The effect of carrageenan-gluten ratio and garlic essential oil concentration on physical, mechanical, and antimicrobial properties of edible film

Edible film can be made from natural polymers, such as fats, proteins, and polysaccharides. The combination of polysaccharides and proteins has the potential to form a film, it can improve the mechanical properties and physical properties of the film based on the complex structure formed. Furthermore, to improve the barrier properties by obstructing water vapour transmission and preventing the proliferation of spoilage microorganisms in food, it is important to present both hydrophobic substances and active compounds extracted from essential oils. This research aimed to find out the best formulation of gluten and garlic essential oil on the physical, mechanical, and bacteria-inhibitory properties of edible film. The method used in this research is the edible film made by combining carrageenan and gluten in several concentrations (7,5%; 10%; and 12,5%) and the addition of garlic essential oil at different concentrations (0%, 2%, and 4%), then observations were made on several test parameters such as solubility in water, thickness, water vapor transmission rate, tensile strength, and antibacterial properties of the film. Results obtained in the research demonstrated that the finest edible film treatment was found in addition of 12.5% gluten and addition of 4% garlic essential oil with the results of solubility parameter of 40.57% thickness of 0.73 mm tensile strength of 0.0009 N/mm2 and WVTR of 21.856 g/h/m2 and antibacterial properties against E. coli bacteria of 26.93 mm and against S. aurerus bacteria of 33.76 mm. This research concluded the addition of gluten and garlic oil could improve physical, mechanical, and antimicrobial properties edible film.


Introduction
Packaging was one of the preventive efforts in preventing food damage.Based on the basic material, the packaging is divided into two types, made from synthetic and natural-based.Synthetic-based packaging is generally derived from synthetic polymer materials such as petrochemicals that are not biodegradable so it can pollute the environment [1].In additions, natural-based packaging generally uses natural biopolymers such as proteins, polysaccharides, fats, and the addition of several other materials such as plasticizers and surfactants [2].To reduce the problem of environmental pollution due to synthetic packaging, it is necessary to develop pertaining to natural-based packaging [3].The development of natural-based packaging is currently a research trend, especially in terms of edible film development.So far, edible film is a research that has been intensified in various countries due to the high level of need and demand for fresh food without preservatives [4].The development of edible film is an effort to preserve the freshness and safety of food products.Unlike conventional packaging that protects food without direct contact, edible film utilizes direct contact with food to preserve the quality of the food because of the active compounds in edible film which act as antibacterial agents.Edible film 2 is a thin layer that serves as a primer packer to protect the product and is environmentally friendly because it can be eaten directly [5].
Edible film can be made from natural polymers, such as proteins, polysaccharides, fats, or the combination of all three materials.The combination of polysaccharides and proteins has the potential to form a film, as it can enchance the physical properties, mechanical properties, and chemical properties of the film based on the complex structure formed [6].The combination of carrageenan and gluten is carried out because it has high bioavailability and is easy to obtain.Carrageenan is used as a filmmaking material because it has good gelling and film-forming properties [7].In addition, carrageenan can form a polymer matrix to allow an increase in the tensile strength between molecules [8].On top of that, the use of gluten as a filmmaking material because it has compactness and elasticity that can help the mechanical strength of the film.Additionally, the application of gluten in films can show significant gas barrier properties and high selectivity [9].
Carrageenan is the most widely used material as a basic ingredient for edible film because it has excellent gelling properties and fairly high bioavailability.Research publications on the use of carrageenan as an edible film have been widely carried out.However, some research results show that the utilization of carrageenan as the basic ingredient of edible film has limitations on its physical properties, whereas carrageenan-based edible film generally has poor gas barrier properties and poor physical properties [10].Previous research showed that edible film derived from carrageenan only has a permeability value to water vapor of 7.5x10 -11 g/m/Pa/s and a permeability value to oxygen of 5.25x10 - 12 cm 3 /m/Pa/s [11].Results that are not much different were showing that edible film derived from carrageenan only has a permeability value to water vapor of 8.32x10 -11 g/m/Pa/s [12].The high permeability value greatly affects a package in protecting foodstuffs, especially from external factors such as water vapor exchange, gas exchange, and moisture from foodstuffs.Various attempts have been made to improve the structure of the film so that it does not have a high permeability value.One of them is the mixing of carrageenan with other compounds.Another previous research investigated that the addition of another biopolymer in order to improve the barrier value of edible film [13].One of the materials that could be used to enhance the barrier properties of the film is gluten.Based on previous research showed that the permeability value of edible gluten film to water is only 0.632x10 -11 g/m/Pa/s [6].In addition to the physical properties of edible film that must be improved, the antimicrobial properties of the film are also very necessary to be able to preserve the quality of the packaged foodstuffs.One of the compounds that can be added is the active compound derived from essential oils for example from the garlic essential oil.Garlic essential oil contain active compound, such as allyl sulfide, dialyl disulfide, methyl allyl disulfide, methyl allyl trisulfide, dialyl trisulfide, α-cadinol isopulegol, s-cadinol, citronella, cyclohexane, β-citronellol, neryl acetate, geraniol, citronelli acetate, βelements, δ-cadinen, dialyl tetrasulfide that have been found to possess various therapeutic properties such as antioxidant, antifungal, and anti inflammatory activities [14].In addition, the content of allisin or allil in garlic essential oil has the potential as an antimicrobial compound [15,16].The usage of garlic essential oil on edible film could damage bacterial cell walls and damage the DNA of bacteria so that they cannot replicate and stop bacterial growth [17].Based on previous research shows that the utilization of garlic oil in edible film as a sausage packer can maintain the quality of sausages from microbiological, chemical, and sensory properties.Therefore a study was carried out to find out the best formulation of gluten and essential oil of garlic against the mechanical properties, physical properties, and antimicrobial properties of edible film [18].This study used a combination of carrageenan (polysaccharide) and gluten (protein) as the basic ingredients in making edible film.This research was carried out to find out the effect of gluten concentration and garlic oil concentration on the edible film properties.

Gluten solution.
Gluten solution was made using gluten according to treatment (7.5%; 10%; and 12.5%) (w/v) dissolved into a mixture of ethanol and aqueous (55%:45%).Then the solution was homogenized while heated to a temperature of 70 o C for 10 minutes, during the stirring process, the addition of NH 4 OH was carried out until it reaches pH 10 [20].

Edible film manufacture.
The manufacture of edible film was carried out using a carrageenan solution that has been made mixed with gluten solution in a ratio of 1:1.The result of mixing the two solutions was heated at a temperature 70 o C.After that, the addition of glycerol (50%) (w / w of the total polymer), then stirring was carried out.Furthermore, garlic oil was added according to the treatment (0%, 2%, and 4%) (v/v), span 80 (0.4%) (v/v), and tween 80 (0.6%) (v/v).Then homogenization was carried out using the help of ultra turrax 24,000 rpm for 2 minutes.The homogenization results were then printed by casting using a petri dish with a diameter of 9 cm and dried in a blower oven with a temperature 50 o C for 16 hours [21].

Observation parameters 2.3.1. Solubility test.
The solubility test of the film was carried out dependent on the modified method from [22], the film was cut into small pieces and weighed 0.5 grams, and soaked in 25 ml of aqueous for 24 hours with a slight stirring, then the filter paper was dried with a temperature of 105 o C for 3 hours until the constant initial weight of filter paper (W0) was obtained.After that, the soaked film is filtered using filter paper of known weight.Then, the filter paper containing the insoluble film is again dried using the oven at a temperature 105⁰C for 5 hours until the constant final weight of the film (W1) was obtained.
: constant final weight of filter paper (g)

Water vapor transmission rate test.
The water vapor transmission rate test was conducted dependent on the modified method from [23], the saucer was prepared in advance, and then the film was cut according to the size of the saucer to be used.Then, 15 ml of aqueous water is put into a saucer, then the saucer is closed using edible film and sealed using plasticine so that there is no air gap.After that, the saucer is weighed at its initial weight.Then, the saucer is put into a desiccator that contains CaCl2 saline solution with 30% relative humidity and at a temperature 25ºC.Then, the dish is weighed every 1 hour for an interval of 5 hours to determine the rate of displacement of moisture.The tensile strength test of the film was carried out dependent on the modified method from [24], the film was cut into 4 x 2 cm size, then attributed to the clamp horizontally on both sides of the tool for measurement.The tensile strength of the film was measured by looking at the maximum pull at the time the film sheet breaks by the maximum force for tearing the film (F) divided with the cross-sectional area of the film (A).
() =   F : maximum force for tearing the film (N) A : cross-sectional area of the film (mm 2 ) 2.3.4.Thickness test.The thickness test was found out dependent on the modified method from [22], the thickness of the film was measured using a micrometer at 5 random points on the film, then calculated the average of the 5 points as the thickness of one film.

Microbial inhibitory test.
The bacterial inhibitory test of the diffusion method was carried out based on the modified method from [25], a film cut measuring 1x1 mm, placed on top of the nutrient agar media surface which the test bacterial culture has been added, namely Escherichia coli and Staphylococcus aureus bacteria.Then the sample was kept in an incubator at 37°C for 48 hours.Subsquently, the clear inhibition zone along with the diameter of the edible film was measured using a digital caliper.The results of the observations are stated in millimeters (mm).

Research design
The design of this study used a randomized design of a group of 2 factorials.Factor A is the gluten concentration has 3 levels, namely: A1 : 7.5% (b/v) gluten A2 : 10% (b/v) gluten A3 : 12.5% (b/v) gluten Factor B is the garlic essential oil concentration has 3 levels namely: B0 : 0% (v/v) garlic oil B1 : 2% (v/v) garlic oil B2 : 4% (v/v) garlic oil Each treatment was repeated 3 times.Each sample was tested for tensile strength, water solubility, film thickness, water vapor transmission rate, and bacterial inhibitory test.

Data analysis
The data gained from the research stage was analyzed with ANOVA (analysis of variance) and continued with Duncan's further tests to see any differences.The software used in processing data is Microsoft Excel 2013 and SPSS.

Results and discussion
The results of this study demonstrate that edible films with different concentration of gluten solution and different concentration of garlic essential oil had different physical, mechanical, and antimicrobial properties.There are several parameters measured and have a difference between each treatment.

Water solubility
Edible film is intended as a primary packer that can ignite foodstuffs directly, therefore the solubility properties of edible film are important to know because it will be in direct contact with foodstuffs.Film solubility is closely related to the digestibility properties as well as the environmentally friendly properties of the film [26,27].On the other hand, solubility is also an indicator to measure the strength of the film in retaining water.When the film has a high solubility, it is feared that it will have low water resistance and will be difficult to apply to foodstuffs that have high humidity, so the percentage of solubility of the film needs to be regulated in such a way as to achieve all the necessary functions.The results of water solubility measurements can be seen in Figure 1 Figure 1.The solubility of edible film with different concentrations of garlic oil.Based on the results of the analysis of variance, it was shown that the difference in gluten concentration did not have a noticeable effect (p>0.05) on the water solubility value of edible film, while the addition of garlic oil had a noticeable effect (p<0.05) on the water solubility value of edible film.The interaction between the difference in gluten concentration and the addition of garlic oil did not exert a noticeable effect (p>0.05) on the value of the water solubility of edible film.Based on this, it is necessary to conduct further tests on Duncan test to see the real differences between treatments.Based on the data obtained in the study, the edible film has a range of water solubility values between 40.57% -52.30%.
In the garlic oil addition treatment, the highest film water solubility was possessed by no garlic oil addition at 51.27%; then the addition of garlic oil at 2% at 45.82%; and the lowest in garlic oil addition of 4% at 42.16%.This shows that the addition of garlic oil with a higher concentration is capable of producing a lower water solubility value.It can occur as a result of garlic oil having hydrophobic properties that prevent the film from dissolving in water, thereby lowering the water solubility of the film.The greater the concentration of oil used, the more hydrophobic groups will result in a decrease in the water solubility of the film.This is aligns with the statement from [28] that the use of higher essential oils results in fewer hydrophilic groups and makes it difficult for water to dissolve the film.

Water vapor transmission rate
Edible film was expected to have the ability to maintain the quality of packaged foodstuffs.One of the parameters for testing such capabilities is the water vapor transmission rate (WVTR) which is a crucial parameter in packaging systems.WVTR shows the transfer of moisture between the environment in the package and the environment outside the package [29].The WVTR value of edible film greatly affects the quality security of foodstuffs.The lower the WVTR value, the higher the ability of a package to extend the shelf life because it is able to become a barrier or barrier from moisture so as not to increase the humidity and water activity of foodstuffs.The results of measuring the rate of transmission of water vapor can be seen in Figure 2 51.27   Based on the results of analysis of variance, shows that the difference in gluten concentration has a noticeable effect (p<0.05) on the value of WVTR of edible film, while the addition of garlic oil does not give a noticeable effect (p>0.05) on the value of the WVTR of edible film.The interaction between the difference in gluten concentration and the addition of garlic oil does not exert a noticeable effect (p>0.05) to the value of WVTR of edible film.Based on this, it is necessary to conduct further tests on Duncan test to see the real differences between treatments.Based on the data obtained in the study that the edible film produced has a range of WVTR values between 20.365 g/h/m 2 -31.510 g/h/m 2 .
In the gluten addition treatment, the highest WVTR was owned by the addition of 7.5% gluten at 30.28 g/h/m 2 ; then the addition of 10% gluten at 27.49 g/h/m 2 ; and the lowest at 12.5% gluten addition of 21.21 g/h/m 2 .This indicate that the higher concentrations of gluten was capable of producing lower WVTR values.It can occur due to the addition of gluten which is getting bigger and bigger causing the increase in the hydrophobic group which is getting bigger and causing water vapor to not be able to transmit easily.Gluten is water-insoluble material or has fewer hydrophilic groups, the complex gluten structure consisting of glutenin and gliadin can give a high barrier effect and selectivity because it has viscoelastic properties that cause water vapor not be easy to transmit [30].On the other hand, the edible film preparation process using the casting method can cause gluten to create an open loose network that has conducive conditions to form hydrophobic chain bonds and cross-linking bonds caused by consistent heating and stirring, where these heating conditions cause the protein to open and expose out the hydrophobic group [31,32].This is in line with the previous research statement, that the application of gluten on films has been shown to be able to show significant gas barrier properties and has high selectivity [9].

Tensile strength
One of the factors that influence the quality of packaging comes from the mechanical properties possessed.Edible film is expected to have good mechanical properties so that it is easy to apply to foodstuffs, one of the commonly used mechanical properties is tensile strength.Tensile strength describes the maximum tensile value that edible film can achieve [33].The tensile strength value becomes very important to know because it describes the strength, stability, and durability of edible film [34].The results of tensile strength values can be seen in Table 1.results of the analysis of variance, it shows that the difference in gluten concentration does not have a noticeable effect (p>0.05) on the tensile strength value of edible film, while the addition of garlic oil, it has a noticeable effect (p<0.05) on the tensile strength of edible film.The interaction between the difference in gluten concentration and the addition of garlic oil gives a noticeable influence (p<0.05) on the tensile strength value of edible film.Based on this, it is necessary to conduct further tests on Duncan test to see the real differences between treatments.Based on the data obtained in the study, the edible film has a tensile strength value range between 0.0009 mPa -0.0163 mPa.
The highest tensile strength value was investigated in the treatment of using 10% gluten with the addition of 2% garlic oil which was 0.0163 mPa, while the lowest tensile strength value was obtained in the treatment of using gluten 12.5% with the addition of 4% garlic oil which was 0.0009 mPa.This suggests that the decrease in tensile strength of edible film is strongly influenced by the addition of garlic essential oil to the blend.The addition of essential oils causes a decrease in intra-and intermolecular interactions in the biopolymer network formed and leads to reduction in the bonds between molecules in the matrix film and result in the formation of weak interaction bonds between biopolymers and oils in the matrix film [35].This is similar with the previous statement from [36], that the addition of garlic essential oil with a greater concentration will result in a very significant decrease in the tensile strength of the film.

Thickness
The thickness of the film becomes one of the macroscopically visible physical properties of the film that must be known because it has an influence on several other parameters of the film.Film thickness is strongly influenced by several factors, especially when printing the film solution in the saucer, such as the size of the mold, the volume of the solution, and the drying temperature used [37].In general, an enhancement in the biopolymer amount is able to increase the thickness of the film, but it is also very dependent on the interaction among the constituent elements of the film.The results of thickness analysis can be seen in Figure 3 Figure 3.The thickness of Edible Film with different concentrations of garlic oil.Based on the results of the analysis of variance, it shows that the difference in gluten concentration does not have a noticeable effect (p>0.05) on the thickness value of edible film, As for the addition of Thickness (mm) Garlic Oil Concentration garlic oil, it has a noticeable effect (p<0.05) on the thickness value of edible film.The interaction between the difference in gluten concentration and the addition of garlic oil did not exert a noticeable effect (p>0.05) on the thickness value of edible film.The data presented in figure 3 shows the differences in thickness value resulting from varying concentrations of garlic oil used.It is evident that further tests are necessary to determine the real differences between treatments, which can be conducted using the Duncan test.The research reveals that the edible film produced had a thickness value range of 0.72 mm -0.84 mm.The addition of garlic oil resulted in different thickness values, with the highest thickness recorded at 0.79 mm for the addition of garlic oil and 2% concentration, and the lowest value of 0.72 mm for the addition of 4% garlic oil.The research demonstrates that the thickness value of the edible film is affected by the concentration of garlic oil added.This suggests that the addition of garlic oil leads to a decrease in the thickness of the film.The film thickness results obtained have not met the Japan Industrial Standard (JIS), which have the maximum edible film standard must be below 0.25 mm.During the study, it was observed that the edible film without the addition of garlic oil when drying the size tended to shrinked and cause the surface of the film to become thicker, compared to edible film with essential oil.This could be due to the presence of essential oils containing phenolic compounds that are able to cause more covalent cross-linking bonds and are able to reduce the tendency of the film to shrink during the drying process [38].In addition, carrageenan also has a role in the occurrence of shrinkage in films.
Based on research conducted from [39] found that the higher temperature used in drying results in an increase in the thickness of the gel film produced as a result of when drying occurs the release of water from the matrix film so that there is a shrinkage of the matrix film.However, when the addition of essential oils is carried out, it is suspected that there is an interaction between biopolymers and essential oils and forms crosslinking that is able to decrease the movement of polymer molecules and reduce the tendency for film shrinkage to occur.

Microbial inhibitory properties
The inhibitory power of microbial film with the addition of garlic oil was tested against two types of bacteria, namely Escherichia coli which is gram-negative, and Staphylococcus aureus which is grampositive.Both are used because they include spoilage bacteria that are most commonly found in fresh meat and are pathogenic or contribute to food-borne diseases [40,41].The test was carried out by measuring the inhibition zone on the media which was characterized by the presence of clear areas that appeared around the film.In general, the greater the active substance added, the greater the inhibitory power formed [42].The results of the inhibitory power of edible film microbes can be seen in Table 2. the results of the analysis of variance, it shows that the increasing concentration of garlic oil has a noticeable effect (p<0.05) on the microbial inhibitory value of edible film in the type of E. coli bacteria, while the increase in garlic oil concentration, it has a noticeable effect (p<0.05) on the microbial inhibitory value of edible film in the type of S. aureus bacteria.In the table, it can be seen the real difference in the inhibitory power value of microbes from two different types of microbes based on the difference in the concentration of garlic oil used.
Based on the results obtained, all concentration treatments of the addition of garlic oil to the film indicate the presence of an inhibitory zone.The presence of inhibitory zone could be categorized in three levels, which is strong, moderate, and weak.The inhibitory level could be measured from the zone of 1230 (2023) 012165 IOP Publishing doi:10.1088/1755-1315/1230/1/0121659 inhibition, strong inhibitory effect has zone of inhibiton more than 28 mm, while moderate inhibitory effect has zone of inhibition about 16 mm to 28 mm, mild/weak inhibitory effect has zone of inhibition about 12 mm to 16 mm, and if it lower than 12 mm it is considered as no inhibitory effect [43].According previous research, the inhibitory zone around the film arises because the antimicrobial agent of garlic oil diffuses into the agar medium, then inhibits the process of germination and growth of test bacteria so that a clear area is formed which is a clue to the inhibitory response of garlic oil compounds.Especially for the phenomenon without the addition of garlic oil which also has inhibitory power, based on observations this occurs due to the disintegration of matrix film, where around the side of the edible film it appears that there is a lipid-like liquid that is suspected to come from edible film additives such as glycerol, tween 80, and span 80 so that the lipids diffuse into the agar media and cause bacteria to be unable to replicate.
The emergence of film inhibition zones against both types of test bacteria shows that the antibacterial activity of garlic oil has a broad spectrum because it is effective in inhibit the growth of gram-positive or gram-negative bacteria.Based on observations, films show that they are more effective at obstruct the growth of gram-positive bacteria compared to gram-negative bacteria, this happens because gram-negative bacteria have lipopolysaccharide cell walls that are able to inhibit the diffusion of film antimicrobial agents toward cells [44].According to latest research, the larger the size of the clear zone around the film, the larger the film inhibition zone [45].The main antimicrobial compound in garlic is alisin, with derivative compounds, namely dialyl sulfide, dialyl disulfide, dialyl trisulfide, and ajoene.Latest research report that the active compound ajoene is responsible for antibacterial activity by inhibiting enzymes containing sulfhydryl groups present in bacteria [41].This microbial inhibition mechanism occurs because essential compounds from garlic oil damage the resistance of microbial cells and break down the cytoplasm of bacteria causing cell death from bacteria [46].The addition of garlic essential oil to edible film can damage bacterial cell walls and damage the DNA of bacteria so that they cannot replicate and stop bacterial growth.Based on previous research stated that garlic oil is able to inhibit the growth of pathogenic microbes by the mechanism of volatile compounds from garlic oil damaging the tissues of bacterial cell membranes by means of compounds from oil entering the cell membrane and causing cell swelling eventually resulting in leakage of bacterial cells so as to damage the entire cell structure and end in the death of the bacteria.In addition, it was also observed at the cellular structure level using SEM that the cell structure that had been damaged by garlic oil had shrunk and changed shape to be more constricted and reduced when compared to control cells [47].Another research also showed that higher amount of garlic oil added could increased the inhibition zone, it happens due to more sulfide compund presents in higher concentration of garlic oil made the microbial cells break and leading to the cell death and damaged the bacterial growth [48,49].

Conclusion
In conclusion, the different in level of gluten concentration could affect the water vapor transmission rate of the film, on top of that the difference level of garlic could affect the solubility, thickness, tensile strength, and microbial inhibitory properties of film.Furthermore, the best formulation of edible film obtained based on physical, mechanical, and microbial inhibitory properties was obtained at the treatment of gluten concentration of 12.5% and the addition of garlic oil by 4% with a water solubility value of 40.57%, a water vapor transmission rate value of 21.856 g/m 2 / hour, a tensile strength value of 0.0009 mPa, a thickness value of 0.73 mm, and microbial inhibitory values in E. coli bacteria of 26.93 mm and S. aureus of 33.76 mm.

Figure 2 .
Figure 2. The WVTR of Edible Film with different concentrations of gluten.Based on the results of analysis of variance, shows that the difference in gluten concentration has a noticeable effect (p<0.05) on the value of WVTR of edible film, while the addition of garlic oil does not give a noticeable effect (p>0.05) on the value of the WVTR of edible film.The interaction between the difference in gluten concentration and the addition of garlic oil does not exert a noticeable effect (p>0.05) to the value of WVTR of edible film.Based on this, it is necessary to conduct further tests on Duncan test to see the real differences between treatments.Based on the data obtained in the study that the edible film produced has a range of WVTR values between 20.365 g/h/m 2 -31.510 g/h/m 2 .In the gluten addition treatment, the highest WVTR was owned by the addition of 7.5% gluten at 30.28 g/h/m 2 ; then the addition of 10% gluten at 27.49 g/h/m 2 ; and the lowest at 12.5% gluten addition of 21.21 g/h/m 2 .This indicate that the higher concentrations of gluten was capable of producing lower WVTR values.It can occur due to the addition of gluten which is getting bigger and bigger causing the increase in the hydrophobic group which is getting bigger and causing water vapor to not be able to transmit easily.Gluten is water-insoluble material or has fewer hydrophilic groups, the complex gluten structure consisting of glutenin and gliadin can give a high barrier effect and selectivity because it has viscoelastic properties that cause water vapor not be easy to transmit[30].On the other hand, the edible film preparation process using the casting method can cause gluten to create an open loose network that has conducive conditions to form hydrophobic chain bonds and cross-linking bonds caused by consistent heating and stirring, where these heating conditions cause the protein to open and expose out the hydrophobic group[31,32].This is in line with the previous research statement, that the application of gluten on films has been shown to be able to show significant gas barrier properties and has high selectivity[9].

Table 1 .
The tensile strength of edible film

Table 2 .
The microbial inhibitory properties of Edible Film with different concentrations of garlic oil.