Probiotic-bioactive ingredient composite film and method of making and use thereof

Abstract

The application belongs to the technical field of packaging materials, and particularly discloses a probiotic-bioactive ingredient composite film as well as a preparation method and application thereof. The composite film takes gelatin and carboxymethyl chitosan as a composite matrix, loads dandelion polyphenols and lactobacillus plantarum, and further comprises a plasticizer, which is a mixture of glycerol and polyethylene glycol. The probiotic-bioactive ingredient composite film, the preparation method and the application thereof can load probiotics and bioactive ingredients in a gelatin-carboxymethyl chitosan composite matrix, form a stable "probiotic-bioactive ingredient-film material" system through a hydrogen bond crosslinking network of the matrix, effectively guarantee the survival of lactobacillus plantarum, and improve the survival rate of lactobacillus plantarum.

Description

Technical Field

[0001] This invention relates to the field of packaging materials technology, and in particular to probiotic-bioactive ingredient composite films, their preparation methods, and applications. Background Technology

[0002] Plastic waste generated by the food packaging industry has become a serious threat to the ecological environment. Furthermore, plastic packaging materials can contaminate the food inside, posing food safety risks. Therefore, developing green, safe, and biodegradable food packaging materials has become a hot research topic in the industry.

[0003] In recent years, the preparation of active food packaging films using biomacromolecules such as polysaccharides, proteins, and lipids as substrates has attracted widespread attention. However, single biomacromolecule substrates have shortcomings such as insufficient mechanical properties and poor barrier properties, which limit their practical applications. Blending two or more film-forming substrates to prepare composite films can achieve complementary advantages and improve the overall performance of the composite films.

[0004] Dandelion is abundant, readily available, and highly safe, rich in phenolic compounds and other active ingredients, possessing various biological properties such as antibacterial and antioxidant effects. Probiotics have positive effects on human health, and the bioactive substances they produce can improve food stability and reduce the risk of spoilage. Studies have shown that co-encapsulating probiotics and bioactive compounds in a film-forming substrate can enhance probiotic activity. Developing a composite food packaging film based on a natural biomacromolecule matrix, synergistically loading bioactive substances and probiotics, exhibiting excellent comprehensive performance and simple preparation, is of great significance for promoting the innovative development and practical application of green food packaging materials. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic-bioactive ingredient composite film, its preparation method, and its application. The prepared composite film has excellent comprehensive performance, is simple to prepare, and is environmentally friendly, solving the problems of poor environmental performance and limited preservation effect of existing food packaging films.

[0006] To achieve the above objectives, the present invention provides a probiotic-bioactive ingredient composite membrane, which uses gelatin and carboxymethyl chitosan as a composite matrix, and loads dandelion polyphenols and Lactobacillus plantarum. The composite membrane also includes a plasticizer, which is a mixture of glycerol and polyethylene glycol.

[0007] Preferably, the mass ratio of gelatin to carboxymethyl chitosan is 1:1, and the amount of dandelion polyphenols added is 2.5% (w / v).

[0008] Preferably, the mass ratio of glycerol to polyethylene glycol is 7:3, and the amount added is 40% of the total mass of gelatin and carboxymethyl chitosan.

[0009] This invention also provides a method for preparing a probiotic-bioactive ingredient composite membrane, comprising the following steps:

[0010] Step 1: Add gelatin and carboxymethyl chitosan to deionized water in a certain mass ratio, and stir in a 60°C water bath until completely dissolved to obtain a mixture;

[0011] Step 2: Add glutamin transferase to the mixed solution cooled to room temperature, react and crosslink at 50°C for 45 min, then place the solution in an 80°C water bath for 10 min, cool to room temperature, add dandelion polyphenols and plasticizer, stir thoroughly to prepare the membrane solution.

[0012] Step 3: Inoculate the activated Lactobacillus plantarum into the membrane solution, mix evenly, pour the solution into a disposable plastic petri dish, and dry it in a controlled incubator at 30°C and 50% relative humidity for 24 hours to prepare a composite membrane.

[0013] This invention also provides the application of probiotic-bioactive ingredient composite membranes in the preparation of meat preservation packaging films.

[0014] The advantages and beneficial effects of the above-mentioned probiotic-bioactive ingredient composite membrane, its preparation method, and its application are as follows:

[0015] 1. This invention selects gelatin and carboxymethyl chitosan as the base materials for film formation. Both are natural macromolecules with characteristics such as non-toxicity, excellent degradation performance, and good biocompatibility, making them ideal green food packaging materials. However, gelatin films and carboxymethyl chitosan alone have defects such as insufficient mechanical properties. The mixed use can synergistically enhance mechanical properties and improve film formation.

[0016] 2. This invention prepares a food packaging composite film with synergistic loading of probiotics and bioactive ingredients. Probiotics (such as *Lactobacillus plantarum*) and bioactive ingredients (such as dandelion polyphenols) are co-loaded into a gelatin-carboxymethyl chitosan composite matrix, forming a stable "probiotic-bioactive ingredient-film material" system through the hydrogen-bonded cross-linking network of the matrix. This not only further improves the overall performance of the composite film but also provides new ideas for the application of probiotics in the food industry.

[0017] 3. This invention provides a method for preparing a polyphenol-probiotic composite membrane. A gelatin-carboxymethyl chitosan mixed membrane solution is prepared, and the membrane solution is modified using glutaminase. Subsequently, dandelion polyphenols, *Lactobacillus plantarum* suspension, and a plasticizer are added to the modified membrane solution, and finally, the membrane is dried. The resulting composite membrane can form a synergistic antibacterial and antioxidant system. Furthermore, relying on the molecular interaction between the polyphenols and the substrate, as well as the bacterial cell filling effect, the overall performance of the composite membrane is superior to that of single-substrate membranes and ordinary membranes.

[0018] 4. The composite membrane prepared by this invention can effectively protect the survival of *Lactobacillus plantarum*. Compared with the composite membrane without added dandelion polyphenols, the survival rate of *Lactobacillus plantarum* is significantly improved after the membrane solution is dried and during the storage of the composite membrane. The polyphenol-probiotic composite membrane prepared by this invention has a good effect on meat preservation, effectively inhibiting meat oxidation and spoilage and microbial growth, and delaying the spoilage process.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 The images are SEM images of the composite membranes, where a represents GEL / CMCS, b represents GEL / CMCS / DP, c represents GEL / CMCS / LP, and d represents GEL / CMCS / DP / LP.

[0021] Figure 2 The image shows the X-ray diffraction pattern of the composite film.

[0022] Figure 3 The Fourier transform infrared spectra of the composite film are shown.

[0023] Figure 4 The results reflect the antioxidant properties of the composite membrane;

[0024] Figure 5 The results show the water vapor transmission rate of the composite membrane;

[0025] Figure 6 The result is related to the water solubility of the composite membrane;

[0026] Figure 7 The survival rate of Lactobacillus plantarum during the drying process;

[0027] Figure 8 The survival rate of *Lactobacillus plantarum* in the composite membrane during storage is given, where A represents storage at 25℃ and B represents storage at 4℃.

[0028] Figure 9 The effect of composite membrane on the pH value of pork;

[0029] Figure 10 The effect of composite film on the color of pork;

[0030] Figure 11 The impact of composite membranes on TBARS;

[0031] Figure 12 The effect of composite membranes on total bacterial count;

[0032] Figure 13 This is a flowchart illustrating the preparation process of a probiotic-bioactive ingredient composite membrane. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0034] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0035] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1

[0037] A probiotic-bioactive ingredient composite membrane, using gelatin and carboxymethyl chitosan as the composite matrix, is loaded with dandelion polyphenols and *Lactobacillus plantarum* ATCC8014. The composite membrane also includes a plasticizer, which is a mixture of glycerol and polyethylene glycol. The mass ratio of glycerol to polyethylene glycol is 7:3, and the amount added is 40% of the total mass of gelatin and carboxymethyl chitosan. The mass ratio of gelatin to carboxymethyl chitosan is 1:1, and the amount of dandelion polyphenols added is 2.5% (w / v).

[0038] Preparation methods of probiotic-bioactive ingredient composite membranes, such as... Figure 13 As shown, it includes the following steps:

[0039] Step 1: Add gelatin (GEL) and carboxymethyl chitosan (CMCS) to deionized water at a mass ratio of 1:1, and stir in a 60°C water bath until completely dissolved to obtain an 8% (w / v) GEL / CMCS mixture.

[0040] Step 2: Add 8 U / g of glutamin transferase (TG) to the mixed solution cooled to room temperature, and react and crosslink at 50°C for 45 min. Then, place the solution in an 80°C water bath for 10 min, cool to room temperature, and add 2.5% (w / v) of dandelion polyphenol (DP) and 40% (based on the total mass of gelatin and carboxymethyl chitosan) of glycerol and polyethylene glycol (mass ratio 7:3). Stir thoroughly to prepare the GEL / CMCS / DP membrane solution.

[0041] Step 3, the activated Lactobacillus plantarum (LP) ATCC8014 ( LPctobacilluspLPntarum The solution was inoculated at approximately 9.5 log CFU / mL into the membrane solution, mixed thoroughly, and then poured into a disposable plastic culture dish with a diameter of 9 cm. The dish was then placed in a controlled incubator at 30°C and 50% relative humidity to dry for 24 hours to prepare the membrane (GEL / CMCS / DP / LP).

[0042] Example 2

[0043] The performance of the composite membrane prepared in Example 1 was tested.

[0044] 1. Performance characterization.

[0045] The surface of the GEL / CMCS / DP / LP (gelatin / carboxymethyl chitosan / dandelion polyphenol / Lactobacillus plantarum composite membrane) was observed using scanning electron microscopy to evaluate its structure. Figure 1 As shown in Figure 1, the surface structures of all membranes are smooth, uniform, non-porous, and crack-free, indicating that dandelion polyphenols and Lactobacillus plantarum have good compatibility in the GEL / CMCS (gelatin / carboxymethyl chitosan) composite membrane.

[0046] The GEL / CMCS / LP (gelatin / carboxymethyl chitosan / Lactobacillus plantarum composite membrane) and GEL / CMCS / DP / LP (gelatin / carboxymethyl chitosan / dandelion polyphenol / Lactobacillus plantarum composite membrane) have small protrusions on their surface due to the addition of Lactobacillus plantarum, resulting in a relatively rough surface structure. When dandelion polyphenol and Lactobacillus plantarum are added to the GEL / CMCS composite membrane, a small amount of aggregates and agglomeration occur, increasing the density of the composite membrane network structure.

[0047] 2. Analysis of X-ray diffraction results of the composite membrane.

[0048] Depend on Figure 2 As can be seen, the XRD patterns of the four composite membranes did not show significant changes, indicating that neither DP nor LP formed new crystals with gelatin or carboxymethyl chitosan, demonstrating good compatibility. All films exhibited a broad peak near 20° 2θ, indicating that the films were in an amorphous state. The results show that the addition of DP and LP affects the structure of the GEL / CMCS composite membrane. LP is inserted between the biopolymer chains, weakening the interactions between molecular chains. Comparison shows that the GEL / CMCS / DP / LP composite membrane has the narrowest half-width at half-maximum and the highest crystallinity.

[0049] 3. Analysis of Fourier transform infrared spectroscopy results of composite membrane.

[0050] Depend on Figure 3It can be seen that no significant peak shift was observed in this region for any of the four samples, indicating that the addition of polyphenols or probiotics did not significantly alter the chemical environment of CN. All four samples exhibited a broad absorption peak at 3540, corresponding to the stretching vibrations of -OH and -NH. After the addition of dandelion polyphenols, the peak of the GEL / CMCS / DP (gelatin / carboxymethyl chitosan / dandelion polyphenol composite membrane) broadened, indicating that the abundant phenolic hydroxyl groups in the polyphenol molecules increased the total amount of free hydroxyl groups in the system, thus deepening the absorption. The peak in the GEL / CMCS / DP / LP samples further intensified, indicating that polyphenols and proteins jointly participated in forming a hydrogen bond network, making the superposition of -OH / -NH absorption more pronounced. The peak at 1430 corresponds to the NH bending vibration, and the peak intensity of GEL / CMCS / DP / LP significantly increased, indicating that the co-existence of polyphenols and probiotics further enhanced the hydrogen bonding in the membrane system. In summary, dandelion polyphenols significantly enhance the hydrogen bond network and provide aromatic ring structural features. Lactobacillus plantarum mainly introduces protein / carbohydrate structures, making the hydrogen bond environment more complex. The GEL / CMCS / DP / LP hydrogen bond network structure is the most stable and stronger, and the interactions between components are the most complete.

[0051] 4. Analysis of the color results of the composite membrane.

[0052] The specific parameters of the four composite films were measured using a colorimeter. The color difference parameter of the white standard calibration plate was L0. =0, a0 =0, b0 =0, the color parameters of the four composite films change as shown in Table 1. The results show that the addition of DP and LP both decrease L. a and b However, the L-type GEL / CMCS composite membrane with both DP and LP added... a and b Both are superior to the GEL / CMCS composite membrane with only dandelion polyphenols added, proving that the addition of Lactobacillus plantarum may offset the adverse effects of adding dandelion polyphenols on the GEL / CMCS composite membrane.

[0053] The opacity of the GEL / CMCS composite membrane was 0.57 ± 0.17 mm. With the addition of DP and LP, the opacity of the GEL / CMCS composite membrane increased. Therefore, although the addition of DP and LP can improve the bioactivity of the GEL / CMCS composite membrane, it negatively affects the membrane's transparency.

[0054] Table 1 Color Change Parameters and Opacity

[0055]

[0056] 5. Analysis of the antioxidant properties of the composite membrane.

[0057] The results are as follows Figure 4 As shown, the GEL / CMCS composite membrane exhibits certain antioxidant properties. The addition of dandelion polyphenols and *Lactobacillus plantarum* enhances the DPPH free radical scavenging ability of the GEL / CMCS composite membrane. Among the four composite membranes, the GEL / CMCS / DP / LP composite membrane demonstrates the strongest DPPH free radical scavenging ability, reaching 41.73 ± 0.92%. This may be because *Lactobacillus plantarum* enhances the antioxidant activity of dandelion polyphenols through biotransformation and also participates in DPPH free radical scavenging. This demonstrates that the combined use of dandelion polyphenols and *Lactobacillus plantarum* in the GEL / CMCS composite membrane provides synergistic antioxidant effects.

[0058] 6. Analysis of the mechanical properties of the composite membrane.

[0059] Films used in food packaging need to have certain tensile strength and ductility. The mechanical properties of the four composite films are shown in Table 2. The GEL / CMCS / DP / LP composite film is thicker than the other three films because the addition of *Lactobacillus plantarum* increases the solids content of the composite film. The addition of dandelion polyphenols improves both the total saturation (TS) and elasticity (EAB) of the GEL / CMCS composite film, possibly because the phenolic hydroxyl groups (-OH) in dandelion polyphenols form hydrogen bonds with the amino groups (-NH2) in gelatin, increasing the crosslinking degree and density in the polymer matrix. The addition of *Lactobacillus plantarum* slightly decreases the TS and EAB of the GEL / CMCS composite film, possibly because the increased *Lactobacillus plantarum* aggregates in the matrix, reducing the film's density.

[0060] The GEL / CMCS / DP / LP composite membrane also exhibited excellent tensile strength and elongation at break, at 38.61±1.48MPa and 76.81±1.63%, respectively.

[0061] Table 2 Mechanical strength of composite membranes

[0062]

[0063] 7. Analysis of the antibacterial properties of the composite membrane.

[0064] As shown in Table 3, the GEL / CMCS composite membrane had almost no inhibitory effect on Escherichia coli and Staphylococcus aureus. However, the GEL / CMCS composite membrane with added dandelion polyphenols and Lactobacillus plantarum showed inhibitory effects on both Escherichia coli and Staphylococcus aureus. The GEL / CMCS / DP / LP composite membrane exhibited the best inhibitory effect on both Escherichia coli and Staphylococcus aureus, with inhibition zone diameters of 14.87±0.24 mm and 15.63±0.19 mm, respectively.

[0065] Table 3. Diameter of the inhibition zone

[0066]

[0067] 8. Analysis of water vapor transmission rate of composite membrane.

[0068] from Figure 5 As can be seen, the WVP value of the GEL / CMCS composite membrane decreased significantly after the addition of DP and LP. The lowest WVP value for GEL / CMCS / DP / LP was 0.68 ± 0.43 g·mm·m. -2 ·Kpa -1 ·h -1 This is because adding DP and LP can effectively increase the density of the GEL / CMCS composite membrane structure, which can effectively reduce the permeation of water. The WVP of the composite membrane is also related to the integrity of the matrix, hydrophilicity, and the interaction between the components.

[0069] 9. Analysis of the water solubility of the composite membrane.

[0070] Figure 6 The results showed that the GEL / CMCS composite membrane had the highest water solubility, which is attributed to the good solubility of the hydrophilic groups in GEL and CMCS. The addition of DP enhanced the network structure density of the GEL / CMCS composite membrane, thus reducing its water volatile protein (WVP). The addition of LP altered the membrane's pH and hydrophobicity by producing lactic acid, leading to a decrease in water volatile protein (WS).

[0071] 10. Survival rate of Lactobacillus plantarum during the drying process.

[0072] Figure 7 The results showed that the addition of DP to the GEL / CMCS composite membrane improved the survival rate of polyphenols (LPs). This is because DP and the membrane can form a denser structure, which restricts the diffusion of moisture, etc., and the functional groups of polyphenols can also adsorb moisture, slowing down the dehydration rate during drying. All of these factors are beneficial for protecting and embedding LPs, thus improving their survival rate during the drying process.

[0073] 11. Survival rate of Lactobacillus plantarum in composite membranes during storage.

[0074] Depend on Figure 8 China A Figure 8As shown in Figure B, the survival rate of LP in the composite membrane decreased throughout the entire storage period. Adding DP improved the survival rate of LP in the GEL / CMCS composite membrane because DP's probiotic ability promotes LP growth, thus increasing its survival rate. After 24 days of storage at 4°C, the viable cell count of GEL / CMCS / DP / LP was 6.53 logCFU / g (initially 8.68 logCFU / g), higher than that after 24 days of storage at 25°C, demonstrating that low temperature effectively delays LP inactivation.

[0075] Example 3

[0076] Application of composite films in meat preservation.

[0077] 1. The effect of composite membrane on the pH value of pork.

[0078] Changes in pH value are mainly caused by the activity of putrefactive bacteria. For example... Figure 9 As shown, the pH value of each group of pork samples showed a trend of first increasing and then decreasing with the extension of storage time. The decrease in pH was due to the production of lactic acid from anaerobic glycolysis of muscle glycogen. However, with further extension of storage time, the various enzymes and microorganisms in the pork produced alkaline nitrogenous compounds, leading to an increase in pH value. The pH value of the control group on day 9 was 7.01±0.03, exceeding the limit of 6.7 in the hygiene standards for livestock and poultry meat. Except for day 0, the pH value of the GEL / CMCS / DP / LP composite membrane was consistently the lowest among all groups. This was mainly attributed to the antibacterial activity of the GEL / CMCS / DP / LP composite membrane, which inhibited the microbial growth of pork.

[0079] 2. The effect of composite film on the color of pork.

[0080] L measured by colorimeter a b In the value, a The value reflects the degree of oxidation of myoglobin in meat, a The higher the value, the more the sample's color shifts towards red, and the lower the degree of oxidation.

[0081] from Figure 10 The data shows that the a content of pork in the control group and the treatment group is different. The values ​​generally showed a downward trend, with the blank group and the plastic wrap group showing the lowest values. The value decreased the fastest. This indicates that the GEL / CMCS composite film with added dandelion polyphenols and Lactobacillus plantarum can, to some extent, slow down the oxidation of oxymyoglobin by inhibiting the growth of microorganisms, thereby delaying the color change of pork during storage and maintaining the good color of pork.

[0082] 3. The impact of composite membranes on TBARS.

[0083] The TBARS value of fresh pork should be less than 0.5 mg MDA / kg. Figure 11 The results showed that the TBARS values ​​of all pork samples gradually increased with prolonged storage time. The pork samples wrapped in the GEL / CMCS / DP / LP composite film exhibited the lowest growth rate among all sample groups, but still exceeded the freshness standard on day 9. The control group and the preservation film group both exceeded the freshness standard on day 6. This indicates that the GEL / CMCS composite film containing DP and LP effectively prevented the oxidation of pork fat. The addition of DP and LP improved the antioxidant and oxygen barrier properties of the GEL / CMCS composite film.

[0084] 4. The effect of composite membrane on total bacterial count.

[0085] The total bacterial count in fresh pork should be less than 6 log CFU / g. Figure 12 The results showed that the total bacterial count in all pork samples increased with prolonged storage time. The control group and the plastic wrap group exceeded the standard value on day 6, while the GEL / CMCS / DP / LP group exceeded the standard value on day 9. This indicates that the GEL / CMCS composite film, combined with the natural antibacterial activity of DP and the antibacterial compounds produced by Lactobacillus plantarum, can effectively inhibit the growth of the total bacterial count in pork.

[0086] Therefore, this invention utilizes the aforementioned probiotic-bioactive ingredient composite membrane, its preparation method, and its application. Probiotics and bioactive ingredients are co-loaded into a gelatin-carboxymethyl chitosan composite matrix, forming a stable "probiotic-bioactive ingredient-membrane material" system through the hydrogen-bonded cross-linking network of the matrix. This effectively ensures the survival of *Lactobacillus plantarum* and improves its survival rate.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A probiotic-bioactive ingredient composite membrane, characterized in that: Using gelatin and carboxymethyl chitosan as a composite matrix, dandelion polyphenols and Lactobacillus plantarum are loaded onto the composite membrane. The composite membrane also includes a plasticizer, which is a mixture of glycerol and polyethylene glycol. The preparation method of the probiotic-bioactive ingredient composite membrane includes the following steps: Step 1: Add gelatin and carboxymethyl chitosan to deionized water in a certain mass ratio, and stir in a 60°C water bath until completely dissolved to obtain a mixed solution; Step 2: Add glutamin transferase to the mixed solution cooled to room temperature, react and crosslink at 50°C for 45 min, then place the solution in an 80°C water bath for 10 min, cool to room temperature, add dandelion polyphenols and plasticizer, stir thoroughly to prepare the membrane solution. Step 3: Inoculate the activated Lactobacillus plantarum into the membrane solution, mix evenly, pour the solution into a disposable plastic petri dish, and dry it in a controlled incubator at 30°C and 50% relative humidity for 24 hours to prepare a composite membrane.

2. The probiotic-bioactive ingredient composite membrane according to claim 1, characterized in that: The mass ratio of gelatin to carboxymethyl chitosan is 1:1, and the amount of dandelion polyphenols added is 2.5% w / v.

3. The probiotic-bioactive ingredient composite membrane according to claim 1, characterized in that: The mass ratio of glycerol to polyethylene glycol is 7:3, and the amount added is 40% of the total mass of gelatin and carboxymethyl chitosan.

4. The application of the probiotic-bioactive ingredient composite membrane according to any one of claims 1-3, characterized in that: It is used in the preparation of meat preservation packaging films.

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