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Antibacterial activities of R-(+)-Limonene emulsion stabilized by Ulva fasciata polysaccharide for fruit preservation
International Journal of Biological Macromolecules 111 (2018) 1273–1280
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International Journal of Biological Macromolecules journal homepage: https://www.journals.elsevier.com/ijbiomac
Antibacterial activities of R-(+)-Limonene emulsion stabilized by Ulva fasciata polysaccharide for fruit preservation Ping Shao a,⁎, Haoya Zhang a, Ben Niu a, Ligang Jiang b a b
Department of Food Science, Zhejiang University of Technology, Hangzhou 310014, PR China R &D Center, Proya Cosmetics Co. Ltd., Hangzhou 310012, PR China
a r t i c l e
i n f o
Article history: Received 3 December 2017 Received in revised form 11 January 2018 Accepted 17 January 2018 Available online xxxx Keywords: Antimicrobial activity Edible coatings Essential oil Fruit preservation
a b s t r a c t It was previously found that R-(+)-Limonene emulsion could be stabilized by Ulva fasciata polysaccharide. In the present study, emulsions of R-(+)-Limonene were developed for coating of strawberries to improve the shelf life and microbiological safety. Edible coatings with at least 0.15% w/w of R-(+)-Limonene improved the microbial stability of the strawberries, resulted effective in the decontamination of external pathogens such as Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Changes in weight loss, total soluble solid (TSS), pH, total phenolic, ascorbic acid, textural properties and surface color of strawberries during storage were evaluated. R-(+)-Limonene emulsions were effective for decreasing water loss of all the fruit samples tested. The decrease in TSS values was higher in the control strawberries. Initial decreases followed by increases of pH during storage of strawberries. The amount of total phenols detected in control fruit sharply increased during the first 6 days then remained steady, whereas the total soluble phenols content of treated strawberries gradually increased during storage. Strawberries coated with emulsion had higher L* and a* values as compared to control fruits. The R(+)-Limonene coatings have demonstrated the potential to inhibit foodborne pathogen contamination of strawberries, and prolong their shelf life. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Fruits have short shelf life due to high perishability, physiological disorders, water loss, and decay [1]. They usually have short ripening and senescent periods that make marketing a challenge. This makes the development of an optimal packaging solution to maintain the quality of these produce during storage of economic interest. Adverse activity of antimicrobial microorganisms is an important task of the fruit preservation. Emulsion-based edible coatings have been widely studied for preservation of fruits and vegetables. Their beneficial effects and easy handling made them broadly applicable to soft fruits such as berries. The main functional advantages attributed to use of edible coatings are slower respiration rate, extended storage periods, firmness retention and controlled microbial growth [2]. In this way, emulsionbased edible coating technology is an alternative method with which to extend the commercial shelf-life of coated products by modifying their internal atmosphere. To meet consumer demands, the application of essential oils as natural antimicrobial compounds in foods has attracted growing interest, in terms of food quality and safety, by substituting chemical preservatives for natural substances with similar properties [3]. R-(+)⁎ Corresponding author. E-mail address: [email protected] (P. Shao).
https://doi.org/10.1016/j.ijbiomac.2018.01.126 0141-8130/© 2018 Elsevier B.V. All rights reserved.
Limonene is the major aromatic compound in essential oils obtained from oranges, grapefruits, and lemons [4]. Moreover the R-(+)-Limonene possess antioxidant, antimicrobial, antifungal, nematocide, antiinflammatory, anti-carcinogenic properties [5]. The improvement of preservation techniques to reduce the growth and activity of spoilage microorganisms in foods is crucial to increase their shelf life and to reduce the losses due to spoilage. Essential oils can be encapsulated in emulsion systems stabilized by Ulva fasciata polysaccharides (UFP). In our previous study, we have successfully extracted Ulva fasciata polysaccharides from Ulva fasciata, which is an amphipathic anionic polysaccharide with the potential of food hydrocolloids and it is able to form gels in presence of borate and calcium ions. We also studied their rheological properties, characteristics, hydrodynamic behavior and dilute solution properties [6]. We have further studied the rheological properties of curcumin coated with UFP stabilized emulsions and the effect of metallic ions on the physicochemical stability of curcumin emulsions stabilized by Ulva fasciata polysaccharide [7]. Not only curcumin has been considered, we also studied R-(+)-Limonene. In our previous study, when R-(+)-Limonene be encapsulated in emulsion stabilized by UFP, it shows that when UFP concentration is 4%, emulsion have homogeneous particle size and the best stability which have high zeta potential (−52.6 mV) and low average droplet diameter (2.45 μm). Otherwise emulsion stabilized by UFP shows better stability
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than Arabic gum and gelatin [8]. The stability of emulsions has been researched deeply, but its application value is still unconsidered. The aim of the present study was to develop an edible coating by incorporating different concentrations of R-(+)-Limonene emulsion into UFP as a carrier matrix. We assessed the antimicrobial effectiveness of emulsions-based edible coatings containing R-(+)-Limonene against inoculated different bacteria, and their capability to improve the shelf life of a highly perishable strawberry. 2. Materials and methods 2.1. Materials Strawberries were purchased from a local wholesale company in Hangzhou, China, and selected for uniformity of size and ripeness, and fruit with apparent injuries were removed. Fruit were stored at 4 ± 1 °C prior to processing. The Ulva fasciata was collected on the coast of Nanji Archipelago (Zhejiang province, China). Soybean oil (60 wt%) linoleic acid, 30 wt% oleic acid, and specific gravity (0.197) was obtained from KLK Oleo, Ltd. (Malaysia). R-(+)-Limonene (95%) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Emulsions preparation Ulva fasciata polysaccharides were extracted using the method described in our previous work [9]. Composition analysis result shows that total content is 99.02%, composed of 42.59% sugars, 20.05% sulfate, and 36.38% uronic acid. The emulsion was prepared according to the method of Shao [10]. The R-(+)-Limonene was added dropwise to the soy oil at a rate of 10% to form the dispersed phase. 4%(w/w) of Ulva fasciata polysaccharide was dispersed in aqueous solution by using to an overhead mixer (IKA Labortechnik, IKA works, Malaysia) for 24 h at room temperature to form the continuous phase. Using a high-speed blender (Kinematica PT-MR 2100, Switzerland) to blending UFP solution and the R-(+)-Limonene dispersed phase in a ratio of 95:5 (v/v) and pre-homogenization at 26,000 rpm for 3 min. Put the mixtures pass through a high pressure homogenizer at 75 MPa (Microfluidics M-110L, USA). 2.3. Determination of MIC of emulsions Minimum Inhibitory Concentrations (MIC) of R-(+)-Limonene against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were determined by a broth microdilution method according to Clinical Laboratories Standards Institute (CLSI) guidelines (CLSI, 2012). Emulsions (20,000 μL/L) were dissolved in agar medium and serially diluted in a 96-well plate. Bacteria inoculum in 50 mL agar was added to the wells to obtain final concentrations of R-(+)-Limonene at 0, 19, 39, 78, 156, 312, 625, 1250, 2500 and 5000 μL/L. The final concentration of bacteria was 1 × 105 CFU/mL. Plates were incubated at 37 °C for 24 h and then measured. The MIC was determined as the lowest concentration of the emulsion that inhibited microbial growth with an absorbance value b0.05 at 595 nm [11]. 2.4. Inoculation of strawberry and application of R-(+)-Limonene emulsions Stock cultures of Staphylococcus aureus, Bacillus subtilis and Escherichia coli at 37 °C for 18 h, followed by washing in PBS for 2 times before inoculation. Strawberries were purchased from a local supermarket. Strawberries weighing 10.0 ± 1.0 g and with a diameter of 2.5 ± 0.1 cm were selected. The selected strawberries were stored at 4 °C and separated randomly into two groups for (1) microbial analysis
and (2) analysis of their physical properties. Stalks were removed from the strawberries before the experiments. One milliliter of individual bacterial suspension in PBS (105 CFU/mL) was coated to strawberry, followed by mixing and drying for 15 min in a Biosafety cabinet. The strawberry was then dipped in 200 mL emulsions with 0.05%, 0.1% v/v and 0.15% of R-(+)-Limonene emulsions for 1 min, dried for 30 min, and then stored in bags at 4 °C. UFP solution (4%, w/w) was used as control. Emulsion concentrations of 0.05% (500 μL/L), 0.1% (1000 μL/L) and 0.15% (1500 μL/L) were selected based on the MIC results [12]. 2.5. Microbial analysis Changes in the numbers of Escherichia coli, Bacillus subtilis and Staphylococcus aureus on the surfaces of strawberry treated with R-(+)-Limonene emulsions were determined during the storage period. On each day (0, 2, 4, 6, and 8 days), one stored strawberry was added to 225 mL of sterilized peptone water (1 g/L) in stomacher bag and homogenized for 2 min. Appropriate serial dilutions were done and then 1.0 mL of every samples were plated by standard microbiological pour plate technique. All the microbiological counts were carried out in triplicates. 2.6. Weight loss To determine weight loss, the same strawberries were weighed at the beginning of the experiment and thereafter each day during the storage period. Weight loss was expressed as the percentage loss of the initial total weight. Ten fruits in three repetitions were used to evaluate the weight loss of every group. 2.7. Total soluble solids (TSSs), and pH determinations To determine soluble solids content and pH values of samples, three replicates of strawberry juice was used. Juice was obtained by homogenizing nine fruits in a blender. Measurements were performed by a digital refractometer (Atago, Tokyo, Japan) and pH meter (Sartorius PP-50, Goettingen, Germany) [13]. 2.8. Total phenolic compound analysis Total phenolic compound of fruits extract were determined by the Folin–Ciocalteu micro-method [14]. Fruit samples of 0.5–1 g were extracted with 10 mL of 80% ethanol and the extract was centrifuged at 10,000 rpm for 20 min. The clear supernatant was collected and allowed to evaporate at room temperature, and the residue dissolved in a known volume of distilled water. 2.5 mL of Folin Ciocelteau's phenol reagent (Merck) (10-fold-diluted) was thoroughly agitated with 0.5 mL of the sample and 2 mL of 7.5% Na2CO3. The mixture was kept at room temperature for 30 min in dark. Then the absorbance was recorded at 760 nm using a UV–Vis spectrophotometer (Shimadzu, UV-1800). 2.9. Ascorbic acid analysis Ascorbic acid content was determined by the method of Gol [15]. The fruit sample (100 mg) was treated in 5% metaphosphoric-glacial acetic acid solution. The extract was centrifuged and a clear supernatant collected. Suitable aliquots of the supernatant were taken in a test tube containing known amounts of 5% metaphosphoric-glacial acetic acid solution, dinitrophenyl hydrazine (2%) and thiourea (10%) solutions. The test tubes were incubated for 3 h at 37 °C in a water bath, after which the reaction was terminated by adding 85% sulphuric acid. The optical density of the reaction mixture was measured against a blank at 540 nm. Levels of ascorbic acid were estimated from a standard curve prepared from pure ascorbic acid.
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2.10. Textural properties
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Escherichia coli
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2.11. Color assessment
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Minolta CR-400 colorimeter (Minolta, Osaka, Japan) was used to express L* (Lightness) and a* (red–green) color parameters of strawberry. Color meter was calibrated using the standard white plate before measurements. Nine strawberries were analyzed and measurements were taken in three different points at the equatorial zone.
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Log CFU/g
Texture profile analysis (TPA) parameters (hardness, cohesiveness, adhesiveness, springiness, gumminess, resilience and chewiness) were determined on fresh strawberry using a Texture Analyzer (Stable Micro Systems, Surrey, England). Measurements were taken along horizontal axis of the nine fruits. A cylindrical plunger with a diameter of 10 mm (SMS-P/10 CYL Delrin) compressed the fruits with pretest speeds of 5 mm/s, test speed of 1 mm/s, post speed of 8 mm/s, penetration distance of 4 mm and trigger force of 1.0 N. Texture Exponent software was used to automatically calculate the TPA parameters of fruits on the basis of fore-deformation curves [16].
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2.12. Statistical analyses 14
Statistical analyses were performed using Origin (ver. 6.0, IBM software, Chicago, USA). One-way analysis of variance (ANOVA) was used to evaluate the significance of differences between sample groups at a level of P ≤ 0.05.
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3. Results and discussions 6
3.1. Minimum inhibitory and minimum bactericidal concentration 4
3.2. Microbial analysis The development of bacteria growth in the in vitro tests is shown in Fig. 1 for different concentrations of emulsion. The use of pure UFP had no effect on bacterial growth. Both concentrations (0.1% and 0.15%) of R-(+)-Limonene emulsions were able to inhibit Escherichia coli and Bacillus subtilis growth significantly (P b 0.05) compared to controls, though a stronger effect was demonstrated by the 0.15% emulsion. The 0.15% emulsion was able to reduce Staphylococcus aureus population. The 0.1% emulsion was able to reduce Escherichia coli population by 0.5 logs at 1 d and then the population increases with time after the
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The MIC of R-(+)-Limonene emulsions was 0.0625% (625 mL/L) for Bacillus subtilis and Escherichia coli and 0.125% (1250 mL/L) for Staphylococcus aureus. Plant essential oils have shown strong antimicrobial effect. However, their application in food protection is still limited partially due to their low solubility in water. Formulating oil emulsions using food-grade emulsifiers increases the solubility and surface area of essential oils, and consequently, enhances their antimicrobial activity. The chemical structure of limonene can carry out active electron transfer, which can interact with ion channel proteins on the surface of microorganisms to change cell membrane permeability and inhibit the growth of microorganisms. Generally, gram-negative bacteria are more resistant to the essential oil treatment than gram-positive bacteria due to the former one having a lipopolysaccharide (LPS) protection from hydrophobic compounds. But the results of MIC are not entirely consistent with this regular pattern. There are some other studies have found gram-positive bacteria less sensitive to essential oils than gram-negative strains [17,18]. This may be due to the differences in the sensitivity of different bacteria strains to essential oils. However, the simple relation involving cell structure and microbial sensitivity to essential oils is not yet well established, and possible antagonistic or synergistic effects among the various active constituents of the oils should be taken into consideration [19].
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Time (days) Fig. 1. Effect of emulsion on the growth and survival of (A) Escherichia coli, (B) Bacillus subtilis and (C) Staphylococcus aureus at 0 d, 2 d, 4 d, 6 d and 8 d after the treatment. Error bars represent standard error of the mean. Measured at 25 °C.
treatment. Increasing concentrations in essential oils caused enhanced inhibition of Escherichia coli by the 0.15% treatment at all testing points. Of the three bacteria, Staphylococcus aureus was the least sensitive to the emulsion treatment. The mechanism of antibacterial and antifungal properties of the essential oil components is connected with the ability of these hydrophobic substances to incorporate into the membrane. Since membranes are the first barrier surrounding the cell, the ability of the essential oil components to successfully incorporate into these structures
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All fruit showed a progressive loss of weight during storage (Fig. 2A). As known, strawberry fruits are susceptible for water loss and thereby shriveling and deterioration because of their thin skin structure [20]. Throughout the storage period, there were significant differences between the control and R-(+)-Limonene emulsions treatments. Weight loss is mainly interested with moisture and moisture evaporation from the skin of fruits. Therefore, their textural properties play role on weight loss. In this study, R-(+)-Limonene emulsions were effective for decreasing water loss of all the fruit samples tested. The increase in oil concentration appeared to affect the weight loss, which could be predominantly caused by moisture loss. The progress of weight loss also could be partly attributed to an increase in the berry's metabolic activity, associated with tissue senescence over long storage times, which is slowed down after coating application [21].
7.5 7.0
Total soluble solid (TSS) is an important parameter that affects fruit quality and consumer acceptability [13]. The results obtained from the TSS analysis are presented in Fig. 2B. TSS levels at the beginning of storage was 5.5% and increased gradually during storage in both emulsions coated and pure UFP uncoated strawberries, that could be partially explained by the conversion of starch to sugars but, since strawberries accumulate very little starch, it may be mainly because of an increase in anthocyanins which contribute to soluble solids. During 4 days of storage, control samples exhibited significantly higher TSS i.e. 7.9%
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Fig. 2. (A) Weight loss (%) and (B) Total soluble solid (%) and (C) pH of strawberries during 8 days of storage. Error bars represent standard error of the mean. Measured at 25 °C.
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and the magnitude of perturbations induced by these compounds at this level may determine the antimicrobial effect of the substance. Using R(+)-Limonene emulsions led to longer-lasting antimicrobial activity.
Fig. 3. (A) Total phenolic content (mg/100 g) and (B) Ascorbic acid (mg/100 g) of strawberries during 8 days of storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
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compared to all other coated samples. In control fruit the TSS content declined from 7.9 to 6.3% during 4 days of storage to 8 days, while the fruit treated by R-(+)-Limonene emulsions showed a gradual increase in their TSS content up to 8 days of storage and thereafter declined. These results are in accordance with the report of Velickova et al. [2] who showed a decrease in the TSS content in strawberries at the end of storage and attributed it to respiration. The decrease in TSS values was higher in the control strawberries. This observed diminishment can be explained by the hydrolysis of sucrose in order to maintain physiological activity and respiration. The effect of different concentrations of R-(+)-Limonene emulsions on pH of strawberries as compared to control samples is presented in Fig. 2C. The pH of strawberry fruit treated by 0.05% R-(+)-Limonene emulsions and UFP treated increased during the entire storage period. These results are in agreement with those of Gol who reported that a higher increase in pH was found in the control samples as compared to edible coated strawberries [15]. The value of pH is related to the
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fruit senescence, increased significantly during the storage due to utilization of organic acids during respiration [22]. The pH of strawberry fruit treated by 0.1% and 0.15% R-(+)-Limonene emulsions was found to have significantly higher pH than the control. The pH of strawberry fruit treated by 0.1% and 0.15% R-(+)-Limonene emulsions, decreased up to 4 days of storage but there is no significant increase on the 8th day. The obtained results are in agreement with similar research conducted by Dhital [23], who observed initial decreases followed by increases of pH during storage of strawberries. 3.5. Total phenolic compound analysis The variation in the total phenolic content as a function of R-(+)Limonene emulsions and storage time for strawberry fruit is presented in Fig. 3A. There was an increasing trend in the total phenolic content for both control and treated fruit up to 8 days of storage. Total phenols, the major antioxidants in plant and fruit tissues, are produced as secondary
Fig. 4. Texture parameters a) firmness, b) adhesiveness, c) springness, d) cohesiveness, e) gumminess, f) chewiness changes of strawberry during storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
P. Shao et al. / International Journal of Biological Macromolecules 111 (2018) 1273–1280
metabolites in response to abiotic and biotic stresses to protect cellular constituents [24]. The change in the concentrations of phenolic compounds in fruits during storage can be attributed to cell structure breakdown [25]. The amount of total phenols detected in control fruit sharply increased during the first 6 days (from 807 to 1552 mg GAE/100 g fresh weight.) then remained steady, whereas the total soluble phenols content of treated strawberries gradually increased during storage. The constant exposure of strawberries to antimicrobial R-(+)-Limonene, which can be considered a controlled stress, might have caused such enhancement at the end of storage time. This outcome could be associated with the different interactions between constituents of essential oils and the food matrix. 3.6. Ascorbic acid analysis Ascorbic acid maintains the physiological functions in the human body and is a measure of the freshness of fruits. The effect of R-(+)-Limonene emulsions on the ascorbic acid content of strawberries is shown in Fig. 3B. The ascorbic acid decreased during the storage period in all treated samples as well as control sample. Treated with R-(+)-Limonene emulsions significantly delayed the rate of ascorbic acid decrease. The initial ascorbic acid content of strawberry fruit was 67.26 mg 100 g−1. Treated with 0.1% and 0.15% R-(+)-Limonene emulsions showed the highest content of ascorbic acid i.e. 23.6 and 27.2 mg 100 g−1, respectively, at 8 days of storage, and was significantly superior to all other samples as well as the control. Ascorbic acid content of strawberries decreased during the storage period and the similar results have been reported in strawberries by Amal [26]. The emulsion evaporates to form a protective film on the surface of the fruit and could inhibit gas exchange between the tissues and the environment, which reduces the O2 concentration that oxidizes ascorbic acid; thus, ascorbic acid oxidation was effectively prevented, thereby keeping a higher amount of ascorbic acid in the treated fruits.
swallowing [30]. Fig. 4F shows the changes in Chewiness value of strawberries during the storage. Control strawberries had a low Chewiness throughout the storage. A possible explanation for texture changed might be that emulsions coating reduced the respiration rate and this resulted in prevention of the cell rupture, water loss and starch to sugar conversion [29]. 3.8. Color assessment Color is one of the main desirable characteristic that might determine consumer acceptance of the product. The changes in the surface color of R-(+)-Limonene emulsions coated strawberries as a function of storage time is presented in Fig. 5. Lightness (L*) of vapor-treated strawberries (Fig. 5A) was significantly affected by storage time reflecting a sharply decrease in the value in the period ranging from 0 to 2 days and a consequent decrease in the final day (day 8). R-(+)Limonene emulsions coated strawberries were also significantly brighter than control samples which might be considered a positive outcome considering that decrease in brightness is usually due to the formation of dark tissues and brown spots, which might be related to inappropriate storage conditions as well as fungus infection [31]. Major changes in strawberry color may be seen by the assessment of
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3.7. Textural properties
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a*
Texture is related to the microstructure of foods such as cell wall, middle lamella and turgor pressure [27]. Fig. 4 shows the changes of the texture parameters including firmness, adhesiveness cohesiveness, springiness, gumminess and chewiness of fruits throughout the storage period. Firmness is associated with the cell wall strength of the tissue [28]. As expected, this parameter decreased in the fruits. However, the observed decrease was significantly higher in untreated fruits compared to R-(+)-Limonene emulsions treated samples. At the end of the storage, the highest firmness value observed in strawberries exposed to 0.15% R-(+)-Limonene emulsions with the value of 667 g. The term adhesiveness is generally described as combination of adhesive and cohesive force [29]. The results obtained from the adhesiveness are presented in Fig. 4B. Control strawberries had a high adhesion throughout the storage. Springiness is generally known as elastically capacity of food which goes back to undeformed structure after force is removed. For strawberries coated by 0.15% R-(+)-Limonene emulsions, springiness decreased slightly (from 0.632 to 0.583) whereas springiness values of control decreased sharply (from 0.632 to 0.527). Use of the term cohesiveness can be explained with difficulty degree in breaking down and strength of the internal bonds of food [21]. We observed from Fig. 4D that, cohesiveness values decreased during the storage for all groups. Higher concentration of R-(+)-Limonene emulsions could inhibit the decrease of cohesiveness. Gumminess is defined as the required force to breakdown food for swallowing. As shown in Fig. 4E, there is a clear trend of decreasing gumminess value throughout the storage. For strawberries coated by 0.15% R-(+)-Limonene emulsions, gumminess decreased slightly (from 432 to 294) whereas gumminess values of control decreased sharply (from 432 to 236). Chewiness refers to the required energy to masticate sample for
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Time (days) Fig. 5. Color parameters (L*, a*) of strawberries during 8 days of storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
P. Shao et al. / International Journal of Biological Macromolecules 111 (2018) 1273–1280
Fig. 6. Effect of different treatment on strawberry, Control: treated with UFP; A: treated with 0.05% emulsion; B: treated with 0.1% emulsion; C: treated with 0.15% emulsion.
a* values (Fig. 5B), a measure of redness. The a* values in strawberries generally decreases over time, losing the fruit their saturated red color. Explanations for this result may be due to anthocyanin degradation by the action of hydrolytic enzymes that, by breaking down the linkage of the glycosidic substituent in the moieties, lead to loss of color during postharvest storage [32]. All indicators in our manuscript reflect fruits quality in different aspects. A single indicator is difficult to explain the problem fully. Weight loss is mainly interested with moisture of fruits. Total soluble solids in fruits are proportional to their sugar content. The value of pH is related to the organic acids of fruit that influence sugar acid ratio. Total phenols, as secondary metabolites are mainly reflect metabolic situation. Ascorbic acid has the physiological function of preventing scurvy and is an important source of nutritional value of fruits. Textural properties and color directly affect the sensory quality of fruits. These indicators are subject to the effects of respiration and transpiration. (See Fig. 6.)
As shown in Fig. 7, R-(+)-Limonene emulsion stabilized by UFP is applied coating on the strawberry surface. Coating a protective thin film on the surface of fruits can separate it from the atmosphere, adverse factors of fruits quality (such as oxygen and microorganisms) can't directly contact with fruits. Without emulsion-coating, there is exchange of CO2 and O2 inside and outside freely through fruit surface. Meanwhile the moisture in the fruit volatile with cell breathing. But when R-(+)Limonene emulsion coating is on the surface of fruit, emulsion-coating can effectively stop fruit gas exchange inside and outside and make a low O2, high CO2 environment in strawberry. It also reduces the generation of endogenous ethylene, inhibit respiratory metabolism [33]. Despite essential oils have good antibacterial properties, they are generally volatile and susceptible to oxidized. With respect to these unexpected phenomena, R-(+)-Limonene emulsion can effectively reduce the volatilization rate and inhibit its oxidation process. Ulva fasciata polysaccharide has water retaining property, it can slow down water evaporates. The structure of the R-(+)-Limonene emulsion will be destroyed as time passed. Moisture in emulsion evaporated gradually, the essential oil is released and show its antibacterial properties. The cell membrane is a barrier to bacteria. If the cell membrane is damaged, the intracellular material will be released, then the growth, reproduction and physiological morphology of bacteria will be damage. Ethylene material in essential oils can make microbial cell lipid structure damage, thereby damaging the cell membrane structure of microorganisms, leading to the release of the cytoplasm, the final cell lysis. We believe it might be the above mechanism has affected a series of indicators we test in preserving quality of strawberry [34]. 4. Conclusions In this study, we investigated the effectiveness of R-(+)-Limonene emulsions at suppressing the growth of microorganisms via the diffusion of its volatile active components. The OEO was encapsulated in a UFP matrix by forming an emulsion. In addition, edible coatings with at least 0.15% w/w of R-(+)-Limonene improved the microbial stability of the strawberries, resulted effective in the decontamination of external pathogens such as Escherichia coli, Bacillus subtilis and Staphylococcus aureus and preserved strawberries outward appearance during the time. As a consequence, the incorporation of emulsions-based edible coatings containing R-(+)-Limonene onto strawberry extended the shelf life of this product. These results evidence the potential advantages of using R-(+)-Limonene as natural antimicrobial within edible coatings acting as preservatives and enhancing the safety, quality and nutritional properties of high perishable R-(+)-Limonene.
Emulsion droplets
coating O2 O2
microorganism H2 O H2 O CO2
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strawberry Fig. 7. Preservation mechanism and antibacterial of strawberry coating by R-(+)-Limonene emulsion stabilized by UFP.
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Acknowledgements This work was supported by National Natural Science Foundation of China (No. 31571833) and Zhejiang province key research and development program (No. 2018C02005, 2017C02G2010946). We confirm that there is no conflict of interest. We also thank Qianjiang special expert position for his supporting. References [1] F. Dong, X. Wang, Int. J. Biol. Macromol. 104 (2017) 821–826. [2] E. Velickova, E. Winkelhausen, S. Kuzmanova, V.D. Alves, M. Moldão-Martins, LWT Food Sci. Technol. 52 (2013) 80–92. [3] Z. Shen, D.P. Kamdem, Int. J. Biol. Macromol. 74 (2015) 289–296. [4] F. Giarratana, D. Muscolino, C. Beninati, G. Ziino, A. Giuffrida, A. Panebianco, Int. J. Food Microbiol. 237 (2016) 109–113. [5] A. Bevilacqua, M.R. Corbo, M. Sinigaglia, J. Food Prot. 73 (2010) 888–894. [6] P. Shao, Y. Zhu, M. Qin, Z. Fang, P. Sun, Carbohydr. Polym. 134 (2015) 566–572. [7] P. Shao, Q. Qiu, H. Chen, J. Zhu, P. Sun, Int. J. Biol. Macromol. (2017) 154–162. [8] P. Shao, H. Ma, Q. Qiu, W. Jing, Int. J. Biol. Macromol. 92 (2016) 926–934. [9] P. Shao, M. Qin, L. Han, P. Sun, Carbohydr. Polym. 113 (2014) 365–372. [10] P. Shao, H. Ma, J. Zhu, Q. Qiu, Food Hydrocoll. 69 (2017) 202–209. [11] M. Ravichandran, N.S. Hettiarachchy, V. Ganesh, S.C. Ricke, S. Singh, J. Food Saf. 31 (2011) 462–471. [12] V. Mikulcová, R. Bordes, V. Kašpárková, Food Hydrocoll. 61 (2016) 780–792. [13] M.S. Aday, C. Caner, F. Rahvalı, Postharvest Biol. Technol. 62 (2011) 179–187. [14] A. Mohammadi, S.M. Jafari, E. Assadpour, A. Faridi Esfanjani, Int. J. Biol. Macromol. 82 (2016) 816–822.
[15] N.B. Gol, P.R. Patel, T.V.R. Rao, Postharvest Biol. Technol. 85 (2013) 185–195. [16] S. Kartal, M.S. Aday, C. Caner, Postharvest Biol. Technol. 71 (2012) 32–40. [17] I. Savarimuthu, J. Manickkam, P. Seenivasan, BMC Complement. Altern. Med. 6 (2006) 39. [18] S. Frassinetti, L. Caltavuturo, M. Cini, C.M. Della Croce, B.E. Maserti, J. Essent. Oil Res. 23 (2011) 27–31. [19] G. Vardar-Ünlü, F. Candan, A. Sökmen, D. Daferera, M. Polissiou, M. Sökmen, E. Dönmez, B. Tepe, J. Agric. Food Chem. 51 (2003) 63–67. [20] H.-M P, A. E, V. VD, V. D, G. R, Food Chem. 110 (2008) 428–435. [21] L. Sánchez-González, C. Pastor, M. Vargas, A. Chiralt, C. González-Martínez, M. Cháfer, Postharvest Biol. Technol. 60 (2011) 57–63. [22] M. Martínezferrer, C. Harper, F. Pérezmuntoz, M. Chaparro, J. Food Sci. 67 (2010) 3365–3371. [23] R. Dhital, P. Joshi, N. Becerra-Mora, A. Umagiliyage, C. Tan, P. Kohli, R. Choudhary, LWT Food Sci. Technol. 80 (2017) 257–264. [24] L. Cisneros-Zevallos, J. Food Sci. 68 (2010) 1560–1565. [25] I. Gulcin, Arch. Toxicol. 86 (2012) 345–391. [26] H.A.S. Amal, M.M. El-Mogy, A. Anean, A.H.W.A. Beatrix, Mogy 33 (2010) 69. [27] I. Fraeye, G. Knockaert, S.V. Buggenhout, T. Duvetter, M. Hendrickx, A.V. Loey, Innovative Food Sci. Emerg. Technol. 11 (2010) 23–31. [28] P.M.A. Toivonen, D.A. Brummell, Postharvest Biol. Technol. 48 (2008) 1–14. [29] L. Sánchez-González, M. Vargas, C. González-Martínez, A. Chiralt, M. Cháfer, Food Eng. Rev. 3 (2011) 1–16. [30] M. Huang, J.F. Kennedy, B. Li, X. Xu, B.J. Xie, Carbohydr. Polym. 69 (2007) 411–418. [31] M. Lacroix, B. Ouattara, Food Res. Int. 33 (2000) 719–724. [32] I. Oey, M. Lille, A. van Loey, M. Hendrickx, Trends Food Sci. Technol. 19 (2008) 320–328. [33] Z. Deng, J. Jung, J. Simonsen, Y. Zhao, Food Chem. 232 (2017) 359–368. [34] S. Dehghani, S.V. Hosseini, J.M. Regenstein, Food Chem. 240 (2018) 505–513.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: https://www.journals.elsevier.com/ijbiomac
Antibacterial activities of R-(+)-Limonene emulsion stabilized by Ulva fasciata polysaccharide for fruit preservation Ping Shao a,⁎, Haoya Zhang a, Ben Niu a, Ligang Jiang b a b
Department of Food Science, Zhejiang University of Technology, Hangzhou 310014, PR China R &D Center, Proya Cosmetics Co. Ltd., Hangzhou 310012, PR China
a r t i c l e
i n f o
Article history: Received 3 December 2017 Received in revised form 11 January 2018 Accepted 17 January 2018 Available online xxxx Keywords: Antimicrobial activity Edible coatings Essential oil Fruit preservation
a b s t r a c t It was previously found that R-(+)-Limonene emulsion could be stabilized by Ulva fasciata polysaccharide. In the present study, emulsions of R-(+)-Limonene were developed for coating of strawberries to improve the shelf life and microbiological safety. Edible coatings with at least 0.15% w/w of R-(+)-Limonene improved the microbial stability of the strawberries, resulted effective in the decontamination of external pathogens such as Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Changes in weight loss, total soluble solid (TSS), pH, total phenolic, ascorbic acid, textural properties and surface color of strawberries during storage were evaluated. R-(+)-Limonene emulsions were effective for decreasing water loss of all the fruit samples tested. The decrease in TSS values was higher in the control strawberries. Initial decreases followed by increases of pH during storage of strawberries. The amount of total phenols detected in control fruit sharply increased during the first 6 days then remained steady, whereas the total soluble phenols content of treated strawberries gradually increased during storage. Strawberries coated with emulsion had higher L* and a* values as compared to control fruits. The R(+)-Limonene coatings have demonstrated the potential to inhibit foodborne pathogen contamination of strawberries, and prolong their shelf life. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Fruits have short shelf life due to high perishability, physiological disorders, water loss, and decay [1]. They usually have short ripening and senescent periods that make marketing a challenge. This makes the development of an optimal packaging solution to maintain the quality of these produce during storage of economic interest. Adverse activity of antimicrobial microorganisms is an important task of the fruit preservation. Emulsion-based edible coatings have been widely studied for preservation of fruits and vegetables. Their beneficial effects and easy handling made them broadly applicable to soft fruits such as berries. The main functional advantages attributed to use of edible coatings are slower respiration rate, extended storage periods, firmness retention and controlled microbial growth [2]. In this way, emulsionbased edible coating technology is an alternative method with which to extend the commercial shelf-life of coated products by modifying their internal atmosphere. To meet consumer demands, the application of essential oils as natural antimicrobial compounds in foods has attracted growing interest, in terms of food quality and safety, by substituting chemical preservatives for natural substances with similar properties [3]. R-(+)⁎ Corresponding author. E-mail address: [email protected] (P. Shao).
https://doi.org/10.1016/j.ijbiomac.2018.01.126 0141-8130/© 2018 Elsevier B.V. All rights reserved.
Limonene is the major aromatic compound in essential oils obtained from oranges, grapefruits, and lemons [4]. Moreover the R-(+)-Limonene possess antioxidant, antimicrobial, antifungal, nematocide, antiinflammatory, anti-carcinogenic properties [5]. The improvement of preservation techniques to reduce the growth and activity of spoilage microorganisms in foods is crucial to increase their shelf life and to reduce the losses due to spoilage. Essential oils can be encapsulated in emulsion systems stabilized by Ulva fasciata polysaccharides (UFP). In our previous study, we have successfully extracted Ulva fasciata polysaccharides from Ulva fasciata, which is an amphipathic anionic polysaccharide with the potential of food hydrocolloids and it is able to form gels in presence of borate and calcium ions. We also studied their rheological properties, characteristics, hydrodynamic behavior and dilute solution properties [6]. We have further studied the rheological properties of curcumin coated with UFP stabilized emulsions and the effect of metallic ions on the physicochemical stability of curcumin emulsions stabilized by Ulva fasciata polysaccharide [7]. Not only curcumin has been considered, we also studied R-(+)-Limonene. In our previous study, when R-(+)-Limonene be encapsulated in emulsion stabilized by UFP, it shows that when UFP concentration is 4%, emulsion have homogeneous particle size and the best stability which have high zeta potential (−52.6 mV) and low average droplet diameter (2.45 μm). Otherwise emulsion stabilized by UFP shows better stability
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than Arabic gum and gelatin [8]. The stability of emulsions has been researched deeply, but its application value is still unconsidered. The aim of the present study was to develop an edible coating by incorporating different concentrations of R-(+)-Limonene emulsion into UFP as a carrier matrix. We assessed the antimicrobial effectiveness of emulsions-based edible coatings containing R-(+)-Limonene against inoculated different bacteria, and their capability to improve the shelf life of a highly perishable strawberry. 2. Materials and methods 2.1. Materials Strawberries were purchased from a local wholesale company in Hangzhou, China, and selected for uniformity of size and ripeness, and fruit with apparent injuries were removed. Fruit were stored at 4 ± 1 °C prior to processing. The Ulva fasciata was collected on the coast of Nanji Archipelago (Zhejiang province, China). Soybean oil (60 wt%) linoleic acid, 30 wt% oleic acid, and specific gravity (0.197) was obtained from KLK Oleo, Ltd. (Malaysia). R-(+)-Limonene (95%) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Emulsions preparation Ulva fasciata polysaccharides were extracted using the method described in our previous work [9]. Composition analysis result shows that total content is 99.02%, composed of 42.59% sugars, 20.05% sulfate, and 36.38% uronic acid. The emulsion was prepared according to the method of Shao [10]. The R-(+)-Limonene was added dropwise to the soy oil at a rate of 10% to form the dispersed phase. 4%(w/w) of Ulva fasciata polysaccharide was dispersed in aqueous solution by using to an overhead mixer (IKA Labortechnik, IKA works, Malaysia) for 24 h at room temperature to form the continuous phase. Using a high-speed blender (Kinematica PT-MR 2100, Switzerland) to blending UFP solution and the R-(+)-Limonene dispersed phase in a ratio of 95:5 (v/v) and pre-homogenization at 26,000 rpm for 3 min. Put the mixtures pass through a high pressure homogenizer at 75 MPa (Microfluidics M-110L, USA). 2.3. Determination of MIC of emulsions Minimum Inhibitory Concentrations (MIC) of R-(+)-Limonene against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were determined by a broth microdilution method according to Clinical Laboratories Standards Institute (CLSI) guidelines (CLSI, 2012). Emulsions (20,000 μL/L) were dissolved in agar medium and serially diluted in a 96-well plate. Bacteria inoculum in 50 mL agar was added to the wells to obtain final concentrations of R-(+)-Limonene at 0, 19, 39, 78, 156, 312, 625, 1250, 2500 and 5000 μL/L. The final concentration of bacteria was 1 × 105 CFU/mL. Plates were incubated at 37 °C for 24 h and then measured. The MIC was determined as the lowest concentration of the emulsion that inhibited microbial growth with an absorbance value b0.05 at 595 nm [11]. 2.4. Inoculation of strawberry and application of R-(+)-Limonene emulsions Stock cultures of Staphylococcus aureus, Bacillus subtilis and Escherichia coli at 37 °C for 18 h, followed by washing in PBS for 2 times before inoculation. Strawberries were purchased from a local supermarket. Strawberries weighing 10.0 ± 1.0 g and with a diameter of 2.5 ± 0.1 cm were selected. The selected strawberries were stored at 4 °C and separated randomly into two groups for (1) microbial analysis
and (2) analysis of their physical properties. Stalks were removed from the strawberries before the experiments. One milliliter of individual bacterial suspension in PBS (105 CFU/mL) was coated to strawberry, followed by mixing and drying for 15 min in a Biosafety cabinet. The strawberry was then dipped in 200 mL emulsions with 0.05%, 0.1% v/v and 0.15% of R-(+)-Limonene emulsions for 1 min, dried for 30 min, and then stored in bags at 4 °C. UFP solution (4%, w/w) was used as control. Emulsion concentrations of 0.05% (500 μL/L), 0.1% (1000 μL/L) and 0.15% (1500 μL/L) were selected based on the MIC results [12]. 2.5. Microbial analysis Changes in the numbers of Escherichia coli, Bacillus subtilis and Staphylococcus aureus on the surfaces of strawberry treated with R-(+)-Limonene emulsions were determined during the storage period. On each day (0, 2, 4, 6, and 8 days), one stored strawberry was added to 225 mL of sterilized peptone water (1 g/L) in stomacher bag and homogenized for 2 min. Appropriate serial dilutions were done and then 1.0 mL of every samples were plated by standard microbiological pour plate technique. All the microbiological counts were carried out in triplicates. 2.6. Weight loss To determine weight loss, the same strawberries were weighed at the beginning of the experiment and thereafter each day during the storage period. Weight loss was expressed as the percentage loss of the initial total weight. Ten fruits in three repetitions were used to evaluate the weight loss of every group. 2.7. Total soluble solids (TSSs), and pH determinations To determine soluble solids content and pH values of samples, three replicates of strawberry juice was used. Juice was obtained by homogenizing nine fruits in a blender. Measurements were performed by a digital refractometer (Atago, Tokyo, Japan) and pH meter (Sartorius PP-50, Goettingen, Germany) [13]. 2.8. Total phenolic compound analysis Total phenolic compound of fruits extract were determined by the Folin–Ciocalteu micro-method [14]. Fruit samples of 0.5–1 g were extracted with 10 mL of 80% ethanol and the extract was centrifuged at 10,000 rpm for 20 min. The clear supernatant was collected and allowed to evaporate at room temperature, and the residue dissolved in a known volume of distilled water. 2.5 mL of Folin Ciocelteau's phenol reagent (Merck) (10-fold-diluted) was thoroughly agitated with 0.5 mL of the sample and 2 mL of 7.5% Na2CO3. The mixture was kept at room temperature for 30 min in dark. Then the absorbance was recorded at 760 nm using a UV–Vis spectrophotometer (Shimadzu, UV-1800). 2.9. Ascorbic acid analysis Ascorbic acid content was determined by the method of Gol [15]. The fruit sample (100 mg) was treated in 5% metaphosphoric-glacial acetic acid solution. The extract was centrifuged and a clear supernatant collected. Suitable aliquots of the supernatant were taken in a test tube containing known amounts of 5% metaphosphoric-glacial acetic acid solution, dinitrophenyl hydrazine (2%) and thiourea (10%) solutions. The test tubes were incubated for 3 h at 37 °C in a water bath, after which the reaction was terminated by adding 85% sulphuric acid. The optical density of the reaction mixture was measured against a blank at 540 nm. Levels of ascorbic acid were estimated from a standard curve prepared from pure ascorbic acid.
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2.10. Textural properties
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2.11. Color assessment
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Minolta CR-400 colorimeter (Minolta, Osaka, Japan) was used to express L* (Lightness) and a* (red–green) color parameters of strawberry. Color meter was calibrated using the standard white plate before measurements. Nine strawberries were analyzed and measurements were taken in three different points at the equatorial zone.
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Texture profile analysis (TPA) parameters (hardness, cohesiveness, adhesiveness, springiness, gumminess, resilience and chewiness) were determined on fresh strawberry using a Texture Analyzer (Stable Micro Systems, Surrey, England). Measurements were taken along horizontal axis of the nine fruits. A cylindrical plunger with a diameter of 10 mm (SMS-P/10 CYL Delrin) compressed the fruits with pretest speeds of 5 mm/s, test speed of 1 mm/s, post speed of 8 mm/s, penetration distance of 4 mm and trigger force of 1.0 N. Texture Exponent software was used to automatically calculate the TPA parameters of fruits on the basis of fore-deformation curves [16].
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Statistical analyses were performed using Origin (ver. 6.0, IBM software, Chicago, USA). One-way analysis of variance (ANOVA) was used to evaluate the significance of differences between sample groups at a level of P ≤ 0.05.
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3. Results and discussions 6
3.1. Minimum inhibitory and minimum bactericidal concentration 4
3.2. Microbial analysis The development of bacteria growth in the in vitro tests is shown in Fig. 1 for different concentrations of emulsion. The use of pure UFP had no effect on bacterial growth. Both concentrations (0.1% and 0.15%) of R-(+)-Limonene emulsions were able to inhibit Escherichia coli and Bacillus subtilis growth significantly (P b 0.05) compared to controls, though a stronger effect was demonstrated by the 0.15% emulsion. The 0.15% emulsion was able to reduce Staphylococcus aureus population. The 0.1% emulsion was able to reduce Escherichia coli population by 0.5 logs at 1 d and then the population increases with time after the
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The MIC of R-(+)-Limonene emulsions was 0.0625% (625 mL/L) for Bacillus subtilis and Escherichia coli and 0.125% (1250 mL/L) for Staphylococcus aureus. Plant essential oils have shown strong antimicrobial effect. However, their application in food protection is still limited partially due to their low solubility in water. Formulating oil emulsions using food-grade emulsifiers increases the solubility and surface area of essential oils, and consequently, enhances their antimicrobial activity. The chemical structure of limonene can carry out active electron transfer, which can interact with ion channel proteins on the surface of microorganisms to change cell membrane permeability and inhibit the growth of microorganisms. Generally, gram-negative bacteria are more resistant to the essential oil treatment than gram-positive bacteria due to the former one having a lipopolysaccharide (LPS) protection from hydrophobic compounds. But the results of MIC are not entirely consistent with this regular pattern. There are some other studies have found gram-positive bacteria less sensitive to essential oils than gram-negative strains [17,18]. This may be due to the differences in the sensitivity of different bacteria strains to essential oils. However, the simple relation involving cell structure and microbial sensitivity to essential oils is not yet well established, and possible antagonistic or synergistic effects among the various active constituents of the oils should be taken into consideration [19].
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Time (days) Fig. 1. Effect of emulsion on the growth and survival of (A) Escherichia coli, (B) Bacillus subtilis and (C) Staphylococcus aureus at 0 d, 2 d, 4 d, 6 d and 8 d after the treatment. Error bars represent standard error of the mean. Measured at 25 °C.
treatment. Increasing concentrations in essential oils caused enhanced inhibition of Escherichia coli by the 0.15% treatment at all testing points. Of the three bacteria, Staphylococcus aureus was the least sensitive to the emulsion treatment. The mechanism of antibacterial and antifungal properties of the essential oil components is connected with the ability of these hydrophobic substances to incorporate into the membrane. Since membranes are the first barrier surrounding the cell, the ability of the essential oil components to successfully incorporate into these structures
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All fruit showed a progressive loss of weight during storage (Fig. 2A). As known, strawberry fruits are susceptible for water loss and thereby shriveling and deterioration because of their thin skin structure [20]. Throughout the storage period, there were significant differences between the control and R-(+)-Limonene emulsions treatments. Weight loss is mainly interested with moisture and moisture evaporation from the skin of fruits. Therefore, their textural properties play role on weight loss. In this study, R-(+)-Limonene emulsions were effective for decreasing water loss of all the fruit samples tested. The increase in oil concentration appeared to affect the weight loss, which could be predominantly caused by moisture loss. The progress of weight loss also could be partly attributed to an increase in the berry's metabolic activity, associated with tissue senescence over long storage times, which is slowed down after coating application [21].
7.5 7.0
Total soluble solid (TSS) is an important parameter that affects fruit quality and consumer acceptability [13]. The results obtained from the TSS analysis are presented in Fig. 2B. TSS levels at the beginning of storage was 5.5% and increased gradually during storage in both emulsions coated and pure UFP uncoated strawberries, that could be partially explained by the conversion of starch to sugars but, since strawberries accumulate very little starch, it may be mainly because of an increase in anthocyanins which contribute to soluble solids. During 4 days of storage, control samples exhibited significantly higher TSS i.e. 7.9%
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Fig. 2. (A) Weight loss (%) and (B) Total soluble solid (%) and (C) pH of strawberries during 8 days of storage. Error bars represent standard error of the mean. Measured at 25 °C.
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and the magnitude of perturbations induced by these compounds at this level may determine the antimicrobial effect of the substance. Using R(+)-Limonene emulsions led to longer-lasting antimicrobial activity.
Fig. 3. (A) Total phenolic content (mg/100 g) and (B) Ascorbic acid (mg/100 g) of strawberries during 8 days of storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
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compared to all other coated samples. In control fruit the TSS content declined from 7.9 to 6.3% during 4 days of storage to 8 days, while the fruit treated by R-(+)-Limonene emulsions showed a gradual increase in their TSS content up to 8 days of storage and thereafter declined. These results are in accordance with the report of Velickova et al. [2] who showed a decrease in the TSS content in strawberries at the end of storage and attributed it to respiration. The decrease in TSS values was higher in the control strawberries. This observed diminishment can be explained by the hydrolysis of sucrose in order to maintain physiological activity and respiration. The effect of different concentrations of R-(+)-Limonene emulsions on pH of strawberries as compared to control samples is presented in Fig. 2C. The pH of strawberry fruit treated by 0.05% R-(+)-Limonene emulsions and UFP treated increased during the entire storage period. These results are in agreement with those of Gol who reported that a higher increase in pH was found in the control samples as compared to edible coated strawberries [15]. The value of pH is related to the
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fruit senescence, increased significantly during the storage due to utilization of organic acids during respiration [22]. The pH of strawberry fruit treated by 0.1% and 0.15% R-(+)-Limonene emulsions was found to have significantly higher pH than the control. The pH of strawberry fruit treated by 0.1% and 0.15% R-(+)-Limonene emulsions, decreased up to 4 days of storage but there is no significant increase on the 8th day. The obtained results are in agreement with similar research conducted by Dhital [23], who observed initial decreases followed by increases of pH during storage of strawberries. 3.5. Total phenolic compound analysis The variation in the total phenolic content as a function of R-(+)Limonene emulsions and storage time for strawberry fruit is presented in Fig. 3A. There was an increasing trend in the total phenolic content for both control and treated fruit up to 8 days of storage. Total phenols, the major antioxidants in plant and fruit tissues, are produced as secondary
Fig. 4. Texture parameters a) firmness, b) adhesiveness, c) springness, d) cohesiveness, e) gumminess, f) chewiness changes of strawberry during storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
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metabolites in response to abiotic and biotic stresses to protect cellular constituents [24]. The change in the concentrations of phenolic compounds in fruits during storage can be attributed to cell structure breakdown [25]. The amount of total phenols detected in control fruit sharply increased during the first 6 days (from 807 to 1552 mg GAE/100 g fresh weight.) then remained steady, whereas the total soluble phenols content of treated strawberries gradually increased during storage. The constant exposure of strawberries to antimicrobial R-(+)-Limonene, which can be considered a controlled stress, might have caused such enhancement at the end of storage time. This outcome could be associated with the different interactions between constituents of essential oils and the food matrix. 3.6. Ascorbic acid analysis Ascorbic acid maintains the physiological functions in the human body and is a measure of the freshness of fruits. The effect of R-(+)-Limonene emulsions on the ascorbic acid content of strawberries is shown in Fig. 3B. The ascorbic acid decreased during the storage period in all treated samples as well as control sample. Treated with R-(+)-Limonene emulsions significantly delayed the rate of ascorbic acid decrease. The initial ascorbic acid content of strawberry fruit was 67.26 mg 100 g−1. Treated with 0.1% and 0.15% R-(+)-Limonene emulsions showed the highest content of ascorbic acid i.e. 23.6 and 27.2 mg 100 g−1, respectively, at 8 days of storage, and was significantly superior to all other samples as well as the control. Ascorbic acid content of strawberries decreased during the storage period and the similar results have been reported in strawberries by Amal [26]. The emulsion evaporates to form a protective film on the surface of the fruit and could inhibit gas exchange between the tissues and the environment, which reduces the O2 concentration that oxidizes ascorbic acid; thus, ascorbic acid oxidation was effectively prevented, thereby keeping a higher amount of ascorbic acid in the treated fruits.
swallowing [30]. Fig. 4F shows the changes in Chewiness value of strawberries during the storage. Control strawberries had a low Chewiness throughout the storage. A possible explanation for texture changed might be that emulsions coating reduced the respiration rate and this resulted in prevention of the cell rupture, water loss and starch to sugar conversion [29]. 3.8. Color assessment Color is one of the main desirable characteristic that might determine consumer acceptance of the product. The changes in the surface color of R-(+)-Limonene emulsions coated strawberries as a function of storage time is presented in Fig. 5. Lightness (L*) of vapor-treated strawberries (Fig. 5A) was significantly affected by storage time reflecting a sharply decrease in the value in the period ranging from 0 to 2 days and a consequent decrease in the final day (day 8). R-(+)Limonene emulsions coated strawberries were also significantly brighter than control samples which might be considered a positive outcome considering that decrease in brightness is usually due to the formation of dark tissues and brown spots, which might be related to inappropriate storage conditions as well as fungus infection [31]. Major changes in strawberry color may be seen by the assessment of
46 44 42 40 38 36 34
3.7. Textural properties
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a*
Texture is related to the microstructure of foods such as cell wall, middle lamella and turgor pressure [27]. Fig. 4 shows the changes of the texture parameters including firmness, adhesiveness cohesiveness, springiness, gumminess and chewiness of fruits throughout the storage period. Firmness is associated with the cell wall strength of the tissue [28]. As expected, this parameter decreased in the fruits. However, the observed decrease was significantly higher in untreated fruits compared to R-(+)-Limonene emulsions treated samples. At the end of the storage, the highest firmness value observed in strawberries exposed to 0.15% R-(+)-Limonene emulsions with the value of 667 g. The term adhesiveness is generally described as combination of adhesive and cohesive force [29]. The results obtained from the adhesiveness are presented in Fig. 4B. Control strawberries had a high adhesion throughout the storage. Springiness is generally known as elastically capacity of food which goes back to undeformed structure after force is removed. For strawberries coated by 0.15% R-(+)-Limonene emulsions, springiness decreased slightly (from 0.632 to 0.583) whereas springiness values of control decreased sharply (from 0.632 to 0.527). Use of the term cohesiveness can be explained with difficulty degree in breaking down and strength of the internal bonds of food [21]. We observed from Fig. 4D that, cohesiveness values decreased during the storage for all groups. Higher concentration of R-(+)-Limonene emulsions could inhibit the decrease of cohesiveness. Gumminess is defined as the required force to breakdown food for swallowing. As shown in Fig. 4E, there is a clear trend of decreasing gumminess value throughout the storage. For strawberries coated by 0.15% R-(+)-Limonene emulsions, gumminess decreased slightly (from 432 to 294) whereas gumminess values of control decreased sharply (from 432 to 236). Chewiness refers to the required energy to masticate sample for
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Time (days) Fig. 5. Color parameters (L*, a*) of strawberries during 8 days of storage. Vertical bars denote standard deviation of three replicates. Measured at 25 °C.
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Fig. 6. Effect of different treatment on strawberry, Control: treated with UFP; A: treated with 0.05% emulsion; B: treated with 0.1% emulsion; C: treated with 0.15% emulsion.
a* values (Fig. 5B), a measure of redness. The a* values in strawberries generally decreases over time, losing the fruit their saturated red color. Explanations for this result may be due to anthocyanin degradation by the action of hydrolytic enzymes that, by breaking down the linkage of the glycosidic substituent in the moieties, lead to loss of color during postharvest storage [32]. All indicators in our manuscript reflect fruits quality in different aspects. A single indicator is difficult to explain the problem fully. Weight loss is mainly interested with moisture of fruits. Total soluble solids in fruits are proportional to their sugar content. The value of pH is related to the organic acids of fruit that influence sugar acid ratio. Total phenols, as secondary metabolites are mainly reflect metabolic situation. Ascorbic acid has the physiological function of preventing scurvy and is an important source of nutritional value of fruits. Textural properties and color directly affect the sensory quality of fruits. These indicators are subject to the effects of respiration and transpiration. (See Fig. 6.)
As shown in Fig. 7, R-(+)-Limonene emulsion stabilized by UFP is applied coating on the strawberry surface. Coating a protective thin film on the surface of fruits can separate it from the atmosphere, adverse factors of fruits quality (such as oxygen and microorganisms) can't directly contact with fruits. Without emulsion-coating, there is exchange of CO2 and O2 inside and outside freely through fruit surface. Meanwhile the moisture in the fruit volatile with cell breathing. But when R-(+)Limonene emulsion coating is on the surface of fruit, emulsion-coating can effectively stop fruit gas exchange inside and outside and make a low O2, high CO2 environment in strawberry. It also reduces the generation of endogenous ethylene, inhibit respiratory metabolism [33]. Despite essential oils have good antibacterial properties, they are generally volatile and susceptible to oxidized. With respect to these unexpected phenomena, R-(+)-Limonene emulsion can effectively reduce the volatilization rate and inhibit its oxidation process. Ulva fasciata polysaccharide has water retaining property, it can slow down water evaporates. The structure of the R-(+)-Limonene emulsion will be destroyed as time passed. Moisture in emulsion evaporated gradually, the essential oil is released and show its antibacterial properties. The cell membrane is a barrier to bacteria. If the cell membrane is damaged, the intracellular material will be released, then the growth, reproduction and physiological morphology of bacteria will be damage. Ethylene material in essential oils can make microbial cell lipid structure damage, thereby damaging the cell membrane structure of microorganisms, leading to the release of the cytoplasm, the final cell lysis. We believe it might be the above mechanism has affected a series of indicators we test in preserving quality of strawberry [34]. 4. Conclusions In this study, we investigated the effectiveness of R-(+)-Limonene emulsions at suppressing the growth of microorganisms via the diffusion of its volatile active components. The OEO was encapsulated in a UFP matrix by forming an emulsion. In addition, edible coatings with at least 0.15% w/w of R-(+)-Limonene improved the microbial stability of the strawberries, resulted effective in the decontamination of external pathogens such as Escherichia coli, Bacillus subtilis and Staphylococcus aureus and preserved strawberries outward appearance during the time. As a consequence, the incorporation of emulsions-based edible coatings containing R-(+)-Limonene onto strawberry extended the shelf life of this product. These results evidence the potential advantages of using R-(+)-Limonene as natural antimicrobial within edible coatings acting as preservatives and enhancing the safety, quality and nutritional properties of high perishable R-(+)-Limonene.
Emulsion droplets
coating O2 O2
microorganism H2 O H2 O CO2
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Emulsion coating
strawberry Fig. 7. Preservation mechanism and antibacterial of strawberry coating by R-(+)-Limonene emulsion stabilized by UFP.
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Acknowledgements This work was supported by National Natural Science Foundation of China (No. 31571833) and Zhejiang province key research and development program (No. 2018C02005, 2017C02G2010946). We confirm that there is no conflict of interest. We also thank Qianjiang special expert position for his supporting. References [1] F. Dong, X. Wang, Int. J. Biol. Macromol. 104 (2017) 821–826. [2] E. Velickova, E. Winkelhausen, S. Kuzmanova, V.D. Alves, M. Moldão-Martins, LWT Food Sci. Technol. 52 (2013) 80–92. [3] Z. Shen, D.P. Kamdem, Int. J. Biol. Macromol. 74 (2015) 289–296. [4] F. Giarratana, D. Muscolino, C. Beninati, G. Ziino, A. Giuffrida, A. Panebianco, Int. J. Food Microbiol. 237 (2016) 109–113. [5] A. Bevilacqua, M.R. Corbo, M. Sinigaglia, J. Food Prot. 73 (2010) 888–894. [6] P. Shao, Y. Zhu, M. Qin, Z. Fang, P. Sun, Carbohydr. Polym. 134 (2015) 566–572. [7] P. Shao, Q. Qiu, H. Chen, J. Zhu, P. Sun, Int. J. Biol. Macromol. (2017) 154–162. [8] P. Shao, H. Ma, Q. Qiu, W. Jing, Int. J. Biol. Macromol. 92 (2016) 926–934. [9] P. Shao, M. Qin, L. Han, P. Sun, Carbohydr. Polym. 113 (2014) 365–372. [10] P. Shao, H. Ma, J. Zhu, Q. Qiu, Food Hydrocoll. 69 (2017) 202–209. [11] M. Ravichandran, N.S. Hettiarachchy, V. Ganesh, S.C. Ricke, S. Singh, J. Food Saf. 31 (2011) 462–471. [12] V. Mikulcová, R. Bordes, V. Kašpárková, Food Hydrocoll. 61 (2016) 780–792. [13] M.S. Aday, C. Caner, F. Rahvalı, Postharvest Biol. Technol. 62 (2011) 179–187. [14] A. Mohammadi, S.M. Jafari, E. Assadpour, A. Faridi Esfanjani, Int. J. Biol. Macromol. 82 (2016) 816–822.
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