Saccharomyces cerevisiae yeast

Source
Saccharomyces cerevisiae yeast is a type of multinuclear monocellular microorganism of
the genus Saccharomyces, class Ascomycetes, mushroom industry. S. cerevisiae has at
least 80 species of the same genus, 600 species of the same family and more than 10,000
different strains. This species can be considered the most useful fungus in human life
from thousands of years ago to the present.
S. cerevisiae has an extensive history of uses in the area of food processing. It is
commonly known as baker’s yeast or brewer’s yeast. S. cerevisiae has been used for
centuries as leavening for bread and as a fermenter of alcoholic beverages and wine
production (Oca et al., 2016). They are very common in nature, wild yeast strains have
been found on the surface of plants (leaves, flowers, fruits) or secretions of plants, body
surfaces and intestinal tracts of insects or crustaceans, soils in temperate, tropical or polar
regions, fresh water or salt water and many other environments (Martini, 2007).
Morphology and physiological properties
S. cerevisiae yeast cells are opaque white or light yellow, spherical, ovan-shaped,
elliptical or egg-shaped. Individual cell size of S. cerevisiae yeast has a large diameter of
5–10μm and a small diameter of 1–7μm (Oca et al., 2016). Their shape and size can vary
during the stages of development, and depending on the ambient conditions.
S. cerevisiae of industrially important due to its ability to convert sugars (i.e., glucose,
maltose) into ethanol and carbon dioxide (baking, brewing, distillery, liquid fuel
industries). S. cerevisiae breaks down glucose through aerobic respiration in presence of
oxygen. If oxygen is absent, the yeast will then go through anaerobic fermentation. The
net result of this process is two adenosine triphosphate molecules, in addition to two by
products; carbon dioxide and ethanol. 98% of glucose is metabolized during
fermentation, while 2% of it is made into cell materials (Oca et al., 2016). S. cerevisiae
are not limited to a particular habitat, they can grow in environments where carbon and
nitrogen levels vary. Optimal yeast growth occurs under aerobic conditions, with an
adequate nutrient supply, at temperatures of 28-30°C (O’Kennedy & Reid, 2008).
Structure
S. cerevisiae are eukaryotic cells containing all the organelles common to animal cells
such as nucleus, endogenous mesh, mitochondria, Golgi apparatus, non-cells, cell
skeletons and many other organelles.
Nucleus occupied 10.5% of the cell volume; cell wall occupied 17%; vacuole occupied
5.8%; cytoplasm occupied 64%; and mitochondria occupied only 1.7% in the G1 phase
(Yamaguchi et al., 2011). Chemical properties of yeast consist of approximately 30–33
%of dry materials, 6.5–9.3 % of nitrogen, 40.6–58.0 % of proteins, 35.0–45.0 % of
carbohydrates, 4.0–6.0 % of lipids,5.0–7.5 % of minerals and various amounts of
vitamins, depending on its type and growth conditions (Bekatorou et al., 2006).
Cell walls and biofilms are the two main components that demonstrate the excellent
protection of yeast cells.

Figure: In the structure of a yeast cell

According to (Coradello & Tirelli, 2021)
Cell Wall
Yeast cells are surrounded by cell walls. On the cell wall there are many holes, through
which nutrients are absorbed and products of metabolism are released. The cell wall
forms the morphology and ensures the integrity of the yeast during cell growth and
division. It gives the cell mechanical durability to be able to withstand changes in
osmotic pressure in the culture environment.
In yeast, the wall accounts for approx. 15–20% of the cell dry mass. Its thickness is very
variable (70–200 nm), since it increases in response to compression or osmotic forces;
structurally, however, it is always a highly polar double-layered matrix (a kind of
hydrogel). Its inner part is mainly composed of branched β-(1,3) and β-(1,6) glucans
(about 50% of the overall wall) hydrogen-bonded to 3–4% of mostly crystalline chitin
(Coradello & Tirelli, 2021). β-1,6-glucan has a high degree of cleft and is water soluble.
They connect the mannoproteins to the insoluble β-1,3-glucan network.
The outer layer, which con- sists of heavily glycosylated mannoproteins emanating from
the cell surface (Klis et al., 2002). The carbohydrate side chains of the cell surface
proteins contain multiple phosphodiester bridges, resulting in numerous negative charges
at the cell surface at physiological pH values (Coradello & Tirelli, 2021). These side
chains are responsible for the hydrophilic properties of the wall, and may be involved in
water retention and drought protection. The outer protein layer accounts for about one
third of the wall dry weight (Klis et al., 2002).
Table: Composition of yeast cell wall S. cerevisiae
Macromolecule % cell wall mass
Mannoprotein 30-50
β-(1,6)-glucan 5-10
β-(1,3)-glucan 30-45
Chitin 1,5-6
According to (Ciamponi, 2011)
Cell walls are highly elastic. When yeast cells are transferred to a superior solution, they
quickly shrink, and depending on the osmotic pressure, they can lose more than 60% of
the original volume. This process is reversible, when transferred back to the original
environment, the cells immediately expand to a normal volume. The elasticity of the cell
wall is due to the elasticity of the β-1,3-glucan chain (Klis et al., 2002). In addition, β-
1,3-glucan along with chitin are also responsible for the mechanical endurance of cells.
The lateral protein layer determines the surface characteristics of the cell such as
immunity, charge, hydrophobicity, cotton binding, and adhesion (Ciamponi, 2011).
Furthermore, through their covalent linkage to the β-glucan layer, mannoproteins
contribute to the wall outer porosity (Coradello & Tirelli, 2021).
Plasma membrane
The structure of the cell membrane is very simple, with a thickness of about 5 nm, mainly
consisting of lipids and proteins in almost equal proportions (Stewart, 2017). It is
composed in equal parts of lipids (mainly glycerophos-pholipids and fatty acids, with a
smaller quantity of sterols such as ergosterol and sphingolipids) and proteins, and is
connected by glycoproteins and glycolipids to the cell wall, from which it is separated by
a non-continuous, enzyme-rich region known as the periplasm. The membrane main
functional role is the regulation of the transport from/to the cell (Coradello & Tirelli,
2021). Therefore, it is the main barrier to permeability for permeable molecules (De
Nobel et al., 1990).
Lipid membrane
There are three types of lipids in the yeast cell membrane S. cerevisiae, including
phospholipid, fatty acids, sterols.
Phospholipids are the main component of cell membrane lipids. They are bipolar,
consisting of a polarized head containing choline, phosphate, glycerol and a non-
polarized tail that contains hydrocarbons derived from fatty acids. Membrane lipids are
arranged into two sheets close together, forming a double lipid layer and acting as a
permeability barrier for most water-soluble molecules. The hydrophilic head of lipid
molecules rotates out of the cell or into the cytoplasm, the water in trending head rotates
into the middle, where the contiguous place of the two lipid layers. The water
environment inside and outside the cell prevents membrane lipids from escaping from the
double layer, but nothing prevents these molecules from moving and changing positions
for each other in the plane of the membrane. Therefore, cell membranes are highly
flexible (Al, 2014).
The main sterol of yeast is ergosterol. The sterols are located between two phospholipid
molecules, the -OH group of sterols opposite the -COOH group of phospholipid circuits.
The interaction between sterols and unsaturated acyl circuits increases the hydrophobic
capacity of the dual lipid layer center and reduces the semi-permeability of the cell
membrane (Subczynski & Wisniewska, 2000).
Protein membrane
Based on how it binds to lipid membranes, membrane proteins are divided into two types:
trans-membrane proteins and peripheral proteins. Transclining proteins make up 70% of
membrane proteins, with one part located throughout the lipid membrane and the two
parts of the head sticking out on either side of the membrane. These proteins float in the
double lipid layer thanks to the hydrophobic bonds. They produce cells that pass through
the cell membrane, whereby molecules and information can enter and exit the cell.
Transclining proteins are fibrous in shape, can penetrate the membrane once or several
times, they do not fix in one place but move back and forth in a flip-flop style.
Peripheral proteins make up about 30% of the membrane protein component, which
appears on the surface or face in the cell membrane. Peripheral proteins bind to
membrane-piercing proteins by electrostatic or by hydrophobic bonds.
When acidic or alkaline agents are used to process cells, they catalyze hydrolysis of
peptide bonds in protein molecules, destroying the structure of the membrane, thereby
altering the properties of the cell membrane.
Hydrophobicity
The hydrophobicity of cells is considered an important property, especially for
pathogenic microorganisms because it determines the cell's ability to adhesion onto their
hosts. At the same time, cells with high hydrophobic properties are also better able to
fight macrophages than cells with a hydrophilic surface (Masuoka & Hazen, 1997).
Hydrophobicity is determined by membrane lipids, of which glycolipids are the most
important because they are found mainly in the single plate outside the membrane.
Glycolipids both contain hydrophobic units such as fatty acid chains, hydrocarbon chains
of sphingosine, and contain hydrophilic units that are one or more sugar roots (Fukui,
1975). It is noticed that if glycolipids on the surface of the cell membrane contain fatty
acid chains and the hydrocarbon chains of sphingosine are mainly then the cell is more
hydrophobic. In contrast, cells that do not show hydrophobicity are due to glycolipids on
its surface containing sugar radicals mainly.
Acid & Base
The acid/base properties of the cell are shown through the cell's electron giving and
receiving. It depends on the carboxyl root (COO–) of the transcene protein and the
peripheral protein. However, membrane-piercing proteins interact closely with membrane
lipid chains, so they can only be separated by agents consistent with these non-polarizing
interactions. Peripheral proteins that bind to the membrane mainly through electrostatic
bonds and hydrogen bonds, when washed with pH = 5-6 phosphate cushions, most of
these weak bonds are broken down. Therefore, the acidity/base of the surface depends
mainly on the properties of the transclining protein. The ends of the membrane-piercing
protein molecule poking out towards the outer surface and inner surface of the membrane
are hydrophilic and may end up being amino groups (NH4+) or carboxyl (COO–).
In addition, the acid/base properties are also shown through the adhesion of the cell to the
solvents. The ability to adhesion to ethylacetate (base solvents) reflects the extreme and
acidity of the cell surface. In contrast, adhesion to chloroform (acidic solvents)
demonstrates the base of the cell surface (Muñoz-Provencio et al., 2009).
Materials, contents and research methods
Materials
Research using yeast S. cerevisiae, Mauripan brand of AB Mauri Vietnam Co., Ltd.
Yeast is packaged vacuum and stored at ambient temperature.
Figure: Dry-yeast Mauripan
Ingredients for beta-carotene oil:
- High purity crystal beta-carotene, produced by Sigma-Aldrich, USA and stored in a
freezer of -20°C.
- Symply soybean oil is produced by Cai Lan Vegetable Oil Co., Ltd.
Chemicals and equipment
Chemicals
Table: Chemicals used in research process
N Chemical Purpose Source
o
1 N-hexan The experiment of determining the content of lipid and x
beta-carotene in yeast cell
2 Polysorbate Creating emulsion between suspension of yeast and oil China
80 of beta-carotene
3 BHT The experiment of determining the content of lipid and China
beta-carotene in yeast cell
4 Ethanol 80oC The experiment of determining the content of lipid and China
beta-carotene in yeast cell
5 HCl Pretreatment China
6 NaOH Pretreatment China
7 NaCl Pretreatment China
8
9
Equipment
Table: Equipments used in research process
N Equipment Manufacturer Source
o
1 Colorimetric Spectrophotometer Jinghua China
2 Weigh 4 odd numbers Sartorius Germany
3 Convection drying cabinet Memmert Germany
4 Thermostatic shaking tank Memmert Germany
5 Centrifuge Hermle Germany
6 Vortex Hwashin Korea
7 Sample shaker cabinet Labtech Korea
8 Ultrasonic Homogenizer Sonis American
Tools
Tools: beaker 100ml, erlenmeyer flask 100ml, volumetric flask 25ml, pipette, centrifuge
tube 15ml & 50ml, thermometer, glass rod, micropipette, glass cuvette, ray flask, rubber
squeeze tube.
Research content
To determine the moisture content
To commit survey of
chemical properties on
material
To determine the lipid content

To survey different time on pre-

treatment

To survey different NaCl on pre-

To commit survey of
treatment
different conditions on yeast
cell processing
To survey different NaOH on pre-
treatment

To survey different HCl on pre-

treatment
To determine hydrophobicity of
yeast cell
To commit survey of cell
properties after pre-
treatment To determine the lipid content of
yeast cell

To determine the beta-carotene

content of yeast cell
To evaluate the efficiency of
To determine the lipid content of
encapsulation process of
yeast cell
yeast cell

To determine the moisture content

of yeast cell

Experiment 1: Determine the chemical composition of the material

Purpose of experiment: Determine the chemical properties of material
Object of analysis: dry yeast.
Parameters surveyed: lipid and moisture content
Method:
- Determine lipid content by extraction method with n-hexane solvent.
- Determine humidity by drying method to constant mass, using drying cabinets
Experiment 2: Surveying the conditions for processing yeast cells S. cerevisiae for
beta-carotene encapsulation
Purpose of experiment: Assess the effect of the treatment of yeast cells under different
conditions on the effectiveness of beta-carotene oil packaging, thereby determining the
appropriate treatment conditions.
Implementation method:
Dried yeast is activated with distilled water within 5 minutes, collects biomass by
centrifugation at 4000 rpm, 5 minutes and washed once again with distilled water at 4oC.
Biomass is dispersed into distilled water in certain proportions and incubate under
different conditions. After that, the biomass is recovered by centrifugation at 4000 rpm.
Biomass after treatment is exposed to beta-carotene oil in the ratio of 1g wet mass: 10mL
distilled water: 1g beta-carotene oil :0,1g surfactant (span80) and incubate at ambient
temperature for 24 hours, shaking speed 200 rpm. Proceed to collect biomass by
centrifuging at 3000 rpm, 10 minutes and wash three times with distilled water.
Yeast microcapsules are then extracted to measure beta-carotene and lipid content with n-
hexane solvents to assess encapsulation effectiveness.
Experiment 2.1: Yeast cell processing time survey
Fixed parameters:
-Treatment biomass rate: 5 g wet biomass: 100 mL of distilled water
- Processing temperature: 50oC.
Parameter survey:
-Processing time: 1h, 2h, 3h, 4h, 5h
Experiment 2.2: HCl concentration survey for yeast cell processing
Fixed parameters:
-Treatment biomass rate: 5 g wet biomass: 100 mL of distilled water: 5ml (HCl: 0M,
0.05M, 0.07M, 0.1M, 0.12M)
- Processing temperature: 50oC.
-Processing time: 3h
Parameter survey:
-Processing concentration: 0M, 0.05M, 0.07M, 0.1M, 0.12M
Experiment 2.3: NAOH concentration survey for yeast cell processing
Fixed parameters:
-Treatment biomass rate: 5 g wet biomass: 100 mL of distilled water: 5ml (NAOH: 0M,
0.05M, 0.07M, 0.1M, 0.12M)
- Processing temperature: 50oC.
-Processing time: 3h
Parameter survey:
-Processing concentration: 0M, 0.05M, 0.07M, 0.1M, 0.12M
Experiment 2.4: NACl concentration survey for yeast cell processing
Fixed parameters:
-Treatment biomass rate: 5 g wet biomass: 100 mL of distilled water: 5ml (NACl: 0%,
10%, 20%, 30%, 35%)
- Processing temperature: 50oC.
-Processing time: 3h
Parameter survey:
-Processing concentration: 0%, 10%, 20%, 30%, 35%
https://www.youtube.com/watch?v=gk4akDW9Py0
HCL
HCl is one of the chemicals that can be used to promote the autolysis of yeast cells. It
catalyzes the hydrolysis of peptide bonds in protein molecules, thereby destroying
mannoproteins, forming holes in the cell wall. As a result, the cell wall becomes more
porous and certain intracellular substances are released into the surrounding medium,
increasing the volume of intracellular free space, so that beta-carotene oil can diffuse
inward more easily.
NAOH
 Porosity of the cell wall in yeast is correlated to the glucan network. There are two main
types of proteins that are covalently bound to the β-glucan network of the cell wall: GPI
protein (Glycosylphosphatidylinositol protein) and PIR protein (Plasmodium interspersed
repeats protein). GPI proteins are normally linked indirectly to β-1,3-glucan through a β-
1,6-glucan moiety. Meanwhile, the PIR protein binds to β-1,3-glucan by alkali-sensitive
binding [9]. When yeast cells are treated with NaOH, the release of GPIs or PIR proteins
creates holes and makes the yeast cell wall more porous, which is expected to facilitate
the diffusion of beta-carotene oil.