Chapter 1:

Product Description

CHAPTER I

Product Description

I.

INTRODUCTION

A rising awareness about health concerns such as diabetes, obesity, and heart diseases has
caused a growing demand for low-calorie products. One contributor of calories in our bodies
comes from added sugar which has little vitamins and minerals. Added sugars, which include raw
sugar, brown sugar, corn syrup, high-fructose corn syrup, malt syrup, maple syrup, and sugar
molecules like dextrose, fructose, lactose, maltose, and sucrose, are the type of sweeteners that are
added to food and beverages during the process. Theses added sugars have many calories but have
little nutritional value. Since there is a need to reduce these added sugars in our diet, the demand
for polyols, also known as sugar alcohols, increased. Polyols, which are often used as sweetener
alternative in low-calorie food, includes sorbitol, mannitol, xylitol, maltitol, lactitol, erythtritol and
isomalt. These polyols have their own sweetness but contains fewer calories.

Erythtritol, a four-carbon sugar alcohol with a chemical formula of C4H10O4, is a lowcalorie substitute for sugar that naturally occurs in fruits such as grapes, pears and watermelons
and in fermented products such as wine, beer, sake, soy sauce, miso bean paste and cheese. It is a
sugar alcohol that has around 60 to 70 percent of the sweetness of sugar but contains less than 0.2
calories per gram. These properties of erythtritol are essential in food and beverage industry.

Mainly, erythtritol is used in candies, chocolates, carbonated beverages, chewing gums, baked
goods, yogurts, jellies and jams.

The application of erythritol is not only limited as an alternative sweetener, it can also serve
as a flavor enhancer, stabilizer, humectant, thickener and formulation aid (Jovanovic, Mach, &
Mach-Aigner, 2014). Erythritol can also be hydrogenated to form hydrocarbons such as butane,
butene, butadiene, and butane-diol (Amada, et al., 2012). This conversion of C4 compound to
hydrocarbons is promising from the perspective of naphtha plants.

In pharmaceutical industry, erythtritol is used as flavoring agents to improve the taste of

medicine tablets and syrups. In cosmetics, it can also be used as preservatives to hinder the growth
of bacteria and is used in creams and lotions because of its moisturizing properties. Erythtritol is
also used as an intermediate in paint industry as well as explosives industry. Erythtritol has been
given a status of generally recognized as safe (GRAS) from the FDA and is broadly used in United
States, Europe, Mexico, Brazil, Russia, Argentina, China, Japan and Canada.

Erythtritol can be produced chemically by hydrolysis of dialdehyde starch, reduction of

meso-tartarate, oxidation and reduction of 4,6-o-ethylidene-D-glucose, reduction of formaldehyde
polymers and hydrogenolysis of sorbitol (United States of America Patent No. 6,060,291, 2000)
(United States of America Patent No. 3,756,917, 1973). Erythtritol can also be produced
synthetically by treating an aqueous alkali carbonate solution with 2-butene-1,4-diol with chlorine
and saponifying the chlorohydrin (United States of America Patent No. 3,756,917, 1973).
Production of erythtritol using chemical routes are often expensive and complex. Hence, many
manufacturers produce erythtritol via fermentation.
3

For industrial production of erythtritol, the most common process is through fermentation
using corn as the major source of carbon. Fermentation is typically used because of its simple
process and with low cost starting materials, the process will offer great potential. In this biological
method, various microorganisms are used as yeasts such as Candida magnolia (Ryu, Park, Park,
Kim, & Seo, 2000),Yarrowialipolytica (Tomaszewska, Rywinska, & Gladkowski, 2012), Torulasp
(Kim, et al., 2000), Moniliella sp. (Lin, Wen, Liau, & Chu, 2001), and Trichodermareesei
(Jovanovic, Mach, & Mach-Aigner, 2014). Erythtritol production on wheat straw using this
T.reesei proved that with strain modification, formation of erythtritol will increase. Strains of
Candida lipolytica may also be used because of its ability to produce erythtritol during the aerobic
fermentation of aqueous media containing a hydrocarbon as a source of carbon. Other yeasts from
the genus of Trichosporonoides and genus of Aureobasidium can also be used in the fermentation
process. Under anaerobic conditions, Leuconostoceonos can also produce erythtritol. There is also
a report on a high erythtritol-producing strain called Pseudozymatsukubaensis that gives a high
yield of erythtritol without producing by-products (Jeya, et al., Isolation of a novel high erythritolproducing Pseudozyma tsukubaensis and scale-up of erythritol fermentation to industrial level,
2009). After the fermentation, impurities are eliminated. The erythtritol will then undergo
crystallization process to obtain a high purity.

With erythtritols advantages in health and in polyol market and by using the inexpensive
and simple process of fermentation, continuous interest about the production of erythtritol will
keep on increasing.

II. PRODUCT PROFILE: ERYTHRITOL

Erythritol is a naturally occurring substance abundant in nature. It is a white crystalline or

powdery polyhydric alcohol and it is a metabolite or storage compound for seaweeds and
mushrooms, same with most of the other polyols. Fruits like melons, grapes and pears also contain
erythritol. It is often produced by bacteria, fungi, and yeast, it also occurs frequently in fermented
food, wines, beer and soy sauce. Since it has a taste very close to sucrose without the bitter
aftertaste, almost no calorie, does not affect blood sugar, does not cause tooth decay and is partially
absorbed by the body, it is an ideal sweetener. Erythritol is widely used a sweeteners in food
production and various industries including beverage production, pharmaceutical, cosmetics and
other industries.

A. Technical Specification

Erythritol Identification
Product Name

Erythritol

Synonym

1,2,3,4 Tetrahydroxybutane; Phycitol; meso-Erythritol

CAS Number

149-32-6

EC Number

205-737-3

Molecular Formula

C4H10O4

Toxicity

May cause eye irritation with susceptible person.

May cause skin irritation in susceptible person.

May cause irritation of respiratory tract.

Sales Specification
Quality

Food Grade

Mesh

30 -60 mesh

Assay (on dry basis)

99.5

Loss on drying

0.20 %

pH

5.0 7.0

Reducing Sugars

0.3%

Ribitol and glycerol

0.1%

Ash

0.1%

Heavy metal (Pb)

0.1 mg/kg

Arsenic (count as As)

0.3 mg/kg

Bacterium total

300 cfu/g

E.Coli

negative

B. Physical and Chemical Properties of Erythritol

Physical State and Appearance

White crystalline/powder solid

Odor

Odorless

Taste

Moderately Sweet

Molecular Weight

122.12 g/mole

Boiling Point

329oC 331oC (642.2oF 627.8oF)

Melting Point

119oC - 123oC (246.2oF 253.4oF)

6

Relative Density

1450 kg/m3

Bulk Density

700 900 kg/m3

Specific Gravity

1.45
Approx.

60g/

100mL

H2O

(at

25C)

Solubility
Slightly Soluble in Ethanol (25oC)
Viscosity

1.200 cP @ 20C

C. Composition and Information on Ingredients

Chemical Name

CAS Number

% by Weight

Erythritol

149-32-6

99.5

Water

7732-18-5

.5

D. Uses of Erythritol
Category

Examples
Used as sweeteners in food to enhance sweetness.
Used as medium in low calorie diet food.

Food
Used as protective agent of dental caries in candy
and dentifrice to protect the mouth.
Used as Flavoring agents in tablets to enhance the
Pharmaceuticals

taste.
7

 Used as excipients in tablets to enhance the taste.

Used as sweeteners in cool drinks to enhance
Beverages
sweetness.
Used as preservatives in cosmetic and personal care
Cosmetics
products to inhibit bacteria.
Used in synthesis of alkyd resin polyester polyether.
Other Industries
Used in manufacture of paint and explosives.

E. Erythritol Transportation, Handling, Storage and Packaging

No special requirements and no restrictions on
Transportation

transportation by land, sea or air.

Keep away from heat and sources of ignition.

Handling

Ground all equipment containing material.

Keep in a dry, cool, well-ventilated and shaded place

Storage

with original packaging.

Avoid moisture and store at room temperature.
Product is available in 25 kg/bag

Product Packaging

Also available in 18.75 mt pallet/ 20gp

F. Erythritol Safety and Disposal.

Safety

Exposure to the chemical may cause eye and respiratory irritation.

Waste should not be disposed of untreated to the sewer.

Incineration or landfill should be considered when recycling is not

feasible.

Dissolve or mix material with a suitable combustible solvent and

Disposal
incinerate in a chemical incinerator equipped with an afterburner
and scrubber.

Avoid dispersal of spilled material and runoff and contact with

soil, waterways, drains and sewers.

III. RAW MATERIALS

Cassava Starch

Cassava starch is made from cassava, which is noted for its low non-carbohydrate
content. It is one of the most important but flexible food ingredients possessing value added
attributes for innumerable industrial applications. The most common sources of food starch
are corn, potato, wheat, cassava/tapioca and rice. Cassava is only second to sweet potato
as the most important starchy root crop of the tropics. (Akpa & Dagde, 2012)

A. Technical specification

Cassava Starch Identification

Product Name

Cassava Starch

Synonym

Amglogen; Amyglodextrin; Tapioca starch

CAS Number

9005-25-8

EC Number

232-679-6

Molecular Formula

(C6H10O5)n

Toxicity

Sales Specification
Compound

Composition by Weight

Starch

83.4%

Ash

0.2%

10

Fats

0.3%

Fibers

0.5%

Proteins (insoluble)

0.3%

Soluble protein

0.3%

Moisture

15%

B. Physical and chemical Properties of Cassava Starch

Appearance

Amorphous powder or granules

Color

White

Odor

Slight characteristic odor

Solubility

Dispersible in hot water

Specific Gravity

Ca. 1.5

%Volatilities by volume @21oC (70oF)

pH

5 to 7 (conc. (%w/w): 1)

C. Composition of ingredients

Compounds

Composition by Weight

Starch

84.5%

Starch as amylose

19.5%

11

Cyanide

0.05%

Sugar

1.5%

D. Uses of Cassava Starch

Use

Main source of starch and glucose.

E. Cassava Starch Transport, Handling and Storage

Transport

Handling

Storage

Not regulated.

Keep in tightly closed container.

Avoid dispersing powder in the air.

Prevent buildup of powder on surfaces.

Store in a cool, dry, well ventilated area away from sources of heat, moisture,
and incompatibilities.

F. Cassava starch Safety and Disposal.

Safety

Use process enclosures, local exhaust ventilation, or other

engineering controls to keep airborne levels below recommended
exposure limits.

12

If user operations generate dust, fume or mist, use ventilation to

keep exposure to airborne contaminants below the exposure limit.

Disposal

Do not dispose untreated in to the sewer.

Avoid dispersal of spilled material and runoff and contact with

soil, waterways, drains and sewers.

12

 Alpha amylase

The amalyses are enzymes that work by hydrolyzing the straight chain bonds between the
individual glucose molecules that make up the starch chain. They are extracellular enzymes that
randomly cleave the 1,4-a-D-glucosidic linkages between adjacent glucose units in the linear
amylase chain.
A. Technical Specification
CAS #

9000-90-2

Chemical Name

endo-1,4-a-D-glucan glucohydrolase

EINECS/ELINCS

232-565-6

IUB

3.2.1.1

B. Physical and Chemical Properties

Physical state

Powder

Appearance

Solid

Solubility in water

10g/L (20C)

Solubility

Liquid

Molecular weight

51.0-54.0 kDa

(Cozzone et al. 1970)

55.4 kDa (SDS-PAGE) (Alkazaz et al. 1996)

Specific Activity

1.314x10-5 mol/mgmin at 40C (48 hours of

fermentation period)

13

Chemical stability

Stable under normal temperatures and pressures

Conditions to avoid

Incompatible material, strong oxidants

Incompatibilities with other materials

Strong oxidizing agents

Hazardous decomposition products

Strong oxidants

Hazardous polymerizations

Will not occur

C. Composition/ Information on ingredients

Chemical Name

CAS#

% by Weight

Alpha Amylase

9000-90-2

100

D. Use of Alpha-amylase
Use

Pancreatic enzyme that aids in the conversion of

starch to maltose and glucose

E. Alpha amylase Transportation, Handling and Storage

Not regulated and not dangerous goods.
Transportation

Alpha amylase will be supplied by Sigma

Aldrich Corporation.

Do not allow contact with water.

Handling

Remove contaminated clothing and wash

thoroughly after handling.

14

Keep from contact with moist air and

steam.

Keep refrigerated (store below 4C) and keep

containers tightly closed.

Storage

Store protected from moisture and store in

cool dry area away from incompatible
substances.

F. Alpha amylase Disposal and Safety

Absorb material with suitable absorbent

and put in a labelled container.

Disposal

Dissolve or mix material with a

combustible solvent and burn in a chemical
incinerator equipped with an afterburner
and scrubber system.

Do not allow product to reach sewage

system or open water.

Safety

Use suitable respiratory protective device

when high concentrations are present.

Refer to the Material Safety Data Sheet for Alpha amylase

15

 Glucoamylase

Glucoamylase (E.C. 3.2.1.3) is an amylolytic enzyme capable to hydrolyze b-D-glucose

units on non-reducing ends of starch molecules. It can also lyse a-1,6 bonds on amylopectin
branching points, thus promoting an almost complete starch hydrolysis into glucose).
A. Technical Specification
CAS #

9032-08-0

Chemical Name

1,4-alpha-D-glucan glucohydrolase

EINECS/ ELINCS

232-877-2

IUB

3.2.1.3

B. Physical and Chemical Properties

Physical state

Powder

Appearance

Solid

Molecular weight

118.17 kDa

Specific Activity

225 U per 75 L / 200 AG units per milliliter

Chemical stability

Stable

Conditions to avoid

Strong oxidizing agents

Incompatibilities with other materials

Strong oxidizing agents

C. Composition/ Information on ingredients

Chemical Name

CAS#

% by Weight

16

1,4-alpha-D-glucan glucohydrolase

9032-08-0

5-10

D. Use of Glucoamylase

Use

Its main application is on the production of

syrups with 96-98 % of glucose. The
hydrolyzates can be used as carbon source in
fermentations

E. Glucoamylase Transportation, Handling and Storage

Store properly within the vehicle to prevent
shifting, spillage, or breakage.
Transportation

Place in coolers to protect them from excessive

heat.

Handle in accordance with good industrial

hygiene and safety practice. Avoid breathing
dust or spray mist.
Handling

Avoid formation of dust and aerosols.

Provide appropriate exhaust ventilation at
places where dust is formed. In case of

17

insufficient ventilation, wear suitable

respiratory equipment.

Storage

Keep tightly closed.

Store in area away from heat. Store at -20C

F. Glucoamylase Disposal and Safety

Contact a licensed professional waste disposal
service to dispose of this material.
Disposal

Dissolve or mix the material with a combustible

solvent and burn in a chemical incinerator
equipped with an afterburner and scrubber.

Safety

Use suitable respiratory protective device when

high concentrations are present.

18

 Pullulanase
Pullulanase is an important de-branching enzyme utilized to hydrolysed the -1-6
glucosidic linkages in starch. Amylopectin, pullulan, and related oligosaccharides, enables a
complete and efficient conversion of the branched polysaccharides into small fermentable sugars
during saccharification process. During saccharification process, pullulanase has been used to
increase the final glucose concentration.

A. Technical Properties
Trade name

Pullulanase

CAS number

9075-68-7

EC number

232-983-9

B. Physical and Chemical properties

Form

Powder, lyophilized

Color

Beige

Odor

Odorless

Activity

0.3 kg per ton dry solids

19

C. Composition of ingredients
Chemical Name

CAS#

% by Weight

Pullullanase

9075-68-7

< 1%

D. Uses of Pullulanase
Pullulanase is used to hydrolysed the 1-6 glucosidic linkages in starch for the
high percentage saccharification of the
Uses

raw material cassava in order to promote

the reaction, improve the content of
glucose and maltose.

E. Pullulanase Transportation, Handling and Storage

Alpha amylase will be supplied by

Sigma Aldrich Corporation.

Transportation

Provide appropriate exhaust ventilation

at places where dust is formed. Normal

Handling
measures for preventive fire protection.

20

 Store in cool place. Keep container

tightly closed in a dry and wellStorage
ventilated place. Recommended storage
temperature: -20 C

F. Pullulanase Disposal and Safety

Offer

surplus

and

non-recyclable

solutions to a licensed disposal company

Disposal

Contaminated packaging should be

disposed of as unused product.

If inhaled, move person into fresh air. If

not breathing, give artificial respiration.

Safety

In case of skin contact, wash off with

soap and plenty of water.

In case of eye contact, flush eyes with

water as a precaution.

21

If swallowed, never give anything by

mouth to an unconscious person. Rinse
mouth with water.

Refer to the Material Safety Data Sheet for Pullulanase

22

 Sodium Hydroxide

Sodium Hydroxide is also known as caustic soda or lye. It is a common reagent for the
adjustment of pH in buffers. It is also used in many industries, mostly in chemical base
manufacturer. Sodium hydroxide reacts violently with water with the liberation of heat when in
contact. When it comes into contact with metals, it liberates flammable hydrogen gas, it is very
corrosive to aluminum alloys and carbon steel. It is hygroscopic and readily absorbs water from
the air, so it should be stored in an airtight container.

A. Technical Specification

Product Name

Sodium Hydroxide

Synonym

Caustic Soda

CAS Number

1310-73-2

Molecular Formula

NaOH

B. Physical and Chemical Properties of Sodium Hydroxide

Physical State and Appearance

White deliquescent solid

Odor

Odorless

Molecular Weight

40 g/mole

pH (1% soln/water)

13.5 [Basic]

Boiling Point

1388C (2530.4F)

23

Melting Point

323C (613.4F)

Specific Gravity

2.13 (Water = 1)

Solubility

Easily soluble in cold water

C. Composition and Information on Ingredients

CAS number

1310-73-2

% by weight

100

D. Uses of Sodium Hydroxide

Uses

Controlling the pH in the pretreatment process

E. Sodium hydroxide Transportation, Handling and Storage

Transportation

Transported via tank truck or in a closed unbreakable container.

Sodium hydroxide will be supplied by Emerald Chemicals Inc.
Do not allow water to get into the container.
Minimize dust generation and accumulation

Handling
Keep container tightly closed and avoid prolonged or repeated exposure

24

Store in an airtight plastic drum containers, in an area that is cool, dry

and well-ventilated, with corrosion resistant concrete floor.

Storage

Store using vent drums to prevent pressure build up

Separated from strong acids, metals, food and feedstuffs.

F. Sodium hydroxide Disposal and Safety

If caustic soda comes in contact with skin, wash the skin under a water
shower and remove the contaminated clothing immediately

Safety

If caustic soda gets into the eyes, immediately rinse it out with a large
quantity of water for at least 15 minutes.

The use of protective equipment like rubber gloves, safety clothing and
eye protection should be always used when handling sodium hydroxide.

It may be necessary to use a corrosion-resistant exhaust ventilation

system separate from other ventilation systems, exhaust directly to the
outside to prevent product release into the workplace.

Disposal

Disposal in the sewages may not post great threat in the aquatic life but
it may alter the pH in water. Therefore only minimal amounts of sodium
hydroxide must be disposed of in the sewage.

Refer to the Material Safety Data Sheet for Sodium hydroxide

25

 Hydrochloric acid

Hydrochloric acid (HCl) or commonly known as muriatic acid is a monoprotic acid used
routinely in chemical research laboratories and manufacturing plants. Despite its acidity, it
produces the less reactive and non-toxic chloride ion. It makes an excellent acidifying reagent and
acid titrant and is also one of the least hazardous strong acid to handle. Intermediate strength
hydrochloric acid solutions are quite stable, maintaining their concentrations over time. Its
applications include the large-scale production of certain compounds, such as vinyl chloride for
polyvinyl chloride (PVC) plastic, removal of rust and scale from metals, petroleum production,
and ore processing. Smaller-scale applications include the production of gelatin and other
ingredients in food, and leather processing. (Hydrochloric acid, n.d.)

A. Technical Specification

Product Name

Hydrochloric acid

Synonym

Muriatic

acid;

Chlorohydric

acid;

Acidechlorhydrique
CAS Number

7647-01-0

EC Number

231-595-7

Molecular Formula

HCl

26

B. Physical and Chemical Properties of Hydrochloric acid

Physical State and Appearance

Colorless to slightly yellow liquid

Odor

Pungent

Taste

Moderately Sweet

Molecular Weight

36.46 g/mole

pH

0.1 (1.0 N), 1.1 (0.1 N), 2.02 (0.01 N)

Boiling Point @ 760 mm Hg

108.58oC (for 20.22% HCl in water)

83oC (for 31% HCl in water)
50.5oC (for 37% HCl in water)

Melting Point

62.25C (20.69% HCl in water)

46.2oC (31.24% HCl in water)

25.4oC (39.17% HCl in water)

Vapor Density (Air = 1)

1.267

Vapor Pressure

16 kPa @ 20oC

Specific Gravity

(20%and 22% HCl solutions)

1.12 (24% HCl solution)

1.15 (29.57% HCl solution)

1.16 (32% HCl solution)

1.19 (37% and 38%HCl solutions)

27

Solubility

Soluble in cold water, hot water, diethyl ether

82.3 g/100 g water at 0oC

67.3 g/100 g water at 0oC

63.3 g/100 g water at 0oC

59.6 g/100 g water at 0oC

56.1 g/100 g water at 0oC

Flammability

Not Flammable

Explosive Limits

Not Explosive

C. Composition and Information on Ingredients

Chemical Name

CAS Number

% by Weight

Hydrogen chloride

7647-01-0

20 38%

Water

7732-18-5

62 80%

D. Uses of Hydrochloric acid

Uses

Hydrochloric acid (HCl) is added in the process

to control the pH of the erythritol crystals

E. Hydrochloric acid Transportation, Handling and Storage

Transportation

Transport in a labeled plastic-coated

glass container.
28

Store properly within the vehicle to

prevent shifting, spillage, or breakage.

Place in coolers to protect them from

excessive heat.

Use non-sparking tools when opening

metal containers

Handling

Observe all warnings and precautions

listed for the product.

Store in cool, dry well-ventilated area.

Keep out of direct sunlight and away

Storage

from heat and water.

Do not wash out container and use it

for other purposes.

F. Hydrochloric acid Disposal and Safety

Wear acid-resistant apron, gloves,

and face shield or protective eyewear.

Safety

Eyewash station and quick drench

shower should be made available
within the work area.

29

Dilute hydrochloric acid to 5 percent

volume or less, and neutralize it with

Disposal

NAHCO3 to a neutral pH before

disposal.

Refer to the Material Safety Data Sheet for Hydrochloric acid

30

 Yeast (Pseudozyma tsukubaensis) strain KN 75

This microorganism was isolated from samples collected from sludge and among the 1,037
strains screened for erythritol production, 21 strains were found to be erythritol producers. One of
these strains, KN75, produced erythritol in an exceptionally high concentration. It was reported
that the characteristics of this novel yeast strain, produces a high yield of erythritol (61% g/g)
without producing by-products such as glycerol and ribitol (Jeya, et al., Isolation of a novel high
erythritol-producing Pseudozyma tsukubaensis and scale-up of erythritol fermentation to industrial
level, 2009).

A. Taxonomy, Classification and Strains

Mnemonic

CANTS

Taxon Identifier

5483

Scientific name

Candida tsukubaensis

Common name

Yeast

Current name

Candida balzeri

Synonym

Pseudozyma tsukubaensis

Other names

Candida tsukunbaensis

Cryptococcus tsukubaensis

Taxonomic Synonyms

Vanrija tsukubaensis

Rank

SPECIES

Classification:

cellular organisms

31

Domain

Eukaryota

Sub domain

Opisthokonta

Kingdom

Fungi

Sub Kingdom

Dikarya

Division

Basidiomycota

Subdivision

Ustilaginomycotina

Class

Ustilaginomycetes

Subclass

Ustilaginales

Order

Ustilaginaceae

Suborder

mitosporic Ustilaginaceae

Genus

Pseudozyma

Type of Strains
Other strains

(UniProt, 2015)

CBS 6389

CBS 422.96, KN 75 (UniProt, 2015) and (Jeya,

et al., Isolation of a novel high erythritolproducing

Pseudozyma

tsukubaensis

and

scale-up of erythritol fermentation to industrial

level, 2009).

B. Morphology

Cell size

1.55.0 m

Cell shape

Globose to spheroidal

Cell Color

White to cream colored

32

Asexual Reproduction

Multilateral budding (Jeya, et al., Isolation of a

novel high erythritol-producing Pseudozyma
tsukubaensis

and

scale-up

of

erythritol

fermentation to industrial level, 2009)

C. Physiological Properties

Growth media

Luria-Bertani (LB) agar

Culture growth media

10% w/v NaCl plus 5% w/v glucose

Temperature growth tests

25 C

Optimum carbon source for cell growth and

300 g/L glucose with erythritol yield of 44.7

erythritol production

4.8%

Optimum nitrogen source for cell growth

Corn steep powder with erythritol yield of 49.7

and erythritol production

5.3

Assimilation growth

Weak growth on 50% glucose

Optimum assimilation growth ph

Optimum assimilation growth temperature

37 C

Fermentations

Erythritol (Jeya, et al., Isolation of a novel high

erythritol-producing Pseudozyma tsukubaensis
and scale-up of erythritol fermentation to
industrial level, 2009)

33

D. Uses of Pseudozyma Tsukabaensis

Pseudozyma Tsukubensis is the erythritol producing microorganism

which will be cultured and fed into the fermenter.
Use

E. Pseudozyma Tsukabaensis Transportation, Handling and Storage

The transport of the yeast strain from the yeast bank

has to be secure, sterile and pure yeast strain KN75 of
Pseudozyma Tsukubaensis depending on the terms

Transportation

provided by the bank as based on the characteristic of

the yeast strain

Handling

Handling must be strictly based on the physiological

properties the yeast possessed as cited above.

A single colony of P. tsukubaensis KN75 was

inoculated in to 5 mL of growth medium in a test tube
and incubated at 30C, 250 rpm for 48 h (Jeya, et al.,

Storage

Isolation

of

novel

high

erythritol-producing

Pseudozyma tsukubaensis and scale-up of erythritol

fermentation to industrial level, 2009).

34

F. Pseudozyma Tsukabaensis Disposal and Safety

If there is any yeast excess settled from the

fermenter or cell separator, offer surplus and non-

Disposal

recyclable

solutions

to

licensed

disposal

company.

Safety

Note proper safety guidelines as prescribed by the

yeast bank depending on the characteristics
possessed by the yeast strain.

Refer to the Material Safety Data Sheet for P. tsukubaensis

35

 Yeast Extract

Yeast extract is a complex and widely used hydrolysate of yeasts. It provides nitrogenous
compounds, carbon, sulfur, trace nutrients, vitamin B complex and other important growth factors,
which are essential for the growth of diverse microorganisms (Sigma-Aldrich Co., 2015). In the
process, these are used for two purposes, as a microbial growth medium and as a culture cell media.

A. Technical Specifications

Product Name

Yeast Extract

CAS Number

8013-01-2

EC Number

232-387-9 (ChemicalBook, 2008)

B. Physical and Chemical Properties

Physical state

Powder

Appearance

Light Yellow to Very Dark Yellow; Light

Beige to Very Dark Beige; Light Brown to
Very Dark Brown

Solubility Color

Yellow to Very Dark Yellow and Yellow

Brown

Solubility in water

100 mg/mL

Solubility turbidity

Clear to slightly hazy

Carbon

35 42%

Nitrogen

10.0 12.0 %
36

6.5 7.5 (Sigma-Aldrich Co., 2015)

ph 10% in water

C. Composition/Information on Ingredients

Components

Composition by weight

Yeast extract (carbohydrates, peptide, water

91- 99%

soluble vitamins, mixture of amino acids)

% Sodium chloride

5 %

% Nitrogen

9-12 %

Grade

For use in microbial growth medium (SigmaAldrich Co., 2015)

D. Uses of Yeast Extract

Use

Yeast extract will be used as one of the constituents of the

growth medium of the yeast that will be used.

E. Yeast Transportation, Handling and Safety

No special requirements and no restrictions on

transportation by land, sea or air (Sigma-Aldrich

Transportation
Co., 2012).

37

Provide appropriate exhaust ventilation at places

where dust is formed.

Handling

Normal measures for preventive fire protection.

Store in cool place.

Keep container tightly closed in a dry and well-

Storage
ventilated place

F. Yeast Disposal and Safety

Disposal

Offer surplus and non-recyclable solutions to a

licensed disposal company.

If contaminated packaging, dispose of as unused

product (Sigma-Aldrich Co., 2012).

Use equipment for eye protection tested and

approved under appropriate government standards

Safety

Gloves must be inspected prior to use. Use proper

glove removal technique without touching glove's
outer surface to avoid skin contact with the yeast
extract.

Refer to the Material Safety Data Sheet for Yeast Extract

38

 Manganese(II) Sulfate Tetrahydrate

This will serve as the culture media of the yeast to be used in the process.

A. Technical Specification

Product Name

Manganese(II) sulfate tetrahydrate

Synonym

Manganese(2+) sulfate tetrahydrate

CAS Number

10101-68-5

EC Number

232-089-9

Molecular Formula

MnSO44H2O

Toxicity

Causes serious eye damage

May cause damage to organs through

prolonged or repeated exposure.

Toxic to aquatic life with long lasting

effects.

B. Physical and Chemical Properties

Physical State and Appearance

Pale pink crystalline solid

Odor

Odorless

Molecular Weight

223.06 g/mol

pH

4.5 - 6.0 (50 g/l, HO, 20 C)

Boiling Point

850 oC
39

Melting Point

26 - 27 C

Solubility w/ water at (20 C) soluble

393 g/L

C. Composition and Information on Ingredients

Chemical Name

CAS Number

Weight %

Manganese(II) Sulfate Tetrahydrate

10101-68-5

>95

Manganese(II) sulfate

7785-87-7

D. Use of Manganese(II) Sulfate Tetrahydrate

It helps in the growth and survival of the cells

Use

in the culture media

E. Manganese(II) Sulfate Tetrahydrate Transportation, Handling and Storage

Transportation

Avoid any release of dust during

transportation, by using air-tight tanks for
powders and covered trucks for pebbles.

Handling

Storage

Keep containers tightly closed in a dry, cool

and well-ventilated place.

Wear personal protective equipment.

Store at +15C to +25C.

40

Store away from oxidizing agents.

Ensure adequate ventilation.

F. Manganese(II) Sulfate Tetrahydrate Safety and Disposal

May cause serious damage to health by

prolonged exposure through inhalation and
if swallowed.

Safety

Avoid release to the environment

Wear

appropriate

personal

protection

equipment

Do not let this chemical enter the

environment.

Disposal

If

the

packaging

is

contaminated,

dispose of this container to hazardous or

special waste collection point.

Refer to the Material Safety Data Sheet for Managanese(II) Sulphate Tetrahydrate

41

 Copper Sulfate Pentahydrate

Copper(II) sulfate pentahydrate (CuSO4.5H2O), is a common salt of copper. Copper

sulfate is a bright blue, odourless crystalline solid which is soluble in water. The archaic name for
copper(II) sulphate is blue vitriol or bluestone. It has numerous applications including as a
fungicide, algaecide, pesticide, laboratory analytical reagent, feed supplement, zinc etching and as
a mordant (Audain, n.d.).

A. Technical Specification

Product Name

Copper Sulfate Pentahydrate

Synonym

Cupric sulfatepentahydrate; Cupric sulphate; sulphuric acid,

Copper salt; Blue vitriol; Bluestone; Sulfate de cuivre;
KupfersulfatPentahydrat;

CAS Number

7758-99-8

EC Number

231-847-6

CB Number

CB6705282

Molecular Formula

CuSO4.5H2O

Toxicity

Harmful by ingestion or if inhaled as dust or mist. It will

severely irritate eyes, skin and mucous membrane.
Very toxic to aquatic organisms. Classed as marine
pollutant PP. Harmful effect due to pH shift. Mutagenic for
mammalian somatic cells.

42

B. Physical and Chemical Properties

Physical State and Appearance

Solid. (Crystalline granules solid. Powdered solid.)

Odor

Odorless

Taste

Nauseous metallic

Molecular Weight

249.69 g/mole

pH

3.7 4.5 at 50 g/l at 25 C

Boiling Point

150C (302F)

Melting Point

110C (230F)

Relative Density

2,284 g/cm3

Bulk Density

1.1~1.3 g/cm3

Specific Gravity

2.28 @ 15.6 deg. C(Water = 1)

Vapor Pressure

7.3 mm Hg @ 25 deg C

Solubility

Easily soluble in hot water. Soluble in cold water,

methanol.

Solubility in water: 31.6 g/100 ml @ 0 deg. C.;

203.3 g/100 ml @ 100 deg. C

Solubility in methanol: 15.6 g/100 ml @ 18 deg. C.

Insoluble in ethanol.

43

C. Composition and Information on Ingredients

Chemical Name

CAS Number

% by Weight

Copper Sulfate Pentahydrate

7758-99-8

100

D. Uses of Copper Sulfate Pentahydrate

Use

Copper sulfate pentahydrate is added in the process for it helps in the

growth and survival of the cells in the culture

E. Copper Sulfate Pentahydrate Transportation, Handling and Storage

Additional markings "Marine Pollutant" - required for bulk

shipments. The words "Marine Pollutant" must be entered on

Transportation
the shipping paper in association with the basic DOT
description for bulk shipments.

Avoid contact with skin and eyes.

Avoid formation of dust and aerosols. Provide appropriate

Handling
exhaust ventilation at places where dust is formed.

44

 Keep container tightly closed.

Keep container in a cool, well-ventilated area.

Air sensitive. Hygroscopic. Handle and store under inert gas.

Storage

F. Copper Sulfate Pentahydrate Safety and Disposal

Use process enclosures, local exhaust ventilation, or other

engineering controls to keep airborne levels below

Safety
recommended exposure limits.

Use splash goggles, lab coat and dust respirator in handling

copper sulfate pentahydrate

Small Spill: Use appropriate tools to put the spilled solid in a

convenient waste disposal container. Clean it by spreading

Disposal
water on the contaminated surface.

Large Spill: Use a shovel to put the material into a convenient

waste disposal container.

Refer to the Material Safety Data Sheet for Copper Sulphate Pentahydrate

45

 Corn Steep Powder

Corn steep solids are soluble and may be used as a nitrogen source in several medium and
cell culture conditions. This can give rise to biomasses that are rich in proteins and poor in acidic
polysaccharides. (Sigma-Aldrich, n.d.)

A. Technical Specifications

Product Name

Corn Steep Powder

Synonym

Corn Steep Liquor

CAS Number

66071-94-1

EC Number

266-113-4

Toxicity

Safe

B. Physical and Chemical Properties

Physical State and Appearance

Yellow Powder

Odor

Characteristic

Taste

Characteristic

pH (1% solution)

4.0 - 4.5

Boiling Point

100-101C

Bulk Density

40-60g/100ml

Specific Gravity

1.15-1.25

Vapor Pressure

17.5 mm Hg at 25C

46

Solubility

Soluble

C. Composition and Information on Ingredients

Components

Composition by Weight

Protein

42%

Nitrogen

6.7%

Phosphorus

1%

Moisture

8%

Ash

5%

Sulfur dioxide

0.2%

D. Use of Corn Steep Powder

Use

Corn steep powder will serve as the nitrogen source of the

medium

E. Corn Steep Powder Transportation, Handling and Storage

Corn steep powder will be supplied by the Sigma-Aldrich

Corporation

Transportation

No special requirements and no restrictions on transportation by

land, sea or air

Handling

Do not stack pallets more than two (2) meters high

47

 Store in plastic or stainless container in a cool, wellventilated, dry place at temperatures above 400F
Storage

F. Corn Steep Powder Disposal and Safety

Disposal

Offer surplus and non-recyclable solutions to a licensed

disposal company.

In case of contact with eyes, rinse slowly and gently with

water for 15-20 minutes

Safety

If ingested, give 2 glasses of water to drink for dilution

Refer to the Material Safety Data Sheet for Corn Steep Powder

48