Journal of Hygienic Engineering and Design

Original scientific paper

UDC 637.12.047:577.161.1/.3(497.751)
636.087.7:577.161.1/.3(497.751)

DIFFERENCES IN THE CONCENTRATION OF VITAMINS A AND E, BETWEEN

MILK AND FODDER CONCENTRATE FROM REGION OF TETOVO, MACEDONIA
Ilmije Vllasaku1*, Julijana Tomovska2, Naser Kamberi1
1
Faculty of Food and Biotechnology Sciences, University of Business and Technology,
Kalabria nn, 10000 Pristina, Kosovo
2
Faculty of Biotechnical Sciences, University ”St. Kliment Ohridski”,
Partizanska nn, 7000 Bitola, Macedonia

*
e-mail: IlmijeVllasaku@hotmail.com

Abstract
Vitamins A and E in have oxidative effects on the health
of dairy cows, as well as on the milk production. Hence
the main goal of this study was to determine the
content of vitamins A and E in animal feed - concentrate
and raw and pasteurized and packaged raw cow milk
from the region of Tetovo, Macedonia.
Analyzed were samples of feed, raw, and packaged raw
cow milk. High performance liquid chromatography
(HPLC) was applied for analysis of the vitamins with
extraction methodology - on Perkin Elmer, pump:
200LC series, self-sample; ISS - 200, detector LC - 135
/ LC -235 C DA. For determination of the gained data
for concentration of vitamins A and E in feed and milk
ANOVA statistical analysis was used.
It was determined that the vitamins A and E
concentrations in the feed concentrate from the region
of Tetovo were 26.21 mcg/100g and 26.234 mcg/kg,
respectively. As for the amount of vitamin A determined
packaged raw cow milk it was 35.82 mcg/100g, and the
amount of vitamin E was 0.87 mcg/100g. A significant
statistical difference was found among the values of
vitamins A and E in milk (p < 0.001).
In conclusion could be stated that the concentrations
of vitamins A and E determined in the concentrates and
the packaged raw milk were low compared to those
found in the packaged raw milk standard samples.
Key words: Fodder, Fodder concentrate, Vitamin A,
Vitamin E.
1. Introduction
The inclusion of antioxidant and lipid sources in the
dairy cows’ diets has been recommended as a method
for increasing the production and quality of milk, and
has beneficial effects on the human immune system
and health [3, 4].
Benefits of milk consumption and the nutrients present
in milk and dairy products are many, but basically these
products represent sources of calcium and vitamin
D, as well as proteins and other essential nutrients. In
addition, dairy products are supply for human body:
phosphorus, potassium, magnesium, and vitamins A,
B12, and riboflavin [6].
The content of fat-soluble vitamins A, D, and E in milk
is strongly influenced by the dairy cows’ food ration
composition [1].
Vitamin A and vitamin E, on the other hand, have to
be obtained from external sources. A daily serving of
244 mL of cow’s whole milk contributes with 7.6% to
the recommended dietary allowance for vitamin A and
only 1% to the recommended dietary allowance for
vitamin E. Despite the lesser nutritional contribution,
vitamin E in milk helps to slow down peroxidation of
lipids and thus enhances the bioavailability of vitamin
A as well as other fat-soluble vitamins [5, 7].
Common signs of vitamin A deficiency in cattle
include: reduced feed intake and growth, rough
hair coat, night blindness, edema, diarrhea, seizures,
increased susceptibility to infection, abnormal

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sperm, abnormal bone growth, low conception rates,
abortion, stillbirths, and weak calves. Vitamin A is a fat-
soluble vitamin which is involved in many important
biological functions. As a lipid soluble antioxidant, it
is considered that it acts primarily on protection the
polyunsaturated fatty acids and related substances -
products from peroxidation and from rancidity [9].
Vitamin A (retinol), E (tocopherol), and the carotenoids
are important micronutrients that are found in various
foods such as vegetables, fruits, meets, and animal
products. Higher content of vitamin A is found in fish
-liver oils, liver, butter, egg yolks, and cream. Rich source of
vitamin E are also nuts and seeds [14]. Some carotenoids
are named as provitamin A that are found in colorful
fruits and vegetables and they are metabolized in the
body to vitamin A. This bioconversion of carotenoids to
vitamin A differ from person to person [10].
The most potent among at least eight naturally
occurring tocopherols is alpha tocopherol. Gamma
tocopherol is less active from of vitamin E that is
plentiful in the diet.
Vitamin E has been also found to be essential for the
integrity and optimum function of reproductive,
muscular, circulatory, nervous, and immune systems,
and it is also involved in arachidonic and prostaglandin
metabolism [2]. Numerous studies have shown that
vitamin E and other antioxidants are absolutely critical
for neutrophils to function properly. When neutrophils
are functioning properly, cows are less likely to
succumb to mastitis and retained placenta [8].
Vitamin E is clearly an integral component of immune
function and is especially critical during the transition
period when cows are more susceptible to retained
placenta and mastitis.
The main objective of this research was to analyze the
vitamins A and E in fodder concentrate and cow milk
from a farm from region of Tetovo, Macedonia, in terms
of their content, and to compare them with a standard.
2. Materials and Methods
2.1 Materials
Samples of cow feed and samples of raw and raw
cow pasteurized and packaged raw milk that were
analyzed were taken from the same cow farm. The feed
samples comprised of two types of concentrates that
were produced by Animal feed factory “Agroinvest”
from Prilep, Macedonia. It was feed for dairy cows was
containing at least 18% raw protein.
The cows were fed three times per day: with the
concentrate in the morning, with alfalfa and straw
at lunch time, and with concentrate in the evening.
Milking was performed with a machine, three times
per day in the summer period, and twice per day
during the winter period.
2.2. Methods
2.2.1 Methods for analysis of vitamin A (retinol) and
vitamin E (tocopherol) in feed concentrates and milk
• Extraction method 1 (Chem Elut)
20 g of the sample (closest to 0.01 g) were weighted
into a 500 mL Erlenmeyer flask, than 1 g of ascorbic acid,
150 mL of ethanol (95%), and 40 mL of a 50% aqueous
solution of potassium hydroxide (KOH) was added. The
condenser was added to the flask and placed in a water
bath with temperature approximately of 95 0C.
The time of hydrolysis was 30 minutes after the start
of the reaction, after what the sample was cooled to a
room temperature. 50 mL of distilled water was added.
Hydrolysate was transferred to a 500 mL volumetric
flask and diluted to the mark with ethanol (50%).
Than 10 mL were transferred to a Chem Elut column
(20 mL) and waited about 10 minutes. The sample
was eluted using 100 mL n-hexane. The eluate was
collected in a 500 mL flask. Evaporation to dryness was
done with rotary centrifugal force (RCF) and by use of
butylhydroxytoluene (BHT) granules. After that, the
sample was dissolved in n-heptane and transferred to
a volumetric flask of 5 mL and was diluted up to the
n-heptane mark.
• Extraction method 2 (separation funnel)
This procedure is similar to that of the Extraction
method 1. 20 g of the sample (closest to 0.01 g) were
weighted into a 500 mL Erlenmeyer flask, than 1 g
of ascorbic acid were added, as well as 150 mL of
ethanol (95%) and 40 mL of a 50% aqueous solution of
potassium hydroxide (KOH) in water.
After finishing the hydrolysis, the sample was cooled to
a room temperature and 50 mL of distilled water were
added and transferred to a 500 mL volumetric flask, and
then diluted with 50% ethanol to the mark. 20 mL were
transferred into a separating funnel and diluted with
100 mL n-hexane. The funnel was shaken for 1 minute,
cleaned and washed (hexane phase, 2 times x 50 mL
1 M potassium hydroxide in ethanol (40%) and with
2 x 50 mL distilled water). Than followed evaporation
to dryness of the hexane phase with a few granules
of BHT, and 8 mL of ethanol (99%). The sample was
dissolved in n-heptane and added to a volumetric flask
of 5 mL and was diluted up to the n-heptane mark.
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After finishing the extraction, analysis by HPLC was that found in the packaged milk was drastically lower
applied (apparatus of Perkin Elmer with pump of series (18.48 μg/100 g). The amount of vitamin E in raw cow
200LC, auto sampler, ISS - 200, detector LC - 135 / LC milk was 0.87 μg/100 g, compared with the packaged
-235 C DA). milk (0.12 μg/100 g) was also drastically lower. Our
results in accordance with results of many studies, such
3. Results and Discussion as Haug [13], who found 280 mcg of vitamin A in per 1
L of milk, as well as 0.6 mcg of vitamin e, per 1 liter of
Many factors such as: the health of mammary gland,
milk, and Mogensen [12], found 23 mg/per kg of dry
age, feed, and milk frequency, affects the milk quality.
matter (DM) of vitamin E.
Milk quality also depends on the mastitis presence, as
well as from milk with a low somatic cell count (SCC),
The determined values of amounts of vitamins A and E
and normal visibly appearance (no clots). The definition
in fodder concentrate and milk from region of Tetovo,
of the milk quality can be expanded according to Weiss
Macedonia, are shown on Figure 1.
[17]. So, the milk quality should also be based on the
amount of antioxidants that milk contains that protect
the milk reducing the oxidation.

In this paper we present the results of determination

the amount of vitamins A and E in fodder concentrate,
packaged raw cow milk, all from the region of Tetovo,
Macedonia.

The value of vitamins A and E were determined

according to Cop Rice [11], as standard that is used
for examination of the concentrates composition in
Australia (Concentrate Au). This standard contains
examination of: dry matter, protein, fiber, fat, urea,
mineral elements such as calcium, phosphorus,
magnesium, manganese, zinc, selenium, as well as
vitamins A, D and E.
In Table 1 are shown the amounts of vitamins A and
E determined in fodder concentrate in Tetovo region.
Amounts of determined vitamins A and E in fodder
concentrate in Tetovo region were 26.21 mcg/100
g, and 27.00 mcg/100 g, respectively. The amount of
vitamin A in raw cow milk was 35.82 μg/100 g, and in
Figure 1. Concentrations of vitamins A and E found in
feed and milk from region of Tetovo, Macedonia
Table 2 present the statistical analysis on concentrations
of vitamins A and E in fodder concentrate and milk made
by the Statistical analysis ANOVA, t-test. The statistical
analysis showed a significant difference between the
values of vitamins A and E in the concentrates and milk
(p < 0.001).
These results are not surprising since many nutrients as
vitamins A and E, and immune-enhancing components

Table 1. Concentrations of vitamins A and E in concentrate and milk from region of Tetovo
Concentrate Concentrate (Au) Raw cow milk Packaged milk
Vitamins
(mcg/g = IU/kg) IU, mg) (μg/ 100g) (μg/ 100g)
А 26.21 26 IU 35.82 18.48
Е 26.234 90 mg 0.87 0.12

Table 2. Statistical data of vitamins A and E determined in concentrates and milk

Comparison parameters Arithmetic mean Standard deviation Significance -
(Vitamins A and E) in t-test
(s) (P)
concentrates and row milk
Vitamin A
Concentrate 35.82:
9.240 0.000
Vitamin A t = -96.333 P < 0.001
17.910 0.127
in packaged milk
18.48
Vitamin A Concentrarte 35.82:
17.910 0.000
Vitamin E 0.87 t = (+inf.) P < 0.001
0.435 0.000
in packaged milk
Legend: t = (+inf ) test Infinity is that which is boundless, endless, or larger than any number.

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Journal of Hygienic Engineering and Design

are degraded or destroyed by exposure to high heat
and the temperatures used during pasteurization.
4. Conclusions
- It can be concluded that the determined concentration
of vitamin A in raw cow milk (35.82 mcg/g) was higher
than that found in fodder concentrate (26.21 mcg/g).
The amount of vitamin E determined in fodder
concentrate (26.234 mcg/g) was higher, while that
found in raw cow milk was lower (0.87 mcl/L).
- The comparison of the amounts of vitamins A and E
determined in feed concentrate with the standard Cop
Rice (2018) showed similar results for vitamin A, while
vitamin E has lower amount in the concentrate.
- A big difference was found between the determined
concentrations of vitamin A in raw milk (35.82 mcg/g)
with that of vitamin E (0.87 mcg/g) in raw milk. It was
also found a big difference between the concentrations
of vitamin A (18.48 mcg/g) found in packaged milk and
with that of vitamin E found also in the packaged milk
(0.12 mcg/g).
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2021.

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