The effect of ozone exposure on spore viability

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Fresh fruits and vegetables constitute a major part of human diet. Spoilage and loss of fresh fruits and vegetables will affects the food industry commercially and can have impacts on human health such as allergic reactions, production of mycotoxins or causing fungal infection (mycosis). Loss rate of fresh fruits and vegetable is particularly high (approximately 50%) in under-developed countries and it is mainly due to plant pathogens . Around 25% of fresh food is lost in storage after harvesting due to spoilage by microbes . Contamination by fungus is one of the most common reasons for spoilage of stored fresh products. The risk of contamination is high when the products are stored for long periods since the physiological changes will encourage plant-pathogens . Penicillum is one of the most notorious fungi known to cause food spoilage . Normally these fungi grow widely in organic materials. It is not surprising that Penicillum species occurs on stored fruits and vegetables; however, they have been also isolated from meat, fish and dairy products. The asexual spores of Penicillium are dry, so their distribution through air is easy and therefore the food products will be contaminated more extensively by this fungus .

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A collection of chemical fungicides are used to preserve fresh produce. However, a number of reasons such as need to reduce the use of chemical compounds in food materials, anxiety about environmental pollution, resistance of microbes against chemicals and fear about potential health problems caused by fungicides have encouraged the development of alternative methods to assist the storage fresh produce .

1.2 Fungal spoilage

Fungi are eukaryotic organisms often associated with spoilage of variety of food materials. Fungal spoilage is an important issue in food industry . Various species such as Aspergillus, Penicillum, and Cladosporium are involved in food spoilage . Penicillum is one of the widespread fungal species isolated from food products. In addition to economic loss they can cause potential health risk on humans such as allergic reactions and mycotoxin production . Most of the food products especially fruits and vegetables contains high moisture, which are highly perishable by microbes . The most common food spoilages are loss of moisture and microbial spoilage. Spoilage of bakery products by fungi represents 90% of total microbial contamination . The fungal diversity and their dominance in food products are depends upon biotic and abiotic factors such as water potential (aw), temperature and competitive microbes . Normally food materials are preserved from fungal spoilage by adding weak organic acid such as propionic acid, benzoic and sorbic acid. Recently pressure has been increased from consumers and legislation to decrease the amount of preservatives in food products, but this may lead to reduction in shelf-life of food products .

Moulds can produce number of enzymes during their growth which may give rise to change in flavours of food materials. The flavour change is due the transformation of 2,4,6-trichlorophenol to tricholroanisol (TCA) by Aspergillus, Penicillum and other species of fungi. Fungal species can also produce viable compounds such as dimethyldisulphaide and geosmin which can affect the quality of food and beverage materials . There is an increasing knowledge and understanding of role played by fungi in food spoilage especially after the discovery of mycotoxin.

Table 1.2: Fungi associated with spoilage of food

Type of Food

Spoilage Fungus

Cream

Ggeotrichum candidum

Butter

Cladosporium butyric

Palm oil

Paecilomyces varioti

Oats

Aspergillus restrictus

Barley

Monilia acremonium

Beef

Penicillium expansum

Bacon

Aspergillus spp., penicillium spp., Mucor spp.

Frozen lamb

Cladosporium pannorum

Fermented sausage, salami, poultry meat

Aspergillus spp.

Sweet potato, radish

penicillium spp.

Source: Food and Beverage Mycology by Larry R. Beuchat (1987)

1.3. Health issues associated with fungal Mycotoxins

Food spoilage by moulds is one of the main problems faced by the food industry. Normally these problems arise when the food products are stored for long time periods . Apart from food spoilage, a number of moulds are famous for their mycotoxin productions. Mycotoxins are chemical compounds secreted by certain filamentous fungi such as Penicillum and they are well known for their immunosuppressive actions . Mycotoxins are secondary metabolic products of fungi which are produced in food as a result of their growth. It is toxic to higher vertebrates and animals causing mycotoxicosis. The mycotoxin enters the human body mainly by the ingestion of Penicillum contaminated plant based food or residues containing animal based food and this contamination could affect the functioning of liver and kidney (Sweeney and Dobson 1998). Quite a few mycotoxins are well known for their antibiotic activity as well, antibiotics such as penicillin have the antibacterial activity .

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Different types of mycotoxins are identified, some of them are neurotoxins, while others will interfere with the protein metabolism lead to immunodeficiency . Among many mycotoxins discovered, some of them are carcinogenic such as aflatoxins and some of them are mutagenic . Many of the mycotoxins are attracting attention because of their association with food and feed and their toxic effects . The mycotoxin problems became a multidisciplinary issue involving biologists and farmers . There are more than 400 mycotoxins are known today and number is increasing rapidly. In this aflatoxins being best known and most intensively researched mycotoxins in the world . Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans. The modern mycotoxicology began with the discovery of aflatoxins after the loss of many livestock in England in 1961. Aflatoxins are broad class of molecules includes Aflatoxin B1, B2, G1, G2 M1and M2.

Fig. 1.3: structural formula of different Aflatoxins

Available at: http://www.food-info.net/uk/tox/afla.htm

The knowledge and understanding of role played by moulds in food spoilage has considerably increased from last 10-20 years after the discovery of mycotoxins. The progress in the prevention of spoilage caused by moulds increased after the international agreements on taxonomy and analytical methods for food born moulds. This agreement led to the discovery of specific groups which are responsible for the spoilage of each kind of food .

Table 1.3: common mycotoxins and associated fungi which contaminating food

Source: Mycotoxins in the food chain: a look at their impact on immunological responses, by Raghubir p. Sharma,

Available at: http://en.engormix.com/MA-mycotoxins/articles/mycotoxins-food-chain-look_290.htm

1.4 Penicillium- a common food contaminant

Fig. 1.4: schematic diagram of Penicillium

Source: Penicillium and acremonium By John F. Peberdy (1987), page no.12

The generic name Penicillium was coined by Link (1809), which means small brush in Latin. The noble identification of Penicillium species were started by Diercks and Thom 1901 . Penicillium has vegetative myceliums that consist of hyphae. The conidial apparatus is divided conidiophores, bearing branches of phialides ends with conidia (asexual spores), in characteristic symmetric or asymmetric broom like structure . Penicillium reproduces asexually, but they are unable to sporulate when submerged. They will reproduce easily when the hyphae comes out into gas phase .

It is noticed that every Penicillium species have not exactly same reproduction method. The conidia development of Penicillium is similar to Aspergillus, but the morphological arrangement of conidiophores is different . Colonies of Penicillium grow slowly, but they rapidly assume a greenish blue colour because of development abundant conidia . Spore production is not much affected by changes in oxygen, CO2, and water level, but it is very much depended on physiological environment at hyphal surface. Many Penicillium species are xerophilic in nature i.e. they are capable of growing at water potential (aw) ≤ 0.85. This nature make them major food spoilage organism which grows on cereal, bread, jam, ham, and fruits . The asexual spores of Penicillium are dry and they can rise above the static level of air adjacent to slid objects , so their distribution through air is easy and therefore the products will be contaminated more extensively by this fungus . So it became necessary to control the spread of spoilage by fungal spores effectively and therefore important to try and find a method to reduce potential food spoilage.

1.5 Different control methods for food spoilage

Infection of Penicillium (and other moulds) normally occurs during storage, transport, commercialisation or after at consumers home. Crops should be handled carefully to avoid the pathogens entry from the environment. Different methods are employed to control the food spoilage. They can broadly classify into physical method and chemical methods.

Physical method includes the temperature treatment, controlled atmosphere, light and water potential. Control of sporulation with temperature treatment has studied earlier. Increase in temperature has a general effect on enzyme and chemical activities of fungi. Temperature change may directly affect the synthesis of vitamins, amino acids and other metabolites . Hot water can also resist the mould growth, but it is very much depended upon the food and fruit type. The temperature range should be decided accurately to prevent the fungal growth and prevent fruit damage as well. The sporulation in many fungi is unaffected by light, but it largely influence their other metabolic processes. Some of fungi required light to initiate sporulation. By considering these factors controlled light can be used for reducing fungal growth to an extent . UV and ionizing radiations are used to control the fungal growth . Although many of fungal species are xerophilic in nature, at very low water potential (aw <0.65) they can't survive . Another option to increase the shelf-life of food is modified atmosphere packing (MAP) and storage in controlled atmosphere (CA). Hoogerwerf et al. (2002) observed the reduction of fungal development under controlled use of oxygen and carbon dioxide.

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Wide ranges of chemicals are used to prevent the fungal food spoilage. Natural Chemical compounds produced by the plants have fungicidal activity. Cinnamaldehyde, a compound in cinnamon has fungicidal activity including Penicillium . Nilson and Rios (2000) showed that the active compounds from the mustard oil have fungicidal activity. In traditional treatment 'iprodione' is used as an antifungal agent to control the disease of fruits and vegitables . Ammonium molybdate has a strong inhibitory effect on Rhizopus which causes lesions in apple. Vapours of short chain organic acid such as acetic acid and formic acid can control the decay of cherry. Propionic acid and its salts used as an antifungal agent in baking industry . Natamycin, a compound isolated from the streptomycetes has a broad spectrum inhibitory effect on fungi and it has more lasting antifungal activity (Samson, 2006).

Several antifungal additives are legally permitted to use in food industry. The use of chemicals to avoid the spoilage of food material is of great interest in food industry but there are limitations in using these synthetic fungicides. There are health issues to reduce the use of chemicals, particularly after harvest. There is great concern on their effect on environment and residual chemicals in food. These problems encouraged the rapid development of alternative approaches. Recent works attract attention to the potential use of ozone to reduce the fungal spoilage of fruits, vegetables and other food materials in storage (Antony-Babu and Singleton, 2009, Das et al., 2006, Tzortzakis et al., 2007, Hildebrand et al., 2008)

1.6 Ozone for controlling of food spoilage

Several methods have been proposed to improve shelf-life during cold storage. Recent studies on ozone highlights its potential application to curb microbial spoilage of food. Ozone (O3) is a toxic air pollutant which is both beneficial and harmful to biosphere on earth. Stratospheric ozone shields harmful UV rays from entering the earth surface. The phytotoxicity of ozone discovered in late 1950s encouraged widespread studies on effect of ozone on cell growth and development . In 1997, ozone was recognized as being safe (GRAS) for food applications in U.S. After this an interest in application of ozone has increased.

1.7 Why use ozone as sterilising agent?

Ozone is the one of most powerful oxidants which destroys the microorganisms by the progressive oxidation of vital cellular compounds such as cell wall and nucleotides and leaves no residues . It can also oxidise polysaturated fatty acids and amino acids of enzymes and proteins into shorter peptides. Ozone degradation of cell wall results in leakage of cytoplasm . Ozone is a tri-atomic form (allotrope) of oxygen which reacts with many cell components and generates active oxygen species (AOS). These AOS mimic the signalling pathways of cell hypersensitive response .

Fig 1.7: ozone molecule formula

Source: http://www.globalwarmingart.com/images/0/0a/Ozone_Molecule_Formula.png

http://commons.wikimedia.org/wiki/File:Ozone-CRC-MW-3D-balls.png

The ozone is formed by the rearrangement of oxygen atoms. When high-voltage electricity passes through the gas, the single oxygen (O) atom rapidly combines with available oxygen (O2) molecules to form ozone (O3) . Commercially, UV based ozone generators are available and they are much more efficient and lower cost . Ozone is a pungent, naturally occurring gas which is highly instable in water. It decomposes continuously to oxygen, but slowly. It has long history of use in disinfection of municipal water, dairy, bottled drinking water, swine effluent, hospital water systems and equipments .

The factors such as temperature, pressure and pH value affects the reactivity of ozone. The temperature difference is directly proportional to the rate of destruction of microorganisms (Van't Hoff- Arrhenius theory) . In the case of ozone, as temperature increases ozone becomes less stable, and the ozone reaction rate with substrate increases gradually. Ozone is more stable in low pH and can deactivate microorganisms through reaction with cellular components .

1.8 Bio-chemical reactions in cells during ozone exposure

The molecular ozone reactions are selective and limited to unsaturated aromatic and aliphatic organic compounds. Oxidation of sulfhydryl group which is present in the microbial enzymes will inactivate microorganism and their spores . Reaction of ozone with polysaccharides is a slow process which leads to the breakage of gycosidic bond and the formation of aldehydes and aliphatic acids . Reaction of ozone with primary and secondary aliphatic alcohols may lead to the formation of hydroxy- hydroperoxides, which strongly reacts with hydrocarbons . Ozone reacts with saturated fatty acids slowly and readily with unsaturated fatty acids. But ozone reacts quickly with nucleotide bases, especially thymine and guanine and release carbonate and phosphate ions .

Inactivation of microorganism by ozone is a complex process. This is because ozone attacks numerous cellular constituents including protein, unsaturated lipids, cellular enzymes and nucleic acid in the cytoplasm. Components of cell wall will be oxidised during the initial ozone exposure. An early event which leads to the lesion formation is the loss of semi permeability of plasma membrane. This scenario leads to plasmolysis followed by rapid unregulated cell death . Khadre (2001) reported, that bacterial coat that acts as a primary protective against ozone, can be disrupted by aqueous ozone.

1.9 Aim of the research

This research concentrate on the way in which ozone affects the spore viability of specific fungi isolates (Penicillium species) that causing spoilage of food materials. The aim and objective of this research project will be the following,

Aim: Ozone control of Penicillium isolates which cause the spoilage of food materials

To accomplish this aim the objectives followed were,

1. Isolation, purification and culturing of Penicillium strains

2. Morphological and molecular identification of Penicillium isolates

3. Examined the effect of two levels of ozone concentration (low level 200ppm and high level 400 ppm) on Penicillium isolates

2: Materials and methods

2.1. Source of isolates and maintenance

The two spoilage fungi used for this investigation were originally isolated from spoiled food produce and previously identified as Penicillium species by morphological and molecular analysis (Allan BSc thesis, 2009). The organisms were stored as spore suspension in ¼ Ringer's solutions at 4°C. The cultures were maintained on Potato Dextrose Agar (Oxoid), incubated at 28°C. A common lab contaminant isolated (isolate LC) from the laboratory by keeping Oxoid media plates in incubator at 28°C was also used in this investigation. These three organisms were aseptically sub cultured and transferred in to ¼ Ringer's solution and stored at 4°C for further use.

2.2. Molecular identification of lab contaminant fungi (isolate LC)

2.2.1 DNA Extraction

Freshly cultured fungal colonies were used for the genomic DNA extraction. Fungal mycelium was scraped using a sterile plastic loop and a loop full of fungal biomass was suspended in sterile tubes containing 500µl of eluate solution (Sigma; 128k6094, B6803 (Appendix2). To this suspension 0.5 g sterile glass beads (100µ) were added and placed in a Ribolyser machine (Fast Prep TM FP120, Thermo electron corperation) and run for 45 seconds at a speed of 4.5 ms-1 to rupture mycelia and release the cellular components. The lysates obtained were then centrifuged at maximum speed of 15996.34 rpm2 (Appendorf Centrifuge; Model: 5415C) for 5 minutes. The supernatant were transferred to a fresh sterile 2.5 ml microfuge tube. Agarose gel electrophoresis was carried (section 2.2.2) out to ensure the presence of genomic DNA in the solution.

2.2.2 Gel electrophoresis

Agarose gel electrophoresis was carried out to ensure the presence of genomic DNA after extraction process. 1% (w/v) agarose gel was prepared using 0.5X TBE buffer and ethidium bromide (10mg/ml) (EtBr) was added before solidifying. Once the gel was set, 4µl of DNA sample of each isolate was mixed with loading dye (Fermentas, 10mg/ml.) and transferred to the wells in the gel. A 100 bp DNA ladder plus (Fermentas) was used as a marker. Electrophoresis was carried out at room temperature, 100V for 30 minutes. The gel was viewed and resulting image was photographed using 'Bio-Rad Fluor-S Multi Imager'.

2.2.3 Polymerase Chain Reaction (PCR) (18S rRNA)

Each PCR reaction mixture was prepared using appropriate concentration of reaction components and each PCR reaction mixture had a total volume of 32µl. The PCR reaction components were mixed in the following concentration into PCR tubes .

Table: 2.2.3 (a): PCR Reaction Components

Components/concentration

Volume (µl)

Final concentration

NH4 Buffer (10x)

3.0

1x

DNTPs

2.0

0.5mM

Forward Primer

NS1(10µM)

0.75

0.2Mm

Reverse Primer

FR1(10µM)

0.75

0.2mM

MgCl2 (50µM)

1.5

3.00mM

Taq(2U µl-1 )

1

2.0 U

Sterile distilled water

22

Template DNA

1.0

Total Volume

32

The following primers were used (18S rRNA gene):

Forward primer-NS1: 5'-GTAGTCATATGCTTGTCTC-3'

Reverse primer- FR1: 5'-CTC TCA. ATC TGT CAA TCC TTA TT-3'

The PCR reaction mixture was prepared by mixing 31µl Master mix (see table: B2.2 (a)) and 1µl of template DNA sample. A negative control reaction mixture was prepared by adding 1µl of sterile distilled water instead of template DNA. The positive control was made by adding known fungal DNA (Rhizopus stolonifer) instead of template. This was done in order to identify the PCR was successful.

The gradient thermal cycles and temperature profiles used for 18S rRNA with NS1 and FR1 as primer set are following,

Table: 2.2.3 (b) PCR reaction conditions

PCR Cycle

Time

Temperature gradient

Number of cycles

Denaturation

5 ninutes

94

1

Annealing

Step1:- 30 seconds

Step 2:- 46seconds

Step 3:- 3minutes

94

47

72

35

Extension

10 minutes

72

1

An agarose gel electrophoresis was carried out as mentioned earlier (see: section 2.2.2) to ensure the amplification of DNA fragments after PCR.

2.2.4 Purification of PCR products via Exo-sap

The PCR products were cleaned up using Exo-sap-IT® before sequencing to remove residual nucleotides. For this end 5µl of PCR products were mixed with 1.5µl of Exo-sap in a separate sterile tube. This mixture was incubated at 37°C for 15 minutes followed by 80°C for 15 minutes. All samples were stored in 4°C until sequencing.

2.2.5 DNA sequencing

Sequencing of DNA samples were carried out using BigDyeTM version 3.1 Terminator Cycle Sequencing Kit (PE Applied Biosystems, MA). Samples were completed using 10 ng of DNA for every 100 bp of template DNA and 3.2 pmol of primer. Post-sequencing reactions were purified away from unincorporated dye terminators using Performa DTR spin-columns (EdgeBiosystems, MD). Sequences were determined using an ABI 3730 Sequencer and sequencing carried out by Genevision, Newcastle University, UK.

2.2.6 Identification of fungal isolate LC

The obtained sequences were analysed using a programme FINCH TV version 1.4. The sequences were blast using the search tool BLAST (Basic Local Alignment Search Tool) offered by NCBI (National Centre for Biotechnology Information) (Available at: http://blast.ncbi.nlm.nih.gov/) and the related sequences were found out. These matching sequences were added to the alignment explorer along with the sequences of Penicillium isolates to be identified in MEGA 4.0.2. These sequences were aligned in order to construct a phylogeny tree.

2.3 Preparation of spore suspensions

The three Penicillum species were cultured on potato dextrose agar and incubated at 28°C for five days. The fungal spores were harvested by adding 10 ml of buffer solution (9ml ¼ strength Ringers and 1ml 1% w/v SDS solution (Merck, Germany)) on the surface of the colony and gently scraped using a sterile plastic loop to displace the spores from mycelia. The resulting spore suspensions were transferred to a universal tube (30 ml). The numbers of spores were determined by using a Neubauer haemocytometer (BS 748, Hawksley, UK) under 40X magnification. The concentration of suspension was adjusted to 106 spores/ml. This spore suspension was used to assess the effect of ozone exposure on spore viability i.e. to determine the potential of ozone gas to be used as a method to kill Penicillium spores and hence be used as a disinfection agent in storage areas.

2.4 Ozone fumigation system (used to assess the effect of ozone exposure on viability of Penicillium spores).

Treatments of both control and ozone sides were prepared in a same method in a controlled laminar air flow chamber. 100µl of the previously prepared spore suspension (section 2.3) was added to each sterilised, dried glass slide. Using a sterile plastic loop a smear was made on the slide and the spore suspension dried in a laminar flow chamber. These dried slides were used for ozone exposure experiments.

The ozone fumigation system consisted of ozone generator (O3 TEXâ„¢, UK), ozone controlling pump (GFG.CORP.USA, model: p101-CD), ozone monitor (APi INC. CA, model 450) and a ozone chamber (see fig2.4 below). The ozone generator uses electricity to produce ozone gas from existing oxygen molecules from air and this ozone gas was transferred to the ozone chamber. A controller in the ozone generator controls the concentration inside the chamber. The ozone monitor draws air from the ozone chamber and determines the concentration of ozone gas in the chamber in parts per million.

Fig.2.4: Image shows the production and transport of oxygen gas in an ozone fumigation system. Ozone generator produces ozone gas from the clean air by electrical discharge method and the ozone gas is then transferred into an airtight ozone chamber (containing slides with spore fungal spore preparation). The concentration of ozone gas to be transferred to the chamber was adjusted by a controller in the generator. The ozone monitor connected to the chamber draws the air from the chamber and analyse the ozone concentration in parts per million.

2.5 Effect of ozone treatment on fungal species

2.5.1Estimation of number of viable spores

2.5.1.1 Ozone exposure

The slides on which dried spore suspensions were made were placed in petri dishes and the dishes then placed in the ozone chamber to allow the ozone exposure. Two levels of ozone was used for this investigation,

1. Low level exposure (200ppm for five minutes)

2. High level exposure (400ppm for one minute)

2.5.1.2 Serial dilution and plating method to assess the effect of ozone exposure on spore viability

Immediately after ozone exposure spore suspensions of ozonated and control spores were made by dipping respective slides in 10 ml of 1/4Ringer's solution in a falcon tube. Serial dilution of each spore sample was made by transferring 1ml spore suspension in to 9ml Ringer's solution. 100µl of diluted samples from each tube were inoculated into plates containing Potato dextrose Agar media and spread using sterile L-shaped plastic speeder. These plates were kept in an incubator at 28°C and growth observed after five days. Spore viability was determined by the number of discrete colonies developing on agar and it was assumed that each colony was derived from a single spore. Results were calculated as the number of spores per ml of Ringers solution.

2.5.2 Effect of ozone exposure on spore viability after storage of spores in buffer

In preliminary experiments it was observed that spores stored in buffer at 4°C had a different viability level to spores that were sampled prior to storage and it was thought that perhaps ozone exposure may decrease the viability of stored spores. To investigate the viability of spores after storage, both ozonated and control spore suspensions were stored in Falcon tube congaing 1/4 Ringer's solution at 4°C. These spore suspensions were inoculated onto the Potato Dextrose Agar media on the 7th and 14th days after ozone exposure. Spore viability was determined as previous.

2.6 Statistical evaluation

All the experiments the assays were performed in triplicate. Standard deviation and standard error of mean of data were determined by using data analysis functions of Microsoft excel. The significant differences between the obtained results were computed by analysis of variance (ANOVA) offered by Minitab 15. All the data were presented in graphs with standard mean of errors. Differences between means were calculated using least significant difference (Minitab 15).

3. Results

3.1. Morphological identification of fungal isolate LC

The colonies of isolate LC was observed on third and seventh day after inoculation. The colonies were white at on the 3rd day and gradually they turned to blue green colour with white border on the 7th day. The opposite side of the colonies were yellow in colour. The colonies appeared to be velvety and grew rapidly. The colonies were stained and observed through light microscope to identify the cellular morphology. The spores (conidia) were held to hyphae by structure known as metulae. All these morphological characters of isolates were identical to that of Penicillium species.

Fig 3.1 (a): shows the growth of isolate LC (lab contaminant) on potato dextrose agar media seven days after incubation at 28°C.

Fig. 3.1 (b): Microscopic image of isolate LC stained with crystal violet

3.2 Molecular identification of fungal isolate LC

Fig.3.2. (a): Photograph 'A' shows the gel electrophoresis of extracted genomic DNA and photograph 'B' is the gel electrophoresis of PCR products. The electrophoresis is carried out using a 100bp DNA ladder. 18 sRNA sequences amplified using PCR and they are cleaned using Exo-sap for sequencing. The expected size of PCR products were 1000bp.

The sequence of isolate LC were analysed using FINCH TV and Mega 4 and identification made in BLAST.

Isolate LC

Penicillium roqueforti strain ATCC 10...

Penicillium expansum strain PSF1 18S...

Penicillium brevicompactum strain ALI...

Penicillium griseofulvum strain 3.519...

Penicillium decumbens strain ML-017 1...

56

47

65

0.0005

Fig.3.2 (b): phylogeny tree created using neighbour joining bootstrap test on Mega 4 and the tree shows that isolate LC is 100% penicillium species

3.3. Effect of ozone treatment on the viability of fungal spores

3.3.1 Visible growth

All three isolates showed reduced spore viability after ozone treatment. Examples are shown in Fig. 3.3.1 (a); 3.3.1 (b).

Fig. 3.3.1(a): These photos shows the plates inoculated with control(non ozone exposed spores) after 5 days incubation

Fig. 3.3.1 (b): These photos shows the plates inoculated with ozonated spores (200 ppm for 5 minutes.): a clear decrease in colonies formed is observed compared to non-ozone exposed spores, shows significant decrease.

3.3.2: Effect of ozone to decrease spore viability

Both control and ozonated spore suspension were inoculated into potato dextrose agar media and observed their growth after 5 days. The reduction in spore viability has shown in Fig.3.3.2 and values are expressed in percentage. Ozone treatment of 200 ppm for five minutes and 400 ppm for one minutes showed significant (P<0.05) reduction of spore viability. However 200 ppm ozone concentration could reduce the spore survival more than that of 400 ppm.

Fig.3.3.2: The effect of different ozone exposure on spores of different species of Penicillum. Three isolates were cultured in similar conditions and they exposed to two different levels of ozone. A high level concentration 400 ppm for 1 minute and low level concentration 200 ppm for five minutes. All ozonated spores and control spores were cultured in potato dextrose agar media separately and their growth were observed after five days

The spores of the different Penicillium isolates showed varying levels of resistance to ozone exposure. The most sensitive isolate was LC (lab contaminant) which showed either 86.1% or 69.04 % reduction (p < 0.05) in spore viability when exposed to 200 ppm ozone for 5 mins or 400 ppm ozone for 1 minute respectively. But isolate 2.1 fond to be some more resistant to ozone exposure compared to isolates 7.1 & LC. It reduced the spore viability only 54.8 % and 32 % (p < 0.05) when exposed 200 ppm ozone for 5 mins and 400 ppm ozone for 1 minute respectively. Ozone exposure significantly reduced (p < 0.05) the spore viability of isolate 7.1 to 23.56 % and 42.29 % when exposed to 200 ppm for 5 minutes and 400 ppm for 1 minute respectively.

3.3.3 Effect of ozone on spore viability through time

To investigate the viability of spores after storage, both ozonated and control spore suspensions were stored and later inoculated onto the potato dextrose agar media on the 7th and 14th days after ozone exposure. It is observed the viability of both control and ozonated spores reduced on seventh day, but at the end of 14th day the viability of ozonated spores remained the same. This phenomenon occurred with all three penicillium isolates used for this investigation.

3.3.3.1: isolate LC

In this experiment Penicillium isolate SX spores were exposed to ozone (200 ppm for 5 min and 400 ppm for 1 min) or not exposed (control) and then stored in buffer at 4°C for up to 14 days before assessing their viability. The experiment initiated with 4.562 log10 viable spores/ml for control sample and 3.95 log10 (200 ppm) & 3.73 log10 (400 ppm) viable spores/ml for ozonated sample respectively. The viability of control spores slowly decreased with time and after 14 days storage no spores appeared to grow under the experimental conditions used. In contrast, the viability of spores exposed to different ozone levels remained at about the same level during storage and the numbers of viable ozone treated spores in buffer after 14 days were significantly higher (P< 0.5) than the numbers of viable control spores.

Fig. 3.3.3.1: The extended effect of ozone exposure on spore viability through time on isolate LC. Two levels of ozone concentration were used- low level 200ppm for 5 minute and high level 400ppm for 1 minute. Both ozonated and control spore suspensions were stored at 4°C up to 14 days and they were inoculated on potato dextrose agar media on 0th, 7th and 14th day. The growth was observed five days after inoculation and recorded. The error bars in the graph denotes the standard error of mean (SEM).

3.3.3.2: isolate 2.1

In this experiment Penicillium isolate 2.1 spores were exposed to ozone (200 ppm for 5 min and 400 ppm for 1 min) or not exposed (control) and then stored in buffer at 4°C for up to 14 days before assessing their viability. The experiment initiated with 4.34 log10 viable spores/ml for control sample and 3.36 log10 (200 ppm) & 3.9 log10 (400 ppm) viable spores/ml for ozonated sample respectively. The viability of control spores slowly decreased with time and after 14 days storage no spores appeared to grow under the experimental conditions used. In contrast, the viability of spores exposed to different ozone levels remained at about the same in buffer after 14 days of storage.

Fig. 3.3.3.2: The extended effect of ozone exposure on spore viability through time on isolate 2.1. Two levels of ozone concentration were used- low level 200ppm for 5 minute and high level 400ppm for 1 minute. Both ozonated and control spore suspensions were stored at 4°C up to 14 days and they were inoculated on potato dextrose agar media on 0th, 7th and 14th day. The growth was observed five days after inoculation and recorded. The error bars in the graph denotes the standard error of mean (SEM).

3.3.3.3: isolate 7.1

In this experiment Penicillium isolate 7.1 spores were exposed to ozone (200 ppm for 5 min and 400 ppm for 1 min) or not exposed (control) and then stored in buffer at 4°C for up to 14 days before assessing their viability. The experiment initiated with 4.34 log10 viable spores/ml for control sample and 3.36 log10 (200 ppm) & 3.76 log10 (400 ppm) viable spores/ml for ozonated sample respectively. The viability of control spores slowly decreased with time and after 14 days storage no spores appeared to grow under the experimental conditions used. In contrast, the viability of spores exposed to different ozone levels remained at about the same level during storage and the numbers of viable ozone treated spores in buffer after 14 days were significantly higher (P< 0.5) than the numbers of viable control spores.

Fig.3.3.3.3: The extended effect of ozone exposure on spore viability through time on isolate 7.1. Two levels of ozone concentration were used- low level 200ppm for 5 minute and high level 400ppm for 1 minute. Both ozonated and control spore suspensions were stored at 4°C up to 14 days and they were inoculated on potato dextrose agar media on 0th, 7th and 14th day. The growth was observed five days after inoculation and recorded. The error bars in the graph denotes the standard error of mean (SEM).

4. Discussion

4.1 Use of ozone to control food spoilage in storage

Fungal contamination is one of the most common reasons for spoilage of stored fresh products. The risk of contamination is high when the products are stored for long periods since the physiological changes will encourage plant-pathogens . A collection of chemical fungicides are used to preserve fresh produce. However, a number of reasons such as need to reduce the use of chemical compounds in food materials, anxiety about the environmental pollution, resistance of microbes against chemicals and fear about the health problems have encouraged the development of alternative method for fresh produce . Several methods have been proposed to improve shelf-life during cold storage. Recent studies on ozone highlights its potential application to curb microbial spoilage of food. Ozone control of microbes is the one method that has been suggested by many researchers . However there has been little knowledge on the effect of ozone on micro-organisms, particularly fungi.

This study about the ozone control of Penicillium isolates showed that ozone could stop spore germination. The significant positive result of this experiment shows that ozone treatment would prevent the nesting of Penicillium on food surface. Outcome of this experiment shows that ozone treatment technology could be used to reduce the food spoilage in storage chambers.

4.2. Identification of fungal isolate

Molecular identification of fungal species is not well established and this is because fungal taxonomy is currently constrained by the rules of botany . Thus fungal species are mostly identified by their morphological characters. Identification by morphological characters is extremely difficult and it requires many years of expertise. The morphological identification was especially not easy in the case of Penicillium . Due to the complications in the morphological identification of Penicillium a PCR based molecular identification had done via 18S rRNA gene sequence data.

In this investigation 18S rRNA showed that the organism isolated was Penicillium, but they could not be identified to species level. 18S rRNA stands for ribosomal RNA of small subunit of ribosome and it is widely used in molecular analysis for reconstructing the evolutionary history of organisms . 18S rRNA sequences are approximately 1,805 bp in length but on average 1-5% of the sites are variable . When it discovered, the 18S rRNA used for large-scale phylogenetic studies but it became difficult during recent years due to increased number of taxa . The taxonomic resolution of 18S rRNA might be insufficient for identifying the fungal species, but it will provide the information about the fungal diversity and dynamics of its related species .

4.3 Effect of ozone on spore's viability

The results shows, the viability of ozonated spores were significantly reduced for all Penicillium isolates when compared to the control samples. Hildebrand et al (2008) and Palou et al (2002) also found in their experiments that the viability of fungal spores were reduced significantly under ozone. The fungal spores contains about 45% proteins, 20%carbohydrates, and 35% hydrocarbonlike compounds . Oxidation reaction of ozone with polysaccharides is a slow process which leads to the breakage of gycosidic bond and the formation of aldehydes and aliphatic acids (Bablon G, 1991). But ozone reacts quickly with nucleotide bases, especially thymine and guanine and release carbonate and phosphate ions (Shinriki et al., 1981). Ozone attacks numerous cellular constituents including, unsaturated lipids, cellular enzymes and nucleic acids in the cytoplasm. Components of cell wall will be oxidised during the initial ozone exposure. An early event which leads to the lesion formation is the loss of semipermeability of plasma membrane. This scenario leads to plasmolysis followed by rapid unregulated cell death (Eva J. Pell, 1997).

The results showed low level ozone exposure more significance in decreasing spore viability than the high level. In previous studies about ozone has justified that the low level ozone exposure for longer duration was more effective than high level exposure for short time (Khadre, 2001, Antony-Babu and Singleton, 2009). In general the effect of ozone exposure is truly depended on type of pathogen, type of material to be treated, storage temperature, and the right concentration of ozone and duration of exposure.

4.4 How ozone exposure affects the spore viability after storage of spores in buffer

An experiment is carried out to investigate if ozone treated spores survived less over time after treatment than control spores. The result showed viability of control spores slowly decreased with time and after 14 days storage no spores appeared to grow under the experimental conditions used. In contrast, the viability of spores exposed to different ozone levels reduced initially and remained at almost the same level during storage in buffer (see Fig:3.3.3.1). This is important for using ozone to treat food storage areas. Further research is needed to confirm this effect.

4.5 How Fungal isolates varied in their response to different ozone concentration

Fig.3.3.2 shows the spores viability of different Penicillium isolates. Here all the isolates behaved differently towards the ozone exposure. Each isolates had shown different levels of inhibitions in different ozone concentration and time of exposure. The recent studies on different strains fungus Penicillium expansum, Mucor piriformis, monilinia fructicolan showed that they behaved differently in exposure to ozone. The 18S rRNA sequencing has showed that the isolate LC appears to be similar to isolate 2.1, but they had shown different behaviour when exposed to ozone exposure. Similar results were reported in Aspergillus nidulans and Aspergillus ochraceus by researchers . Although the research supports the application of ozone in controlling food spoilage, it is truly depended on factors such as on type of pathogen, type of material to be treated, storage temperature, and the right concentration of ozone and duration of exposure.

4.6 How could ozone revolutionize the future industry?

This experiment has proved that the ozone can significantly reduce the spore viability of Penicillium isolates. Many researchers have already mentioned the industrial application of ozone. The bactericidal effects of ozone have been well documented , but in the case of fungi more study has to be carried out. Since ozone is a safe and powerful disinfectant, it can be used to control the biological growth of organisms in equipments and products in the food industry. Ozone can act as a preservative for fruits, vegetables, fish and meat .

The food industry is more interested in the applications of ozone in decontamination of water, storage chambers and surface sterilization of processing equipments to decrease the chemicals and biological oxygen demand (BOD) . If these results confirm the success of ozone application, this technology can be successfully employed as an eco-friendly method for control of wide range of microbes.

5. Conclusion

Ozone is a potent purifier with promising application in the food industry. Ozone is effective against a wide spectrum of microorganisms including Penicillium and it is more eco-friendly than any fungicides . Stability of ozone in chilling temperatures, easy production and handling constitute attractive savings to industry. Chlorine and other fungicides which are currently used in the industry can successfully decontaminate the process environment, equipment surface and surface of fruits and vegetables, but the concerns about the health and safety of these chemicals are increasing.

In this experiment ozone was significantly reduced the spore viability of Penicillim isolates and the reduction is more significant under low level ozone exposure for longer period. However the reduction of spore viability depends on factors such as type of pathogen, type of material to be treated, storage temperature, and the right concentration of ozone and duration of exposure. In conclusion, ozone is the most effective and economical choice for controlling the fungal spore viability, but further researches are needed explore the application of ozone in food industry.

6. Acknowledgement

This research project was carried out in Ridley Building 4.0 laboratory, Newcastle University. I would like to express my sincere gratitude to Dr. Ian Singleton for offering me the opportunity to work this project and providing me the supervision and support. I especially want to thank my advisor Dr. Sanjay Antony Babu, for his guidance throughout the project. I would like to extend my gratitude to Roselyn Brown and Miriam Earnshaw for their technical and practical support, and all my laboratory buddies in RB 4.0 made it a convivial place to work. Finally, I would like to take opportunity to thank my parents who provided me a great support and encouragement throughout my studies.