This study focuses on investigation of milk fermentation process and the development of antioxidant activity measured by the lipid peroxidation
assay. Raw cow milk was fermented using symbiosis of the cultures Streptococcus thermophilus and Lactobacillus delbrueckii
ssp. bulgaricus, as well as the monocultures Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium bifidus. Inhibition of
lipid peroxidation (LPI) was determined by a method based on developing of a dyed system using the thiobarbituric acid and iron ions.
Titrable acidity (0
SH) was also assayed by Soxlet’s extractor. The results obtained from the investigation showed that the values of LPI
were increased at the end of the investigation with all the strains of the cultures applied, ranging from 52,87%; 65,85%; 76,07% and
65,67%.. The maximum inhibition capacity of lipid peroxidation possessed Lactobacillus acidophilus, than Bifidobacterium bifidus.
Milk fermented with Lactobacillus acidophilus also demonstrated the maximum degree of titrable acidity with a value of 40,06 оSH
what proved that Lactobacillus acidophilus possessed the most proteolytic ability among the all strains used. This is of great importance
for the production of yogurt where these two cultures are applying for, providing health benefits for the consumers.
Milk, Fermentation, Antioxidant Activity, Lipid Peroxidation, Titrable Acidity, Microbiological Cultures.
The metabolism in living organisms is a continuous process of oxidation
and reduction when some food components are oxidized
with the releasing of energy, while some other components are reduced.
During these complex processes, there is always a process
of formation of potentially dangerous free radical species.
The oxidative stress reflects the internal balance between systemic
manifestations of reactive oxygen species (ROS) and the ability of
the biological system to detoxify these reactive intermediates or to
remedy the damage from their activity. Disruption in the normal
cell redox status may cause toxic effects through the production
of peroxides and free radicals that damage all parts of the cell,
including proteins, lipids and DNA. In humans, oxidative stress is
thought that is involved in the development of various fatal diseases
such as cancer , Parkinson’s disease, Alzheimer’s disease ,
atherosclerosis, death of heart muscle and degenerative processes
of the myocardial .
Production of ROS represents a particular aspect of the destructive
oxidative stress. These ROS include free radicals and peroxides.
Any free radicals in which the oxygen is turned on, can be referred
to as reactive oxygen species (ROS). The radicals with oxygen as a
central atom contain two unpaired electrons in the last electronic
layer. When free radicals perform the “taking “ of an electron from
a compound in the immediate vicinity, new radical occurs in its
place. This is followed by tending of a newly formed radical to return
to its neutral or “grounded” form, what makes it through the
“seizure”- of another electron from the molecules being around it.
Thus arises a chain reaction that can be infinite . The chain electron
transport which is located in the membrane of mitochondria
in each cell uses oxygen to create energy from ATP. Oxygen acts as
a terminal electron acceptor in the chain electron transport.
The natural antioxidants are divided into intracellular antioxidant
and antioxidants introduced through the food. Intracellular antioxidants
may be bioactive peptides, amino acids, vitamins and other
intracellular antioxidant systems. Antioxidant protection systems
protect cells and organ systems in the body from the harmful effects
of ROS. The human body has developed a highly sophisticated
and complex defensive strategy that includes a number of antioxidants
and antioxidant systems. In defense of the body a number
of components of endogenous and exogenous origin are included,
which operate interactively and synergistic in the neutralization of
the free radicals. In reference to our study these systems besides other include the antioxidants from food, including tocopherols
and tocotrienols (vitamin E), carotenoids and other substances of
low molecular mass such as glutathione (GSH) and lipoic acid.
Here also belong the antioxidant enzymes, including superoxide
dismutase (SOD), glutathione peroxidase and glutathione reductase
that catalyze the reactions to neutralize free radicals.
Antioxidant activity and the ability to perform the inhibition of
lipid peroxidation are important because many diseases in humans
begin just by insufficient activity of these processes in the body.
Lack of antioxidant capacity in certain cells cause oxidative damage
of the cell structure and its abnormal function what is called
oxidative stress on the body. Therefore very important question
arises of consuming functional foods that possess exactly the capabilities
to perform an antioxidant function and inhibition of lipid
Oxygen reacts with many organic substrates forming
hydroproxides and other oxidized compounds. This reaction
mostly represents a chain reaction of production
of free radicals and it undergoes several phases:initiation
reaction, reaction of developing when more and
more molecules are included in the reaction with oxygen
and reaction of termination. These reactions are
The production of free radicals can be carried out through a direct
thermal dissociation, hydroperoxide decomposition, reactions catalyzed
by metal ions and by light exposure (photolysis) with or
without photosensitive substances.
The production of free radicals is through the reactions (2) and (3):
Organic substances are susceptible to auto oxidation what depends
on their relative affinity for giving away hydrogen atoms through
the reaction (3).
Unsaturated lipids are susceptible to auto oxidation what depends
on accessibility of allyl hydrogen atoms for reaction with peroxide
The valence bonds in the structure A can be represented as a hybrid
B with partial free radical at each end of the allyl system. The
reaction of oxygen occurs at the last atom of C of the allyl system
when different isomeric hydroperoxides are produced.
The most important process of the termination of secondary peroxide
radicals at room temperatures is proposed by  including
tetroxide intermediate and results in production of ketones, alcohols
There is evidence that the oxygen produced through the reaction
(7) can be activated in its excited state (‘O2
Fermentation of milk extends milk durability, and gives a possibility
to obtain functional food with acceptable and good taste.
Fermentation is important in the production of dairy products
because the bacteria used in this process to perform decomposition
of the main components of milk (mainly lactose and proteins)
and yields new components such as acids, free amino acids, peptides
and peptones which have different health contribution to the
body of man. Namely, the free amino acids and peptide sequences
of amino acids have an antihypertensive effect , increasing
the anti-oxidative capacity and inhibition of lipid peroxidation 
achieving numerous benefits to human health.
The aim of this research was to monitor the dynamics of development
of antioxidant activity and capacity to inhibit lipid peroxidation of milk fermented by different microbial cultures.
Material and Methods
As was already mentioned in the text , a medium used for this
survey was sterilized whole cow milk having the average chemical
composition shown in Table 1.
The milk was fermented with different microbial cultures: three
monocultures (Lactobacillus casei, Lactobacillus acidophilus and
Bifidobacterium bufidus).and a symbiosis of Streptococcus thermophilus
and Lactobacilus delbrueckii ssp. Bulgaricus. Antioxidant
activity (AOA) was expressed in (%) of the neutralization
of free radical as was previously reported .
Inhibition of lipid peroxidation (LPI) was expressed in (%) of LPI.
Besides AOA and LPI pH and titratable acidity was also monitored
and statistical processed (ANOVA test) which are closely related
to metabolic degradation of proteins present in milk due to bacterial
cells, as was previously demonstrated . Namely, how the
pronounced proteolitic activity of the cultures is, the higher titrable
acidity, what means a greater release of amino acids and thereby a
positive correlation between AOA and LPI.
The parameters were monitored before fermentation, immediately
after completion of the fermentation and at the first, third, 5 th, 10
th and 15th day after the fermentation process.
Preparation for Fermentation
Before fermentation process milk was heated in a sterilized container
at temperature of 35°C. Inoculation was done by direct
seeding of culture in the required amount of milk with concentration
of 0,01% w/v, and that represents the industry standard for
fermented products manufacture. After seeding the contents were
mixed 5 to 10 minutes with sterile blender for achieving better
dispersion of the culture in the medium. Before use the edge of
the bag where the cultures were sterilized with ethanol. Also the
equipment for cutting and weighing container was sterilized with
ethanol. Fermentation was performed in a sterile disposable container
and thermal chamber at temperature of 40°C. The length
of the fermentation was 4 hours using a starter culture consisted
of the symbiosis of Streptococcus thermophilus and Lactobacillus
delbruescii ssp. Bulgaricus, and 12 hours with samples fermented
by Lactobacillus casei, L. Acidophilus and Bifidobacterium bifidus.
The fermentation was completed when casein reaches its
isoelectric point at 4,6 pH. After completing of the fermentation
process the all samples were stored at refrigerator temperature of
4°C up to the next control measurement .
Results and Discussion
In Table 2 are presented the parameters of the sterilized whole milk
which were analysed.
From the Chart 1 can be noticed, similar to АОА , drastically
increasing of the LPI as result of milk fermentation with the all
kinds of cultures investigated. From the point of view of the lowest
and highest values of LPI, from the chart can be seen that the
highest LPI had milk fermented by Lactobacillus acidophilus and
the lowest value had milk fermented with Streptococcus thermophilus
and Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus
casei. The highest LPI value was achieved at the 15th day
of the experiment (L. acidophilus) when the samples were kept at
temperaturature of 4оС. The lowest value was noticed at the 10th
day of the experiment.
Inhibition of lipid peroxidation had a trend of steadily developing
with small ups and downs between the control points. Immediately
after completion of the fermentation, the observed values for LPI
from 39,19% are noticed with the symbiosis of Streptococcus thermophilus
and Lactobacillus delbrueckii ssp. bulgaricus, than 46,49
% with Lactobacillus casei, 61,30 % with Lactobacillus acidophilus
and 42,15% with Bifidobacterium bifidus. LPI values at the
end of the study showed an increase with the all cultures species,
ranging from5 52,87%; 65,85%; 76,07% to 65,67%. The obtained
results have clearly shown that the greatest capacity for inhibition
of lipid peroxidation owned Lactobacillus acidophilus. It was followed
by Bifidobacterium bifidus. This is of particular importance
because in production of probiotic yoghurt these cultures are used.
So, these results confirmed the positive effect of probiotics on the
balance of all systems in the human body. According to a statistical
data processing it was noticed that between the cultures species
there was a significant statistical difference in LPI values at the
level of p <0,05 what is shown in Table 3.
As a culture that showed the highest degree of titrable acidity
confirming thus its proteolytic characteristics, Lactobacillus acidophilus
had the lowest value of 34,8 oSH measured at the end of
fermentation and the highest one of 40.06 oSH . These data
confirmed the highest AOA and LPI because Lactobacillus acidophilus
possessed the greatest proteolytic ability among the all
cultures investigated. The lowest values of titratable acidity during
this research showed the symbiosis of Streptococcus thermophilus
and Lactobacillus delbrueckii ssp. bulgaricus of 27,87оЅН at the
end of the fermentation of a maximum one of 33,73 оЅН. This
contributes to the fact why this symbiosis showed the lowest values
for AOA and LPI.
In conclusion can be stated the following:
1.Fermentation of milk does much more than a simple extension
of its life.
2.The highest capacity for LPI has shown milk fermented with Lactobacillus
3.The highest value of LPI was measured on the 15th day of keeping
the fermented product and is 76,07%.
4.The lowest capacity of LPI has shown milk fermented with the
symbiosis of Streptococcus thermophilus and Lactobacillus delbrueckii
ssp. bulgaricus, with the average values of 43,01% and the
lowest value of 31,61% which was measured on the 10th day.
5.The results of this research can justify the use of fermented dairy
products as functional food and prevention of the oxidative stress
and diseases related to.
- Barry, H. (2007). Oxidative stress and cancer: have we moved
forward?. Biochem. J.,401 (1): 1-11.
- Valko, M., Morris H. and Cronin, M.T. (2005). Metals, toxicity
and oxidative stress. Curr. Med. Chem.,12 (10): 1161-208.
- Singh, N., Dhalla, A.K., Seneviratne, C. and Singal, P.K. (1995).
Oxidative stress and heart failure. Molecular and Cellular Biochemistry,147
- Goldfarb, A., H. (1999). Nutritional antioxidants as therapeutic
and preventive modalities in exercise-induced muscle damage.
Can. J. Appl. Physiol., 24: 249-266.
- Frankel, E.N.(1980). Lipid oxidation. Prog.Lipid. Res., 19:1.
- Russell, G. A.(1957). Deuterium isotope effects in the autoxidation
of aralkyl hydrocarbons. Mechanism of the interaction of peroxy radicals. J. Am. Chem. Soc., 79: 3871-3877.
- Howard, J.A. and Ingold, K.U. (1968).The self-reaction of sec-butyl-peroxyl
radicals: confirmation of the Russell mechanism. J. Am.Chem. Soc., 90: 1056-1058.
- Tomovska, J., Presilski, S., Gjorgievski, N., Tomovska, N.,
Qureshi, M.N. and Bozinovska, N.P., (2013). Development of a
spectrophotometric method for monitoring angiotensin-converting
enzyme in dairy products. Pak. Vet. J., 33(1): 14-18.
T., Pihlanto A., Akkanen S. and Korhonen, H. (2007). Development
of antioxidant activity in milk whey during fermentation
with lactic acid bacteria. J. appl. microbial., 102: 106-115.
- Gjorgievski, N., Tomovska, J., Dimitrovska, G., Makarijoski, B.
and Shariati, M. A. (2014). Determination of the antioxidant activity
in yogurt. Journal of Hygienic Engineering and Design, Original
scientific paper UDC 637.146.3:615.272: 88-92.
- Tomovska, J., Gjorgievski, N. and Makarijoski, B. (2016). Examination
of pH, titratable acidity and antioxidant activity in fermented
milk. Journal of Materials Science and Engineering A 6(11-12):326-333. doi:10.17265/2161-6213/2016.11-12.006.