MINI PROJECT part 2 Essay

CHAPTER 1 INTRODUCTION

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

radiation burns.

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.

Copy rights: hortorumcultus.actapol.net

Fig 1: Structure of ? -glucan with ?(1 -6)D -glucose branch and ?(1 -3)D -glucose

chains

CHAPTER 2 LITERATURE REVIEW

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

industry.

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).

CHAPTER 3 METHODOLOGY

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

rpm.

A B

Fig 3: Yeast cell ( Sac charomyces cerevisiae ) cultures ; A – Control, B – Yeast

culture.

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

A B

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.

3.3.2. SONICATION:

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.

3.6. QUANTIFICATION:

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.

CHAPTER 4

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

was obtained.

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

concentration

Glucan quantification by DNSA assay

CHAPTER 5 CONCLUSION

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.

CHAPTER 6 REFERENCE

1. Gordon D. Brown, Philip R. Taylor, Delyth M. Reid, Janet A.

Willment, David L. Williams, Luisa Martinez -Pomares, Simon Y.C.

Wong, Siamon Gordon (2002), Dectin -1 Is A Major ? -Glucan Receptor On

Macrophages, Journal of Experimental Medicine, Vol. 19 6, No . 3, pp. 407

412 .

2. Kusmiati , Dhewantara FX (2016), Cholesterol -Lowering Effect of Beta

Glucan Extracted from Saccharomyces cerevisiae in Rats , Scientia

Pharmaceutica, Vol. 84, N o. 1, pp.153 -65 .

3. Deepak Mudgil (2017), Dietary Fiber for the Prevention of Cardiovascular

Disease , Chap.3: Fibers Interaction between Gut Micoflora, Sugar

Metabolism, Weight Control and Cardiovascular Health, Academic press, pp.

35 -37.

4. Vassilios Raikos *, Shannon B. Grant, Helen H ayes, and Viren Ranawana

(2018), Use of ? -glucan from spent brewers yeast as a thickener in skimmed

yogurt: Physicochemical, textural, and structural properties related to sensory

perception , Journal of Dairy Science , Vol. 101 , No. 7 .

5. Krzysztof s obieralski, Ma rek siwulski, Jolanta lisiecka, Ma?gorzata

j?dryczka, Iiwona sas -golak, Dorota fru?y??ska -j??wiak (2012), Fungi –

derived ?-glucans as a component of functional food, Acta sci. Pol.,

Hortorum Cultus , Vol.11, No. 4, pp.111 -128.

6. Vassileios Varelas *, P. Tataridis, M. Liouni, E. T. Nerantzis (2016),

Application of different methods for the extraction of yeast ?-glucan, e-

Journal of Science & Technology (e -JST), Vol. 11, No. 1, pp. 75 -89 .

7. Hojjatollah Shokri , Farzad Asadi & Ali Reza Khosravi (2008), Isolation of

? -glucan from the cell wall of Saccharomyces cerevisiae, Natural Product

Research, Vol. 22, No. 5, pp. 414 -421 .

8. Andrea Poli, Francesco Visioli (2019), Pharmacology of Nutraceuticals with

Lipid Lowering Properties, High Blood Pressure & Cardiovascular

Prevention.

9. Ana Vitlic, Sohaib Sadiq, Hafiz I. Ahmed, Elisa C. Ale, Ana G. Binetti,

Andrew Collett, Paul N. Humpreys, Andrew P. Laws (2019), Isolation and

characterization of a high m olecular mass ? -glucan from Lactobacillus

fermentum Lf2 and evaluation of its immunomodulatory activity,

Carbohydrate Research.

10. Rahmani J, Miri A, C? ernevic?iu?te? R, Thompson J, de Souza NN, Sultana

R, Kord Varkaneh H, Mousavi SM, Hekmatdoost A (2019), Effects of cereal

beta -glucan consumption on body weight, body mass index, waist

circumference and total energy intake: a meta -analysis of randomized

controlled trials, Complementary Therapies in Medicine.

11. Sandra M. Vasquez Mej? a*, Alicia de Francisco, Raceli Sandrin, Tiago da

Silva, and Benjamin M. Bohrer ( 2019 ), Effects of the incorporation of ?-

glucans in chicken breast during storage .

12. No? Medina -C?rdova, Martha Reyes -Becerril, Felipe Ascencioa, Thelma

Castellanosa, Angel I. Campa -C?rdova , Carlos Angulo (2019),

Immunostimulant effects and potential application of ? -glucans derived from

marine yeast Debaryomyces hansenii in goat peripheral blood leucocytes ,

Biological macromolecules.

13. Ji-Young Hong, Sung -Ho Son, Sang -Pil Hong, Sung -Hun Yi , Sun Hee Kang,

Na -Kyoung Lee, Hyun -Dong Paik (2018), Production of ? -glucan,

glutathione, and glutathione derivatives by probiotic Saccharomyces

cerevisiae isolated from cucumber jangajji , LWT – Food Science and

CHAPTER 1

INTRODUCTION