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Kefir is a fermented beverage originating form the Caucasian regions composed of a number of bacteria and yeasts living together in polysaccharide grains secreted by them. Kefir can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. Aiming to appraise a possible anti-inflammatory effect of kefir we conducted cotton-induced granuloma and paw oedema assays in rats, the latter using carrageenan, dextran and histamine as stimuli. Kefir samples were thawed and continuously cultured during 15 days both in a molasses solution (50 g/l) and in cow's milk. A polysaccharide extract isolated from the grains (kefiran) was also tested in cotton-pellet experiments. The results showed significant inhibition in the formation of granuloma tissue for all the test groups, as compared to the blank group. Kefir suspensions in molasses presented an inhibition of 41 +/- 3% for the inflammatory process, fermented milk prepared from kefir showed 44 +/- 6% inhibition and kefiran extract 34 +/- 15%. Rat paw oedema also showed significant decreases with the mediators. Dextran-induced oedema was completely inhibited at 1 h after input, with a 76% inhibition after 2 h. Carrageenan stimulus was inhibited 62% after the 3rd hour, and histamine by 52% after the 2nd hour. These results points to the existence of anti-inflammatory prebiotic compounds present in symbiotic cultures of kefir growing in both aqueous and milky suspensions.
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Inflammopharmacology, Vol. 13, No. 5–6, pp. 485492 (2005)
VSP 2005.
Also available online - www.vsppub.com
Short communication
Anti-inflammatory properties of kefir and its
polysaccharide extract
K. L. RODRIGUES, J. C. T. CARVALHO and J. M. SCHNEEDORF
Laboratório de Fitofármacos, Unifenas, Rod MG 179 Km 0, POB 23, CEP 37130-000, Alfenas,
Minas Gerais, Brazil
Received 8 April 2005; revised 5 May 2005; accepted 7 May 2005
Abstract—Kefir is a fermented beverage originating form the Caucasian regions composed of a
number of bacteria and yeasts living together in polysaccharide grains secreted by them. Kefir
can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and
immunomodulatory properties. Aiming to appraise a possible anti-inflammatory effect of kefir we
conducted cotton-induced granuloma and paw oedema assays in rats, the latter using carrageenan,
dextran and histamine as stimuli. Kefir samples were thawed and continuously cultured during 15
days both in a molasses solution (50 g/l) and in cow’smilk. A polysaccharide extract isolated from the
grains (kefiran) was also tested in cotton-pellet experiments. The results showed significant inhibition
in the formation of granuloma tissue for all the test groups, as compared to the blank group. Kefir
suspensions in molasses presented an inhibition of 41 ±3% for the inflammatory process, fermented
milk prepared from kefir showed 44 ±6% inhibition and kefiran extract 34 ±15%. Rat paw oedema
also showed significant decreases with the mediators. Dextran-induced oedema was completely
inhibited at 1 h after input, with a 76% inhibition after 2 h. Carrageenan stimulus was inhibited 62%
after the 3rd hour, and histamine by 52% after the 2nd hour. These results points to the existence of
anti-inflammatory prebiotic compounds present in symbiotic cultures of kefir growing in both aqueous
and milky suspensions.
Key words: Kefir; kefiran; probiotic; prebiotic; inflammation.
1. INTRODUCTION
Kefir is a beverage consisting of gelatinous and irregular grains formed by a consor-
tium of yeasts and lactic acid bacteria which causes an acid-alcoholic fermentation
in sugar and milk solutions (Schneedorf and Anfiteatro, 2004). This microflora is
embedded in a resilent polysaccharide matrix, called kefiran, composed of branched
chains of glucose and galactose as metabolic products of souring milk lactose and
To whom correspondence should be addressed. Tel.: (55-31) 353-299; Fax: (55-31) 353-239;
e-mail: jose.silva@unifenas.br
486 K. L. Rodrigues et al.
other substrates (Micheli et al., 1999). There are approximately 30–50 bacteria
and yeast strains living together in kefir grains, although this number may change
depending on the culture media used (sugar, milk, fruit juices) and the country of
the grain source (Hallé et al., 1994). Kefir can be considered to be a probiotic
resource as it can improve a variety of health conditions besides its nutritional sta-
tus. There are several studies investigating immunomodulatory, pathogenic barrier,
anti-neoplastic and digestive effects caused by kefir intake (Schneedorf and An-
fiteatro, 2004). The digestive benefits of kefir have been investigated by Cardoso
et al. (2003) who found an improvement of the peristaltic activity in the intesti-
nal tract of rats treated with sugary kefir for 15 days. Liu and co-workers (2005)
have recently demonstrated that milky kefir possesses significant anti-mutagenic
and anti-oxidant activity in Salmonella mutagenicity and radical scavenging as-
says. Kefir also has anti-microbial activity against a wide range of bacteria and
fungi. A pathogen barrier mediated by kefir was found in some studies with poul-
try, chickens and human subjects, including resistance against enterohaemorragic
Escherichia coli (Ota, 1999), Helicobacter pylori (Bohmler, 1996) and Staphylo-
coccus aureus (Rodrigues et al., 2005). Immunological stimulation using kefir and
sphingomyelins isolated from its lipid content has also been demonstrated (Osada,
1993). Aiming to evaluate in vivo anti-inflammatory properties of this symbiotic
association growing in milky and aqueous suspensions, we conducted induction of
granulomatous tissue (cotton pellet test) and paw oedema experiments in rats.
2. MATERIALS AND METHODS
2.1. Kefir culture
5 g of starter grains were continuously cultured in 50 g/l of molasses and cow’s
milk for 15 days before experiments up to the achievement of a sigmoidal profile in
growth curves (5th day of culture). The grains were placed in polyethylene vessels
containing the nutrient media and allowed to grow up to 48 h. The suspensions were
withdrawn from the containers, and the grains were gently washed in distilled water
and settled again with a fresh nutrient preparation.
2.2. Kefiran isolation
A matrix of polysaccharide nature was extracted from the grains cultured in
molasses, following an adaptation of Micheli et al. (1999). Briefly, samples of
100 g kefir grains were mechanically disintegrated with a tissue grinder and allowed
to keep in boiling water during 2 h. After centrifugation at 1500 rpm for 45 min
the supernant was allowed to precipitate in ethanol at 5C overnight. The formed
pellet was collected by centrifugation at 1500 rpm for 15 min and resuspended in
0.9% NaCl (10 ml final volume). The final sample was assayed for reducing sugars
(Fehling’s reagent) and mono- and di-saccharides (thin-layer chromatography with
Anti-inflammatory properties of kefir 487
glucose, fructose, galactose, sucrose and lactose as standards, Reagen, São Paulo,
Brazil) and amide content (biuret reagent) were determined.
2.3. Animals
Wistar rats of both genders, weighing between 180 and 200 g, were used in the ex-
periments for the assessment of the anti-inflammatory activity. The rats were kept
in groups of 5 animals in polyethylene boxes at 27C and 12 h of dark/light cycle,
and fed on water ad libitum during 24 h before the experiments. After the exper-
iments the animals were killed by carbon dioxide inhalation. All procedures were
conducted following the recommendations of the Ethical Commitee of University
Jose do Rosario Vellano, Unifenas, MG, Brazil.
2.4. Induction of granulomatous tissue
Pellets weighing approximately 40 mg each were made with 5 mm of dental
cotton tampon. The pellets were sterilized and impregnated with 0.4 ml of
5% ampicillin solution at the moment of implantation. After the animals were
anaesthetized, the pellets were introduced subcutaneously through abdominal skin
incisions, in accordance with Meier’s method (Meier et al., 1950). The following
was administered daily: 1 ml of 0.9% NaCl (CN, orally) 0.2 mg/kg dexamethasone
(DM, topically). Test groups received 1 ml kefir suspension in molasses (SK) and
cow’s milk (MK) fermented, and 1 ml of 0.1% polysaccharide extract (PE). This
treatment was initiated 2 h following the implantation of the pellets. On the 7th
day the animals were killed with ether overdose, the granulomas were removed, left
to dry for 24 h at 60C and the weight was determined. The difference between
the initial weight and the final weight was the weight of the granulomatous tissue
sample thus produced.
2.5. Rat paw oedema
The right rear plantar region of the rats was injected with either 1 µg/paw (0.1 ml)
carrageenan (Iota-Fluka Biochemika), dextran (T-70, MW 70 ×103, Pharmacia)
50 µg/paw (0.1 ml) or histamine 50 µg/paw (0.1 ml). The left rear paw of each
animal receiving any of the three drugs listed above was injected with an equal
volume of 0.9% saline solution. Thirty minutes before experiments the animals
received 1 ml of 10 mg/kg indomethacin (p.o.) (carrageenan group), the same
amount of cyproheptadine (dextran or histamine group), 1 ml of 48-h fermented
kefir, or 1 ml of disintegrated grains. For the measurement of the volume of the
paws, the diameter of each paw was measured with an analogue pakimeter (Vernier,
Beaverton, OR, USA) after stimulations.
2.6. Statistical analysis
Statistical analysis was done using one-way analysis of variance. The data are
expressed as mean ±SEM. Differences between the controls and the treated groups
488 K. L. Rodrigues et al.
Figure 1. Effect of administration of kefir preparations, polysaccharide extract and dexamethasone
during 6 days, on the formation of granulomatous tissue. Values are mean ±SEM of granuloma
weight (P<0.05; Student’s t-test). CN, control (0.9% NaCl, 1 ml); DM, dexamethasone
(0.2 mg/kg, topical application); SK, kefir in sugar (aqueous molasses suspension, 50 g/l, 1 ml);
PE, polysaccharide extracted from the grains (0.1%, 1 ml); MK, kefir in cow milk (1 ml).
of animals in theses experiments were tested for statistical significance by Student’s
t-test (P<0.05).
3. RESULTS AND DISCUSSION
3.1. Kefir growth
Biomass production of typical kefir cultures fermenting in molasses or in cow’s
milk was obtained. After a 2-day period of adaptation, kefir grains showed a
sigmoidal profile for biomass production, assuring a culture media replacement
at 48-h intervals (data not shown). After the 5th day kefir was used for the
inflammatory experiments, since a linear trend of biomass production was achieved.
3.2. Cotton pellet test
Kefir suspensions, both in molasses and cow’s milk, and a polysaccharide extract
isolated from kefir grains cultured in aqueous solution were able to reduce the
inflammatory process of granuloma formation in rats after the 6th day of treatment,
as related to control samples (Fig. 1). The inhibiton was more effective for the
suspension (41 ±3% for molasses and 44 ±6% for milk fermentation, P<0.05)
than the polysaccharide extracted from the grains (34 ±2%). Although the
inhibition with dexamethasone was 52 ±4%, this substance is well-known to
Anti-inflammatory properties of kefir 489
induce immunosuppressive effects (Carvalho, 1998). Sugary kefiran presented
a lower anti-granuloma effect as compared to kefir suspensions; even so, the
extract seems to exhibit a significant antimicrobial response (minimal inhibitory
concentration of 460 µg/ml against Streptococcus strains; Rodrigues et al., 2005).
3.3. Rat paw oedema
The anti-oedematogenic activity of kefir suspensions and disrupted kefir grains
was evaluated by the rat paw oedema test (Carvalho et al., 1999). This test
is used to evaluate anti-inflammatory drugs and has been used to test the anti-
oedematogenic effect of various substances (Ferreira, 1979). The administration
of carrageenan (1 µg/paw, 0.1 ml), dextran (50 µg/paw, 0.1 ml) and histamine
(50 µg/paw, 0.1 ml) induced significant oedema in the rat paws (Figs 2–4, P<
0.05). The inhibition of rat paw oedema induced by the mediators dispensed
(carrageenan, dextran and histamine) was significantly different between control
and test groups (P<0.05). Kefir suspensions in aqueous molasses (50 g/l),
as well as kefir grains that were mechanically disintegrated (1 g/ml, 0.1 ml),
caused a decrease in the inflammation induced by the described compounds.
Kefir suspensions orally administered were found to be more effective than kefir
grains that were mechanically disintegrated. Oedema induced by carrageenan was
gradually observed 30 min after administration (Fig. 2), with an inhibition of 62%
using the suspension and 40% with kefir grains after the 3rd hour. The carrageenan-
Figure 2. Effect of p.o. administration of 1 ml 48-h fermented kefir in molasses (50 g/l) and
disintegrated grains (1 g/ml, 1 ml) on the rat paw oedema induced by intraplantar injection of
carrageenan (1 µg/paw, 0.1 ml). P<0.05 (Students’s t-test), n=8/group.
490 K. L. Rodrigues et al.
Figure 3. Effect of p.o. administration of 1 ml 48-h fermented kefir in molasses (50 g/l) and
disintegrated grains (1 g/ml, 1 ml) on the rat paw oedema induced by intraplantar injection of dextran
(50 µg/paw, 0.1 ml). P<0.05 (Students’s t-test), n=8/group.
Figure 4. Effect of p.o. administration of 1 ml 48-h fermented kefir in molasses (50 g/l) and
disintegrated grains (1 g/ml, 1 ml) on the rat paw oedema induced by intraplantar injection of
histamine (50 µg/paw, 0.1 ml). P<0.05 (Students’s t-test), n=8/group.
Anti-inflammatory properties of kefir 491
induced inflammatory process is referred in the literature as comprising three phases
(DiRosa et al., 1971), the second and third ones associated with kinins (2–6 h)
and prostaglandin-like compounds (Van Arman et al., 1965). The initial phase
(90 min) is attributed to histamine and serotonin release. Therefore, the data
presented in Fig. 2 suggest a participation of prostaglandins mediators more than
just histamine and serotonin, though these latter are released only in the initial phase
after inflammatory challenges (DiRosa et al., 1971). Kefir suspension administered
30 min before dextran stimulation, however, showed a complete inhibition of the
inflammation after 1 h (Fig. 3). Furthermore, a maximum peak of stimuli obtained
with dextran was found after 2 h, with 76% of the inhibitory effect of orally
administered kefir suspension. Nonetheless, ground grains were found to inhibit
only 12% with dextran at the same period. Dextran-induced oedema is considered
a consequence of histamine and serotonine liberation from mast cells (Rowley and
Benditt, 1956). Even though administered orally, kefir suspension did not show
the same significant reduction of the histamine-induced oedema (Fig. 4). The anti-
histaminic effect of kefir was observed with small changes during the first 2 h after
stimulation, with a maximum peak of 52% for inhibition of the kefir suspension
after the 2nd hour and 43% with disintegrated kefir grains at the same time (Fig. 4).
Curiously there was no inhibitory activity of kefir grains after this period. These
overall results point to a mechanism of serotonine release taking into account
the prebiotic compounds in kefir suspensions parallel to prostaglandin mediators.
Although the suggested mechanisms need to be sustained, this work presents the
ancient culture of kefir as a potential resource for anti-inflammatory therapeutics
besides its digestive, anti-biotic and anti-mutagenic properties (Schneedorf and
Anfiteatro, 2004).
Acknowledgements
The authors thank the Brazilian Research Concil (CNPq), Research Support Foun-
dation of Minas Gerais (FAPEMIG) and University of Alfenas for financial support.
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Kefir is traditionally produced by fermenting cow’s milk using kefir grains as a starter culture. As the viability of microbes within kefir grains is limited and preparing the grains for kefir fermentation is laborious, here, a single starter that ferments lactose and produces ethanol is developed. For this purpose, it is important to isolate yeasts that can ferment lactose and subsequently produce alcohol. This study aimed to isolate and identify yeasts from kefir and characterise their ability as single starters to produce kefir. Based on morphological and physiological evaluations, 15 presumptive yeast isolates were obtained, 10 of which grew well on lactose-containing media. Those that were able to grow on lactose using only carbon sources were subjected to molecular identification based on the internal transcribed spacer (ITS) of the 5.8 rDNA using PCR technology. Molecular identification confirmed four isolates—namely, KFA 3, KFA 7, KFA 9 and KFB 1—as belonging to Kluyveromyces marxianus. The batch fermentation data of these strains were fitted on a logistic model to obtain the carrying capacity coefficients and strain performances were compared. The kinetic modelling revealed that KFA 9 had the highest values for the carrying capacity coefficient, biomass yield and product yield, indicating that, among the four K. marxianus strains, this was superior due to its relatively fast growth and good ethanol productivity.
... Many studies have shown Kefir and its kefiran extract to possess anti-inflammatory activity. They exhibit this activity by preventing the formation of granuloma tissue (Rodrigues et al. 2005). They exhibit variable levels of anti-inflammatory activity such as in case of suspensions with molasses, fermented milk and kefiran extract an inhibition of 41, 44, and 34%, respectively have been reported. ...
... A wide variety of health-promoting benefits have been associated with its use [28][29][30][31][32][33] and have expanded its popularity beyond its traditional borders within northeaster Europe and Asia. Anti-inflammatory effects [34], antimicrobial activity [35], strengthening of the immune system [36], antioxidant activity [37], and the inhibition of pathogenic microorganisms [24,38] have been reported as a result of kefir consumption. In addition, the topical application of a gel made from a non-microbial fraction of kefir showed an improvement in the wound healing capacity [39]. ...
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Diet has a fundamental role in the homeostasis of bodily functions, including the skin, which, as an essential protective barrier, plays a crucial role in this balance. The skin and intestine appear to share a series of indirect metabolic pathways, in a dual relationship known as the “gut–skin axis”. Hence, the gut–skin axis might be receptive to modulation via dietary modification, where probiotics can be included, thus representing a potential therapeutic target in inflammatory skin diseases, such as atopic dermatitis (AD), in order to control and/or ameliorate symptoms. Kefir is one of the most ancient fermented foods, with probiotic characteristics that have been associated with a wide variety of health-promoting benefits, and it presents a microbiological diversity that makes its application as a probiotic in the gut–skin relationship of the utmost interest. However, the impact of a diet containing kefir on skin health has yet to be reported in scientific literature. This study aimed to assess the impact of the intake of homemade kefir in the skin of healthy and atopic volunteers. The intervention resulted in a boost on barrier function in both skin types verified only in the respective kefir intake groups. An improvement in the degree of severity of AD was also confirmed for the kefir intake group. Atopic individuals may benefit from kefir intake, especially in regard to their skin hydration. Finally, the effects observed on skin barrier function in this study probably culminate from the effects of all the ingredients in kefir, including the complex microbiota, its metabolites and macro- and micronutrients resulting from the fermentation. This work opens the way for more advanced research on the impact of the probiotic kefir on cutaneous health, further clarifying its mechanism of action namely via gut–skin axis.
... Similarly, orally administrated kefiran in mice increased the number of IgA+ cells and macrophages in small and large intestine lamina propria and peritoneal cavity, suggesting its use in intestinal pathologies [149]. Additionally, Rodrigues et al. [150] examined the anti-granuloma and anti-oedematogenic effect of kefiran extract in rats using carrageenan, dextran and histamine as stimuli and highlighted a possible anti-inflammatory effect. ...
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