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Study of anti-inflammatory activity of Tibetan mushroom, a symbiotic culture of bacteria and fungi encapsulated into a polysaccharide matrix

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Tibetan mushroom (TM) is a fermented beverage composed by a dozen of bacteria and yeasts living together into polysaccharide grains secreted by them. TM is similar to kefir, a probiotic beverage originated in the Caucasian mountains exhibiting some anti-bacterial, anti-mycotic, anti-neoplastic and immunomodulatory effects. Aiming to evaluate a plausible anti-inflammatory property of TM we conducted cotton-induced granuloma and paw edema assays in rats, the latter using carrageenin, dextran and histamine as stimuli. TM samples were thawed and continuously cultured during 15 days into molasses solutions (50 g/l). The experiments used TM suspensions after 24h fermentation and TM grains mechanically disintegrated. The results showed a significant inhibition on the formation of granuloma tissue for the test group as compared to the negative control group. TM suspensions presented an inhibition of 43% for the inflammatory process. Rat paw edema also showed significant decreases with the mediators. The edema induced by carrageenin was inhibited 62% at the 3rd hour. The edema dextran-induced was completely inhibited at 1h and antagonized the histamine edema 52% at 1h.
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Pharmacological Research 47 (2003) 49–52
Study of anti-inflammatory activity of Tibetan mushroom, a symbiotic
culture of bacteria and fungi encapsulated into a polysaccharide matrix
R.O. Diniz, L.K. Garla, J.M. Schneedorf, J.C.T. Carvalho
Laboratório de Fitofármacos, Universidade de Alfenas, Rod MG 179 Km 0, P.O. Box 23, Alfenas, Minas Gerais, Brazil
Accepted 4 September 2002
Abstract
Tibetan mushroom (TM) is a fermented beverage composed by a dozen of bacteria and yeasts living together into polysaccharide
grains secreted by them. TM is similar to kefir, a probiotic beverage originated in the Caucasian mountains exhibiting some anti-bacterial,
anti-mycotic, anti-neoplastic and immunomodulatory effects. Aiming to evaluate a plausible anti-inflammatory property of TM we con-
ducted cotton-induced granuloma and paw edema assays in rats, the latter using carrageenin, dextran and histamine as stimuli. TM samples
were thawed and continuously cultured during 15 days into molasses solutions (50g/l). The experiments used TM suspensions after 24h
fermentation and TM grains mechanically disintegrated. The results showed a significant inhibition on the formation of granuloma tissue
for the test group as compared to the negative control group. TM suspensions presented an inhibition of 43% for the inflammatory process.
Rat paw edema also showed significant decreases with the mediators. The edema induced by carrageenin was inhibited 62% at the 3rd
hour. The edema dextran-induced was completely inhibited at 1h and antagonized the histamine edema 52% at 1h.
© 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Anti-inflammatory; Tibetan mushroom; Granulomatous tissue; Rat paw edema; Probiotic
1. Introduction
Tibetan mushroom (TM) are gelatinous and irregular
grains formed by a symbiotic association of yeasts and lactic
acid bacteria which causes an acid-alcoholic fermentation
in sugar and milk preparations. This association is some-
times mistaken with mushrooms originated from Tibet like
Camellia assamica [1] and Cordyceps sinensis [2]. Further-
more TM is also misidentified with symbiotic associations
of the same nature like kombucha [3] and kefir [4], both
originated from Asian countries. Kombucha and kefir are
popular health promoting beverages and natural folk reme-
dies made by fermenting green or black tea (kombucha),
milk or molasses (kefir). TM can be distinguished from
kombucha and kefir samples from their products, microbi-
ological content and morphological structure. Furthermore
TM is the only symbiotic association able to produce a
leaflike lamellae membrane around the culture grains. The
microflora of TM is embedded in a resilent polysaccharide
matrix similar to kefiran matrix presented in kefir [5]. Kefir
Corresponding author. Tel.: +55-21-35-3299-3239;
fax: +55-21-35-3299-3239.
E-mail address: fitofarmacos@unifenas.br (J.C.T. Carvalho).
can be considered as a probiotic resource because it can en-
joy a variety of health claims besides their nutritional status.
There are several studies investigating immunomodulatory,
pathogenic barrier, anti-neoplastic and pro-digestive effects
leading through kefir intake [6]. Although kefir and TM
are very similar in structure, microbial content, cultivation
procedures and fermentation products, only kefir is usually
reported to lead health benefits of probiotic nature [7].In
this sense, the authors conducted induction of granuloma-
tous tissue (cotton pellet test) and paw edema experiments
in rats aiming to evaluate a plausible anti-inflammatory
activity of TM cultured into molasses solutions.
2. Material and methods
2.1. TM culture
Tibetan mushroom samples were kindly given by
Dominiq Anfiteatro, a TM producer from Australia (10B
Harrow Avenue, Magil, 5072 Adelaide SA, Australia).
Starter grains (5g) were continuously cultured in 50 g/l of
molasses during 15 days before experiments. The grains
were placed into plastic bottles containing the nutrient media
1043-6618/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S1043-6618(02)00240-2
50 R.O. Diniz et al./Pharmacological Research 47 (2003) 49–52
and allowed to grow up at room temperature during 48h.
Soured suspensions were withdrawn from the containers
and discarded, the grains were gently washed in mineral wa-
ter and settled again into a fresh nutrient preparation. After
the 6th day fermentation TM was used for the inflammatory
experiments. Disintegrated TM grains were obtained with
a tissue grinder and resuspended in 0.9% NaCl (1g/ml).
2.2. Anti-inflammatory activity assessment
2.2.1. Animals
Wistar rats (males and females) weighing between 180
and 200g were used in the experiments for the assess-
ment of the anti-inflammatory activity. The rats were kept
in five-animal groups in polyethylene boxes at room tem-
perature, and fed on water ad libitum during 24h before the
experiments.
2.2.2. Induction of granulomatous tissue
Pellets weighing approximately 40mg each were made
with 5mm of dental cotton tampon. The pellets were ster-
ilized and then impregnated with 0.4ml 5% ampicillin
aqueous solution at the moment of implantation. Having
the animals anesthetized, the pellets were subcutaneously
introduced through abdominal skin incisions, in accordance
with Meier’s method [8]. The following was administered
daily, 4ml/kg of 5% Tween solution (orally), and 0.2mg/kg
dexamethasone (topically). Test groups were carried out
using 1ml TM suspension 24 h fermented. The treatment
was initiated 2h following the implantation of the pellets
and continued until the 6th day. On the 7th day, the ani-
mals were killed with ether overdose. The granulomas were
removed, left to dry for 24h at 60C temperature and the
weight were then determined. The difference between the
Fig. 1. Growth curve of TM fermented in aqueous solution (5g starter grains) containing 50g/l molasses. Numbers are represented by mean ±S.E.M.
of biomass produced.
initial weight and the final weight was the weight of the
granulomatous tissue sample thus produced.
2.2.3. Rat paw edema
An amount of 1ml TM suspension and 1 g disintegrated
grains resuspended in 0.9% NaCl (1g/ml) were adminis-
tered to the animals 30min before the experiments. The
right rear plantar region of the rats were injected with
1mg perpaw (0.1ml) carrageenin (Iota-Fluka Biochemika),
dextran (T-70; MW 70,000; Pharmacia) 50gper paw
(0.1ml), and histamine 50 g perpaw (0.1ml). The left
rear paw of each animal receiving any of the three drugs
listed above was also injected with an equal volume of
0.9% saline solution. The edema produced in each paw was
determined by measuring the paw diameter using an ana-
logic pakimeter (Vernier, Beaverton, OR) after stimulations
[9].
2.2.4. Statistical analysis
The statistical analysis were done using one way ANOVA.
The data are expressed as mean ±S.E.M. Differences be-
tween the controls and the treated groups of animals in
theses experiments were tested for statistical significance
by Student’s t-test for non-parametric data.
3. Results and discussion
The biomass production of a typical TM culture fer-
menting into molasses is presented in Fig. 1. After a 6-day
period, TM grains underwent a linear profile in biomass
production assured a media replacement at 48h intervals.
After the 6th day fermentation, TM was used for the
inflammatory experiments.
R.O. Diniz et al./Pharmacological Research 47 (2003) 49–52 51
Fig. 2. Effect of administration of TM suspension and dexamethasone during 6 days on the formation of granulomatous tissue. Numbers are represented
by mean ±S.E.M.of granuloma weight (P<0.05; Student’s t-test). CN—negative control (0.9% NaCl, 1ml), DM—dexamethasone, positive control
(0.2mg/kg, topical application), TM—Tibetan mushroom suspension (50g/l, 1ml).
TM suspensions were able to reduce the inflammatory
process of granuloma formation in rats after the 6th day
treatment at 42 ±5% as related to negative control samples
(Fig. 2). Although the inhibition with the positive control
dexamethasone was at the same degree as TM inhibition,
this substance is well-known to induce immunosuppressive
effects [10].
The anti-edematogenic activity of TM suspensions and
disrupted TM grains was evaluated by the rat paw edema test.
The administration of carrageenin (1mg per paw, 0.1 ml),
Fig. 3. Effect of p.o. administration of TM suspension with molasses (50g/l) and disintegrated grains cultured in the same media (1g/ml, 0.1ml), on the
rat paw edema induced by intraplantar carrageenin injection (1mgperpaw, 0.1ml). Numbers are represented by mean ±S.E.M.of edema lengthiness.
P<0.05 (Students’s t-test), n=8 per group.
dextran(50 g perpaw,0.1ml) and histamine (50 gper paw,
0.1 ml) showed significant edema in the rat paws (Figs. 3 and
4,P<0.05). The inhibition of rat paw edema induced by
the inflammatory agents was significantly different between
the negative control and test groups (P<0.05). TM sus-
pensions in aqueous molasses (50g/l) as well as TM grains
mechanically disintegrated (1g/ml, 0.1ml) presented mean-
ingful decreases in the inflammation response induced by
those compounds. TM suspensions orally administered were
found to be more effective than TM grains mechanically
52 R.O. Diniz et al./Pharmacological Research 47 (2003) 49–52
Fig. 4. Effect of p.o. administration of TM suspension in molasses (50g/l) and disintegrated grains cultured in the same media (1g/ml, 0.1ml), on
the rat paw edema (50gperpaw 0.1ml) after 1h of intraplantar injection of dextran (panel A) and histamine (panel B). Numbers are represented
by mean ±S.E.M.of edema lengthiness. P<0.05 (Students’s t-test), n=8 per group. CN—negative control, TM—Tibetan mushroom suspension,
TG—disintegrated TM.
disintegrated. Edema induced by carrageenin was gradually
observed after 30 min administration (Fig. 3), with an inhibi-
tion of 62% using the suspension and 40% with TM grains at
the 3rd hour. The inflammatory process carrageenin-induced
is referred in the literature as comprising three phases [11]
with the presence of histamine, serotonin, kinin system,
leucotriens (LTC4and LTD4), and prostaglandins [12–14].
Therefore the data presented in Fig. 3 suggested a partici-
pation of prostaglandins mediators more than histamine and
serotonin, though these latter are released only in the initial
phase after inflammatory challenges [11]. TM suspensions
administered 30min before dextran stimulation, however,
showed 100% inhibition for the inflammation at the 1st
hour. Nonetheless ground grains were found to inhibit
53 ±18% with dextran at the same period (Fig. 4A). The
dextran-induced edema is considered as a consequence of
histamine and serotonin release from mast cells [15].Even
though orally administered TM suspension showed the
same significant reduction for the histamine-induced edema
(Fig. 4B). The antagonized histaminic effect of TM was ob-
served with small changes during the 1st hours after stimuli,
with 52±26% inhibition using TM suspension and 43±22%
with disintegrated grains at the same period (Fig. 4B). In-
triguing there was no inhibitory activity of TM grains after
this period. Although the suggested mechanisms need to be
sustained, this work presented the ancient culture of TM as
a potential resource for anti-inflammatory therapeutics.
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Effect of lactic acid bacteria and yeasts on the structure and fermentation properties of Tibetan kefir grains, International Dairy Journal, https://doi. ABSTRACT 24 25 To better understand the effects of lactic acid bacteria (LAB) and yeasts on the 26 structure and fermentation properties of Tibetan kefir grains (TKGs), we treated 27 TKGs for 1 month with high doses of mycillin and cycloheximide to inhibit LAB and 28 yeasts, respectively. The results showed that in each group of TKGs, Lactobacillus 29 kefiranofaciens was the only dominant and stable bacterial species, and it exhibited 30 two distinct morphologies in TKGs, short rods in the LAB-inhibited group, and long 31 rods in the yeast-inhibited and control groups, which might directly result in the 32 inability of the grains in the LAB-inhibited group to grow further. Compared with 33 bacteria, the relative abundance of yeasts in TKGs was very low. However, even 34 though yeast had no effect on the structure and growth characteristics of TKGs, they 35 had a great influence on the fermentation characteristics of Tibetan kefir, such as acid 36 production and aroma. 37 _____________________________________________________________________ 38 J o u r n a l P r e-p r o o f 1.
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