International Journal of Food Microbiology 211 (2015) 44–50

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified

from a newly isolated Lactococcus sp.
Kontham Kulangara Varsha a, Leena Devendra a, Ganesan Shilpa b, Sulochana Priya b,
Ashok Pandey a, Kesavan Madhavan Nampoothiri a,⁎
a
Biotechnology Division, CSIR—National Institute for Interdisciplinary Science and Technology (NIIST), Trivandrum 695 019, Kerala, India
b
Agroprocessing Division, CSIR—National Institute for Interdisciplinary Science and Technology (NIIST), Trivandrum 695 019, Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: The volatile organic compound 2,4-di-tert-butyl phenol (2,4 DTBP) was purified from the cell free supernatant of a
Received 23 March 2015 newly isolated Lactococcus sp. by solvent extraction and chromatographic techniques. Molecular characterization
Received in revised form 6 June 2015 of the compound by ESI-MS, 1H NMR and FTIR analysis revealed the structure, C14H22O. Fungicidal activity was
Accepted 25 June 2015
demonstrated against Aspergillus niger, Fusarium oxysporum and Penicillium chrysogenum by disc diffusion assay.
Available online 2 July 2015
Among the cell lines tested for cytotoxicity of this compound (normal cell line H9c2 and cancer cell lines HeLa
Keywords:
and MCF-7), a remarkable cytotoxicity against HeLa cells with an IC50 value of 10 μg/mL was shown. A biocontrol
Antifungal experiment with 2,4 DTBP supplemented fraction prevented growth of the abovementioned fungi on wheat
Antioxidant grains. The study further strengthens the case for development of biopreservatives and dietary antioxidants
Biopreservation from lactic acid bacteria for food applications.
Cytotoxicity © 2015 Elsevier B.V. All rights reserved.
2,4-Di-tert-butyl phenol
Lactic acid bacteria

1. Introduction acid, p-coumaric acid, malic acid and ferulic acid along with 5% v/v
ethanol in MRS led to the production of volatile phenolic compounds,
The food industry is in a continuous search for new biopreservatives 4 vinyl phenol and 4 ethyl phenol by Lactobacillus through p-coumaric
where antifungal lactic acid bacteria (LAB) and their metabolites can be acid metabolic pathway (Silva et al., 2011a, 2011b). Production of 4VP
used effectively. LAB have received the GRAS (generally recognized as and 4EP by Lactobacillus in wine conditions was detected by Fras et al.
safe) and the QPS (qualified presumption of safety) status and represent (2014).
the microbial group most commonly used as protective cultures (Gaggia Cereals comprise a major food source and contribute more than 60%
et al., 2011). It is estimated that over 3400 tonnes of pure LAB cells to the world food production providing dietary fiber, proteins, minerals
is consumed every year in Europe alone (Franz et al., 2010). An and vitamins required for human health. Beneficial effects of cereals can
array of antifungal compounds produced by LAB have been identified be exploited in different ways leading to the design of novel functional
and characterized and include organic acids, reuterin, proteinaceous foods based on cereals or cereal ingredients that can target specific
compounds, cyclic dipeptides (Oliveira et al., 2014; Schnurer and populations (Charalampopoulos et al., 2002). One of the major causes
Magnusson, 2005), methylhydantoine, mevalonolactone (Niku-Paavola of cereal spoilage during storage is contamination by fungi which,
et al., 1999) and 2-propenyl ester (Wang et al., 2012). apart from diminishing the quality and nutritive value, may produce
Phenolic compounds as food additives are of particular interest when mycotoxins that can cause severe health problems. In response to
considering health and diet due to their antioxidant properties. The consumer demands, biopreservation technologies are being favored
advantage of LAB metabolites having both antifungal and antioxidant to improve the safety, nutritional value and the organoleptic properties
properties is that they can be developed as food preservatives with of cereals (Oliveira et al., 2014). Previous investigations showed that
dietary antioxidant properties once incorporated into food. Production if cereals are fermented with LAB strains, the shelf life can be further
of phenolic compounds by LAB and their antifungal activity is not extended (Blandino et al., 2003).
meticulously researched and remains to be explored. LAB are capable A pool of different LAB strains was screened to synthesize antioxidant
of producing volatile phenolic compounds according to the availability peptides during sourdough fermentation of cereal flours (Coda et al.,
of suitable substrates. It is reported that supplementation of caffeic 2011). However, reports on antioxidant phenolic compound production
and their contribution towards antifungal and antioxidant properties of
⁎ Corresponding author. LAB were not addressed well. As a continuation of our earlier study
E-mail address: [email protected] (K.M. Nampoothiri). (Varsha et al., 2014), here we report the purification and characterization

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.025
0168-1605/© 2015 Elsevier B.V. All rights reserved.
 K.K. Varsha et al. / International Journal of Food Microbiology 211 (2015) 44–50 45

of the phenolic compound 2,4 di-tertiary butyl phenol (2,4 DTBP) from was checked against A. niger (KACC 42589), and P. chrysogenum
Lactococcus sp. To the best of our knowledge, this is the pioneer report (NII 08137) by disc diffusion method on PDA plates which were already
of a phenolic compound extracted from the culture broth of a lactic acid swabbed with individual fungal suspension (1 × 104 spores/mL). The
bacterium showing a wide spectrum of biological functions including control was 50% acetonitrile and the plates were incubated at 30 °C
antifungal, antioxidant and anti tumor properties. for four days to check the inhibition zones.

2. Materials and methods 2.5. Total antioxidant and ferric Ion (Fe3+) reducing activities

2.1. Microorganisms, cell lines and culture conditions Active B1 fraction (1 mg/mL) containing 2,4 DTBP was checked for
its total antioxidant and Ferric Ion (Fe3 +) reducing activities by the
Lactococcus sp. BSN307 (NCBI accession KM261818), previously methods previously reported by Prieto et al. (1999) and Oyaizu
denoted as BSN in Varsha et al. (2014) was cultivated in MRS (de Man (1986) respectively and the activities were expressed as ascorbic acid
Rogosa Sharpe, Himedia, India) medium at 30 °C for 24 h and preserved equivalents.
at −20 °C in MRS broth with 20% (v/v) glycerol. The fungal test strains,
Aspergillus niger (KACC 42589), Penicillium chrysogenum (NII 08137), 2.6. Assay of free radical scavenging
Fusarium oxysporum (KACC 42109), Fusarium moniliforme (KACC
08141), Fusarium graminearum (MTCC 1893), Fusarium chlamydosporum Free radical scavenging activity of the B1 fraction as well as 2,4 DTBP
(MTCC 2399) were maintained on potato dextrose agar (Himedia, (≥90% purity) purified from BSN307 culture medium were determined
India) at 30 °C. Mammalian cell lines, H9c2 (myoblast cell line), HeLa by the method of Shimada et al. (1992) which is based on the principle
(cervical adenocarcinoma cell line) and MCF-7 (breast carcinoma cell of scavenging the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. DPPH
line) were obtained from ATCC (American Type Culture Collection). was prepared in 95% ethanol to a final concentration of 0.2 mg/5 mL.
Samples were prepared in methanol with the concentration of B1 fraction
2.2. Chemicals and solvents as 5 mg/mL and 2,4 DTBP as 2 mg/mL and the radical-scavenging
activities of samples were expressed as percentage scavenging of
All the organic solvents and H2SO4 used in this study were obtained DPPH. Each mixture was kept in the dark for 30 min and the absorbance
from Merck (India), DPPH (1,1-diphenyl-2-picryl-hydrazyl), MTT (3-(4, was measured at 517 nm against a blank. The scavenging ability was
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), Dulbecco's defined as:
Modified Eagle's Medium (DMEM) and trichloroacetic acid, streptomycin,

penicillin and amphotericin B were purchased from Sigma (India). Scavenging activity ð%Þ ¼ AControl −ASample =AControl  100:
Analytical grade sodium sulfate, ammonium molybdate, potassium
ferricyanide, ferric chloride, fetal bovine serum (FBS) and ascorbic
acid were purchased from Himedia (India). 2.7. Cytotoxicity to mammalian cell lines

2.3. Purification and characterization of the bioactive Cytotoxicity against mammalian cells was determined by MTT assay
as described previously by Mosmann (1983), where the conversion
For purification, 10 L of MRS medium was inoculated (5% (v/v)) with of tetrazolium salt MTT to blue colored formazan by the mitochondrial
24 h old culture and incubated at 30 °C for 48 h under static conditions. enzyme succinate dehydrogenase which is active in living cells was
After incubation, the supernatant was collected by centrifugation at measured to quantify cell survival and proliferation. In brief, mammalian
8590 ×g for 15 min and extracted with double volume of chloroform. cell lines, H9c2, HeLa and MCF-7 were maintained in DMEM supple-
The solvent was evaporated under reduced pressure in a Rotavapor mented with 10% FBS, 10 mg/L streptomycin, 100 U/L penicillin and
(Buchi, Switzerland) and the concentrated sample was further dissolved 25 μg/mL amphotericin B. The cells were maintained at 37 °C in a humid-
in acetonitrile (B1 fraction). The B1 fraction was purified by high pressure ified atmosphere of 5% CO2 (SANYO CO2 Incubator, Japan) until a conflu-
liquid chromatography (Shimadzu UFLC, Japan) using Prep-C18 column ent monolayer was obtained. Cells were seeded at a concentration of
(Phenomenex, US) and acetonitrile: water (90:10) as mobile phase with 1 × 104 cells into each well of a 96-well tissue culture plate followed by
a flow rate of 4 mL/min. Fractions with antifungal activity were pooled incubation at the same conditions for 24 h. The medium was removed
and purified further by analytical HPLC (C18 column, Phenomenex, US) and the cells were treated with varying concentrations of purified 2,4
using acetonitrile as mobile phase with a flow rate of 1 mL/min. DTBP prepared in DMEM without FBS and incubated in the CO2 incubator
Antifungal activity of the purified product (400 μg/disc) was confirmed for 24 h. After incubation, the medium was removed from each well with
against F. oxysporum (KACC 42109) by disc diffusion assay as described a pipette and 200 μL of MTT (0.5 mg/mL) dissolved in DMEM was added
in Section 2.4. followed by incubation in the dark for 3 h. The reaction was stopped by
addition of 100 μL DMSO into the wells. Plates were further incubated
2.3.1. Structure analysis for 30 min in a shaker and the absorbance was read at 570 nm. The
Structure of the antifungal compound was determined by 1H nuclear experiments were performed in triplicate. Cytotoxicity (%) and IC50
magnetic resonance (NMR) spectroscopy using Bruker Avance II 500 (effective concentration of drug resulting in 50% of maximal toxicity)
spectrometer (US). Liquid chromatography–mass spectrometry (Thermo were calculated as follows:
Orbitrap LC/MS, US) employing electrospray (ESI) ionization method was
used to determine molecular mass of the compound. The sample was Cytotoxicityð%Þ ¼ ½1−ðATest =AControl Þ  100:
prepared in acetonitrile with 0.1 N formic acid and acetonitrile was
used as mobile phase. Fourier transform infrared spectroscopy (FTIR)
was performed with PerkinElmer model Spectrum 100 (US) to obtain IR 2.8. Biocontrol of fungi on wheat grains
spectrum of the compound.
The wheat grains were surface disinfected with 4% sodium
2.4. Antifungal activity assay of the pure compound hypochlorite and thoroughly washed with sterilized distilled water
followed by air drying. The active fraction (B1) was extracted from
As well as F. oxysporum (KACC 42109), the wide spectrum antifungal 1 L of BSN307 culture filtrate using double volume of chloroform,
activity of the purified compound (400 μg) dissolved in 50% acetonitrile concentrated in a rotary evaporator and dissolved in 20% acetonitrile.
46 K.K. Varsha et al. / International Journal of Food Microbiology 211 (2015) 44–50

Grains were treated with active fraction (25 mg/mL) for 15 min and sample (Fig. 1A). The sample was then further purified to 99.5% by
air dried along with those treated in 20% acetonitrile as control. The analytical HPLC using C18 column. Fig. 1B shows the HPLC chromatogram
dried grains were distributed into sterile glass vials and inoculated of crude B1 fraction and the purified compound (Fig. 1C) at 270 nm.
with 2–3 day old mycelial plugs (~ 5 mm) of A. niger, F. moniliforme, NMR and IR were performed. IR spectra of a purified sample showed
F. graminearum, F. chlamydosporum and F. oxysporum and incubated a peak at 3533 cm− 1 indicating stretching of O–H phenolic group.
at room temperature. Fungal growth on the grains was monitored Peaks at 2871–2959 cm− 1 indicated C–C stretching of alkyl group.
every day. C–O stretching of phenols was seen at 1252 cm − 1. Presence of
aromatic C_C stretching was identified by peaks at 1506–1600 cm−1.
2.9. Statistical analysis Thus the presence of this functional group provided suggestive evidence
that the compound is phenolic in nature. NMR data of the purified sample
All the experiments were performed in triplicate and results were revealed the presence of aromatic hydrogen corresponding to 7 to
expressed as mean values ± standard deviation. One-way ANOVA and 7.3 ppm, a doublet at 7.07 ppm indicate aromatic metahydrogen.
Dunnet's test were performed to test the differences between test and Singlets of 9 hydrogen atoms at 1.309 and 1.431 suggested presence
control groups (p b 0.05). Statistical analyses were performed using of di substituted tertiary butyl group. A downfield singlet at 4.662 indi-
the Minitab statistical package v. 17 (Minitab Inc., USA). cates the presence of phenolic hydrogen (Fig. 2A). Thus, after careful
interpretation and co-relation of IR, NMR and LC–MS data, the com-
3. Results and discussion pound was structurally elucidated as C14H22O (Fig. 2B) known as 2,4
di-tert butyl phenol (2,4 DTBP), a volatile organic compound (VOC)
3.1. Purification and characterization and discovered to play a major role in the antifungal activity of chloro-
form fraction along with other unidentified antifungal compounds.
Solvent extraction was followed in a sequential order, from hexane– The final yield of 2,4 DTBP (≥ 90% purity) was 2 mg/L. Using purified
chloroform–ethyl acetate. No antifungal activity was observed in 2,4 DTBP a clearing zone of 3 cm against F. oxysporum (Fig. 1D) was
the hexane fraction and the antifungal compounds identified in ethyl obtained.
acetate have been previously reported (Varsha et al., 2014). The 2,4 DTBP is used as an intermediate in for the preparation of UV
remaining solvent fraction with activity was the chloroform fraction stabilizers and antioxidants as well as in the manufacture of pharma-
and this was further purified to identify the antifungal compounds. ceuticals and fragrances (Choi et al., 2013). The possible precursor and
HPLC analysis of prep-C18 purified sample revealed a major component the pathway that leads to the production of this compound is not yet
of 90% and few minor impurities contributing the remaining 10%. The elucidated clearly although the gene clusters and enzymes required for
sample was then subjected to LC–MS analysis which indicated a molecu- shikimate and mevalonate pathway have been identified in Lactobacillus
lar weight of 206 at positive mode corresponding to the major peak in the (Bolotin et al., 2001; Smeds et al., 2001).

Fig. 1. Purification and antifungal activity of 2,4 di-tert-butyl phenol. MS chromatogram of 2,4 DTBP (standard) (A), HPLC chromatogram of B1 fraction (B) purified 2,4 DTBP (C) and
antifungal activity of 2,4 DTBP against F. oxysporum (D).
 K.K. Varsha et al. / International Journal of Food Microbiology 211 (2015) 44–50 47

Fig. 2. Structure elucidation of 2,4 di-tert-butyl phenol. 1H NMR spectrum (A) structure of 2,4 DTBP (B).

3.2. Antifungal and antioxidant activity assay of 2,4 DTBP of production of other VOC with various biological activities from
LAB.
2,4 DTBP purified from the culture medium of BSN307 exhibited Evaluation of total antioxidant capacity (TAC) by the phos-
fungicidal activity against all the fungal strains tested. Three differ- phomolybdenum method showed that 40 μg of B1 fraction had 80%
ent genera of fungi that often cause contamination in various food antioxidant activity where the same amount of ascorbic acid had
products were selected to study the antifungal activity of 2,4 DTBP 83% activity. Ferric ion reducing power assay revealed that 1 mg of
and all were found to be sensitive. A 400 μg/disc produced a clearing this fraction held 33% of reducing power and the same amount of
zone of 0.8 cm against A. niger and 1.5 cm against P. chrysogenum. ascorbic acid showed 50% activity.
Among the fungi tested F. oxysporum was most sensitive to 2,4
DTBP. The minimum inhibitory concentration against F. oxysporum 3.3. Assay of free radical scavenging
was 62 μg/disc and produced a clearing zone of 0.6 cm.
Our result supports and confirms previous research on the anti- The percentage of free radical scavenging activity of the B1 fraction
fungal activity of VOC produced by bacteria (Yuan et al., 2012; and 2,4 DTBP is shown in Fig. 3. Fourty micrograms of purified 2,4
Weisskopf, 2014). 2,4 DTBP has been reported to be present in fruits DTBP had 77.5% free radical scavenging ability. 2,4 DTBP was reported
as well as seeds and imparts antioxidant properties (Choi et al., to be associated with the antioxidant effect of the methanolic extract
2013). This volatile phenolic compound is reported to have in vitro
antimalarial activity where the plasmodial growth was arrested
with an amount of 100 mM compound (Kusch et al., 2011). One
recent report indicates the presence of 2,4 DTBP in the culture filtrate
(CF) of an antifungal Lactobacillus strain where the compound was
detected by GC–MS analysis (Sangmanee and Hongpattarakere,
2014). Dharni et al. (2014) reported prevention of spore germination
of F. oxysporum with 100 μg/mL of 2,4 DTBP. The proposed mechanism of
antifungal activity as described by this group was the inhibition of spore
germination by preventing the emergence of normal germ tube leading
to abnormal swelling and branching of hyphae. It has been demonstrated
that 2,4 DTBP inhibits the assembly of spindle microtubules and disturbs
the chromosomal alignment at the metaphase plate and microtubule–
kinetochore interactions, causing chromatid loss, which may reduce the
mycelia growth and the germination of spores. Our study confirms the
earlier reports as well as describes the efficiency of this compound
in prevention of A. niger and P. chrysogenum growth along with Fig. 3. DPPH scavenging activity of different concentrations of B1 fraction and pure 2,4
growth inhibition of F. oxysporum and points towards the possibility di-tert-butyl phenol (p b 0.05).
48 K.K. Varsha et al. / International Journal of Food Microbiology 211 (2015) 44–50

from dried Scolopendra subspinipes mutilans (Scolopendridae), a In a study by Choi et al. (2013), sweet potato extract is reported to
centipede that has been utilized as a traditional Chinese and Korean have protective effect against hydrogen peroxide-induced oxidative
medicine for a variety of diseases, such as spasm, seizures, poisonous stress and cytotoxicity on the pheochromocytoma cell line (PC12). The
nodules, childhood convulsions, diphtheria and tetanus. The antioxidant active component in this extract was purified and identified as 2,4
effects displayed by this compound include prevention of LDL oxidation DTBP and administered to check the effects on amyloid-beta peptide
and DPPH scavenging ability (Yoon et al., 2006). In a study by Choi et al. (Aβ1–42) induced learning and memory impairment in mice and
(2011), the ethanol extract of pomegranate had antioxidant and neu- shown to have a protective effect against Aβ1–42 by decreasing neuronal
ronal protective effects and diminished hydrogen peroxide induced cell damage and increased spontaneous alteration behavior in mice
oxidative stress in PC-12 cells. The protective effect against learning when provided a diet supplemented with 2,4 DTBP. A previous study
and memory impairment induced by Aβ1–42 is due to the antioxidant (Chen et al., 2007), reported the effect of six day old extracts of
effect of 2,4 DTBP where ABTS radical assay was followed to confirm fermented milk product kefir reduced the growth of human mammary
the antioxidant activity of 2,4 DTBP. The disease prevention roles of cancer cells (MCF-7) in a dose-dependent manner, showing 29% inhibi-
fruits, vegetables and red wine have been attributed, in part, to the tion of proliferation at a concentration of 0.63% without any anti-
antioxidant properties of their constituent polyphenols (vitamins E proliferative effect against normal human mammary epithelial cells
and C, and the carotenoids) and the antioxidant property of plant (HMECs). Even though it has certain degree of cytotoxicity in normal
materials was found to increase with an increase in the amount of cell lines, studies show that 2,4 DTBP is a common ingredient in many
total phenols (Rice-Evans et al., 1997; Velioglu et al., 1998). Apart medicinal preparations and can be helpful during disease conditions.
from the excellent antifungal activity, the antioxidant activity assays The presence of 2,4 DTBP in the cell free supernatant (CFS) of LAB
revealed the potential of this fraction to act as reducing agent and may be one of the reasons behind their many health benefits including
thus inhibit the oxidation of molecules as well produce free radicals. anticancer properties. LAB represent a major component of the human
Based on this study and also the previous studies 2,4 DTBP has been microbiota and are common starter cultures in food fermentations.
shown to be a natural antioxidant with very good protective effect Since LAB are normal part of our daily diet, their intake may carry
against oxidative damage. therapeutic effects as they are source of natural antioxidants and may
be particularly useful when long term treatment is required.
3.4. Assay of cytotoxicity to cancer cell lines
3.5. Biocontrol of fungi on wheat grains
With the crude supernatant no cytotoxic activity was observed.
However, 2,4 DTBP purified from the culture medium displayed
In all vials of wheat grains treated with B1 fraction (approximately
remarkable cytotoxic activity against HeLa cell lines with an IC50 value
2 mg/25 mL of 2,4 DTBP) (Fig. 5) fungal mycelial growth was completely
of 10 μg/mL. IC50 against MCF-7 was achieved with 16 μg/mL. Against
prevented. The inoculated mycelia plug failed to start growing and dried
the normal cell line H9c2, a higher amount of 19 μg/mL was required
off. In the control vials, fungal mycelia started growing on the third day
to bring about IC50. The values are represented in Fig. 4. The cytotoxic
of inoculation and by seven days infested the grain leading to spoilage.
property of 2,4 DTBP has been previously reported by different research
F. chlamydosporum and F. oxysporum formed a mat on the grain surface
groups. Malek et al. (2009) purified 2,4 DTBP from the leaves of Pereskia
(Fig. 5). As microbial contaminants are found mostly on the grain surface
bleo (Kunth) where it represents a major component and found it to
(Oliveira et al., 2014), the compounds produced by LAB may control their
have a cytotoxic effect on the human cancer cell lines KB, MCF-7,
attack in a natural way. Due to the GRAS status, it is possible to obtain
CasKi, HCT 116, A549 and in normal human cell line MRC-5. However,
regulatory approval for the compounds produced by LAB because of
HeLa and H9c2 cell lines were not included there and were covered in
their origin and incorporation into food products. Biocontrol activity of
this study. In the abovementioned study, IC50 against MCF-7 was obtain-
2,4 DTBP produced by Flavobacterium johsoniae strain GSE09 against
ed with 5.75 μg/mL and in MRC-5 with 20 μg/mL. At the same time
Phytophthora capsici in pepper was reported by Sang and Kim (2012).
29 μg/mL was required in HCT 116 cell line and below 6 μg/mL was
Growth of the pathogen was reduced to half with 100 μg/mL of this
enough to bring about IC50 in other cell lines used. Leaves of Kunth are
bioactive compound and the study demonstrated that radicle formation
used traditionally in Malaysia for the treatment of cancer, diabetes,
in pepper seeds was not affected by 2,4 DTBP treatment which
high blood pressure and diseases associated with rheumatism and in-
protected against the infection by P. capsici. These authors also showed
flammation as well as remedy for the relief of gastric pain, ulcers and
that pepper seeds could germinate after treatment with 2,4 DTBP.
for revitalizing the body. Presence of this compound in plants is not
Recently antimicrobial peptides from LAB were used to prevent
yet explained biogenetically but the same authors (Malek et al., 2009)
fungal attack on wheat grains along with maintaining the health of
reported the presence of 2,4 DTBP in the termite-associated
grains under storage (Gupta and Srivastava, 2014). Thus, research by
fungus Termitomyces heimi and the edible fungus Hericium erinaceus.
various groups has shown that 2,4 DTBP is not an unusual compound
and occurs naturally in fruits, seeds and also is produced by microorgan-
isms. LAB producing this compound can be explored and developed
as starter cultures for fermented food and may also be used to prevent
fungal contamination of food.

4. Conclusion

Antifungal and antioxidant property of 2,4 DTBP, obtained from a

Lactococcus sp. demonstrate the potential of this compound for
development as a food additive which may improve the food safety
as well as having health promoting characteristics. This would be of
particular interest in grain or cereal based food products where fungal
contamination is a major cause of food spoilage. Cytotoxicity against
cancer cell lines revealed the diverse biological potential possessed by
this bioactive and provides evidence on the therapeutic potential of
Fig. 4. IC50 of 2,4 di-tert-butyl phenol in μg/mL against different cell lines (p b 0.05). lactic acid bacteria.
 K.K. Varsha et al. / International Journal of Food Microbiology 211 (2015) 44–50 49

Fig. 5. Biocontrol of fungi on wheat grains (14th day). Antifungal activity of B1 fraction containing 2,4 di-tert-butyl phenol against A. niger (A), F. chlamydosporum (B), F. moniliforme (C),
along with untreated control (C) on right side.

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