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Antimicrobial effects of β-glucans and pectin and of the agaricus. Blazei-based mushroom extract, andosan™ examples of mouse models for pneumococcal-, fecal bacterial-, and mycobacterial infections



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Antimicrobial effects of β-glucans and pectin and of the Agaricus blazei-
based mushroom extract, AndoSanTM. Examples of mouse models for
pneumococcal-, fecal bacterial-, and mycobacterial infections.
G. Hetland1, E. Johnson2,3, D.M. Eide4, B. Grinde5, A.B.C. Samuelsen6 and H. G. Wiker7,8
1Depts of Immunology and Transfusion Medicine 2Gastroenterological and Pediatric Surgery, Oslo University Hospital,
3Inst of Clinical Medicine, Medical Faculty, and 6School of Pharmacy, Faculty of Mathematics and Natural Sciences,
University of Oslo, 5Depts of 4Environmental Medicine and 5Mental Health, Norwegian Inst of Public Health, Oslo, and
7Dept of Microbiology, Haukeland University Hospital, Bergen and 8The Gade Research Group for Infection and
Immunity, Dept of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen, Norway.
The increasing occurrence of multi drug-resistant (MDR) pathogenic microbes is a threat to the public health and prompts
a call for novel antimicrobial strategies. In Eastern traditional medicine edible mushrooms have been used for over 3000
years against a range of diseases including infections. β-glucans from yeast and mushrooms and pectin from Plantago
major L. have anti-infectious properties in rodent models against different microbes, including mycobacteria. The
medicinal mushroom Agaricus blazei Murill, used traditionally against cancer and hepatitis, has been found to have anti-
tumor effects in mouse models. An Agaricus extract, AndosanTM, also containing two related mushrooms has been shown
to protect against both Gram-positive and -negative sepsis in mice and has been tested against viral infections, as reviewed
here. Thus, in the future, biologically active substances isolated from medicinal mushrooms and plants, may prove useful
alternatives in the fight against serious infections by MDR pathogens.
Keywords MDR microbes, pneumococci, fecal bacteria, mycobacteria, β-glucan; Plantago major L., Agaricus blazei
Murill, AndosanTM
1. Introduction
Besides the well-known hospital tormentor, methicillin-resistant Staphylococcus aureus (MRSA), life-threatening
bacteria such as Mycobacterium tuberculosis, Streptococcus pneumoniae and others are becoming MDR, suggesting
that the microbes may win the battle over antibiotics. That would not be surprising because microbes have been on this
planet far longer than humans and have survived huge climate changes and are today adopted to life in quite different
habitats, e.g. thermic bacteria in Yellowstone geysers and in volcanoes that live on sulphur, pressure (>1000x
atmospheric pressure)-resistant bacteria in the sub-sea Mariana depression in the Pacific ocean near Guam [1],
anaerobic bacteria in the gut etc. The mechanisms for development of MDR in microbes under repression of
bacteriostatical or bactericide drugs, include lateral transfection of other strains and species of bacteria by plasmids
containing genes for drug resistance. One reason is unwise over-use of antibiotics for infections, also most probably
viral ones such as those causing otitis media in small children, or under-use - too low doses or shortened antibiotics
administration. The MRD infection epidemic is of great concern to the public health and new strategies are called for to
regain the upper hand in this battle.
In Eastern and African traditional medicine edible mushrooms and medicinal herbs have been used for thousands of
years against a range of diseases including infections, which is still the major health threat in Africa. This empiric
knowledge is very little tapped into by Western medicine and it is therefore pertinent to exploit this field in search of
drugs novel to Western societies that may supplement or adjuvate our current hospital therapies for infectious diseases
and also in other disciplines. Especially, it is of interest to activate the innate arm of the immune system because it is
evolutionary old and shared with sea urchin, sebra fish and banana fly, and has thus been successfully exploited in other
and more ancient species than ours. Many fungi and mushrooms are lethal to insects, animals and humans and we have
therefore developed specific receptors, e.g. toll-like receptor 2 (TLR2), on immune cells for the common fungal
signature molecule, β-1,3-D-glucan, that is the main structural ingredient of the cell wall of fungi and mushrooms. β-
glucan are also found in some bacteria and plants [2]. We share TLR with the banana fly, Drosophila melanogaster, in
which these receptors were detected in 1985 [3] and with rodents and other mammals. Other non-TLR receptors that
may be involved are dectin-1 and the lectin-binding site in CD11b/18 (complement receptor 3). Most probably fungi
and mushrooms also contain other such so-called pathogen-associated molecular pattern substances that may similarly
activate a native immune response in the host against potential danger. Therefore, β-glucans and other
immunomodulating molecules in fungi and mushrooms represent danger signals that trigger cells of the innate immune
system against potentially lethal attack from the outer world. β-glucans are D-glucose polymers linked by β-glycosidic
bonds. The structures of two different types of β-glucans are shown in Fig. 1. SSG (scleroglucan) is a soluble, although
viscous, gel-forming and highly branched β-glucan with high molecular weight (>5x106 kD) from the culture broth of
the fungus Sclerotinia sclerotiorum [4]. In Japan similar β-1,3-glucans from mushrooms such as lentinan, have been
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
used in combination with chemotherapy to treat cancer patients for more than 30 years [5]. MacroGard® is a β-1,3-
glucan extracted from baker’s yeast with less frequent side-chains but that contain more than two glucose molecules. It
is a potent immuno-stimultant produced in both a soluble and particular form [6]. Others and we have found that
harmless substances such as β-glucans and the edible Bacidiomycetes mushroom, Agaricus bM, can be exploited to
enhance the immune response against invading and dangerous microbes both of extracellular, e.g. E.coli [7] and
pneumococci [8,9], and obligate intracellular nature, e.g. mycobacteria. The anti-mycobacterial properties of β-glucans
have been proven both for M. tuberculosis-infected macrophages in vitro [10] and in a M. bovis, BCG model in mice
[11]. Similar to β-glucans, Agaricus bM is also found to stimulate TLR2 [12].
Actions of innate immunity are swift, powerful and general and may thus be effective against different infections -
MDR ones or not. β-glucan is a structural polyglucose in the cell wall of yeast and mushrooms with known
immunomodulatory, antitumor and anti-infection effects in rodent models. Plantago major L. is a plant used
traditionally for wound healing world-wide [13], and we have shown that isolated biologically active pectin
polysaccharides from it have anti-infection effects in a mouse model for pneumococcal sepsis [14]. The medicinal
Basidiomycetes mushroom Agaricus bM (Fig. 2) is a.k.a. A. brasiliensis because of its Brazilian origin, but has also
been designated A. rufotegulis and A. subrufescens, already described in 1893 by CH Peck, [15]. It is closely related to
the common champignon, A. bisporus, which has also been found to have benefial health effects [16]. Since the
mushroom was used in traditional medicine in Brazil against cancer, chronic hepatitis and other serious conditions, it
was taken to Japan in the mid 1960-ties and cultivated commercially as health food. Scientists have since then
documented immunomodulatory and antitumor effects of Agaricus bM in mouse models and we have found that an
extract of it, AndosanTM, that also contains two other related Basidiomycetes mushrooms from Japan, i.e. Hericium
erinaceus and Grifola frondosa, can protect against both Gram positive and Gram negative coliform sepsis in mice
[9,17]. This was basis for Dr. SV Bernardshaw’s PhD thesis in 2007 at the University of Oslo, Norway. Effect of
Andosan was also examined against influenza infection in mice as well as against chronic hepatitic C virus infection in
humans. Here, we review antimicrobial findings with bioactive polysaccharides such as β-glucans and Plantago major
L. pectin and a combined extract of medicinal Bacidiomycetes mushrooms in in vitro systems and in examples of
infection models in the mouse.
Fig. 1 Structure of two different types of β-1,3-glucan: Structural composition of SSG (scleroglucan) and MacroGard® showing the
β-1,3-linked backbone and its β-1,6-attached side-chains, which are responsible for the binding to glucan receptors (CD11b/18,
TLR2, dectin-1) and the resulting immunomodulatory effect. The figure, which also shows another SSG-like β-1,3-glucan, lentinan,
is modified from an illustration in the publication: “MacroGard®: structural aspects and basic mode of action on phagocytes” from
Biotec Pharmacon ASA, Tromsø, Norway by R.E. Engstad. Also see [6].
Fig. 2 Agaricus blazei Murill. Photo NutriCon.
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
2. Infection models in which β-glucans and pectin have been used:
2.1. β-glucans and M. tuberculosis – infected macrophage cultures
Since β-glucans stimulate innate immune cells such as monocytes and macrophages via binding to TLR2 and other
receptors, it was pertinent to examine whether these host cells for obligate intra-cellular pathogens such as
mycobacteria, could be activated to intracellular killing of these parasites. We used both a highly virulent strain of M.
tuberculosis, the culprit of the cardinal bacterial infection, tuberculosis, and its attenuated live vaccine; M. bovis,
Bacillus Calmette-Guerin (BCG).
Peritoneal macrophages were harvested from Balb/c mice and cultured in vitro. The cells were then infected with the
highly virulent M. tuberculosis strain H37Rv in presence or absence of PBS control, scleroglucan (SSG) or particulate
yeast β-glucan MacroGard®(MG). After 24 h of co-incubation extracellular bacteria were washed away, the
macrophages lysed and the lysate with the intracellular bacteria cultured for 3 weeks on Løwenstein-Jensen egg
medium and examined by immunofluorescence microscopy after auramin O-staining of the acid-fast bacilli. Although
SSG at 0.5 mg/ml gave a significant 40% reduction in the number of M. tuberculosis colony-forming units (CFU),
particulate MG (but not soluble MG; not shown) was 50x more efficient at inhibiting M. tuberculosis growth dose-
dependently (Fig. 3) [10].
Fig. 3 Effect on M. tuberculosis growth of yeast β-glucan (MacroGard® =MG) and scleroglucan (SSG) incubated simultaneously for
24 h with the tubercle bacteria in macrophage cell cultures, previously published [10].
2.2. β-glucan and M. bovis, BCG– infected Balb/c mice
Instead of hazardous animal studies with highly virulent M. tuberculosis bacteria, we chose to establish a model for M.
bovis, BCG, in the susceptible Balb/c mice by i.v. injection of viable bacteria into the tail vein. The animals were
injected i.v. either 100 µg of soluble β-glucan from barley (G-6513, from Sigma) or vehicle (PBS). At the peek of
infection 4 weeks after challenge, the mice were sacrificed and major organs homogenized and cultured or stained for
immunofluorescence microscopy. We found significantly lower bacterial counts in the spleen (p=0.01) of β-glucan-
treated than of PBS-treated mice when given pre-challenge (Fig . 4). Similar findings were done in liver homogenates
(not shown). When the β-glucan was injected post-challenge there was also a significantly lower bacterial load
(p<0.05) in the spleen (Fig. 4), but not in the liver [11].
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
Fig. 4 Immunofluorescence microscopy of M. bovis, BCG, bacteria (formaldehyde-fixed and treated with auramin O) in spleen of
Balb/c mice (n=8) sacrificed 4 weeks after challenge. Animals were given 100 µg of barley β-glucan i.v. 3 days pre- or 7 days post-
challenge. P<0.05, **P<0.001. Adapted from figure in [11].
2.3. β-glucan and pneumococcal sepsis
NIH/OlaHsd mice infected i.p. with S. pneumoniae serotype 6B were also injected i.p. with SSG (4 µg (low dose)-200
µg (high dose)) or PBS either 3 days before or 3 h, 24 h and or 72 h after bacterial challenge. Tiny blood samples were
collected daily from the lateral femoral vein and plated, and the number of bacteria (CFU) in the animals’ blood and
their survival were recorded. Pre-challenge SSG administration protected against S. pneumoniae sepsis as shown by a
dose-dependent inhibition of bacteremia and increased survival rates up to 50% with 200 µg of SSG as compared with
10% survival after 14 days of the PBS-treated mice (P=0.005) [8] (figs not shown). This high dose of SSG injected once
post-challenge after 24 h had curative effect against S. pneumoniae 6B as demonstrated by 40% survival at end of
experiment compared with none of the PBS controls (P=0.02) (Fig. 5).
Fig. 5 Survival (median values, n=8) from peritonitis and sepsis of NIH/OlaHsd mice challenged with S. pneumoniae serotype 6B
and treated with PBS or SSG β-glucan (L=low and h= high dose) 3 h, 24 h and/or 72 h later (adapted from [8]).
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
2.4. Pectin: Plantago major L. pectin PMII and pneumococcal infection in mice
Plantago major L., large plantain leaves, have been used as a wound healing remedy in traditional medicine for
centuries and in most parts of the World [13]. The purposed wound healing effect is not well documented. Never-the-
less, one might regard this plant as a potential source of immunomodulatory components triggering the healing process
or other processes involving the innate immune system. This was supported by Lithander [18] who reported
prophylactic effects of a P. major aqueous extract on mammary cancer in mice, indicating immunomodulatory
activities. A complex pectin fraction, PMII, that was isolated from the leaves of P. major showed anti-complementary
(complement-fixing) activity in vitro and was also shown to induce TNFα secretion after stimulation of human
monocytes [19,20,21].
Pectin polysaccharides are water soluble compounds found in the cell wall of dicotyledons. In general, pectins are
composed of unbranched homogalacturonan regions and regions with different types of side chains such as
arabinogalactans, galactans or arabinans linked to rhamnogalacturonan sequences of the backbone in so called
rhamnogalacturonan I (RGI) structures. In addition, single xylose residues and well defined side chains called
rhamnogalacturonan II (RGII) are found linked to the galacturonan backbone. For review, see [22]. The fine structures
of RGI vary with regard to monosaccharide composition, linkages, ramification and chain length. Structurally, PMII
was composed of galacturonic acid (71.7 %), rhamnose (4.2%), arabinose (8.8 %), galactose (8 %) and glucose and had
a molecular weight of 46-48 kDa. PMII was highly methylesterified (67 %), and contained both smooth and ramified
regions. Structure-activity studies revealed that the RGI-like structures of PMII containing 1,4- and 1,3,6-linked
galactose residues had the highest anti-complementary activity [23,20]. The fine structure and bioactivity of pectins
from different sources vary. For instance, cabbage (Brassica oleracea) leaves, which are also used to aid the healing of
wounds in folk medicine, were found to contain pectin fractions with lower complement-fixing activity than PMII [24]
even when the same isolation procedure was applied. Multivariate statistical analysis suggested that pectin activity is
enhanced by the content of 1,6- and 1,3,6- galactose side chains and low amounts of homogalacturonan regions [25]. It
was also found that isolated single side chains of white cabbage pectin did not affect the complement system, side
chains were only active when attached to the rhamnogalacturonan backbone [26]. The isolation procedure also affect
the structure of isolated pectins as well as their activity [27]. Due to modest activity in the complement system, Brassica
pectins were not subjected to further testing.
Due to its high complement-fixing activity, PMII was subjected to an in vivo study revealing a protective effect
against bacterial infection in mice. Inbred NIH/OlaHsd and Fox Chase SCID mice were pretreated with i.p. 12, 120 or
1200 µg PMII, 1.2 µg LPS or PBS 3 days before infection with S. pneumoniae serotype 6B. In PBS treated mice,
bacteremia levels increased after one day (see Fig. 6), and after 3 days none of the PBS treated mice were alive
compared to 50 % in the PMII and LPS-treated groups. In PMII treated mice bacteremia rose moderately until
reaching PBS levels at day 9, whereas bacteremia levels in LPS treated mice reached lethal levels after 4 days. PMII
had no effect after established infection, and there was not found any correlation between levels of anti-6B
pneumococcal IgM or IgG antibodies and the dose of PMII given indicating that the protective effect was due to
stimulation of the innate rather than the adaptive immune system [14].
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
Fig. 6 Colony-forming units (CFU) in peripheral blood from NIH/OlaHsd female mice pretreated with PBS, PMII low dose (PM L):
12 µg, median dose (PM m) 120 µg, high dose (PM h) 1200 µg or E.coli LPS (1.2 µg) i.p. 3 days before challenge with 10
pneumococci 6B i.p. The data points represent median values from eight animals. Reprinted from [14] with permission from John
Wiley & Sons, Inc.
3. Infection models in which Agaricus blazei extract has been used:
3.1. Antimicrobial effects of the Agaricus blazei Murill-based mushroom extract AndoSanTM.
The potential anti-bacterial effect of the Agaricus blazei Murill (AbM) (82%)-based Basidiomycetes mushroom extract,
AndoSan™ (Immunopharma AS, Høvik, Norway), including Hericeum erinaceus (15%) and Grifola frondosa (3%),
was studied in mice given monobacterial or fecal polymicrobial peritonitis. AndoSan™ was introduced by orogastric
intubation to NIH/OlaHsd mice prior to (24 h or 2 h) or simultaneously with induction of peritonitis by intraperitoneal
inoculation with moderately virulent S. pneumoniae serotype 6B [9]. End points were bacteremia and survival rate.
Controls were mice treated likewise but given PBS instead of AbM. The number of CFU was significantly reduced in
the AbM group compared to the PBS group (Fig. 7A). Furthermore, the effect was comparable and more pronounced
when given 24 h before relative to 2 h before or simultaneously with the induction of pneumococcal peritonitis. The
survival of the mice was improved in the three AbM treated groups of mice, but was also most pronounced (50%) after
treatment of AbM 24 h prior to induction of peritonitis (Fig. 7B). Since cultivation of the bacteria in the presence of
AbM on agar plates indicated no detectable reduction of number of CFU, AbM per se had no antimicrobial effect on the
pneumococci. Increases in the level of pro-inflammatory cytokines MIP-2 (murine equivalent to human IL-8) and
TNFα in the serum of mice receiving AbM once but more pronounced when received twice, indicated that the
protective effect of AbM was mediated by involvement of the native immune system. In order to study further the
potential protective effect of AbM in a more physiologically relevant setting for clinical and secondary aerobic
peritonitis, an experimental and reproducible model for induction of fecal peritonitis was developed in Balb/c mice [17].
Dilutions 1/4, 1/8 and 1/12 of mouse feces inoculated i.p. in the mice lead to severe, moderate and mild peritonitis,
respectively. In this model, using AbM compared with control (PBS) introduced orogastrically 24 h before bacterial
inoculation, a significant protection was revealed as measured by significantly improved overall survival for all degrees
of peritonitis (45% vs 28%) and particularly, severe peritonitis (25% vs 0%) (Fig. 8). Similarly, there was as significant
reduction of CFU in AbM-treated mice with severe and moderate peritonitis [17]. The temperature measurements
showed a negative correlation with the degrees of septicemia conditions and higher CFU, which is normal for septic
mice. These animal experiments took place at The Norwegian Institute of Public Health, Oslo. Quantitative and
qualitative characterization of the bacteremia revealed that both Gram-positive streptococci and Gram-negative
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
coliform bacteria dominated. In both studies the protective effect of AbM was demonstrated by use of two different
strains of mice expressing either Th-1/Th-2 balanced immunity (NIH/OlaHsd) or pronation towards Th-2 immunity
(Balb/c). A moderately virulent S. pneumoniae 6B [9] or fecal bacterial flora [17] were used in these studies. Since
AndosanTM seems to inhibit TLR4 (the LPS receptor)-mediated cellular stimulation of NF-κB activation, this may
partly explain the observed protection against Gram negative sepsis in the mouse model [12].
Fig. 7A Number of CFU of S. pneumoniae serotype 6B in blood of NIH/OlaHsd mice after treatment intragastrically with AbM and
PBS before (24 h) or simultaneously (0 h) with intraperitoneal bacterial inoculation (from [9] with permission).
Fig. 7B Survival in NIH/OlaHsd mice given AbM or PBS intragastrically prior to (24 h) or simultaneously (0 h) with intraperitoneal
inoculation with S. pneumoniae serotype 6B (from [9] with permission).
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
Fig. 8 Survival of severe fecal peritonitis in Balb/c mice after orogastric introduction of Andosan (AbM) or PBS control 24 h prior to
challenge. The results are based on 2 separate experiments with 8 mice in each group (from [17] with permission).
Table 1 Comparison of minimum inhibiting concentration (MIC) of the polysaccharide and mushroom products reviewed.
Polysaccharide or
mushroom product
Antimicrobial action in type
of infection model
MIC of compound given
pre-challenge or with
MIC of compound
given post-
-glucan from barley
β-glucan from baker’s yeast
M. bovis, BCG-infection in
100 μg/ mouse (i.v.)
10 μg/ mouse (i.v.)
100 μg/ mouse (i.v.)
MacroGard® (soluble)
M. tuberculosis (strain
H37Rv)-infected mouse
macrophage cultures
500 μg/ml* in vitro
10 μg/ml* ”
No effect* ”
No effect
S.pneumoniae (type 6B)-
infection in mice
4 μg/ mouse (i.p.)
12 μg/ mouse (i.p.)
4 μg/ mouse (i.p.)
(effect on
200 μg/ mouse (i.p.)
(effect on survival)
No effect (i.p.)
S.pneumoniae (type 6B)-
infection in mice
Fecal (Gram negative)
bacteria-infection in mice
0.9 μg/ mouse (p.o.),
*also with challenge
0.9 μg/ mouse (p.o.)
There was no direct antimicrobial effect of AndoSanTM against S. pneumoniae serotype 6B in bacterial cultures. Each
mouse was given 200 μl of AndoSanTM by orogastric installation, which is equivalent to 0.9 μg according to the dry
weight of 4.5 mg/ml after lyophilation of the extract (Samuelsen, unpublished results). The table shows that AndoSanTM
Time after challenge (days)
Survival (%)
Ab M
Numbers at risk 0 d 1 d 2 d 3 d 4 d 5 d 6 d 7 d p* me an **
Seve re
peri tonitis
Ab M 16 9 5 5 4 4 4 4 0.005 3
PBS 16 2 1 0 0 0 0 0 1
* log- rank test ** mean su rvival time in d ays
Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
was the product with the lowest MIC even though it was given enterally, in contrast with the parenteral administartion
of β-glucans and PMII. Hence, it proved to be the product with the highest efficacy in the comparison above.
3.2. Viral infections
Agaricus blazei extracts have previously been reported to inhibit certain viruses. More specifically, in vitro studies on
cell cultures have found antiviral effects against Western equine encephalitis virus [28], poliovirus type 1 [29], and
bovine herpes virus 1 [30]. As the extract has traditionally been used in connection with liver diseases, including
chronic hepatitis, we were allowed to test the in vivo effect of AbM on five patients with chronic hepatitis C virus
infection [31].
The patients did not respond to interferon treatment and were not given other anti-viral therapy. Daily, oral doses of
AbM were administrated for one week. Blood samples were obtained before and after treatment. The viral load was
slightly, but not significantly, decreased after treatment (5.3 compared to 5.8 million copies of virus per ml plasma).
The experimental setup allowed us to examine changes in gene expression in leucocytes from the patients prior to and
after treatment [30]. As might be expected, the changes were less pronounced compared to previous studies looking at
similar effects on monocytes treated in vitro [32]. Moreover, the cytokine genes most strongly induced in vitro were not
induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled
receptor signaling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the β-glucans of
the extract, which presumably are responsible for cytokine induction, did not readily enter the blood; while other
components, such as substances proposed to have anticancer effects, were active. The treatment did, however,
upregulate the gene for IFNα-receptor. Consequently, a study examining AbM intake combined with regular IFNα
treatment, might have been of interest. However now, other antiviral treatment than IFNα is used against HCV
The AbM extract was also tested on a mice model for influenza. No antiviral effect was demonstrated (unpublished
results). The discrepancy between the previously published in vitro antiviral effects, and the in vivo results on hepatitis
C and influenza virus, may be explained by the antiviral ingredients in the extract not readily entering the blood upon
oral administration.
4. Conclusions
The time has come to exploit novel and alternative strategies to combat MDR resistant harmfull and potentially lethal
microbes. Since the mixed Basidiomycetes mushroom product AndoSanTM has proven to be the most efficient of the
polysaccharide and mushroom-related products tested and reviewed here, we recommend this extract or components
thereof for future investigative clinical studies in patients with hard-to-cure bacterial infections inflicted by MDR
microbes. One such attempt is a planned clinical study, albeit awaiting ethical approval and financing, in which
AndoSanTM can be used against MDR-tuberculosis in patients at Armauer Hansen Research Institute (AHRI) in Addis
Abeba, Ethiopia. Regarding viral infections, we have so far not observed any significant effects of Basidiomycetes
mushroom extracts on viral load.
Acknowledgements We thank prof Berit Smestad Paulsen, School of Pharmacy, University of Oslo, for valuable discussions and
help with lyophilization of AndoSanTM for preservation, prof Ketil K. Melby at Department of Microbiology, Oslo University
Hospital for microbial quality control of AndoSanTM batches. Sponsor: AndoSanTM was supplied by Immunopharma AS, Høvik,
Conflict of interest GH is a stock holder of Immunopharma AS.
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Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
... Recently, Hetland et al. (2013) published a review which aimed to compare the antimicrobial effects of the following types of polysaccharides: ␤-glucans, pectin and commercial A. blazei mushroom extract (AndoSan TM ). These researchers demonstrated that ␤-glucans are present in the structure of yeasts and mushrooms and that pectin is present in Plantago major L., these having anti-infection properties in various models of mice against microorganisms, including bacteria. ...
... According to Hetland et al. (2013), this extract was notable among the other polysaccharides investigated in this study, that is, ␤-glucans and pectin, since it was more effective in terms of its antimicrobial action, based on previously reported results (Bernardshaw et al., 2005(Bernardshaw et al., , 2006. However, Soković et al. (2014) hypothesized that the mechanism of action associated with the antimicrobial property promoted by the mushroom A. blazei is not based on the stimulation of innate immunity, but could be explained by the induction of antiquorum (anti-QS). ...
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Infectious diseases associated with antimicrobial resistance are considered to represent an important public health problem. In this regard, the mushroom Agaricus blazei Murrill contains several bioactive substances that promote significant functional properties, among them, antimicrobial activity, which has attracted the interest of the scientific community. Thus, the aim of this study was to determine whether evidence of the antimicrobial action of A. blazei has been reported in the literature. In this integrative review, manuscripts held in research databases available online were examined with a view to answering the question “Does the mushroom A. blazei exert antimicrobial activity against Gram-negative and/or Gram-positive bacteria?” Only eight scientific articles that have addressed the antimicrobial properties of A. blazei, in vitro and in vivo, were found, all characterized as pre-clinical, i.e., with level VII evidence. Most authors have found that the A. blazei extract promotes an antimicrobial effect against peritonitis, as well as deadly oral infections, especially those caused by Gram-positive bacteria. However, the scientific data currently available are not sufficient to verify the antimicrobial aspect of the mushroom A. blazei and thus further investigation is required.
... The addition of S. cerevisiae and the content of β-glucan in tempe is expected to improve the functional properties of tempe so that the health benefits of tempe are also increased. This is supported by the opinion of Hetland et al. [10], that β-glucan has various biological activities including as an anticholesterol and immune system enhancer. ...
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Premium tempeh starter is a tempeh starter containing a mixed inoculum of Rhizopus oligosporus and Saccharomyces cerevisiae. Previously, premium tempeh starter was made in the form of liquid culture. This study aims to produce premium tempeh starter in powder form with the best type of substrate and incubation time so that it can be used practically. In this study, the effect of substrate type and incubation time on microbial viability of instant premium tempeh starter was studied. The study was arranged in a Completely Randomized Block Design with two factors and three replications. The first factor was the type of substrate: tapioca flour and rice flour, while the second factor was the incubation time at room temperature: 0, 24, 48, 72, 96 and 120 hours. The instant premium tempeh starter was analyzed for pH value, water content, number of fungi, yeast and bacteria. The microbial viability of tempeh starter was indicated by the growth of fungi, yeast and bacteria during incubation. The data obtained were analyzed by analysis of variance and further tested with the Honest Significant Difference (HSD) test at a 5% significance level. The results showed that rice flour and incubation time of 96 hours produced the best premium tempeh instant starter with the number of fungi of 9.02 Log CFU/g, 9.17 Log CFU/g yeast, 7.81 Log CFU/g bacteria, pH 4.2 and 7.75% water content. Tempeh made using the best premium tempeh instant starter has a chemical composition in accordance with the tempeh product standard (SNI 3144:2015).
... The addition of S. cerevisiae and the content of β-glucan in tempe is expected to improve the functional properties of tempe so that the health benefits of tempe are also increased. This is supported by the opinion of Hetland et al. [10], that β-glucan has various biological activities including as an anticholesterol and immune system enhancer. ...
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The aim of the research was to study the effect of inoculum type and fermentation time on microbial growth patterns (yeast, fungi and bacteria), β-glucan formation and antioxidant activity during soybean fermentation into tempe. The research was conducted using factorial Completely Randomized Block Design with 3 replications. The first factor was the types of inoculum: commercial inoculum of tempe, Raprima (3%), a single inoculum of S. cerevisiae (3%), a single inoculum of R. oligosporus (3%), and mixed inoculum of 1.5% S. cerevisiae and 1.5% R. oligosporus. The second factor was the length of fermentation which consisted of 0, 8, 16, 24, 32 and 40 hours at room temperature. Regarding the number of fungi, yeasts and bacteria, the observational data were presented descriptively in the form of graphs, while for the data from the analysis of β-glucan and antioxidant activity, the data obtained were analyzed for variance with analysis of variance (ANOVA) and then analyzed further by the Least Significant Difference (LSD) at the 5% significance level. The results showed that the type of inoculum and duration of fermentation had an effect on increasing the growth of fungi, yeasts and bacteria, as well as increasing β-glucan content and the antioxidant activity of tempe. Yeast growth had a more dominant effect on increasing β-glucan content and antioxidant activity compared to fungi and bacteria. Tempe inoculated with a mixed inoculum of 1.5% R. oligosporus + 1.5% S. cerevisiae, resulted in the highest β-glucan content of 0.58% and the highest antioxidant activity at 82.42%. In conclusion, a mixed inoculum of 1.5% R. oligosporus + 1.5% S. cerevisiae with 36−40 hours of fermentation produced tempe with the highest β-glucan content and antioxidant activity. Therefore, the β-glucan content causes tempe to have better potential health benefits than tempe without the addition of S. cerevisiae.
... Numerous natural antibiotics and antiviral substances have been isolated from mushroom fruiting bodies, including triterpenes, ganodermadiol, ganodermic acid, and lucidol, showing activity against herpes virus, influenza, and HIV [142][143][144]. Polysaccharides, mainly β-glucans, are also responsible for their microbial inhibitory properties [145,146]. The mechanism of action of glucans against microorganisms mainly involves the activation of several different immunomodulatory mechanisms, including phagocytosis, in which the phagocytic cells of the immune system, neutrophils, and macrophages participate [134,147]. ...
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Beta-glucans comprise a group of polysaccharides of natural origin found in bacteria, algae, and plants, e.g., cereal seeds, as well as microfungi and macrofungi (mushrooms), which are characterized by diverse structures and functions. They are known for their metabolic and im-munomodulatory properties, including anticancer, antibacterial, and antiviral. Recent reports suggest a potential of beta-glucans in the prevention and treatment of COVID-19. In contrast to β-glucans from other sources, β-glucans from mushrooms are characterized by β-1,3-glucans with short β-1,6-side chains. This structure is recognized by receptors located on the surface of immune cells; thus, mushroom β-glucans have specific immunomodulatory properties and gained BRM (biological response modifier) status. Moreover, mushroom beta-glucans also owe their properties to the formation of triple helix conformation, which is one of the key factors influencing the bioac-tivity of mushroom beta-glucans. This review summarizes the latest findings on biological and health-promoting potential of mushroom beta-glucans for the treatment of civilization and viral diseases, with particular emphasis on COVID-19.
... In another set of experiments, mice were injected iv. with-Mycobacteria bovis (2x10 5 /mouse in 0.1 ml of PBS) [9]. The optimal dilution was determined in preliminary experiments. ...
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Background: Glucans are known for their effects on anti-infection immunity. However, the individual papers always focused on one glucan, and no attempts were ever made to use one particular glucan in numerous infection. Methods: In our study, we use yeast-derived glucan #300, previously shown to ameliorate the effects of two different infections, to evaluate its anti-infection effects on mouse model of Mycobacteria bovis, Aeromonas hydrophila and Escherichia coli infection. Effects of supplementation was evaluated after 14 days of oral suppelentation with glucan. Results: In case of E. coli infection, we found glucan treatment achieved strong improvements in lowering numbers of bacterial in the spleen. Similar effects were found in the number of specific antibodies and survival. Similarly, glucan supplementation caused significant reduction of tissue bacterial load in Aeromonas infection and the spleen of Mycobacterium bovis infected mice. Conclusions: Our results clearly demonstrated that once the glucan significantly improved anti-infection reaction of mice.
... 포유류의 경우 체내로 유입된 β-glucans은 다양한 사이토카 인의 분비를 촉진시키고, monocytes나 macrophages의 증식 을 유도하거나 바이러스, 진균 및 세균 등 감염성 질환에 대한 비특이 면역 기작을 강화하는 것으로 알려져 있다 (Tzianabos, 2000;Hetland et al., 2013). 이러한 β -glucan의 면역 증강 효과는 무척추동물에서도 나타나고 있 다. 특히 최근 연구에 의하면 해산 이매패 역시 PRR으로서 β 1,3-glucan-binding proteins을 갖고 있는 것으로 보고 된 바 있다 (Review, Allam and Raftos, in press (Diao et al., 2013), 틸라피아 (Sirimanapong et al., 2015, 잉어 (Brogden et al., 2014), 챠넬메기 (Ainsworth, 1994), 무지개 송어 (Ghaedi et al., 2015) Table 1. ...
{\beta}-Glucan is a polysaccharide that is widely used as an adductive in fish feed to facilitate immune stimulation. This study aimed to investigate the effect of {\beta}-glucan on immune responses in the Manila clam Ruditapes philippinarum. For this purpose, three groups of R. philippinarum were exposed to 0%, 0.1%, or 1% {\beta}-glucan in sea water for 1 hr/day for 2 weeks using an immersion method. Thereafter, two immune parameters-phagocytic rate and antibacterial activity-were measured. R. philippinarum exposed to 1% {\beta}-glucan showed an approximate 30% significant increase in phagocytic rate. In addition, {\beta}-glucan significantly limited the growth of the pathogenic bacteria Vibrio tapetis, V. parahaemolyticus, and V. ordalii. Moreover, the mortality rates of {\beta}-glucan-treated clams decreased during a 17-day experiment. Our study suggests that treatment with {\beta}-glucan significantly increases the immune responses in R. philippinarum, and that immersion is a simple and effective method for immune stimulation in this species.
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Generally, the microorganism involved in soybean fermentation for the production of tempeh is Rhizopus oligosporus. However, Saccharomyces cerevisiae, a type of β-glucan-producing yeast, is known to be present and grow in the fermentation process. This study was aimed at investigating yeast and fungal growth dynamics, β-glucan formation, and antibacterial activity against Escherichia coli during the fermentation after adding S. cerevisiae as an inoculum. The Randomized Complete Block Design (RCBD) was applied with two treatments and three repetitions. Three types of starter culture were S. cerevisiae, R. oligosporus, and the combination of both. The second treatment was fermentation time at room temperature (30±2°C) for 0, 8, 16, 24, 32, and 40 hours. The dynamics were observed every eight hours. The obtained data were tested using Tukey’s Honestly Significant Difference (HSD) test. The results indicated that yeast grew during this process from a single S. cerevisiae culture and a mixture of R. oligosporus and S. cerevisiae, but not from R. oligosporus alone. The yeast grew during and until the end of fermentation and decreased after 32 hours in the mixed cultures. The β-glucan formed in tempeh with all types of inoculum, but the antimicrobial activity against E. coli increased with fermentation time and was significantly different between treatments. The highest β-glucan content and antibacterial activity of tempeh are from the mixed culture. In conclusion, the addition of S. cerevisiae and R. oligosporus in soybean fermentation produced tempeh with the highest β-glucan content and antibacterial activity against E. coli. The presence of β-glucans suggests higher health benefits of tempeh.
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Kapang, R. oligosporus merupakan mikroba utama yang berperan dalam fermentasi tempe. Penambahan khamir selama fermentasi tempe mempengaruhi kandungan aroma tempe dan diduga menghasilkan beta-glukan dalam tempe. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan Saccharomyces cerevisiae terhadap sifat organoleptik dan kandungan betaglukan pada tempe. Perlakuan terdiri dari 2 faktor yaitu konsentrasi S. cerevisiae yang terdiri dari 1% dan 3%, perlakuan kedua adalah cara pemasakan terdiri dari 2 taraf yaitu tempe digoreng dan dikukus. Sebagai kontrol dilakukan pengamatan terhadap tempe mentah. Pengujian organoleptik, sampel tempe diambil 15 g lalu dimasak sesuai perlakuan kemudian diamati secara organoleptik dengan uji skoring menggunakan 25 orang panelis. Data dianalisis dengan sidik ragam untuk mendapat penduga ragam galat dan uji signifikansi untuk mengetahui pengaruh antar perlakuan. Perbedaan antar perlakuan dianalisis menggunakan Duncan Multiple Range Test (DMRT) pada taraf 5% untuk pengamatan terhadap sifat organoleptik tempe. Pengamatan sifat organoleptik dilakukan terhadap aroma langu, aroma khas tempe, rasa asam dan rasa pahit, dan penerimaan keseluruhan tempe. Hasil penelitian menunjukkan tempe yang dibuat dengan penambahan S. cerevisiae 1% dan digoreng memiliki sifat organoleptik terbaik, yakni aroma khas tempe, bau langu lebih rendah, tidak berasa asam, dan tidak pahit. Meskipun berdasarkan skor penerimaan keseluruhan organoleptik, tempe yang diberi penambahan S. cerevisiae 1% dan digoreng lebih disukai panelis dibandingkan perlakuan lainnya, akan tetapi, penambahan S. cerevisiae 3% menghasilkan tempe dengan kandungan beta-glukan lebih tinggi (0,250%) dibanding penambahan S. cerevisiae 1% (0,181%).
To analyze BRCA1 and BRCA2 genes using a cost-effective and rapid approach based on next generation sequencing (NGS) technology. A population of Spanish cancer patients with a personal or familial history of breast and/or ovarian cancer was analyzed for germline mutations in BRCA1 and BRCA2 genes. The methodology relies on a 5 multiplex PCR assay coupled to NGS. Ten pathogenic mutations (four in BRCA1 and six in BRCA2 gene) were identified in a Spanish population. The deletion c.1792delA, in exon 10, and the duplication c.5869dupA, in exon 11 of BRCA2 gene were not previously reported and should be considered as pathogenic due to its frameshift nature. Two novel frameshift mutations in BRCA2 gene were detected using the multiplex PCR-based assay following by NGS.
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Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also play an important role in the defence mechanisms against plant pathogens and wounding. As constituents of plant cell walls and due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport, the porosity of the walls and in this way in the control of the permeability of the walls for enzymes. They also determine the water holding capacity. The amount and composition of pectic molecules in fruits and vegetables and other plant produce strongly determine quality parameters of fresh and processed food products. Pectin is also extracted from suitable agro-by-products like citrus peel and apple pomace and used in the food industry as natural ingredients for their gelling, thickening, and stabilizing properties. Some pectins gain more and more interest for their health modulating activities. Endogenous as well as exogenous enzymes play an important role in determining the pectic structures present in plant tissue, food products, or ingredients at a given time. In this paper functional and structural characteristics of pectin are described with special emphasis on the structural elements making up the pectin molecule, their interconnections and present models which envisage the accommodation of all structural elements in a macromolecule. Attention is also given to analytical methods to study the pectin structure including the use of enzymes as analytical tools.
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Mushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells. Mushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR. Semi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked β-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of β-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1β and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract. The polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly β-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.
Agaricus subrufescens Peck was cultivated first in the late 1800s in eastern North America. The type consists partly of cultivated material and partly of field-collected specimens. Once a popular market mushroom, the species faded from commerce in the early 20th century. More recently, a mushroom species growing wild in Brazil has been introduced into cultivation in Brazil, Japan and elsewhere. This Brazilian mushroom has been referred to by various names, most commonly as A. blazei Murrill (sensu Heinemann) and most recently as A. brasiliensis Wasser et al. The author first cultivated A. subrufescens in 1981 and has grown and studied Brazilian isolates since 1992. The species has an amphithallic pattern of reproduction. Based on DNA sequence analysis of the rDNA ITS region and on mating studies and genetic analysis of hybrid progeny, there is a strong case for conspecificity of the Brazilian mushrooms with A. subrufescens. Based on a study of the type and other data, the recent lectotypification of A. subrufescens is accepted. Data are presented on mushrooms of diverse geographical origins, including A. rufotegulis Nauta from western Europe, another apparent con-specific. A possible role for interpopulational hybridization in current populations of A. subrufescens is proposed. The agronomic history of the species is reviewed.
Scarce food supplies could hinder biological activity in the ocean's depths. However, measurements at Mariana Trench point to an unexpectedly active microbial community in the deepest seafloor setting on the planet.
The leaves of large plantain (Plantago major L.) are used for wound healing in the traditional medicine. The effect might be due to biologically active polysaccharides. A pectin, PMII with anti-complementary activity has been isolated from the leaves by water extraction and ion exchange chromatography (1).Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides.Oligosaccharides consisting of galacturonic acid and rhamnose with dp 3–5 and a series of 1,4 linked galacturonic acid oligosaccharides of dp 4–10 were isolated.
Polysaccharide fractions were isolated from Plantago Major L. leaves by extraction with cold water, hot water and with dimethylsulphoxide (DMSO). Each of the three crude extracts obtained was further separated by ionexchange chromatography. Only the two acidic fractions from the 50°C crude extract were biologically active, demonstrated by in vitro testing of activation of the complement system and induction of tumour necrosis factor alpha (TNF-α) production from human monocytes. One of the fractions, PM I50, contains polysaccharide material comprising arabinose, galactose and xylose with smaller proportions of galacturonic acid, glucose, mannose and rhamnose. This fraction was further fractionated by size exclusion chromatography to give three fractions PM Ia, PM Ib and PM Ic. PM Ia is probably an arabinogalactan and PM Ib is mainly a 1,4-linked xylan. The other biologically active fraction, PM II50 is composed of 75% galacturonic acid, 3% rhamnose, 6% arabinose and 8% galactose. Structure analysis indicates that PM II50 is a pectin type polysaccharide.
PMII isolated from the leaves of Plantago major L. is a pectin type polysaccharide with anti-complementary activity. It is highly esterified and partly O-acetylated with regions of 1,4 linked polygalacturonic acid and at least two different hairy regions. The galactose side chains are linked to position 4 of rhamnose in the main chain. The structure of the galactan side chains is complex, but 1,3,6 linkages are dominating in one of the isolated hairy regions. Arabinose is attached to position 3 and 6 of galactose. In the other hairy region arabinose is attached to position 3 of galacturonic acid.De-esterification and de-acetylation do not alter the anti-complementary activity of PMII. Different parts of PMII were shown to have different activities. The smooth regions are only slightly active in contrast to the hairy regions which had significantly higher activity. The hairy regions of highest molecular weight (PVa) with 1,3,6 linked galactose side chains were found to be the most active fraction. The importance of arabinose for the activity seems to depend on the site of substitution. Removal of arabinose terminally linked to galactose increases the activity slightly while removal of arabinose linked to the galacturonic acid backbone decreases the activity.
The purpose of this study was to isolate and perform chemical analyzes as well as biological testing of pectic material from white cabbage isolated by sequential aqueous ionic solutions (SEQAIS) or a simple pure water extraction (PW). Water extraction was aimed at yielding water-soluble pectins only, while the harsher conditions in SEQAIS aimed at extracting proto pectin as well. The pectic material resulting from the various extraction steps was characterized and tested, in order to determine whether structural and biological activity were influenced through different isolation procedures. The SEQAIS fractions obtained were one water-soluble and six partly water-soluble extracts, whereas PW yielded two water-soluble extracts. Sugar composition analysis, linkage analysis, HPSEC molecular weight distribution, HPAEC and 13C NMR were run to obtain structural characteristics of the extracted material. Both extraction procedures resulted in degradation of pectin. Pectin containing highly methyl esterified GalpA probably underwent β-elimination due to neutral pH during PW, while hydrolysis of Araf occurred in the first step of SEQAIS in 50 mM acetic acid pH 4.5. Water-soluble extracts were tested for complement-fixing activity and acidic extracts with degraded side chains showed reduced activity. Authors suggest that extraction conditions at neutral pH should be used in order to withhold side chain structure and immuno-activity.
Natural products are an inexhaustible source of compounds with promising pharmacological activities, including antiviral action. In the present study, the antiviral potential of polysaccharide-peptide (PLS) and an extracted β-glucan from Agaricus brasiliensis were investigated in the replication of bovine herpesvirus 1 (BoHV-1) in HEp-2 cell cultures. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the plaque reduction assay. To study the possible mode of action of PLS and β-glucan, the following protocols were performed: the virucidal assay, adsorption assay and the time-of-addition assay. The PLS presented a selectivity index (SI) higher than 12.50 and β-glucan 9.19. The antiviral inhibition (67.9%) in cells treated with PLS during virus infection was higher than that in cells treated prior to or post infection. The β-glucan presented high inhibition of virus replication by plaque assay (83.2%) and by immunofluorescence assay (63.8%). Although the mechanism has yet to be defined, we suggest that PLS and β-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. Additional studies are required for a better understanding of the mechanism of action of PLS and β-glucan.
A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting beta-elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by beta-eliminative treatment were composed of alpha-1,5 linked arabinosyl residues with 2- and 3-linked alpha-arabinosyl side chains, and, or beta-1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (8 kDa) obtained from beta-elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity.