Ozone treatment is one among many existing processes that contribute to the improvement of the safety and quality of food products.

1. Ozone: A Potent Antimicrobial Agent for
Application in Food Industry of Livestock
Origin
Bariya Akshay R.
akshaybariya196@gmail.com
DEPARTMENT OF LIVESTOCK PRODUCTS TECHNOLOGY
COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDARY
1

2. 2
9
8
Advantages and disadvantages
7
6
5
Production and method of application
4
Ozone and its properties
3
History
2
Introduction
1
Working principle
Cost analysis
Applications of ozone in Food Industry of
Livestock Origin
Conclusions
10 Future Prospect

3. Introduction
Currently, the livestock sector is one of the fastest-growing
agricultural subsectors in developing countries and its growth is
driven by the rapidly increasing demand for livestock products.
The globalization of food trade increases the potential to spread
foodborne hazards around the world.
3
(Bhat, 2004; Thornton, 2010)
The pathogenic organisms of public health importance may be
transmitted through contaminated foods comprise bacteria, viruses,
protozoa, trematodes, cestodes, and nematodes.

4. Among these, the most important organisms which cause foodborne
diseases include bacteria and virus-
• Staphylococcus aureus, Bacillus cereus, Campylobacter jejuni, Clostridium
botulinum, Escherichia coli, Listeria monocytogenes, Salmonella typhi and
paratyphi, Vibrio cholera,
• norovirus, etc.
4 (Vemula et al., 2012; Behravesh et al. 2012)
Selection of methods for food decontamination depends on factors
inherent to the product, common pathogenic and spoilage
microorganisms, and cost.
Introduction…..
Routinely used thermal processing may cause some adverse effects
on the nutritional and organoleptic attributes of food.

5. 5
Hypochlorite salts of chlorine having been used for decades to
sanitize utensils and equipment in dairy and other food industries.
(Younis et al., 2019)
Introduction…..
But chlorine compounds have some drawbacks that limit their use in
the food industry because toxic and carcinogenic compounds are
formed in water as a result of chlorination,
• Trihalomethanes as it is formed by the reaction of free chlorine with
soluble organic compounds.
Safety is one of the crucial part of the food industry and consumers
don't want chemical preservatives in their food.

6. 6
(Sharma and Bhattacharya, 2017; Brodowska et al., 2018)
Innovative, easy to implement, and rapid technologies are required
for the modern food industry to meet the never ending demands of
consumers.
Introduction…..
Ozone treatment is one among many existing processes that
contribute to the improvement of the safety and quality of food
products.
It is a chemical method of food decontamination that encompasses
exposing contaminated foodstuffs (fruits, vegetables, beverages,
spices, herbs, meat, fish, etc).

7. 7
Potent sanitizer
Effective against a wide range of
microorganisms
Spontaneously decomposes to oxygen leaving no
residue
Permitted for food use as regulated by the U.S.
FDA and several other countries
Ozone has great potential for enhanced food safety
because of four benefits:
(Predmore et al., 2015)
Introduction…..

8. 8
As a potent antimicrobial substance it has been used to
 Disinfect
• Drinking water, swimming pools, spas, marine aquaria,
municipal water and sewage.
 Treat
• Meat, dairy, fish, as well as egg industries.
(Gonçalves et al., 2016)
Introduction…..

9. History
9
1750-1837: Martinus Van Marum- First person to detect the
ozone through the smell
1840: First time ozone was mentioned by its name by
Schönbein
1856: Thomas Andrews exhibited that ozone was formed
only by oxygen
1863: Soret found that three volumes of oxygen produce two
volumes of ozone
1816 – 1892: Siemens- The first ozone generator was
manufactured in Berlin

10. Ozone and its properties
Ozone (O3) is an allotropic form of oxygen
(O2).
10
In nature ozone is continuously
produced in the stratosphere (at 25–30
km from the Earth surface) by UV
radiation (<183 nm) by splitting an
atmospheric oxygen molecule into two
highly reactive oxygen atoms.
Ozone is a gas that is naturally present in the stratosphere; it is water-
soluble and has a high oxidative power.

11. It is a metastable gas with a temperature-dependent half-life, it can
be stored in liquid form at a temperature below -111.9°C with a
specific weight of 1.571 g/mL.
11
Among oxidant agents, it is the third strongest (E° = +2.076 V), after
fluorine and persulphate.
Oxidizing potential of various reagents
Oxidant agent Oxidizing
Potential (mV)
Reactive power
of oxidation
Fluorine 3.06 2.25
Ozone 2.07 1.52
Hydrogen Peroxide 1.77 1.30
Hypochlorous acid 1.49 1.10
Chlorine gas 1.36 1.00
(Guzel-Seydim et al., 2004; Ozone Solutions, 2007)
Ozone and its properties…..

12. The half-life of ozone molecules in the air is relatively long and
spans for ca. 12 hours; in solutions it depends on the content of
organic matter.
12
Ozone dissolves in water at pH below 7.0; at this level, it does not
react with water and remains present in the form of molecules.
(Holcman and Domoradzki, 2003)
Ozone and its properties…..
The half-life of ozone in distilled water at 20°C is about 20 to 30
min.

13. 13
It has the capacity of absorption of
flavors and strange smells in the
water and helpful in destruction of
organic compounds responsible for
the smell.
In the same way, ozone has a deodorization role of the air.
(Khadre et al., 2001)
Ozone and its properties…..

14. 14
In water also, it is useful for the elimination of heavy metals like iron
and manganese that precipitate quickly in oxide form.
Ozone decomposes in solution in a stepwise fashion, producing in
turn hydroperoxyl (˙HO2), hydroxyl (˙OH), and superoxide (˙O2-)
radicals.
(Cuerda-Correa et al., 2013 and Trombete et al., 2016)
Ozonation also reduces
toxicity of the mycotoxins
and pesticide due to their
molecular degradation.
Ozone and its properties…..

15. Production and method of application
Ozone is formed naturally in the stratosphere in small amounts (0.05
mg/liter) by the action of solar UV irradiation on oxygen.
15
Ozone is generated by the exposure
of air or another gas containing
normal oxygen to a high-energy
source, convert molecules of oxygen
to molecules of ozone.
A small amount of ozone is also formed in the troposphere as a by-product
of photochemical reactions between hydrocarbons, oxygen, and nitrogen
released from automobile industries, forests, and volcanic action.
(Cuerda-Correa et al, 2013)

16. 16
For commercial use ozone can be generated by
Electrical Discharge Method
(Corona Discharge)
Electrochemical Method
(Cold plasma Method)
Radiation Method
Production and method of application….

17. 17
1. Electrical Discharge Method (Corona Discharge)
Oxygen is forced between high voltage plates
to simulate corona discharge. The oxygen is
broken apart and recombines into ozone
The discharged gas mixture normally contains
1%–3% of ozone when dry air is used and 3%–
6% of ozone when pure oxygen gas is used.
(Gonçalves, 2009)
Production and method of application….

18. 18
2. Electrochemical Method (Cold plasma Method)
Design of electrochemical ozone generator with
oxygen cathode
Advantages:
• Use of low-voltage DC current,
• No feed gas preparation, and
reduced equipment size
• Possible generation of ozone at
high concentration
Disadvantages:
• The output is low
• cost is several times more than
that of the corona
(Pushkarev et al., 2016)
Production and method of application….
Electrolytic solution
containing water and a
solution of highly
electronegative anions

19. 19
3. Radiation Method
Radiation method: Oxygen turns into ozone after it is hit with
UV light from a UV generating bulb
Due to poor yields of Ozone, this method has very limited uses.
(Gonçalves, 2009)
Production and method of application….

20. 20
Method of
application of
Ozone
• Gas (gaseous phase)
• Ozonated water (aqueous
phase)
Production and method of application….

21. 21
Ozone in the Aqueous phase
Ozone dissolved in water degenerate much faster than that dissolved
in oxygen or air.
It dissolves in water at pH below 7 and at this pH, it does not react
with water. > 7.5 pH, spontaneous ozone decomposition will take
place.
(Manousaridis et al., 2005; Greene et al., 2012)
Production and method of application….
The rate of ozone degradation to oxygen in impure solutions is
higher than in pure water.

22. 22
(Pirani, 2010; Brodowska et al., 2018)
Ozone solubility- water temperature its solubility
Ozone stability in the aqueous phase is also affected by alkalinity
(concentration of carbonates) as well as organic matter content.
Liquid ozone, in contrast to the gaseous ozone which is relatively
safe, may explode even at very low temperatures.
Production and method of application….

23. 23
Ozone in the gaseous phase
Pure gaseous ozone is relatively safe and kinetically stable under the
pressure of several kPa at room temperature.
The half-life of ozone decreases with an increase of ozone dose in
the gaseous state and with an increase of temperature.
(Dietrich et al., 2000; Brodowska et al., 2018 )
Ozone decay in the gaseous phase may be initiated by some gaseous
substances, including nitrogen oxides (NO, NO2), chlorine, sulfur
dioxide and trioxide, as well as hydrogen sulphide.
Production and method of application….

24. Time line of use of ozone for water and food
24
Time Event
1902 Cologne used to disinfect frozen beef to prolong its
shelf life
1906 Nice city in France installed ozone to disinfect raw
water supplies coming into this community
1918 Used to sanitize swimming pools in the USA
1936 Used to treat shellfish in France.
1942 Used in egg storage rooms and in cheese storage
facilities in the United States
1977 Used to reduce Salmonella in shell eggs in Russia

25. 25
Time Event
1982 Declared GRAS for bottled water in the United States
1997 Expert Panel convened by Electric Power Research Institute
(EPRI) declared ozone GRAS in food processing in the
United States.
2000 Food Additive Petition filed with the FDA
2001 FDA officially approved media containing ozone for use in
the food industry, for direct contact with food products,
including fish, meat, and poultry
2004 FDA issues industrial guidance and recommendations to
processors of apple juice or cider on the use of ozone for
pathogen reduction purposes.
(Gonçalves, 2009; Carletti et al, 2013)

26. Working principle of Ozone
26
Ozone possesses a broad spectrum of antimicrobial activities, which
results from its high reactivity and its oxidizing power of free
radicals.
As it is unstable in both the aqueous and gaseous phases, it
decomposes into hydroxyl, hydroperoxy, and superoxide radicals.
It is claimed that molecular ozone is the main inactivator of
microorganisms, researchers indicated the mentioned highly reactive
by-products created during ozone decomposition as a source of
potential antimicrobial activity.
(Pirani, 2010)

27. 27
Inactivation of microbes by ozone is a complex process because
ozone attacks numerous cellular constituents including
 Cell membranes- Proteins, unsaturated lipids and respiratory
enzymes
 Cell envelopes- Peptidoglycans
 Cytoplasm- Enzymes and nucleic acids
 Spore coats- Proteins and peptidoglycan
 Virus capsids.
(Guzel-Seydim et al., 2004a; Pirani, 2010)
Working principle of Ozone….

28. 28
The first step includes:
Oxidation of unsaturated fatty acids
formation of peroxides compounds.
Peroxidation of polyunsaturated fatty acids
due to the ozone
Bacterial cell wall- Cytoplasmic-membrane
consisting of phospholipids.
A hydrogen molecule is peeled off from the rest
of the unsaturated fatty acid molecule and a
free alkyl radical with an unpaired electron.
Working principle of Ozone….

29. 29
The destruction of the membrane barrier is the main factor leading to
the secondary DNA damage and, finally, death of the cell.
Series of chemical reaction leads to
peroxidation of lipid completely
The peroxidation products change the
physical properties of the cell membranes,
causing their depolarization and inhibition
of the activities of the membrane enzymes
and transport proteins.
(Antoszewski et al., 2004; Brodowska et al., 2018)
The Second step includes:
Working principle of Ozone….
Breakdown of Cell
Wall

30. 30

31. 31
The type of microorganism and the age of culture
The density of the treated population
The presence of compounds in solution with demand for O3
Forms of application of O3 and dose and treatment time
Accuracy of methods for quantification of ozone
Procedure to assess the antimicrobial effectiveness
(Guzel-Seydim et al., 2004; Pascual et al., 2007)
The effectiveness of ozone to reduce the number microorganisms
is influenced by several factors:
Working principle of Ozone….

32. Applications of ozone in Food Industry
of Livestock Origin
32
Meat
Industry

33. Meat Industry
33
Survival population of E. coli O157:H7 and aerobic bacteria on fresh beef
after aqueous ozone spray chill application (The concentration of aqueous
ozone was 12 ppm)
Treatment Number Survivors (log CFU/cm2)b
O157 APCc
Control 24 5.19 ± 0.18 A 5.40 ± 0.08 A
Water 24 4.54 ± 0.29 B (0.60)d 5.31 ± 0.15 A (0.09)
Control 72 5.24 ± 0.13 A 5.42 ± 0.10 A
Ozone 72 3.78 ± 0.38 C (1.46) 4.43 ± 0.24 B (0.99)
a- Control, no treatment of any kind; water, water spray chill for 90 s every 30 min for
12 h; ozone, treated with aqueous ozone for 90 s every 30 min for 12 h.
b- Means Means with different superscript differ significantly (p<0.05)
(Kalchayanand et al., 2019)

34. 34
(Cho et al., 2015)
Bacterial counts on chicken breast inoculated with Salmonella typhimurium (st)
exposed to gaseous ozone during storage (continuous flux
of ozone (10 × 10−6 kg O3/m3/h) and negative ions)
Bacteria Inoculation
with ST
Ozone
treatment
Storage (days)
0 1 2 3
ST Yes NO *7.64 ±
0.38aB
7.84 ±
0.29aB
8.05 ±
0.18aAB
8.30 ±
0.04aA
Yes 7.64 ±
0.38aA
7.24 ±
.14bA
7.32 ±
0.04bA
7.51 ±
0.03bA
Total
aerobic
bacteria
Yes NO 8.05 ±
0.19aB
8.08 ±
0.27aB
8.49 ±
0.17aA
8.82 ±
0.36aA
Yes 8.05 ±
0.19aA
7.26 ±
0.18bC
7.53 ±
0.24bBC
7.75 ±
0.27bAB
Total
anaerobic
bacteria
Yes NO 7.98 ±
0.06aC
8.16 ±
0.21aC
8.56 ±
0.11aB
9.18 ±
0.28aA
Yes 7.98 ±
0.06aA
7.43 ±
0.14bB
7.60 ±
0.29bB
8.17 ±
0.02bA
Means in the same column and row followed by different superscript lowercase and
superscript uppercase letters are significantly different (P<0.05) respectively
Meat Industry…

35. Applications of ozone in Food Industry
of Livestock Origin
35
Dairy Industry

36. 36
Microbiological situation (log cfu/ml) of cow's milk as affected by alternative
conventional heat or ozonation at the rate of 400 mg O3/h for different times
Microbiological
count (log cfu/ml)
Raw Treatment of cow's milk
Heat at
(72°C/15s
ec)
Ozonation time (min)
5 10 15 20 25 30
Total bacteria 7.22ª 4.32d 6.15ᵇ 5.27c 4.12d 3.45e 3e 2.40ᶠ
Yeasts & Molds 3.11ª 1.05d 2.9ᵇ 2.45c 1.11d 1d 0.9d 0.5e
Enterobacteriacae 2.18 <1 2.00 1.50 <1 <1 <1 <1
Psychrotrophes 3.15ª 1.60e 2.9ªᵇ 2.71ᵇ 2.50c 2.01d 1.8e 1.17ᶠ
Means with the same letter at any position did not significantly differ (p>0.05).
(Younis et al., 2019)
Dairy Industry

37. 37 (Sert et al., 2020)
Microbiological characteristics of butter produced from ozone-treated
cream for different treatment times (3.5 g ozone/h)
Coliform Salmonella Staphylococci Yeast and
mould
TMAB
Control 4.71* 3.71 5.01 5.01 6.37
OT-5 1.26 (2.45) 0.33 3.89 2.00 4.71
OT-15 1.20 (3.50) - x 4.03 - x 4.35
OT-30 - - 4.24 - 4.39
OT-60 - - 3.00 (2.01) - 3.80 (2.57)
PSD 0.13 0.26 0.11 0.79 0.16
TMAB: total mesophilic aerobic bacteria.
Control: from non-ozone-treated cream; OT-5: from ozone-treated cream for 5
min; OT-15: from ozone-treated cream for 15 min; OT-30: from ozone-treated
cream for 30 min; OT-60: from ozone-treated cream for 60 min.
Dairy Industry….

38. 38 (Torlak and Sert, 2013)
Inhibitory effect of gaseous ozone at two different concentrations ( , 2.8 mg/ L; -
5.3 mg L/1) against Cronobacter in whole milk powder (A) and skimmed-milk
powder (B) during 120 min of treatment
(Error bars denote standard deviations)
2.8 ppm- 1.16
5.3 ppm- 2.71
2.8 ppm- 1.65
5.3 ppm- 3.28
Dairy Industry….

39. Applications of ozone in Food Industry
of Livestock Origin
39
Egg Industry

40. 40
Egg Industry
(Braun et al., 2020)
Effect of Relative Humidity (rh) during ozonation (0.5% wt/wt for 20
min) on the reduction of Salmonella
Enteritidis (105–106 cfu/shell) on Egg Surfaces
rh n Control eggs (log10)
Mean and SD
Ozonated eggs
(log10) Mean and
SD
Reduction
(log10)
<30 % 30 5.95 ± 0.08 5.95 ± 0.04 0.04
>70 % 30 5.82 ± 0.09 5.13 ± 0.07 0.70∗
Significant reduction (p < 0.01).

41. 41
(Braun et al., 2020)
Influence of Different Ozone Doses (0.5, 1, 3, 5%) and Exposure Times
(120 or 180 min) on the Reduction of Salmonella
Enteritidis (105–106 S. E./Shell ) on Egg Surfaces
Ozone (%wt/wt)
/Exposure time
(min)
n Control eggs
(log10)
Mean and SD
Ozonated eggs
(log10)
Mean and SD
Reduction
(log10)
0.5%/120 50 5.33 ± 0.04 0.21 ± 0.09 5.12
1.0%/120 50 5.26 ± 0.03 0.35 ± 0.11 4.91
3.0%/180 50 5.45 ± 0.10 0.00 ± 0.00 5.45
5.0%/180 50 6.23 ± 0.07 0.00 ± 0.00 6.23
Egg Industry….

42. 42 (Rodriguez and Yousef, 2005)
Inactivation of Salmonella Enteritidis on shell eggs when treated with
gaseous ozone at atmospheric pressure and 4 to 8 °C for up to 8 min
Treatmenta Treatment
time (min)
Count
(log CFU/g of eggshell)b
Log
reductionc
Contaminated
untreated (control)
0 6.3 ± 0.2 A NA
Air-treated 3 5.2 ± 0.1 B 1.1
5 5.3 ± 0.1 B 1.0
8 5.4 ± 0.1 B 0.9
Ozone-treated 3 3.2 ± 0.3 C 3.1
5 4.0 ± 0.1 D 2.3
8 3.7 ± 0.1 D 2.6
Egg Industry….

43. Applications of ozone in Food Industry
of Livestock Origin
43
Aquaculture
industry

44. 44
Aquaculture industry
Data within control group with different lowercase letters and within treated group
with different uppercase letter are significantly (p<0.05) different
Microbiological changes in Nile Tilapia (Oreochromis niloticus) fillets
sterilized by ozonated water pre treatment during frozen storage (4.5
mg/L ozone-containing water)
(Zhao et al., 2017)
x

45. 45
Arrows indicated % reduction of bacterial loads compared to the starting bacterial
concentration. Bars represent standard deviation from 3 replicates.
Bacterial counts of Streptococcus agalactiae (A) and Aeromonas veronii (B)
upon exposure to NB-O3 for 10 min, three times (orange lines), compared to
that of the control water without NB-O3 (blue lines).
(Jhunkeaw et al., 2017)
Aquaculture industry…

46. 46
Mobile ozone surface sanitization system CIP system using ozonated water

47. 47 (FSSAI, 2020)

48. 48

49. Advantages and disadvantages
49
The prime advantage is most likely a non-residual nature of the
process.
It is a promising substitute for the conventional fumigation in use.
Can be applied to all types of foods, from fruits, vegetables, spices,
meat, egg, milk and seafood products to beverages.
Reduces the microbial contamination of a food without having an
unfavourable effect on its visual, textural and nutritional quality and in
extending its shelf life.
Can be used for both fresh and frozen foods.

50. 50
No need to store hazardous substances compared to other sanitation
methods.
No heat requirement and no heat generation in treatment and thus
saves the need of input energy.
Saves transport of disinfectant chemicals and storing of gas cost.
Beneficial due to lower costs of the purchase and maintenance of the
supply units compared to the cost of the other food disinfectants.
Ozone destroys the microorganism through cell lysis, the development
of resistance to ozone disinfection is not found.
(Pascual et al., 2007; Brodowska et al., 2014)

51. 51
As effectiveness of ozone treatment is strongly determined by so
many factors may cause some limitations in the selection of a
sufficiently effective ozone dose.
At higher concentrations it can negatively affect food quality and
leads reduction of vitamin, polyphenol, and volatile compound
contents, color changes, and loss of firmness etc.
Chance of recontamination problems in clean in process pipes as
ozone decomposes completely within a short duration.
Storage of ozone is not possible as it decomposes quickly.

52. Cost analysis/ Cost on the adoption of technology
52
• Plumrose USA Inc. employs ozonated water for sanitising work
areas and for equipment used for slicing and packaging ham, chicken
and other meats.
• It has a centralised system that produces ozonated water on demand
(28 g ozone/h) and delivers it automatically to the work areas
through closed piping under low pressure.
• Company also uses ozone instead of chlorine to rinse its stainless
steel transportation racks in a three-stage process.
• Ozonated water from the final rack rinse can be re-ozonated and
used for the first rinse, reducing water usage and disposal costs.
Examples at industrial scale

53. 53
• Before installing ozone, the company kept a large stock of 30%
sodium hypochlorite.
• Throughout the day, 100 ppm chlorine rinses were used on equipment
surfaces.
• Now, water containing 1 ppm ozone is used.
• The total cost of the system was $ 73,800, but it has already reduced
the $ 9000 per quarter expenditure on hypochlorite.
(Pascual et al., 2007)
Cost analysis/ Cost on the adoption of technology…

54. 54
Financial study conducted in an American food plant (20 processing lines
operating 24 h/day, 300 days a year) that introduced a mobile ozonated
water disinfection system. The new system enabled the processor to cut
the previous four disinfection steps to only two and thereby reduce water
use from 56.8 m3 to 22.7 m3. annual savings of $ 18,981 were achieved.
Financial study
Cost of
chemical
products
($/year)
Wastewater
discharges
(m3/day)
Discharge
tax ($/m3)
Monthly
discharge
tax ($)
Annual
discharge
tax ($)
Annual
total ($)
Without
ozone
6000 56.775 12,702 1802 21,635 27,635
With ozone 0 22.710 12,702 721 8654 8654
Total annual
saving
18,981
(Rice et al., 2002)
Cost analysis/ Cost on the adoption of technology…

55. Conclusions
55
 The methods and ozone concentration used to reduce the
number of microbes in livestock products could vary
depending the condition of the medium.
 Ozone has proven to be a great opportunity for the
environment and it is considered an excellent ecological
disinfectant because it has no negative impact on the
environment.
 Due to the microbial destruction by cell lysis, the
development of resistance to ozone disinfection is not found.
 Ozone can be used as effective sanitizer with great potential
applications in the food industry.

56. Future Prospect
56
 Degradation products, formed after ozonation, have not exactly been
determined and its detailed evaluations is require.
 In vivo and in vitro toxicological tests are needed to be conducted to
screen the effects of degradation products on human and animal
health.
 Ozone generating and its application systems are needs to be
evaluated more effectively in future.
 Further research is required to ascertain the interaction of food
constituents with ozone and role of resulting compounds in the
inactivation process.

57. Future Prospect
57
 Further more studies are require to understand the mechanism of
actions at various applications.
 It is important that further studies are warranted to:
 Involve other commercially available domestic ozone facilities
 To test it on a wide range of economically important livestock
products.
 To authenticate and validate the preservation potential of the
domestic facility in food processing technology.

58. 58