneous Schiff base reaction between the amide bond on the carbon skeleton of the bacterial cellulose film and the carbonyl group of the vanillin molecule to obtain a bacterial cellulose-aloe-emodin-vanillin composite film. The temperature of the cross-linking in the present invention is preferably 30 - 110 °C, more preferably 40 °C; the time of the cross-linking is preferably 4 - 8 h, more preferably 6 h; the rotation speed of the cross-linking is preferably 80 - 120 rpm, more preferably 100 rpm.

[0048] The preparation method of the bacterial cellulose-aloe-emodin-vanillin composite film of the present invention is simple and easy to implement.

[0049] The present invention provides the application of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution in food preservation.

[0050] As an alternative embodiment, the food in the present invention is meat and / or fruit, and the meat includes but is not limited to beef, mutton, chicken, pork and seafood; the seafood includes fish; the fruit includes but is not limited to apples, peaches, grapes and pears. In the embodiments of the present invention, salmon is used as the research object to verify the application effect of the bacterial cellulose-aloe-emodin-vanillin composite film.

[0051] As an alternative embodiment, the application method preferably includes: wrapping the food with the composite film. When used for food preservation, the application amount of the composite film in the present invention is preferably 10-15 cm 2 / g, and can also be 12 cm 2 / g.

[0052] The composite film of the present invention can effectively inhibit the growth of spoilage bacteria, and its antioxidant effect effectively prevents the oxidation of lipids and proteins, maintains the color of the food, avoids the generation of peculiar smells, effectively maintains the sensory quality of the food, and extends the shelf life.

[0053] The present invention provides the application of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution in antibacterial. As an alternative embodiment, the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria include Staphylococcus aureus (S. aureus), and the Gram-negative bacteria include Escherichia coli (E. coli). In the specific embodiments of the present invention, Staphylococcus aureus numbered ATCC 29213 and Escherichia coli numbered MG1655 are used for effect verification.

[0054] The application method of the present invention has been discussed above and will not be elaborated here. As an alternative embodiment, when used for antibacterial, the application amount of the composite film in the present invention is preferably 30-40 mg / mL or 30-40 mg / g. The composite film prepared by the present invention has a strong antibacterial effect, has broad-spectrum antibacterial properties, and can achieve food preservation.

[0055] The present invention provides the application of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution in food antioxidant. The technical features of the food in the present invention have been described above and will not be repeated here. The food antioxidant in the present invention is achieved through free radical scavenging. As an alternative embodiment, the free radical scavenging in the present invention preferably includes ABTS· + (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging and ·OH (hydroxyl radical) scavenging.

[0056] The application method of the present invention has been described above and will not be repeated here. As an alternative embodiment, when used for free radical scavenging, the application amount of the composite film is preferably 30-40 mg / mL or 30-40 mg / g. The composite film prepared by the present invention has a strong free radical scavenging ability and can achieve food antioxidant.

[0057] The present invention provides the application of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution as an intelligent packaging. The intelligent packaging in the present invention can indicate the freshness of food.

[0058] The present invention provides the application of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution as a food freshness detection reagent.

[0059] The composite film of bacterial cellulose-aloe emodin-vanillin prepared by the present invention also has pH responsiveness due to the presence of aloe emodin, showing different color reactions under different pH conditions. At the beginning of storage, the composite film is light brown, and as the pH increases, the color of the composite film gradually changes to reddish brown, dark red, and finally dark red under alkaline conditions, that is, the color of the composite film gradually deepens as the pH increases. The increase in pH indicates that the freshness of the food has decreased compared to when it was first stored.

[0060] The present invention provides an application method of the composite film described in the above technical solution or the composite film prepared by the preparation method described in the above technical solution, including: packaging food with the composite film.

[0061] Although traditional plastic polymer packaging has good mechanical strength, it is usually made of high molecular organic compounds that are difficult to degrade, with low recovery rates and is prone to environmental pollution. Such packaging materials cannot be completely degraded in a short period of time, causing a long-term environmental burden. In intelligent food packaging, natural pigments are needed to replace synthetic chemical dyes to avoid toxicity problems. However, natural pigments (such as aloe-emodin) themselves have poor water solubility and are difficult to form a stable structure, which limits their effectiveness in practical applications. Although BC, as an ideal food packaging material, has good biocompatibility, mechanical properties and degradability, it does not have antibacterial activity itself. Therefore, it needs to be modified to enhance its antibacterial, free radical scavenging and other functions. To solve this problem, the present invention provides a bacterial cellulose-aloe-emodin-vanillin composite film. Based on the characteristics of the Enterobacter sp. FY-07 strain, which has strong environmental competitiveness, strong tolerance, a unique deep fermentation method, and a fast growth and production rate, using N-acetylglucosamine as a carbon source, the present invention introduces amide bonds into bacterial cellulose (BC) by a sustainable in-situ modification method, adding amide bonds in-situ at the 2nd carbon of bacterial cellulose. At the same time, using the suspension effect of xanthan gum, aloe-emodin is suspended in the HS-XGK medium to make the bacterial cellulose and aloe-emodin mix evenly to form a suspension system, obtaining an in-situ modified bacterial cellulose-aloe-emodin composite film. Then, through a green, environmentally friendly, simple and effective Schiff base reaction, vanillin molecules are cross-linked to the bacterial cellulose-aloe-emodin composite film. The prepared bacterial cellulose-aloe-emodin-vanillin composite film has multiple effects for food preservation, such as extending the shelf life of salmon, antioxidant capacity and antibacterial properties, providing important reference significance for the preparation of food packaging composite films in the future.

[0062] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0063] Example 1

[0064] The composition of the LB solid medium is: using distilled water as a solvent, 5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of NaCl, and 15 - 20 g / L of agar, with a pH value of 7.4 - 7.6.

[0065] The strain Enterobacter sp. FY-07 was isolated from the produced fluid of oilfields and deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. Its deposit number is CGMCC No. 6103, and the deposit date is May 11, 2012. Enterobacter sp. FY-07 is described in Chinese Patent CN102690773A, "A strain of Enterobacter FY-07 and its method for static liquid deep fermentation to produce bacterial cellulose". The xanthan gum was purchased from Meihua Group.

[0066] I. Preparation of Bacterial Cellulose-Aloe-emodin Composite Membrane (BCA)

[0067] 1. Add Congo red to the LB solid medium, denoted as LB-Congo red. The mass concentration of Congo red in the LB solid medium is 0.1% (w / v), and prepare an LB-Congo red medium plate.

[0068] Streak the Enterobacter sp. FY-07 bacterial solution on the LB-Congo red medium plate, and then incubate the plate at 30 °C for 24 h.

[0069] 2. Pick the large and red single colonies obtained in step 1 and streak them densely on the LB solid medium. The solid medium solidifies in a test tube to form an inclined plane, that is, an inclined plane medium, and incubate at 30 °C for 24 h.

[0070] 3. Pick some bacterial cellulose films from the inclined plane medium obtained in step 2 and streak them densely on the LB medium. The bacterial cellulose films contain a large number of active bacteria. Then incubate at 30 °C for 24 h to obtain an inclined plane medium containing bacterial cellulose membranes.

[0071] 4. Rinse the inclined plane medium obtained in step 3 with 100 mL of sterilized distilled water. Rinse repeatedly until the bacterial cellulose membrane on the inclined plane medium detaches. The bacterial cellulose membrane contains a large number of bacteria. Place the detached bacterial cellulose membrane in the 100 mL of distilled water used for the above rinsing, mix well, and use it as a seed solution. The viable count of the seed solution is 1×10 7 CFU / mL.

[0072] 5. Prepare the HS-XGK-A fermentation medium: Using distilled water as the solvent, the composition of the medium is: 0.5 g / L aloe-emodin, 1.0 g / L xanthan gum, 7.5 g / L yeast powder, 10 g / L peptone, 10 g / L Na 2 HPO 4 ,1.0 g / L KNO 3And 25 g / L N-acetylglucosamine, without pH adjustment. When preparing the HS-XGK-A fermentation medium, xanthan gum and aloe-emodin should be added last. Specifically: when adding xanthan gum, it should be added slowly with stirring. After the addition, it should be placed in a shaker at 30 °C and mixed until completely dissolved. Subsequently, aloe-emodin should be added and mixed evenly. After autoclaving, the HS-XGK-A fermentation medium is obtained.

[0073] 6. Take the seed solution prepared in step 4 and inoculate it into the HS-XGK-A fermentation medium prepared in step 5 at a ratio of 1% by volume. After mixing evenly, place it in an incubator and cultivate statically at 30 °C for 24 h.

[0074] 7. Scoop out the bacterial cellulose membrane produced by the fermentation in step 6, wash it with water to remove most of the bacteria and impurities, and then keep it in a lysozyme solution at 30 °C for 2 h. The mass concentration of the lysozyme solution used is 0.2% (w / v). After that, wash and soak it with deionized water multiple times until the pH value of the deionized water is neutral, obtaining a bacterial cellulose-aloe-emodin composite wet membrane. Perform air drying treatment on the bacterial cellulose-aloe-emodin wet membrane at room temperature to obtain a bacterial cellulose-aloe-emodin composite dry membrane, denoted as BCA.

[0075] II. BCA@Van 1 BCA@Van 2 and BCA@Van 3 Preparation

[0076] 1. The same as step 1 in the preparation of BCA;

[0077] 2. The same as step 2 in the preparation of BCA;

[0078] 3. The same as step 3 in the preparation of BCA;

[0079] 4. The same as step 4 in the preparation of BCA;

[0080] 5. The same as step 5 in the preparation of BCA;

[0081] 6. The same as step 6 in the preparation of BCA;

[0082] 7. Scoop out the bacterial cellulose membrane produced by the fermentation in step 6, wash it with water to remove most of the bacteria and impurities, use a lysozyme solution with a concentration of 0.2% (w / v) of itself, immerse the bacterial cellulose membrane in the lysozyme solution, and then keep it at 30 °C for 2 h. After that, wash and soak it with deionized water multiple times until the pH value of the deionized water is neutral, obtaining a bacterial cellulose-aloe-emodin wet membrane;

[0083] 8. The purified membrane obtained in step 7 was immersed in 100 mL of vanillin solutions with different concentrations. The vanillin solutions submerged the membrane, and the concentrations of the vanillin solutions used were 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) respectively. Then, it was gently shaken at 40 °C and 100 rpm for cross-linking reaction for 6 h. Subsequently, it was thoroughly washed with deionized water to remove any unreacted vanillin reagent, and BCA@Van was obtained. 1 Wet membrane, BCA@Van 2 Wet membrane, BCA@Van 3 Wet membrane, BCA@Van 1 Wet membrane, BCA@Van 2 Wet membrane, BCA@Van 3 The wet membranes represent vanillin concentrations of 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) respectively.

[0084] 9. For BCA@Van 1 Wet membrane, BCA@Van 2 Wet membrane, BCA@Van 3 The wet membranes were air-dried at room temperature (20 - 35 °C) to obtain BCA@Van 1 Dry membrane, BCA@Van 2 Dry membrane and BCA@Van 3 Dry membrane, denoted as BCA@Van 1 , BCA@Van 2 and BCA@Van 3 .

[0085] III. Preparation of bacterial cellulose BC

[0086] 1. The same as step 1 in the preparation of BCA;

[0087] 2. The same as step 2 in the preparation of BCA;

[0088] 3. The same as step 3 in the preparation of BCA;

[0089] 4. The same as step 4 in the preparation of BCA;

[0090] 5. Preparation of BC fermentation medium: xanthan gum 1.0 g / L, yeast powder 7.5 g / L, peptone 10 g / L, Na 2 HPO 4 10 g / L, KNO 3 1.0 g / L and N-acetylglucosamine 25 g / L, pH does not need to be adjusted. When preparing the medium, xanthan gum was added last. Specifically: when adding xanthan gum, it should be added slowly with stirring. After adding, it was placed at 30 °C and shaken and mixed until completely dissolved. After autoclaving, BC fermentation medium was obtained;

[0091] 6. Take the seed solution prepared in step 4 and inoculate it into the BC fermentation medium prepared in step 5 at a ratio of 1% by volume. After mixing evenly, place it in an incubator and perform static fermentation at 30 °C for 24 h.

[0092] 7. Fish out the bacterial cellulose membrane produced by fermentation in step 6, wash it with water to remove most of the bacteria and impurities, add a lysozyme solution with a mass concentration of 0.2% (w / v). After maintaining it at 30 °C for 2 h, wash and soak it with deionized water multiple times until the pH value of the deionized water = 7, thereby obtaining a bacterial cellulose membrane with amide bonds introduced in-situ; perform natural air drying treatment on the bacterial cellulose membrane with amide bonds introduced in-situ at room temperature (20 - 35 °C) to obtain a dry bacterial cellulose membrane with amide bonds introduced in-situ, namely BC.

[0093] IV. Preparation of BCV Membrane

[0094] 1. The same as step 1 in the preparation of BC.

[0095] 2. The same as step 2 in the preparation of BC.

[0096] 3. The same as step 3 in the preparation of BC.

[0097] 4. The same as step 4 in the preparation of BC.

[0098] 5. The same as step 5 in the preparation of BC.

[0099] 6. The same as step 6 in the preparation of BC.

[0100] 7. Fish out the bacterial cellulose membrane produced by fermentation in step 6, wash it with water to remove most of the bacteria and impurities, add a lysozyme solution with a mass concentration of 0.2% (w / v). After maintaining it at 30 °C for 2 h, wash and soak it with deionized water multiple times until the pH value of the deionized water = 7, thereby obtaining a bacterial cellulose membrane with amide bonds introduced in-situ.

[0101] 8. Immerse the purified membrane obtained in step 7 into 100 mL of vanillin solutions with different concentrations. The vanillin solution submerges the membrane, and the concentrations of the vanillin solutions used are 0.3% (w / v). Then gently shake it at 40 °C and 100 rpm for cross-linking reaction for 6 h. Subsequently, wash it thoroughly with deionized water to remove any unreacted vanillin reagent, and perform natural air drying treatment at room temperature (20 - 35 °C) to obtain the BCA membrane.

[0102] BC, BCV, BCA, BCA@Van prepared in Example 1 1 and BCA@Van 2 and BCA@Van 3The dry film was used in the experiments of Examples 2 to 6 below.

[0103] Example 2 Free Radical Scavenging Experiment

[0104] 1. ABTS + Free radicals were formed by the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The membrane specimens (BC, BCA, BCA@Van 1 , BCA@Van 2 and BCA@Van 3 ) were respectively cut into 2×2 cm 2 specifications, then immersed in 4.5 mL of distilled water, centrifuged at 8000 rpm for 10 min, and the supernatant was collected for detection.

[0105] The ABTS detection was carried out according to the kit. The catalog number of the kit was Cat#R24146, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0106] The scavenging activity of ABTS + was calculated according to formula (1):

[0107] ABTS· + scavenging activity (%) = [(A c - A t ) / A c × 100 (1)

[0108] In formula (1), A c and A t respectively represent the absorbance values of the control group and the sample.

[0109] 2. The detection of hydroxyl radical scavenging ability was determined by the Fenton colorimetric method. The detection principle is that H 2 O 2 / Fe 2+ generates hydroxyl radicals through the Fenton reaction and oxidizes Fe 2+ to Fe 3+ , resulting in the oxidation of the red o-phenanthroline-Fe 2+ to the colorless o-phenanthroline-Fe 3+ , causing the disappearance of the maximum absorption peak of o-phenanthroline-Fe 2+ at 536 nm. The change in absorbance at 530 - 540 nm can be measured by a spectrophotometer, and based on this, the change in the content of hydroxyl radicals can be calculated, and then the hydroxyl radical scavenging rate or scavenging ability of the sample can be calculated. The membrane specimens (BC, BCA, BCA@Van 1 , BCA@Van 2 and BCA@Van 3)(2×2 cm 2 ) was immersed in 4.5 mL of distilled water. After centrifugation at 8000 rpm for 10 min, the supernatant was collected for subsequent detection.

[0110] The detection was carried out using a kit with the product number R30345, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0111] The absorbance value at 530 - 540 nm was measured to calculate the hydroxyl radical scavenging rate or scavenging ability of the sample. The calculation formula is as follows:

[0112] Hydroxyl radical scavenging rate of tissue sample (%) = [(C 3 - C 3 ’) - (C 2 - C 0 )] / [(C 1 - C 0 ) - (C 2 - C 0 )] × 100 (2)

[0113] In formula (2), C 0 is the absorbance value of the blank tube, C 1 is the absorbance value of the undamaged tube, C 2 is the absorbance value of the damaged tube, C3’ is the absorbance value of the control tube, and C 3 is the absorbance value of the measurement tube.

[0114] The results are shown in Figure 1 , and it can be seen that the BC sample showed limited antioxidant activity due to the lack of antioxidant functional groups. BCA had a certain free radical scavenging ability, scavenging 20.83% of ABTS· + , and scavenging 35.45% of the hydroxyl radical (·OH). This is because the chemical structure of aloe-emodin has a conjugated aromatic structure with antioxidant properties, which can disperse the energy of free radicals. Through conjugation, free radicals form a relatively stable resonance structure in the molecule, reducing their oxidative damage to biomolecules. And BCA@Van 1 had scavenging activities against ABTS· + and ·OH of 40.28% and 52.31% respectively. This efficiency increased with the increase in the concentration of vanillin. BCA@Van 2 had scavenging activities against ABTS· + and ·OH of 58.79% and 67.07% respectively. BCA@Van 3 had scavenging activities against ABTS· +The scavenging rate of free radicals was 69.44%, and the scavenging rate of ·OH was 80.69%. This is because vanillin, like aloe-emodin, has an aromatic ring with a conjugated π-electron system. This structure can disperse the energy of free radicals through resonance effects, stabilize free radicals, and reduce their oxidative damage to cells or biomolecules. In addition, the phenolic hydroxyl group (-OH) in the vanillin molecule can provide hydrogen atoms to reduce free radical molecules to relatively stable molecules, while itself being transformed into a stable phenoxy free radical, avoiding the expansion of the free radical chain reaction. These results all indicate that the BCA@Van composite film has a strong free radical scavenging ability. Free radicals are an important factor leading to the oxidative deterioration of food. It can be seen that the BCA@Van composite film has great potential to inhibit the oxidation reactions of lipids, proteins and other components in substances such as meat, thereby delaying food spoilage and flavor deterioration.

[0115] Study on the antibacterial activity of the composite film in Example 3

[0116] The antibacterial activity of the composite film was evaluated by the agar diffusion method and colony-forming unit (CFU) counting.

[0117] 1. Agar diffusion method.

[0118] (1) The common Gram-positive bacterium Staphylococcus aureus (S. aureus, ATCC 29213) and the Gram-negative bacterium Escherichia coli (E. coli, MG1655) were selected to verify the broad-spectrum antibacterial activity of the film.

[0119] (2) The bacterial suspensions of Staphylococcus aureus ATCC 29213 and Escherichia coli MG1655 cultured overnight were respectively adjusted to an OD 600 value of 1.0; then 1 mL of each bacterial suspension was added to 100 mL of melted LB solid medium with an agar mass concentration of 1% for the LB solid medium; after the LB solid medium solidified, the ATCC 29213 plate and the MG1655 plate were obtained.

[0120] (3) The BCA, BC, BCA@Van 1 , BCA@Van 2 and BCA@Van 3 prepared in Example 1 were pre-cut into composite film samples with a diameter of 9 mm; 5 composite film samples were respectively taken and placed on the ATCC 29213 plate and the MG1655 plate, and then the culture medium plates were incubated at 37 °C for 12 h, photographed and the diameter of the inhibition zone was measured. Three parallel experiments were set up.

[0121] 2. Colony-forming unit (CFU) counting

[0122] Treatment 1: Add BC, BCA, and BCA@Van prepared in Example 1 1 , BCA@Van 2 and BCA@Van 3 composite membranes into the Staphylococcus aureus (ATCC 29213) bacterial suspension at a concentration of 30 mg / mL respectively. The volume of the bacterial suspension is 30 mL, and the viable bacteria count in the bacterial suspension is 1×10 6 cells / mL; the ATCC 29213 bacterial suspension without any composite membrane sample is used as Control Group 1.

[0123] Treatment 2: Add BC, BCA, and BCA@Van 1 , BCA@Van 2 and BCA@Van 3 composite membranes into the bacterial suspension of Escherichia coli (MG1655) at a concentration of 30 mg / mL respectively. The volume of the bacterial suspension is 30 mL, and the viable bacteria count in the bacterial suspension is 1×10 6 cells / mL; the MG1655 bacterial suspension without any composite membrane sample is used as Control Group 2.

[0124] For Treatments 1 - 2 and Control Groups 1 - 2, three parallel experiments are set up. After Treatments 1 - 2 and Control Groups 1 - 2 are incubated at 37°C and 180 rpm for 6 h, the bacterial suspension is serially diluted, and 100 μL is taken and spread on the agar plate. After incubation at 37°C for 24 h, the viable bacteria count is counted, and the survival rate is calculated. The survival rate formula is:

[0125] Survival rate (%) = (N e / N c )×100 (3)

[0126] In the formula, N e and N c are the viable bacteria counts of the treatment group and the control group respectively; the average value and standard deviation are calculated from the results of the three parallel experiments for each treatment.

[0127] 3. Antibiofilm ability

[0128] Experimental group: Bacterial suspensions of Staphylococcus aureus ATCC 29213 and Escherichia coli MG1655 (10 6 CFU mL -1) Inoculate into 24-well plates and incubate at 37 °C to form mature biofilms. Add the composite membranes of samples BC, BCA, BCA@Van1, BCA@Van2, and BCA@Van3 into the wells respectively, with one kind of composite membrane added to each well. After incubating for 24 h, discard the samples and wash the wells with PBS. Fix the bottom biofilms with 95% ethanol and stain them with 0.1% crystal violet solution; set up 3 parallel experiments.

[0129] Bacterial control group: Only contains bacterial suspension, without adding composite membranes, and the rest are all the same as the experimental group. Set up 3 parallel experiments.

[0130] Bacteria-free control group: Only add 95% ethanol and 0.1% crystal violet solution; the addition amounts are the same as those in the experimental group. Set up 3 parallel experiments.

[0131] For each group, measure the OD 595 using a microplate reader (PerkinElmer, USA).

[0132] Calculate the biofilm formation index (BFI) using the following equation: BFI = (OD i - OD c ) / OD s (4)

[0133] In formula (4), the OD values of the experimental group, bacteria-free control group, and bacterial control group are OD i , OD c and OD s . According to the calculated BFI values, the intensity of biofilm formation is classified into the following categories: no biofilm (<0.35), weak biofilm (≈0.35 - 0.69), medium-strength biofilm (≈0.70 - 1.09), and strong biofilm (>1.10).

[0134] The results are shown in Figure 2 and Tables 1 - 2. In Figure 2 , A is the agar diffusion diagram of the prepared membranes against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). (a) - (e) represent the samples of BC, BCA, BCA@Van 1 , BCA@Van 2 , BCA@Van 3 , BCA@Van 3 respectively. It can be seen that the inhibition zone diameters of BCA@Van 3 against Escherichia coli and Staphylococcus aureus are 23.86 ± 0.15 mm and 22.73 ± 0.17 mm respectively.

[0135] Table 1 Inhibition zone diameters of BC, BCA, and BCA@Vann composite membranes after co-culturing for 24 h

[0136]

[0137] Figure 2 In it, B to C respectively represent those related to BC, BCA, BCA@Van 1 , BCA@Van 2 and BCA@Van 3 Photographs and quantitative analysis graphs of the colonies of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) that survived after incubation with the composite film for 6 h. The bar graph represents the quantitative analysis graph. The survival rate of E. coli after treatment with BCA@Van within 6 h was 2.52%, and the inhibition rate against E. coli was 97.48%; within 6 h, BCA@Van 3 completely eradicated S. aureus. These findings highlight the significant potential of the BCB@PEI composite film in eliminating microorganisms in water. 3

[0138] Table 2 Quantitative analysis results (%) of the colonies of E. coli and S. aureus that survived in the culture solution after co-incubation for 6 h

[0139] MG1655 viability error ATCC29213 viability error Control Group 1 100 3.00996 Control Group 2 100 3.61798 BC 99.44 5.68098 BC 100.37 3.06913 BCA 63.49 1.43742 BCA 57.77 5.66643 BCA@Van1 24.09 2.0681 BCA@Van1 20.60 2.70691 BCA@Van2 13.63 4.27878 BCA@Van2 10.49 1.58474 BCA@Van3 2.52 0.80112 BCA@Van3 0 0

[0140] Figure 2 In it, D shows the anti-biofilm abilities of BC, BCA, BCA@Van 1 , BCA@Van 2 and BCA@Van 3 The composite film. Since BC lacks active functional groups for inhibiting biofilms, it did not show any ability to weaken biofilms. However, the BCA composite film showed a reduced ability to form biofilms of E. coli and S. aureus, showing a medium-strength biofilm; after co-treatment with BCA@Van 1 , both bacteria formed medium-strength biofilms; after co-treatment with BCA@Van 2 , both bacteria formed weak biofilms; while after co-treatment with BCA@Van 3 , no biofilm formation occurred.

[0141] These results indicate that the BCA@Van n composite film can not only effectively inhibit bacterial growth, but also show the potential for long-term antibacterial applications by inhibiting biofilm formation.

[0142] Example 4 pH responsiveness of the composite film

[0143] ​Protein-rich foods are susceptible to the effects of enzyme and microbial metabolism during storage and are extremely perishable, which not only poses a threat to human health but also causes significant economic losses. The internal environment of the packaging changes due to food spoilage. Therefore, if food packaging materials can detect and indicate the pH value of salmon to ensure its freshness and safety, it will bring the best experience to consumers.

[0144] 1. pH responsiveness of aloe-emodin solution and composite film

[0145] Accurately weigh 1 mg of aloe-emodin into 10 mL of water and dissolve it with ultrasonic assistance to a final concentration of 0.1 mg / mL. Use 0.1 mol / L NaOH solution or 0.1 mol / L HCl solution to accurately adjust the pH value of the aloe-emodin solution (pH = 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Take pictures and record the color changes of the solution at room temperature (25 °C). Use a UV-visible spectrophotometer (UV-Vis) to detect the UV-visible absorption spectrum (350 - 750 nm) of the aloe-emodin solution under different pH conditions.

[0146] Use 0.1 mol / L NaOH solution or 0.1 mol / L HCl solution to accurately adjust solutions with different pH values (pH = 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), and immerse the BCA@Van 3 composite film into the solutions with different pH values and quickly take it out. Start timing when the composite film is taken out of the solution, and take pictures and record the color changes of the solution and the accurate time of color response at room temperature (25 °C). Use a colorimeter (Model LS171, Linshang Technology Co., Ltd., China) to measure the color index with air as the blank. The color difference (ΔE) is calculated according to the equation:

[0147]

[0148] In formula (5), L represents the brightness of the material. The larger the L value, the higher the brightness; a represents the red-green intensity of the material, with positive values representing red and negative values representing green; b represents the yellow-blue intensity of the material, with positive values representing yellow and negative values representing blue. Among them, L 0 、a 0 and b 0 are the color parameters of the standard whiteboard.

[0149] 2. Reversibility of acid-base color switching in response to volatile ammonia

[0150] Add 80 mL of ammonia water (ammonia water concentration is 0.8 mol / L) to a 250 mL beaker. After it has fully volatilized, immerse the BCA@Van 3The composite film was placed 1 cm above the liquid surface, and the beaker was sealed. Then, 80 mL of volatile hydrochloric acid solution (0.8 mol / L) was prepared and added to a 250 mL beaker and sealed. After the BCA@Van had completed the color response, 3 it was taken out, and after measuring the color index using a color difference meter, it was quickly placed 1 cm above the hydrochloric acid liquid surface, and the beaker was sealed. After the composite film had completely responded to the color change, it was quickly taken out and placed 1 cm above the ammonia liquid surface again. A total of six repeated experiments were carried out, and the color index was recorded using a color difference meter (Reference: https: / / www.sciencedirect.com / science / article / pii / S0144861719306976).

[0151] The results are shown in Figure 3 and Table 3. Figure 3 In A of [reference], the color changes of the aloe-emodin solution under different pH conditions are shown. Under natural conditions (pH = 7), the composite film is light brown, and as the pH increases, the color of the composite film gradually changes to reddish-brown, dark red, and finally dark red under alkaline conditions. The color change of aloe-emodin under different pH conditions is mainly related to its chemical structure. Aloe-emodin is an anthraquinone compound with the molecular formula C 15 H 10 O 5 and a molecular weight of 270.23. When this compound is reduced under acidic conditions, it forms anthranol and its tautomer anthrone, resulting in a color change from orange to yellow. Specifically, under acidic conditions, the hydroxyl groups (-OH) and carbonyl groups (C=O) in the molecule of aloe-emodin react with hydrogen ions (H + ) to form positively charged ions. This ionic form of aloe-emodin absorbs different wavelengths in the absorption spectrum, thus showing yellow. Under alkaline conditions, aloe-emodin shows red. This is because in an alkaline environment, the anthraquinone structure of aloe-emodin becomes an anthranol structure, forming a conjugated system, enabling the compound to absorb light of longer wavelengths, thus showing red. The stronger the alkalinity of the environment, the more complete the reaction. The ratio of the anthraquinone and anthranol structures is affected by the environmental pH value. Therefore, it shows different colors macroscopically ( Figure 3 C of [reference]). And the BCA@Van 3 composite film also has pH responsiveness due to the presence of aloe-emodin and shows different color reactions under different pH conditions. And because aloe-emodin is evenly distributed, the presence of the bacterial cellulose membrane does not affect its color reaction ( Figure 3 B of [reference]).

[0152] The aloe-emodin solution was scanned for wavelength using a UV-visible spectrophotometer. The results showed that the UV absorption spectrum of the aloe-emodin molecule underwent a red shift with the increase of the solution pH and exhibited a macroscopic color change, demonstrating its excellent pH responsiveness. Figure 3 In D) of 3 . In the reversibility test of the acid-base color reaction, even after being repeatedly exposed to ammonia water and hydrochloric acid solutions 5 times, the BCA@Van 3 film was still very sensitive to color changes. Each acid-base conversion could occur within <2 min (dark purple-red → light yellow). The color indices a * , b * of the BCA@Van Figure 3 composite film are shown in E-F of * . The results showed that before and after each color change, the color indices a * (red-green intensity), b 3 (yellow-blue intensity) of the composite film remained basically stable, proving that BCA@Van

[0153] had excellent pH-responsive reversibility and exhibited unique reversible color changes. 3 Table 3 Color parameters (L * , a * and b * ) of the BCA@Van

[0154]

[0155] Example 5 Application of the composite film in salmon preservation and freshness indication

[0156] The prepared BCA@Van 3 composite film was used to detect the ability of salmon preservation and freshness indication. Commercially available salmon slices with uniform appearance and freshness were divided into five groups of the same weight (30.0 g per group) and placed in plastic shallow dishes. The salmon samples wrapped in commercially available polyethylene (PE) food wrap and the salmon samples without any treatment were set as the control group (PE group) and the blank control (control group, Control group), respectively. The samples wrapped with BC film, BCA film and BCA@Van 3 film were used as the experimental groups. All samples were stored in a 4 °C refrigerator, and the physicochemical parameters of the salmon samples were measured on the 3rd, 5th, 7th, 9th, and 11th days.

[0157] 1. pH determination of salmon samples

[0158] Weigh 5.0 g of salmon meat from each group separately, grind it, and add 45 g of deionized water. Then, the mixture was homogenized thoroughly using a high-pressure homogenizer and placed in a refrigerator at 4 °C for 30 min. Record the pH value of the supernatant using a pH meter, repeat three times, and take the average value.

[0159] 2. Determination of total volatile basic nitrogen (TVB-N)

[0160] The TVB-N detection method for each group of salmon was determined according to GB 5009.228-2016.

[0161] 3. Determination of total viable count (TVC)

[0162] The TVC detection method for each group of salmon was determined according to GB4789.2-2022.

[0163] 4. Sensory evaluation

[0164] For the sensory evaluation of salmon during storage, consumers evaluated five types of models: unpackaged (Control group), commercially available plastic food wrap (PE group), BC group, BCA group, and BCA@Van 3 group.

[0165] Twenty trained experts (10 males and 10 females, aged between 20 and 40 years) from the general population conducted a sensory evaluation of the salmon. A 5-point descriptive scale (5 = excellent, 4 = good, 3 = acceptable, 2 = unacceptable, 1 = very poor) was used to evaluate the color, odor, and overall acceptability of the meat samples.

[0166] The salmon was taken out at regular intervals, photographed, and the tissue state and spoilage condition of the salmon surface were observed. See Figure 4 A. Black spots were found on the surface of the control group (Control group) on the 5th day, and the overall color became darker and lost its luster. This was caused by the oxidative spoilage of fat, indicating that the surface of the salmon was contaminated. The salmon packaged with PE was slightly better than the control group, with a lower degree of surface blackening. This was because the PE packaging isolated the package from the air, thus slowing down the spoilage caused by aerobic spoilage microorganisms. The freshness preservation ability of the BC group was roughly the same as that of the PE group. However, due to the rich hydroxyl groups in BC, it had good water retention, maintaining the water content in the salmon, making the color change of the fish meat in this group better than that of the PE group and the control group. The BCA group had the effect of extending the freshness preservation period of the salmon, and color changes only began to appear on the 7th day. This was because aloe-emodin had certain antibacterial ability, effectively inhibiting the growth of spoilage-causing microorganisms and thus slowing down the spoilage and deterioration of the salmon. And BCA@Van 3The group did not show blackening and deterioration at any stage, indicating a delay in the spoilage process of salmon. The combination of vanillin can enhance the antibacterial effect and greatly inhibit the growth of microorganisms. The strong oxidizing property of vanillin can also inhibit the oxidation reactions of fats, proteins, etc. occurring in salmon, delaying the spoilage and deterioration of salmon. Moreover, vanillin is reported to be a generally recognized safe food additive and has functions such as stabilizing food components. The phenolic hydroxyl group (-OH) in the vanillin molecule can neutralize free radicals by providing hydrogen atoms, thus slowing down the lipid oxidation reaction in salmon. This antioxidant property can effectively prevent fatty acid rancidity in food and extend the shelf life of food. In addition, vanillin can have weak interactions with certain components in salmon (such as proteins and fats), reducing color changes and flavor deterioration caused by oxidation or enzymatic reactions, thereby maintaining the sensory quality of food. Therefore, BCA@Van 3 The composite film can maximize the preservation of the state of salmon while keeping it fresh.

[0167] The pH value is one of the key indicators for judging food spoilage, and the pH value changes of salmon during storage were measured. During the spoilage process of meat, the pH value generally shows a trend of first decreasing and then increasing. As Figure 4 shown in B, the pH values of the control group, PE group, and BC group decreased within 0 - 3 days. The initial decrease in pH was due to the production of a small amount of lactic acid by the somatic cells in salmon through respiration using oxygen and acidic substances produced by aerobic microbial metabolism. However, the pH of the BCA group and BCA@Van 3 group did not start to decrease until the 5th day because the composite film can not only isolate oxygen, thus limiting the rate of aerobic respiration, but also inhibit the growth of aerobic microorganisms, thereby delaying the decrease in pH. In the BCA@Van 3 group, since the phenolic hydroxyl group (-OH) in the vanillin molecule can neutralize free radicals by providing hydrogen atoms, thus slowing down fatty acid rancidity, and its enhanced antibacterial activity also further delays the decrease in pH during the storage of salmon. The subsequent increase in pH is due to the production of some alkaline substances by microbial growth and metabolism. In the later stage, the increase rate of the pH value of the BCA and BCA@Van 3 groups is slower than that of the control group, indicating that the BCA@Van 3 composite film effectively inhibits the spoilage of spoilage microorganisms and slows down the production of amines and other alkaline substances. In addition, in the later stage of storage, especially when salmon enters the spoilage stage, proteins are decomposed by microorganisms or autologous enzymes, releasing alkaline amino compounds (such as ammonia, amines, etc.), which will also cause an increase in the pH value. BCA@Van 3The combination of vanillin and aloe-emodin in the composite film has enhanced antioxidant properties, which can reduce protein degradation caused by oxidation reactions, thus maintaining the acid-base balance inside the salmon and reducing the phenomenon of increased pH during the spoilage process. Figure 4 Figure C shows the color diagrams of the films of each group at different storage days. There is no color change in the PE group and the BC group; for BCA and BCA@Van 3 Starting from the 5th day, as the pH increases, the color of the film gradually darkens, indicating that the composite film has great potential for application in salmon preservation and has a color indication effect.

[0168] Total volatile basic nitrogen (TVB-N) is one of the key indicators for measuring the freshness of fresh meat. The increase in TVB-N is usually closely related to the metabolic activities of microorganisms (especially spoilage bacteria), which decompose the proteins in salmon and produce volatile basic nitrogen compounds such as ammonia and amines. TVB-N is an important indicator for evaluating the quality of salmon meat. The maximum TVB-N of fresh salmon is 20 mg / 100 g (Reference: https: / / doi.org / 10.1080 / 10942912.2018.1451343). As Figure 4 shown in Figure D, the TVB-N of the control group, the PE group, and the BC group exceeded the freshness range on the 5th day, indicating that the fish had spoiled. The TVB-N value of the BCA group reached the critical standard on the 7th day, and the TVB-N value of the BCA@Van 3 group was 20.32 mg / 100 g on the 9th day, slightly exceeding the critical value. It can be seen that the synergistic effect of vanillin and aloe-emodin reduces the volatile basic nitrogen compounds produced by the metabolism of spoilage bacteria by inhibiting bacterial growth. In addition, the strong antioxidant effect of the BCA@Van 3 film can reduce the decomposition of nutrients such as fat and protein in salmon, reduce the production of volatile basic nitrogen compounds such as ammonia and amines, and thus slow down the increase in the TVB-N value.

[0169] Total viable count (TVC) is one of the key indicators for measuring food spoilage. The maximum edible TVC of salmon is 6 lg (CFU / g) (Reference: https: / / doi.org / 10.3390 / foods10020401). On the 7th day, the TVC of the control group reached 6.51 lg (CFU / g), and that of the PE group was 6.03 lg (CFU / g); this result may be due to the encapsulation effect of PE, which reduces the contact between salmon and external bacteria, so the TVC value is reduced. BCA has a certain antibacterial effect due to the presence of aloe-emodin, so it shows a reduced TVC value, and its TVC value was 5.48 lg (CFU / g) on the 9th day. For BCA@Van 3The group had enhanced antibacterial activity, and the TVC value was 4.98 lg (CFU / g) at the 9th day. This indicated that BCA@Van 3 film could effectively inhibit the growth of spoilage bacteria and had the effect of extending the shelf life of salmon.

[0170] During storage, salmon undergoes microbial and metabolic changes, leading to physical and chemical changes. Therefore, sensory evaluation is also a conventional method for detecting food freshness. We conducted a comprehensive rating, and the results are shown in Figure 4 F - I below, including color (F), odor (G), texture (H), and overall acceptability (I). The scores of color, odor, texture, and overall acceptability of salmon gradually decreased with the extension of storage time. After 3 days of storage, the gap between groups gradually widened, and the BCA@Van 3 group scored the highest in all evaluations. In contrast, the scores of the control group and the PE group were within the unacceptable range (close to 2 points) on the 5th day. At this time, the color of the surface meat of salmon in the control group and the PE group darkened, and slight color spots due to bacterial spoilage appeared on the surface. Amino acids and proteins in salmon were decomposed by bacteria, emitting a slight strange smell, the texture softened, and the overall was unacceptable. While the BCA@Van 3 composite film could effectively inhibit the growth of spoilage bacteria, and its antioxidant effect effectively prevented the oxidation of lipids and proteins, maintained the color of salmon, avoided the generation of strange smells, effectively maintained the sensory quality of salmon, and extended its shelf life.

[0171] Example 6 Biocompatibility Evaluation

[0172] 1. The cell compatibility of all samples was evaluated by the CCK - 8 method

[0173] Experimental group: Human skin fibroblasts (HSF) were cultured in HSF complete medium, then digested with trypsin and inoculated into 24 - well plates, and the cell density was adjusted to 1×10 5 cells / well, and cultured in an incubator at 37℃ and 5% (v / v) CO 2 for 24 h to allow the cells to adhere. Subsequently, the cells were washed with PBS, and after discarding the medium, ultraviolet - sterilized BC, BCA, and BCA@Van n composite film samples (30 mg / mL) were added respectively, and the cells were cultured for another 24 h. After the treatment, 10 μL of CCK - 8 reagent was added to each well, gently mixed, and incubated for 2 h under the same conditions until sufficient color development occurred, and finally the absorbance value was measured at a wavelength of 450 nm.

[0174] Control group: The same as the experimental group, except that the composite film samples were not added, the composite film samples were replaced with HSF medium, and then the CCK - 8 reagent was not added.

[0175] Blank control group: Similar to the experimental group, except that the composite membrane sample was not added and there were no cells. The composite membrane sample was replaced with HSF medium, and then CCK-8 reagent was added.

[0176] Calculate the cell viability based on the absorbance data. The calculation method of cell survival rate is as follows:

[0177] Cell survival rate (%) = [(A e - A b ) / (A c - A b )] × 100 (6)

[0178] In formula (6), A e , A b and A c represent the OD 450 values of the experimental group (cells treated with the sample and CCK-8 reagent), the control group (cells treated only with CCK-8 reagent), and the blank control group (medium and CCK-8 reagent), respectively.

[0179] To more intuitively observe the survival state of HSF cells after co-incubation with the sample, after incubation, the medium was aspirated, and the cells were gently washed twice with sterile phosphate buffer saline (PBS). Prepare calcein-acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) staining solution according to the instructions of the manufacturer (BestBio, Cat.#BB-4126). Add 1 mL of the staining solution to each well and incubate in the dark at room temperature for 15 min. Wash the cells three times with PBS to terminate the staining process. A confocal microscope (Olympus FV1200) was used to examine the cell morphology of HSF.

[0180] 2. Evaluate the blood compatibility of the composite membrane with mammalian cells using mouse whole blood. Sample group: Store the mouse whole blood sample in a centrifuge tube containing sodium heparin, wash it with an equal volume of 0.9% saline, and then centrifuge at 1500 rpm for 10 min. This washing process was repeated three times until the final supernatant became clear. After discarding the supernatant, add 400 μL of precipitated red blood cells (RBCs) to 20 mL of 0.9% saline to prepare a 2% RBC suspension. Add the composite membrane to the RBC suspension to make the concentration of the composite membrane 30 mg / mL. Subsequently, the RBC suspension of each group was incubated at 37 °C and 5% CO 2Cultivate for 1 h under the conditions. After cultivation, centrifuge the samples at 1500 rpm for 10 min. The RBC suspension in the positive control group was resuspended with PBS, and the rest was the same as the sample group; the cells in the negative control group were resuspended with deionized water, and the rest was the same as the sample group. Transfer 100 μL of the supernatant to a 96-well plate. Set three parallel samples for each experimental group, and measure the absorbance at 540 nm using a multifunctional microplate reader (BioTek Cytation 5). The hemolysis rate was calculated according to the following formula:

[0181] Hemolysis rate (%) = [(E e - E n ) / (E p - E n )] × 100(7)

[0182] In formula (7), E e represents the absorbance value of the sample supernatant, and E p and E n are the absorbance values of the positive control group and the negative control group, respectively.

[0183] During the process of food packaging materials contacting food, their components may migrate into the food. Therefore, determining the biocompatibility of food packaging materials is an important measure to ensure food safety. Figure 5 Figure A shows the survival rate of HSF cells under the condition of co-culturing with different composite membranes. The results show that all samples have no toxicity to HSF cells. Even for the BCA@Van 3 composite membrane with the highest Van concentration, the cell survival rate is above 93%, indicating the good biocompatibility of the composite membrane. Figure 5 Figure B shows the results of the cell viability and cytotoxicity staining experiment. Observe the active state of HSF cells after treatment through a fluorescence microscope. Green fluorescence represents live cells, and red fluorescence represents dead cells. The results show that in most sample groups, the cells show green fluorescence and almost no red fluorescence, further proving that the material has no obvious toxicity to cells. According to the literature report, materials are considered blood-compatible when the hemolysis rate is < 5% (Reference: https: / / pubs.acs.org / doi / abs / 10.1021 / acsami.8b02527). Figure 5 Part C in the figure evaluates the blood compatibility of the samples, including hemolysis rate tests and observation of the plasma color after hemolysis. The hemolysis rate results are all far lower than the safety threshold of 5%. At the same time, the supernatant after centrifugation of both the control group and the sample group is transparent light yellow, and no obvious red hemoglobin release is observed, indicating that the material has low hemolytic activity on red blood cells and has good blood compatibility. These results together indicate that BC, BCA, and BCA@Van 3 composite membranes show excellent biocompatibility in terms of cell compatibility and blood compatibility and are suitable for use in the field of food packaging.

[0184] Example 7 Natural degradability of composite films

[0185] Soil burial was used to investigate the degradability of the composite film under natural conditions. The degradation behavior of the cellulose film followed the method reported in the literature with slight modifications (https: / / www.nature.com / articles / s41598-024-57436-w). The specific method is as follows:

[0186] A plastic tray (length × width × height of approximately 20 × 12 × 15 cm) was selected and holes were punched in the bottom for drainage. Then, pre-moistened organic potting soil was added to prepare a 10 cm deep soil bed. The soil bed was placed at a humidity of (12 ± 2)% for 7 days, and water was added as needed to maintain the soil moisture at (12 ± 2)%. BC and BCA@Van were placed in the tray. 3 Cellulose films were cut into the same size (length × width of about 3.5 × 2.5 cm), and after weighing the initial weight, they were buried in different moist soil beds, 5 cm from the soil surface, and the soil was gently compacted by hand to ensure good contact between the sample and the soil. Commercially available degradable starch film (i.e., edible starch film) was used as a degradation control group of cellulose-based composite films under the same treatment conditions.

[0187] The soil moisture content of each tray was recorded daily using a digital soil detector (TES-1360A, Qingdao Xinye Environmental Protection Technology Co., Ltd.), and water was added with a watering can as needed to maintain a soil moisture level of 12 ± 2%. Five sets of replicates were set for each composite film. After each sampling, the composite film sample was gently cleaned with a brush to remove attached soil particles and photographed for record. The cleaned film was then placed at room temperature for at least 48 h, and the weight measurement was recorded.

[0188] The percentage weight change due to soil burial-induced degradation was calculated for each sample using the following formula:

[0189] Weight loss (%) = [(initial weight - weight after recovery) / initial weight] × 100 (8)

[0190] The natural degradation results of all samples are shown in Figure 6 As shown. After 10 days in soil, BC and BCA@Van 3 The composite film surface was obviously damaged and cracked, and its mass loss was 23.68±2.32% and 19.55±3.16%, respectively. In contrast, the starch film only had blurred edges and a slightly rough surface. The soil degradation time was further extended. At the 20th day, BC and BCA@Van 3The composite film was severely broken, and the film within the red dotted box was significantly decomposed, and in some areas, the presence of the film was hardly visible. The mass loss of both was about 90%; while the mass loss of the degradable starch film was only 4.88 ± 0.95%. BC and BCA@Van 3 Both composite films could be completely degraded by environmental microorganisms in the soil within 30 days without causing any environmental damage. These composite film materials demonstrated excellent degradation performance in the soil environment and were suitable for further application as environmentally friendly degradable materials in the food packaging field.

[0191] Characterization of the properties of the composite film in Example 8

[0192] The chemical composition and bonding information of the composite film were analyzed using a Fourier transform infrared spectrometer (Thermo Fisher Scientific, Inc., USA, Nicolet iS50) in the attenuated total reflection mode (ATR-FTIR), and the spectral data were collected in the range of 4000 cm -1 to 400 cm -1 The surface microtopography was observed using a field emission scanning electron microscope (FE-SEM, Apreo S, FEI, Czech Republic). The thermal stability of the composite material was studied using a thermogravimetric analysis system (Netzsch, Germany). Under a nitrogen atmosphere, the temperature was increased from 25 °C to 800 °C at a rate of 10 °C / min. The crystal structure of the sample was characterized using an X-ray diffractometer (XRD, Rigaku SmartLab), with the scanning angle range from 5° to 80°, the scanning speed of 5° / min, and the step size of 0.02°. The surface wettability of the film was tested using a JC2000C1 contact angle measuring instrument (Zhongchen Digital Technology Equipment Co., Ltd.), and the droplet volume was 2 μL.

[0193] The BC, BCA, and BCA@Van n composite films were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray crystallography (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and water contact angle (WCA). The results are shown in Figure 7 and Figure 8 . Figure 7 In A - B, the ordinate represents the transmittance (%), the abscissa represents the wavelength, and A - B is the FTIR graph; Figure 7 C and D in are obtained using a thermogravimetric analyzer. Figure 7 In C, the ordinate represents the mass; the abscissa represents the temperature, and C is the TGA graph; Figure 7 In D, the ordinate represents the mass derivative; the abscissa represents the temperature, and D is the DTG graph. Figure 7 In E, the ordinate represents the intensity; the abscissa represents the angle, and E is the XRD graph; Figure 7The ordinate of F represents the water contact angle; the abscissa represents the sample name, and F is the WCA diagram. Figure 8 (A - F) are BC film, BCA film, BCV film (film of pure BC crosslinked with vanillin), BCA@Van 1 、BCA@Van 2 、BCA@Van 3 film.

[0194] The chemical composition and properties of purified and functionalized BC films were studied by FTIR spectroscopy. BC films fermented with glucose as the carbon source (i.e., BC - Glu) and BC films fermented with GlcNac as the carbon source (i.e., BC - GlcNAc) both showed almost the same peak patterns. Characteristic absorption peaks were observed at 3345 cm -1 , corresponding to the stretching vibration of the -OH group; the peaks at 2904 cm -1 and 1422 cm -1 were attributed to the stretching vibration of the -CH bond. The absorption peaks in the range of 1170 cm -1 to 1000 cm -1 represented the polysaccharide structure of BC, and the peak at 1053 cm -1 was attributed to the stretching of the C - O - C bond. The peak at 898 cm -1 corresponded to the bending vibration of the β-(1 - 4) glycosidic bond. Different from that, the BC - GlcNAc film showed an additional peak at 1660 cm -1 , which was caused by the stretching vibration of C - O in the NH - CO group

Reference: https: / / doi.org / 10.1016 / j.ijbiomac.2023.124831

Reference: https: / / doi.org / 10.3390 / foods11111613

[0195] Original BC and BCA@Van n The thermal properties of the samples were tested by thermogravimetric analysis (TGA). The TGA curves and the derivative thermogravimetry (DTG) curves are shown in Figure 7 C and D in Figure 7 respectively. Due to the evaporation of absorbed water, each sample had a slight weight loss around 50 °C - 100 °C. However, the BCA@Van n composite film required a lower temperature for the evaporation of free water than BC. This may be because after the introduction of aloe-emodin and vanillin, the pore structure of the BC film material was increased, thereby improving the water diffusion channels, making these weakly bound water molecules easier to diffuse to the surface of the sample and be evaporated and released. The weight loss between 100 - 300 °C was due to the loss of bound water. The significant mass loss of the BC film between 330 - 410 °C was due to the dehydration, depolymerization, and thermal decomposition of the pyranose glucose units. BCA and the BCA@Van n composite film showed similar thermal degradation behaviors. It is worth noting that the initial weight loss temperature of the BC film was about 273 °C, and the initial thermal degradation temperature of the BCA@Van 1 film was 286 °C, and that of the BCA@Van 3 film was 297 °C. This difference is attributed to the fact that the aloe-emodin and vanillin molecules contain aromatic rings and conjugated structures with higher thermal stability, which delays the heat transfer inside the BC film, thereby inhibiting the initial thermal decomposition. And vanillin crosslinks with BC through a Schiff base structure with higher stability, increasing the energy requirement for decomposing the material in the initial stage, improving the thermal resistance of the composite film, and making it maintain relative stability under heating conditions. The thermal stability of the composite film has significant advantages in protecting food quality, improving the strength and durability of food packaging bags, and meeting diverse food packaging requirements.

[0196] The XRD results are shown in Figure 7As shown in Fig. E, diffraction peaks appeared at 2θ values of 14.8°, 16.7°, and 22.6° for all samples, indicating the same position, corresponding to the (100), (010), and (110) crystal planes of BCN. In addition to showing the main peak of BC, BCA showed additional peaks at 8.5° and 11.5°, corresponding to the hydrated crystal structure of aloe-emodin. This indicates that aloe-emodin has been successfully modified into the porous network structure of BC [Reference: https: / / www.sciencedirect.com / science / article / pii / S0268005X24003047?via%3Dihub]. BCA@Van n In addition to showing the characteristic peaks of BC and AE, the composite film showed strong diffraction peaks at 13.1°, 17.2°, 27.3°, 39.4°, and 42.3°, belonging to the typical phase structure of vanillin (standard XRD pattern (PDF 40-1938) [Reference: https: / / www.sciencedirect.com / science / article / pii / S0141813015005863].

[0197] The water contact angle results of the composite film are as Figure 7 shown in Fig. F. WCA is usually defined as 90° as the hydrophilic / hydrophobic critical point. The contact angle of the BC film after air drying was 64.4°, indicating the hydrophilic characteristics of BC, which is due to the presence of a large number of hydrophilic hydroxyl groups in BC. After composite with aloe-emodin, the hydrophilicity of the BCA film decreased, and the WCA was 79.3°, which was attributed to the non-polar aromatic ring structure of the anthraquinone skeleton of aloe-emodin, giving it a certain degree of hydrophobicity [Reference: https: / / www.sciencedirect.com / science / article / pii / S0308814624003352?via%3Dihub]. After crosslinking with vanillin, the BCA@Van 1 composite film became hydrophobic, and the WCA increased with the increase in the amount of vanillin. BCA@Van 3The WCA of the composite membrane is 123.2°. This is mainly due to the hydrophobic benzene ring structure in vanillin, and the van der Waals force between vanillin molecules (mainly contributed by the non-polar part of the aromatic ring) is stronger than the polar force such as hydrogen bonds, which makes the self-assembly ability and dispersibility of vanillin in water poor, and thus shows a certain hydrophobicity [reference: https: / / advanced.onlinelibrary.wiley.com / doi / abs / 10.1002 / adfm.202415952Hyperbranched Vanillin-Based Composite Coating:Achieve Efficient Icephobicity in High Humidity and Dynamic Environments]. This shows that the addition of aloe-emodin and the modification of vanillin enhance the hydrophobicity of the BC membrane. Hydrophobic films can effectively prevent the penetration of moisture, oxygen and other gases, thereby preventing the oxidation reaction and moisture deterioration of packaged foods [reference: https: / / doi.org / 10.1016 / j.foodchem.2024.141116].

[0198] The microstructure of the composite membrane was observed by scanning electron microscopy. Figure 8 , Figure 8 A to F in the figure represent BC, BCA, BCV, and BCA@Van respectively. 1 、BCA@Van 2 and BCA@Van 3 BC membranes show a highly porous three-dimensional network structure, such as Figure 8 As shown in A, BC presents a three-dimensional network structure. In addition to the cellulose network structure, the BCA composite membrane contains aloe-rhodiola rosea particles that fill the interwoven nanofiber network. This uniform distribution is conducive to the rapid and efficient pH response and color change of the composite membrane, confirming the expected preparation of the BCA composite membrane. In the BCV membrane formed by cross-linking vanillin with BC alone, the cellulose nanofibers are cross-linked by vanillin, and the vanillin filled in it makes the surface morphology between the BC nanosheets smoother and more uniform. Similarly, in BCA@Van n In the composite film, aloe-emodin filling inside the cellulose nanofibers can also be observed. In addition, with the increase of vanillin addition, the contact between the cellulose nanofibers and vanillin is closer, resulting in a more stable internal structure. This phenomenon may help to improve the overall mechanical properties of the composite film.

[0199] according to Figure 8It can be seen that the basic property detection indicates that aloe-emodin has been successfully incorporated into the bacterial cellulose membrane, and vanillin has been successfully cross-linked to the composite membrane through the Schiff base reaction with the BC amide bond. Fourier transform infrared spectroscopy shows the presence of typical functional groups of aloe-emodin, vanillin, and bacterial cellulose in the membrane, and the appearance of the Schiff base bond ( Figure 8 in A–B). The addition of aloe-emodin and vanillin can increase the thermal stability of the composite membrane ( Figure 8 in C–D). X-ray diffraction presents the crystal form interface of aloe-emodin and vanillin ( Figure 8 in E). Moreover, the BCA@Van membrane after the combination of aloe-emodin and vanillin has the effect of enhancing hydrophobicity ( Figure 8 in F).

[0200] In summary, the present invention prepares a BCA@Van composite membrane for food packaging by an innovative method. The analysis results of FTIR, XRD, TGA, and SEM show that the composite membrane is successfully prepared. The composite membrane has strong free radical scavenging ability and broad-spectrum antibacterial activity, and can effectively inhibit the growth of Gram-negative bacteria and Gram-positive bacteria. And it can effectively extend the shelf life of salmon at 4 °C. The composite membrane has good cell compatibility and blood compatibility, and can degrade under natural soil conditions. The BCA@Van composite membrane of the present invention has great potential as a food packaging film, and the research results provide valuable insights for the modification and application of BC-based composites.

[0201] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bacterial cellulose-aloe-rhodiola rosea-vanillin composite film, characterized in that: The invention comprises bacterial cellulose-aloe-emodin and vanillin; the vanillin is cross-linked to the bacterial cellulose-aloe-emodin; and the bacterial cellulose-aloe-emodin is formed by suspending the bacterial cellulose through xanthan gum and the aloe-emodin.

2. The composite membrane according to claim 1, characterized in that The preparation method of bacterial cellulose-aloe-emodin comprises: culturing Enterobacter sp. FY-07 to obtain bacterial cellulose-aloe-emodin; the components of the culture medium comprise N-acetylglucosamine, xanthan gum and aloe-emodin.

3. The composite membrane according to claim 2, characterized in that With distilled water as solvent, the culture medium comprises: 0.01-5 g / L aloe-emodin, 0.1-1.0 g / L xanthan gum, 0.1-7.5 g / L yeast powder, 0.1-10 g / L peptone, 0.1-10 g / L Na2HPO4, 0.1-1.0 g / L KNO3 and 0.1-25 g / L N-acetylglucosamine.

4. The composite membrane according to claim 2, characterized in that The culture temperature is 28-35° C. and the culture time is 22-26 hours.

5. The method for preparing the composite membrane according to any one of claims 1 to 4, characterized in that: The following steps are involved: The bacterial cellulose-aloe-emodin and vanillin solutions are cross-linked to obtain a bacterial cellulose-aloe-emodin-vanillin composite film.

6. The preparation method according to claim 5, characterized in that: The volume concentration of the vanillin solution is 0.1% to 1% (w / v).

7. The preparation method according to claim 5, characterized in that: The cross-linking temperature is 30-110° C.; the cross-linking time is 4-8 hours; and the cross-linking speed is 80-120 rpm.

8. Application of the composite film according to any one of claims 1 to 4 or the composite film prepared by the preparation method according to any one of claims 5 to 7 in one or more of the following 1) to 5); 1) Food preservation; 2) Food antioxidants; 3) As smart packaging; 4) Antibacterial; 5) As a food freshness detection reagent.

9. A method for applying the composite film according to any one of claims 1 to 4 or the composite film prepared by the preparation method according to any one of claims 5 to 7, characterized in that: include: The composite film is used to package food.

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