Composite biofilm chitosan-microcrystalline cellulose for tomato preservation

The demand for packaging has caused a surge in non-biodegradable plastic waste. To tackle this issue, biofilms provide a safe and effective alternative for packaging and preservation. This research focused on combining chitosan and microcrystalline cellulose (MCC) to produce composite biofilms to preserve fresh fruits. The study involved adding varying quantities of MCC, ranging from 0g to 11g, to chitosan using a glycerol plasticizer. The results showed that adding MCC reduced the adhesion of the chitosan-based film, resulting in a more intact film. The surface morphology of the film showed uniform dispersion of MCC particles. The water adsorption and solubility of the MCC-added films increased while biodegradability decreased. The best biofilm for preservation application was the chitosan film supplemented with 3g of MCC. This film helped limit weight loss, vitamin C content, total acid content, and soluble solids loss in tomatoes during storage. Essentially, the chitosan-MCC film helped to reduce water evaporation, respiration, metabolism with the external environment, and penetration of microorganisms on tomatoes, thus extending their shelf life.


Introduction
Food is a crucial need for human survival, and fresh fruit and vegetables are an essential source of nutrients [1].They provide vitamins A, B, C, and E, phytosterols, fibre, and minerals that help improve health and prevent chronic diseases like hypertension and heart disease [2][3][4][5].Tomatoes are a popular fruit that contains minerals, antioxidants, vitamins C and E, lycopene, β-carotene, lutein, and flavonoids like quercetin [5].Regular consumption of tomatoes can reduce the risk of cancer, cardiovascular disease, and age-related macular degeneration [5][6][7][8][9].Due to the increasing demand for tomatoes, developing better cultivation and post-harvest preservation techniques is necessary.
Recently, the chemical preservation of fruits has become less popular due to its negative impact on human health.Instead, packaging films are being used to preserve fresh fruits.Unfortunately, synthetic packaging has led to a surge in solid waste.Consequently, sustainable fruit preservation methods are being explored as viable and practical options for the well-being of humans and the environment.
Preserving environmentally friendly and safe fruit for human health is currently trending.Biofilms with highly biodegradable properties, such as chitosan, are used for this purpose.Chitosan is a biopolymer with good film-forming ability, antibacterial ability, and excellent resistance to oxygen [10].It is composed of β-(1-4)-2-acetamido-d-glucose and β-(1-4)-2-amino-d-glucose units with low acetyl content, abundant and abundant in nature with the second largest yield after cellulose [10][11].Chitosan has antioxidant, antibacterial and antifungal properties, making it a preferred material for preservation [12][13].However, the mechanical properties of chitosan films are not high, which limits their application in packaging.To improve the mechanical properties of chitosan films, scientist Song C.C. and colleagues reported that adding MCC to chitosan could help [1].MCC is becoming increasingly popular for application in many fields due to its availability, biodegradability, large aspect ratio, low cost and good mechanical properties [14].Reinforcing MCC significantly improves the mechanical properties of films [15][16][17][18].However, there are almost no studies investigating the effects of fortification of MCC into chitosan on the properties related to preservation.
Therefore, this study aimed to obtain chitosan films supplemented with different contents of MCC.The properties of the obtained films, such as film-forming ability, morphological surface, water adsorption, water solubility and biodegradation properties of biofilms, were investigated.Based on their properties, a suitable film formulation for preserving fresh tomatoes was proposed.The preservation process of fresh tomatoes was assessed by observing the state of the fruit, the degree of weight loss, the loss of acid ascorbic content, the total acid content, and the soluble solids content of the fruit during the storage process.

Materials
Two primary materials were used to create a composite biofilm: microcrystalline cellulose (MCC) and chitosan.The chitosan, a light yellow powder, was purchased from Chitosan Vietnam Co., Ltd in Vietnam and has a deacetylation degree of over 90%.The MCC is an opaque white fine powder with a purity of 99% and was purchased from Shandong Zunhong Biotechnology in China.Other chemicals were also used in this study, such as acetic acid (Vietnam), glycerol (China), potassium iodide (China), potassium iodate (China), hydrochloric acid (Vietnam), sodium hydroxide (Vietnam), and phenolphthalein (France).

Fabrication of composite biofilm chitosan-MCC (CM).
The CM biofilms were obtained according to the description of the author from Can Tho University (Vietnam) [19] with some adjustments to fit real-world conditions.First, 2 grams of chitosan were fully dissolved in 100 millilitres of 2% acetic acid solution.Then, various amounts of MCC (0, 1, 3, 5, 7, 9, and 11 grams) were added to the mixture, which was stirred continuously at 500 rpm for two hours at room temperature using a stirrer (Velp, Arec.X, Italia).Next, 3 grams of glycerol were added and stirred for 30 minutes until homogeneous.Air bubbles were removed by sonication with an ultrasound machine (Skymen, JP-008, China) for 10 min.Finally, 15 grams of the film mixture were shaped into circular Petri dishes with an 11 cm diameter and dried at 50°C until a composite film formed.The films were collected and stored in a sealed bag at room temperature.

2.2.2.
Water uptake.The water absorption capacity of the obtained film was performed according to the description of method ASTM D570-98 [20][21].To prepare biofilm samples, cut them to approximately 1x1cm in size.Then, place the sample in 100 ml of water at room temperature.Remove, dry and weigh the sample after 1, 2, 3, 4, 5, and 6 hours.The water absorption (H, %) of the research sample was calculated according to the formula (1): Where, m1: the initial sample mass, g; m2: the sample mass after water adsorption, g.

Solubility.
The studied films were prepared in advance with the same initial mass and size.Then the samples were dried to constant weight at 50ºC and weighed.Next, the samples were placed in a beaker containing 100 ml of water.After 24 hours, the samples were removed from the beaker, lightly wiped with blotting paper and dried to constant weight at 50ºC [21].The solubility (W, %) was calculated according to formula (2): .100 (2) Where, m1: the mass of the film before immersion, g; m2: the mass of the film after immersion, g.

Compostability.
The compostability of the sample was carried out by the landfill method [20,22].Samples of equal size and weight were prepared and buried in the soil at 5-7 cm depth.Then, land the sample in the soil at a 5-7 cm depth.After 5, 10, and 15 days, the samples were taken out, cleaned, and dried at 50°C until their weight remained the same.The compostability (P, %) of the sample was determined using formula (3): Where, m1: the mass of the samples before landfill, g; m2: the mass of the samples after langdfill, g.

Surface morphology.
The surface morphology of biofilms was observed using a scanning electron microscope model (Optika, Italy) with 100-500 magnification.

Tomato preservation using studied composite biofilm.
For the preservation study, the ripe tomatoes that were carefully chosen based on their uniform size and colour, absence of spoilage, and freshness without any signs of wrinkling were utilized.The preservation process was monitored in two groups: the coating group was tomatoes preserved by the studied biofilm, and the control group was tomatoes without any preservation method.For film-preserved tomatoes: a film is applied to the fruit by dipping.Samples were stored under the same conditions [20].Observe the colour and state of the two fruit groups during storage.
The degree of weight loss (Pw, %) of the fruit during the storage period was calculated according to the formula (4): Where, m1: the initial mass of fruit, g; m2: the mass of fruit after preservation, g.The ascorbic acid content in the fruit was determined by titration with iodine [23].The degree of loss of ascorbic acid content (Paa, %) over storage time was calculated according to the formula (5): . 100 (5) Where, aa1: the ascorbic acid content in fruit before preservation, mg/100g; aa: the ascorbic acid content in fruit after preservation, mg/100g.
The total acid content of the fruit was measured by the alkaline acid titration method [24].The loss of total acids (Pa, %) in the fruit over storage time was calculated by the formula (6): . 100 (6) Where, a1: the total acids content in the fruit before prevervation, mEq/l; a2: the total acids content in fruit after prevervatuon, mEq/l.
The soluble solids content in the fruit was measured using a Brix refractometer (Master-T, Atago, Japan) at 20ºC [25].The degree of loss in the soluble solids content (PBx, %) in the fruit according to the storage time was calculated by formula (7): . 100 (7) Where, Bx1: the soluble solids content of fruit before preservation, ºBx; Bx2: the soluble solids content of fruit after preservation, ºBx.

Film-forming ability
The experiment of creating a composite biofilm revealed that the mixture containing solely chitosan had a high viscosity and adhesion.Meanwhile, when MCC was added to the chitosan, the resulting filmforming solution had a lower viscosity and improved adhesion and consistency than the mixture without MCC.Increasing the MCC content did not affect the viscosity and consistency of the film-forming mixture.
In this study, it was discovered that all formulations were capable of creating films.However, after drying the chitosan film without adding MCC was difficult to remove from the plate and resulted in an incomplete film.Films containing MCC were easily removed from the plate with less film loss than chitosan films.The amount of MCC added did not affect the ability to obtain films after drying.The visual image of the magnetic film showed a uniform surface without cracks or roughness.However, as the amount of MCC increased, the opacity of the film also increased.Up to 5g of MCC did not significantly change the transparency of the film, but adding 7g or more resulted in an opaque white film.When MCC was added to chitosan, the film appeared thicker despite using the same volume of the film-forming solution.

Surface morphology
The films obtained with different formulations were observed for their surface morphology by optical electron microscopy.Figure 2 indicates that MCC is evenly distributed in the chitosan film.The film surface is relatively flat when adding 1g to 5g of MCC.However, increasing the MCC content to 7g results in a slightly rougher surface.Adding 9g and 11g of MCC leads to a significantly harsher surface, with angular crystals forming on the films.

Water uptake
Most foods, especially fruits, contain a high percentage of water in their composition.Fresh fruits undergo biochemical processes like living organisms even after being harvested from the mother plant.
Water evaporation can lead to the growth of food spoilage microorganisms and a decrease in fruit quality, which creates water vapour on the fruit surface.Therefore, the fruit packaging film needs to have suitable water adsorption.Figure 3 indicates that the water absorption capacity of the studied films increases as the immersion time increases.After one hour of immersion, all MCC-added films, except those with 11g of MCC, had similar water adsorption capacity as those without MCC addition.After two hours, the membranes supplemented with MCC had better water absorption capacity than those without.This increased water absorption is due to hydroxyl groups in the chitosan and MCC molecular formula, which forms hydrogen bonds with water molecules.However, excessive water absorption can harm food, especially fresh fruits, as it can drain the fruit's juices and cause it to wilt.Additionally, high water absorption can lead to low mechanical strength.Therefore, the complementary membranes of 1g, 3g, and 5g of MCC are still preferred in this study.

Solubility
The films were tested for solubility by immersing them in water for 24 hours.Figure 4 indicates that adding MCC to the films increases the solubility of composite biofilm.However, there is no significant difference in solubility between films with varying amounts of MCC.In comparison, chitosan films have lower solubility than MCC-supplemented films, possibly due to the solubility of chitosan in acidic environments.

Biofilm compostability
After the use of biofilms is over, for conventional waste disposal, the landfill method is used [22].Therefore, this study investigated the compostability of the studied films in the landfill.Figure 5 shows that Adding MCC to chitosan reduced the compostability of the films compared to the original chitosan films, possibly due to the formation of polymer chains between chitosan and MCC through hydrogen bonds between OH-and NH2-groups.The bulkier structure of the chains resulted in a slower breakdown into simpler organic compounds.Figure 5 also shows that the higher the MCC content, the lower the compostability.However, the degree of attenuation was not significant between films with different addition contents.The compostability process was observed for up to 30 days, and by the twentieth day, the studied films were not intact and had almost disintegrated into tiny pieces.It proves that the films in this study have high compostability within 20 days.It indicated that biofilms can be disposed of quickly but do not negatively affect the environment.It is a simple, economical, practical, and environmentally friendly treatment method without additional chemicals or equipment.After analyzing some critical properties of the composite biofilms produced, it was discovered that using MCC supplementary films that measure 7g or more can negatively impact the colour and surface of the final film.As a result, it is undesirable using MCC contents of 7g or more.Regarding the three other MCC contents (1g, 3g, and 5g), the chitosan-3g MCC film showed the highest level of compostability.It had a solubility similar to the 1g MCC film but lower than the 5g MCC film.Additionally, its water uptake was higher than the 1g MCC sample but comparable to the 5g MCC film.Therefore, within the scope of this study, the chitosan-3g MCC film is deemed suitable for researching its potential use in preserving fresh fruits.

Tomato preservation using composite biofilm chitosan-MCC
Preserving food and fresh fruit is one of the most important and widely used biofilm applications.Biofilm is considered the safest, most environmentally friendly, and most effective method for preserving fresh fruit.Previous parts have shown that chitosan films supplemented with 3 g of MCC have optimal properties compared to other studied films.Therefore, this study selected chitosan films with 3 g MCC for application research.
Tomatoes are a common fruit in the human diet and a significant source of vitamins A and C, lycopene, and β-carotene [26].The global consumption of tomatoes is rapidly increasing, estimated at 37 million tons/year [27].The increasing demand for tomatoes has led to a significant increase in crop and post-harvest fruit production requirements.The post-harvest yield depends significantly on the storage process.
To evaluate the process of preserving tomatoes with a study film, the external morphology of the fruit, vitamin C content, soluble substances content (SSC), total acid content, and the natural weight of the fruit was observed.
After observing the appearance of two different groups of tomatoes, it was found that the control group, which had no preservation film, became wrinkled due to evaporation during storage.Starting from day 6, most fruits in the control group began to wilt.In contrast, the coating group preserved by the developed film had a white layer on their surface due to the colour of MCC.The film became more white as the preservation time increased, but it could be easily removed by rinsing it with water.The size of the film-preserved tomatoes changed from the ninth day, while the control group's fruit size changed from the sixth day.Additionally, the freshness of the film-preserved fruits remained the same until the ninth day, while the control group's fruit freshness decreased from the sixth day.These findings indicate that the chitosan-MCC film provided a protective coating that prevented water evaporation from the fruit.During storage, biochemical changes in the fruit still occur that can affect its quality.In the case of ripe tomatoes, these changes occur more quickly, leading to spoilage.The main processes involved in weight loss are the evaporation of water and respiration [21,28].Therefore, the natural weight loss of the fruit is a crucial factor in determining its quality during storage [28].Figure 7a shows that the group of fruits preserved with chitosan-MCC film experienced significantly less weight loss than the control group.After 12 days of storage, the average weight loss of the film-preserved group was 8.14%, while the weight loss of the control group was up to 10.38%.It is related to the fact that chitosan-MCC film can prevent water evaporation and limit respiration, thus preserving the fruit's quality.
During the post-harvest storage of fresh fruit, usually, the polysaccharides will be hydrolyzed to form monosaccharides, causing an increase in the fruit's soluble solids content.Therefore, if the dissolved solids content remains unchanged from the original, the fruit retains its freshness and has a longer shelf life [21].According to figure 7b, after 12 days of storage with a chitosan-MCC film, the soluble solids content in the fruit changed, but not as much as in the control sample.The dissolved solids content of the control group changed to 14.20%, while the group of samples preserved with the developed biofilm changed only 6.77%.It suggests that the chitosan-MCC complex membrane can effectively inhibit the hydrolysis of carbohydrates into simple sugars, thereby increasing the fruit's shelf life [29].The total acid content determines the unique quality and flavour of the tomato.As the fruit ripens, its titratable acidity decreases due to respiration activity [28,[30][31].Additionally, changes in acid content during fruit storage are caused by increased citrate and decreased maltase and citric levels during metabolism [32].This study showed that the group of fruits preserved by a biofilm lost 59.18% in total acid content after 12 days, while the control sample lost up to 84.27%.These results suggest that the biofilm studied may slow down respiration and chemical metabolism, thus helping to maintain the acid content of fruit during storage.Ascorbic acid is an essential nutrient found in tomatoes but is easily degraded during processing and storage [33].Typically, the ascorbic acid content in ripe tomatoes will decrease with storage time [28].This loss of nutritional composition is undesirable; therefore, it is necessary to investigate the failure of vitamin C in tomatoes over storage time.Figure 7d shows that the group of fruits preserved by the study film had a significantly lower loss of vitamin C content than the control group.After 12 days of storage, the control group lost up to 63.89% of the vitamin C content, while the coating group lost only 30.56%.Thus, the studied biofilm can help to retain the ascorbic acid present in the fruit.

Conclusion
In this study, composite biofilms were prepared based on chitosan and MCC with different tonic contents.The study found that adding MCC to chitosan reduced the adhesion of the film and helped in obtaining intact film samples.The surface analysis showed that MCC molecules were evenly distributed in the chitosan matrix.The higher the MCC content, the higher the water uptake and solubility of the film, but it had a negligible impact on biodegradability.The results suggested that chitosan films supplemented with 3g MCC were the most effective.Applying chitosan-3g MCC film to preserve fresh tomatoes showed a reduction in weight loss, soluble solids, total acid, and vitamin C content.The composite biofilm of chitosan and MCC could limit water evaporation, chemical metabolism, fruit respiration, and spoilage microorganisms invasion, thereby extending the shelf life of tomatoes.

Figure 1 .
Figure 1.Physical image of the composite biofilms based on chitosan and MCC with different content.

Figure 3 .
Figure 3. Water uptake of studied biofilm at the different immersion time.

Figure 6 .
Figure 6.The external morphology of the tomatoes during storage.

Figure 7 .
Figure 7. Degree of weight loss (a), soluble substances content (SSC) loss (b), total acid content loss (c), and ascorbic acid content loss (d) of tomatoes over storage time.