CyTA - Journal of Food

ISSN: 1947-6337 (Print) 1947-6345 (Online) Journal homepage: https://www.tandfonline.com/loi/tcyt20

Characterization of pine bud syrup and its effect

on physicochemical and sensory properties of
kefir

Cristina Anamaria Semeniuc, Ancuţa Rotar, Laura Stan, Carmen Rodica Pop,
Sonia Socaci, Vioara Mireşan & Sevastița Muste

To cite this article: Cristina Anamaria Semeniuc, Ancuţa Rotar, Laura Stan, Carmen Rodica Pop,
Sonia Socaci, Vioara Mireşan & Sevastița Muste (2016) Characterization of pine bud syrup and its
effect on physicochemical and sensory properties of kefir, CyTA - Journal of Food, 14:2, 213-218,
DOI: 10.1080/19476337.2015.1085905

To link to this article: https://doi.org/10.1080/19476337.2015.1085905

© 2015 The Author(s). Published by Taylor & Published online: 28 Sep 2015.
Francis.

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CyTA – Journal of Food, 2016
Vol. 14, No. 2, 213–218, http://dx.doi.org/10.1080/19476337.2015.1085905

Characterization of pine bud syrup and its effect on physicochemical and sensory properties
of kefir
Caracterización del sirope de pino y sus efectos en las propiedades fisicoquímicas y sensoriales
del kéfir
Cristina Anamaria Semeniuc a, Ancuţa Rotar b
*, Laura Stan b
, Carmen Rodica Pop a
, Sonia Socaci b
,
Vioara Mireşanc and Sevastița Muste b
a
Department of Food Engineering, Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary
Medicine of Cluj-Napoca, 3-5, Mănăştur Street, 400372 Cluj-Napoca, Romania; bDepartment of Food Science, Faculty of Food
Science and Technology, University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, 3-5, Mănăştur Street, 400372
Cluj-Napoca, Romania; cDepartment of Fundamental and Biotechnology Sciences, Faculty of Animal Science and Biotechnology,
University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, 3-5, Mănăştur Street, 400372 Cluj-Napoca, Romania
(Received 20 May 2015; final version received 4 August 2015)

This study aimed to develop a value-added kefir by the addition of pine bud syrup. For this purpose, different concentrations of pine bud
syrup [2, 4, 6, 8, and 10% (w/w)] were added to kefir formulation after the fermentation. The pine bud syrup used for kefir fortification is
rich in polyphenols and terpenes and has a high antioxidant activity. The study was designed to evaluate the influence of pine bud syrup on
the physicochemical and sensory properties of kefir. The addition of pine bud syrup resulted in an increase in total solids and a decrease in
fat content, proteins, and pH. The kefir sample with 10% pine bud syrup was the most appreciated by sensory panelists. Its overall
acceptability score was higher (6.71 points) than regular kefir (5.57 points). The addition of 10% pine bud syrup improved the texture and
consistency of regular kefir.
Keywords: kefir; processing; fortification; physicochemical properties; sensory analysis

Este estudio tiene como objetivo el desarrollo de un kéfir con un valor adicional añadiéndole sirope de pino. Para conseguir este propósito, se
añadieron diferentes concentraciones de sirope de pino [2, 4, 6, 8 y 10% (w/w)] a la formulación del kéfir después de la fermentación. El sirope
de pino que se utilizó para la fortificación del kéfir es rico en polifenoles y tarpanes, además de tener una actividad antioxidante alta. Este
estudio se diseñó para evaluar la influencia del sirope de pino en las propiedades fisicoquímicas y sensoriales del kéfir. La adición de sirope de
pino resultó en un aumento del total de sólidos y una disminución del contenido en grasa, proteínas y pH. La muestra de kéfir con un 10% de
sirope de pino fue la que los panelistas sensoriales valoraron con mayor puntuación. Su puntuación total de aceptabilidad fue mayor (6,71
puntos) que la del kéfir normal (5,57 puntos). Al añadir un 10% de sirope de pino se mejoró la textura y la consistencia del kéfir normal.
Palabras clave: Kéfir; Procesamiento; Fortificación; Propiedades fisicoquímicas; Análisis sensorial

Introduction In Romanian traditional medicine, pine bud syrup is used

Kefir is obtained by the acidification of milk with kefir grains.
Kefir grains contain different species of bacteria (Lactobacillus
kefiri and other species of the genera Leuconostoc, Lactococcus,
and Acetobacter) and yeasts (Kluyveromyces marxianus,
Saccharomyces unisporus, Saccharomyces cerevisiae, and
Saccharomyces exiguus). The microorganisms from the starter
culture must be viable, active, and abundant in the product
during its shelf life (CODEX STAN 243, 2003).
Kefir is widely produced and consumed in the Middle East,
Eastern Europe, and Central Asia, especially, Turkey, Russia,
Ukraine, Poland, and the Czech Republic (Farnworth &
Mainville, 2008; Hutkin, 2006). It originated in the Caucasus
Mountains and traditionally was fermented in animal skin bags
(Ŏzer & Kirmaci, 2014). Presently, kefir is one of the most
popular fermented milk products in Romania. The literature
mentions some of the health benefits of regular kefir consump-
tion. These include antibacterial, antifungal, and antineoplastic
effects (Cevikbas et al., 1994), as well strengthening the immune
system (Ahmed et al., 2013).
against colds and flu. However, few studies were carried out on
the coniferous bud syrup (pine, fir, and spruce). In our analysis
of the literature, we found only the study of Deac, Fărcaş,
Vodnar, Tofană, and Socaci (2014) that investigated the antiox-
idant and antimicrobial properties of fir bud syrup.
In previous studies, the fortification of kefir was carried out
with inulin (Ertekin & Guzel-Seydim, 2010; Montanuci,
Pimentel, Garcia, & Prudencio, 2012; Pilipenko, Burliaeva,
Shakhovskaia, & Isakov, 2009; Tratnik, Bozanic, Zoran, &
Drgalić, 2006), Dary-Lo (Ertekin & Guzel-Seydim, 2010),
brewer’s yeast (Păucean, Rotar, Jimborean, Mudura, &
Socaciu, 2009), and Propionibacterium freudenreichii strains
(Van Wyk, Witthuhn, & Britz, 2011).
Various parts of the pine tree (i.e., buds, needles, twigs,
cones, and nut shells) are used to extract essential oil. There
are several studies on the chemical composition, antioxidant, and
antimicrobial activities of the essential oil from pine
needles, pine cones, pine nut shells, and pine branches
(Judžentienė, Šližytė, Stiklienė, & Kupčinskienė, 2006;
Koukos, Papadopoulou, Patiaka, & Papagiannopoulos, 2000;

*Corresponding author. Email: anca.rotar@usamvcluj.ro

© 2015 The Author(s). Published by Taylor & Francis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
214 C.A. Semeniuc et al.
Liu & Xu, 2012; Mimoune, Mimoune, & Yataghene, 2013;
Tumen et al., 2010; Yang et al., 2010; Zeng, Zhang, Gao, Jia,
& He, 2012). Yet, the composition of pine bud essential oil is
poorly known. In general, the essential oil from conifers is
rich in terpenes (Lavabre, 1996). Monoterpenes are the most
commonly found terpenes in plant volatile oils; they have
antiseptic, bactericidal, analgesic, expectorant, and stimulating
properties (Price, 2012).
There are also studies that show the antioxidant, anti-inflam-
matory, and antinociceptive effects of alcoholic and aqueous
extracts from pine pollen, pine needles, pine buds, and pine
bark (Apetrei et al., 2011; Choi, 2007; Feng et al., 2010; Lee,
Kim, & Choi, 2009; Park et al., 2011).
The main purpose of this research was to obtain a fortified
kefir by the addition of pine bud syrup. Five different levels of
pine bud syrup [2, 4, 6, 8, and 10% (w/w)] were added to kefir
formulation after the fermentation. The current study was
designed to (i) advance scientific knowledge of pine bud syrup
phytocompounds in order to highlight their contribution to pos-
sible health benefits, (ii) evaluate the effect of fortification on
kefir physicochemical and sensory properties, and (iii) establish
the level of added ingredients based on consumer acceptance.
This study may be useful to dairy producers in the strategy of
widening their product range considering that the consumption
of functional food is increasing.
Materials and methods
Preparation of pine bud syrup
Pine tree buds were harvested when they reached 3–4 cm in
length, washed, and then boiled in water (2:1 ratio of water to
pine buds, v/w) for 1 h. After 48 h of storage at room tempera-
ture (20°C), the extract was filtered and mixed with white sugar
(1.5:1 ratio of white sugar to pine buds, w/w), and then was
boiled until it reached the desired consistency. Once the syrup
cooled down, 200 mL of lemon juice was added. The lemon
juice is added to decrease the pH of pine bud syrup to an acidic
level to prevent microorganism growth and fermentation. Pine
bud syrup was then stored at room temperature in sealed color
glass bottles.
Preparation of kefir added with pine bud syrup
Frozen kefir grains come from the collection of the Walloon
Agricultural Research Center (CRA-W, Gembloux, Belgium).
The bacteria and yeast strains present in kefir grains include
Lactobacillus kefiranofaciens subsp. kefirgranum, Lactobacillus
kefiri, Lactobacillus parakefiri, Lactococcus lactis subsp. lactis,
Leuconostoc mesenteroides, Acetobacter sp., and Kazachstania
exigua.
Reactivation of kefir grains
For thawing, frozen kefir grains were kept in the refrigerator at
+4°C for 2 h, then at room temperature for another 2 h.
Subsequently, they were washed with distilled water and inocu-
lated (5%, w/v) in pasteurized cow milk (1.5% fat). After fer-
mentation was completed (24 h), kefir grains were separated
from fermented milk and washed with distilled water for the
next inoculation.
Inoculation of milk with kefir grains
Standardized cow milk (3.5% fat) was inoculated with 5% (w/v)
kefir grains and incubated at 25°C for approximately 24 h. Next,
kefir grains were separated from kefir, washed with distilled
water, and stored at −18°C until further use. The kefir was then
fortified with different levels of pine bud syrup [2 (K2), 4 (K4), 6
(K6), 8 (K8), and 10% (K10)]. The control sample (K0) consisted
of kefir without pine bud syrup. Samples were collected and
analyzed on the same day. Until analysis, all kefir samples
were kept in the refrigerator (at +4°C).
Pine bud syrup analysis
Total soluble content was measured in an ABBE-refractometer
AR2 (Krüss Optronic GmbH, Hamburg, Germany). On the
refractometer prism, a drop of syrup was placed for measure-
ment. The reading was taken in Brix degree (°Bx). Two readings
per sample were run.
Measurement of total phenolic content was based on the
Folin–Ciocalteu method described by Mureşan-Cerbu et al.
(2012) with some modifications. An aliquot of 25 μL pine bud
syrup (diluted 1:1 with distilled water, v/v) was transferred into a
glass test tube with a screw cap. Then, 1.8 mL of distilled water
and 120 μL of 2 N Folin–Ciocalteu phenol reagent were added
and immediately vortexed. After 5 min, 340 μL of sodium
carbonate aqueous solution (7.5%, w/v) was added to the mix-
ture. The incubation of the test sample was carried out in the
dark, at room temperature for 90 min. The absorbance value was
read at 750 nm against methanol with a double-beam UV-VIS
spectrophotometer (PharmaSpec UV-1700, Shimadzu, Kyoto,
Japan). The standard curve range was 0–1.0 mg/mL gallic acid
(y = 0.6859x – 0.0098, r2 = 0.9997). Three readings per sample
were taken. The concentration obtained from the standard curve
was multiplied by the dilution factor and by 100. Total phenolic
content is expressed as mg gallic acid equivalents (GAE)/
100 mL.
The antioxidant activity was determined using the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) method according to
Mureşan, Muste, Borşa, Vlaic, and Mureşan (2014) with
some modifications. An aliquot of 10 μL pine bud syrup was
transferred into a glass test tube with a screw cap, then 90 μL
of distilled water and 3.9 mL of DPPH solution in methanol
(0.025 g/L) were added. The incubation of the test sample was
carried out in dark, at room temperature for 30 min. The
absorbance value was read at 515 nm against methanol with
a double-beam UV-VIS spectrophotometer (PharmaSpec UV-
1700, Shimadzu, Kyoto, Japan). Three readings per sample
were taken. In the same manner as the sample, both controls
were treated. The positive control was prepared using a gallic
acid solution (0.5 mg/mL). The negative control was prepared
using methanol. The DPPH free radical scavenging activity
(RSA) was calculated using the following equation and
expressed in percentage:
AbsDPPH  Abssample
ð%Þ RSA ¼  100 (1)
AbsDPPH
where AbsDPPH is the absorbance of DPPH free radical in
methanol and Abssample is the absorbance of DPPH free radical
solution mixed with the sample/standard.
Qualitative ITEX/GC-MS analysis of the volatile compounds
was performed using an analytical technique based on in-tube

extraction (ITEX). One gram of pine bud syrup was weighed
into a 20 mL sealed-cap headspace vial and kept at 60°C for
20 min with continuous agitation. A Combi PAL AOC-5000
autosampler (CTC Analytics, Zwingen, Switzerland) with a
headspace syringe ITEX-II equipped with a microtrap (ITEX-
2TrapTXTA, Tenax TA 80/100 mesh, Switzerland) was used for
the adsorption (30 strokes) of volatile compounds from the
gaseous phase of the sample. The analytes were released by
thermal desorption into the injection port of the GC-MS-
QP2010 system (Shimadzu, Kyoto, Japan); the microtrap was
then flash-heated with N2.
The chromatographic separation of volatile compounds was
performed on a Zebron ZB-5 ms capillary column
(30 m × 0.25 mm i.d. × 0.25 μm film thickness; Phenomenex,
USA). The column oven temperature program was set as fol-
lows: from 40°C (kept at this temperature for 5 min) to 180°C at
7°C/min, then raised to 240°C at 15°C/min (kept at this tem-
perature for 5 min). The ion source, injector, and interface
temperatures were set at 250°C. The carrier gas was helium, at
a constant flow rate of 0.84 mL/min. The split ratio was 2:1. The
MS mode used was Electron Impact (EI) ionization. A mass
range from 40 to 650 m/z was scanned.
The identification of volatile compounds was performed by
comparing their mass spectra with those in the NIST27 and
NIST147 libraries and by retention indices drawn from www.
pherobase.com or www.flavornet.org (for columns with a similar
stationary phase to ZB-5 ms). The result is expressed as a
percentage of the total area.
Physicochemical analysis of kefir with pine bud syrup
Determination of fat content was performed using the Gerber
method described by SR ISO 488:2009. Total protein content
was determined using the Kjeldahl method described in SR EN
ISO 8968-1:2014 and total solids content according to the SR
ISO 13580:2009 method. The results are expressed in percen-
tage. Titratable acidity was determined according to the ISO/TS
11869:2012 method and expressed as percent lactic acid. pH was
measured at room temperature using a Consort C535 pH meter
(Consort nv, Turnhout, Belgium). For each physicochemical
parameter, two replicates per sample were run.
Sensory evaluation of kefir with pine bud syrup
Sensory characteristics of samples were evaluated by a panel of
26 trained assessors (13 female and 13 male), with a mean age of
23. Kefir samples were presented in a random order in plastic
cups. The nine-point hedonic scale test (1 was the worst and 9
was the best) was used to evaluate all kefir samples. Overall
acceptability, texture, consistency, color, smell, taste, and flavor
were the sensory attributes evaluated.
Statistical analysis
To perform statistical tests, Minitab statistical software (version
16.1.0; LEAD Technologies, Inc., Charlotte, NC, USA) was
used. One-way analysis of variance (ANOVA) was applied to
determine whether there are any significant differences between
treatments. Tukey’s test (α = 0.5) was used to determine the level
of significance. Correlations among data were calculated using
Pearson’s correlation coefficient.
CyTA – Journal of Food 215
Results and discussion
Characterization of pine bud syrup
The total soluble solids content in pine bud syrup was 61.75°Bx
and the total phenolic content was 123.20 mg GAE/100 mL. The
RSA of pine bud syrup was 68.88%; for the negative control it
was 2.82% and for the positive control it was 70.90%. In fir bud
syrup, Deac et al. (2014) have found values of 21.12 mg GAE/
100 mL and 18.59% RSA, approximately six times lower than
those in pine bud syrup. The high antioxidant activity of pine
bud syrup used in this study is most probably due to its increased
total phenolic content.
Table 1 lists the volatile compounds detected in pine bud
syrup (in their order of elution) and their percentage composi-
tion. To the best of our knowledge, this is the first study on the
volatile fingerprinting of pine bud syrup. The volatile profile of
pine bud syrup contains 91.81% terpenes, 3.77% aldehydes,
2.59% ketones, 0.84% alcohols, 0.46% organic acids, and
0.28% hydrocarbons. The terpene fractions include monoterpene
hydrocarbons (80.44%), sesquiterpene hydrocarbons (1.36%),
monoterpene alcohols (7.38%), monoterpene esters (2.41%),
and monoterpene bicyclic ketones (0.22%). The monoterpene
hydrocarbons class is the main terpene fraction also found in
pine needle essential oil (Judžentienė et al., 2006) and pine cone
essential oil (Tumen et al., 2010; Yang et al., 2010). The major
volatile compounds found in pine bud syrup are α-pinene
(28.82%) and d-limonene (24.84%), which belongs to the mono-
terpene hydrocarbons class. Alpha-pinene, an organic monoter-
pene, is usually found in essential oils from conifer trees (Aydin,
Türkez, & Geyikoǧlu, 2013). Other monoterpenes identified in
pine bud syrup are 3-carene (7.75%), α-terpineol (6.30%), cam-
phene (4.01%), β-pinene (3.15%), o-cymene (2.57%), terpino-
lene (2.52%), α, p-dimethylstyrene (2.31%), and β-trans-ocimene
(2.12%). The sesquiterpene hydrocarbons class is poorly repre-
sented by caryophyllene (1.02%) and α-caryophyllene (0.34%).
Within the terpene oxidation products, bornyl acetate (2.13%) is
the main volatile compound found. Among the identified volatile
compounds, α-pinene, β-pinene, 3-carene, 4-carene, 1-terpinen-
4-ol, terpinolene, α,p-dimethylstyrene, and linalool acetate are
responsible for the pine-like aroma. d-Limonene, 3-carene,
o-cymene, and α,p-dimethylstyrene are responsible for the citrus
aroma.
Effect of fortification on physicochemical properties
Fortification with pine bud syrup affected the physicochemical
composition of kefir. Table 2 presents the values of the physico-
chemical parameters for the kefir samples tested. The addition of
pine bud syrup had a significant effect on kefir fat content
(p < 0.05), proteins (p < 0.001), total solids (p < 0.001), and
pH (p < 0.001), but no significant effect on titratable acidity
(p > 0.05). The fat content, proteins, and pH decrease and total
solids increase with the amount of added pine bud syrup. The
strong negative correlations found between total solids and fat
content (r2 = −0.812; p = 0.001), total solids and proteins
(r2 = −0.955; p = 0.000), and total solids and pH (r2 = −0.867;
p = 0.000) confirm the above information. The fat content ranged
from 3.05% to 3.45%, protein content from 2.65% to 3.83%,
total solids from 12.30% to 16.61%, lactic acid from 0.918% to
0.927%, and pH from 4.23 to 4.34. According to the Codex
standard for fermented milks (CODEX STAN 243, 2003), the
composition of kefir must contain a maximum of 10% milk fat, a
Table 1. Volatile compounds detected in pine bud syrup and comparison to literature data.
Tabla 1. Compuestos volátiles detectados en el sirope de pino y comparación con los datos de la literatura. 216
No. Class Compound Concentration (%) Characteristic odor Literature

1 Terpenes α-Pinene 28.82 Pine, turpentine Pine twig essential oil (Koukos et al., 2000), pine needle litter (Isidorov, Vinogorova, & Rafałowski, 2003),
pine needle essential oil (Judžentienė et al., 2006; Koukos et al., 2000; Mimoune et al., 2013; Zeng
et al., 2012), pine cone essential oil (Tumen et al., 2010; Yang et al., 2010), pine nut shell essential oil
(Liu & Xu, 2012)
2 Camphene 4.01 Camphoraceous Pine twig essential oil (Koukos et al., 2000), pine needle litter (Isidorov et al., 2003), pine needle essential
oil (Judžentienė et al., 2006; Koukos et al., 2000), pine cone essential oil (Tumen et al., 2010; Yang
et al., 2010), pine nut shell essential oil (Liu & Xu, 2012)
3 β-Pinene 3.15 Pine, resin, turpentine Pine twig essential oil (Koukos et al., 2000), pine needle litter (Isidorov et al., 2003), pine needle essential
oil (Judžentienė et al., 2006; Koukos et al., 2000; Zeng et al., 2012), pine cone essential oil (Tumen
C.A. Semeniuc et al.

et al., 2010; Yang et al., 2010), pine nut shell essential oil (Liu & Xu, 2012)
4 3-Carene 7.75 Lemon, resin Pine needle litter (Isidorov et al., 2003), pine cone essential oil (Yang et al., 2010), pine needle essential oil
(Zeng et al., 2012), pine nut shell essential oil (Liu & Xu, 2012)
5 4-Carene 0.54 Sweet, pine, cedar, woodsy, pungent
6 o-Cymene 2.57 Citrus Pine cone essential oil (Yang et al., 2010)
7 d-Limonene 24.84 Lemon, orange Pine needle litter (Isidorov et al., 2003), pine needle essential oil (Judžentienė et al., 2006; Zeng et al.,
2012), pine cone essential oil (Tumen et al., 2010; Yang et al., 2010)
8 β-trans-Ocimene 2.12 Sweet, herb Pine needle litter (Isidorov et al., 2003), pine needle essential oil (Judžentienė et al., 2006)
9 γ-Terpinene 0.37 Turpentine, herbaceous, fruity, sweet Pine needle litter (Isidorov et al., 2003), pine needle essential oil (Judžentienė et al., 2006), pine cone
essential oil (Yang et al., 2010)
10 Terpinolene 2.52 Pine Pine needle litter (Isidorov et al., 2003), pine needle essential oil (Judžentienė et al., 2006)
11 α,p-Dimethylstyrene 2.31 Citrus, pine
12 β-Linalool 0.24 Flower, lavender, muscat, sweet, green Pine needle litter (Isidorov et al., 2003)
13 1,3,8-p-Menthatriene 0.15 Turpentine
14 Fenchol 0.94 Camphoraceous, earthy, dry, rooty Pine cone essential oil (Yang et al., 2010)
15 Borneol 0.58 Camphoraceous, musty Pine needle essential oil (Judžentienė et al., 2006), pine cone essential oil (Yang et al., 2010)
16 1-Terpinen-4-ol 0.26 Woody, sweet, herbaceous, pine, musty, fruity, Pine needle essential oil (Judžentienė et al., 2006; Zeng et al., 2012), pine cone essential oil (Tumen et al.,
nutmeg 2010)
17 α-Terpineol 6.30 Oily, anise, minty, peach-like Pine twig essential oil (Koukos et al., 2000), pine cone essential oil (Tumen et al., 2010), pine needle
essential oil (Zeng et al., 2012)
18 l-Verbenone 0.22 Minty, spicy Pine needle litter (Isidorov et al., 2003), pine cone essential oil (Tumen et al., 2010), pine needle essential
oil (Mimoune et al., 2013; Yang et al., 2010)
19 Carvone 0.33 Minty Pine cone essential oil (Yang et al., 2010)
20 Bornyl acetate 2.13 Caraway-like, peppermint Pine twig essential oil (Koukos et al., 2000), pine needle essential oil (Judžentienė et al., 2006; Koukos
et al., 2000), pine cone essential oil (Tumen et al., 2010; Yang et al., 2010)
21 Linalool acetate 0.28 Sweet, fruity, bergamot-like, resinous
22 Caryophyllene 1.02 Wood, spicy Pine needle essential oil (Zeng et al., 2012), pine nut shell essential oil (Liu & Xu, 2012)
23 α-Caryophyllene 0.34 Oily, fruity, woody Pine cone essential oil (Yang et al., 2010), pine nut shell essential oil (Liu & Xu, 2012)
24 Aldehydes Hexanal 0.19 Green, fruity, grassy, herbal, leafy
25 Furfural 0.91 Almond, sweet
26 Heptanal 0.17 Fat, citrus, fruity, solvent
27 Benzaldehyde 0.53 Almond, burnt sugar
28 Octanal 0.69 Lemon, green, flower, orange
29 Nonanal 1.01 Fat, green
30 Decanal 0.18 Orange peel, tallow, burnt, green, waxy, floral
31 Undecanal 0.09 Oil, pungent, sweet
32 Ketones Acetophenone 1.05 Must, floral, almond
33 3ʹ-Methyl- 1.54 Floral
acetophenone
34 Alcohols 1-Octanol 0.84 Chemical, metal, burnt
35 Organic acids Benzoic acid 0.46 Wine-like, very weak, balsamic odor
36 Hydrocarbons Toluene 0.28 Caramel, ethereal, fruity, rubbery, solvent-like Pine needle litter (Isidorov et al., 2003)
37 – n.i. 0.25 –

Note: n.i., not identified.

Nota: n.i, sin identificar.
 CyTA – Journal of Food 217

Table 2. Physicochemical properties of kefir samples.

Tabla 2. Propiedades fisicoquímicas de las muestras de kéfir.

Formulations Fat (%) Proteins (%) Total solids (%) Titratable acidity (% lactic acid) pH

K0 3.45 ± 0.071a 3.83 ± 0.071a 12.30 ± 0.234a 0.927 ± 0.000a 4.34 ± 0.007a
K2 3.35 ± 0.071a 3.70 ± 0.014ab 12.62 ± 0.182a 0.927 ± 0.013a 4.32 ± 0.007ab
K4 3.35 ± 0.071a 3.56 ± 0.064bc 13.83 ± 0.066b 0.923 ± 0.006a 4.32 ± 0.007ab
K6 3.25 ± 0.071ab 3.42 ± 0.064c 14.70 ± 0.016c 0.923 ± 0.006a 4.31 ± 0.007bc
K8 3.25 ± 0.071ab 2.98 ± 0.071d 16.31 ± 0.326d 0.918 ± 0.013a 4.29 ± 0.007c
K10 3.05 ± 0.071b 2.65 ± 0.042e 16.61 ± 0.144d 0.918 ± 0.000a 4.23 ± 0.007d

Note: aK0, kefir without pine bud syrup; K2, kefir with 2% pine bud syrup; K4, kefir with 4% pine bud syrup; K6, kefir with 6% pine bud syrup; K8, kefir
with 8% pine bud syrup; K10, kefir with 10% pine bud syrup.
b
Different letters (on column) indicate statistically significant differences at p < 0.05 (Tukey’s test).
Nota: aK0-kéfir sin sirope de pino; K2- kéfir con 2% de sirope de pino; K4- kéfir con 4% de sirope de pino; K6- kéfir con 6% de sirope de pino; K8- kéfir
con 8% de sirope de pino; K10- kéfir con 10% de sirope de pino.
b
Las diferentes letras (en la columna) indican diferencias estadísticamente significativas a p < 0,05 (Test de Tukey).

Table 3. Hedonic scores of sensory attributes in kefir samples.

Tabla 3. Resultados hedónicos de los atributos sensoriales en las muestras de kéfir.

Formulations Texture Consistency Color Smell Taste Flavor Overall acceptability

K0 5.56 ± 2.18ab 5.52 ± 1.87bc 7.28 ± 1.28a 5.68 ± 1.95a 4.60 ± 1.94a 4.76 ± 1.71a 5.57 ± 1.09bc
K2 4.24 ± 2.07b 4.12 ± 2.05c 6.88 ± 1.09a 5.04 ± 2.21a 4.64 ± 2.33a 4.80 ± 2.45a 4.95 ± 1.54c
K4 4.92 ± 2.12b 5.00 ± 2.08bc 6.72 ± 1.21a 5.08 ± 1.98a 4.96 ± 2.07a 5.04 ± 2.15a 5.29 ± 1.47bc
K6 5.08 ± 2.45b 5.12 ± 2.40bc 6.92 ± 1.26a 5.60 ± 1.71a 4.64 ± 2.56a 4.72 ± 2.46a 5.35 ± 1.26bc
K8 6.76 ± 1.68a 6.56 ± 2.00ab 7.32 ± 0.75a 6.28 ± 1.37a 5.24 ± 2.30a 5.40 ± 2.27a 6.25 ± 1.16ab
K10 7.04 ± 1.31a 7.16 ± 1.25a 7.28 ± 0.98a 6.40 ± 1.58a 6.20 ± 1.91a 6.24 ± 1.98a 6.71 ± 1.12a

Note: aK0, kefir without pine bud syrup; K2, kefir with 2% pine bud syrup; K4, kefir with 4% pine bud syrup; K6, kefir with 6% pine bud syrup; K8, kefir
with 8% pine bud syrup; K10, kefir with 10% pine bud syrup.
b
Different letters (on column) indicate statistically significant differences at p < 0.05 (Tukey’s test).
Nota: aK0-kéfir sin sirope de pino; K2- kéfir con 2% de sirope de pino; K4- kéfir con 4% de sirope de pino; K6- kéfir con 6% de sirope de pino; K8-kéfir con
8% de sirope de pino; K10- kéfir con 10% de sirope de pino.
b
Las diferentes letras (en la columna) indican diferencias estadísticamente significativas a p < 0,05 (Test de Tukey).

minimum of 2.7% milk protein, and 0.6% lactic acid. All kefir
formulations meet these requirements.
Effect of fortification on sensory properties
Fortification of kefir with pine bud syrup also influenced its
sensory properties. Table 3 lists the hedonic scores for sensory
attributes (texture, consistency, color, smell, taste, and flavor)
of kefir samples. The addition of pine bud syrup had a
significant effect on the texture (p < 0.001) and consistency
(p < 0.001) of kefir, but was not statistically significant on
color, smell, taste, and flavor. The kefir sample with 10% pine
bud syrup was the most appreciated by panelists in terms of
consistency, smell, flavor, and taste. The highest overall score
was also found in K10 (6.71 points), followed by K8 (6.25
points), K0 (5.57 points), K6 (5.35 points), K4 (5.29 points),
and K2 (4.95 points). The paired t-test shows no significant
difference (t(5) = 2.35; p = 0.065) between male mean total
scores [5.69 ± 1.51(K0); 6.28 ± 0.95(K2); 6.53 ± 0.95(K4);
5.64 ± 1.64(K6); 6.69 ± 1.39(K8); 6.67 ± 1.09(K10)] of this
study’s panelists and female mean total scores [5.53 ± 1.90
(K0); 4.54 ± 2.09(K2); 4.89 ± 1.96(K4); 5.25 ± 2.28(K6);
6.12 ± 1.80(K8); 6.74 ± 1.63(K10)]. The strong positive cor-
relations between total solids and overall score (r2 = 0.834;
p = 0.039), total solids and texture (r2 = 0.817; p = 0.047),
and total solids and consistency (r2 = 0.822; p = 0.045) show
a significant improvement of the sensory attributes of kefir by
the addition of pine bud syrup. The scores of overall accept-
ability and consistency were significantly higher for the kefir
with 10% pine bud syrup than for the other formulations.
Therefore, a 10% (w/w) level of pine bud syrup is recom-
mended for the kefir fortification.
Conclusions
This study is the first that reports the fortification of kefir with
pine bud syrup. The results of the current research reveal
new perspectives for the innovative application of pine bud
syrup in fermented dairy products. The kefir fortified with
10% pine bud syrup was the best in all aspects of quality.
These findings may be useful to dairy producers to obtain new
types of kefir.
Acknowledgements
The authors are grateful to Alexandra Şandru, BSc. Eng., for her
valuable help in the acquisition of plant material.
Disclosure statement
No potential conflict of interest was reported by the authors.
218 C.A. Semeniuc et al.
Funding
This study was partly supported by the European Social Found
[grant POSDRU/89/1.5/S/62371], [grant POSDRU/159/1.5/S/
132765].
ORCID
Cristina Anamaria Semeniuc http://orcid.org/0000-0002-
9721-4560
Ancuţa Rotar http://orcid.org/0000-0002-4544-5754
Laura Stan http://orcid.org/0000-0002-9999-1101
Carmen Rodica Pop http://orcid.org/0000-0002-7798-3397
Sonia Socaci http://orcid.org/0000-0003-0594-4628
Sevastița Muste http://orcid.org/0000-0002-7352-6957
References
Ahmed, Z., Wang, Y., Ahmad, A., Khan, S. T., Nisa, M., Ahmad, H., &
Afreen, A. (2013). Kefir and health: A contemporary perspective.
Critical Reviews in Food Science and Nutrition, 53, 422–434.
doi:10.1080/10408398.2010.540360
Apetrei, C. L., Tuchilus, C., Aprotosoaie, A. C., Oprea, A., Malterud, K.
E., & Miron, A. (2011). Chemical, antioxidant and antimicrobial
investigations of Pinus cembra L. bark and needles. Molecules, 16,
7773–7788. doi:10.3390/molecules16097773
Aydin, E., Türkez, H., & Geyikoǧlu, F. (2013). Antioxidative, anticancer
and genotoxic properties of α-pinene on N2a neuroblastoma cells.
Biologia, 68, 1004–1009. doi:10.2478/s11756-013-0230-2
Cevikbas, A., Yemni, E., Ezzedenn, F. W., Yardimici, T., Cevikbas, U., &
Stohs, S. J. (1994). Antitumoural antibacterial and antifungal activ-
ities of kefir and kefir grain. Phytotherapy Research, 8, 78–82.
doi:10.1002/(ISSN)1099-1573
Choi, E.-M. (2007). Antinociceptive and antiinflammatory activities of
pine (Pinus densiflora) pollen extract. Phytotherapy Research, 21,
471–475. doi:10.1002/(ISSN)1099-1573
CODEX STAN 243-2003. (2003). Codex standard for fermented milks.
New Zealand: Codex Alimentarius, Commodity Committee for Milk
and Milk Products. Retrieved from http://www.codexalimentarius.org/
committees-and-task-forces/en/?provide=committeeDetail&idList=19
Deac, L. M., Fărcaş, A., Vodnar, D. C., Tofană, M., & Socaci, S. A.
(2014). Antioxidant and antimicrobial properties of the fir buds syrup.
Bulletin of University of Agricultural Sciences and Veterinary
Medicine Cluj-Napoca. Food Science and Technology, 71, 77–78.
Ertekin, B., & Guzel-Seydim, Z. B. (2010). Effect of fat replacers on kefir
quality. Journal of the Science of Food and Agriculture, 90, 543–548.
Farnworth, E. R., & Mainville, I. (2008). Kefir-a fermented milk product.
In E. R. Farnworth (Ed.), Handbook of fermented functional foods
(2nd ed., pp. 90–118). Boca Raton, FL: CRC Press/Taylor and
Francis Group.
Feng, S., Zeng, W., Luo, F., Zhao, J., Yang, Z., & Sun, Q. (2010).
Antibacterial activity of organic acids in aqueous extracts from pine
needles (Pinus massoniana Lamb.). Food Science and
Biotechnology, 19, 35–41. doi:10.1007/s10068-010-0005-2
Hutkin, R. W. (2006). Microbiology and technology of fermented foods.
Ames, IA: Blackwell Publishing.
Isidorov, V. A., Vinogorova, V. T., & Rafałowski, K. (2003). HS-SPME
analysis of volatile organic compounds of coniferous needle litter.
Atmospheric Environment, 37, 4645–4650. doi:10.1016/j.
atmosenv.2003.07.005
ISO/TS 11869:2012 (IDF 150). (2012). Fermented milks –
Determination of titratable acidity – Potentiometric method.
London: International Organization for Standardization.
Judžentienė, A., Šližytė, J., Stiklienė, A., & Kupčinskienė, E. (2006).
Characteristics of essential oil composition in the needles of young
stand of Scots pine (Pinus sylvestris L.) growing along aerial ammo-
nia gradient. Chemija, 17, 67–73.
Koukos, P. K., Papadopoulou, K. I., Patiaka, D. T., & Papagiannopoulos,
A. D. (2000). Chemical composition of essential oils from needles
and twigs of balkan pine (Pinus peuce Grisebach) grown in Northern
Greece. Journal of Agricultural and Food Chemistry, 48, 1266–
1268. doi:10.1021/jf991012a
Lavabre, M. (1996). Aromatherapy workbook (Revised ed.). Rochester,
VT: Healing Arts Press.
Lee, K.-H., Kim, A.-J., & Choi, E.-M. (2009). Antioxidant and antiin-
flammatory activity of pine pollen extract in vitro. Phytotherapy
Research, 23, 41–48. doi:10.1002/ptr.v23:1
Liu, R., & Xu, B. (2012). Characterization of essential oil in pine nut
shells from commodity waste in China by steam distillation and GC-
MS. Food Analytical Methods, 5, 435–440. doi:10.1007/s12161-011-
9264-7
Mimoune, N. A., Mimoune, D. A., & Yataghene, A. (2013). Chemical
composition and antimicrobial activity of the essential oils of Pinus
pinaster. Journal of Coastal Life Medicine, 1, 54–58.
Montanuci, F. D., Pimentel, T. C., Garcia, S., & Prudencio, S. H. (2012).
Effect of starter culture and inulin addition on microbial viability,
texture, and chemical characteristics of whole or skim milk kefir.
Food Science and Technology (Campinas), 32, 580–865.
doi:10.1590/S0101-20612012005000119
Mureşan, E. A., Muste, S., Borşa, A., Vlaic, R. A., & Mureşan, V.
(2014). Evaluation of physical-chemical indexes, sugars, pigments
and phenolic compounds of fruits from three apple varieties at the
end of storage period. Bulletin of University of Agricultural Sciences
and Veterinary Medicine Cluj-Napoca. Food Science and
Technology, 71, 45–50.
Mureşan-Cerbu, E. A., Muste, S., Borşa, A., Sconţa, Z., Crainic, D., &
Mureşan, V. (2012). Total phenolic content changes during apple
growth as a function of variety and fruit position in the crown.
Journal of Agroalimentary Processes and Technologies, 18, 341–344.
Ŏzer, B., & Kirmaci, H. A. (2014). Products of Eastern Europe and Asia.
In C. A. Batt & M. L. Tortorello (Eds.), Encyclopedia of food
microbiology (2nd ed., pp. 900–907). London: Elsevier.
Park, Y. S., Jeon, M. H., Hwang, H. J., Park, M. R., Lee, S.-H., Kim, S.
G., & Kim, M. (2011). Antioxidant activity and analysis of proantho-
cyanidins from pine (Pinus densiflora) needles. Nutrition Research
and Practice, 5, 281–287. doi:10.4162/nrp.2011.5.4.281
Păucean, A., Rotar, M. A., Jimborean, M., Mudura, E., & Socaciu, C.
(2009). A study on sensory characteristics of a kefir type produced
using starter cultures and brewer’s yeast. Journal of Agroalimentary
Processes and Technologies, 15, 267–272.
Pilipenko, V. I., Burliaeva, E. A., Shakhovskaia, A. K., & Isakov, V. A.
(2009). Efficacy of using inulin fortified fermented milk products in
patients with functional constipation. Voprosy Pitaniia, 78, 56–61.
Price, L. (2012). Chemistry of essential oils. In S. Price & L. Price (Eds.),
Aromatherapy for health professionals (4th ed., pp. 19–35).
Edinburgh: Churchill Livingstone/Elsevier.
SR EN ISO 8968-1:2014. (2014). Milk and milk products –
Determination of nitrogen content – Part 1: Kjeldahl principle and
crude protein calculation (ISO 8968-1:2014). Bucharest: Romanian
Standard Association.
SR ISO 13580:2009. (2009). Yogurt – Determination of total solids
content (Reference method). Bucharest: Romanian Standard
Association.
SR ISO 488:2009. (2009). Milk – Determination of fat content – Gerber
butyrometers. Bucharest: Romanian Standard Association.
Tratnik, L., Bozanic, R., Zoran, H., & Drgalić, I. (2006). The quality of
plain and supplemented kefir from goat’s and cow’s milk.
International Journal of Dairy Technology, 59, 40–46. doi:10.1111/
j.1471-0307.2006.00236.x
Tumen, I., Hafizoglu, H., Kilic, A., Dönmez, I. E., Sivrikaya, H., &
Reunanen, M. (2010). Yields and constituents of essential oil from
cones of Pinaceae spp. natively grown in Turkey. Molecules, 15,
5797–5806. doi:10.3390/molecules15085797
Van Wyk, J., Witthuhn, R. C., & Britz, T. J. (2011). Optimisation of
vitamin B12 and folate production by Propionibacterium freudenrei-
chii strains in kefir. International Dairy Journal, 21, 69–74.
doi:10.1016/j.idairyj.2010.09.004
Yang, X., Zhao, H. T., Wang, J., Meng, Q., Zhang, H., Yao, L., . . .
Ding, Y. (2010). Chemical composition and antioxidant activity
of essential oil of pine cones of Pinus armandii from the south-
west region of China. Journal of Medicinal Plants Research, 4,
1668–1672.
Zeng, W.-C., Zhang, Z., Gao, H., Jia, L.-R., & He, Q. (2012). Chemical
composition, antioxidant, and antimicrobial activities of essential oil
from pine needle (Cedrus deodara). Journal of Food Science, 77,
C824–C829. doi:10.1111/jfds.2012.77.issue-7