Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice

Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice

Accepted Manuscript Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergi...

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Accepted Manuscript Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice Suradeep Basak, Proshanta Guha PII:

S0023-6438(17)30169-X

DOI:

10.1016/j.lwt.2017.03.024

Reference:

YFSTL 6097

To appear in:

LWT - Food Science and Technology

Received Date: 17 October 2016 Revised Date:

23 January 2017

Accepted Date: 12 March 2017

Please cite this article as: Basak, S., Guha, P., Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.03.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf

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(Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its

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antifungal activity in raw apple juice

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Author names

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Authors’ Affiliation address:

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Suradeep Basak∗ and Proshanta Guha

Indian Institute of Technology Kharagpur

Agricultural and Food Engineering Department,

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Kharagpur - 721302 West Bengal

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India.

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Corresponding author. Tel.: +91-9476481537 e-mail address:

[email protected]

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ACCEPTED MANUSCRIPT Abstract

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The present study describes the effect of betel leaf (Piper betle L.) essential oil

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microemulsion (BLEO-ME) on spore inactivation and cell viability of Aspergillus flavus and

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Penicillium expansum using predictive model and its antifungal activity in raw apple juice.

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Survival curve of the spores under minimum fungicidal concentration of BLEO-ME was

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modelled by a modified Weibull model: log

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scatter parameters of flow cytometer suggested differences in size and complexity of spores.

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Propidium iodide was used as fluorescent stain to distinguish between viable and non-viable

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spores. Scanning electron microscopic images revealed morphological alterations of treated

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spores. The cell viability effect of BLEO-ME on mycelial matrix of A. flavus and P.

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expansum was measured using tetrazolium salt MTT and the inhibition was fitted to dose-

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response curves. Mycelial biomass of a selected mould in raw apple juice treated with BLEO-

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ME was determined based on ergosterol content. Formulated BLEO based microemulsion

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exhibited antifungal efficacy in raw apple juice, which suggests its potential as natural

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preservative.

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Keywords: betel leaf essential oil; Weibull model; spore inactivation; MTT assay; ergosterol

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− ( ⁄ ) . The forward and side-

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= log

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1. Introduction Essential oils are secondary metabolites of plants which can be extracted from herbs and

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spices. Bioactive phenolic components of essential oil possess a wide range of biological

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functionalities that have large variety of application in food processing industries. Deep green

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heart-shaped leaves of betel vine are popularly known as Paan (Piper betle L.) in India

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possess essential oil which can be used as an antimicrobial agent in food. Previous work by

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Basak & Guha (2015) revealed 46 different major and minor chemical components

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comprising 99.7% of total oil composition. Major components included chavibetol (22.0%),

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estragole (15.8%), β-cubebene (13.6%), chavicol (11.8%), and caryophyllene (11.3%), and

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components like eucalyptol (1.1%), α-Cubebene (2.0%), β-elemene (2.7%), γ-Muurolene

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(6.0%), Elixene (3.2%), δ-Cadinene (2.1%), 4-Allylphenyl acetate (1.2%) were present in

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trace amount.

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Spore inactivation or inhibition of spore germination is mandatory to restrict fungal

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infection and mycotoxin production in food. Most articles in present literature have

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approached linear inactivation with classical first order equation to describe fungal spore

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inactivation (Bayne & Michener, 1979; Dao et al., 2008; Begum, Hocking, & Miskelly,

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2009), but there are a few reports that have modelled conidial inactivation using nonlinear

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approach (Fujikawa & Itoh, 1996; Dao et al., 2010; Coronel et al., 2011). Fungal spore

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inactivation kinetics under the influence of essential oil has not been as widely explored as

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bacteria so far in current scientific literature.

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Tetrazolium salt, 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT)

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have been used to measure the effect of antifungal agents on cell viability of a number of

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fungal species (Krishnan, Manavathu, & Chandrasekar, 2005; Patel et al., 2013). In this

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assay, yellow coloured salt MTT is reduced to purple coloured MTT–formazan crystals by

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mitochondrial dehydrogenases of live cells. The stain determines metabolic activity of the

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fungal mycelia but not growth. While MTT assay is very useful to establish the efficacy of

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antifungal agents, to our knowledge no report was found in present literature that evaluated

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the effect of essential oil on cell viability of food spoilage filamentous fungi using

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tetrazolium salts. Apple juice is a rich and popular source of various components that are always in high

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consumer demand around the world. But, toxigenic and psychrotrophic spoilage

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microorganisms like Penicillium expansum poses a major threat to this apple juice industry

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(Liewen & Bullerman, 1992). However, in the food system, antimicrobial efficacy of

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essential oils often gets substantially reduced as compared to the model system due to the

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presence of interacting and interfering components of food (Hyldgaard, Mygind, & Meyer,

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2012). A solution to this problem can be nanoemulsification of essential oil that will provide

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uniform distribution of the antimicrobial agent throughout the product, reduce ingredient

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interaction, and improve quality and safety of food (Weiss et al., 2009; Donsì et al., 2011).

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Use of betel leaf essential oil based microemulsion (BLEO-ME) as food preservative in

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unfiltered or raw apple juice can be a promising alternative to chemical preservatives.

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In view of the above research gap, this study was taken up to describes the effect of betel

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leaf (Piper betle L.) essential oil on spore inactivation and cell viability of Aspergillus flavus

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and Penicillium expansum using predictive model and its antifungal activity in raw apple

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juice.

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2. Materials and methods

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2.1. Essential oil based microemulsion

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Freshly harvested betel leaves (Piper betle L.) var. Tamluk Mitha were purchased from the

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local market of Kharagpur. Extraction of essential oil from the leaves by hydrodistillation

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(100 0C, 2 h 30 min) was done on the same day of purchase using Betel Leaf Oil Extractor

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designed and developed at IIT Kharagpur (Guha, 2007). Betel leaf essential oil based

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ACCEPTED MANUSCRIPT microemulsion was formulated according to Basak & Guha (2017). The low-energy method

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of emulsification was used to formulate betel leaf essential oil based microemulsion with

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BLEO as the organic phase, non-ionic surfactant Tween 20 as emulsifier and water as the

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continuous phase.

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2.2. Fungal strain used

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The filamentous fungal strain Penicillium expansum (MTCC 4485) and Aspergillus flavus

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(MTCC 6750) was obtained from Microbial Type Culture collection and Gene Bank

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(MTCC), India. It was maintained on potato dextrose agar (PDA; HiMedia, India) at 4 0C and

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sub-cultured at regular interval.

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2.3. Spore inactivation kinetics

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The minimum fungicidal concentration (MFC) of BLEO for P. expansum and A. flavus

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spores were determined using broth dilution method as reported by Shukla et al. (2008) with

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modification. Briefly, spores from 7 days old culture obtained on PDA at 25 0C and were

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harvested with 10 ml of potato dextrose broth (PDB; HiMedia, India). BLEO based

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microemulsion was added to 2 ml sterile tubes containing PDB inoculated with fungal spores

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(107 spores/ml) to obtain different concentrations (0 – 50 µl/ml) of BLEO and were incubated

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at 25 0C for 30 days. Tubes that did not show any visible mycelial growth was selected and

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centrifuged (4,000 g for 3 min) to harvest the spores. Supernatant was discarded and the

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pellet was washed three times with phosphate buffer saline (PBS; pH 7.4) to remove traces of

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essential oil. Washed pellets were finally resuspended in 1 ml of the same buffer and

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subcultured on fresh PDA medium (with no essential oil) to check if the inhibition was

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reversible. Lowest concentration of BLEO at which growth was not reverted was considered

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as MFC.

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For spore inactivation kinetic study, initial spore count was determined using a Neubauer’s

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Chamber (depth 0.1 mm, 0.0025 mm2) under a biological microscope (Olympus CX31,

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ACCEPTED MANUSCRIPT Tokyo, Japan) and expressed as spores per millilitre which was further confirmed using total

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plate count on fresh PDA medium. MFC of the essential oil was similarly prepared using

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BLEO-ME in PDB with known initial spore count and incubated at 25 0C. At regular interval

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(24 h), tubes were taken out to determine the number of surviving spores. Spores were

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harvested by washing using a centrifuge and were re-suspended in 1 ml PBS buffer (pH 7.4).

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The concentration of viable spores was determined by pouring the harvested spore suspension

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and its three subsequent decimal dilutions on Petri dishes containing PDA as a growth

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medium. After incubation at 25 0C for 72 h, the colony forming units (spores/ml) were

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determined on plates that have colonies ranging 10 and 100.

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To describe the survival of P. expansum and A. flavus spores the decimal logarithmic form

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of Weibull distribution model (Mafart et al., 2002) was fitted to the data and parameters were

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estimated accordingly: log

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This model has three parameters (N0, δ and β) where N0 is spore concentration at the t = 0.

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Scale parameter δ (h) corresponds to the time that directs to one decimal reduction of

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surviving spores. The shape of the curve is characterized by β (dimensionless). The value of β

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depends on the shape of inactivation curve (β >1 produces a concave curve, β = 1 gives linear

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curve and β <1 will describe convex curves).

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2.4. Spore viability

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2.4.1. Fluorescence assisted cell sorting using propidium iodide stain

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Sporicidal activity of BLEO at its MFC was established using flow cytometry. Propidium

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iodide (PI) fluorescence intensity was used to assess spore viability. The method of Mesquita

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et al. (2012) was followed to assess the viability of the spores. Spores treated with MFC of

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BLEO were harvested by centrifugation followed by washing with PBS as described above

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and resuspended in 1 ml distilled water. 100 µl of this spore suspension was pipetted to flow 6

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Sigma, St Louis, USA) and final volume was made up to 2 ml with distilled water and

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incubated at 30 0C for 15 min and analysed in Fluorescence Assisted Cell Sorter (BD

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FACSAria™ III, BD Biosciences, USA).

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2.4.2. Scanning electron microscopic analysis of inactivated spores

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The modified method of Tyagi & Malik (2010) was followed to study ultrastructure of

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spore treated with MFC of BLEO. All the treated spores were harvested by centrifugation

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followed by washing with PBS (pH 7.4) as described above and were prefixed with 2.5 %

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glutaraldehyde solution overnight at 4 0C. The fixed spores were harvested and dried under a

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laminar air flow at 25 0C and mounted on SEM stubs using conductive carbon tapes. The

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stubs were sputter-coated with gold by cathode spraying (POLARON, UK) and imaged using

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scanning electron microscope (Zeiss MERLIN Field Emission SEM). The SEM analysis was

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done under following analytical conditions: EHT = 5.00 kV, WD = 4.0 – 10 mm and Signal

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A = InLens.

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2.5. Cell viability study

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The method of Pierce et al. (2009) and Meletiadis et al., (2000) was used to determine

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sessile cell minimum inhibitory concentration (SMIC) of BLEO using tetrazolium salt MTT.

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Briefly, 200 µl of spore suspension (106 spores/ml) was pipetted into selected wells of a flat-

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bottom 96-well plate and incubated for 48 h at 25 0C for mycelial mat formation. After

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incubation, the remaining medium was aspirated and non-adherent cells were removed by

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washing the mycelial mat three times with PBS (pH 7.4) using gentle pipetting. 200 µl of

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highest concentration of the essential oil was added to a first well of column 1 followed by

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two-fold serial dilution in adjacent wells containing pre-washed mycelial mats to obtain

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concentration range (0.0 – 6.8 µl/ml) of BLEO and incubated at 25 0C for 7 days. The

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mycelial mats were re-washed three times in a similar way using PBS after incubation to

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ACCEPTED MANUSCRIPT remove traces of antifungal used and MTT assay was carried out. 200 µl of freshly prepared

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MTT solution (5 mg/ml; SRL, India) was added to each well and incubated at 37 0C. After 3

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h of incubation, the supernatant was carefully discarded and 200 µl of dimethyl sulfoxide

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(DMSO) was added to each well to solubilize the formazan complexes formed due to the

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reduction of MTT by mitochondrial enzymes of metabolically active cells. Antifungal free

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wells and mycelial mat free wells were considered as a positive control and negative control,

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respectively. From each well, 100 µl of the resulting coloured supernatant was transferred

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into wells of a new microtiter plate and absorbance was measured at 565 nm (A565) using

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microtiter plate reader (Epoch 2, BioTek, USA).

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percentage with respect to control (untreated) by using formula (Rautenbach et al., 2006): % inhibition = 100 −

× (

!" #$%&#%' (%))* +%$&,%

+%$&,%

!" 0!1.#.+% /!-#$!)* +%$&,%

!" -%,&#.+% /!-#$!))

!" -%,&#.+% /!-#$!)

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(3)

The inhibition percent data were fitted to dose-response curves described by Rautenbach

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et al. (2006) to determine BLEO concentration (x) at which 50% inhibition (IC50) and MIC,

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respectively using GraphPad Prism® v5.01: Y=

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4!##!56(#!0*4!##!5)

[89: (;< = >?)×@ABCDCBE F89GH]



(4)

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Top and bottom represent growth at high and low antifungal concentration, respectively. The

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slope of the curve is defined by activity slope.

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2.6. Antifungal activity of BLEO in raw apple juice

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Raw apple juice was selected as food system to study the antifungal efficacy of BLEO

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against the growth of a selected postharvest food spoilage microorganism. Although,

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occurrence of P. expansum (FAO, 2001) is not prevalent in pasteurized apple juice because

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the spores are heat inactivated. Primarily, the aim of this study is to determine antifungal

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activity of BLEO in raw apple juice. Hence, freshly harvested juice was pasteurized to

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eliminate initial microbial load and then was inoculated with P. expansum spores. 8

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2.6.1. Sample preparation

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Apples (cv. Red delicious) at commercial maturity were purchased from local market of

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IIT Kharagpur. After being cleaned with 70% ethanol and distilled water, each apple was cut

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and made into pulpy raw apple juice (Total soluble solids (TSS): 13 0Brix; pH 4.5 ± 0.2 at 25

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0

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pasteurized by heating at 72 0C for 14 seconds (centre temperature) in thermostat water bath

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(Mak, Ingham, & Ingham, 2001). Pasteurized raw apple juice was kept at 4 0C and used same

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day of the experiment. 30 ml of the juice containing 3 ml of P. expansum spores (106

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spores/ml) and 27 ml of raw apple juice having BLEO-ME to produce concentration range

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(0.0 – 0.7 µl/ml) of BLEO in Erlenmeyer flasks were incubated at 25 0C for 10 days. An

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indirect method to estimate the mould growth in raw apple juice was carried out by analysing

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ergosterol content.

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2.6.2. Ergosterol analysis

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The method of Marín et al. (2008) was followed to extract ergosterol from mycelial mats

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of P. expansum grown on raw apple juice treated with different concentrations of BLEO.

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Filtration of the mycelial mats from the juice was not conducted prior to ergosterol analysis,

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so as to avoid the loss of newly growing mycelia fragments at the bottom of the flask.

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Briefly, the mycelial mat was extracted with 40 ml of 10% KOH in methanol with continuous

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stirring for 30 min followed by heating in a hot water bath (55 – 60 0C) for 20 min. After

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cooling to room temperature, 3 ml of water and 2 ml of hexane were added and mixed in a

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vortex for 1 min. After layer gets separated, the upper layer (hexane) was transferred to a

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screw cap tube. The hexane extraction step was repeated twice with 2 ml each time. The

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extracts were combined and evaporated under vacuum. The dried extracts were dissolved in 2

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ml methanol and syringe filtered (0.45 µm).

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ACCEPTED MANUSCRIPT The samples were injected into HPLC using Agilent 1100 Infinity series containing

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Eclipse XDB- C18 Zorbax analytical column (4.6×150 mm) and the wavelength was set at

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282 nm. The mobile phase was methanol passed at flow rate of 1.0 ml/min. Ergosterol

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(Sigma, St Louis, USA) standard was used to obtain calibration line (R2 = 0.99). The

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resulting ergosterol was expressed in mg of ergosterol per sample of raw apple juice.

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Determined ergosterol (mg) in treated (et) raw apple juice was converted to inhibition

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percentage with respect to untreated (ec) raw apple juice by using formula (Tripathi, Dubey,

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& Shukla, 2007):

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(JK *JL ) JK

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Treated juices which didn’t show any visible mycelial growth at 10th day of incubation

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were incubated till 30 days.

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2.7. Statistical analysis

Modified Weibull model and dose-response curves were fitted to individual experimental

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data and the model parameters were estimated by Levenberg-Marquardt algorithm for

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nonlinear least square minimization using GraphPad Prism® version 5.0 for Windows

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(GraphPad Software, San Diego, CA, USA). The performance of models was assessed using

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Root Mean Square Error (RMSE) and coefficient of determination (R2). Models having

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RMSE values close to zero and R2 values close to one were considered as a good fit

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(Ratkowsky, 2003). All experiments were conducted in triplicates and repeated thrice to

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establish repeatability.

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3. Results and discussion

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3.1. Spore inactivation kinetics

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Minimum concentration of BLEO that exhibited sporicidal activity against both fungal

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strains was found to be 15 µl/ml among the range (0 – 50 µl/ml) selected for the study. The

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modified Weibull model explained the inactivation kinetics of the spores treated with MFC of 10

ACCEPTED MANUSCRIPT BLEO. The surviving spores decreased gradually with increase in treatment time (Fig. 1).

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Time (δ) required to achieve 1 log10 reduction of viable spores was estimated to be 69.14 ±

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4.85 and 60.7 ± 6.95 hours for A. flavus and P. expansum, respectively. It can be suggested

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that MFC of BLEO can achieve 1 log10 reduction of both fungal spores at significantly similar

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(P = 0.163) time. But, the value of shape parameter (β = 0.963) suggested an almost linear

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inactivation of surviving spores with time for P. expansum, which indicates initiation of spore

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inactivation as soon as spores are exposed to BLEO. On the contrary, A. flavus spores

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exhibited concave shape (β = 1.486) curves that suggested delay in sporicidal activity initially

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but escalation of spore inactivation happened eventually. The model was fitted to the data

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with satisfactory statistical indices (Table 1). The current study includes spore inactivation

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using single hurdle (BLEO) keeping other rate limiting factors at the optimum level for P.

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expansum growth (pH: 5.6 and incubated at 25 0C), so as to determine the efficacy of the

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essential oil only.

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3.2. Spore viability study

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Spore viability was also checked using flow cytometry to confirm the inactivation under

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MFC of BLEO. Forward (FSC) and side (SSC) scatter were used as an indicator to assess the

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viability of treated and untreated spores. As compared to untreated P. expansum spores, the

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treated spores showed 50% decline in mean FSC value and 11% increase in mean SSC value,

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respectively (Table 2). Similarly, treated A. flavus showed 4% decline in mean FSC value and

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36% increase in mean SSC value with respect to untreated spores. Spore inactivation occurs

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due to loss of membrane integrity which is responsible for the release of internal cellular

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components that eventually reduced the size (mean FSC value) of the treated spores. It can be

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hypothesized that increase in granulation of inactivated spores resulted in more internal

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complexity that further increased the mean SSC value as compared to low SSC values of

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healthy spores. This supported the fact that essential oil causes physical damage of spore

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ACCEPTED MANUSCRIPT which leads to loss of viability. SEM images also suggested similar mode of spore

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inactivation under influence of BLEO. It revealed cytoplasmic coagulation, shrinkage,

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granulation and serious damage to the morphology of the treated spores with respect to the

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untreated spores which had absolutely healthy ultrastructure (Fig. 2). The observations made

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are consistent with earlier studies that reported abnormalities in fungal spores exposed to

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lethal treatments (Evrendilek et al., 2008; Dasan, Mutlu, & Boyaci, 2016). Components of

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essential oil usually interact and penetrate through the cell membrane and interfere with

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enzymatic reactions of cell wall synthesis that eventually leads to a loss in germination,

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growth and morphogenesis of the fungus (Ghfir, Fonvieille, & Dargent, 1997; Carmo, Lima,

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& De Souza, 2008).

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There was also a major increase in PI fluorescence intensity of almost 98% in treated

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spores as compared to untreated spores of both the moulds (Table 2). Higher PI fluorescence

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intensity of treated spores suggested the presence of more non-viable spores in the

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population. Therefore, an increase in a number of PI–stained spores in the BLEO treated

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population supported the sporicidal activity of the essential oil.

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3.3. Cell viability study

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Metabolic activity of mycelia was determined using intensity of purple colour which is

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directly related to the reduction of the stain. Inhibition percent of mycelial growth

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exponentially increased with dose of BLEO (0.0 – 6.8 µl/ml). Estimated values of activity

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parameters of dose-response curve were found to have wide confidence interval (Table 3)

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that suggests good fit of the equation with raw data (Fig. 3). Accordingly, 0.29 µl/ml and 0.18

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µl/ml of BLEO were found to be IC50 value against A. flavus and P. expansum mycelial cells,

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respectively. Maximum or 90% mycelial inhibition of A. flavus and P. expansum was

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estimated to be 0.67 µl/ml and 0.41 µl/ml of BLEO, respectively. According to the estimated

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MIC in the present study, it can be inferred that BLEO is more effective against P. expansum

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than A. flavus. Stević et al. (2014) have also reported low MIC values, hence lower antifungal

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efficacy of savory, oregano and rose oil against A. flavus as compared to Penicillium spp.

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3.4. Antifungal activity in raw apple juice The effect of BLEO on the ergosterol content in the plasma membrane of P. expansum has

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grown on raw apple juice is shown in Table 4. Ergosterol content decreased from 12.7 ± 1.0

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mg to 1.3 ± 0.1 mg for 0.1 and 0.4 µl/ml of BLEO, respectively. According to Eq. 4, the

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inhibition of ergosterol increased from 23.4 ± 4.6 % to 92.2 ± 0.5% for 0.1 and 0.4 µl/ml of

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BLEO, respectively. There was no visible mycelial growth recorded on raw apple juice

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treated with 0.5, 0.6 and 0.7 µl/ml of BLEO during 30 days of incubation. Juneja, Dwivedi, &

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Yan (2012) have reviewed the potential of combined treatment of natural antimicrobials and

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physical treatments in food preservation. Similarly, reason associated to mycelial growth

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inhibition could be synergistic antifungal effect of BLEO (≥0.5 µl/ml) and low pH (4.5 ± 0.2)

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in raw apple juice on germination of P. expansum spores during the incubation period.

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Suggestions by Pauli (2006) support the fact that specific inhibition of ergosterol biosynthetic

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pathway due to the presence of BLEO in the juice may have resulted in a reduction of

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ergosterol content. It can also be inferred that reduction in spore germination resulted in low

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mycelial mass that eventually resulted in low ergosterol content as it is an indirect method of

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measuring fungal growth (Gibson & Hocking, 1997; Gutarowska & Zakowska, 2009). A

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similar correlation between ergosterol content and fungal growth under the influence of

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essential oil has been reported earlier (Dhingra et al., 2009; Tian et al., 2012; Kedia et al.,

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2014). Among a few previous reports on essential oil based microemulsion, Wang et al.

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(2014) and He et al. (2016) showed antifungal activity of cinnamon and clove essential oil

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based microemulsion against B. cinerea and P. digitatum on pears and orange, respectively.

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However, these studies have used ethanol as continuous phase in the formulated

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microemulsions, which might have added to antimicrobial activity of the emulsions, whereas

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our study involves BLEO-ME with water as continuous phase.

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4. Conclusion It can be concluded from the current study that, use of BLEO based microemulsion has

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high fungitoxic activity at lower concentration whereas sporicidal activity is exhibited only at

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higher concentration. This study also provides an insight into kinetics of spore inactivation

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under influence of essential oil using nonlinear inactivation model. It has shown that cells of

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A. flavus and P. expansum lose its viability when treated with the essential oil. On this

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premise of antifungal activity of BLEO on the growth of P. expansum in raw apple juice, we

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can conclude it as a potential natural food preservative. Further study on overall acceptability

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of sensory attributes of the essential oil treated food stuffs is warranted so as to avoid market

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failure of the product.

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5. Acknowledgements

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The authors are grateful to Indian Institute of Technology Kharagpur for financial

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assistance, infrastructure and facilities to conduct the research. They also thank Dr. S. L.

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Shrivastava, Dr. Jayeeta Mitra and Ms. Ruby Pandey Agricultural and Food Engineering

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Department, IIT Kharagpur for their continual support throughout.

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Fig. 1. Survival curves of (a) A. flavus (b) P. expansum spores treated with MFC of BLEO.

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Values are mean ± S.E.

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Fig. 2. SEM images showing morphological alteration of untreated spores: A. flavus (a), P.

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expansum (d) and spores treated with MFC of BLEO: A. flavus (b and c), P. expansum (e and

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f).

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Fig. 3. Dose-response curve representing percent growth inhibition of (a) A. flavus and (b) P.

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expansum mycelial matrix under influence of different concentration of BLEO. Values are

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mean ± S.E.

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ACCEPTED MANUSCRIPT Table 1 Parameter estimates and 95% confidence interval of the fit to modified Weibull model to the inactivation curves of A. flavus and P. expansum spores under influence of BLEO (15 µl/ml) Estimated parameters

P. expansum

δ (h)

β

8.19a (7.89, 8.37)

69.1a (59.4, 78.9)

1.49a (1.33, 1.65)

b

6.94 (6.66, 7.11)

a

60.7 (46.7, 74.7)

b

RMSE

0.977

0.33

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A. flavus

LogN0

R2

0.96 (0.82, 1.11)

0.959

0.25

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The values followed by same letter in same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests.

ACCEPTED MANUSCRIPT Table 2 Mean FSC, SSC and Propidium iodide (PI) fluorescence values of untreated and BLEO treated (15 µl/ml) A. flavus and P. expansum spores. Values are presented in arbitrary units (AU). Approximately, 10,000 particles were analysed in each run. A. flavus

P. expansum

Treated

Untreated

FSC

15,569

14,886

20,138

10,015

SSC

18,846

25,556

15,779

17,494

PI

30

1,041

27

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ACCEPTED MANUSCRIPT Table 3 Parameter estimates and 95% confidence interval of dose-response curve of A. flavus and P. expansum mycelial cells grown in vitro under influence of BLEO (µl/ml) Estimated parameters

P. expansum

IC90

0.29a (0.24, 0.34)

0.67c (0.61, 0.73)

b

0.18 (0.16, 0.20)

d

RMSE

0.991

2.7

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A. flavus

IC50

R2

0.37 (0.31, 0.44)

0.986

3.5

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ACCEPTED MANUSCRIPT Table 4 Effect of different concentration of BLEO on ergosterol content and its dose-dependent inhibition. The values represented here are means with 95% confidence interval. Ergosterol (mg) 16.6a (15, 18.2) 12.7b (8.3, 17.2) 6.1c (4.3, 7.8) 4.5c (3.4, 5.7) 1.3d (0.9, 1.6) 0.0

Inhibition of ergosterol synthesis (%) 0.0 23.4c (3.8, 43.1) 63.3b (50.9, 75.6) 72.7b (68.3, 77.2) 92.2a (89.9, 94.6) 100a

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The values followed by same letter in same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests.

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ACCEPTED MANUSCRIPT Highlights The modified Weibull model described spore inactivation under influence of BLEO in vitro antifungal activity was established using MTT assay Antifungal effect in real apple juice was estimated in terms of ergosterol content

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BLEO is a promising natural preservative to inhibit food spoilage