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Characterisation of the exopolysaccharide kefiran produced by lactic acid

bacteria entrapped within natural kefir grains

Article  in  International Journal of Dairy Technology · November 2011

DOI: 10.1111/j.1471-0307.2011.00704.x

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 doi: 10.1111/j.1471-0307.2011.00704.x

ORIGINAL
RESEARCH Characterisation of the exopolysaccharide kefiran
produced by lactic acid bacteria entrapped within natural
kefir grains
KATJA ZAJŠEK,* MITJA KOLAR and ANDREJA GORŠEK
Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova 17, SI-2000 Maribor, Slovenia

The main goal of this work was to characterise and quantify the exopolysaccharide kefiran and to dis-
cover an effective procedure for its isolation from kefir grains, originating from the Caucasian Moun-
tains. Capillary electrophoresis was used for the characterisation and quantification of the D-glucose and
D-galactose in our samples at a mass ratio of 1:0.7. The effect of fermentation time on growth of kefir
grains and the content of kefiran within the grains were determined. The pH profiles were monitored
dynamically. In addition, the influence of fermentation temperature on kefir grains mass concentration
(cKG) and the content of kefiran within the grains (wKEF ⁄ KG) were studied. The highest values for both
were obtained at 30 C.
Keywords Natural kefir grains microflora, Kefiran, pH, Kefir grains growth, Exopolysaccharides.

become popular in many countries (Muir et al.

INTRODUCTION
Bacterial growth is often accompanied by the pro-
duction of exopolysaccharides (EPS), which have
significant ecological and physiological functions
(Cheirsilp et al. 2001). Microbial EPS are a wide
group of secreted polymers that can be tightly
attached to the cell surface or released as extracel-
lular slime throughout the surroundings of the cell
(Cerning et al. 1994; Duboc and Mollet 2001). An
increasing interest in the study of these molecules
exists (Duboc and Mollet 2001; Lin and Chang
Chien 2007) because of their potential application
for the food, pharmaceutical and chemical indus-
tries, and their function as bioflocculants, bioabsor-
bents, heavy metal removal agents, drug delivery
agents, etc. Industrially, the most important EPS
are dextrans, xantan, gellan, pullalan, yeast glucans
and bacterial alginates (Wang et al. 2008).
To date, EPS produced by lactic acid bacteria
(LAB) have received increasing interest, mainly
because of their GRAS (generally recognised as
safe) status and their rheological properties in food,
which improve the textures of fermented dairy
*Author for products, such as yoghurt, cheese, kefir or fer-
correspondence. E-mail: mented milks (Faber et al. 2001; Rimada and
katja.zajsek@uni-mb.si
Abraham 2006).
 2011 Society of Kefir, an ancient Caucasian beverage, is an
Dairy Technology acidic and mildly alcoholic fermented milk that has
1999; Güzel-Seydim et al. 2000). Traditionally, it
is produced from milk fermented with an original
native starter – kefir grains (Rodrigues et al. 2005;
Bosch et al. 2006). Kefir grains are soft, gelatinous
irregular particles, composed of LAB, acetic acid
bacteria and yeasts that are surrounded with pro-
teins, lipids and a slimy EPS matrix named kefiran.
It is produced mainly by Lactobacillus kefiranofac-
iens and several other undefined species of Lacto-
bacillus present in the grains (Frengova et al.
2002; Liu et al. 2002). Kefiran is a water-soluble
branched glucogalactan containing approximately
equal amounts of glucose and galactose (Cheirsilp
et al. 2001; Maeda et al. 2004). It is quite resistant
to hydrolysis and forms gels in aqueous solutions
containing ethanol (Micheli et al. 1999). This
polymer is widely used as a thickener, stabiliser,
emulsifier, fat substitute and gelling agent and also
exhibits antibacterial, antifungal and antitumour
activities (Welman and Maddox 2003; Tada et al.
2007). In addition, kefiran enhances the production
of interferon b-cortisol and noradrenaline in human
cell lines and is used as a stress-reducing food sup-
plements (Wang et al. 2008).
To utilise kefiran in many applied fields such as
food, cosmetics and the pharmaceutical industries,
it may be logical to isolate a large quantity of the
polysaccharide from the native kefir grains. The

Vol 64 International Journal of Dairy Technology 1

Vol 64
yield of kefiran, produced with lactobacilli within the grains, in
milk medium is fairly low (within the range of mg ⁄ L). In some
previous studies, researchers have demonstrated that growth
conditions (temperature, medium composition, incubation time
and source of carbon) have a considerable influence on LAB
exopolysaccharides yield and composition (Cerning et al.
1992; Taniguchi et al. 2001; Liu et al. 2002). Despite a wide
range of published methods for EPS isolation (Rimada and
Abraham 2006; Lin and Chang Chien 2007; Wang et al. 2008),
more advanced ones are still indispensable.
The goals of this study were (i) to develop a simple and
effective procedure for isolating kefiran from original kefir
grains cultures, (ii) to determine the quantity and chemical
structure of this specific exopolysaccharide and (iii) to deter-
mine optimum growth conditions for the kefir grains, by fol-
lowing the growth, pH of fermentation media and the content
of kefiran within the kefir grains in response to changing the
incubation times and temperatures during fermentation. An
optimal temperature was established for kefiran production.
M AT E R I A L S A N D M E T H O D S
Kefir grains and culture conditions
Kefir grains, which were used as kefiran producers during this
research, were obtained from the existing local dairy Kele &
Kele doo (Logatec, Slovenia), having originated from the Cau-
casian Mountains. In the laboratory, they were propagated at
room temperature (22–25 C) for 24 h with daily transfers into
1 L of fresh ultra high temperature–treated (UHT) full-fat
cows’ milk (Ljubljanske mlekarne d.d., Ljubljana, Slovenia) to
keep their viability. Milk from the same origin was also used as
a fermentation medium.
Fermentation procedure
The fermentation procedure was the same for all experiments.
Individual experiment was performed by means of first charg-
ing the reactor (RC1 reaction calorimeter, Mettler-Toledo Inter-
national Inc., Greifensee, Switzerland) by 1L of fresh milk
(Zajšek and Goršek 2009). The medium was heated-up to the
working temperature, as defined later. After establishing a
steady-state temperature, the medium was inoculated with
active kefir grains. The addition of grains caused the fermenta-
tion of milk. Fermentation medium pH values were monitored
during the process.
Determination of the mass concentration of final kefir
grains
At the end of each fermentation, the kefir grains were sepa-
rated from the fermentation product by filtration, using a
plastic household sieve, washed with cold water and then
dried carefully on paper towelling. The kefir grains mass con-
centration was determined by weighting on a Mettler Toledo
analytical balance (PG5002-S) (Mettler-Toledo International
Inc., Greifensee, Switzerland). After determining the grains
2  2011 Society of Dairy Technology
mass, the grains were used during the kefiran isolation proce-
dure.
Isolation of kefiran
The content of kefiran within the kefir grains was determined
based on the methods of Wang et al. (2008), Lin and Chang
Chien (2007) and Rimada and Abraham (2006), with some
modifications resulting in a shorter time required for isolation.
The total procedure lasted for approximately 3 days (compared
to the reported 5 days). The final amount of kefir grains was
first treated in 300 mL of boiling water for 3 h with continuous
stirring at 200 rpm. One volume of 20% trichloroacetic acid
solution was added to the cooled sample, and the overnight pre-
cipitated proteins and microbial cells were removed by centri-
fugation (17 700 g, 20 min, 4 C). The kefiran in the
supernatant was precipitated by the addition of an equal volume
of chilled >99.5% acetone and left in a refrigerator overnight.
The mixture was centrifuged at 17 700 g for 20 min at 4 C.
An isolated sample was washed with acetone and dried over
48 h at 42 C.
Standard solutions, calibrations, hydrolysis and
derivatisation of kefiran
Standard stock solutions of D-glucose (Glc), D-galactose (Gal)
and D-xylose (Xyl) (Fluka) were prepared at concentrations of
1 g ⁄ L. The stock solutions were diluted for calibration, using
Milli Q water. Typical calibration range for monosaccharides
was from 10 to 200 mg ⁄ L.
Approximately 50 mg of kefiran was diluted in 4 mL of Milli
Q water and mixed for 24 h. The solution was sonificated for
30 min at 40 C before hydrolysis. Hydrolysis was performed
by adding 2 mL of H2SO4 (c = 0.5 mol ⁄ L) to the sample solu-
tion, which was heated in autoclave (CV-EL12LGS, Certoklav)
at 120 C for 40 min. The hydrolysates were then cooled down
to room temperature and diluted to a final volume of 50 mL.
The derivatisation of hydrolysates was performed through
reductive amination, using sodium cyanoborohydride according
to Doliška et al. (2009).
All buffers, samples, Milli Q water and other solutions were
filtrated through syringe filters (0.2 lm) before use.
Determination of monosaccharides in kefiran using
capillary zone electrophoresis
A detailed explanation and optimisation of capillary electropho-
resis experiments was described by Dahlman et al. (2000). In
our work, separations were carried out using a G-1600 agilent
CE3D Instrument equipped with DAD (190–600 nm). Separa-
tions were performed in borate buffer (c = 0.1 mol ⁄ L) at
pH = 10.5, using 30% of acetonitrile as an electro-osmotic flow
modifier.
Samples were injected hydrodynamically at 50 mbar for 5 s,
followed by a plug of buffer solution at P = 50 mbar for 2 s.
A voltage of 25 KV was applied, the temperature being constant
at 20 C, and UVabsorbance (A) was measured at 306 nm.
Vol 64
Influences on kefiran production
Incubation time
A 2-L batch reactor with a working volume of 1.8 L was used
for studying the production of kefiran during the milk fermenta-
tion with kefir grains. First, 1 L of UHT full-fat cows’ milk
was poured into the reactor. The pure milk was inoculated with
active kefir grains with a mass concentration of 42 g ⁄ L and
allowed to proceed at intervals of (0, 5, 14, 20, 30, 40, 50, 60
and 70) h. The reactor was operated at 30 C. Slow agitation
(fm = 60 rpm) was periodically maintained to keep the fermen-
tation broth homogeneous, and no air was added.
Temperature
Fermentations were carried out in a glass vessel (V = 2 L).
After filling with 1 L of full-fat cows’ milk, active kefir grains
(cKG,0 = 42 g ⁄ L) were added into the vessel. Fermentations
occurred at (25, 30 and 37) C for 24 h without agitation.
R E S U LT S A N D D I S C U S S I O N
Characterisation of monosaccharides in kefiran
After the hydrolysis and derivatisation of hydrolysates, capil-
lary electrophoresis was used for the characterisation and quan-
tification of monosaccharides within the kefiran. The peaks of
the products were identified by spiking the samples with
glucose and galactose (c = 25 mg ⁄ L) and quantified using
calibration plots. Xylose (c = 25 mg ⁄ L) was used as the inter-
nal standard. The typical working range applied for
glucose and galactose determination in kefiran was from
20 to 100 mg ⁄ L (AGlc = 1.3643cGlc + 16.85, R2 = 0.9982;
AGal = 0.8586cGal + 12.35, R2 = 0.9947). Figure 1 shows the
Xyl
Glc Gal
10 14 18
t/min
Figure 1 Electropherograms of standards, and the kefiran obtained using a
0.1 mol ⁄ L borate buffer and 30% acetonitrile at pH 10.5: Standard A:
25 mg ⁄ L Xyl, 25 mg ⁄ L Glc and 25 mg ⁄ L Gal. Standard B: 50 mg ⁄ L Xyl,
50 mg ⁄ L Glc and 50 mg ⁄ L Gal. Standard C: 100 mg ⁄ L Xyl, 100 mg ⁄ L Glc
and 100 mg ⁄ L Gal. Kefiran sample (1–1) D: 25 mg ⁄ L Xyl added as internal
standard.
 2011 Society of Dairy Technology
electropherograms of the three standards solutions (A, B and C)
of glucose, galactose and xylose at different mass concentra-
tions, c = (25, 50, 100) mg ⁄ L, and kefiran D is shown as an
example. Process parameters for sample D were as follows:
T = 37 C; t = 24 h; cKG,0 = 42 g ⁄ L; and fm = 0 g. Only glu-
cose and galactose were found within all the analysed kefiran
samples. The average content of monosaccharides was
w = 63.5 ± 5.0%, whilst other monosaccharides, such as
xylose and mannose, were not presented.
Influences on the production of kefir grains biomass and
kefiran
Incubation time
Figure 2 shows the time courses of kefir grains growth, pH pro-
file and kefiran production by naturally mixed kefir grains
microflora in a batch reactor, using a milk medium at a fermen-
tation temperature of S = 30 C. It is evident from the kefir
grains growth curve that growth increased during 30 h of fer-
mentation. Afterwards, the grains micro-organisms entered into
a stationary phase on the growth curve, which took up the time
between (30 and 40) h of fermentation. After 40 h, the number
of micro-organisms within the kefir grains started to decrease
(death phase).
The process of acidification is also reported in Figure 2. The
pH of the pure milk prior to inoculation with kefir grains was
around 6.6. The pH progressively decreased during fermenta-
tion until the death phase began (40 h). A final pH of 3.6 was
reached after (40–70) h of incubation.
The content of kefiran within the grains between (0–70) h of
fermentation time ranged from wKEF ⁄ KG = (3.0–4.5) %. The
experimental data in Figure 2 show that the content of kefiran
was almost constant during the first 40 h of fermentation,
which includes the lag, exponential and stationary phases of the
growth curve (the measurements at 5 and 20 h deviated from
the other ones, probably due to experimental error). After 40 h
of fermentation, the number of micro-organisms within the kefir
22
Figure 2 Changes in kefir grains growth, pH, and kefiran during milk
fermentation by mixed kefir grains microflora in full-fat cows’ milk at 30 C.
3
Vol 64
grains started to decrease (death phase), whilst the content of
kefiran within the kefir grains started to increase. This phenom-
enon is the result of more kefiran being produced to protect the
microbial cells themselves. The content of kefiran within the
kefir grains after 60 h decreased, probably due to the presence
of glycohydrolases, capable of hydrolysing the kefiran and
liberating the monomer (Sutherland 1999).
Temperature
Kefir grains in 1 L of milk medium at temperatures ranging
from 25 to 37 C for 24 h were incubated to investigate the
effect of the fermentation temperature on kefir grains growth
and content of kefiran within the kefir grains (Figure 3).
As indicated in Figure 3, the optimum temperature for kefir
grains growth was found to be 30 C. The production of kefir
grains biomass was near its optimal temperature over the range
from 25 to 30 C, but declined sharply outside this temperature
range. Meanwhile, the content of kefiran within the kefir grains
increased as the fermentation temperature increased from 25 to
30 C. The highest content of kefiran was obtained at the same
temperature as the kefir grains growth (S = 30 C). Although
microbial amylases are generally active at ambient temperatures
of up to 60 C (Yeesang et al. 2008), the optimal temperature
for the cell growth and kefiran production from lactose by
Lactobacillus kefiranofaciens, which is responsible for kefiran
production (Yeesang et al. 2008), was the same as found for
kefiran production during this study. At temperatures higher
than 30 C, the production of kefir grains biomass and kefiran
started to decrease.
Influence of temperature on the monosaccharide
composition of kefiran
The strains, the culture conditions and the type of carbon source
influence the composition of microbial exopolysaccharides pro-
duced by a certain species (Celik et al. 2008). The monosaccha-
ride compositions of kefiran produced within the selected
Figure 3 Effect of temperature on the kefir grains growth, and the content of
kefiran within kefir grains after 24 h of fermentation.
4  2011 Society of Dairy Technology
Table 1 Relative monosaccharide composition of the kefiran produced
by mixed kefir grains microflora in 1 L of milk containing lactose
(c = 46 g ⁄ L) as a carbon source, after 24 h of fermentation at selected
temperatures
Composition of kefiran (%) Ratio
S (C) Glucose Galactose Glucose ⁄ Galactose
25 59.5 40.5 1:0.68
30 59.8 40.2 1:0.67
37 58.6 41.3 1:0.70
temperature range from 25 to 37 C by mixed kefir grains micro-
flora in milk containing lactose as the sole carbon source were
analysed using capillary zone electrophoresis after acid hydro-
lysis and derivatisation. The results are presented in Table 1.
Glucose and galactose were the basic structural units of our
kefiran. The lactobacilli synthesised biopolymers with composi-
tion contained glucose and galactose (Frengova et al. 2002).
The content of glucose (58.6 to 59.8%) in the kefiran was
higher than the content of galactose (40.2 to 41.3%). The high-
est amount of glucose was achieved at 30 C. The level of
galactose was higher at 37 C. No xylose and mannose were
found in the kefiran produced at selected temperatures. In all
samples of kefiran (Table 1), the glucose ⁄ galactose mass ratio
was around 1:0.7. Different sugar compositions of kefiran were
observed in previous studies where exopolysaccharides were
produced by the kefir grains of other origins or by single-strain
cultures isolated from kefir grains (Frengova et al. 2002; Liu
et al. 2002).
CONCLUSION
This paper presented a modified method for the quantitative
isolation of EPS produced by kefir grains originating from the
Caucasian Mountains. It resulted in a shorter time required for
isolation and in relatively high EPS concentrations (0.85 g ⁄ L
media). Using capillary electrophoresis, we identified a D-
glucose ⁄ D-galactose ratio within the kefiran samples (1:0.7). As
growth environment (temperature, pH, carbon and nitrogen
content) influences EPS production, its optimisation is impor-
tant. Therefore, we established the optimal fermentation tem-
perature to be S = 30 C, where EPS production from the kefir
grains of our origin was maximal. Changes in kefir grains
growth, pH and kefiran concentrations during milk fermenta-
tions at different temperatures were also examined and
explained. The optimisation of other growth parameters will be
the subject of our future research.
ACKNOWLEDGEMENTS
This work was supported financially by the Slovenian Research
Agency.
Vol 64
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