Essential Oils Antagonism against Three Hygiene Significant Yeasts and Juice Spoilage by Saccharomyces Cerevisiae

Antifungal antagonism of different fourteen plant essential oils was examined as natural agents against economic and hygienic effective three yeasts; the preservative efficacy of most potent anti-yeast essential oil in food sanitary was also tested. Study involved oils antifungal bioactivity screening against Saccharomyces cerevisiae, Candida albicans, and Candida utilis. Study also included selection and in vitro extraction of most bioactive oil, and evaluation of its antifungal minimal inhibitory concentrations (MICs). Control of juice spoilage by Saccharomyces cerevisiae under the effect of in vitro oil extract different concentrations was also screened. Among the tested essential oils, apricot seed oil was the most bioactive anti-yeast agent. Two MIC values of apricot oil in vitro extract, 12.5μgml and 25μgml were recorded. In juice samples, oil extract bioactivity increased gradually up to concentration 100μgml. Highest oil preservative ability was observed at oil concentration of and above 125μgml. Higher oil concentrations needed for juice preservation were found more than in vitro assay to give the same effect. Applying of apricot oil and some other plant essential oils could be used as an environmental safety mode in osmophilic food preservation and in Candidate diseases biocontrol. DOI : 10.14302/issn. 2576-9383.jhhr-17-1768 Corresponding author: Sahar Yassin Ibrahim, Botany Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, EgyptTel.: 00201006556388, E-mail: sahar_moussa@women.asu.edu.eg


Introduction :
Antimicrobial activities of plant extracted essential oils (EOs) and their components play a detectable role in environment hygiene as alternative natural substances.EOs or aromatic plant essences are volatile and fragrant substances with oily consistency typically produced by plants.EOs have been long recognized for their antimicrobial, antiviral, insecticidal, antiparasitic, antidermatophytic, cytotoxic activities, antioxidant properties and as flavoring agents in foods [1][2][3][4].They are widely used in medicine and in food industry as natural antimicrobial agents to control foodborne bacteria and other pathogenic microorganisms [5 -9].In food industry, food preservation with natural antimicrobial products is being popularly used due to increase of consumer perception and concern regarding synthetic chemical additives [10].This supports applying of EOs as natural agents in food preservation.
Apricot seed EO (Prunus armeniaca) was rarely investigated as antimicrobial agent [8,9].It showed a strong antimicrobial bioactivity and was recommended to be used as a natural food preservative and as an environmental safety mode of diseases control.
Yeasts are significant microorganisms that have positive and negative effects within food industry and microbiology.Some osmophilic yeasts are potent food spoilage agents responsible for large economic losses of some food products according to their ability to survive under special environmental conditions of low pH and water activity, and in the presence of some common chemical preservatives [11][12][13].Moreover, foodassociated yeasts could be an underestimated source of infections and public health risks [14].As a result, yeasts have been considered as an important and attractive research area.Saccharomyces cerevisiae is one of the most remarkable yeasts in food industries; as a starter for several common food products; as a spoilage causative microorganism of osmophilic foods [15][16][17].
Candida is a genus of yeasts which is the most not unusual purpose of fungal infections global.About 20 out of over 150 known species of Candida they have been found to cause problems for the health of humans.During the last three decades the number of fungal infections caused by Candida species has increased dramatically [18].Candidiasis is a mycosis caused by different Candida species, which can promote superficial and systemic opportunist diseases around the world [19].Candida utilis is anamorphic form of Pichia jadinii, known for its industrial applications and rarely associated with disease [18].
The increasing clinical importance of mycoses in veterinary medicine in addition to the emergence of more severe presentations prompts to the development of new diagnostic procedures and treatments [19&20].
Most antifungals currently available for the treatment of different clinical forms of this disease have limitations that hinder their use, which makes the search for safe, efficient antimycotic products or molecules necessary [19].Essential oils have long been utilized to help eradicate Candida from the body; moreover, many researches indicated that EOs have potent activity against Candida species [19 &21-25].
Current study was designed to assess the fungicidal antagonism of 14 plants commercial EOs against three economic and hygienic important yeast strains.Likewise, to select and test the most significant fungicidal oil in controlling juice spoilage caused by S. cerevisiae.

Study Design
Fourteen EOs were screened for their bioactivity against three yeast strains.Most significant bioactive EO was selected and re-extracted in vitro; then studied for its minimal inhibitory concentration (MIC) value against three yeasts and for its effectiveness in shelf-life extending of osmophilic fresh juice.Sugar cane juice was examined as a suspected suitable target environment for S. cerevisiae growth.

Anti-Yeast Bioactivity Screening
Bioactivity of EOs was screened using agar-well diffusion test (ADT), growth inhibition zone evaluation diameters, YEMEA and Glucose peptone broth (GPB) (diffco) [26][27][28].For each target yeast strain, precultured inoculum on GPB of 24 h age at 35 o C incubation temperature was prepared and adjusted to final density of 10 4 CFU/ml.A set of sterile Petri dishes was prepared, each containing 25 ml sterile YEMEA seeded with 100 μL target yeast inoculum.A well of 1.0 cm diameter/Petri dish was aseptically made centrally in the solidified seeded YEMEA and subsequently 1.0 ml of EO under investigation was added in each well.Negative and positive controls were prepared, 1.0 ml of sterile saline solution (0.85% NaCl) instead of oil for negative control and 1.0 ml of standard anti-yeast nystatin reference (10 mg/ml sterile water) for positive one.The plates were kept at 4 o C for 2-3 h to allow oil agar diffusion then incubated for 24 h at 35 o C. Anti-yeast oil bioactivity was evaluated by measuring diameters of confirmed inhibition zones (included well diameter) in mm.

In Vitro Extraction of Prunus Armeniaca Seed Oil
Apricot seed oil was extracted by hydrodistillation and preserved in a sealed vial at 4 o C prior to further studies [8][9].

Evaluation of Minimal Inhibitory Concentration
MIC evaluation of in vitro extracted apricot seed oil against each of the three yeast targets was assayed as follow: extracted oil stock solution was prepared; 1.0 gm extracted apricot seed oil was dissolved in 10 ml 5% dichloromethane [8].Oil stock solution was added to sterile YEMEB to get final concentrations (100, 75, 50, 25 and 12.5μgml -1 ).Agar-well diffusion test was applied as proceeded before using YEMEA medium seeded with one yeast strain; 1.0 ml of the tested concentration of

Osmophilic Juice Preservative Efficacy
In vitro extracted Pru.armeniaca seed oil was tested for its preservative efficiency against S. cerevisiae spoilage of osmophilic juice.Sugar cane fresh juice, Saccharum spp., was the target osmophilic juice.Juice sample was collected, examined biologically according to the Laboratory Methods in Food Microbiology [29]

Results :
Anti-Yeast Bioactivity Screening The representative three yeasts showed variable responses to the 14 tested oils as mentioned in Table 1.
The widest yeast inhibition spectrum (23-25 mm) was confirmed with Pru.armeniaca EO, followed by M. piperita, S. aromaticum, O. basilicum, O. vulgare and O. europaea in ascending order.On contrast, Pru.amygdoles exhibited the lowest antifungal activity with 11-12 mm inhibition zones against three tested yeasts.

Evaluation of Minimal Inhibitory Concentration
On comparing zone diameters evaluation data in the dose response, a negative correlation between yeast growth and oil concentration value of Pru.armeniaca EO was observed; where the growth of the three yeasts were decreased with increasing oil concentration (Fig. 1).As a result of achieved data, 12.5μgml -1 was recorded as the Pru.armeniaca oil MIC against C. utilis (Fig. 1).While 25μgml -1 was confirmed as the Pru.armeniaca MIC against both S. cerevisiae and C. albicans.

Osmophilic Juice Preservative Efficacy
Control of S. cerevisiae juice spoilage was effectively impressed by apricot oil extract at room temperature (Table 2 and Fig. 2).According to estimated data, it was found that high oil concentrations were needed for effectively juice preservation against yeast fermentation even for 10 days of incubation.Also yeast VC was markedly declined at high and relatively high concentrations.At the same time, low oil extract concentrations showed low tendency in juice preservation.In negative controls of oil-free juice, yeast VC was over counting and vigorous juice spoilage symptoms were observed; extreme alcohol odor and gassy appearance appeared at the end of the second day of storage at room temperature.It was noticeable in Fig 1 and Fig. 2 that the anti-yeast bioactivity of oil extract in juice samples were detected at higher oil concentrations than in vitro assay.

Statistical Analysis
Statistical analysis detected high significant difference among the incubation periods, oil concentrations and the interaction between incubation period and oil concentration.

Discussion
Current study objectives were designated in accordance with several studies who examined plants derived EOs for their various biological properties.EOs were tested as natural antimicrobial active agents in food sanitary [31][32][33][34][35][36][37][38] and as a natural therapeutic treatment of Candida and other dermatophyte infections [9,[18][19][20][21][22][23][24][25].The antagonistic property detected for EOs from Pru. armeniace, M. piperita, S. aromaticum, O. basilicum, O. vulgare and O. europaea were also proved by Abd El Salam and Ibrahim (2014) [8] and Ibrahim and Abd El Salam (2015) [9] who studied the efficacy of different EOs against foodborne, food spoilage and pathogenic bacteria and fungi [8,9].As well, O. vulgare Furthermore, they recorded EOs as antioxidants; and in some cases, EOs can be associated with their capacity to exert antigenotoxic effects.Burt (2004) [36] added that the hydrophobicity of EOs enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents.
Antimicrobial activity of apricot seed oil was rarely investigated before, only two studies was published [8,9]; in agreement with these two researches, present assay concluded that apricot seed oil could be applied as natural antimicrobial and save its juice preservation effectiveness.Also, it is expected that under refrigeration, needed EO concentrations will be lower for juice preservation than registered in this study.Screening evaluation data of oil concentration needed for controlling juice spoilage were found in accordance with many investigators results; Burt (2004) [36], who searched antibacterial properties of EOs and their and potential applications in foods, recorded that a higher concentration is needed to achieve the same effect in foods; Bassolé and Juliani (2012) [5] mentioned that in food systems, higher concentrations of EOs are needed to exert similar antibacterial effects as those obtained in in vitro assays; Abd El Salam and Ibrahim (2014) [8] oil extract was added in each well.Positive and negative controls were also involved.Triplicates of Petri dishes were performed for each concentration and controls.All Petri dishes were thereafter incubated at 35 o C for 24 h.Diameters of differentiated inhibition zones in addition to well diameters were measured in mm.Diameter mean values were calculated and MIC value was specified.The MIC was determined as the lowest concentration of anti-yeast showing a zone of growth inhibition and expressed in μgml -1 .
and sterilized by filtration.A set of nine flasks containing 100 ml sterile juice sample/flask was prepared and tested for sterility using sterile YEMEA medium.Extracted Pru.armeniaca seed oil stock was added to each flask to prepare seven concentrations in addition to the negative and positive controls.The seven tested concentrations were 200, 175, 150, 125, 100, 75 and 50μl oil stock/100ml sample.All flasks were then inoculated under aseptic conditions with 100μl yeast inoculum/100 ml juice sample and mixed well.A broth culture of S cerevisiae adjusted to final density of 10 4 CFU/ml was used as the target inoculum.Samples were incubated at room temperature and inspected daily for 10 days.Yeast juice spoilage was followed via monitoring of sample fermentation for alcohol odor and gassy appearance and evaluation of yeast VC expressed in CFU/ml on YEMEA medium.Data of juice fermentation following up and VC evaluation in juice samples were collected daily.Statistical Analysis After testing the data for normality, two way analysis of variance (ANOVA) was used to assess the significance of variations of yeast viable count (CFU/ml) under different incubation time and concentration of oil according to SPSS software (SPSS, 2006) [30].CC-license DOI : 10.14302/issn.2576-9383.jhhr-17-1768Vol-1 Issue 1 Pg.
recorded a remarkable antifungal activity against Candida species when investigated by Cleff et al (2010) [19].In present assay, bioactive EOs confirmed their effectiveness in controlling yeast overgrowth and inhibition of yeast formation; The EOs antagonism was clarified by Bakkali, et al (2008) [31] who worked on EOs inhibitory effect, they explained that because of the mode of EOs extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol derived aromatic components and aliphatic component.These oil constituents are responsible for developing the antimicrobial oil activity.They also added that in eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles.

Figure 1 Figure 2
Figure 1 Effect of different concentrations of in-vitro extracted Prunus armeniace oil on the growth inhibition zones diameters against three yeast strains

Table 2 . Spoilage symptoms of 100 ml sugar cane juice sample inoculated with 100μl Saccharo- myces cerevisiae in relation to different concentrations of Prunus armeniace oil in vitro extract at different incubation periods under room temperature Prunus ar- meniace oil concentra- tion (µl/100ml) Spoilage symptoms (fermentation appearance) Incubation period (day)
tested in vitro extracted Pru.armeniaca oil against microbial spoilage of raw foods, they estimated high oil concentrations needed for raw food preservation more than needed in vitro assay.Spoilage incidents caused by yeasts are controlled by many preservative systems as were discussed by Stratford and James (2003 ( [42]; chilled storage effect on extending the open shelf life of foods and fruit juices were examined in variuos searches; Ghalfi et al. (2007) 37] examined EOs effectiveness in pork meat during cold storage; Belletti [38]l.(2008)[38]applied refrigeration in addition to EO for efficacy enhancement in fruit-based salads preservation during storage.As a result, it is recommended to use refrigeration with apricot oil extract to enhance