sustainability

Article
Assessment of Water Buffalo Milk and Traditional
Milk Products in a Sustainable Production System
Zsolt Becskei 1, *, Mila Savić 1 , Dragan Ćirković 2 , Mladen Rašeta 3 , Nikola Puvača 4 ,
Marija Pajić 5 , Sonja Ðord̄ević 6 and Snežana Paskaš 7
1 Department of Animal Breeding and Genetics, Faculty of Veterinary Medicine, University of Belgrade,
18 Bulevar Oslobod̄enja, 11000 Belgrade, Serbia; [email protected]
2 Department of Chemical and Technological Sciences, State University of Novi Pazar, bb Vuka Karadžića,
36300 Novi Pazar, Serbia; [email protected]
3 Institute of Meat Hygiene and Technology, 13 Kaćanskog, 11000 Belgrade, Serbia; [email protected]
4 Department of Engineering Management in Biotechnology, Faculty of Economics and Engineering
Management in Novi Sad, University Business Academy in Novi Sad, 2 Cvećarska, 21000 Novi Sad, Serbia;
[email protected]
5 Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad,
8 Trg Dositeja Obradovića, 21000 Novi Sad, Serbia; [email protected]
6 Department of Animal Science, Faculty of Agriculture, University of Priština, bb Kopaonička,
38219 Lešak, Serbia; [email protected]
7 Department of Animal Science, Faculty of Agriculture, University of Novi Sad, 8 Trg Dositeja Obradovića,
21000 Novi Sad, Serbia; [email protected]
* Correspondence: [email protected]; Tel.: +381-65-991-1101




Received: 22 July 2020; Accepted: 14 August 2020; Published: 15 August 2020


Abstract: Water buffalo (Bubalus bubalis) conservation in Serbia is under an in situ program,
but additional efforts are needed to ensure the development of this animal’s genetic resources
biodiversity. This research aims to describe challenges and possible strategies for sustainable water
buffalo milk production. In this study, the physicochemical characteristics of buffalo milk and buffalo
dairy products (cheese, butter, and kajmak) were determined. Furthermore, amino and fatty acids
composition and the related health lipid indices (atherogenic and thrombogenic) were assessed.
The findings support the fact that buffalo milk is a reliable source of high-quality nutrients (dry matter:
16.10%, fat: 6.02%, protein: 4.61%). Leucine, lysine, and valine content were found to be high
in buffalo milk and cheese. A substantial quantity of non-essential glutamic and aspartic amino
acids was observed in milk, as well as glutamic acid and tyrosine in cheese. It was established
that milk protein of buffalo cheese had a favorable proportion of essential and non-essential amino
acids (61.76%/38.24%). The results revealed significant differences (p < 0.05) in fatty acid profiles
among the three dairy products for saturated short-chain, n-3, and n-6 fatty acids. Conversely,
no significant difference (p < 0.05) was observed in monounsaturated fatty acids content. Kajmak
showed the most favorable anti-atherogenic and anti-thrombogenic properties due to lower saturated
and higher polyunsaturated fatty acid content. These results confirmed that buffalo milk could
be successfully used in producing high-quality traditional dairy products with added value and
beneficial characteristics from the aspect of a healthy diet. Furthermore, it could actively contribute
to the promotion of sustainable production of buffaloes and strengthen the agricultural production of
rural areas and their heritage.

Keywords: sustainable livestock production; added value; buffalo milk; dairy products; chemical
composition; nutritional properties

Sustainability 2020, 12, 6616; doi:10.3390/su12166616 www.mdpi.com/journal/sustainability

Sustainability 2020, 12, 6616 2 of 13

1. Introduction
Water buffaloes (Bubalus bubalis) are distributed worldwide, with a total population of about
180 million. The majority of populations are located in Asia (around 97%). It is considered that the water
buffalo is an efficient converter of poor quality forages into high-quality products [1]. Buffaloes play an
essential role in the heritage of the local rural populations and their economy in South-Western Serbia,
where these animals are raised under extensive husbandry and sustainable principles. In previous
decades, control of productivity and selection for milk production were insufficient. As a result,
milk production is lower in comparison with selected populations of buffalo in some Asian countries [1].
The mainly conventional livestock production in Serbia and the popularization of imported, highly
productive breeds of livestock contributed to a significant reduction in the number of autochthonous
breed populations, and the genetic diversity is eroded. Buffalo population, as well, showed a tendency
to decrease. Since the 2000s, a program for the in situ conservation of buffalo population started in
Serbia [2], but still, according to the Domestic Animal Diversity Information System of the Food and
Agriculture Organization of the United Nations (FAODAD-IS), the estimated population of buffalo is
low, and their local risk status is “endangered”, with a decreasing trend.
As reported by the same database, there are about 30 bulls and 724 cows of buffalo evidenced in
the breeding program in Serbia [3]. Furthermore, the main challenges facing the buffalo breeding sector
is low profitability. On the other hand, in many countries, an increasing demand for buffalo dairy
products has been noticed, and accordingly, most farms are undertaking a progressive intensification
of rearing techniques [4,5]. Besides, non-cattle dairy systems, whether intensive or not, could be a
sustainable alternative and potentially a significant added value for producers and the dairy sector [6].
Sheep and goats are essential species in non-cattle milk production. Thus, the polymorphism of
selected genes affecting milk composition in small ruminants is well documented [7–9], but efforts are
needed in the genetic assessment of buffalo populations.
Buffaloes of the Mediterranean region are phenotypically different from country to country
because of the different environment and management practices. Still, the fundamental objective of
breeding buffaloes in Europe and the Near East is milk production [5]. Dairy farms have valuable
input to a resilient and sustainable food system. They possess the ability to provide not just basic
nutrition, but also healthy nutrition. For developed and developing countries, dairy has the potential to
reinvigorate rural economies, providing sustainable livelihoods for smallholder farmers and a resilient
source of economic growth [10]. According to Borghese and Moioli [5], there is a need to more deeply
study dairy products made from buffalo milk in the Mediterranean area as well the variability of their
technologies because they represent an important part of global biodiversity.
Buffalo milk, like cow milk, can be used for the manufacture of a wide variety of dairy products [11].
It has long been valued for its fortunate chemical composition which determines its nutritive properties
and suitability for the production of traditional as well as industrial dairy products [12]. Compared
with cow milk, it contains less water and more fat and is especially useful for the production of
fat-based dairy products such as butter and kajmak. Kajmak is an artisanal Serbian dairy product
mainly produced from cow’s milk, and based on its physicochemical characteristics may be placed
between cheese and butter [13]. The specific fatty acid composition of buffalo milk contributes to the
unique features of its products. Butter, for example, made from buffalo milk due to its higher level of
saturated fatty acids is much harder than that made from bovine milk [11].
Moreover, the high variability in triglyceride and fatty acid composition of buffalo milk makes it
possible to separate milk fat into various fractions based on its melting characteristics [14]. Higher
proportions of high-melting triglycerides in buffalo milk contribute to its higher density [15] and
make it suitable for cheese making. Buffalo cheese is highly prized for its pure white appearance
and smooth texture [11]. In Italy, fresh and Pasta Fialata cheeses, especially Mozzarella and Borelli
cheeses, are traditionally prepared from buffalo milk. In Balkan countries, several types of white
brined cheese and pickled cheese are made from buffalo milk [16]. Because of differences between
buffalo and other ruminant milk in compositional and physicochemical properties, some modifications
Sustainability 2020, 12, 6616 3 of 13

of processing technology and equipment designed for different kinds of milk are necessary for buffalo
milk processing [12].
The advantages of buffalo milk compared to cow milk are not only in terms of physicochemical,
compositional, and sensory attributes, but also in its nutritional and health aspects [17]. Buffalo milk
proteins are complete proteins of high biological value, and they contain all the essential amino acids
in the proportions required by the human body [15]. Furthermore, buffalo milk and its products could
represent a good source of favorable conjugated linoleic acid (CLA) in human nutrition [18]. In recent
decades, many studies have been devoted to improving milk fatty acid (FA) composition by increasing
the amount of FA with beneficial effects on human health and with more appropriate technological
properties [19]. Findings of FA and triglyceride profiles of low melting fractions of milk fat have
suggested that the therapeutic value of low melting fractions of buffalo milk fat was higher than native
milk fat [14].
There are insufficient research data on the production potential and dairy product quality of
water buffalo in sustainable farming systems in Serbia. In particular, the chemical characteristics of
buffalo milk and its dairy products in Serbia have been studied in a limited fashion [2]. In this paper,
the composition and physicochemical properties of major constituents of buffalo milk are presented,
with particular emphasis on amino and fatty acids. Furthermore, buffalo dairy products are analysed,
compared, and discussed in terms of their nutritional, technological, and health aspects to find the
added value of the traditional products from sustainable production.

2. Materials and Methods

2.1. Sample Collection

Samples of bulk, raw buffalo milk and dairy products (cheese, butter, and kajmak) were taken in
early spring 2017, from small households of the Rashka district (42.989400, 20.332684) in South-Western
Serbia. Ten households participated in the study, with an average water buffaloes herd size of 5 milking
buffaloes. All the herds were reared in a sustainable, extensive management system, grazing on
pastures and meadows. The milking animals each received 0.5 kg oats daily as supplementary feed
during milking.

2.2. Milk Chemical Analysis

The standard chemical composition and milk urea (MU) analysis of bulk, raw buffalo milk
samples were conducted using a MilkoScan FT + analyzer for routine compositional raw milk analysis
employing Fourier Transform Infrared “FTIR”. The MilkoScanTM FT + techniques comply with:
ISO 9622/IDF 141:2013 [20] and the AOAC official method 972.16 [21]. The energy value of milk was
estimated, according to Popović-Vranješ [22].

2.3. Buffalo Milk Dairy Products Physicochemical Analysis

Samples of buffalo dairy products (cheese, butter, and kajmak) were analyzed for chemical
composition (dry matter, fat, protein, and moisture) following standard methods of the Association
of Official Analytical Chemists [23]. The dry matter was determined with a standard method of
measuring weight loss after drying (AOAC 926.08-1927). Protein content was measured using the
Kjeldahl-Van Slyke method for the determination of total N (AOAC 2001.14) with a Kjeltec Auto
Analyzer (Model 2400, Tecator, Hoganas, Sweden) and converted by a multiplication factor of 6.38.
The fat content was quantified according to the Van Gulik method [24]. Fat in dry matter (FDM, %)
was calculated as fat/(100 − moisture)×100, moisture in nonfat substance (MFFB, %) was calculated as
moisture/(100 − fat) × 100. The active acidity of the dairy product was measured applying a pH meter
(WTW, tip inoLab pH 720), and titratable acidity was expressed in terms of Soxhlet-Henkel/◦ SH value.
Sustainability 2020, 12, 6616 4 of 13

2.4. Amino Acid Profile

Amino acid profile of buffalo milk and dairy products was performed following the protocol
of Henderson [25]. The prepared samples were analyzed for the content of amino acids after acid
hydrolysis using the HPLC method (High-Performance Liquid Chromatograph Chromaster, Zorbax
Eclipse-AAA column (4.6 × 250, 5 µm) with DAD-3000 (diode array detector).

2.5. Fatty Acid Profile

To determine the milk fatty acids, a gas chromatograph (GC) with flame ioniyation detector
(FID) was used. Determination of the free fatty acids (FA) in the buffalo milk and dairy products
was done after methylation with boron trifluoride in methanol, using Shimadzu’s gas chromatograph
with FID on InterCap WAX (length 30 m, inner diameter 0.25 mm, film thickness 0.25 µm) column
(AOAC 996.06). Atherogenic (AI) and thrombogenic indices (TI) were calculated according to Ulbricht
and Southgate [26].

2.6. Statistical Analysis

Statistical analysis of the data was carried out using GraphPad Prism v6 (GraphPad, San Diego,
CA, USA) software. All data was normally distributed (Shapiro-Wilk normality test p > 0.05).
The homogeneity of variances was analyzed using Levene’s test and then the parametric model of
analysis of variances (ANOVA) followed by Tukey’s multiple comparison test, and t test. Significance
was established at p < 0.05.

3. Results

3.1. Milk Chemical Composition

The chemical composition of the buffalo milk samples is presented in Table 1. Results indicated
that fat and ash were the most inconsistent components, whereas lactose and MU content showed
minimum variation. As a result of a higher degree of fat variation, wide range of fat/protein ratio
(CV: 28.36%) was also found. The analysis confirmed that buffalo milk is rich in fat, protein, and lactose.
Therefore, the estimated energy value of buffalo milk varied from 81.81 to 106.62 (kcal/100 mL).

Table 1. Chemical composition of buffalo milk.

¯
Parameters x ± SD CV (%) Min–Max
Dry matter (%) 16.60 ± 1.12 6.75 14.91–17.80
Solid nonfat (%) 10.48 ± 0.52 4.96 9.97–11.51
Fat (%) 6.02 ± 1.27 21.10 4.26–7.16
Protein (%) 4.61 ± 0.52 11.28 4.12–5.62
Lactose (%) 5.36 ± 0.16 2.98 5.10–5.63
Ash (%) 0.60 ± 0.14 23.33 0.37–0.80
MU (mg/dL) 35.80 ± 0.88 2.46 33.90–36.70
F/P 1.34 ± 0.38 28.36 0.77–1.72
Energy value (kcal/100 mL) 96.90 ± 10.52 10.86 81.81–106.62
Milk density (g/cm3 ) 1.037 ± 0.002 0.19 1.036–1.042
x—arithmetic mean; SD—standard deviation; CV (%)—coefficient of variation; minimal X (min) and maximal X
(max) values of variables; F/P—fat/protein ratio; MU—milk urea.

3.2. Milk Amino Acid Composition

Table 2 summarizes the composition of amino acids in buffalo milk. The results of the amino
acid analysis revealed that leucine (0.41%) was the major amino acid, while lysine (0.33%) was second
among all essential amino acids. A substantial quantity of other branched amino acids (valine and
isoleucine) was also observed. Of the non-essential amino acids (NEAA), glutamic and aspartic acids
Sustainability 2020, 12, 6616 5 of 13

were found at a high level. Methionine is the first limiting amino acid. Higher variations of NEAA,
especially of tyrosine content (CV: 36.93%), were also observed.

Table 2. Amino acids composition of buffalo milk (%).

Essential Non-Essential
¯ ¯
Amino Acid x ± SD CV (%) Min–Max Amino Acid x ± SD CV (%) Min–Max
(EAA) (NEAA)
Histidine 0.15 ± 0.02 13.33 0.13–0.17 Asparatic acid 0.32 ± 0.02 5.71 0.29–0.33
Threonine 0.14 ± 0.01 7.14 0.12–0.15 Glutamic acid 1.01 ± 0.01 0.99 0.99–1.02
Valine 0.26 ± 0.01 3.85 0.25–0.27 Serine 0.09 ± 0.02 22.22 0.07–0.11
Methionine 0.05 ± 0.004 8.00 0.05–0.06 Glycine 0.08 ± 0.003 3.75 0.08–0.09
Phenylalanine 0.21 ± 0.01 4.76 0.19–0.23 Arginine 0.11 ± 0.01 9.09 0.10–0.12
Isoleucine 0.24 ± 0.01 4.17 0.23–0.25 Alanine 0.14 ± 0.01 7.41 0.13–0.16
Leucine 0.41 ± 0.01 2.44 0.39–0.42 Tyrosine 0.11 ± 0.04 36.36 0.08–0.16
Lysine 0.33 ± 0.02 6.06 0.30–0.35
Total 1.86
Total 1.79
x—arithmetic mean; SD-standard deviation; CV (%)—coefficient of variation; minimal X (min) and maximal X (max)
values of variables.

3.3. Milk Fatty Acid Composition

Saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acid (PUFA) content of
buffalo milk was found to be 69.04%, 28.46%, and 2.50%, respectively (Table 3). The values found
for fatty acid composition also showed that investigated samples contained a considerable amount
of saturated long chain fatty acid (SLCFA) content (C16:0, C18:0, C20:0; in total: 45.99 g/100 g). The
AI and TI amounted to 2.72 and 3.02, respectively, whereas the n-6/n-3 ratio was 2.42. Results also
showed that SLCFAs were the most stable in buffalo milk and varied least (CV: 9.41%), while SSCFA
and SMCFA possessed much higher coefficients of variation (CV: 33.05% and 27.48%, respectively).

Table 3. Fatty acid composition of buffalo milk (g/100 g).

¯
Parameters x ± SD CV (%) Min–Max
MUFA 25.43 ± 4.38 17.22 19.01–32.09
SSCFA 2.36 ± 0.78 33.05 1.21–3.31
SMCFA 13.32 ± 3.66 27.48 8.04–17.52
SLCFA 45.99 ± 4.33 9.41 40.98–53.58
SFA 61.68 ± 5.81 9.42 52.23–70.35
PUFA 2.23 ± 0.36 16.14 1.7–2.67
PUFA/SFA 0.04 ± 0.01 25.00 0.02–0.05
Total omega-6 fatty acids (n-6) 1.78 ± 0.22 12.36 1.37–2.06
Total omega-3 fatty acids (n-3) 0.77 ± 0.20 25.97 0.45–1.00
n-6/n-3 2.42 ± 0.59 24.38 1.85–3.69
Atherogenicity index (AI) 2.72 ± 1.05 38.60 1.56–4.71
Thrombogenicity index (TI) 3.02 ± 0.76 25.24 2.03–4.47
x—arithmetic mean; SD-standard deviation; CV (%)—coefficient of variation; minimal X (min) and maximal X (max)
values of variables; MUFA—monounsaturated fatty acids (C18:1); SSCFA—saturated short-chain fatty acids (C 4:0,
C8:0); SMCFA—saturated medium-chain fatty acids (C10:0, C12:0, C14:0); SLCFA—saturated long-chain fatty acids
(C16:0, C18:0, C20:0); SFA—total saturated fatty acids; PUFA—polyunsaturated fatty acids (C18:2, C18:3).

3.4. Chemical Composition of Buffalo Dairy Products

Mean composition values of cheese, butter, and kajmak produced from buffalo milk are shown in
Table 4. According to Serbian regulations [27], buffalo cheese is classified as a semi-fat and semi-hard
cheese, due to FDM and MFFB values of 43.5% and 60.1%, respectively. Moreover, kajmak samples met
the Serbian requirements for mature kajmak, and contained 77.1% of FDM, on average, and appropriate
values for pH and moisture (6.6 and 34.0%, respectively). On the other hand, butter did not meet
Serbian regulations for solids non-fat (SNF) content (>2%), but fat and moisture contents were within
the acceptable range. The results of chemical composition also showed that kajmak represents a special
Sustainability 2020, 12, 6616 6 of 13

and unique dairy product. It contains less fat compared with butter, as well as less protein than
cheese. Cheese is the most varied regarding fat content (CV: 14.51%), while SNF content was the most
inconsistent component in butter and kajmak (CV: 63.79% and 25.39%, respectively).

Table 4. Chemical composition of buffalo dairy products.

Cheese Butter Kajmak

Parameters
¯ ¯ ¯
x ± SD CV (%) x ± SD CV (%) x ± SD CV (%)
Moisture (%) 46.0 ± 1.20 2.61 14.2 ± 0.28 1.97 34.0 ± 1.30 3.82
Dry matter (%) 54.0 ± 1.20 2.22 85.8 ± 0.28 0.33 66.0 ± 1.30 1.97
Protein (%) 27.0 ± 1.62 6.0 0.9 ± 0.08 8.89 5.7 ± 0.34 5.96
Fat (%) 23.5 ± 3.41 14.51 83.0 ± 1.67 2.01 50.8 ± 2.56 5.04
FDM (%) 43.5 ± 5.22 12.0 96.7± 3.93 4.06 77.1 ± 5.39 6.99
MFFB (%) 60.1 ± 1.11 1.85 84.0 ± 8.12 9.67 69.3 ± 6.26 9.03
SNF 30.5 ± 2.21 7.24 2.9 ± 1.85 63.79 15.2 ± 3.86 25.39
pH 5.4 ± 0.12 2.22 6.7 ± 0.08 1.19 6.6 ± 0.12 1.82
◦ SH 60.0 ± 2.78 4.63 14.0 ± 0.44 3.14 14.0 ± 1.20 8.57
x—arithmetic mean; SD—standard deviation; CV (%)—coefficient of variation; FDM—Fat on a dry matter basis;
MFFB-Moisture on a fat-free basis; SNF—solids non-fat.

3.5. Amino Acid Composition of Buffalo Dairy Products

Buffalo cheese represents a good source of amino acids, and, as expected, it is significantly different
(p < 0.05) compared with kajmak and butter (Table 5.). In particular, buffalo cheese is abundant in
essential amino acids: lysine, leucine, and valine (4.42%, 3.90%, and 2.45%, respectively). The primary
non-essential amino acids were glutamic acid (5.09%) and tyrosine (1.97%). Results also revealed that
EAA/NEAA ratio in buffalo cheese is very favorable (61.76%/38.24%) and together with a high amount
of branched amino acids (leucine, valine, and isoleucine) confirmed the tremendous nutritional value of
buffalo cheese. Kajmak and butter, on the other hand, as high-fat dairy products contain a low amount
of protein and, consequently, amino acids. Compared with butter, kajmak contained a significantly
higher (p < 0.05) amount of threonine, leucine, and arginine. However, minor differences were noticed
in the isoleucine and glutamic acid content of butter and kajmak, whereas histidine, phenylalanine,
valine, and threonine were not detected.

Table 5. Amino acid composition of buffalo dairy products.

Cheese Butter Kajmak

Parameters
¯ ¯ ¯
x ± SD x ± SD x ± SD
Essential Amino Acid (EAA)
Histidine 0.75 ± 0.05 ND ND
Threonine 1.13 ± 0.01 a 0.02 ± 0.004 c 0.13 ± 0.02 b
Valine 2.45 ± 0.17 a ND 0.16 ± 0.04 b
Methionine 1.68 ± 0.08 a ND 0.10 ± 0.01 b
Phenylalanine 1.81 ± 0.04 a ND 0.12 ± 001 b
Isoleucine 2.03 ± 0.03 a 0.04 ± 0.004 c 0.13 ± 0.02 b
Leucine 3.90 ± 0.09 a 0.06 ± 0.007 c 0.26 ± 0.03 b
Lysine 4.42 ± 0.18 a 0.09 ± 0.07 b 0.26 ± 0.03 b
Total 18.17 0.21 1.16
Non-Essential Amino Acid (NEAA)
Aspartic acid 1.46 ± 0.14 a ND 0.23 ± 0.05 b
Glutamic acid 5.09 ± 0.20 a 0.19 ± 0.01 c 0.74 ± 0.05 b
Serine 0.99 ± 0.16 a 0.04 ± 0.01 b 0.17 ± 0.01 b
Glycine 0.42 ± 0.01 a 0.01 ± 0.002 c 0.07 ± 0.01 b
Arginine 0.90 ± 0.01 a 0.01 ± 0.001 c 0.20 ± 0.01 b
Alanine 0.42 ± 0.01 ND ND
Tyrosine 1.97 ± 0.20 a ND 0.11 ± 0.01 b
Total 11.25 0.25 1.52
x—arithmetic mean; SD—standard deviation; ND—Non-detectable; a,b,c —different letters indicate statistical
differences (p < 0.05), while same letters indicate no statistical difference.
Sustainability 2020, 12, 6616 7 of 13

3.6. Fatty Acid Composition of Buffalo Dairy Products

The fatty acid content of buffalo dairy products is presented in Table 6. Significant differences
(p < 0.05) were established between butter and kajmak regarding all groups of saturated fatty acids.
In particular, kajmak contained more SSCFA and SMCFA, whereas butter was more abundant in SLCFA.
MUFA content did not show a significant difference (p < 0.05) between all investigated dairy products.
The data showed that the nutritionally most beneficial fatty acids are found in kajmak. Thus, the fatty
acid composition grouped as, SFA, MUFA and PUFA were: in cheese: 66.85%, 29.06%, 4.09%; in butter:
70.82%, 26.28%, 2.09%; and in kajmak: 65.30%, 30.52%, 4.18%, respectively. Concerning health-related
factors (AI), significant differences (p < 0.05) were established between butter and kajmak. Furthermore,
it was found that kajmak had the most favourable AI and TI (1.84 and 1.82, respectively). Furthermore,
kajmak possessed the highest content of n-3 fatty acids, while cheese contained the highest amount of
n-6 fatty acids and had the highest n-6/n-3 ratio.

Table 6. Fatty acid composition of buffalo dairy products (g/100 g).

Cheese Butter Kajmak

Parameters
¯ ¯ ¯
x ± SD x ± SD x ± SD
MUFA 26.85 ± 2.82 a 24.06 ± 0.17 a 26.89 ± 1.56 a
SSCFA 2.53 ± 0.02 c 3.5 ± 0.22 b 5.41 ± 0.23 a
SMCFA 13.35 ± 0.51 b 14.88 ± 1.08 b 21.48 ± 0.65 a
SLCFA 45.90 ± 2.62 b 46.46 ± 2.48 b 30.09 ± 0.73 a
SFA 61.78 ± 3.15 a 64.84 ± 3.78 a 57.52 ± 1.51 a
PUFA 3.78 ± 0.21 a 2.66 ± 0.10 b 3.68 ± 0.31 a
PUFA/SFA 0.06 ± 0.01 a 0.04 ± 0.001 b 0.06 ± 0.005 a
Total omega-6 fatty acids (n-6) 3.77 ± 0.21 a 1.92 ± 0.05 c 2.65 ± 0.26 b
Total omega-3 fatty acids (n-3) 0.31 ± 0.03 c 0.74 ± 0.05 b 1.30 ± 0.08 a
n-6/n-3 12.16 ± 0.54 a 2.60 ± 0.11 b 2.04 ± 0.07 b
Atherogenicity index (AI) 2.31± 0.34 b 3.01 ± 0.16 a 1.84 ± 0.06 b
Thrombogenicity index (TI) 2.99 ± 0.37 a 3.12 ± 0.11 a 1.82 ± 0.08 b
x—arithmetic mean; SD-standard deviation. MUFA—monounsaturated fatty acids (C18:1); SSCFA—saturated
short-chain fatty acids (C 4:0, C8:0); SMCFA—saturated medium-chain fatty acids (C10:0, C12:0, C14:0);
SLCFA—saturated long-chain fatty acids (C16:0, C18:0, C20:0); SFA—total saturated fatty acids; PUFA—
polyunsaturated fatty acids (C18:2, C18:3); a,b,c —different letters indicate statistical differences (p < 0.05), while same
letters indicate no statistical difference.

4. Discussion
Dairy possesses an important role in the livestock sector and global healthy diets. It makes
a significant contribution towards meeting the challenges of nutritional security, sustainability,
and reduction in diseases related to poor quality diet [10]. On the other hand, it is very important
to consider the economic sustainability of primary production in such a chain, as profitability is a
precondition for the food chain to be relevant and sustainable [28]. To achieve economic sustainability
dairy farms and producers should meet consumers demand. In particular, for the realization of
sustainable non-bovine breeding in Serbia, it is necessary to take into account the habits and preferences
of consumers to ensure market supply chains [29]. The study conducted by Paskaš et al. [30] examined
the behaviour of consumers in Serbia and confirmed that healthiness and nutritional benefits are
amongst the most important factors for consuming non-bovine milk and dairy products.
Even within the same species, milk composition can vary, due to genetics, physiology, nutrition,
and environment [31]. According to Borghese and Moioli [5], buffalo milk possesses a higher fat
(6–9.5%) and protein (4–5%) content than cow milk. The present study has shown the mean content of
fat (6.02%) to fall within this range, but it was considerably less compared with the results of Tiezzi
et al. [32] and Liotta et al. [33] (7.56% and 8.78%, respectively). On the other hand, the obtained
mean protein content (4.61%) was in agreement with those researches (4.69% and 4.61%, respectively).
Sustainability 2020, 12, 6616 8 of 13

The energy value of milk is closely related to the concentration of certain compounds in dry matter,
especially the amount of fat [34]. Among the common dairy species, the energy density of buffalo
milk is remarkably high [11]. In the research of Mane and Chatli [16], the energetic value of buffalo
milk amounted to 117 kcal/100 g while presented results showed a lower value (96.90 kcal/100 mL).
Furthermore, our findings have shown that lactose is the second major constituent of buffalo milk,
with a minimum and maximum of 5.10% and 5.63%. On the contrary, Gantner et al. [31] reported that
the content of lactose in buffalo milk is ≤5%. In general, buffalo milk is a richer source of lactose than
cow, goat, sheep, and camel milk. From the health aspect, lactose could be a good source of energy for
body functions, particularly for the brain and hormonal regulation [12]. Many factors influence milk
quality, and for traditional cheese production milk fat, protein, and lactose content, as well as their
ratio, are essential [35]. The optimal fat/protein ratio in buffalo milk is 2:1 [17], while the presented
results showed an approximate value of 1.34.
Milk urea measured at the group level can be used to monitor the efficiency of nitrogen utilization
in commercial buffalo herds and as an indicator of the protein feeding situation in buffaloes [36].
Observed MU levels in this study ranged between 33.90–36.70 mg/dL and were lower compared with
the findings of Di Francia et al. [36] (40.8 mg/dL). Furthermore, Liotta et al. [33] recorded higher MU
value in intensive (40.68 mg/dL) than in semi-intensive buffalo herds (37.50 mg/dL). In contrast, Santillo
et al. [37] concluded that levels of MU were not different among buffalo groups that were fed low
protein diets and diets with flaxseed supplementation.
Buffalo milk can be considered a good source of essential amino acids, and lysine content was
found to be the highest, followed by valine and isoleucine. These findings correspond to Ren et al. [38].
Studies also have shown that buffalo milk contained methionine in traces, which is comparable with
the findings of Barlowska et al. [34].
The majority of fatty acids in ruminant milk are saturated [19]. Obtained results especially showed
a considerable amount of SLCFA (45.99 g/100 g). Buffalo milk contains almost three times more C14:0
(myristic) acid and two times less C16:0 (palmitic) acid than cow, sheep, or goat milk [34]. Regarding
fatty acids content, the previous research works are very diverse. Bustamante et al. [39] reported a
higher amount of MUFA (33.1% vs. 28.46%) and, at the same time, lower content of n-6 acids than the
current study (0.96% vs. 1.78%). In contrast, Pegolo et al. [40] compared with our results, found in
Mediterranean buffalo milk less amount of n-3 (0.46%) but approximately the same content of n-6 fatty
acids (1.78%), whereas findings of Gantner et al. [31] were in line with our data. Comparative studies
indicate that dairy breeds with a high milk fat content often have a less desirable milk fat composition,
have higher levels of saturated and hypercholesterolaemic fatty acids, and a lower proportion of PUFA
than breeds with a lower milk yield or fat content [41]. The importance of FA profile is reflected in
terms of the technological quality of raw milk. It possesses the potential to contribute to the production
of dairy products with added value. From a usability point of view, higher proportions of unsaturated
fatty acids are preferred (as they increase the spreading ability of butter), but their milk fat content
could also cause lower stability, oxidation, and possible sensory changes [19]. Nutritional effects of milk
and dairy products are highly influenced by fatty acid profile [42], as well, and FAs possess diverse
implications on human health [19]. Considering the two health-related indices, the ratio of essential FA
(n-6/n-3), and AI estimated values were higher than those reported by Varricchio et al. [43] (2.42 and
2.71 vs. 2.15 and 2.61, respectively). According to Claeys et al. [44], the n-6/n-3 ratio varies from 1.0 to
4.0 in ruminant milk. Consuming large amounts of n-3 fatty acids is beneficial and contributes to a
lower risk of coronary diseases and some types of cancer [34].
Considerable diversity traits of buffalo milk result in various directions of milk utilization. Each
buffalo dairy product varies in composition, and the present study discussed their different production,
composition, and potential health properties. Thanks to its specific characteristics, buffalo milk needs
to be treated differently in the cheese-making process. It is less suitable for the manufacture of hard
varieties of cheese, such as Cheddar cheese. Some problems that can occur are slow development of
acidity, higher curd tension, shorter renneting period, hard, dry, crumbly, corky body and texture, and
Sustainability 2020, 12, 6616 9 of 13

slower proteolysis [16]. The buffering capacity, pH, and viscosity of buffalo milk are higher than those
of cow milk, while the fermentation and ripening process of buffalo milk is generally slower [11]. Some
procedural improvements are necessary for hard cheese production from buffalo milk, such as higher
heat treatment, Mucor rennet, and a greater amount of Streptococcus thermophilus and Lactobacterium
bulgaricus cultures [16]. Results of the present study showed the good quality of semi-hard buffalo
cheese. Still, certain varieties including Mozzarella and white pickled Domiati cheese possess superior
quality when made from buffalo milk [15]. One of the main problems in the production of buffalo
semi-hard and hard cheese is the difficulty of converting milk into naturally ripened cheese [45].
Buffalo milk is also very desirable for the manufacture of fat-rich dairy products due to its higher
fat content, the bigger size of the globule, and the higher proportion of solid fat [17]. Industrial butter
is produced by the churning of cream, often after pasteurization. The homemade product is obtained
simply by churning acidified milk. A peculiarity of buffalo butter is the colour, which is much whiter
than cows’ milk butter, due to the lack of carotenoids [5]. Furthermore, buffalo milk produces butter
with a significantly higher yield, and displays more stability than that from cow cream, due to the
more solid fat and a slower rate of fat hydrolysis in the former cream [17]. The results of this study
confirm the results of [46,47] for butter chemical composition (pH, fat, and protein). Kajmak is a
homemade product, it is produced based on the traditional manufacturing procedure [13]. Kajmak
is a specific product, characterized by high-fat content, the presence of proteins, and its peculiar
ripening process [48]. During the ripening process, which lasts 3–4 weeks, kajmak partly loses the
continuity of its moisture phase, while limited fat phase continuity appears, and a specific flavor is
developed [13]. Therefore, the ripened kajmak made from cow milk published by Pudja et al. [13] had
values of chemical composition in the following ranges: moisture: 15–35%, fat: 50–70%, FDM: 75–90%
and proteins: 2–7%, and do not differ from values in the present study.
The present investigation revealed that in cheese, lysine was the major amino acid but also
a substantial quantity of branched-amino acids (valine, isoleucine, and leucine) was observed.
Branched-amino acids promote protein synthesis in muscle cells, and they are metabolized to generate
energy in muscles rather than in the liver [49]. Glutamic acid content was found to be the highest
of non-essential amino acids in cheese. Similarly, glutamic acid was the predominant non-essential
amino acid in butter made from cow milk, but in a much smaller amount. At the same time, leucine
and isoleucine were the main essential amino acids [50]. On the contrary, leucine was not detected in
the present study in buffalo butter.
Dairy products, such as butter, ghee, and cream, have been considered as basic nutrient-dense
foods that can deliver many energy-rich nutrients [46]. It was reported by Popović et al. [47] that
traditional Serbian dairy products, cheese, and kajmak, made from bovine milk, possessed a high
content of SFAs (70%), mainly palmitic acid. In contrast, the present study showed lower SFA contents
in buffalo milk cheese and kajmak and a more desirable fatty acid pattern. However, saturated fatty
acids were the predominant fraction in buffalo butterfat (70.49%). Kwak et al. [50] reported that
butter has double the concentration of saturated fat in comparison with that in cream and other dairy
products. Therefore, dairy products such as butter very often have been criticized for their unfavorable
FA profile [19]. However, stearic acid could be beneficial to health and contribute to the level of
low-density lipoprotein (LDL) in the blood [50]. Compared with butter, cheese possesses a more
favourable FA profile: in particular, higher proportions of MUFA and PUFA.
The manufacturing process could also affect the nutritional and health characteristics of the
cheeses [51]. Even though ripened cheeses contain more fat on a wet basis, their fatty acid profile
is more desirable. In particular, the Blu cheese shows a healthier fatty acid profile than Mozzarella
and the cheese-making process, and ripening contribute to reducing atherogenic (C12:0 and C14:0)
and increasing some beneficial fatty acids (C18:3 n-3, cis-9, trans-11 conjugated linoleic acid) [52].
Furthermore, it is supposed that milk fat with high AI and TI values may be more likely to contribute
to the development of atherosclerosis or coronary thrombosis in humans [53]. The value of AI in milk
and dairy products is around 2, whereas AI = 1.5 is considered as low, and 2.5 is high [54]. Accordingly,
Sustainability 2020, 12, 6616 10 of 13

our dairy products, kajmak, and cheese appeared to exhibit stronger anti-atherogenic activities
compared with milk and butter. The thrombogenic indices take into account the relationship between
the pro-thrombogenic (saturated) and anti-thrombogenic fatty acids (unsaturated) [26]. Despite being
a high-fat product, kajmak also showed a low TI value (1.82).

5. Conclusions
The buffalo production under sustainable principles and extensive husbandry plays an essential
role in the heritage of local rural populations and their economy in South-West Serbia. Buffalo milk is
used for manufacturing of traditional milk products, such as cheese, butter, and kajmak. The results
of the chemical composition of buffalo milk showed that it could be utilized for making a variety of
good quality traditional dairy products with added value. Consequently, they are important for the
dairy industry and especially useful for traditional artisanal production. The analysed parameters
of milk and dairy products showed considerable diversity, and nutritional value varies from one
product to the other. In particular, the nutritional content of ripened kajmak and cheese was the
most favourable. Primarily, kajmak represents a valuable source of essential fatty acids, while cheese
was abundant in important branched amino acids (leucine, valine, and isoleucine). These results can
contribute to the promotion of the value-added buffalo dairy products from sustainable production
systems. Although buffaloes are successfully bred with traditional methods on pastures, to achieve
more consistent and sustainable milk production, some husbandry improvements should be required.
In particular, more attention should be paid to the uniform quality of the raw milk, selective breeding,
and productivity of animals. Thus, these would improve the farm economy and the quality of
derived products, which could actively contribute to rural development of the region and more
effective conservation of buffalo genetic resources. Furthermore, to enhance buffalo production in more
sustainably and holistically, producers should be encouraged to access appropriate market information
and take into account more sustainable initiatives in the dairy industry, such as the nutritional and
health benefits of dairy products.

Author Contributions: Conceptualization, Z.B., S.P. and M.S.; methodology, S.P.; software, S.P.; validation, S.P.;
formal analysis, Z.B., M.R., N.P. and M.P.; investigation and resources, Z.B., M.S., D.Ć., S.Ð. and M.P.; data curation,
S.P.; writing—original draft preparation, S.P., Z.B.; writing—review and editing, S.P., Z.B., N.P.; visualization,
M.S.; supervision, Z.B.; project administration, M.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Ministry of Education, Science and Technological Development of the
Republic of Serbia, grant number TR31085.
Acknowledgments: This study was supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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