Next Article in Journal
Brewing Efficacy of Non-Conventional Saccharomyces Non-cerevisiae Yeasts
Next Article in Special Issue
Fruit Juices: Technology, Chemistry, and Nutrition 2.0
Previous Article in Journal
The Effect of Carbonation Level on the Acceptability and Purchase Intent of Muscadine and Fruit Wines
Previous Article in Special Issue
Short Wave Ultraviolet Light (UV-C) Effectiveness in the Inactivation of Bacterial Spores Inoculated in Turbid Suspensions and in Cloudy Apple Juice
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Beverages
Volume 7
Issue 3
10.3390/beverages7030067
beverages-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Antimicrobial Activity of Nanoencapsulated Essential Oils of Tasmannia lanceolata, Backhousia citriodora and Syzygium anisatum against Weak-Acid Resistant Zygosaccharomyces bailii in Clear Apple Juice

by
Fahad Alderees
1,
Saleha Akter
1,
Ram Mereddy
2 and
Yasmina Sultanbawa
1,*
1
ARC Industrial Transformation Training Centre for Uniquely Australian Foods, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Long Pocket Campus, Brisbane, QLD 4068, Australia
2
Department of Agriculture and Fisheries, Queensland Government, Health and Food Sciences Precinct, Coopers Plains QLD, Brisbane, QLD 4108, Australia
*
Author to whom correspondence should be addressed.
Beverages 2021, 7(3), 67; https://doi.org/10.3390/beverages7030067
Submission received: 23 July 2021 / Revised: 10 September 2021 / Accepted: 14 September 2021 / Published: 17 September 2021
(This article belongs to the Special Issue Fruit Juices: Technology, Chemistry, and Nutrition 2.0)
Browse Figures
Versions Notes

Abstract

:
The anti-yeast activity of oil-in-water encapsulated nanoemulsion containing individual or a combination of the three essential oils of Tasmanian pepper leaf (Tasmannia lanceolata), lemon myrtle (Backhousia citriodora), and anise myrtle (Syzygium anisatum) against weak-acid resistant Zygosaccharomyces bailii in clear apple juice was investigated. The effectiveness of the shelf-life extension of Z. bailii-spiked (1 × 103 CFU/mL) clear apple juice was evaluated and compared between natural (essential oils) and synthetic (sodium benzoate) antimicrobial agents. Essential oils showed an immediate reduction in the Z. bailii cell population at day-0 and exerted a fungicidal activity at day-4 of storage, with no further noticeable growth at the end of the experiment (day-28). At lower concentrations, Tasmanian pepper leaf oil of 0.0025% had >6 log CFU/mL at day-12 of storage. For lemon myrtle essential oils, the yeast population reached >6 log CFU/mL at day-24 and day-20 for concentrations of 0.02% and 0.01%, respectively. The fungicidal activity of Tasmanian pepper leaf oil reduced from 0.005% to 0.0025% v/v when mixed at a ratio of 1:1 with anise myrtle oil. The results of the present study suggest that these three native Australian herbs have the potential to be used in the beverage industry by controlling Zygosaccharomyces bailii in clear apple juice products.

Graphical Abstract

1. Introduction

Controlling the growth of food spoilage causing microorganisms is mainly achieved by the use of synthetic preservatives; however, they have been linked to negative health effects such as carcinogenicity, acute toxicity, teratogenicity, attention deficit hyperactivity disorder, asthma, and allergy, and to environmental pollution due to their slow degradation [1,2,3,4,5]. Even though synthetic preservatives are consumed regularly on a daily basis as effective food preservatives, their usage is limited by the U.S. Food and Drug Administration (FDA) due to safety issues [6]. The use of natural antimicrobial agents in food preservation is becoming an area of interest among researchers and food industries due to the great awareness and concern of consumers regarding what they perceive as harmful synthetic antimicrobial and preservative agents in food [7,8,9,10,11,12]. In addition, the emergence of weak-acid resistant microorganisms that are capable of growing in and spoiling beverage products even at the maximum permitted levels of synthetic preservatives; this turned the focus toward investigating new potential natural alternatives to synthetic preservatives to extend the shelf-life of beverage products [4,13,14,15,16]. The growth of the majority of spoilage, causing microorganisms in fruit juice products, is well controlled through the use of synthetic preservatives (such as potassium sorbate and sodium benzoate) and through lowering the juice pH to 2 to 4 (by the addition of acidulants) [15,17,18,19]. A low pH of <4.5 prevents the spore germination and growth of the great majority of bacteria; however, there are some reported yeasts belonging to the Zygosaccharomyces genus capable of growing in a low pH and resisting the action of synthetic preservatives [16,20,21,22]. Z. bailii is a food and beverage spoilage yeast and is described as being notorious for its extreme resistance to weak-acid preservatives [22,23,24]. This yeast can grow above the legally permitted levels of preservatives, thus becoming a dangerous yeast in the food and beverage industries [22,23,24]. Sodium benzoate is a monocarboxylate weak-acid preservative (E211) commonly used in a low-pH (<4.5) beverage system; however, it has been found to react with ascorbic acid in drinks producing a carcinogen benzene, which occurs during storage due to the decarboxylation of benzoate [19,22,25]. The inhibition action of sodium benzoate against the growth of microorganisms is active only in a low-pH environment. Sodium benzoate is soluble in water and converted into benzoic acid once dissolved in water, which then becomes undissociated to its protonated acid form (–COOH) at a low pH; however, once it has entered into the microbial cells, reaching the neutral pH of the cellular cytosol (intracellular fluid), it then dissociates, producing an anion (–COO−) and releasing a proton (H+) inside the cell [26]. The accumulation of anions (–COO−) inside a cell creates a turgor pressure, which causes oxidation stress by affecting the free radical production inside the cell through the mitochondrial respiratory chain, while the accumulation of protons (H+) acidifies the cytosol, which delays or prevents essential metabolic functions of the cell [27,28,29]. Z. bailii cells are able to resist the action of sodium benzoate by pumping out anions (–COO−) and protons (H+), preventing their accumulation inside the cells. Z. bailii cells consume energy (ATP) to pump out anions (–COO−) through membrane protein Pdr12 and to pump out protons (H+) via membrane protein Pma1. Non-resistant yeasts lack the membrane protein Pdr12 and fail to eliminate or prevent the accumulation of anions (–COO−) within cells, which eventually lead to their death. Z. bailli yeasts are considered to be preservative-resistant and ethanol-tolerant (spoiling of alcoholic beverages) species [17,21,30,31,32]. They are also considered to be a fermentative spoilage organism of fruits, fruit juices, and syrup concentrates (osmotolerant) [17,21,30,31,32], and they are known for spoiling acidic food sauces (acid-tolerant) and carbonated soft drinks [17,21,30,31,32]. Z. bailli cells have been reported to resist the action of benzoic acid at 1000 ppm [33,34]. With the current status of limiting the use of synthetic preservatives in food products, it has become a challenge for the beverage industry to increase the dosage of weak-acid preservatives to control the growth of resistant yeasts. This provides an opportunity for plant essential oils to be used as a natural source of antimicrobial agents in beverage products. Currently, plant essential oils are gaining much attention for their use as an alternative solution to control the growth of weak-acid resistant yeast and, at the same time, satisfying modern consumers by the application of natural antimicrobial agents in food products instead of synthetic preservatives [35,36,37]. The hydrophobicity of essential oils is their major limitation, which lowers their antimicrobial action and prevents their application into liquid-food or beverage products [38]. To overcome this hurdle, nanoemulsion technology could be utilized to reduce the size of essential oil particles to nanoscale and, with the aid of surfactant, to allow essential oils to be effectively dissolved in liquid while time maintaining their antimicrobial activity in liquid.
Tasmanian pepper (Tasmannia lanceolata) leaf, lemon myrtle (Backhousia citriodora), and anise myrtle (Syzygium anisatum) are three native Australian herbs reported to have antibacterial and antifungal activities [39,40,41,42,43,44]. Tasmanian pepper leaf belongs to the family Winteraceae, found in forested regions in Tasmania, Victoria, and New South Wales in Australia and it is characterized by a high content of sesquiterpene and monoterpene essential oils [45,46,47,48]. The essential oil of Tasmanian pepper leaf contains polygodial as its major bioactive compound responsible for its reported antimicrobial and antifungal activities [39,47,49,50]. Lemon myrtle is a member of the Myrtaceae family; its essential oil contains citral (82–91%) as its major bioactive component possessing strong antimicrobial activity [51,52,53]. Anise myrtle belongs to the Myrtaceae family, where its essential oil is dominated by either anethole (94.97%) or methyl chavicol (77.54%) depending on the chemotype of anise myrtle trees [43,54,55]. The essential oil of Tasmanian pepper leaf and lemon myrtle has shown in vitro antimicrobial activity against many yeasts, including the most weak-acid resistant Z. bailii. Therefore, the inhibition action of encapsulated nanoemulsion of Tasmanian pepper leaf, lemon myrtle, and anise myrtle (individually or in synergy) were evaluated in an in vitro setting using a commercialized clear apple juice system free of chemical preservatives and microbial contamination. The clear apple juice was challenged with Z. bailii cells during the experiment and the inhibition action of oil-in-water encapsulated nanoemulsions and sodium benzoate at low, medium, and high concentrations were evaluated during 28 days of storage at 25 °C.

2. Materials and Methods

2.1. Oils, Reagents, and Apple Juice

Pure 100% essential oils of lemon myrtle and anise myrtle are supplied from Australian Rainforest Products Pty Ltd. (New South Wales, Australia), while the essential oil of Tasmanian pepper leaf was supplied by Essential oils of Tasmania Pty Ltd. (Devonport, TAS, Australia). The essential oils were kept at their original bottles protected from light exposure and stored at 4 °C until further use. Sunflower oil (100% pure, Crisco, Sydney, Australia) was purchased from a retail supermarket and stored at room temperature. Non-ionic surfactant Tween 80 was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Double distilled water purified in a Milli-Q system (Millipore Co., Bedford, MA, USA) was used in all experiments. Pasteurized clear apple juice (Golden circle, Coles, Melbourne, Australia) with no added preservatives, no added sugar, and a long shelf-life was used in the study.

2.2. Culture Preparation and Inoculation

The antimicrobial activity of oil-in-water nanoemulsion containing essential oils of Tasmanian pepper leaf, lemon myrtle, and anise myrtle was evaluated against a weak-acid resistant yeast, Zygosaccharomyces bailii (ATCC 38923). The Z. bailii culture was grown in tryptone soya yeast extract broth for 48 h at 25 °C prior to the day of experiment. The yeast culture suspension was measured at optical density (OD540) = 0.5 McFarland to produce approximately 2.5 × 106 CFU/mL, where 420 µL of this suspension was added per 1050 mL of apple juice to provide a final concentration of 1 × 103 CFU/mL.

2.3. Addition of Essential Oil Nanoemulsion and Sodium Benzoate in Apple Juice

Apple juice treatment was divided into the following: Negative control (without yeast or treatment, sterility test); positive control (spiked with yeast and without treatment); sodium benzoate treatment at 500, 750, and 1000 ppm; Tasmanian pepper leaf oil nanoemulsion at 0.00125%, 0.0025%, and 0.005%; lemon myrtle oil nanoemulsion at 0.01%, 0.02%, and 0.04%. To evaluate the synergistic effects between oils, apple juice was also treated with a combination of Tasmanian pepper leaf oil and lemon myrtle oil (1:1) nanoemulsion at 0.01% and 0.02% and a combination of Tasmanian pepper leaf oil and anise myrtle oil (1:1) nanoemulsion at 0.00125%, 0.0025%, and 0.005%. After the application of a treatment, the apple juice samples were transferred into 50 mL sterile bottles and incubated at 25 °C. Microbial growth was monitored on the day of the experiment (day-0) and every fourth day afterward, lasting for 28 days, where two bottles were removed from each apple juice treatment on the measurement day and duplicate measurements were taken for each bottle using yeast mold agar (YMA) consisting of 3 g/L of yeast extract (Oxoid, LP0021), 3 g/L of malt extract (Oxoid, LP0039), 5 g/L of peptone (Oxoid, LP0037), 10 g/L of dextrose (Oxoid, LP0071), and 20 g/L of agar (Oxoid, LP0011). The pH of YMA was adjusted to 6.2 ± 0.2 using hydrochloric acid (1 M). Enumeration of inoculated Z. bailii growth in the apple juice samples was determined after 48 h of incubation at 25 °C.

2.4. Statistical Analysis

The experiment was repeated twice (n = 2), and measurement performed in duplicate (n = 2). Cell counts were converted into logarithm values to calculate significant differences between the treatments over time. Yeast cell counts between treatments were considered significant at the 95% confidence limit (p ≤ 0.05). Statistical analysis of the results was performed using two-way ANOVAs, followed by Tukey’s multiple comparison post-hoc tests, with significant differences observed at p ≤ 0.05 using GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA, USA), and figures were generated in Microsoft Excel (Office 2016).

3. Results and Discussion

Effect of Sodium Benzoate and Essential Oils on Zygosaccharomyces bailii Cell Counts in Apple Juice during Storage

The clear apple juice samples from the negative-control group confirmed that the juices were free of microbes by showing no microbial growth initially (day-0). The apple juice had a pH average of 3.37. The treatment effect of nanoemulsion essential oils, either as a single oil or a combination of two oils, sodium benzoate on Z. bailii cell growth during storage (28 days) at 25 °C is graphically presented in Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5.
The positive-control group of clear apple juice samples spiked with yeast but without treatment had 3.2 log CFU/mL initially at day-0, but experienced a significant (p < 0.05) yeast growth at day-4 of 6.8 log CFU/mL. There were no significant differences within the results of the sodium benzoate treatment groups and the positive-control group. After the addition of yeast cells at day-0, microbial counts from sodium benzoate treatment groups of 500, 750, and 1000 ppm were observed to be 3.2, 3.1, and 3.2 log CFU/mL, respectively, which is similar to the microbial counts of the positive-control group (Figure 1).
Sodium benzoate did not show a fungicidal effect against Z. bailii inocula, but instead exerted a fungistatic effect. However, for treatment of Tasmanian pepper leaf oil at 0.005%, lemon myrtle oil at 0.04%, a combination of Tasmanian pepper leaf oil and lemon myrtle oil (1:1) at 0.02%, and a combination of Tasmanian pepper leaf oil and anise myrtle oil (1:1) at 0.005% caused a significant (p < 0.05) reduction in microbial counts at day-0 to 2.1 log CFU/mL, 2.9 log CFU/mL, 2.1 log CFU/mL, and 2.2 log CFU/mL respectively. Unlike sodium benzoate, essential oils, either alone or in synergy, caused an immediate inhibition of yeast cells at day-0. In addition, the essential oil nanoemulsion concentrations mentioned above not only caused an immediate reduction in yeast cell counts at day-0, but also caused a total elimination (inactivation) of yeast cells at day-4 and continued to show no signs of growth until the last day of the experiment (day-28). These concentrations exhibited biocidal activity against weak-acid resistant Z. bailii, which reduced yeast cell counts from 3.1 log CFU/mL to 0 log CFU/mL (in comparison to the positive-control group) by day-4 of storage at 25 °C. In our experiment, the time between apple juice inoculation with Z. bailii cells and the addition of nanoemulsion essential oils was determined to be around 30 min. During this period of exposure (30 min), there was a noticeable decrease in yeast cells ranging from 0.2 log CFU/mL to 1.1 log CFU/mL from the treatments of Tasmanian pepper leaf oil at 0.005%, lemon myrtle oil at 0.04%, a combination of Tasmanian pepper leaf oil and lemon myrtle oil (1:1) at 0.02%, and a combination of Tasmanian pepper leaf oil and anise myrtle oil (1:1) at 0.005%. Other reports found a complete inactivation of yeast cells within 24 h of exposure to essential oils. For instance, Souza et al. [56] reported on the anti-yeast property of Origanum vulgare essential oil and found it to cause a prominent biocidal effect against Candida albicans and C. krusei cells at 24 h of exposure, as well as a biostatic effect against C. tropicalis at 12 h of exposure. Furthermore, Loeffler, Beiser, Suriyarak, Gibis, and Weiss [13] evaluated oil-in-water emulsions containing a combination of essential oil components, cinnamaldehyde, perillaldehyde, and citral against Z. bailii in diluted 2% apple juice and found these compounds to have biocidal activity, causing an inactivation of Z. bailii cells in apple juice within 24 h of storage at 25 °C. In addition, Friedman et al. [57] evaluated the antibacterial activity of 17 plant essential oils and nine essential oil compounds in a clear apple juice system (pH = 3.7) and found that citral, geraniol, eugenol, carvacrol, oregano oil, cinnamon leaf oil, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil (in the range of 0.018–0.093%) caused inhibition against 50% of the tested Escherichia coli inocula within 1 h of exposure. In addition, the essential oils Melissa, oregano, lemon, lemongrass, and cinnamon leaf and the essential oil compounds citral, terpeineol, geraniol, carvacrol, and linalool (in the range of 0.004–0.011%) caused inhibition against 50% of the tested Salmonella enterica inocula within 1 h. Moreover, Friedman, Henika, Levin, and Mandrell [57] observed that the antimicrobial activity of essential oils could be categorized as slow- or fast-acting, where a biocidal effect could occur within 1 h of treatment. The major bioactive compounds found in Tasmanian pepper leaf oil, polygodial, lemon myrtle oil, and citral were reported to possess biocidal activity not only toward yeasts, but also against bacteria [49,50,58,59,60,61,62,63,64,65,66,67,68]. Citral was among the essential oil components that Souza, Stamford, Lima, and Trajano [56] and Friedman, Henika, Levin, and Mandrell [57] reported to be effective against Z. bailii, Z. bisporus, C. albicans, E. coli, and S. enterica in beverage model systems. Lemon myrtle essential oil has been reported to contain approximately 82–91% citral as its main bioactive compound, responsible for its strong antimicrobial activity [51,52,53]. In a previous study, the yeast cells of Saccharomyces cerevisiae exposed to lemon myrtle extract for a period of 30 min showed morphological changes, where the cells became swollen with a round-shaped structure; however, the cells showed membrane rupture when treated for 1 h. In addition, in our previous study [69], we found that the essential oils of lemon myrtle and Tasmanian pepper leaf have a fungicidal effect through targeting yeast cell membranes, causing membrane lysis, membrane pore formation, and cell content leakage. This could explain the phenomenon where the Z. bailii cell count was significantly lower than the positive-control group at day-0 shortly after being exposed (30 min) to a nanoemulsion of Tasmanian pepper leaf and lemon myrtle essential oils.
Among all of the tested concentrations (0.00125%, 0.0025%, and 0.005%) of Tasmanian pepper leaf nanoemulsion (Figure 2), only the 0.005% concentration was able to reduce the yeast growth to 0 log CFU/mL (cell inactivation) at day-4, which is a 7-log reduction in comparison to the positive-control group, and no further yeast growth was observed at any day throughout the storage period of 28 days. Tasmanian pepper leaf nanoemulsion oil of 0.005% is considered the minimum fungicidal concentration (MFC) needed to kill all inocula yeast cells in clear apple juice. When the concentration of Tasmanian pepper leaf nanoemulsion oil lowered to 0.0025%, it did not kill the yeast cells, but instead delayed their growth and reduced their population significantly to 3.3 log CFU/mL at (<0.05) day-4 (3.7 reduction compared to the positive-control group), to 4 log CFU/mL at day-8, and to 6 log CFU/mL at day-12 of storage. A further decrease in the Tasmanian pepper leaf nanoemulsion oil concentration to 0.00125% reduced the yeast population to 5 log CFU/mL at day-4 of storage and to 7 log CFU/mL at day-8.
The effect of different concentrations of lemon myrtle nanoemulsion essential oil (0.01%, 0.02%, and 0.04%) on the growth rate of Z. bailii cells during storage is presented in Figure 3. The MFC of lemon myrtle nanoemulsion essential oil against Z. bailii in clear apple juice was 0.04%, which resulted in complete inactivation, where the yeast population reached 0 log CFU/mL at day-4 of storage. They did not show any further growth afterward. As the concentration of lemon myrtle nanoemulsion essential oil decreased, its antimicrobial activity decreased as well. When its concentration lowered to 0.02%, the yeast population reached 1.8 log CFU/mL at day-4 of storage, which was a significant (<0.05) decrease in yeast growth by 5.2 log CFU/mL compared to the positive-control group; however, the yeast growth reached 3.1 log CFU/mL, 4.4 log CFU/mL, and 5.5 log CFU/mL at day-8, day-12, and day-16 of storage. Moreover, the yeast growth from lemon myrtle nanoemulsion essential oil of 0.02% was significantly (<0.05) lower than that of the positive-control group from day-4 to day-28. Lemon myrtle nanoemulsion essential oil of 0.01% had a similar growth pattern of 0.02%, but was higher in yeast growth between log CFU/mL of 0.29, 1.17, and 0.72 at day-4, -8, and -12, respectively. Still, lemon myrtle nanoemulsion essential oil of 0.01% was able to maintain a yeast population below 5 log CFU/mL until day-8 of storage and was significantly (<0.05) lower than that of the positive-group at day-4, -8, -12, and -16 of storage.
In the case of anise myrtle nanoemulsion essential oil, it did not show antimicrobial activity against Z. bailii when tested up to a 2% concentration (p = 0.9391). Anise myrtle has been found to consist of two chemotypes that vary in essential oil compositions [43,55]. The first chemotype contains E-anethole (94.97%), methyl chavicol (4.43%), α-pinene (0.09%), 1, 8-Cineole (0.02%), and α-farnesene (0.07%), while the other chemotype consists of methyl chavicol (77.54%), E-anethol (19.95%), 1,8-Cineole (0.80%), α-pinene (0.40%), and α-farnesene (0.11%) [55]. Mainly, both chemotypes of anise myrtle contain the same components but at different amounts. The differences in the amount of E-anethol (C10H12O) and its isomer methyl chavicol, known as estragole, between the two chemotypes could influence the antimicrobial property of anise myrtle essential oil [70]. For instant, Wilkinson and Cavanagh [71] evaluated the antimicrobial activity of two chemotypes of anise myrtle essential oil, where the first sample exhibited no activity against E. coli and C. albicans, while the second sample showed inhibition against E. coli, S. aureus, and C. albicans. In addition, a study conducted by Hood et al. [72] reported antimicrobial activity of anise myrtle essential oil against E. coli and S. aureus; however, another report by Nirmal et al. [73] found no activity against E. coli and S. aureus. In addition, anise myrtle essential oil did not work in synergy with lemon myrtle essential oil, but it enhanced the antimicrobial activity of Tasmanian pepper leaf essential oil. Thus, anise myrtle was included in this experiment to evaluate if it enhances the antimicrobial activity of Tasmanian pepper leaf in clear apple juice during the storage period. The growth rate pattern of Z. bailii cells when treated with anise myrtle and Tasmanian pepper leaf oils (1:1) at 0.00125%, 0.0025%, and 0.005% is shown in Figure 4. Anise myrtle and Tasmanian pepper leaf of 0.005% was the MFC that inactivated the Z. bailii cells at day-4 of storage. Since the two oils were mixed, this means that half of the concentration of Tasmanian pepper leaf oil of 0.0025 in the mixture was able to inactivate Z. bailii cells at day-4 of storage. The anise myrtle oil worked in synergy by reducing the MFC of Tasmanian pepper leaf oil from 0.005% (when tested alone) to 0.0025%. When the concentration of anise myrtle and Tasmanian pepper leaf nanoemulsion oils (1:1) lowered to 0.0025%, it significantly (<0.05) reduced the yeast population to 1.64 log CFU/mL at day-4 and 5.21 log CFU/mL at day-8 of storage compared to the positive-control group. Anise myrtle oil enhanced the Tasmanian pepper leaf oil inhibition action and significantly (<0.05) reduced the yeast growth compared to Tasmanian pepper oil when tested alone at 0.00125% at day-4 by 3.55 log CFU/mL and day-8 by 2.0 log CFU/mL. Anise myrtle and Tasmanian pepper leaf oils at a concentration of 0.00125% was not enough to cause a major reduction is yeast growth, except at day-4, where the yeast population reached 5.97 log CFU/mL, which is significantly (<0.05) lower than the positive-control group.
There was no synergistic effect found between lemon myrtle and Tasmanian pepper leaf essential oils. They performed better when tested as individual oils over their mixture in reducing the growth of Z. bailii cells during storage. The combination of lemon myrtle and Tasmanian pepper leaf nanoemulsion essential oils at 0.02% was the MFC that caused cell inactivation at day-4 of storage, and no further yeast growth was observed at any time until the end of the storage period (Figure 5). However, their combination at 0.01% did significantly (<0.05) lower yeast growth compared to the positive-control group at day-4, -8, and -12 to log CFU/mL of 2.3, 4.6, and 5.6, respectively.

4. Conclusions

The action of natural antimicrobial agents, essential oils, is dose- and time-dependent, which showed biocidal activity at fungicidal concentrations against the weak-acid resistant Z. bailii in a clear apple juice. A reduction in Z. bailii cells was observed to be immediate (at day-0) after a short period (30 min) of exposure to essential oils at biocidal concentrations. In addition, essential oils exerted biostatic activity instead of biocidal activity when added to clear apple juice at lower concentrations, which caused a delay in the lag phase in the growth of Z. bailii cells. Sodium benzoate did not exhibit biocidal activity, but only showed biostatic activity during storage. The results of this study clearly showed that essential oils of lemon myrtle and Tasmanian pepper leaf as individual oils and the combination of anise myrtle and Tasmanian pepper leaf were effective in controlling the growth of Z. bailii in clear apple juice within the studied storage period (28 days) and temperature (25 °C). These results indicate the great potential of native Australian herbs in controlling one of the most resistant yeasts to the action of weak-acid preservatives in clear apple juice.

Author Contributions

Conceptualization, R.M. and Y.S.; methodology, F.A.; software, F.A.; validation, S.A., R.M. and Y.S.; formal analysis, F.A.; investigation, F.A.; resources, Y.S.; data curation, F.A. and S.A.; writing—original draft preparation, F.A.; writing—review and editing, S.A., R.M. and Y.S.; visualization, F.A.; supervision, R.M. and Y.S.; project administration, Y.S.; funding acquisition, Y.S. All authors read and agreed to the published version of the manuscript.

Funding

This research was supported by the Public Authority for Applied Education and Training, Kuwait, and the Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Australia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data presented in this study is available in this article.

Acknowledgments

The authors thank Andrew Cusack and Margaret Currie for their expert technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Faleiro, M. The mode of antibacterial action of essential oils. In Science against Microbial Pathogens: Communicating Current Research and Technological Advances; Formatex Research Center: Norristown, PA, USA, 2011; Volume 2, pp. 1143–1156. [Google Scholar]
  2. McCann, D.; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.; Grimshaw, K.; Kitchin, E.; Lok, K.; Porteous, L.; Prince, E. Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: A randomised, double-blinded, placebo-controlled trial. Lancet 2007, 370, 1560–1567. [Google Scholar] [CrossRef]
  3. Gortzi, O.; Lalas, S.; Chinou, I.; Tsaknis, J. Reevaluation of antimicrobial and antioxidant activity of thymus spp. Extracts before and after encapsulation in liposomes. J. Food Prot. 2006, 69, 2998–3005. [Google Scholar] [CrossRef] [PubMed]
  4. Burt, S. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol. 2004, 94, 223–253. [Google Scholar] [CrossRef] [PubMed]
  5. Piper, P. Potential safety issues surrounding the use of benzoate preservatives. Beverages 2018, 4, 33. [Google Scholar] [CrossRef] [Green Version]
  6. Dehghan, P.; Mohammadi, A.; Mohammadzadeh-Aghdash, H.; Ezzati Nazhad Dolatabadi, J. Pharmacokinetic and toxicological aspects of potassium sorbate food additive and its constituents. Trends Food Sci. Technol. 2018, 80, 123–130. [Google Scholar] [CrossRef]
  7. Calo, J.R.; Crandall, P.G.; O’Bryan, C.A.; Ricke, S.C. Essential oils as antimicrobials in food systems—A review. Food Control 2015, 54, 111–119. [Google Scholar] [CrossRef]
  8. Holley, R.A.; Patel, D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol. 2005, 22, 273–292. [Google Scholar] [CrossRef]
  9. Prakash, B.; Kujur, A.; Yadav, A.; Kumar, A.; Singh, P.P.; Dubey, N.K. Nanoencapsulation: An efficient technology to boost the antimicrobial potential of plant essential oils in food system. Food Control 2018, 89, 1–11. [Google Scholar] [CrossRef]
  10. Siddique, S.; Perveen, Z.; Nawaz, S.; Shahzad, K.; Ali, Z. Chemical composition and antimicrobial activities of essential oils of six species from family Myrtaceae. J. Essent. Oil Bear. Plants 2015, 18, 950–956. [Google Scholar] [CrossRef]
  11. Nabet, N.; Boudries, H.; Loupassaki, S.; Souagui, S.; Madani, K.; Carbonell-Barrachina, Á.A. Chemical composition, antimicrobial and antioxidant activities of Thymus fontanesii Boiss. et Reut. and Origanum glandulosum Desf. essential oils. Int. Food Res. J. 2017, 24, 2518–2525. [Google Scholar]
  12. Almadiy, A.A.; Nenaah, G.E.; Al Assiuty, B.A.; Moussa, E.A.; Mira, N.M. Chemical composition and antibacterial activity of essential oils and major fractions of four Achillea species and their nanoemulsions against foodborne bacteria. LWT—Food Sci. Technol. 2016, 69, 529–537. [Google Scholar] [CrossRef]
  13. Loeffler, M.; Beiser, S.; Suriyarak, S.; Gibis, M.; Weiss, J. Antimicrobial efficacy of emulsified essential oil components against weak acid–adapted spoilage yeasts in clear and cloudy apple juice. J. Food Prot. 2014, 77, 1325–1335. [Google Scholar] [CrossRef] [PubMed]
  14. Tyagi, A.K.; Gottardi, D.; Malik, A.; Guerzoni, M.E. Chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil. LWT—Food Sci. Technol. 2014, 57, 731–737. [Google Scholar] [CrossRef]
  15. Azeredo, D.R.P.; Alvarenga, V.; Sant’Ana, A.S.; Sabaa Srur, A.U.O. An overview of microorganisms and factors contributing for the microbial stability of carbonated soft drinks. Food Res. Int. 2016, 82, 136–144. [Google Scholar] [CrossRef]
  16. Rawat, S. Food spoilage: Microorganisms and their prevention. Asian J. Plant Sci. Res. 2015, 5, 47–56. [Google Scholar]
  17. Fleet, G.H. Yeast spoilage of foods and beverages. In The Yeasts, 5th ed.; Boekhout, C.P.K.W.F., Ed.; Elsevier: London, UK, 2011; pp. 53–63. [Google Scholar]
  18. Roberts, T.; Cordier, J.-L.; Gram, L.; Tompkin, R.; Pitt, J.; Gorris, L.; Swanson, K. Soft drinks, fruit juices, concentrates, and fruit preserves. In Micro-Organisms in Foods 6; Springer: Boston, MA, USA, 2005; pp. 544–573. [Google Scholar]
  19. Piper, J.D.; Piper, P.W. Benzoate and sorbate salts: A systematic review of the potential hazards of these invaluable preservatives and the expanding spectrum of clinical uses for sodium benzoate. Compr. Rev. Food Sci. Food Saf. 2017, 16, 868–880. [Google Scholar] [CrossRef] [Green Version]
  20. Pitt, J.I.; Hocking, A.D. The ecology of fungal food spoilage. In Fungi and Food Spoilage; Springer: Boston, MA, USA, 2009; pp. 3–9. [Google Scholar]
  21. Karaman, K.; Sagdic, O.; Yilmaz, M.T. Multiple response surface optimization for effects of processing parameters on physicochemical and bioactive properties of apple juice inoculated with Zygosaccharomyces rouxii and Zygosaccharomyces bailii. LWT—Food Sci. Technol. 2016, 69, 258–272. [Google Scholar] [CrossRef]
  22. Piper, P.W. Resistance of yeasts to weak organic acid food preservatives. In Advances in Applied Microbiology; Laskin, A.I., Sariaslani, S., Gadd, G.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2011; Volume 77, pp. 97–113. [Google Scholar]
  23. Stratford, M.; Steels, H.; Nebe-von-Caron, G.; Novodvorska, M.; Hayer, K.; Archer, D.B. Extreme resistance to weak-acid preservatives in the spoilage yeast Zygosaccharomyces bailii. Int. J. Food Microbiol. 2013, 166, 126–134. [Google Scholar] [CrossRef] [Green Version]
  24. Stratford, M.; James, S.A. Non-alcoholic beverages and yeasts. In Yeasts in Food: Beneficial and Detrimental Aspects; Boekhout, T., Robert, V., Eds.; Woodhead Publishing: Sawston, UK, 2003; pp. 309–345. [Google Scholar]
  25. Dos Santos, S.; Paula, V.; Medeiros Salgado, A.; Guedes Torres, A.; Signori Pereira, K. Benzene as a chemical hazard in processed foods. Int. J. Food Sci. 2015, 2015, 545640. [Google Scholar] [CrossRef]
  26. Mollapour, M.; Shepherd, A.; Piper, P.W. Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives. Yeast 2008, 25, 169. [Google Scholar] [CrossRef]
  27. Piper, P.W. Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives. Free Radic. Biol. Med. 1999, 27, 1219–1227. [Google Scholar] [CrossRef]
  28. Ullah, A.; Orij, R.; Brul, S.; Smits, G.J. Quantitative analysis of the modes of growth inhibition by weak organic acids in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 2012, 78, 8377–8387. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Piper, P.; Mahe, Y.; Thompson, S.; Pandjaitan, R.; Holyoak, C.; Egner, R.; Mühlbauer, M.; Coote, P.; Kuchler, K. The Pdr12 abc transporter is required for the development of weak organic acid resistance in yeast. EMBO J. 1998, 17, 4257–4265. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Zuehlke, J.; Petrova, B.; Edwards, C. Advances in the control of wine spoilage by Ygosaccharomyces and Dekkera/Brettanomyces. Annu. Rev. Food Sci. Technol. 2013, 4, 57–78. [Google Scholar] [CrossRef]
  31. Kalathenos, P.; Sutherland, J.; Roberts, T. Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine. J. Appl. Bacteriol. 1995, 78, 245–250. [Google Scholar] [CrossRef]
  32. Tapia de Daza, M.; Argaiz, A.; López-Malo, A.; Díaz, R. Microbial stability assessment in high and intermediate moisture foods: Special emphasis on fruit products. In Food Preservation By Moisture Control–Fundamental and Applications; Barbosa-Canovas, G.V., Welti-Chanes, J., Eds.; Techtonic: Lanchester, PA, USA, 1995; pp. 575–601. [Google Scholar]
  33. López-Malo, A.; Palou, E. Modeling the growth/no-growth interface of Zygosaccharomyces bailii in mango puree. J. Food Sci. 2000, 65, 516–520. [Google Scholar] [CrossRef]
  34. Stratford, M.; Capell, C. Soft drinks: Microbiology. In Encyclopedia of Food Sciences and Nutrition, 2nd ed.; Caballero, B., Finglas, P., Toldra, F., Eds.; Academic Press: Oxford, UK, 2003; pp. 5358–5366. [Google Scholar]
  35. Reyes-Jurado, F.; Franco-Vega, A.; Ramírez-Corona, N.; Palou, E.; López-Malo, A. Essential oils: Antimicrobial activities, extraction methods, and their modeling. Food Eng. Rev. 2015, 7, 275–297. [Google Scholar] [CrossRef]
  36. Pandey, A.K.; Kumar, P.; Singh, P.; Tripathi, N.N.; Bajpai, V.K. Essential oils: Sources of antimicrobials and food preservatives. Front. Microbiol. 2017, 7, 2161. [Google Scholar] [CrossRef] [Green Version]
  37. Leyva Salas, M.; Mounier, J.; Valence, F.; Coton, M.; Thierry, A.; Coton, E. Antifungal microbial agents for food biopreservation—A review. Microorganisms 2017, 5, 37. [Google Scholar] [CrossRef] [Green Version]
  38. Donsì, F.; Annunziata, M.; Vincensi, M.; Ferrari, G. Design of nanoemulsion-based delivery systems of natural antimicrobials: Effect of the emulsifier. J. Biotechnol. 2012, 159, 342–350. [Google Scholar] [CrossRef]
  39. Clarke, M. Australian Native Food Industry Stocktake; RIRDC Publication No. 12/066; Union Offset Printing: Canberra, Australia, 2012. [Google Scholar]
  40. Scheman, A.; Scheman, N.; Rakowski, E.-M. European directive fragrances in natural products. Dermatitis 2014, 25, 51–55. [Google Scholar] [CrossRef]
  41. Jones, G.L. Asia and Australia–essentially a well oiled connection. In Aromatic Plants from Asia, Their Chemistry and Application in Food and Therapy; Jirovetz, L., Dung, N.X., Varshney, V.K., Eds.; Har Krishnan Bhalla and Sons: Uttarakhand, India, 2007; p. 247. [Google Scholar]
  42. Ress, N.; Hailey, J.; Maronpot, R.; Bucher, J.; Travlos, G.; Haseman, J.; Orzech, D.; Johnson, J.; Hejtmancik, M. Toxicology and carcinogenesis studies of microencapsulated citral in rats and mice. Toxicol. Sci. 2003, 71, 198–206. [Google Scholar] [CrossRef] [Green Version]
  43. Blewitt, M.; Southwell, I.A. Backhousia anisata Vickery, an alternative source of (e)-anethole. J. Essent. Oil Res. 2000, 12, 445–454. [Google Scholar] [CrossRef]
  44. Sultanbawa, Y. Tasmanian pepper leaf (Tasmannia lanceolata) oils. In Essential Oils in Food Preservation, Flavor and Safety; Preedy, V.R., Ed.; Elsevier: Amsterdam, The Netherlands, 2016; pp. 819–823. [Google Scholar]
  45. Smyth, H.E.; Sanderson, J.E.; Sultanbawa, Y. Lexicon for the sensory description of Australian native plant foods and ingredients. J. Sens. Stud. 2012, 27, 471–481. [Google Scholar] [CrossRef]
  46. Netzel, M.; Netzel, G.; Tian, Q.; Schwartz, S.; Konczak, I. Sources of antioxidant activity in Australian native fruits. Identification and quantification of anthocyanins. J. Agric. Food Chem. 2006, 54, 9820–9826. [Google Scholar] [CrossRef]
  47. Pengelly, A. Indigenous and naturalised herbs: Tasmannia lanceolata: Mountain pepper. Aust. J. Med Herbal. 2002, 14, 71–74. [Google Scholar]
  48. Menary, R.C.; Dragar, V.A.; Thomas, S.; Read, C.D. Mountain Pepper Extract, Tasmannia Lanceolata: Quality Stabilisation and Registration: A Report for the Rural Industries Research and Development Corporation; RIRDC: Barton, Australia, 2003. [Google Scholar]
  49. Fujita, K.-I.; Kubo, I. Naturally occurring antifungal agents against Zygosaccharomyces bailii and their synergism. J. Agric. Food Chem. 2005, 53, 5187–5191. [Google Scholar] [CrossRef]
  50. Kubo, I.; Fujita, K.i.; Lee, S.H.; Ha, T.J. Antibacterial activity of polygodial. Phytother. Res. 2005, 19, 1013–1017. [Google Scholar] [CrossRef]
  51. Forbes-Smith, M.; Paton, J. Innovative products from Australian native foods. In Rural Industries Research and Development Corporation; RIRDC: Barton, Australia, 2002; pp. 1–78. [Google Scholar]
  52. Pengelly, A. Antimicrobial activity of lemon myrtle and tea tree oils. Aust. J. Med. Herbal. 2003, 15, 9. [Google Scholar]
  53. Southwell, I.A.; Russell, M.; Smith, R.L.; Archer, D.W. Backhousia citriodora f. Muell. (myrtaceae), a superior source of citral. J. Essent. Oil Res. 2000, 12, 735–741. [Google Scholar] [CrossRef]
  54. Southwell, I.; Russell, M.; Smith, R. Chemical composition of some novel aromatic oils from the Australian flora. In Proceedings of the International Conference on Medicinal and Aromatic Plants (Part II), Budabest, Hungary, 8–11 July 2001; pp. 79–89. [Google Scholar]
  55. Brophy, J.J.; Boland, D.J. The leaf essential oil of two chemotypes of Backhousia anisata Vickery. Flavour Fragr. J. 1991, 6, 187–188. [Google Scholar] [CrossRef]
  56. Souza, E.L.; Stamford, T.L.M.; Lima, E.O.; Trajano, V.N. Effectiveness of Origanum vulgare l. Essential oil to inhibit the growth of food spoiling yeasts. Food Control 2007, 18, 409–413. [Google Scholar] [CrossRef]
  57. Friedman, M.; Henika, P.R.; Levin, C.E.; Mandrell, R.E. Antibacterial activities of plant essential oils and their components against Escherichia coli o157: H7 and salmonella enterica in apple juice. J. Agric. Food Chem. 2004, 52, 6042–6048. [Google Scholar]
  58. McCallion, R.F.; Cole, A.; Walker, J.; Blunt, J.; Munro, M. Antibiotic substances from New Zealand plants. Planta Med. 1982, 44, 134–138. [Google Scholar] [CrossRef]
  59. Himejima, M.; Kubo, I. Fungicidal activity of polygodial in combination with anethole and indole against candida albicans. J. Agric. Food Chem. 1993, 41, 1776–1779. [Google Scholar] [CrossRef]
  60. Fujita, K.-I.; Kubo, I. Multifunctional action of antifungal polygodial against Saccharomyces cerevisiae: Involvement of pyrrole formation on cell surface in antifungal action. Bioorganic Med. Chem. 2005, 13, 6742–6747. [Google Scholar] [CrossRef] [PubMed]
  61. Kubo, I.; Taniguchi, M. Polygodial, an antifungal potentiator. J. Nat. Prod. 1988, 51, 22–29. [Google Scholar] [CrossRef]
  62. Lim, T.K. Tasmannia lanceolata. In Edible Medicinal and Non-Medicinal Plants: Volume 6, Fruits; Springer: Dordrecht, The Netherlands, 2013; pp. 493–499. [Google Scholar]
  63. Yano, Y.; Taniguchi, M.; Tanaka, T.; Oi, S.; Kubo, I. Protective effects of Ca2+ on cell membrane damage by polygodial in Saccharomyces cerevisiae. Agric. Biol. Chem. 1991, 55, 603–604. [Google Scholar] [CrossRef]
  64. Kubo, I.; Fujita, K.i.; Lee, S.H. Antifungal mechanism of polygodial. J. Agric. Food Chem. 2001, 49, 1607–1611. [Google Scholar] [CrossRef] [PubMed]
  65. Machida, K.; Tanaka, T.; Taniguchi, M. Depletion of glutathione as a cause of the promotive effects of polygodial, a sesquiterpene on the production of reactive oxygen species in Saccharomyces cerevisiae. J. Biosci. Bioeng. 1999, 88, 526–530. [Google Scholar] [CrossRef]
  66. Kubo, I.; Fujita, K.i. Naturally occurring anti-salmonella agents. J. Agric. Food Chem. 2001, 49, 5750–5754. [Google Scholar] [CrossRef]
  67. Marques, A.M.; Lima, C.H.; Alviano, D.S.; Esteves, R.L.; Kaplan, M.A.C. Traditional use, chemical composition and antimicrobial activity of Pectis brevipedunculata essential oil: A correlated lemongrass species in brazil. Emir. J. Food Agric. 2013, 25, 798–808. [Google Scholar] [CrossRef] [Green Version]
  68. Aoudou, Y.; Léopold, T.N.; Michel, J.D.P.; Franccedil, E.; Moses, M.C. Antifungal properties of essential oils and some constituents to reduce foodborne pathogen. J. Yeast Fungal Res. 2010, 1, 001–008. [Google Scholar]
  69. Alderees, F.; Mereddy, R.; Webber, D.; Nirmal, N.; Sultanbawa, Y. Mechanism of action against food spoilage yeasts and bioactivity of Tasmannia lanceolata, Backhousia citriodora and Syzygium anisatum plant solvent extracts. Foods 2018, 7, 179. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Moghaddam, M.; Alymanesh, M.R.; Mehdizadeh, L.; Mirzaei, H.; Ghasemi Pirbalouti, A. Chemical composition and antibacterial activity of essential oil of Ocimum ciliatum, as a new source of methyl chavicol, against ten phytopathogens. Ind. Crop. Prod. 2014, 59, 144–148. [Google Scholar] [CrossRef]
  71. Wilkinson, J.M.; Cavanagh, H. Antibacterial activity of essential oils from Australian native plants. Phytother. Res. 2005, 19, 643–646. [Google Scholar] [CrossRef] [PubMed]
  72. Hood, J.R.; Wilkinson, J.M.; Cavanagh, H.M. Evaluation of common antibacterial screening methods utilized in essential oil research. J. Essent. Oil Res. 2003, 15, 428–433. [Google Scholar] [CrossRef]
  73. Nirmal, N.P.; Mereddy, R.; Li, L.; Sultanbawa, Y. Formulation, characterization and antibacterial activity of lemon myrtle and anise myrtle essential oil in water nanoemulsion. Food Chem. 2018, 254, 1–7. [Google Scholar] [CrossRef] [Green Version]
Beverages 07 00067 g001 550
Figure 1. Effect of sodium benzoate (SB) on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Figure 1. Effect of sodium benzoate (SB) on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Beverages 07 00067 g001
Beverages 07 00067 g002 550
Figure 2. Effect of Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Figure 2. Effect of Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Beverages 07 00067 g002
Beverages 07 00067 g003 550
Figure 3. Effect of lemon myrtle (LM) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Figure 3. Effect of lemon myrtle (LM) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Beverages 07 00067 g003
Beverages 07 00067 g004 550
Figure 4. Effect of combining anise myrtle (AM) with Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Figure 4. Effect of combining anise myrtle (AM) with Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Beverages 07 00067 g004
Beverages 07 00067 g005 550
Figure 5. Effect of combining lemon myrtle (LM) with Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Figure 5. Effect of combining lemon myrtle (LM) with Tasmanian pepper leaf (TPL) nanoemulsion essential oil on the growth rate of Zygosaccharomyces bailii in clear apple juice during 28 days of storage at 25 °C. Means with different letters on the same day are significantly different at p < 0.05.
Beverages 07 00067 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Alderees, F.; Akter, S.; Mereddy, R.; Sultanbawa, Y. Antimicrobial Activity of Nanoencapsulated Essential Oils of Tasmannia lanceolata, Backhousia citriodora and Syzygium anisatum against Weak-Acid Resistant Zygosaccharomyces bailii in Clear Apple Juice. Beverages 2021, 7, 67. https://doi.org/10.3390/beverages7030067

AMA Style

Alderees F, Akter S, Mereddy R, Sultanbawa Y. Antimicrobial Activity of Nanoencapsulated Essential Oils of Tasmannia lanceolata, Backhousia citriodora and Syzygium anisatum against Weak-Acid Resistant Zygosaccharomyces bailii in Clear Apple Juice. Beverages. 2021; 7(3):67. https://doi.org/10.3390/beverages7030067

Chicago/Turabian Style

Alderees, Fahad, Saleha Akter, Ram Mereddy, and Yasmina Sultanbawa. 2021. "Antimicrobial Activity of Nanoencapsulated Essential Oils of Tasmannia lanceolata, Backhousia citriodora and Syzygium anisatum against Weak-Acid Resistant Zygosaccharomyces bailii in Clear Apple Juice" Beverages 7, no. 3: 67. https://doi.org/10.3390/beverages7030067

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop