Exopolysaccharides is microbial polysaccharides secreted extracellularly,
whose amou nt and structure depends on the particular microorganism and substrate
available. One such exopolysaccharide is ?-Glucan a water -soluble polysaccharide
consists of glucose units obtained from oats, barley, bacte ria, yeast, algae, and
mushrooms . The glycosidic bonds found in ? -glucan varies depending upon the
source from which they are obtained. In bacteria and algae g lucose monomers of ? –
glucan are linked via ? -(1?3) glycosidic bonds which is a linear structure . In yeast
and mushrooms glucose monomers of ? -glucan which exhibits branched structure
are linked via ? -(1?3) and ? -(1?6) glycosidic bonds.
In oats and barley, glucose
monomers are link ed via ? – (1?4) and ? -(1?3) gly cosidic bonds , whose resultant
?-glucan also exhibit a branched structure .
?-glucans are of greater medical significance. They used for treating a wide
range of diseases namely, high cholesterol , diabetes , cancer , HIV, colds (common
cold ), flu (influe nza ), H1N1 (swine) flu, allergies , hepatitis , Lyme disease , asthma , ear
infections , aging, ulcerative colitis and Crohn’s disease, fibromyalgia , rheumatoid
arthritis , and multiple sclerosis .
It is also used to boost the immune system in people
whose body defenses have been weakened by adverse physiolo gical conditions such
as chronic fatigue syndrome, phys ical and emotional stress or when subjected to
treatments such as radiation or chemotherapy. People apply ?-glucan s to
the skin for dermatitis , eczema , wrinkles , bedsore s, wounds, burns, diabetic ulcers, and
Besides all its medical uses , ?-glucans are widely reported for lowering
cholesterol levels and acting as an effective immunomodulator. Water -solubility and
molecular weight of ? -glucan is considered to have a gre ater impact in controlling
its hypo cholester olemic effect. High molecular weight and high solubility in water
of ? -glucan have a high capacity of reducing serum cholesterol as compared to low
molecular weig ht and low soluble ? -glucan . It has also been repo rted that viscosity
of ? -glucan in the gut is mainly responsible for its lowering effects on chole sterol
(bad) . The v iscosity of ? -glucan is directly related to its molecular weight, molecular
structure, solubilit y in water, and food matrix . This may be due t o the higher
intestinal viscosity of ? – glucan, which reduces the reabsorption of bile acids and
leads to hig her excretion of bile acids . Synthesis of bile acids from cholesterol is
enhanced by higher excretion of bile acids , which ultimately increase s the c ho –
lesterol uptake and thus redu ces the LDL serum cholest erol. Concentration of ?-
glucan concentration consumed in the diet and its molecular weight, controls the
vis cosity of the digesta , hence the glycemic response is reported to have a significant
corre lation with concentration and mole cular weight of ? -glucan (Deepak Mudgil ,
2017). ?-1,3 -Glucan molecules are almost resistant to the acidic secretions in the
human stomach. After ingestion, ? -glucans gradually passes into the first section of
the small intestine (duodenum) and are trapped by macrophage receptors located on
the intestinal wall. Activation of macrophage receptor by glucan binding generates
bacter icidal compounds, such as lysozyme, reactive oxygen radicals, and oxides.
After that the cells commence to yield numerous cytokines, which activate the sur –
rounding phagocytes and leukocytes tha t lead to specific immunity (Gordon D.
Brown, et. al., 2002 ).
Cell wall of Saccharomyces cerevisiae (bakers yeast) is rich in ? -glucan,
composed of ?-(1?6) Glycosidic side chains interconnect ed with the ? -(1?3)
glucan chains to create a rigid network and water -soluble because of its structure.
The objective of this project is to mass cultivate yeast cells and isolate ? -glucan from
yeast cell wall by alkaline extraction and acid treatment.
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Fig 1: Structure of ? -glucan with ?(1 -6)D -glucose branch and ?(1 -3)D -glucose
2.1. SOURCES OF ? -GLUCAN:
? -glucans are components of cell walls of plants, algae, bacteria , and fungi.
Widely exploited plant sources for ?-glucan isolation are oats and barl ey. Bacteria –
derived ?-glucans , such as, xanthan, dextran, pollulan and gellan are used on the
large scale in food industry, which are com monly named as bacterial
exopolysaccharides. These ? -glucans are secretions of different microorganisms,
such as Cellulomonas flavigena strain KU, Bacillus curdlanolyticus , Bacillus
kobensis , Bacillus , and Micromonospora , Agrobacterium sp. ATCC 31749,
Bradyrhizobium , Rhizobium spp., and Sarcina ventriculi. Yeast (Saccharomyces
cerevisiae ) has been a valuable source as 55 -60% of its cell wall constituent is ?
glucan. Species, that have been used for many years in traditional medicine of the
Far West, are, Ganoderma lucidum , Lentinula edodes , Grifola frondosa , Hericium
erinaceus , Trametes versicolor , Schizophyllum commune , Phellinus linteus ,
Inonotus obliquus , and Pleuortus ostreatus .( Krzysztof Sobieralski, 2012).
2.2 . COMMERCIAL APPLICATIONS:
?-glucan are exploited from various sources and with its varying properties
they find applications in diverse industrie s namely pharm, food, beverage and textile
In recent years they are studied to analyze the effect on physical, chemical,
textural, microbial and thermal properties of the food product imparted with ? –
glucan. The application of ?-glucans in chicken meat ( ?-Glucan and SALT+ ?-
Glucan treatments) presented improved cooking loss compared with control samples
but did not present additional advantages with respect to the SALT treatment. Under
the conditions evaluated, incorporation of ?-glucans into the whole muscle chicken
did not present detrimental effects to product quality or safety. However, after
cooking, the fiber concentration in the final product was reduced to levels where it
would not be sufficient for health benefits or for the product to be declared as a
source of dieta ry fiber . (Sandra M. Vasquez Mej?a, et. al., 2019).
In yogurt fortification with ?-glucan, it has imposed an incremental effect on
the textural properties that were more detectable at 0.8% concentration, with an
adverse effect on the over all liking of th e yogurt. ( Vassilios Raikos, et. al., 2018)
2.3 . MEDICAL USES OF ? -GLUCAN:
Besides all the commercial application of ? -glucan, it is highly noted for its
medi cal significance too , notably as an immunomodulator and lowering cholesterol
levels . Cardiovascular disease due to LDL cholesterol is known to ruin most of the
life. Use of ? -glucan is said to significantly lower the LDL levels, thereby lowering
the cardiovascular disease for greater extent. A daily dose of 3 g reduces LDL
cholesterol by 5 6% without significantly affecting the plasma concentrations of
other lipids. Glucomannan, P lantago/psyllium, and chitosan are also effective. Other
potentially healthful effects of beta -glucan include modulation of glycemia
(probably at a higher daily do sage) and prebiotic actions. ( Andrea Poli , Francesco
Visioli , 2019)
?-glucan from Lactobacillus fermentum Lf2 imparts immunotolerance in
peripheral blood mononuclear cells (PBMC). An ability to modulate the release of
the proinflammatory mediators, such as TNF -?, is an important goal in the
development of therapies for the treatment of diseases, such as Crohns disease and
ulcerative colitis, associated with excessive release of inflammatory mediators (Ana
Vitlic et. al ., 2019) .
Studies reveal that, by promoting cell viability, phagocytic activity,
antioxidant immune response , and immune -related gene expression , ?-glucans are
able to modulate the immune system . Indeed, activated stronger immune responses
of Dectin -1 and Toll -like receptor 4 (TLR4) could be activated by ?-glucans derived
from D. hansenii . (No? Medina -C?rdova, et. al., 2019)
Cereal ? -glucan is said to impart a significant reduction in body weight and
body mass index (BMI) following ?-glucan consumption (weighted mean difference
[WMD]: -0.77 kg, 95% CI: -1.49, -0.04) and (WMD: -0.62 kg/cm2, 95% CI: -1.04,
-0.21), respectively. And a subgroup analysis showed that a beta -glucan dose of ?
4g/day tends to increas e the amount of energy intake ( Ra hmani J et. al., 2019).
3.1. CELL CULTURING:
Commercially available Bakers yeast (Saccharomyces cerevisiae ) was
purchased and 2 g of granule was inoculated in 2 00 ml of Potato Dextrose A gar
(PDA) with Chlora mphenicol which has antibacterial activity over a wide range of
bacteria under aseptic condition. Cultured media was incubated with continuous
agitation at 120 rpm for 6 days, at room temperature. It was followed with plating
them in PDA plates and was left under in cubation around 35 ?C for 7 days to obtain
pure colony. On the other hand , slants were prepared to preserve them for later use.
Fig 2. Culture plates of Saccharomyces cerevisiae ; A – Control plate, B – Culture plate
Colonies of Saccharomyces cerevisiae was then again cultured in 200ml of
PDA, and incubated for 7 days at room temperature with continuous agitation at 120
Fig 3: Yeast cell ( Sac charomyces cerevisiae ) cultures ; A – Control, B – Yeast
3.2. CELL HARVEST:
6-day old media was centrifuged at 10,000 rpm for 20 min to separate the
grown culture cells from the media. Media (supernatant) was removed and cell
(pellet) was washed thrice with distilled water over mild centrifugation. Cells were
stored by suspending in 0.1 M sodium phosphate buffer of pH 7.2.
Fig 4: Harvested yeast cells suspended in sodium phosphate buffer
3.3. CELL DISRUPTION BY SONICATION:
In order to ease the sonication process just before sonication induced cell lysis
and hot water treatment was carried out.
3.3.1. INDUCED LYSIS AND HOT WATER TREATMENT:
Stored cell s were centrifuged to remove the buffer and was suspended in 3%
NaCl and 0.1N HCl to adjust the pH to 5.3. It was left incubated for 1 day with
agitation at 120 rpm, at room tempe rature. Then was subject ed to hot water
treatment, by placing it in hot water bath at 100 ?C for 2 hours.
Hot treated cells w ere subjected to sonication at 60% amplitude and pulse -on:
pulse -off ratio to be 30: 30 sec for 6 min. This would bring about cell disruption.
3.4. CELL WALL SEPARATION:
The sonicated product was diluted with distill ed water and was centrifuged at
5000 rpm for 2 min. sequentially it was again washed with distilled water by
centrif ugation. Then it was again suspended in sodium phosphate buffer and
incubated in hot water bath at 80 ?C for 2 min to inactivate the lytic enzyme.
3.5. ALKALINE EXTRACTION AND ACID TREATMENT:
The r esidue obtained after cell wall separation was suspended in 2% sodium
hydroxide at 90 ?C for 2 hours. And it was centrifuged at 11,000 rpm for 20 min. The
supernatant obtained was neutralized with 2M acetic acid and thrice its volume of
ethanol. As a result crude ? -glucan gets precipitated. And was separated out by
filtering and the crude residue was preserved.
The crude precipitate of ? -glucan was subjected to DNSA assay to quantify
the product obtained. It was performed for 1 ml of the crude sample. To it , 3ml of
DNS reagent was added and was incubated at 90 ?C in hot water bath for 10 min. It
was cooled to room temperature then 1 ml of Rochelles salt was added and read
under spectroscope at 560nm in order to determine the amount of product obtained.
Similarly, for a standard 1% glucose stock was diluted to different concentration and
was analyzed to obtain the standard plot and extrapolation of obtained sample OD
in standard plot gives out the concentration value of sample ?-glucan.
RESULTS AND DISCUSSION
4.1. CELL HARVEST:
As a result of culturing Saccharomyces cerevisiae cells for 7 days , 6.4 g of
the cell mass was obtained.
Fig 5: Harvested yeast cell
4.2. ISOLATION OF ? -GLUCAN:
At the end of isolation process, a trace amount of crude ? -glucan precipitate
Fig 6 : Crude ? -glucan isolated
4.3. QUANTIFICATION BY DNSA ASSAY:
The results obtained from DNSA assay is tabulated below,
CONCENTRATION Blank 0.5 1.0 1.5 2.0 2.5 Sample
OD VALUES 0 3.69 6.59 9.98 11.97 16.99 2.50
Table 1: DNSA assay values
Fig 7 : Graph for DNSA assay
From the graph obtained it could be interpreted that the concentration of ? -glucan
was estimated to be 0.384 mg.
Extraction method employed to isolate ?-glucan has got a significant impact
on the yield and its property. With the isolation process followe d above the net
quantity of ?-glucan isolated was 0.384 mg from 6.4 g of cells. Yie ld could be
increased by o ptimiz ing the isolation procedure .
y = 6.5178x
R? = 0.9887
0 0.5 1 1.5 2 2.5 3
OD @ 560 nm
Glucan quantification by DNSA assay
Wide range of sources exist s for the extraction of ? -glucan, out of which yeast
are the significant choice due to its high glucan content and ease of mass cultivation
at any scale under controlled conditions. Meanwhile, the nature and prop erty of ? –
glucan isolated vary depending upon the source and method of isolation.
Optimization in the isolation me thod may be performed to increas e the yield.
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