(M) Basic Practical Manual

Basic Practical Manual on Industrial Microbiology
Basic
Practical
Manual on
Industrial
Microbiology
ISBN 978-1-365-11491-5
90000
ID: 18844176
Basanta Kumar Rai
Dil Kumar Subba
www.lulu.com
9 781365 114915
Basic Practical Manual on
INDUSTRIAL
MICROBIOLOGY
Basanta K. Rai
Dil K. Subba
First Edition: 2016

Note to the users
This practical manual is basically meant for Food Technology

courses where Industrial Microbiology is one of the subjects. However,
because of the very general nature of the practicals/experiments, it can
be equally useful in other streams where food biotechnology course is
offered.
Each practical described in this manual consists of following

parts/sections:
1. A concise background
2. Objectives
3. Working principle
4. Requirements
5. Procedure
6. Observation
7. Results and discussion
8. Questions
9. Helpful drawings
Every student must get a copy of this manual well before the
practical session. The instructor must inform the students about the
upcoming/ensuing experiment or practical and the students must come
prepared accordingly before carrying out the experiment. Planning an
experiment in advance and telling the students to come prepared for the
same is both effective and time-saving.
Although the experiments given in this manual follow some sort of

order, most of them are of standalone nature. This means that the
instructor can plan the experiment according to his convenience,
availability of raw materials, etc.
Students should be encouraged to find answers to the thought-

provoking questions given at the end of each practical in this manual. If
possible, similar questions should be planned for the viva-voce in
practical examination. To aid the user, several helpful diagrams have also
been provided in this manual. Extensive bibliography and glossary are
other useful features of this manual.
iii
Preface
This practical manual has been prepared for students of the

developing countries where, in general, laboratory facilities are scarce.
The manual contains 6 chapters and 23 practicals. The chapters are
arranged in a logical sequence, starting with prerequisites to ending with a
few slightly involved practicals. Each chapter begins with a concise
background and very carefully worked-out objectives. Great care has
been taken to present the practicals in as lucid a manner as possible. The
chapters are extensively cross-referenced because of the highly
interrelated nature of the practicals. Profuse illustrations (about 58) have
been provided in the form of helpful diagrams. A bibliography has been
appended for further reading. One of the important features of this
manual is the inclusion of glossary of almost all technical terms
encountered during the use of this manual. Another unique feature is the
thought-provoking questions given at the end of each practical. We hope
that the quest for answers to these questions will definitely hone the
analytical skill on the part of the students and teachers alike.
This manual has evolved from several years of practical classes and
in-house researches we have conducted at Central Campus of
Technology and Dharan Multiple Campus, Dharan, Nepal. During the
course of practicals (and sometimes a bit of an experimentation!) some
of our students have been of great help, particularly in the sections
dealing with amylolytic starters and UV-mutation. In particular, we
would like to acknowledge Jeny Subba and Sangen Ruma Rai for their
contributions on yeasts and Manikala Rai for kinema.
The manual includes many non-standard methods also, some of

which were developed by the authors themselves (for example, the
comparison of yeast cells by juxtaposition method, use of cellotape for
smears, molasses agar for yeast growth, etc.). The use of these non-
standard methods may well invite criticisms from different scientific
quarters as regards their merit, but we believe that it definitely opens
avenues for the exploration of alternative approaches when resources
are scarce.
Central Campus of Technology Basanta K. Rai

Dharan, NEPAL Dil K. Subba
v
Contents
Note to the users iii

Preface v
List of figures ix
SECTION I: Prerequisites
Practical 1: Micrometry 2
Practical 2: Direct microscopic count of yeast cells 8
Practical 3: Replica plating 12
SECTION II: Screening and culture preservation

Practical 4: Screening of amylolytic molds from murcha 16
Practical 5: Screening of fermentative yeasts from murcha 21
Practical 6: Screening of antibiotic producers from soil 27
SECTION III: Fermentation and fermented products

Practical 7: Preparation of ethanol from molasses 32
Practical 8: Single column distillation of ethanol 40
Practical 9: Preparation of rum from single-column distillate 47
Practical 10: Preparation of Red Table Wine 54
Practical 11: Preparation of beer by decoction mashing 60
SECTION IV: Amylolytic starters

Practical 12: Propagation of murcha cake 66
Practical 13: Preparation of bran koji 70
Practical 14: Preparation of murcha using pure cultures 73
Contents …
SECTION V: Traditional fermented foods

Practical 15: Preparation of millet jand 82
Practical 16: Preparation of sauerkraut 87
Practical 17: Preparation of kinema 91
SECTION VI: Miscellaneous practicals

Practical 18: Comparison of baker’s yeast activity 96
Practical 19: Preparation of restructured fruit alginate 101
Practical 20: Continuous fermentation using immobilized yeast 106
Practical 21: Detection of RDMs in brewing yeast 111
Practical 22: Demonstration of UV-radiation lethality in yeasts 115
Practical 23:Determination of amylase activity of amylolytic 122
starter
Bibliography 130
Glossary 134
List of figures
Fig. 1.1 Calibration of ocular micrometer for a given objective 6

lens
Fig. 1.2 Preparation of slide for negative staining 7
Fig. 1.3 Fixing of cellotape on smear 7
Fig. 1.4 Appearance of cell through ocular micrometer 7
Fig. 2.1 Preparation of slide for DMC 11
Fig. 2.2 Vortex mixing of cells 11
Fig. 3.1 Replica plating technique 14
Fig. 3.2 Replica plating for sugar assimilation property 14
Fig. 4.1 Some common molds 20
Fig. 4.2 Preparation of tape culture 20
Fig. 5.1 Four-way streak 24
Fig. 5.2 Correct and incorrect ways of spread-plating 25
Fig. 5.3 Drying of agar surface 25
Fig. 5.4 Yeast cells as seen under microscope (oil-immersion) 25
Fig. 5.6 Schematic diagram of spread-plating 26
Fig. 6.1 Simplified protocol for the screening of antibiotic 30
producing microorganisms from soil and subsequent
testing for antibiotic spectrum
Fig. 7.1 Pathway for alcohol synthesis 33
Fig. 7.2 A typical glass pycnometer 39
Fig. 7.3 A typical distillation assembly 39
Fig. 8.1 A typical single-column distillation system 44
Fig. 8.2 Traditional Nepalese pot still for raksi production 44
Fig. 8.3 Schematics of open-fired kettle distillation (for brandy) 45
Fig. 8.4 Locally fabricated simple distillation unit 45
Fig. 8.5 Typical alcohol hydrometer 46
Fig. 9.1 An example of label for alcoholic beverage 51
Fig. 9.2 Specimen of score card for hedonic rating 51
Fig. 9.3 Genstat® statistical package 52
Fig. 9.4 Tabulation of sensory data in MS-Excel (for Genstat®) 52
Fig. 9.5 Example of output from Genstat® 53
List of figures ….
Fig. 10.1 Equipment needed for wine making 59

Fig. 10.2 In-bottle pasteurization of wine 59
Fig. 11.1 Fresh hop cones 64
Fig. 11.2 Structure of α-acid 64
Fig. 12.1 Murcha cakes spread on fern fronds for incubation 69
Fig. 13.1 Outline of bran koji preparation 72
Fig. 14.1 Outline of murcha making using pure culture 79
Fig. 14.2 Determination of bulk density 79
Fig. 14.3 IR moisture meter 80
Fig. 14.4 Alignment of moisture meter scale 80
Fig. 14.5 Cooling in a desiccator 80
Fig. 15.1 A typical tongba 86
Fig. 16.1 Commercial sauerkraut fermentation tank 90
Fig. 17.1 Simplified diagram for laboratory preparation of 94
kinema
Fig. 18.1 Hypothetical scattered plot of volume vs time 99
Fig. 18.2 Result obtained by using Excel add-in Analysis 99
Toolpak for paired t-test.
Fig. 18.3 Experimental set up for comparison of baker’s yeast 100
activity
Fig. 19.1 Basic structure of alginic acid 104
Fig. 19.2 Biosynthesis of alginic acid in A. vinelandii 105
Fig. 20.1 Set-up for continuous ethanol fermentation using 109
immobilized cells
Fig. 20.2 Different immobilization techniques 110
Fig. 22.1 Absorption of UV radiation by DNA 115
Fig. 22.2 Thymine dimer formation and repair 116
Fig. 22.3 Wrapping of the spread-plated base plate with Al foil 120
Fig. 22.4 Irradiation of seeded plates 120
Fig. 22.5 Effect of UV radiation duration on yeast cells 120
Fig. 22.6 Calculation of radiation intensity 121
Fig. 22.7 A hypothetical survival curve after UV irradiation of 121
yeast cells
Fig. 23.1 Action of β- and α-amylase on starch molecule 129
Section I
PREREQUISITES
Practical 1
MICROMETRY
Background
In microscopy, micrometry refers to the measurement of the size of

microorganisms. Bacteria, yeasts and viruses on average (respectively)
are 2 millionths, 10 millionths, and a few tenths of a billionth of a meter.
It is therefore customary to measure the sizes of bacteria and yeasts in
micrometer (also called micron; notations µm or µ) and that of viruses
in nanometer (notation: nm). The units are related thus: 1 nm = 10-9 m,
1 µm = 10-6 m, and 1 µm = 1000 nm. In general, the relative sizes of
different microorganisms are: yeasts (~ 10 µ) > ordinary bacteria (~ 2 µ)
> small bacteria (~ 1 µ) > rickettsia (~ 0.2 µ) > poliovirus (~ 0.01 µ).
There are several exceptions, though. The cell size of a given organism
does not remain constant. It is dependent on species, nutrition, the
stages in its growth cycle, and even the environmental conditions.
A light microscope cannot resolve sizes less than 0.2 µ. At best, it

can measure the size of a bacterium. Despite defying size, however,
bacteria can be measured with relative ease and accuracy using
micrometric method. The method employs two scales, viz.,:
1. Ocular micrometer
2. Stage micrometer
The graduation inscribed in ocular micrometer is arbitrary while

that of stage micrometer is absolute (0.01 mm spacing). In operation, the
ocular micrometer is calibrated by superimposing it with stage
micrometer (Fig. 1.1). For example, using oil immersion objective, if 25
Ocular Divisions (OD) = 3 Stage Micrometer Divisions (SD), then:
3 × 0.01 mm
Every OD = = 1.2 μ ( 1 SD = 0.01 mm)
25
The ocular micrometer must be recalibrated every time micrometric

measurement is carried out because the calibration factor does not
remain constant: it depends on the powers of the objective and ocular
lenses, as well as the microscope used.
2
In micrometry, specimens are usually stained because the cells
themselves are transparent (and hence cannot be seen). Staining the cells
with nigrosine or India ink produces a better estimate of the cell size
because:
1. Cells appear less shriveled and distorted because no heat-fixing is

done
2. Capsulated bacteria that are difficult to stain can be rapidly
observed by this technique
Objectives
1. To become familiar with micrometry and related calculations

2. To measure cell sizes
Principle
Measurement of dimensions of microorganisms is done under

microscope with the help of two micro-scales, viz., ocular micrometer
and stage micrometer. Both the micrometers have microscopic
graduations etched on their surfaces. The ocular micrometer is a circular
glass disc, which fits into the circular shelf inside the eyepiece. It has
arbitrary graduations etched on its surface. However, the distance
between the etched graduations is constant for a particular ocular
micrometer. The stage micrometer is a special glass slide, which is
clipped to the stage of the microscope. It has standard graduations
etched on its surface, which are 10 µ apart. For calibration, both the
micrometer etchings are superimposed by rotating the eyepiece. The
number of ocular divisions (OD) coinciding with the number of stage
divisions (SD) is found out. Once calibrated, the stage micrometer is
replaced with the specimen and measurements taken with the calibrated
ocular micrometer.
Requirements
1. Stage and ocular micrometer

2. Culture suspension (e.g., yeast suspension)
3. Microscope slides
4. Microscope with oil-immersion objective
5. Immersion oil
6. Inoculating loop
3
7. Spirit lamp / Bunsen burner
8. Nigrosine dye (1% aqueous)
9. Cellotape
Procedure
1. Calibrate the ocular micrometer (for the oil immersion objective

used). You can follow your instructor’s directions but the general
process is as follows:
i. Remove the eyepiece from the microscope, unscrew its
top lid and insert the ocular micrometer carefully into the
slot. Put back the top lid.
ii. Clip stage micrometer to the stage and center the
graduated etchings by moving it with the help of
mechanical stage. Do not forget to use immersion oil!
iii. Take oil-immersion objective to position.
iv. Once the stage micrometer (the scales) appears in the
field, rotate eyepiece till etchings on both micrometers
superimpose. Search for lines on both micrometers
coinciding with each other. Count the number of ocular
divisions (OD) equivalent to the number of coinciding
stage divisions (SD). Calculate the calibration factor (for
example, if 10 OD = 2 SD, then 1 OD = 2 µ and so the
calibration factor is 2 µ).
v. During superimposition, the stage micrometer scale
appears several times thicker than the ocular scale. The
scale should be measured either from center to center or
from edge to edge of the thick scale as shown in Fig. 1.1.
2. Prepare specimen for microscopic observation for negative
staining (modified process) as follows:
i. Add a loopful of broth near the edge of a clean, briefly
flamed slide.
ii. Add a drop of 1% nigrosine (aqueous) and mix well with
the loop.
iii. Spread the mixture into a smear by running across the
length the edge of another slide (Fig. 1.2).
iv. Allow the smear to air-dry.
v. Carefully stick a cellotape over the smear, avoiding
formation of air bubbles. The cellotape technique has been
used (developed by the author) to overcome poor quality
4
immersion oil (which often washes away the smear) (Fig.
1.3).
vi. Add a drop of immersion oil over the cellotape.
3. Replace stage micrometer with the prepared slide (do not forget to
use immersion oil). Record the dimensions of the cells in terms of
ocular division. You may need to manipulate ocular micrometer
(by rotating the eyepiece) and position of the cells (by sliding the
mechanical stage) before you can record the dimension (length
and breadth of the cells). See Fig. 1.4 also.
4. Determine the size of the microbe by multiplying the number of
ocular divisions covered by the microbe with the calibration
factor (for example, if the cell covers 6 OD, the dimension is 6 ×
2 µ = 12 µ, taking 2 µ as the calibration factor).
5. Take at least 10 readings each for length and breadth of the cells.
Rotate the ocular if needed (by rotating the eyepiece) for aligning
the scale.
6. To account for variations in cell sizes due to differing phases of
growth cycle, express the result as arithmetic mean ± standard
deviation. The method for determining standard can be found in
any standard textbook of statistics. Scientific calculators also
have programs for direct calculation of standard deviation.
Briefly, the formula for calculating standard deviation is:
1 N

2
σ n −1 = (xi − x )
N − 1 i =1
Where, σn-1 = sample standard deviation, N = number of
samples taken (readings), N-1 = Bessel’s correction, for unbiased
estimate), x = arithmetic mean of observation, xi = individual
observations.
Observation
1. End-to-end or center-to-center superimposed readings of ocular

and stage micrometer
2. Relative sizes of the ocular and stage micrometer scales when
viewed through the microscope
3. Dimensions (length and width) of the cells (at least 10 readings
in a tabular form)
4. Two-dimensional geometry of the cells (circular, elongated,
oblong, etc.), arrangement, budding, etc.
5
Results and discussion
1. Calculation for calibration of the ocular division

2. Calculation of cell size
3. Statistical inference from the data
4. General conclusion about the yeast cell morphology (size, shape,
arrangement, etc.)
Questions
1. Can you use micrometry as an aid in the determination of

microbial load of samples?
2. What is smallest cell size that you can measure by micrometry,
using a standard bright field microscopy?
Sample calculation
Calibration
Let, on average, 1 SD (0.01 mm) = 8 OD = 10 μ
∴ 1 OD = (10/8) μ = 1.25 μ
Cell size measurement

Let the lengths (in μ) of 10 randomly selected yeast cells be 5, 7, 4,
6, 6, 10, 5, 6, 5, 6.
Average length ± sample standard deviation = 6 ± 1.63 μ
Proceed in similar way for breadth of the yeast cells.
Helpful diagrams
Ocular scale
Center-to-center End-to-end
alignment Stage scale alignment
Fig. 1.1 Calibration of ocular micrometer for a given objective lens
6
Slide movement
Slide 2
Culture + Nigrosine
Slide 1
Smear
Fig. 1.2 Preparation of slide for negative staining
Cellotape Mineral oil
Fig. 1.3 Fixing of cellotape on smear
Stage scale
Ocular Cells
scale
Fig. 1.4 Appearance of cells through ocular micrometer
7
Practical 2
DIRECT MICROSCOPIC COUNT OF YEAST CELLS
Background
The standard method of determining microbial population is serial

dilution and growth on suitable agar medium. The greatest disadvantage
of this method is the long duration needed for the growth (24-48 hrs).
Under conditions where rapid results are required (for example, cell
count in milk before receipt, fermentation kinetic study under laboratory
environment, etc.) the standard method is of limited value. Advanced
methods are of course available but these come at a cost. Besides, these
are not suitable for the demonstration of principles underlying microbial
protocols. A simple and rapid alternative method for determining
microbial load is Direct Microscopic Count (DMC). There are many
variations of DMC method, some of which having the ability to
differentiate live and dead cells (by use of special dyes, e.g., methylene
blue). Use of hemocytometer is fairly general in many laboratories, including
breweries. Even where hemocytometer is not available, DMC is still
possible, using a standard slide. If viability is not important, the relatively
simple negative staining approach can also be used.
Objectives
1. To carry out micrometric calculations

2. To carry out serial dilution
3. To calculate cell concentration
Principle
A uniform smear of the test organism on a standard area (on the

slide) corresponding to a standard volume of culture is observed under
oil-immersion objective and the number of cells in the field is recorded.
The area of the field, in turn, is calculated by measuring the diameter
with a calibrated ocular micrometer. Once the number of cells in a given
area (of the field) is known, back-calculation can be done to obtain the
number of cells in the standard area (and therefore the corresponding
volume of culture).
8
Requirements
1. Stage and ocular micrometer

2. Culture suspension (e.g., yeast suspension)
3. Microscope slides
4. Microscope with oil-immersion objective
5. Immersion oil
6. Pipette – 0.1 ml graduation
7. Vortex mixer
8. Nigrosine dye (1% aqueous)
9. Cellotape or masking tape
10. Ruler (scale)
11. Marker pen (0.5 mm)
12. Razor blade
13. Inoculating loop
Procedure
1. Take 1 ml of the actively growing culture (yeast) in a clean test

tube and subject it to vortex mixing for 1 min at medium speed
2. Transfer the culture to 99-ml blank (distilled water) and mix well.
This gives 100-fold dilution.
3. Carry out other necessary serial dilutions (with the help of your
instructor) so that the number of cells in the microscopic field is
less than 50.
4. Take a clean slide and mark an area (2 cm × 2 cm) with a marker
pen.
5. Stick a cellotape (or masking tape) on the outside of the square
(see Fig. 2.1) to make a boundary. Trim off the excess with a
sharp razor blade.
6. Take 1 ml each of diluted sample and a drop of nigrosine dye in
a test tube and subject it to vortex mixing (Fig 2.2) for a 30 s.
7. Put 0.1 ml of mixture at the center of the slide and quickly
spread it over the entire bounded area. You can tilt or jerk the
slide to affect the spreading.
8. Place the slide on a perfectly horizontal rack and allow the smear
to air-dry.
9. In the meantime calibrate the ocular micrometer (see Practical
1) and record the diameter of the field.
10. As in Practical 1, put a cellotape over the smear and observe the
cells under oil-immersion objective.
9
11. Count cells in at least 10 fields and calculate standard deviation
(the lower the standard deviation, the better). You may have to
dilute the sample further if the cell count in the field is more
than 50 (it is difficult to count large numbers under the
microscope without the grid guide).
Observation
1. Calibration readings
2. Count of cells in 10 microscopic fields
3. Diameter of the field
1. Calculation of area of the microscopic field

2. Calculation of cell concentration
Questions
1. Using this method, is it possible to count only the viable cells?

Why was vortex mixing done in steps 1 and 6?
2. What is the purpose of step 8?
Sample calculation
Let us assume that we got following data:
The dilution performed before preparing the slide is 1000 folds

The calibration is: 1 OD = 2 μ
The average field diameter = 100 OD = 200 μ
The area of the smear is 4 cm2 = 4×108 μ2
The volume of dilute broth delivered in the smear area = 0.1 ml
The average number of cells recorded in 10 fields = 18
Calculation:
Area of the field = (π×2002/4) μ2 = ~ 31416 μ2

Number of fields in 4 cm2 area = (4×108)/31416 = ~ 12732
Number of cells in 4 cm2 area = 12732×18 = 229176
Number of cells in 1 ml = 2291760
Number of cells/ml of original broth = 2291760×1000 = 2.29×109
10
Helpful diagrams
Trim this portion with Bounded area for

a sharp razor blade receiving sample
Cellotape
Fig. 2.1 Preparation of slide for DMC
2
1 3
Fig. 2.2 Vortex mixing of cells
11
Practical 3
REPLICA PLATING
Background
Replica plating is a microbiological technique in which one or more

secondary Petri plates (agar-based) are imprinted with microbial colonies
from a primary plate (or master plate), reproducing the original spatial
pattern of colonies. The technique involves pressing a velveteen-covered
disk, and then imprinting secondary plates with cells in colonies
removed from the original plate by the material.
The purpose of replica plating is to be able to compare the master

plate and any secondary plates, typically to screen for a desired
phenotype. The objective of replica plating may range from negative
screening (e.g., to screen antibiotic-sensitive colonies), auxanography (e.g., to
study sugar assimilation property), to screening of Respiratory Deficient
Mutants (RDMs). Depending on the objective, the secondary plate may
contain the same media used for the master plate, or may contain some
chemicals that aid in the selection and/or differentiation (phenotypic
and biochemical) of colonies. By increasing the variety of secondary
plates with different selective growth media, it is possible to rapidly
screen a large number of individual isolated colonies for as many
phenotypes as there are secondary plates. Replica plating was first
described by Esther Lederberg and Joshua Lederberg in 1952 and same
technique (sometimes with modifications) is still used for many
microbiological studies.
Objectives
1. To study the working principle of replica plating

2. To assemble and carry out replica plating
Principle
A culture plate (master plate) containing well developed colonies is

inverted over sterile velveteen of the replica plating unit to produce an
imprint of the master plate. Thereafter, one or more secondary agar
plates are inverted over the imprint (on the velveteen) to copy (replicate)
12
the colony patterns. Upon incubation, the copied colonies will grow in
exactly the same pattern (a replica) as in the master plate.
Requirements
1. Master plate (containing well-developed, 30-300 colonies)

2. Replica plating unit (wooden or metal block, sterile velveteen,
clamp, etc.)
3. Secondary agar plates (normal media) – 3-4 plates
4. Incubator
5. Marker pen
Procedure
1. Assemble the replica plating unit. Maintain asepsis during fixing

the velveteen (Follow your guide’s instruction, Fig. 3.1).
2. On the back side of your master plate, put a mark with marker
pen (to help identify the position of colonies in replica plates).
3. Positioning the mark to the North (away from you), invert the
master plate on the velveteen and press gently with finger tips.
4. Slowly take out the master plate (vertically up), put on the lid
and store.
5. Take a secondary plate and put a mark on the back with a
marker pen.
6. Positioning the mark to the North (away from you), invert the
secondary plate on the velveteen and press gently with finger tips.
7. Slowly take out the secondary plate (vertically up), put on the lid
and incubate.
8. Continue in similar way for other secondary plates.
9. Observe the growth of colonies in replica plates and compare
their patterns and positions with the master plate.
Observation
1. Imprint on the velveteen

2. Spatial pattern in the replica plates
Questions
1. How can you carry out test of sugar assimilation (auxanography)

with replica plating? Hint: prepare separate secondary plates
13
containing glucose, fructose, lactose, raffinose, etc., and copy the
imprint from the velveteen. Observe the extent of growth (no
growth, weak, good, luxuriant, etc.) of individual colonies and
interpret the results by comparing with standard keys (reference
chart). See also Fig. 3.2.
2. How can you screen Respiratory Deficient Mutants (RDMs)
using replica plating? Hint: refer to Practical 21. Carry out
replica plating, develop the colonies, and add TTC overlay agar.
RDMs will give colorless colonies while the healthy cells will give
pink colonies. You can now select the RDMs in the replica plate
and then isolate the cells from the corresponding colonies in the
master plate.
Helpful diagrams
Imprint
Master plate Fresh media transfered to
with colonies plate fresh media
Velveteen with
imprint of all
Velveteen colonies
(sterilized)
Metal ring
(clamp)
Wooden
block
Fig. 3.1 Replica plating technique
Master plate
No
growth
Glucose Fructose Maltose Raffinose Lactose
Fig. 3.2 Replica plating for sugar assimilation property

14
Section II
SCREENING AND CULTURE PRESERVATION

Practical 4
SCREENING OF AMYLOLYTIC MOLDS FROM MURCHA
Background
Molds are found in virtually every environment, and can be

detected both indoors and outdoors, year round. There are more than
100,000 species of molds, all of them either saprophytes or parasites.
In food fermentation industries, saprophytic molds with amylolytic

and proteolytic activities are of great importance. Such molds are found
in many traditional starter cultures (such as murcha and manapu of Nepal)
and commercial koji cultures. Koji is an amylolytic and/or proteolytic
fungal starter culture indigenous to Japan.
The dominant amylolytic molds found in murcha are species of

Mucor, Rhizopus and Aspergillus. These molds produce extracellular
amylases which subsequently hydrolyze starch substrate (in the cooked
cereals and starchy roots/tubers) into simple sugars. These sugars are in
turn used by the native yeasts and bacteria of murcha to produce alcohol,
acid, and other characteristic congeners.
Objectives
1. To carry out spot culture

2. To carry out tape culture
3. To become familiar with mold colonies
4. To subculture molds by modified hypheal tip technique
5. To identify mold at the genus level
Principle
When small fragments (~3 mm ×3 mm) of murcha are planted on

MYGP agar or molasses agar, a cottony growth will develop upon
incubation at ~26-28°C for 2-3 days. The molds can be identified
microscopically by preparing tape culture. The ability to produce
amylolytic enzymes can be demonstrated easily by carrying out starch-
agar-iodine test. A relatively simple (and probably more reliable) method
is to carry out liquefaction test by inoculating a small lump of cooked
rice with the mold isolate and incubating at room temperature for a
week (in suitable glass container, e.g., cotton-plugged conical flask or
16
beaker). Appearance of profuse watery liquid confirms the presence of
amylolytic activity in the isolate. Mold colonies are simultaneously
subcultured in MYGP plates, allowed to develop profuse mycelia (at 28-
30°C), and stored in refrigerator after sealing the edges of the plates with
a masking tape. This culture serves as a stock for further study.
Requirements
1. Murcha sample
2. Rice
3. MYGP plates (or molasses agar plates)
4. Tweezers
5. Cellotape (clear tape)
6. Masking tape
7. Microscope
8. Inoculating needle
9. 70% alcohol
10. Spirit lamp or Bunsen burner
Procedure
1. Break murcha sample into small pieces (~3 mm × 3 mm). Make

sure that the pieces are free from murcha dust. Dust particles
complicate the isolation process.
2. Sterilize the tweezers tip by dipping in alcohol and brief flaming.
3. With the help of tweezers, place murcha particles on the MYGP
agar surface and slightly press it into the medium. A maximum
of 3 particles can be planted on a single plate. Make sure that the
particles are sufficiently apart (the mold colonies should not
overlap).
4. Invert the plate and incubate at 27-28°C for 2-3 days. Follow the
course of growth by observing the plate every day. At some
point of time, you should be able to see cottony growth around
the murcha particle, with or without minute, colored spores
(black, yellow, green, etc.). Take photographs or make drawings
of the colonies (Fig 4.1).
5. Make tape cultures from morphologically distinct colonies for
microscopic examination as follows:
i. Follow asepsis in every step.
ii. Cut a small piece of clear tape and lightly touch the mold
colony with the sticky surface. Mycelial fragments and
17
some spores should stick to the tape (this is called tape
culture, see Fig. 4.2).
iii. Stick the tape culture on a microscope slight and observe
the specimen in 20× objective. You may also use 45×
for detailed view.
iv. Observe carefully the details of spores (conidiospore or
sporangiospore), rhizoids, zygospores. Take photographs
and make drawings (see Fig. 4.1).
v. Make drawings and identify the molds at genus level
6. Subculture the identified mold colony on a fresh MYGP agar
plate or slant. For agar plates, you can use following simple
technique:
i. With the help of sterile inoculating needle, tease out a
small portion of mycelium and place it over the fresh
MYGP agar plate.
ii. Alternatively, stab the inoculating needle in the fresh
medium and then roll the needle over the mold spores.
This causes some spores to stick to the agar coating in
the needle. Next, stab the needle in the previous medium
making sure that the mold spores are transferred to the
fresh medium.
7. Invert the plates and incubate at 27-28°C for 4-5 days until very
dense colonies with profuse spores are seen.
8. Reserve one of the plates for test of amylolytic activity.
9. Use another plate for stock culture. Preserve/maintain the stock
culture as follows:
i. Seal the plate with a masking tape by running the tap
around the plate perimetrically.
ii. Store the plates in refrigerator.
10. Carry out liquefaction test as follows:
i. Cook about 100 g of rice in a beaker (you can use
autoclave or electrical heater).
ii. Allow the rice to cool to room temperature.
iii. Transfer the entire mold colony from the selected plate
to the cooked rice and mix well.
iv. Crimp aluminum foil to cover the mouth of the beaker
and incubate at 27-28°C for 1 week.
v. Observe daily for evidence of liquefaction (formation of
watery liquid). Production of profuse extract indicates
that the mold is amylolytic.
18
Observations
1. Colony characteristics of the mold (cottony/velvety colony,

spore color, etc.)
2. Mycelial and spore details (microscopic examination)
3. Extent of liquefaction, color of extract, taste of extract
1. Success or failure in the isolation, along with reasons

2. Description of colonial and spore characteristics
3. Discussion on why you considered the mold amylolytic or non-
amylolytic
Questions
1. Amylases produced by molds are extracellular enzymes, why?

2. What is the difference between liquefaction and saccharification?
3. Can both liquefaction and saccharification proceed together?
4. How does the subculturing you carried out differ from hypheal
tip technique?
5. How does mold contribute in alcoholic fermentation of starchy
material?
6. Why do molds lead either parasitic or saprophytic lifestyle?
7. Is there chance of pathogenic molds (e.g., Aspergillus flavus) being
isolated in the above experiment?
8. Can toxic molds survive in traditional fermented foods?
9. Is it possible to prepare murcha using pure fermentative yeasts
and amylolytic molds?
10. How does Mucor differ from Rhizopus?
Medium composition
Molasses agar (pH ~5.6)
High test cane molasses (75-80% soluble solids) : 4 g

Agar-agar : 20 g
Distilled water : 1000 ml
If pH needs to be reduced (e.g., pH 3 or 4), 1% citric acid solution can

be added aseptically in the sterilized medium.
19
Malt Yeast Extract Glucose Peptone Agar (MYGP, pH 4.5)
Malt extract :3g

Yeast extract :3g
Peptone :5g
Glucose : 10 g
Agar agar : 20 g
Distilled water : 1000 ml
Dissolve the ingredients in water by boiling. Autoclave at 121°C for
15 min. Add calculated amount of 1% citric acid solution aseptically
while the medium is still hot and mix. If you maintain pH before
autoclaving, agar will be hydrolyzed and will not set.
For MYGP broth, omit agar in the composition.
Note
On the left-side plain page of the exercise book, make drawings of

all relevant things.
Helpful diagrams
Ascospore Conidiospores
Rhizoid
Rhizopus sp. Aspergillus sp. Penicillium sp.

Fig. 4.1 Some common molds
Cellotape
Plate Mold colony

Fig. 4.2 Preparation of tape culture
20
Practical 5
SCREENING OF FERMENTATIVE YEASTS FROM

MURCHA
Background
Yeasts are ubiquitous in nature. They occur in soil, plants, berries,

fermenting foods, and even in animals. However, only a small number
of yeast types are actually associated with fermented and microbial
foods. Some of those that enjoy a special status in food and
fermentation industries are species of Saccharomyces, Schizosaccharomyes,
Candida, etc. Today, there are 86 genera of yeasts consisting of 597
species. Saccharomyces cerevisiae, which is the most familiar yeast in
alcoholic fermentation, has around 125 synonyms.
Murcha, an amylolytic starter cake indigenous to Nepal, is

extensively used as a source of fermenting yeasts and amylolytic molds
in the production of traditional, cereal-based alcoholic beverages, namely
jand (undistilled) and raksi (distilled). Murcha functions both as a
substrate and carrier for the indigenous yeasts, molds, and bacteria.
From this perspective, murcha can also be considered as a simple stock
culture.
Objectives
1. To carry out enrichment of fermentative yeasts

2. To separate molds (if present)
3. To carry out isolation of yeasts by spread-plating on Malt Yeast
extract Glucose Peptone (MYGP) or molasses agar
4. To study the morphology (microscopic examination, negative
staining) and colony characteristics of the isolates
5. To subculture the isolates in slants and plates
6. To carry out test fermentation on molasses broth
7. To preserve/maintain stock culture
Principle
When murcha sample is inoculated in molasses medium containing

about >10% fermentable sugar and incubated for a week at 28-30°C, the
yeast flora are enriched due to selective pressures of lowered pH, alcohol
content, reduced nutrient and reduced O/R potential that results from
21
the fermentation. It is expected that only the fermentative yeasts (as
opposed to oxidative yeasts) will survive the selective pressure. The
broth can now be tested for the presence of yeast by microscopic
examination (negative staining). If the yeasts are present, a loopful of the
suspension can be spread-plated sequentially on 5-6 MYGP or molasses
agar plates for isolation. The sequential spreading has the effect of serial
dilution, and at some point some plates will yield sufficiently well-
isolated colonies. The plates are inverted and incubated at 28-30°C for
2-3 days. The most well-isolated colonies are examined microscopically
(negative staining) to ascertain the presence of yeast and then
subcultured in slants and plates (four-way streaks). At the same time, a
loopful of culture is inoculated in a 15% molasses broth (250 ml, boiled
and cooled, pH ~ 4 with H2SO4) for test-fermentation.
Requirements
1. Conical flasks
2. Microscope
3. Materials needed for routine microbiological work
4. Dallying rod (bent glass rod) or similar spreader (Fig. 5.2)
5. MYGP plates and slants (or molasses agar plates and slants)
6. Murcha sample
7. Enrichment broth (10% molasses broth, pH 4 with H2SO4)
8. Medium for test-fermentation (250 ml of 15% molasses broth,
pH 4 with H2SO4)
9. 70% alcohol
Procedure
1. Suspend ~ 5 g murcha powder in ~ 100 ml sterile enrichment

broth (15% molasses broth, pH 4 with H2SO4, boiled and
cooled) in 500-ml conical flask.
2. Agitate well and incubate at 28-30°C for 1 week.
3. Observe the broth, second day onwards, for helpful indications
such as CO2 evolution, alcoholic smell, flocculation, etc.
4. If mold pellicles are present on the surface, carefully take it out
with a bent inoculating needle.
5. Next week, ascertain the presence of fermentative yeasts by
microscopic examination of the broth (by negative staining, see
Practical 1). Observe at least 10 fields. Take photographs for
drawing.
22
6. Take a loopful of suspension and spread-plate on 5 to 6 MYGP
or molasses agar plates using inoculation turntable or with the help
of sterile dallying rod (bent glass rod) or suitable glass spreader as
follows:
i. Dip the bent rod in 70% alcohol for a few minutes.
ii. Take out the rod and quickly pass over Bunsen flame or
spirit lamp to pick up the flame.
iii. Move the rod away from the burner (but still in the
sterile zone) and allow the blue flame to die out.
iv. Wait for a minute to cool the rod.
v. Place the bent rod on the well (the inoculum on the
MYGP plate) and spread the culture uniformly all over
the surface of the agar. Do not lift the plate lid wide
open (See Fig. 5.2 and 5.6).
vi. Take out the rod and quickly place on the surface of the
next MYGP agar plate. Take care not to flip the dallying
rod in the opposite direction.
vii. Spread the residual inoculum thoroughly on the agar
surface.
viii. Continue spreading the residual culture on to remaining
plates sequentially.
Note that the plate surface should be dry to affect isolation.
If the plate surface is not dry, dry it at 60°C for 15-20 min in an
inverted position as shown in Fig. 5.3. As an alternative, the
agar surface can also be briefly flamed on Bunsen burner or
spirit lamp by quickly swaying the agar plates (inverted position)
directly over the flame (Fig. 5.5). However, resist overdoing it.
7. Incubate the plates (inverted) at 28-30°C for 2 to 3 days.
8. Determine the colony characteristics of the well-isolated
colonies. Take photographs (close view).
9. Carry out microscopic examination (aseptically) of the well-
isolated colonies. Take photographs for drawing (Fig. 5.4).
10. Subculture the morphologically distinct yeast colonies in MYGP
or molasses agar plates (four-way streak) and slants (Fig. 5.1).
10. Incubate at 28-30°C for 2-3 days, observe the growth, take
photographs, pack in plastic bags (aseptically), and store in
refrigerator. This will be used as stock culture for further study.
11. Simultaneously, carry out test-fermentation in 250-ml molasses
broth (prepared earlier) by inoculating a loopful of culture from
the selected colony. Shake well and incubated at 28-30°C for 1
23
week. Determine the TSS of the broth (with refractometer)
before and after fermentation.
Observations
1. Behavior of the culture during enrichment (gas evolution,

alcoholic smell, sedimentation, etc.)
2. Presence or absence of mold mycelia
3. Morphology of yeasts
4. Colony characteristics (do not forget to mention the color)
5. Decrease in TSS
1. Success or failure in the isolation, along with reasons

2. Description of cell morphology (micrometric, if possible) and
colony characteristics
3. Response of the isolates to test-fermentation
4. Success or failure in subculturing, along with reasons
Questions
1. Why do you think malt extract was included in the medium?

2. Why is pH of the medium important?
3. What do you understand by selective pressure?
4. What are the factors that make MYGP agar medium relatively
selective?
Helpful diagrams
Lid
Inoculating
loop
Agar plate
Fig. 5.1 Four-way streak
24
Dallying rod Lid wide open
Lid partially open (bent glass rod)
(a) Correct technique (b) Incorrect technique

Fig. 5.2 Correct and incorrect ways of spread-plating
Oven at 60oC
Inverted Dry for 15-20 min
Lid agar plate
Solidified
medium
Fig. 5.3 Drying of agar surface
Culture
Culture
Fig. 5.4 Slant culture
Yeast cell
Budding
Fig. 5.4 Yeast cells as seen under microscope (oil-immersion)
25
Sterile zone
Inverted plate
Medium
Flame
Fig. 5.5 Alternative way of drying agar surface
bent glass rod MYGP plate
broth
1 2 3 6
Fig. 5.6 Schematic diagram of spread plating
26
Practical 6
SCREENING OF ANTIBIOTIC PRODUCERS FROM SOIL
Background
Soil is an inexhaustible source of microorganisms, including

antibiotic producers. Antibiotics are secondary metabolites produced by
one type of microorganism that in low concentrations act against other
organisms. In a more general sense, antibiotics represent a category of
chemotherapeutic compounds used to combat plants and animal diseases.
Antibiotics are elaborated by bacteria as well as fungi. For example,

Penicillium chrysogenum (a mold) produces penicillins; Streptomyces species
(bacteria) produce streptomycins and tetracyclines. Streptomyces are the
most widely distributed antibiotic-producing soil microorganisms. Out
of 340 species and 39 subspecies, close to 50% of them are antibiotic
produces. Over 500 distinct antibiotic substances have been shown to
be produced by streptomycetes. Streptomyces prevail in soils which are
alkaline or neutral, and well-drained, e.g., sandy loam, or soils covering
limestone.
Antibiotics have been variously classified. The major bases of

classification and the corresponding examples are given in Table 6.1.
Table 6.1 Schemes for the classification of antibiotics
Scheme Examples of class Examples of antibiotics

Extent of effect o Bacteriostatic Penicillins
o Bactericidal Tetracycline
o Fungicidal Griseofulvin
o Fungistatic Nystatin
Chemical nature o β-lactams Penicillin, Cephalosporin
o Aminoglycoside Streptomycin
o Tetracycline Tetracycline
o Polypeptide Gramicidin
Target o Antiviral Interferon
o Antitumor Nalidixic acid
o Antibacterial Streptomycin
o Antifungal Nystatin
27
Table 6.1 Schemes for the classification of antibiotics …..
Scheme Examples of class Examples of antibiotics

Mode of action o Inhibition of cell Penicillin, Cephalosporin
(the manner in wall synthesis
which the o Inhibition of Tetracycline, Streptomycin,
effect is protein and nucleic Gentamicin, Erythromycin,
manifested) acid synthesis Chloramphenicol
o Inhibition of Sulphonamides
specific enzymes
o Damage to Nystatin
cytoplasmic
membrane
Objectives
1. To prepare crowded plate culture

2. To subculture the colonies exhibiting zones of inhibition
3. To demonstrate the antibiotic spectrum
Principle
Primary screening of antibiotic-producing microorganisms from soil

is relatively easy: a suspension of soil in sterile water is spread-plated on
a suitable agar medium and incubated at 25°C for 5-7 days. Evidence of
antibiotic production is often seen in the crowded plate (300-400
colonies): the colonies that produce antibiotic show clear zones around
them (called zone of inhibition). Since the zone of inhibition is more
conspicuous in a crowded plate, the method is called crowded plate
technique. Once the antibiotic producers have been detected, they can be
subcultured and tested with test organisms such as Escherichia coli,
Staphylococcus aureus, Bacillus subtilis, and Saccharomyces cerevisiae for the
antibiotic spectrum.
Requirements
1. Soil sample (~ 100 g, from a loamy place)

2. Dallying rod (see Fig. 5.2 of Practical 5)
3. Plate Count Agar (PCA) plates
4. Test organisms (e.g., E. coli, Staph. aureus, B. subtilis, S. cerevisiae)
28
5. Routine equipment (autoclave, incubator, balance, magnetic
stirrer, etc.) and glasswares (Petri plates, measuring cylinder,
conical flasks, etc.) used in microbiology laboratory
Procedure
1. Take 10 g soil sample and make 10-fold dilution with sterile

water. Mix properly by shaking (manually or in a mechanical
shaker)
2. Prepare 10 PCA plates in advance and label them (1, 2, 3, ….,
10).
3. Transfer with a sterile inoculating loop, a loopful of suspension
to the first plate.
4. Spread the inoculum sequentially on 6 other plates using the
guideline given in Practical 5. Spreading can be done manually
or with inoculation turntable.
5. Incubate (inverted) all the plates at 25°C for 5-7 days.
6. Observe daily for growth and zone(s) of inhibition (see Fig. 6.1
for an idea).
7. Subculture the selected colonies (those exhibiting zone of
inhibition) in PCA by streaking and incubating at 25°C for 2-3
days.
8. Carry out the test of antibiotic spectrum (see Fig. 6.1) as
follows:
i. Streak suspected antibiotic producer across one side of
the agar plate.
ii. Incubate at 25-30°C for 2-3 days. Allow growth of
microorganism and diffusion of the elaborated
antibiotic.
iii. Streak across (right-angle to the previous streak) the
plate with test-organisms (Fig. 6.1).
iv. Incubate at 30°C for 2-4 days.
v. Observe and interpret the degree of inhibition on the
growth of the test organisms.
Observation
1. Zone(s) of inhibition (and the radius) in the crowded plate

2. Colony characteristics of the suspected colonies
3. Pattern of inhibition on the test organisms
29
1. Success or failure, along with reasons

2. Reasons for variation in the inhibition pattern
Questions
1. Microorganisms not showing zone(s) of inhibition are not necessarily non-

producers of antibiotics, explain.
2. What do you mean by broad spectrum antibiotics?
3. What are the different modes of action of antibiotics?
4. What are the requirements for an antibiotic to qualify as a
chemotherapeutic agent?
Helpful diagrams
Dally rod Zone of inhibition
Incubation Subculturing
25oC/5-7 days
Zone of inhibition
Growth
a b c d a b c d
Incubation Incubation
30oC/3 days 30oC/3 days
Cross-streaking of Diffusion of Streaking of
antibiotic producer antibiotic test organisms
Test organisms:
a = Bacillus subtilis; b = Staphylococcus aureus; c = Escherichia coli;
d = Saccharomyces cerevisiae
Fig. 6.1 Simplified protocol for the screening of antibiotic producing

microorganisms from soil and subsequent testing for antibiotic spectrum
30
Section III
FERMENTATION & FERMENTED PRODUCTS

Practical 7
PREPARATION OF ETHANOL FROM MOLASSES
Background
Ethanol can be produced microbiologically as well as chemically.

The chemical production of ethanol accounts for above 90% of the total
volume but because of the associated potential health risks in chemically
produced ethanol, it is not used for beverage purpose. This is where
microbial production of ethanol comes in. The commercial production
of beverage alcohol normally utilizes improved strains of Saccharomyces
cerevisiae. Schizosaccharomyces species are also used elsewhere. The choice of
substrate depends on the type of beverage intended. Both saccharine
materials (molasses in particular) and starchy materials (cereals, starchy
roots/tubers, etc.) can be used but the processes are very different.
Blackstrap molasses, a byproduct of sugar refinery, is universally used
for large-scale production of beverage alcohol. Both batch and
continuous processes can be used for the production, the choice
depending on the ready availability of raw materials and the technical
know-how. The batch process is very simple to carry out. It involves
dilution of blackstrap molasses to about 15°brix with water, addition of
H2SO4 to give a pH of 4.5, and fermentation with yeast culture at 27-
32°C for 40-48 hrs. The final concentration of ethanol in the wash is
about 7% abv (alcohol by volume). The medium is not pasteurized
because the fermentation process by itself is a protected process (because of
hurdles such as low pH, alcohol content that accumulates overtime,
anaerobic condition, etc.). The nominal capacity of the industrial batch
fermentor (= fermenter) is 500 HL (hectoliter).
Objectives
1. To prepare fermentation medium

2. To study fermentation kinetics
3. To determine alcohol content of wash
4. To observe flocculation characteristics of the yeast
32
Principle
When a fermentative strain of Saccharomyces cerevisiae is grown in a

suitable medium containing most readily assimilable sugar source (e.g.,
glucose, invert sugar, etc.) at concentrations greater than 5%, the
respiratory activity of the yeast cells is suppressed. This phenomenon is
called glucose effect or Crabtree effect. This effect leads to two important
consequences, viz.: (1) Shifting of microorganism from respiratory mode
to fermentative mode (and therefore alcohol production), and (2) cell-
economy for the microorganism (because it does not have to operate the
energy-requiring aerobic pathway).
During fermentation, the organism uses Embden-Meyerhoff-Parnas

(EMP) pathway, generating 2 ATPs per mole of glucose utilized.
Ethanol, which is a primary metabolite, is a partially oxidized product
formed without the involvement of molecular oxygen as the terminal
electron acceptor. For the same reason, ethanol is a growth-associated
metabolite implying that growth and alcohol production occurs
simultaneously.
Starting from glucose, yeast uses a set of 12 enzymes (collectively
called zymase) for the conversion. The stoichiometry is:
zymase
C6 H12O6 ⎯⎯⎯⎯→ 2CH3CH2OH + 2CO2 + energy
(Glucose ) ( Ethanol )
The theoretical conversion of glucose into ethanol is therefore
51.11% (m/m). In other words, the formation of 51.11 g of ethanol
from 100 g of glucose represents 100% theoretical conversion (100%
efficiency). In practice, however, only 90-95% efficiency can be
achieved. This is because some portion of the carbon source is
simultaneously utilized by the yeast for its cell build-up. See Fig. 7.1 for
the truncated sequence of the metabolic pathway leading to ethanol
formation.
4[ADP+Pi] 4ATP
2ATP 2ADP
Glucose 2[1,3-di- P glycerate] 2[Pyruvate]
2[NAD+] 2[NADH+H+]
2CO2
2[Ethanol] 2[Acetaldehyde]
Fig. 7.1 Pathway for alcohol synthesis
33
Ethanol content in the wash can be determined by a number of
methods. The chemical method depends on the oxidation of ethanol
with potassium dichromate in sulfuric acid medium and determination
of excess dichromate by ferrous ammonium sulfate in the presence of
ferrous-1,10 phenanthroline as indicator. The physical method is based
on the determination of apparent specific gravity of distillate obtained
from standard amount of wash and then referring to standard alcohol
specific gravity charts. Hydrometers can also be used for the same.
Requirements
1. Yeast culture (Active Dry Yeast or screened fermentative yeast)

2. Blackstrap or high-test molasses (2 kg)
3. Sugar (2 kg)
4. Yeast food (ammonium phosphate or ammonium sulfate)
5. Fermentation jar (20 L capacity)
6. Conc. H2SO4
7. pH meter or multi-range pH paper
8. Refractometer (able to measure up to 25°brix)
9. Laboratory glassware (general)
10. Glass pycnometer
11. Electronic balance (± 1 mg)
12. Distilled water
13. Volumetric flask (100 ml)
14. Blotting papers
15. Thermometer (digital or mercury)
16. Alcohol chart
17. Distillation set (Fig.7.2)
18. Ca(OH)2 solution (~ 2N)
Procedure
1. Dissolve sugar and molasses in ~ 10 L water in a large container

2. Dilute with water until the solution attains a TSS (Total Soluble
Solids) of 15°brix, as measured by refractometer (do hit and
trial). Note down the total amount of water needed.
3. Add conc. H2SO4 until the pH is 4.5. Be careful when using
conc. H2SO4. Use pH meter or multi-range pH paper to check
the pH. Note down the amount of acid needed.
4. Add yeast food at the rate of 0.5 g/L and mix.
5. Prepare yeast inoculum as follows:
34
i. Weigh Active Dry Yeast (ADY) at the rate of 0.5 g per
liter of medium.
ii. Transfer ADY to 500-ml beaker containing 250 ml of
lukewarm water. Stir with a glass rod to disperse the
yeast.
iii. Leave the beaker for ~ 10 min to activate the yeast. A
thick foam that looks like cauliflower head should
develop within 10 min.
6. Add the activated yeast to the medium and mix vigorously.
7. Transfer the contents to 20-L jar. Put on the lid and leave for
fermentation at ~ 30°C. If the temperature is too low (e.g., in
winter), place the jar in the sun until fermentation begins to
occur vigorously.
8. If you are using screened yeast culture, the inoculum must be
built-up until the amount is suitable for pitching. Use following
guidelines for inoculum build-up:
i. Take slant or plate stock culture of the screened yeast.
ii. Add 2-3 ml of sterile water or molasses medium and
agitate gently to get yeast suspension.
iii. Transfer the suspension to 100 ml of pre-sterilized
molasses medium (kept in cotton-plugged conical flask)
containing ~ 4% sugar (pH 4.5). Pre-sterilization of
medium can be carried out by boiling the medium for 10
min. Make sure that the flask is cotton-plugged during
boiling. Use the medium after it has cooled to room
temperature.
iv. Shake the contents vigorously and carry out fermentation
at 27-30°C for 2 days. Shake the flask occasionally to
facilitate aeration.
v. Transfer the contents to 1-L flask containing 500 ml
medium (similar to previous) and carry out fermentation
for 2 days (as previously done). Shake the contents
occasionally to facilitate aeration.
vi. Use the contents for pitching the main medium for
alcohol production (follow step 7).
9. Follow the course of fermentation by measuring TSS before and
during the fermentation.
10. Record TSS every 12 hrs (or daily, if not possible) until no
further drop in TSS is observed (this may take 2-6 days, as the
case may be).
35
11. Plot a graph of Time vs TSS, using 15°brix (initial observation)
for time = 0 hrs.
12. Make observations for following:
i. Evidence of gas evolution
ii. Crackling sound when you place your ears against the
wall of the jar (the sound indicates that fermentation is
occurring)
iii. Yeast flocculation (top or bottom)
13. Terminate the fermentation after TSS ceases to drop further.
The wash (beer) should not taste sweet when you taste a drop or
two. It generally tastes lightly sour.
14. Determine alcohol content by pycnometer as follows:
i. Distillation of alcohol:
a. Take 200 ml of wash (beer) in a beaker.
b. Neutralize it with Ca(OH)2 solution. You can use
multi-range pH paper or phenolphthalein for this.
c. Transfer the contents to distillation flask (500-ml
capacity, Fig. 7.3).
d. Carry out distillation and collect 80-90 ml of the
distillate. Observe appearance of the oily droplets
in the condenser. This indicates presence of
alcohol.
e. Transfer the distillate quantitatively to a 100-ml
volumetric flask. Make up the volume with distilled
water, mix well, stopper, and store until used.
ii. Preparation of pycnometer:
a. Take a 50-ml pycnometer (with the plug, Fig. 7.2).
b. Clean it with cleansing agent. You can use cleaning
solution (a saturated mixture of K2Cr2O7 and conc.
H2SO4 if the pycnometer is dirty).
c. Wash repeatedly with distilled water until
absolutely clean.
d. Dry the pycnometer (and the plug) in hot air oven
at above 130°C for an hour. Note that it is very
difficult to remove water from inside the
pycnometer.
e. After the pycnometer has cooled, you may/may
not see moisture condensing on the walls again.
Introduce 1 ml of acetone, shake the bottle (i.e.,
36
pycnometer), and drain. Once again, dry the bottle
in hot air over for ~ 10 min.
f. Cool the bottle (along with the plug) and weigh in
an electronic balance.
g. Record the weight of the bottle + plug (say w1 g).
h. Take out the distillate you have stored and record
the temperature of the contents (say T°C).
i. Pour distillate carefully into the pycnometer until
almost full. Stop at mid-way through the neck of
the pycnometer. Insert the plug slowly and allow
the distillate to rise through the capillary, until it is
full to the tip. If the plug bottom does not touch
the distillate, add more distillate (drop-wise) until it
does. It is likely that the plug is either under-filled
or over-filled. If under-filled, add few drops until
the distillate reaches the tip. If over-filled, soak the
extra liquid by quickly running a blotting paper
over the spilt liquid.
j. When finished, quickly wipe the entire surface of
the bottle with blotting paper. Do not hold the
bottle in in the hand for long (the heat transferred from
the hands to the bottle will expand the liquid more than the
glass, thereby pushing the liquid out of the capillary in the
plug).
k. Weigh the bottle + distillate + plug quickly (say, w2 g).
l. Empty the contents and rinse the bottle thoroughly
with distilled water.
m. Wipe the surfaces of bottle and the plug with
blotting paper.
n. Fill the bottle with distilled water, following the
procedure used for distillate.
o. Weigh the bottle + distilled water + plug quickly
(say w3 g).
15. Calculate the apparent specific gravity and density of distillate (at
temperature T°C) as follows:
w 2 - w1
i. Apparent specific gravity =
w 3 - w1
37
ii. Density at T°C = Apparent specific gravity × density of
w 2 − w1
water at T°C = ×ρ
w 3 − w1 T
ρT can be obtained from standard charts.
16. Calculate alcohol content by referring to standard charts (for
example, AOAC International [2005], REFERENCE
TABLES Appendix C. p. 18). Divide the alcohol content by 2
to get the actual alcohol content by volume at 15.5°C (60°F).
Observations
1. TSS of the broth before and after fermentation

2. Duration of fermentation
3. Flocculation characteristics of the yeast cells
4. Temperature of distillate and distilled water
5. Density of water at the observed temperature
6. Pycnometer readings
7. Appearance of alcohol droplets in the condenser during
distillation
1. Success or failure in carrying out fermentation, along with

reasons
2. Graph of time versus TSS and its explanation
3. Alcohol content of wash
Questions
1. What do you understand by the term fermentation kinetics?

2. Why was alcohol content divided by 2 to get the actual alcohol
content?
w 2 − w1
3. Can you prove Density at T° = ×ρ ?
w 3 − w1 T
4. How does density change with alcohol content?
5. What is density of absolute alcohol?
6. What do you understand by the terms wash and spent wash?
7. What is the difference between apparent specific gravity and true
specific gravity?
38
Helpful diagrams
plug capillary
50cc
Fig. 7.2 A typical glass pycnometer
Water out
Condenser
Alcoholic Still
broth
Water in
Heat
Receiver
Fig. 7.3 A typical distillation assembly
39
Practical 8
SINGLE COLUMN DISTILLATION OF ETHANOL
Background
Single column distillation is perhaps the oldest operation used for

separation of liquid mixtures. For centuries and also today, batch
distillation is widely used for the production of fine chemicals and
specialized products such as alcoholic beverages, essential oils, perfumes,
pharmaceutical and petroleum products. It is the most frequent
separation method in batch processes. At the simplest, the single column
distillation takes the form of pot stills used traditionally for the
production of whisky and brandy.
The essential features of a conventional batch distillation (CBD)

column are as follows:
1. A bottom receiver/reboiler which is charged with the feed to be

processed and which provides the heat transfer surface
2. A rectifying column (either a tray or packed column)
superimposed on the reboiler, coupled with either a total
condenser or a partial condenser system
3. A series of product accumulator tanks connected to the product
streams to collect the main and/or the intermediate distillate
fractions
A schematic diagram of single column distillation is given in Fig.

8.1. Operation of such a column involves carrying out the fractionation
until a desired amount has been distilled off. The overhead composition
varies during the operation and usually a number of cuts are made. Some
of the cuts are desired products (main-cuts) while others are intermediate
fractions (off-cuts) that can be recycled to subsequent batches to obtain
further separation. A residual bottom fraction may or may not be
recovered as product.
The main advantages of batch distillation over a continuous

distillation lie in the use of a single column (as opposed to multiple
columns) and flexibility in operation. With CBD, only one column is
necessary and there is only one sequence of operation (with or without
the production of off-specification materials) to separate all the
components in a mixture. The only requirement here is to divert the
40
distillate products to different product tanks at specified times. Some
batch still types are shown in Fig. 8.2, 8.3 and 8.4.
Objectives
1. To study the parts of single column distillation system

2. To carry out single column distillation
3. To obtain different cuts (main cut, heads and tails)
4. To determine alcohol content of the product streams by alcohol
hydrometer
5. To study temperature profile during distillation
Principle
When a mixture of liquids is heated up, the more volatile

components tend to come off first. In fermented alcoholic wash,
methanol, aldehydes and esters will be the first to come off. This
fraction is called heads. Heads are followed by the main fraction (main-cut)
that consists of ethanol-water binary azeotrope. The last to come off are
the higher alcohols (called tails). Tails consist of propanol, butanol, amyl
alcohol, etc., collectively called fusel oils.
Pure ethanol has a boiling point of 78.5°C while pure water boils at
100°C. A binary azeotrope of water and ethanol boils at a temperature
lower than either of these two extremes (78.2°C), which forms the basis
of ethanol distillation. The ethanol-water azeotrope has 95.6% ethanol
and 4.4% water. Since this is a constant boiling mixture, no amount of
further distillation will give distillate of ethanol content greater than
96.5%.
Requirements
1. Single column distillation system or pot-still system

2. Alcohol hydrometer
3. Heating arrangements
4. Jars for collecting distillates (different cuts)
5. Measuring cylinder (250-ml capacity)
6. Tools for dismantling the distillation equipment
41
Procedure
1. Agitate the alcoholic wash thoroughly to drive away gases.

2. Transfer the contents to still/reboiler, not exceeding 75% of the
volume capacity.
3. Add some boiling chips (porcelain chips can be used) to avoid
bumping during boiling.
4. Connect the still to reflux column and condenser.
5. Insert thermometer to the reflux column (if there is provision
for one).
6. Open heating (electric or gas).
7. Turn on the tap to run condenser.
8. If possible, watch through sight glass the enrichment process in
the column.
9. Supposing that you are using 10 L of wash, remove the first 100
ml of the distillate. This fraction obviously contains heads. Do
not taste it!
10. Continue distillation and collect the distillate in a different
container until you collect about 100 ml.
11. Collect the next 100 ml distillate in another container.
12. Determine alcohol content of the distillate with alcohol
hydrometer every 100-ml portion collected (Fig. 8.5).
13. Continue distillation until the alcohol content of the distillate is
less than 50%.
14. Record the temperature profile during distillation.
15. Further distillation will lead to distillation of higher alcohols.
This can be confirmed by adding water to the distillate. Presence
of higher alcohols turns the distillate turbid white.
16. Terminate the distillation and pool the 100-ml fractions you have
collected.
17. Determine the final alcohol content of the pooled distillates (it
should be greater than 50%) and the volume.
Observations
1. Arrangement of different parts of the distillation equipment

2. Movement of distillate in the reflux/enrichment column
3. Smell of heads, main cut and tails
4. Alcohol content of different cuts
5. Quantities of different cuts produced
6. Time taken for the heads to appear in the collection jar
42
7. Time taken to complete the distillation
8. Any peculiar observation
9. Temperature profile
1. Success or failure in carrying out distillation, along with reasons

2. Simple or complicated nature of experiment?
3. Alcohol content (hydrometric reading) of different cuts
4. Quantities of different cuts produced
5. Alcohol content of different cuts
Questions
1. What do you mean by positive- and negative azeotrope?

2. Why do higher alcohols turn milky when mixed with water?
3. How is methanol produced during fermentation?
4. Why is methanol poisonous? What is its antidote and how does
it work in principle?
5. What do the following terms mean?
i. Rectified spirit
ii. Industrial spirit
iii. Denatured spirit
iv. Extra neutral alcohol
v. Absolute alcohol
vi. Congeneric and non-congeneric alcoholic beverages
vii. Degree Proof
6. What is the fatal dose of ethanol in human adult?
7. What happens when methanol is ingested?
8. Where do higher alcohols come from?
Note:
You may sometimes observe turbidity in the heads portion. This

turbidity is caused by the displacement of water-insoluble residual long
chain fatty acids and their esters, which have remained on the inner
surfaces of the still from the previous distillation.
The turbidity that you see in the tail fraction is due to higher
alcohols, which are sparingly soluble in water.
43
Helpful diagrams
Condenser
Reflux
Main-cut Off-cut Off-cut

Feed
Reboiler
Fig. 8.1 A typical single column distillation system
Cold water
Copper pot for batch-
condensation (bata)
Condensate
(raksi)
Earthen column (paini)
Vapor
Condensed alcohol (raksi)
Holes at the bottom Receiving pot (nani)
of paini for letting out
alcoholic vapor
Wash
Copper still
(phonsi)
Fire
Fig. 8.2 Traditional Nepalese pot still for raksi production
44
Swan neck Pre-heater
Head
Coiled pipe
Condenser
Boiler
Fire
Barrel
Fig. 8.3 Schematics of open-fired kettle distillation (for brandy)
Thermometer
Water out
Condensing coil
Reflux
column
Condenser
Water in
Distillate
Still
Fig. 8.4 Locally fabricated simple distillation unit
45
100 Read the lower
95 %
meniscus
90 100
95
85 90
80 85
80
70 70
60
60 50
40
30
50 10
0
40
30
20
10
0
distillate
Fig. 8.5 Typical alcohol hydrometer
46
Practical 9
PREPARATION OF RUM FROM SINGLE-COLUMN
DISTILLATE
Background
Rum is a congeneric (containing more than 500 flavor compounds

originating from fermentation), distilled alcoholic beverage made from
sugarcane byproducts, such as molasses, or directly from sugarcane
juice, by a process of fermentation and distillation. The majority of the
world’s rum production occurs in the Caribbean and Latin America.
There are various grades of rums, such as dark rums, flavored rums,
gold (amber) rums, light (silver or white) rums, overproof rums,
premium rums and spiced rums. Light rums are usually used in cocktails.
Golden and dark rums are consumed straight or used in cooking recipes.
Rums generally have alcohol content between 40 to 50% abv but
overproof rums such as Bacardi can have as high as 75% abv. It is also
common to express alcohol content of distilled alcoholic beverages in
terms of degree proof (°Proof). In the American system, 1% abv at 60°F
(15.56°C) equals 2°Proof. Thus an alcoholic solution of 50% abv equals
100°Proof, and is called proof spirit. Strengths above 100°Proof are
denoted by OP (overproof), which is the number of degree proofs above
100°Proof. Therefore, a 125°Proof spirit equals 25 OP. Strengths below
100°Proof are denoted by UP (underproof), which is the number of degree
proofs below 100°Proof.
Rums can be produced by distillation in continuous as well as batch

mode. The pot still method is considered classical and produces more
flavorful rum. In dark rums, caramel is added for the characteristic color
and flavor. Gold rums acquire their color from aging in oak barrels.
Light rums have no color at all. Spice rums are generally based on gold
rums. Among the spices added are cinnamon, rosemary, aniseed, or
pepper.
Objectives
1. To prepare caramel color

2. To carry out blending
3. To prepare dark rum
47
4. To design labels for the product
5. To carry out hedonic ratings of the prepared rum
6. To carry out statistical analysis of data using Genstat®
Principle
The main cut of the single-column distillate, assumed to contain

above 55% abv, and from which heads and tails have been removed, can
be blended with distilled water (to eliminate foreign taste originating
from water) and required amount of spirit caramel (Class I caramel)
color to give rum of 40 to 50% abv. Either Pearson square or the
general formula S1V1 = S2V2 can be used to calculate the proportion of
water required. The caramel color needed for the experiment can be
easily prepared in the laboratory by burning sugar. Alternatively, suitable
grades of caramel (plain caramel / spirit caramel) can be purchased from
the store. The proper sensory evaluation of rum is quite involved
because of the complex flavor and lexicons used to describe them.
However, a simple comparison of product types based on preference
can be carried by hedonic rating test and the data statistically analyzed
through ANOVA of attributes (color, taste, smell, overall). Data analysis
can be carried out using either the common MS-Excel Program or
dedicated statistical softwares like Genstat®, IBM-SPSS®, Sigmaplot®,
etc.
Requirements
1. Single-column distillate (main-cut, >55% abv)

2. Distilled water
3. Alcohol hydrometer
4. Caramel color (or arrangements for preparing caramel)
5. Volume measuring arrangements (graduated jars, etc.)
6. Glass bottles (square type) for bottling
7. Statistical software package
Procedure
1. Measure the volumes of the distillates (two types).

2. Measure the alcohol content of the distillate (with alcohol
hydrometer). Note that the alcohol content should be greater
than 50% abv.
48
3. Calculate the amount of distilled water needed to make rum of
45% abv (90°Proof) using following formula:
VD
VW = (S − S )
SR D R
where,
VW = volume of water needed, VD = volume of distillate
given (supplied),
SD = strength of distillate (% abv), SR = strength of rum to
be prepared (% abv),
4. Mix the distillate and water.
5. Check the alcohol content with alcohol hydrometer (if necessary,
readjust alcohol content).
6. Add caramel solution to give the desired, dark color. If you do
not have caramel color, prepare it in the laboratory as follows:
i. Take a teaspoonful of table sugar in a sauce pan.
ii. Apply gentle heat until the sugar caramelizes to the color
of a copper penny (darker than coffee color).
iii. Add some water and continue heating until the caramel
lump dissolves.
iv. Cool and store the solution in a dark bottle.
7. Prepare labels for the rum (see Fig. 9.1 for idea). The label
should contain following items:
i. PDP (prominent display panel, front side of the
container):
a. Statement of identity
b. Brand display
c. Net quantity statement
ii. Information panel (right side of PDP):
a. Name and address of manufacturer, packer or
distributor
b. Date of manufacture
c. Batch number
d. Other information
8. Carry out sensory evaluation (hedonic rating) using score card
given in Fig. 9.2 for taste, smell, color and overall.
9. Carry out statistical analysis (ANOVA) for attributes (color,
taste, smell, overall) using Genstat 12th edition (a statistical
package, Fig. 9.3, 9.4 and 9.5). You may also use Excel Add-in of
Microsoft Office for the statistical analysis.
49
Observations
1. Events observed during caramel making

2. Volume of the supplied distillate and final rum
3. Alcohol content of supplied distillate and the final rum
4. Color of the rum (descriptive)
5. Caramel color and flavor
6. Appearance of the product after labeling
1. Success or failure in preparing rum, along with reasons

2. Simple or complicated nature of experiment?
3. Alcohol content (hydrometric reading) of distillate and rum
4. Quantities of distilled water needed to prepare rum
5. Comment on color and flavor, along with plausible reasons
6. ANOVA tables of statistical test and discussion
7. Photographs of the final product
Questions
1. What type of caramel is used in distilled alcoholic beverages?

How and why does it differ from the one used in beer?
2. You have two distillate stocks: 20 L of Stock “A” with 65% abv
and 25 L of Stock “B” with 45% abv. Use all of Stock B and
make 40 L of blended product with 50% abv. [Ans: 25 L of
Stock B, 13.08 L of Stock A, and 1.92 L of distilled water].
3. What is dunder yeast?
4. Why is rum categorized as a congeneric alcoholic beverage?
5. How is rum aged?
6. What is cachaca?
7. Calibration in the alcohol hydrometer is not linear (see Fig. 9.4),
why?
8. What is tequila?
50
Helpful diagrams
DMC's
Produced since 2014
DARK RUM
Dharan Multiple Campus

Dharan-16
A Product of Nepal
Net Vol. 720 ml Finest rum produced using indigenous yeast

Alcohol content: 45% abv Batch No. 2070/3
PDP Information Panel
Fig. 9.1 An example of label for alcoholic beverage
Panelist: ...................................
Date: ...................................
Product: Rum
HEDONIC
Evaluate the samples presented. Write the code number of sample

on the appropriate scale provided below that best describes your
degree of liking or disliking towards the product
*************************************************************************
9 = Like extremely 5 = Neither like nor dislike
8 = Like very much 4 = Dislike slightly
7 = Like moderately 3 = Dislike moderately
6 = Like slightly 2 = Dislike very much
1 = Dislike extremely
************************************************************************
Comments: -------------------------------------------------------------------------------------
Attribute Rum 1 2 3
Taste
Smell
Color
Overall
Fig. 9.2 Specimen of score-card for hedonic rating

51
Fig. 9.3 Genstat® statistical package
Fig. 9.4 Tabulation of sensory data in MS-Excel (for Genstat®)
52
Fig. 9.5 Example of output from Genstat®
The output (Fig. 9.5) shows that there is no significant difference

in the color of different rums at 5% level of significance. The output
also shows good agreement between the panelists (Roll No. of students).
In case difference is observed, you can use additional

measures/tests to identify the magnitude and direction of the difference.
The common measures are LSD (Least Significant Difference) and
DMRT (Duncan Multiple Range Test). The description of the tests is
beyond scope of this manual. You can take your instructor’s help for the
same. An example of how a summarized output may look like is shown
in Table 9.1.
An example of summarized ANOVA output
Table 9.1 Statistical analysis of sensory attributes of rum (Panelist = 10)
Rum type Color Taste Smell Overall

A 4.0 (0.34)a 4.8 (0.33)a 5.0 (0.32)a 4.9 (0.21)a
B 4.2 (0.45)ac 4.1 (0.10)b 4.6 (0.25)a 4.0 (0.09)b
C 3.3 (0.25)b 3.4 (0.12)c 3.3 (0.11)b 3.0 (0.45)c
D 3.8 (0.40)c 4.1 (0.22)b 3.8 (0.32)c 3.6 (0.43)d
LSD (5%) 0.21 0.65 0.45 0.23
Values are arithmetic means and standard deviations (in

parenthesis) of scores of 5-point preference rating given by 10 panelists.
Values in the column bearing similar superscripts are not significantly
different at 5% level of significance. LSD represents Least Significant
Difference.
53
Practical 10
PREPARATION OF RED TABLE WINE
Background
Wine, without a modifier, means the end product of complete or

partial alcoholic fermentation of fresh grape juice. The distilled product
from wine is called brandy. Wines from fruits/berries other than grapes
are required to indicate the source on the label. For example, pineapple
wine, orange wine, apple wine (cider or cyder), etc.
The basic components required for wine making are yeast (selected
strains of Saccharomyces cerevisiae) and grape juice (called must). Grapes
with sugar content ≥ 20% are preferred. The yeast culture can be either
obtained from a commercial supplier or isolated from grape berries
(natural flora, that is). SO2 is routinely added during crushing and racking
to control wild yeasts and bacteria. Fermentation is carried out at 10-
20°C for white wines and 27°C for red wines. The duration of
fermentation is variable, taking at least a month for the completion of
fermentation alone. Thereafter follows aging process that may take
several months to several years.
White wines are prepared from white grapes whereas red wines are
prepared from purple grapes. The red color of wine is due to
anthocyanins of purple grapes that leach out in the wine during
fermentation/maceration. Typically, red wine production is carried out
in 2 to 3 stages. The first stage, called primary fermentation, is carried out
along with skins to promote extraction of color. Thereafter, the liquid
portion is separated from the pomace and a secondary fermentation is carried
out until the sugar level drops to less than 1% (for dry wine).
Fermentation may take a few to several months. The lees that settle
down are removed by a process called racking. Depending on the acidity
of wine, a final bacterial fermentation called malo-lactic fermentation (ML
fermentation) may be needed. ML fermentation helps reduce the
titratable acidity of wine. The process also mellows or ages the wine.
Finally, the wine is blended, filtered, pasteurized, and bottled in colored
bottles.
For home-scale production of wine, a good quality baker’s yeast can

also be used. Since baker’s yeast is not trained to tolerate SO2, it is better
54
to avoid this preservative. The quality of wine, however, will be
compromised.
Objectives
1. To prepare must
2. To carry our primary and secondary fermentation
3. To perform racking operation
4. To carry out in-bottle pasteurization
5. To determine physicochemical properties of red wine
6. To carry out bottling and labeling
7. To carry out organoleptic test
Principle
Selected purple grapes (≥ 15°brix TSS) are crushed lightly and

ameliorated to give a must of TSS ≥ 20°brix and pH ~ 3.5. Yeast is
added to the must, mixed well, and primary fermentation carried out in a
jar at 28°C for 4-5 days. The must is shaken occasionally to mix the skin
cap to facilitate color extraction. Once the primary fermentation is over,
the entire contents are strained through muslin cloth and the free-run
liquid collected. The remainder of the liquid is extracted by pressing and
the residue and fermented separately for press wine. The free-run and the
pressed extracts are fermented in jars separately for 1 month. The clear
liquids are separated by siphoning (racking) and bottled. The wine is
then pasteurized by dipping the bottles in hot water bath and holding
until the internal temperature of wine reaches 65°C. The bottles are
taken out, surface dried, labeled, and stored.
Requirements
1. Purple grapes (20 kg)

2. Sugar ~ 2 kg
3. Wine yeast (if not available, use baker’s yeast)
4. Demijohns, 15-20 L capacity (if not available, water carboy)
5. Racking pipe (transparent labeling pipe, ~ 2 m long)
6. Muslin cloth
7. Pycnometer (for alcohol determination)
8. Weighing and volume-measuring arrangements
9. Heating arrangement
10. Refractometer
55
11. pH meter
12. Wine bottles (20 pcs)
13. Bucket with cover (25 L capacity)
Procedure
1. Prepare yeast inoculum by activating 5 g dry yeast in 100 ml of

lukewarm grape juice for 10 min. This pitching rate roughly equals
to 0.25 g dry yeast per liter of must.
2. Destem the grapes and sort out immature/spoiled berries.
3. Weigh the berries.
4. Rinse the berries in clean water.
5. Rupture the berries with hands (avoid mashing, as this will
complicate clarification).
6. Measure TSS (> 16°brix is preferred). The must should have at
least 24°brix for good wine. This level can be maintained by
adding sugar (the process is called gallization). Some countries do
not allow addition of sugar in the must.
7. Measure pH of the must.
8. If needed, bring the pH of the must to 3.5 by adding 2% citric
acid solution (by hit and trial). Do not be tempted to increase the
pH if already low. Adding neutralizers will blacken the must
color. Alternatively, you can use proprietary deacidifying
chemicals, e.g., Acidex®.
9. Add the yeast slurry (prepared earlier) and mix well.
10. Transfer the contents to bucket and put on the cover (Fig. 10.1).
11. Carry out primary fermentation for 4 days at ~ 27°C. Push the skin
cap down twice a day to facilitate color extraction.
12. Strain the contents through double-layer muslin cloth and collect
the free-run liquid. Do not be tempted to press the residue to
recover the juice! This produces poor quality wine.
13. Measure the TSS (probably down to <6°brix) as well as the
amount of free-run wine obtained.
14. Add potable water to make 15 L (total volume). This is not
allowed in many countries as this will produce poor quality wine!
15. Measure the TSS again.
16. Add sugar to get 24°brix equivalent of the original must (see
sample calculation later). This step helps avoid high TSS in the
primary fermentation as it can be inhibitory to the yeast. This
56
manipulation also produces wine that is stronger (and may be
sweeter).
17. Mix the contents and transfer to Demijohn (or water carboy,
Fig. 10.1).
18. Plug the jar with cotton and leave for secondary fermentation for 1
month at 27°C. Follow the progress of fermentation by
measuring TSS every 5 days and watching the gas bubbles. After
the fermentation has completed, the TSS ceases to decrease
further and the gas bubbles disappear.
19. Press the residue in the muslin strainer and collect the extract.
Add a small amount of water to finish off the extraction. Add
sugar as in step 16 and carry out secondary fermentation of the
extract in a separate jar (for 1 month at 27°C) to produce press-
wine, which is supposed to be of inferior quality.
20. After fermentation, rack the wines by siphoning with a labeling
pipe.
21. Fill into bottles, loosely cap them and pasteurize in hot water
bath until the internal temperature of the bottled wine reaches
65°C (Fig. 10.2).
22. Take out the bottles, tighten the caps, and allow the surface to dry
by its own heat.
23. Label the bottle to reflect the identity of the product, net
content, manufacturer, batch number, place and year of
manufacture, etc. and store for a week.
24. Carry out sensory evaluation of free-run- and press-wines.
25. Determine alcohol content (pycnometric), pH and acidity (as %
lactic acid).
Observations
1. TSS and pH of the grape

2. Behavior of cap
3. Bubbling vigor, smell, etc., during primary fermentation
4. Volume of free-run juice
5. TSS of free-run juice after primary fermentation
6. Status of lees and clarity of wine during secondary fermentation
7. Color, alcohol content, TSS, acidity, and pH of wine
8. Color of wine before and after pasteurization
57
1. Success or failure of the experiments, along with reasons

2. Physicochemical properties of wine (pH, alcohol content, TSS,
acidity)
3. Sensory quality (descriptive) of free-run and press-wines
Questions
1. Define the terms: amelioration, gallization, chaptalization,

binning, and fortification
2. How does ML fermentation occur and when is it undesirable?
How do you control it?
3. How does SO2 control wild yeasts and bacteria?
4. What happens if you add more than 200 ppm of SO2 in the
must?
5. What events occur during aging?
6. What are the fundamental differences between white table wine
and red table wine?
7. What is the scientific name of grapes used in wine production?
8. Is it good to add sugar in the must?
9. What do you know about fermentation lock or air lock system used
in commercial wine fermentation?
10. What is microoxygenation?
11. What is sur lie storage of wine?
Note:
Sample calculation for sugar adjustment
Let us suppose the amount of crushed grapes = 20 kg

TSS observed = 20°brix
Amount of free-run wine obtained = 12 kg
Extra water to be added = (15-12) kg = 3 kg
24°brix equivalent for 15 kg must = 15×24/100 = 3.6 kg
TSS in 12 kg of must (before fermentation) = 12×20/100 = 2.4 kg
TSS of free-run wine = 7°brix (i.e, TSS left = 12×7/100 = 0.84 kg)
∴ Sugar to be added = 3.6-(2.4-0.84) = 0.36 kg
58
Helpful diagrams
Cork
Lid Cap
Outlet
Bucket Demijohn
Fig. 10.1 Equipment needed for wine making
Thermometer
Hot water
Fig. 10.2 In-bottle pasteurization of wine
59
Practical 11
PREPARATION OF BEER BY DECOCTION MASHING
Background
Beer is an undistilled, carbonated, alcoholic malt beverage that has

been flavored and preserved with hops. With the exception of zero
alcohol- and extra strong beers, a typical beer generally contains about 5%
alcohol by volume and about 0.5% CO2 (m/m). Beers are of two main
types, viz. (i) Lager, and (ii) Ale, each of which has several sub-types.
Lager beers are produced by fermenting the wort with Saccharomyces
carlsbergensis (syn. uvarum) at 8-10°C for 12-14 days. Saccharomyces
carlsbergensis is also dubbed bottom yeast. In contrast, ales are fermented
with Saccharomyces cerevisiae (also dubbed top yeast) at comparatively higher
temperatures for shorter duration (12-18°C for 7-8 days). The
fundamental differences between a lager and an ale are given in Table 11.1.
Table 11.1 Some fundamental differences between lager and ale beers
Characteristic Lager Ale

Yeast S. uvarum S. cerevisiae
Fermentation Bottom Top
Temperature, °C 8-12 12-18
Time cycle, days 8-14 7-8
Mashing process Decoction Infusion
Yeast cropping Bottom crop Top crop
Hops Bitter hops Aroma hops
Carbonation More Less
The common ingredients of all beer types are (i) barley malt, (ii) hops,
(iii) adjuncts, (iv) potable water, (v) carbon dioxide, and (vi) yeast. Hops
refer to dried hop cones (or products thereof) of female hop plant
Humulus lupulus (see Fig. 11.1). Hops give the characteristic bitterness of
beer. The hop compound responsible for the bitterness is α-acids (see
Fig. 11.2). Barley malt is used in beer making (brewing) for amylase
enzymes (that break down the starch into simple sugars, which in turn
are utilized by yeast during fermentation), characteristic color and flavor,
and as a substrate for the fermentation. Adjuncts are unmalted
60
carbohydrate sources that beneficially supplement and complement
barley malt.
Objectives
1. To carry out decoction mashing

2. To study fermentation kinetics of beer fermentation
3. To prepare beer
4. To carry out sensory and physicochemical analysis
Principle
Ground barley malt is mashed along with adjuncts in a stepwise

manner to increase the temperature of mash from ~40°C to a final of
about 70°C. The stepwise increase in temperature is achieved by taking
out about 1/3rd portion of the mash and returning it to the main mash
after bringing it (the portion taken out) to boil. Following a brief rest
period, ~ 1/3rd portion is taken out again, brought to boil, and returned
to the main mash. The process is continued until the main mash attains
a temperature of about 70°C whereupon the mash is filtered to obtain
sweet wort. During the rest period, the solid residue settles to the bottom
to give what is called thick mash and it is this portion that is taken out for
boiling. The extract (sweet wort) is boiled vigorously for 1-2 hrs along with
hops to extract hop principles. The wort is then strained, cooled to
room temperature (~ 25°C), TSS adjusted to ~ 15°brix, and fermented
with brewer’s yeast for 8-10 days to obtain green beer. The yeast is
removed by decantation and the product is pasteurized at 65°C for 8-10
min. The product is then bottled, cooled to ~ 5°C and carbonated to
obtain beer ready for consumption.
Requirements
1. Barley malt – 1 kg (if not available, use 500 g malt extract)

2. Sugar – 1 kg
3. Malt extract – 100 (optional)
4. Hops (pellets, extracts or powder)
5. Clean water (boiled and cooled)
6. Brewer’s yeast (if not available, use baker’s yeast)
7. Lactic acid
8. Cooking utensil
9. pH meter
61
10. Muslin cloth
11. Beer bottles (colored) with crown cap
13. Arrangements for direct microscopic count (DMC, see Practical
2)
Procedure
1. Grind 1 kg of barley malt into coarse particles. If barley malt is

not available, use 500 g of malt extract powder, in which case
mashing is not needed (steps 3 and 4 can be skipped).
2. Add warm water (4 L) to the grist to make a slurry with
temperature around 40°C.
3. Mix and stand the mixture for 15 min.
4. Separate the lower portion of the mash (thick mash) and bring to
boil. This portion should represent about 1/3rd of the total mash
volume.
5. Return the boiled mash to the main mash, mix, and allow it to
stand for another 15 min.
6. Repeat steps (4) and (5) until the temperature of the main mash
reaches 70°C.
7. Stand the mash for 15 min and then strain through muslin cloth.
8. Soak the residue in a minimal amount of hot water (~ 80°C) and
strain again to recover as much soluble matter as possible.
9. Add malt extract (optional).
10. Boil the extract for 1.5 hr. Add hops to the required degree of
bitterness (you can taste the wort!) during the latter part of the
boiling. Follow supplier’s guidelines for the hopping rate.
11. Cool the wort to ~ 25°C.
12. Add boiled (cooled) water to make 4-5 L.
13. Add sugar to make wort of ~ 14°brix.
14. Add brewer’s yeast and mix vigorously for 15 min. The pitching
rate should be 1 million cells/ml of wort/°brix. Alternatively,
use 0.5 g Active Dry Yeast (ADY) per liter of wort.
15. Carry out fermentation at ~ 25°C in a cotton-plugged Demijohn
for 8-10 days. Leave headspace of about 25% volume.
16. Follow the course of fermentation by measuring TSS, pH, and
cell number every 12 hrs.
17. Observe the high krausen stage (heavy foaming).
18. Note the flocculation properties of yeast.
62
19. After the fermentation has completed, separate the green beer
from the lees by siphoning into colored beer bottles.
20. Plug or cork the bottles and pasteurize at 65°C (internal
temperature) for 10 min.
21. Allow the bottles to cool and then store in refrigerator for 2
weeks.
22. Carbonate the cold beer with a carbonator (follow the
equipment manual) and crown-cork the bottle. If carbonator is
not available, simply crown-cork the beer (this will give still beer,
which obviously lacks the characteristic carbonic bite of the beer).
23. Carry out sensory and physicochemical analysis (color, taste,
smell, turbidity, acidity as lactic acid, alcohol content, and
specific gravity).
Observation
1. Hops characteristics
2. Observations made in mashing step
3. Flocculation characteristics of the yeast (top or bottom)
4. Time to reach high krausen
5. Kinetic data (decrease pH, increase in cell number, and decrease
in °brix, resulting from substrate utilization)

2. Alcohol content and acidity of beer
3. The sensory quality of beer
4. Graphical presentation of changes in °brix, pH and cell number
(add secondary axis for multiple plot)
Questions
1. What is ruh beer?

2. Define sugar pause and protein pause.
3. Why is wort boiling necessary?
4. What compounds are responsible for the bitter taste in beer?
5. What is malt?
6. What is proanthocyanidin-free barley?
7. What is chill-proofing?
8. What is diacetyl rest period?
63
9. What is lagering?
10. What are infusion- and decoction mashing?
11. What is diastatic power of malt?
12. What is degrees Lintner?
13. What is high-gravity brewing?
14. How do you define alcohol-free beer and low-alcohol beer?
15. How do you assess vigor and vitality of yeast cells?
Helpful diagrams
Fig. 11.1 Fresh hop cones
O O
R
HO OH
OH
α-acid
Fig. 11.2 Structure of α-acid
64
Section IV
AMYLOLYTIC STARTERS
Practical 12
PROPAGATION OF MURCHA CAKE
Background
Murcha is an amylolytic starter culture used in the preparation of

traditional alcoholic beverages like jand (undistilled) and raksi (distilled)
in Nepal and parts of North India. Microbiologically, murcha is a mixed
culture containing saccharifying / amylolytic molds (species of Mucor,
Rhizopus and Aspergillus), fermentative yeasts (mainly Saccharomyces
species) and lactic acid bacteria. Murcha preparation at a traditional level
involves the use of more than 40 wild plants, such as Polygala arillata, P.
abyssinica, Vernonia cinerea, Inula sp. Piper chaba, etc. After collection, the
plants are sundried, pounded into coarse powder and stored until actual
murcha preparation. During preparation, a requisite amount of plant
powder is taken, mixed with steeped cereals (usually rice) and pounded
into flour. A small amount of mother culture from the previous batch of
murcha is also added. A requisite amount of water is added and the
mixture is kneaded into stiff dough. The dough is then divided into
small lumps or patted into flat cakes. These are next placed on a bedding
of fern fronds, covered with fern fronds again, and incubated in a warm
place for 2-3 days.
The well-fermented cakes (or balls/lumps) become puffy and give a

sweet, alcoholic smell after incubation. Finally, the cakes are sundried to
a moisture content of about 14%, packed, and stored. Murcha has a shelf
life of about 1 year. Hence, murcha cake is a type of stock culture.
Plants used in murcha making serve as the source of yeasts, molds
and bacteria, all of which are indispensable for the fermentation of
starchy substrate. They also serve as a support material in the solid state
fermentation of murcha. Their role as a nutrient for microorganisms
during fermentation is negligible but they do play an important role in
the flavor quality of the beverage.
Objectives
1. To propagate murcha from selected murcha as the mother culture

2. To evaluate the quality of the prepared murcha
3. To study physicochemical properties of the prepared murcha
66
Principle
In laboratory, murcha can be prepared using either pure isolates of

essential microorganisms (yeasts and molds) or mother culture (murcha
of good quality). The need for lactic acid bacteria can be obviated by
using lactic acid or citric acid for maintaining the favorable pH. Cereals
such as broken rice, whole rice, maize, etc., can be used as the carrier
plus nutrient medium. A small amount of wheat bran can be used as the
support material.
Requirements
1. Rice (broken or whole, coarse variety)

2. Wheat bran
3. Mortar and pestle (or electric grinder/blender)
4. Fern leaves
5. Muslin cloth
6. Murcha cake (mother culture)
7. Plastic bags (reclosable)
8. Acidulated water (pH 3-3.5 with citric acid)
Procedure
1. Take 250 g of rice and 25 g of wheat bran.

2. Steep / soak the materials for 2-3 hrs in acidulated water (see
requirements).
3. Drain the water completely.
4. Spread the material to hasten removal of surface moisture
(important!).
5. Pulverize / grind to make medium-fine powder.
6. Take 10 g of mother culture (supplied murcha) and crush it into
powder.
7. Sprinkle the powder over the pulverized rice/bran and mix.
8. Add some water and knead to make a very stiff dough.
9. Line up fern fronds on a tray to make a thick bedding.
10. Divide the dough into small lumps/balls (~ 50 g). Flatten the
balls by patting into circular cakes (4-5 cm thick).
11. Place the cakes on a bedding of fern fronds (Fig. 12.1).
12. Cover the cakes with a blanket of fern fronds.
13. Cover the frond blanket with yet another blanket of moist muslin
cloth.
67
14. Incubate the cakes in a warm place for 2-3 days.
15. Observe daily for appearance of cottony mycelia.
16. Terminate the fermentation before spores (black, brown or
yellow dots) begin to appear.
17. Dry the cakes in the sun until about ~ 10-14% moisture content
18. Pack in polyethylene bags and store in refrigerator.
19. Test murcha quality as follows:
i. Cook ~ 50 g of rice (as you would do for bhaat).
ii. Cool and spread on a tray.
iii. Sprinkle ~ 0.25-0.5 g of murcha powder over the rice and
mix well.
iv. Transfer the mixture to a reclosable plastic bag (100 g
capacity) and incubate at 28-30°C for 1 week.
v. Observe for liquefaction and alcoholic smell.
vi. Taste a little to see if the product has the characteristic
taste of jand.
Observations
1. Appearance of the cakes / balls during fermentation (swollen,

puffy)
2. Smell of the cake
3. Presence of cottony mycelia
4. Alcoholic smell, sweet smell
5. Presence / absence of spores, spore color, etc.
6. Properties of dried murcha after drying
i. Tap murcha lightly with your forefinger and listen for
hollow sound.
ii. Take a small chunk and try crushing between the thumb
and the forefinger and see if it can be easily broken into
free-flowing grains.
1. Success or failure in preparing murcha, along with reasons

2. Brewing quality of murcha prepared (good, bad, satisfactory).
What can be done to improve the quality?
3. Physical quality of murcha cake you prepared (shape, size, sound
on tapping, breakability, etc.)
68
Questions
1. What is the purpose of using fern fronds in murcha making? Can

you use other materials or methods to replace fern fronds?
2. What is the purpose of using wheat bran in this experiment?
3. Why does the cake sound hollow?
4. Can you devise a method for determining bulk density of the
cakes (this becomes important in commercial production)?
5. Suggest a method for determining amylase activity of murcha
sample.
6. Why is surface drying of the steeped mixture important?
7. Ferns contain plethora of phylloplane microorganisms, both
desirable and undesirable. What sanitary measures can you adopt
to prevent contamination?
8. Can you suggest an improved method of murcha preparation?
Hint: pure culture, aseptic operation, control of fermentation
conditions, etc.
9. What do you know about similar starter cultures of Asian
countries? Hint: ragi, bubod, tape ketan, chu, etc.
Helpful drawings
Fern fronds
Murcha cake
Fig. 12.1 Murcha cakes spread on fern fronds for incubation
69
Practical 13
PREPARATION OF BRAN KOJI
Background
Koji is a Japanese term for fungal culture used as an inoculum for

solid-state fermentation. The Chinese counterpart for the word koji is
‘qu’, meaning bloom of mold. Koji is generally made by inoculating the
fungal culture (selected strain or strains) in either rice- or wheat bran,
which serves both as a carrier and a substrate. Cereals (rice, barley,
wheat) or soybeans may also be used as the substrate for specific koji
types. Fermentation is carried out under controlled condition
(temperature, RH, aeration) for a few days in shallow trays or rotating
drum fermenters. Most industrial molds grow well at 30-40°C, pH 5-6,
and RH above 90%.
In East Asia, koji is used as a source of extracellular hydrolytic

enzymes in the manufacture of soy sauce and rice wine. The most
important enzymes are amylases, proteases and cellulases. The
microorganisms of importance are Aspergillus oryzae, Rhizopus oryzae and
certain Mucor species.
Objectives
1. To prepare substrate for koji culture

2. To carry out solid-state fermentation
3. To preserve koji for future use
4. To test hydrolytic (liquefaction/saccharification) property of koji
Principle
Koji can be prepared by inoculating steam-sterilized wheat- or rice

bran with selected mold culture(s) and carrying out solid-state
fermentation for a few days under controlled conditions of temperature,
RH and aeration.
Requirements
1. Pure culture of amylolytic mold (e.g., Rhizopus oryzae) grown and

preserved in a Petri plate, from Practical 4.
70
2. Wheat bran – 250 g
3. Muslin cloth
4. Conical flask – 1000 ml capacity
5. Shallow trays
6. Electric dryer or solar dryer
7. Plastic bags or jars for packaging
8. Autoclave
9. Dilute citric acid solution (pH 5 to 6)
10. pH meter
11. Cooked rice
12. Beaker – 100-ml size
13. Aluminum foil
Procedure
1. Prepare 100 ml of dilute citric acid solution (pH 5-6).

2. Moisten (thoroughly, but not like a paste) 250 g of wheat bran
with citric acid solution.
3. Transfer the moist bran into 1000-ml conical flask, cotton plug,
and sterilize in autoclave at 121°C for 15 min.
4. Take out the flask and allow it to cool spontaneously.
5. Aseptically transfer a small amount of the sterilized bran to the
Petri plate containing the mold (see Fig. 13.1).
6. Vigorously jerk the plate to mix the mold with the bran.
7. Aseptically transfer the mixture to the main bulk of the sterilized
bran and shake well to affect mixing.
8. Incubate the culture at 30-35°C for 2-3 days until profuse
mycelial growth or incipient spore production occurs. Prevent
drying by placing water (in beaker) in the incubator.
9. After growth, transfer the contents to a shallow tray and dry at
45°C in an electric dryer until about 5-6% moisture. Cover the
tray with muslin cloth to prevent spreading of spores.
10. Collect the dried koji, pack in plastic bags or jars and store in
refrigerator.
11. Test the amylolytic property of koji as follows:
i. Take ~ 50 g of cooked rice and mix ~ 2 g of koji.
ii. Transfer the mixture to a clean beaker. The beaker must
be almost full.
iii. Cover the beaker by crimping an aluminum foil over the
mouth and incubate the whole at 30°C for 3-4 days.
71
iv. Observe daily for signs of liquefaction/saccharification.
Appearance of limpid liquid in the rice is an indication of
liquefaction and a sweet taste is an indication of
saccharification.
Observation
1. Abundance of spores and mycelia in the bran

2. Color of spores
3. Liquefaction/saccharification of cooked rice by the koji
1. Success or failure
2. Discussion on amylolytic property (or absence of it) observed in
the practical
Questions
1. What is the difference between liquefaction and saccharification?

2. What enzymes are involved in the hydrolysis of starch?
3. How do you differentiate between the hydrolysis due to
preformed enzymes (in the koji) and enzymes formed during
fermentation?
Helpful diagrams
Mold culture
Inoculation of Incubation
autoclaved, moist (3 days/35oC
bran (20% mc)
Electrical dryer Reclosable

(45oC until 5-6% mc) plastic pack
(for storage)
Fig. 13.1 Outline of bran koji preparation
72
Practical 14
PREPARATION OF MURCHA USING PURE CULTURES
Background
Description of murcha as an amylolyic starter cake has already been

given in Practical 12. Traditional murcha is a mixed flora of a large
number of microorganisms, some of which may be undesirable. Use of
pure culture for the preparation amylolytic starters (except murcha) in
semi-commercial and commercial scale has been described by many
authors. Thus, it is possible to apply similar approach to prepare
Nepalese murcha.
The quality of murcha is contingent on many factors, first and

foremost being the properties of microflora that the murcha cake
harbors. The next important point to consider is the ratio of amylolytic
molds to fermentative yeasts. It has been found that murcha with higher
mold to yeast ratios lead to faster solid-state fermentations, for example,
jand fermentation. This is because the biomass build-up time is greatly
shortened. The type of amylase elaborated by the mold is also important.
Higher α-amylase activity causes faster liquefaction (but the mash may
or may not be sweet) whereas higher β-amylase activity produces a
sweeter mash.
Objectives
1. To prepare pure cultures for murcha making

2. To prepare substrate-cum-carrier for murcha
3. To carry out test fermentation using laboratory murcha
4. To study physicochemical properties of the prepared murcha
Principle
Pure cultures of amylolytic molds and fermentative yeasts are

inoculated in cereal flour, kneaded (with added water) and shaped into
cakes (or balls). The cakes (or balls) are then incubated for 2-3 days at
30-35°C to obtain green murcha, which are later dried to 10-14%
moisture content at ~ 45°C and packed in plastic bags.
73
Requirements
1. Rice – 600 g
2. Yeast broth – You are going to use your own yeast, so refer to
Practical 5
3. Amylolytic mold – You are going to use your own mold, so refer
to Practical 4. It is better to use koji that you have prepared and
tested in Practical 13
4. Mortar-pestle and electric grinder
5. Nylon mesh
6. Acidulated water (pH 3-3.5 with citric acid) – 1 L
7. Distilled water
8. Petri plate
9. Muslin cloth
10. Incubator
11. Electric dryer or solar dyer
12. Plastic bags
13. Meshed rack (or a porous tray)
14. Weighing arrangement
15. Standard utensil (pots, bowls, etc.)
17. pH meter
18. Thermometer
19. Marker pen (0.5 mm)
Procedure
1. Soak 500 g rice in ~ 1 L of warm (40-45°C) acidulated water (see

requirements) for 2-3 hrs. The acidic environment maintained
during soaking discourages/inhibits the growth of contaminants.
2. At the end of the soak period, pressure-rub between your thumb
and forefinger a few grains of rice. If it crumbles into powder,
the soaking is complete.
3. Drain the soak-water through the nylon mesh, as completely as
possible.
4. Pulverize the soaked rice in mortar-pestle into course powder.
5. Transfer this relatively dry powder to electric grinder and finish off
the milling process. Note that the flour should not be too fine.
Finally, transfer the flour to a bowl for mixing and kneading.
6. Add 10 g koji and 5 ml yeast slurry to the flour, add distilled
water, and begin kneading. For every 100 g of moist flour, you
74
may need ~ 30-35 ml of water (this is for guidance only). The
dough must be stiff, yet easily moldable into cakes or balls.
7. Take a piece of muslin cloth and place over a Petri plate to make
an internal lining (the Petri plate is for molding purpose only).
8. Divide the dough and place it on the lined Petri plate. Press
gently to obtain a cake bearing the perfect circular shape.
9. On a meshed tray spread muslin cloth for receiving the formed
cake. See Fig. 14.1 for an outline of the method.
10. Transfer the molded cake to the meshed tray by carefully
inverting and releasing the cake from the plate (first gently
remove the plate and then then retrieve the muslin piece).
11. Prepare cakes similarly from the rest of the dough.
12. Cover the cakes with muslin cloth and incubate at 30°C for 2
days. Cottony mycelial growth will have occurred by now.
13. Take out the cakes and dry in an electric dryer (sun-drying may
also be done but there is risk of contamination by airborne
spores) at 45°C until 10-14% moisture content.
14. Pack the cakes in a polythene bag and store in the refrigerator
15. For testing the quality, follow the procedure given in Practical
12.
16. Determine pH on 10% slurry (aqueous) as follows:
i. Weigh 10 g murcha and make powder.
ii. Transfer to a volumetric flask and make 100 ml with
distilled water.
iii. Mix well and determine pH with a calibrated pH meter
(sensitivity ± 0.1 unit).
17. Determine bulk density (g/L) as follows (see Fig. 14.2 also):
i. Choose a beaker with diameter slightly greater than that
of the cakes.
ii. Select 3-4 cakes and take their compounded weight (g),
say W.
iii. Put some mustard seeds in the beaker, about the
thickness of the murcha cake.
iv. Lay down murcha cake over the seeds and press it a little
so that about half the cake is immersed.
v. Add more mustard seeds over the cake until it is fully
covered. Add a little excess as in step (iii).
vi. Follow steps (iv) and (iii) until all the cakes have been
covered.
75
vii. Jerk the beaker to level off the top surface (of the seed
layer). Make sure that the cakes do not come out and
there are no spaces between the cakes.
viii. Mark the level of the top surface on the outside of the
beaker with a marker pen.
ix. Take the cakes out carefully, without spilling the seeds.
Alternatively, empty the beaker and refill it with the same
seed and level off the top surface.
x. Mark the level of the seed again with a marker pen.
xi. Empty the beaker and fill it with water up to the lower
mark.
xii. Record the volume of water needed, say V1 L.
xiii. Add more water up to the second mark (upper mark)
and similarly record the volume, say V2 L.
xiv. Calculate the bulk density as follows:
W
Bulk density = g/L
V2 − V1
18. Determine the moisture content of the murcha cake by routine
hot-air oven method or IR moisture meter. In either case, follow
your instructor’s directions. A general process for hot-air oven
method is as follows:
Hot-air oven method
i. Turn on the hot-air oven so that it reaches a temperature

of around 100°C.
ii. Take a piece of murcha sample and grind it into powder.
Weigh 10 g of the pulverized murcha in a tared Petri dish
by difference. Note that you clean, dry and tare the Petri
dish beforehand.
iii. Dry the sample in the hot-air oven at 105 ± 5°C until
constant weight. It may take 2-3 hrs and you may weigh
the sample every 45 min or so. You must cool the sample in
the desiccator before weighing (see Fig. 14.5).
iv. Once you get the constant weight (tolerance of ± 5 mg),
calculate the % moisture content as follows:
W − WF
Moisture (%) = I × 100
WI
Where, WI = initial weight of murcha (g), WF = final
weight (after drying) of murcha (g).
76
IR method
IR method depends on make of the equipment. The method for

a simple equipment as shown in Fig. 14.3 is as follows:
i. Turn on the equipment (follow the manual and your

instructor’s direction).
ii. Align the red needle (pointer) and “100” of the
graduated scale with the reference mark. You can rotate
both the knobs (to your right and left) for this. See Fig.
14.4.
iii. Turn off the equipment and rotate the knob (to your
right) to align the zero mark of the scale to the reference
mark. Note that the directions of the movements of scale and knob
are opposite.
iv. Open the cover of the equipment and begin slowly
adding murcha powder in the pan until the red needle
aligns with the reference mark (the zero mark of the
scale has already been aligned). Make sure you add the
sample on all sides. Heaping on one side only will tilt the pan!
v. With a spatula, spread out the sample uniformly so that
the pan is free and balanced.
vi. Close the cover, turn on the lamp (medium intensity)
and watch the red needle gradually move up.
vii. Manipulate the right knob (don’t use the left knob!)
occasionally to bring back (down) the red needle to the
reference mark (original position).
viii. Record the reading when the needle remains stagnant
(does not move) for at least 5 min. This gives the %
moisture content of the sample.
Sometimes, due to sample size constraints, the quantity of

sample will not be adequate enough to make 100 units of the
scale. In such cases, you will have to make some manipulations
in calculation.
For example, while adding samples to the pan, your sample

quantity could only be aligned with 45 units of the scale. This
means that the sample quantity is (100 – 45) units = 55 units.
After IR-drying, the needle could be aligned at 75 units of the
77
scale. This means that the moisture removed is (100 – 75) units
= 25 units.
The moisture content (%) is therefore: (25/55)×100%

= 45.45%
Observation
1. Same as in Practical 12
2. Average weight of murcha cake
3. Average volume of each murcha cake
4. Moisture content data
1. Same as in Practical 12
2. Bulk density
3. Moisture content
Questions
1. Why was mustard seed used for the determination of bulk

density? Can you suggest an alternative material or method?
2. Why do you think meshed rack was used for the incubation?
3. Do the pre-formed enzymes (amylases) have relevance in jand
fermentation?
4. What types of amylases are produced by molds?
5. Which type of rice do you think is better: glutinous or non-
glutinous? New stock or old stock? Why?
6. How do you judge/assess the quality of murcha as a (i) producer,
and (ii) customer?
7. What will be the effect variation in the amount of koji and yeast
slurry on the quality of murcha?
8. What is the shelf life of murcha? Do you know about insect
infestation of murcha? Does this have relevance to Sanitary and
Phytosanitary measures (SPS)?
78
Helpful diagrams
Soaking of rice in
acidulated water
(45oC/~3 hrs) Slant culture
Grinding Broth culture
Water
Kneading Koji
(43% mc)
Dough
Muslin lining
Petri plate
Meshed rack
Cake
Incubation
Drying
Murcha
Fig. 14.1 Outline of murcha making using pure culture
Beaker
Mustard seed
Murcha cake
Fig. 14.2 Determination of bulk density
79
Movable cover
Calibrated wheel
Filter glass and pointer
IR lamp
Plate and
sample
Adjustment
knob
Power
switch
Fig. 14.3 IR moisture meter
Reference Reference
mark Red mark
Red 100 needle
needle Graduated 100
95
scale Graduated
95 scale
90
90
Before alignment After alignment
Fig. 14.4 Alignment of IR moisture meter scale
Desiccator
Plate with
sample
Silica gel
Fig. 14.5 Cooling in a desiccator
80
Section V
TRADITIONAL FERMENTED FOODS

Practical 15
PREPARATION OF MILLET JAND
Background
Jand (also spelt jaand, jnaar or njard) is a traditional, sour-sweet

alcoholic beverage indigenous to Nepal and some parts of Northern
India. The product is based on solid-state fermentation of starchy
materials like cereals and starchy roots and tubers by the use of
amylolytic starter culture called murcha. Murcha contains amylolytic
molds, fermenting yeasts, and acidifying lactic acid bacteria, all of which
are essential to the fermentation.
Finger-millet (Eleusine coracana L. Gaertn), locally called kodo, is the

material of choice for jand preparation. Traditionally, jand fermentation is
carried out in earthen pots (called ghyampo) but these days, plastic
containers are being increasingly used for the same.
Jand is intimately associated with cultures and traditions of many

ethnic groups of Nepal. For kiranti people (a nationality of Nepal), jand
(undistilled) and raksi (distilled, congeneric product from jand, similar to
whisky) are indispensable ritual items. Small wonder, we find today
renewed attention from the scientific community in researches on varied
aspects of jand and raksi.
The term jand is often used interchangeably to mean both the

fermented mash and the strained, cloudy, aqueous liquor. Tongba is
another variation of serving jand, where the fermented mash is
soaked/steeped in a barrel-type container called dhungro and the extract
sucked in through a small pipe called peepa (see Fig. 15.1).
Objectives
1. To prepare finger-millet for fermentation

2. To carry out primary fermentation (biomass build-up)
3. To carry out anaerobic fermentation
4. To carry out physicochemical tests of jand
5. To carry out sensory analysis of jand
82
Principle
The actions of essential organisms (yeasts, molds and bacteria) from

murcha on cooked millet occur in a sequence. Amylolytic molds (and
sometimes bacteria) are the first to act. They produce extracellular
amylases and convert starch into simple sugars. Thereafter the action of
yeasts and bacteria follow. Yeasts convert simple sugars into alcohol and
other congeneric metabolites. Bacteria produce organic acids and lower
the pH, thereby creating favorable environment for the growth and
metabolism of yeast. In the latter phase of fermentation, saccharification
and alcohol fermentation proceeds simultaneously, leading to parallel
fermentation. The entire fermentation is essentially a solid-state culture.
Since the size of inoculum used is usually small, a period of biomass
build up is necessary before anaerobic fermentation is carried out. The
anaerobic fermentation is carried out for few weeks to several months.
Prolonged fermentation leads to accumulation of a limpid, alcoholic
extract called nigaar.
Requirements
1. Finger-millet (~ 2 kg)
2. Murcha cake
3. Cooking arrangement (e.g., gas stove)
4. Cooking pot, tray, trough/tub, etc.
5. Plastic or glass jar (~ 5 kg capacity)
6. Muslin cloth
7. Plastic sheet
8. Strainer (plastic mesh bag)
9. Beakers (250- and 500 ml)
10. Equipment / instruments for the determination of alcohol
content (pycnometric method, see Practical 7)
11. Titration arrangement (for acidity)
12. pH meter
13. Standard NaOH, 0.1N
14. Phenolphthalein indicator
15. Ca(OH)2 ~ 2N
Procedure
1. Clean finger-millet (remove husk, immature seeds, stones and

other debris). The loose husk can be removed by winnowing and
83
the attached husk can be removed by pounding in a mortar-and-
pestle after moistening the seeds. Stones can be easily removed
by repeated gravity settling during washing.
2. Cook finger-millet (just as you would cook rice) until well-
cooked. Adjustment of the amount of water is critical.
3. Spread the cooked millet on a shallow tray to cool to ~ 30°C.
4. Sprinkle about 50 g of murcha powder and mix well (the quantity
of murcha may vary depending on the season; you will require
smaller quantity in the summer).
5. Pull moist muslin cloth over the edges to cover the tray. Tighten
the cloth with a string to keep away drosophila and flies.
6. Incubate in a warm place for 2 days for biomass build-up (until
cottony mycelial growth is evident).
7. Transfer the contents to a narrow-necked jar of appropriate size
(you can also use pickle jar). Press with palms in-between to
compress the mass. The container must be almost full when
packed.
8. Pull a plastic cover over the edges, secure it with a rubber band
and ferment at room temperature (~ 25-28°C) for at least 20
days.
9. Carry out sensory evaluation of strained jand as follows:
i. Take ~ 1 kg of fermented mash and add 1 L of water.
ii. Leach out the extracts by squeezing between hands.
iii. Strain the turbid extract and drink.
iv. Note down your feeling regarding color, taste, smell,
body, etc.
10. Carry out sensory evaluation of tongba as follows:
i. Take a 500-ml beaker half-filled with lukewarm water.
ii. Add fermented mash to the beaker until nearly full.
iii. Steep the mash for 10-20 min.
iv. With the help of peepa suck in the extract and swallow it a
couple of times.
v. Note down your feeling regarding taste, body, etc.
11. Determine alcohol content as follows:
i. Take 200 g of well-mixed mash and squeeze using 200
ml of water.
ii. Neutralize the slurry with Ca(OH)2 solution. You can
use phenolphthalein or universal indicator for the test.
iii. Distill to obtain about 90 ml of distillate. Use heating
mantle (not burner).
84
iv. Make up the volume to 100 ml with distilled water.
v. Determine apparent specific gravity and alcohol content
by pycnometric method as described in Practical 7. You
should divide the tabulated result by 2 to get the true
value (because you have taken 200 g to get 100 ml of
distillate).
12. Determine acidity as follows:
i. Take 10 g of well-mixed mash.
ii. Grind the mash into fine slurry in mortar and pestle.
Add a small amount of water to assist grinding.
iii. Add about 25 ml of water and mix well.
iv. Titrate the suspension against 0.1N NaOH using
phenolphthalein indicator and calculate acidity using
following expression:
Titer (ml) × N of NaOH × 9

Acidity (as % lactic acid) =
Wt of sample (g)
Observation
1. Appearance of jand mash (e.g., discrete or mushy, presence of

nigaar, etc.)
2. Sensory attributes (smell, taste and after-taste, astringency/
sweetness/sourness, cloudiness of strained jand, body and taste
of tongba as observed when sucked in, etc.)
1. Success or failure of the experiment (along with plausible

reasons)
2. Summary of observed data and explanation
3. Alcohol content
4. Acidity
Questions
1. What is the purpose of aerobic fermentation (1st stage

fermentation) in jand fermentation?
2. Why is millet preferred for jand making?
3. Why is finger-millet called poor man’s cereal?
4. Can you make tongba using other materials, e.g., rice? Why?
85
5. What are the nutritional benefits and adverse health implications
of jand ? Hints: Jand contains fibers and resistant starch. Jand also
contains yeasts and the associated problems arising from their
nucleic acids. Finger-millet does not contain gluten.
6. Jand is considered a category of beer (and not wine) why? What
is the difference between nigaar and the Japanese wine sake?
7. Name some commercial fermented cereal beers similar to jand?
8. What are the drawbacks in traditional jand making process? How
can you improve it to produce consistent quality?
9. Do you think the ratio of population of mold to yeast in murcha
have influence on the quality of jand? Why?
10. Drinking of jand by people with gout problem is not advisable,
why?
11. How is raksi produced?
Helpful diagrams
"Peepa" for
sucking the extract
Lid
Metal brace
"Dhungro" (barrel)
(~ 1/2 kg capacity)
Fig. 15.1 A typical tongba
86
Practical 16
PREPARATION OF SAUERKRAUT
Background
Most horticulture products can be preserved by lactic acid

fermentation. In the occident, the most important commercially are
cabbage, cucumbers and olives. Fermented vegetables, commonly
cabbage, in Korea is known as kimchi. The two most common lactic-
fermented vegetable products of Nepal are gundruk and sinki. Edible
bamboo shoot that has undergone similar lactic fermentation is called
mesu or tama.
Sauerkraut, literally meaning sour cabbage is defined as a clean, sound

product of characteristic flavor, obtained by full fermentation, chiefly
lactic, of properly prepared and shredded cabbage in the presence of not
less than 2%, nor more than 3% salt. It contains, upon completion of
fermentation, not less than 1.5% of acid, expressed as lactic acid.
Sauerkraut is traditionally served with pork, sausages, bacon and

similar meat dishes. It can also be used as condiments and in soups.
The commercial production of sauerkraut is technically simple but

involves some interesting and complex chemistry and biochemistry.
Commercial sauerkraut production uses special cabbage cultivars. In
essence, the production of sauerkraut involves removal of outer leaves,
decoring, shredding into slaw, salting (2-3%, w/w), and fermenting in a
vat at around 20°C for few days.
The microbiology of sauerkraut fermentation is very complex.

Although commercial cultures for sauerkraut fermentation are available,
they are used less often than in other food fermentations. Generally,
fermentation occurs spontaneously by natural microflora of the cabbage.
The fermentation is initiated by Leuconostoc mesenteroides, which is among
the less acid-and-salt tolerant heterofermenting lactic acid bacteria (LAB)
but grows the fastest during the early stages. As the pH drops due to
acid production, Leuconostoc is inhibited and replaced, first by Lactobacillus
brevis, and then by the homofermentative Lactobacillus plantarum. Acid
accumulation continues in the form of lactic acid although the pH
stabilizes at somewhere around 3.6 (the pKa of lactic acid) because of
weak dissociating property of this organic acid.
87
Objectives
1. To prepare sauerkraut by spontaneous fermentation

2. To observe the change in microbial profile by microscopic
examination
3. To carry out physicochemical and sensory analysis
Principle
Sauerkraut production is essentially a spontaneous, lactic-acid

fermentation of cabbage. When shredded cabbage is salted and packed
in a partially aerobic environment, the native lactic acid bacteria are
encouraged to grow in succession, heterofermentative followed by
homofermentative. Although the fermentation is initiated by Leuconostoc
mesenteroides, the bacterium that is to be found at the end is Lactobacillus
plantarum. The optimum temperature of fermentation is 18-20°C. The
organism utilizes sugar and other components of cabbage for growth
and metabolism thereby producing a highly appetizing sour end product.
The product is perishable but can be preserved by pasteurization.
Requirements
1. Sound cabbage
2. Salt
3. Shredding equipment
4. Transparent jars - 2 kg capacity
5. Polythene bags
7. pH meter
8. Microscope
9. Arrangement for acidity determination (titrimetric)
10. 100-ml beakers - 4 nos.
Procedure
1. Remove the outer leaves of cabbage.

2. Trim and decore the cabbage.
3. Shred the cabbage into 2-3 mm thickness to produce thin, long
strands.
4. Add salt at the rate of 2.5% (w/w) and mix uniformly.
88
5. Fill in clean jars. Use pressure with hands to make it slightly
compact. Leave ~ 1/5th of the jar for headspace.
6. Cover the jar with polythene bag (Fig. 16.1). Press the bag to fit
in the headspace.
7. Pour 10% brine over the polythene bag to make a brine seal.
8. Tie the overhanging edges of the polythene bag to the neck of
the jar.
9. Prepare sauerkraut similarly in four 100-ml beakers.
10. Leave the preparation for spontaneous fermentation for about
10 days under ambient condition (25-30°C).
11. Analyze the contents of beakers every second day for sensory
properties (crispness, sourness, color), pH, acidity (as % lactic
acid), and microscopic details (cell morphology, population, etc.)
as follows:
Acidity: Take 10 g sample → Grind into paste in mortar-and-

pestle → Transfer the contents quantitatively to conical flask
with ~ 25 ml of water → Titrate with 0.1N NaOH using
phenolphthalein indicator → Express acidity as % lactic acid
using following expression:
Titer (ml) × N of NaOH × 9

% Acidity =
Wt. of sample (g)
pH: Extract juice of the sample and immerse pH meter → Note

the reading.
Microscopy: Carry out negative staining (see Practical 1) of a

loopful of the extracted juice. Note the cell morphology and cell
density in the field (sparse, dense, very dense, etc.).
Sensory analysis: Bite a few shreds of sauerkraut → Note down the

feeling (crispy, rubbery, tart, flat, etc.).
12. Prepare a compound graph of acidy and pH against time (days).

You can use MS Excel Add-in for preparing the graph (your
instructor will help you).
89
Observation
1. Changes in morphology and population of bacteria over time

2. Color, flavor, acidity, pH, spoilage, etc., of sauerkraut
3. Any abnormalities (discoloration, contamination, etc.)

2. The sensory quality of sauerkraut
3. Changes in microbial profile (from microscopic examination)
4. Changes in acidity and pH
5. Summary of observed data and explanation
Questions
1. Compare and contrast sauerkraut with kimchi.

2. What is the lowest pH (and the highest acidity) you can obtain in
sauerkraut fermentation?
3. Can you increase acidity by adding sugar?
4. How is lactic acid formed?
5. What are the purposes of salt addition in sauerkraut fermentation?
Helpful diagrams
Slaw (shredded cabbage)

Brine
Plastic cover
Fermentation tank
Fig. 16.1 Commercial sauerkraut fermentation tank
90
Practical 17
PREPARATION OF KINEMA
Background
Kinema is an indigenous bacterial fermented soybean food

commonly consumed as side-dish in the East hills of Nepal and parts of
North hills of India. It is traditionally prepared by fermenting boiled
soybeans under warm condition for 2-3 days. The resulting product,
which is ammoniacal in smell and stringy/mucilaginous in appearance, is
either consumed fresh as curry or sundried for storage. Fresh kinema has
shelf-life of only a day or two.
Kinema is mostly prepared by Limbus (an ethnic group of Nepal).

The traditional production, in essence, involves: cooking of soybean,
draining, light crushing, mixing with a small amount of firewood ash,
wrapping in banana or other leaves, and fermenting near fireplace for 2-
3 days.
It has been observed that details of traditional kinema production

method differ slightly, depending on the availability of raw material,
region and community involved. Black soybean (Nepali bhatmas) is
generally preferred but other soybeans can also be used. Some
communities prepare kinema from roasted and ground soybeans.
The essential microorganisms come from the leaves (used for

wrapping) and the environment. Researches have shown that the
traditionally produced kinema contains mixed cultures of bacteria, molds
and yeasts, viz., Bacillus subtilis, Enterococcus faecium, Candida parapsilosis, and
Geotrichum candidum. Bacillus subtilis is the dominant and the most
important kinema organism. Although the traditional kinema production
relies on spontaneous fermentation recent efforts on standardization of
the process has shown considerable promise, for instance, study on the
preparation of pulverized kinema starter.
Kinema resembles natto, a similar fermented soybean product of

Japan. Natto is prepared by using Bacillus natto (a strain of Bacillus subtilis).
Studies have revealed that fermentation markedly improves the
nutritional value of soybean. Proteins, carbohydrates and lipids are
broken down to simple and readily assimilable forms. There is an
increase in amounts of water-soluble vitamins.
91
Objectives
1. To prepare kinema using good quality kinema as the starter

culture
2. To carry out physicochemical and sensory analysis of kinema
Principle
Kinema can be prepared in the laboratory by using starter culture

instead of depending on spontaneous fermentation: this produces kinema
of consistent quality. Instead of banana leaves (or other leaves) paper
bags may be used to wrap/contain the beans for fermentation.
Temperature and humidity can be easily controlled by carrying out
fermentation under controlled condition in an incubator. The essential
steps are: boiling soybean until cooked → draining completely → light
crushing → mixing with 0.5% of good quality dried kinema starter →
loose wrapping in paper bags → incubating at 40°C for 2 days → fresh
kinema → sun-drying → dried kinema.
Requirements
1. Soybean (black or white) – 1 kg

2. Cooking utensil
4. Mortar-pestle
5. Paper bags
6. Incubator
7. Reclosable plastic bags
8. pH meter
Procedure
1. Soak soybeans for 3-4 hrs. See Fig. 17.1 for simplified scheme
for the preparation of kinema in the laboratory.
2. Cook or steam until fully cooked.
3. Drain away the excess water (make sure that the beans are
almost dry).
4. Crush the beans lightly in mortar and pestle. Do not be tempted
to make it a paste. This decreases surface area and interferes with
the highly aerobic nature of the fermentation.
5. Lightly pack in paper bags.
92
6. Incubate at 40°C for 2 days.
7. Take out the product and make observations.
8. Measure pH on 10% aqueous solution (grind and macerate 1 g
kinema in 10 ml water).
9. Dry in the sun until dry.
10. Pack in reclosable plastic bags and store.
11. Carry out sensory analysis (dried kinema).
Observation
1. Smell (ammoniacal, nutty, etc.)

2. Appearance (sticky, stringy/ropy, etc.)
3. Taste (meaty, revolting, tasty, etc.) of the dried kinema grains
4. pH of the product

2. The sensory quality of kinema
3. pH of the product and explanation
Questions
1. What is the contribution of firewood ash used in traditional

fermentation?
2. Why is kinema fermentation called an alkaline fermentation?
3. What do you know about natto kinase?
4. The protein content of kinema (on dry basis) is higher than the
protein content of the soybean from which it was prepared,
why?
5. Kinema is a product resulting from incomplete microbial oxidation.
Explain
6. Any food containing microbial load greater than 108 cfu/ml (or
g) can be regarded as spoiled. What view do you hold as regards
kinema?
7. In the light modern understanding, what is the compound
responsible for stringiness/ropiness in kinema?
8. Many researchers claim that fermented soybean products like
kinema can help solve food insecurity (in terms of protein
malnutrition). How is this possible?
9. How do you relate kinema with Japanese natto (itihiko)?
93
Sobeans
Bowl Soaking
Cooker Cooking
Good kinema
(mother culture)
Strainer Draining Paper
~ 0.5% bag
Fermentation
Mixing (~ 2 days, warm place)
Water
Crushing
Fresh kinema
Drying
Dried kinema
Fig. 17.1 Simplified diagram for laboratory preparation of kinema
94
Section VI
MISCELLANEOUS PRACTICALS
Practical 18
COMPARISON OF BAKER’S YEAST ACTIVITY
Background
Baker’s yeast is used in bakery for two main purposes: (i) leavening,
and (ii) flavor. Leavening power of baker’s yeast (also called baker’s yeast
activity or gassing power) is the most important test reflecting the
performance of yeast in a bakery operation.
There are several tests proposed by different investigators (White,

1954; Schultz et al., 1942; Peppler, 1972; Brummer, 1977; Borzani, 2004).
However, each of these methods has its own advantages and limitations,
which in turn affect the accuracy and reproducibility of the test.
Preparation of dough and baking under standard conditions to

measure the volume of the bread may appear to be an attractive
proposition but the laboratory results, which are typically based on one or
several simple bake tests, cannot adequately reflect all the variations that
occur in baker’s processes. The bakers have a wide range of processes to
choose from (bread from straight doughs, sponge doughs, lean doughs,
sweet doughs, etc.).
A very simple estimate of the yeast activity can be carried by

preparing dough and measuring either (a) time required by the dough to
proof to a given height, or, (b) volume of the bread for given proof time.
Generally, proof times are between 45 and 60 min. According to
Borzani (2004), the gassing power of yeast is preferably calculated by
Eq. 18.1.
V − Vo ΔV
P= = ………….. (18.1)
m ×t p m ×t p
where,
P = gassing power of the yeast; V = dough volume corresponding

to an incubation proof time tp, Vo = initial volume of the dough, m
= mass (dry matter) of the yeast used in the test
Another approach suggested for the determination of activity of

baker’s yeast is based on the measurement of CO2 evolved in a given
96
time period. This can be done in solutions of various sugars in a simple
fermentometer. The gas is determined by bubbling the gas through an
alkaline solution followed by back titration. For more accurate results, it
is advisable to measure CO2 evolution in actual doughs. In such cases,
suitable instruments may be used to give automatic measurements. The
results of baking tests are often expressed in terms of proof minutes,
and the results of gassing power tests in terms of ml of CO2 evolved.
Objectives
1. To carry out thin dough fermentation

2. To derive empirical equations for plots of dough volume vs
incubation time
3. To calculate gassing power
4. To carry out statistical test for the comparison of yeast activity
Principle
Based on work by Borzani (2004), comparison of gassing powers of

compressed baker’s yeasts can be carried out by incubating for ~ 90 min
a specified volume of thin doughs inoculated with standard amount of
yeast. The recommended incubation temperature is 30-31°C. For active
dry yeast (ADY) a slight modification can be done in which the yeast is
rehydrated in lukewarm water for about 5 min. Graphs of dough
volumes vs incubation times can be plotted and equation fitted. The
gassing power can be calculated by the use of Eq. 18.1. The statistical
comparison of yeast activity can be carried out by paired t-test (for two
samples) or ANOVA (for 3 or more samples). MS-Excel or other
dedicated statistical package can be used for the statistical part.
Requirements
1. Distilled water
2. 100-ml graduated measuring cylinder
3. 250-ml Erlenmeyer flask (conical flask)
4. 500-ml beaker
5. Incubator
6. Wheat flour
7. Hot air oven or IR moisture meter for determining moisture
content of yeast
8. Active dry yeast (2 types for comparison)
97
9. Computer (for statistical analysis and graphing)
Procedure
1. Take the two types of ADY and note down the details
(producers, production date, etc.).
2. Set separate sets of experiment for two different types of yeasts.
3. To a 250-ml conical flask containing 120 ml of lukewarm (about
35°C) distilled water, add 0.3 g of active dry yeast.
4. Agitate the flask for 5 min to disperse the yeast cells.
5. Add the yeast suspension slowly to a 500-ml beaker containing
120 g of wheat flour while stirring the mixture for 5 min to
obtain a thin dough.
6. Transfer 50 ml of the dough to 100-ml glass measuring cylinder,
without touching the walls. To facilitate the transfer, you can
first transfer the thin dough to a plastic bag and then squeeze the
dough through a small hole.
7. Incubate the system at 30°C and measure the volume of the
dough at time intervals of 15-30 min for up to 90 min.
8. Plot the graph of time (hrs) vs volume (ml). See Tables 18.1 and
18.2, and Figs. 18.1 and 18.2 in the sample calculation section.
Note that data are hypothetical.
9. Calculate the gassing rates for each measurement of volume.
Plot a graph of incubation time vs gassing power
10. Carry out paired t- test of data to compare gassing power of the
two ADYs
Sample calculation
Table 18.1 Hypothetical test data for yeast types ADY-I and ADY-II
Incubation Dough volume (ml) for Dough volume (ml) for
time (hr) ADY-I ADY-II
0 54 50
0.25 55 51
0.5 58 56
0.75 63 64
1 72 73
1.25 83 85
1.5 96 100
98
Table 18.2 Gassing powers yeasts based on Table 18.1 and Eq. 18.1
Gassing power P (ml/g/hr)
ADY-I ADY-II
13.75 13.75
27.49 41.24
41.24 64.15
61.86 79.04
79.73 68.73
96.22 114.55
Please note that the moisture content of yeast has been taken as 7%
in this calculation. This means that 0.3 g ADY taken = 0.291 g dry yeast
mass.
ADY-I ADY-II
100
90
Volume (ml)
80
70
60
50
40
0 0.25 0.5 0.75 1 1.25 1.5 1.75
Time (hrs)
Fig. 18.1 Hypothetical scattered plot of volume vs time based on Table 8.1
Fig. 18.2 Result obtained by using Excel add-in Analysis Toolpak for
paired t-test. Data given in Table 8.2 were used.
99
From the statistical output (Fig. 18.2) it can be concluded that there
is no significant difference in gassing powers of two yeast types ADY-I
and ADY-II.
Observations
1. Rise in volume of the thin dough every 15 min of incubation at 30°C

2. Any interesting observation (gas bubbles, air pocket, etc.)
1. Graphical comparison of the yeast activity (see Fig. 18.1)

2. Tabulated results of gassing power
3. Statistical comparison (paired t-test) of gassing power
4. Conclusion (also relate this to age of yeast, manufacturing company,
integrity of the package, storage condition, expiry date, etc.)
Questions
1. Based on Eq. 18.1, what is the unit of gassing activity?

2. Is the increase in volume in sponge dough similar to that of thin
dough? Why?
3. What is proofing?
4. What is gluten window?
5. What is the shelf-life of ADY? What is the basis of its
calculation?
Helpful diagrams
100 100
90 100-ml measuring 90
80
cylinder 80
70 70
60 60
50 50
40 40
30 30
20 20
10
Thin dough 10
Initial state Final state
Fig. 18.3 Experimental set up for comparison of baker’s yeast activity

100
Practical 19
PREPARATION OF RESTRUCTURED FRUIT ALGINATE
Background
Alginate is a structural carbohydrate found in marine brown algae

(Phaeophyceae) (see Fig. 19.2 for the biosynthesis of alginic acid by
Azotobacter vinelandii). Alginates of microbial origin are industrially
produced by using Azotobacter species (soil bacteria). Chemically, alginate
is a family of unbranched binary copolymers (see Fig. 19.1) made up of
blocks of (1→4) β-D-mannuronic acid (M-block) and α-L-guluronic acid
(G-block).
Alginate has been used in a wide variety of areas including, but not
limited to, the food, pharmaceutical, biomedical, personal care, water
treatment, and textile industries. Alginate is a popular ingredient for
food processors because of its many unique colloidal properties,
including thickening, stabilizing, suspending, film forming, gel producing
and emulsion stabilizing. While alginate does not provide calories to the
body, as a soluble fiber, it can influence digestion.
One of the popular uses of alginate in food industries is as calcium

alginate gels for preparing restructured food products. These are
products in which pieces of foodstuffs are bound together to make it
resemble the original product. Onion rings, meat products, pimento
olive fillings, crabsticks, and cocktail berries are examples of alginate-
based restructured products. Recently, there has been increasing interest
in the use of alginate in the field of molecular gastronomy, e.g., for
spherification of juices.
Objectives
1. To produce restructured fruit alginate (globs and cubes)

2. To carry out sensory analysis
Principle
Alginate is able to form gels in the presence of certain polyvalent

metal cations. Calcium is commonly used to gel alginate although
barium, strontium, cobalt, zinc, etc., have also been used. Since the
cations preferentially bind to the carboxyl groups in the G-blocks the
101
proportion of guluronic and mannuronic acid in the alginate significantly
affects the gel property. Higher amounts of guluronic acid produce
strong, brittle and heat-stable gels whereas higher amounts of
mannuronic acid produce weaker, elastic and less heat-stable gel. The
amount of monovalent salts in solution, the solution temperature,
degree of polymerization and the polyvalent ion itself will influence the
behavior of the reacted alginate.
The toughening of the skin of the molded/restructured fruit pulp

occurs due to crosslinking of alginate molecules by calcium ion. During
the curing/submersion stage, exchange of Na+ from sodium alginate
with Ca++ from calcium chloride occurs as follows:
2Na-alginate + Ca++ → Ca-alginate + 2Na+
The ionically linked gel structure is thermostable over the range 0-

100°C. Therefore, heating will not disintegrate the gel. However, care
must be taken not to allow too long a curing period. This will result in
very thick skin, which is not desirable. Also, excess use of calcium
chloride (> 5M) can result in a bitter taste thereby spoiling the
characteristic sensory properties of the fruit.
Requirements
1. Fruit juice or pulp

2. Sugar
3. Citric acid
4. Calcium chloride solution (2% aqueous)
5. Beakers (50 ml and 250 ml)
6. Cutting knife
7. Sodium alginate (Important! molecular biology grade)
8. Blender / mixer
9. Reclosable plastic bags (250 g capacity)
Procedure
2. Take 250 ml of fruit juice or pulp.

3. Add sugar to taste (to give a TSS of > 20°brix).
4. Add citric acid to taste.
5. Take 5 g sodium alginate in a beaker (to give ~ 2% of the final
product). Now add a small amount of juice or pulp and stir well
102
to make a uniform suspension. If lumps are formed, break them
with a glass rod.
6. Transfer the contents to the bulk juice and mix well.
7. While stirring, heat the mixture until sodium alginate has
completely dissolved.
8. Cool the mixture to room temperature.
9. Divide the contents into two portions.
10. Take one of the portions and do the following:
i. Add 1 g CaCl2 (previously dissolved in a small amount of
water) and blend briefly.
ii. Quickly pour the contents in a tray in a thick layer (~ 1
cm thick).
iii. Allow to set for an hour.
iv. Cut into cubes.
v. Let the surface dry for few minutes and then pack the
product in reclosable plastic bags.
11. Take another portion and do the following:
i. Prepare 200 ml of 2% CaCl2 solution in a beaker.
ii. With the help of 100-ml beaker, pour the mixture in the
form of blobs (about 1 cm dia) into CaCl2 solution. It
requires considerable skill to form a perfect sphere. The
trick to success is: be fast, pour at the surface of CaCl2
solution, break the stream quickly. Alternatively, you can
also use a large syringe for dispensing the gel into the
CaCl2 solution.
iii. Allow the curing reaction to proceed for 1-2 min.
iv. Fish out or strain the globs.
v. Wash the globs under running water.
vi. Let the surface dry for few minutes. You can spread the
globs on filter paper, provided the paper is free from
toxic chemicals and additives.
vii. Pack the product in reclosable plastic bag and store.
12. Carry out sensory analysis (for taste, color and texture).
Observation
1. Dispersibility characteristics of sodium alginate

2. Shapes of the globs and cubes
3. Taste and texture of the restructured fruit
103
1. Success or failure of the experiment, along with reasons and

suggestions
2. Level of difficulty in preparing the product
3. Taste and texture of the product
Questions
1. What happens if you keep the gels in CaCl2 solution for a long
time?
2. Name some common fruit alginate products.
3. Why is sodium alginate a hydrocolloid?
4. Can you prepare alginate gel of juices having very low pH?
5. What do you know about spherification of foods using alginate?
6. What is the role of alginate in molecular gastronomy?
Helpful diagrams
OH
OH
HOOC HOOC O
OH OH
O
HO
HO OH HO
β-D-mannopyranuronate (M) α-L-gulopyranuronate (G)

4C conformation 1C conformation
1 4
HOOC OH OH
HOOC HOOC
O O O O
HO
OH HO O OH
O O
O OH O O
OH HOOC HO
O O O
OH HOOC OH HOOC OH
G G M M G M
Fig. 19.1 Basic structure of alginic acid (M = mannuronic acid,

G = guluronic acid)
104
Sucrose
Invertase
Glucose Fructose
Glucokinase Phosphomannose Fructokinase
isomerase
Glucose-6-phosphate Fructose-6-phosphate
Phosphoglucose
isomerase
Mannose-6-phosphate
Phosphomannomutase
Mannose-1-phosphate
GDP-massose
pyrophosphorylase
GDP-Mannose
GDP-massose
dehydrogenase
GDP-Mannuronic acid
Polymerase
Polymannuronic acid
Polymannuronic acid
5-epimerase
Alginic acid
Fig. 19.2 Biosynthesis of alginic acid in A. vinelandii (Pindar and Bucke,
1975)
105
Practical 20
CONTINUOUS FERMENTATION USING IMMOBILIZED
YEAST
Background
Immobilization, in the context of enzymes and cells, refers to

confinement of the enzyme or cell to a distinct phase from one in which
the substrates and the products are present. This is achieved by fixing
the enzyme or cell to or within some suitable material. It is critical that
the substrates and products move freely in and out of the phase to
which the enzyme or cell is confined. Immobilization of enzyme or cell
does not necessarily render them immobile. Immobilization can be
carried out by either bonding (chemical or physical) to a solid support
material (ceramics, cellulose, etc.) or by physical entrapment (gelation
and encapsulation) (see Fig. 20.2). The simplest example is the gelation
of live cells in alginate and agar gel. The greatest potential for
immobilized cell system (vis-à-vis enzyme) lies in its suitability for
fermentation involving multi-enzyme pathways (because cells are a bag
of multitude of enzymes).
Objectives
1. To prepare entrapped yeast cells

2. To assemble apparatus and glassware for continuous fermentation
3. To produce ethanol from sugar solution by continuous
fermentation
4. To study fermentation kinetics (substrate utilization and product
formation rates)
5. To calculate the dilution rate
Principle
The set-up of a prototype continuous laboratory fermenter using

entrapped yeast cells is quite straightforward. Matured yeast (S. cerevisiae)
cells can be entrapped in alginate beads or agar cubes and placed in a
side-arm flask (with arrangement for preventing washout of the beads).
When sugar solution is continuously passed through these cubes or
beads, some of the sugar will be converted into ethanol, which can be
collected in a separate vessel.
106
Requirements
1. Mature yeast culture (S. cerevisiae) or Active Dry Yeast (baker’s

yeast)
2. Molasses solution (18°brix) – 2 L
3. Agar-agar – 2% aqueous solution, ~ 300 ml and ~ 20 ml
prepared separately
4. Demijohns – 2 nos., 2.5 L capacity
5. Side-arm flask – 500 ml capacity
6. Rubber tubes/pipes
7. Flow regulators (e.g., roller clamp of an IV set)
8. Rubber corks
9. Refractometer
10. Alcohol meter or hydrometer
11. Shallow trays (steel)
12. Cutting knife
13. Filter paper circles
Procedure
1. Prepare 2 L of molasses solution (18°brix)

2. Weigh 8 g of Active Dry Yeast and prepare slurry in a small
amount of luke-warm water.
3. Prepare 300 ml and 20 ml of 2% agar separately in conical flasks.
Dissolve, autoclave to sterilize and cool to 50°C.
4. Add the yeast suspension to 300-ml agar solution and mix well.
5. Pour the mixture in shallow steel tray (to produce ~ 5 mm thick
agar layer) and allow it to set.
6. Once set, cut the agar into small cubes with a sharp cutting knife
7. Collect the cubes and wash in running tap water.
8. Fill the side-armed flask with immobilized yeast cubes (until
about 1 cm below the side arm, Fig. 20.1).
9. Add molasses medium over the cubes until nearly submerged.
Note the volume of the molasses solution used, say V ml.
10. Push in the filter paper and manipulate with a glass rod to cover
the agar cubes (don’t bother about the wetting of the paper but
take care not to punch holes).
11. Add the melted agar (20 ml of agar that was kept separately)
over the filter paper and allow it to set. The agar layer (slab)
should not be above the side arm!
107
12. With a glass tube, punch two holes through the agar slab (and
also through the moist filter paper), one at the center and
another at off-center. The punching should be delicate (by gentle
twisting).
13. Insert a short glass tube through the off-centered hole and leave
it embedded. Note that the agar plug must be removed from the
tube (to allow free passage of the fermented broth across the
slab to the surface).
14. Assemble the apparatus as shown in Fig. 20.1 and place it in a
warm place (e.g., in the sun) for faster fermentation.
15. Start feeding of molasses solution (prepared in step 1) at a very
slow rate (for example, 3 ml/min). You can connect roller clamp
of an IV set (available in drug stores) for controlling the flow.
16. Note down the TSS and Hydrometer reading of the outlet
stream after about 20 min. You will have to manipulate the roller
clamp to maintain a constant flow rate. Note that the flow rate
may slowly decrease because of the reduced pressure in the feed
tank (because it is slowly emptying).
17. Observe CO2 evolution and difference in the smell of the
solution (before and after passing through the continuous
fermenter).
18. Calculate the dilution rate by dividing the medium flow rate by
operating volume of the fermenter. Express the calculation in per
hour.
Observation
1. Smell of the feed and the product

2. Evolution of CO2
3. Time before reaching the steady state
4. TSS and hydrometric reading of the wash

2. Compound graph of TSS and hydrometer reading against time
(hr)
3. Time before reaching steady state
4. Dilution rate: ratio of feed rate (ml/hr) to operating volume (V
ml, see procedure steps 9 and 15)
108
Questions
1. What are the benefits of continuous fermentation?

2. Is it possible to carry out continuous ethanol fermentation using
immobilized enzyme(s)? Why?
3. What do you mean by short-circuiting and wash-out in continuous
fermentation? Do you see any arrangement being used in Fig.
20.1 to avoid short-circuiting?
4. What corrections would you suggest if the conversion is found
to be too slow? Hints: alter cell density, feed flow rate,
concentration of sugar in the feed, fermentation temperature,
etc.
5. Suggest a method for using sodium alginate instead of agar-agar
for immobilizing the yeast cell.
Helpful diagrams
Cotton plug
Feed tank
Roller clamp
Molasses
solution
(Feed)
Rubber
connector
Agar plug
Side-armed
flask
Agar cubes
(Immobilized
yeast cells) Receiver
vessel
Fig. 20.1 Set-up for continuous ethanol fermentation using immobilized

cells
109
Semi-permeable
Entrapment membrane
Charge network
distribution
E
E Enzyme E E
E E E E E
molecule E
E E E
E E E
E E
Adsorption Entrapment Encapsulation
E Bond
E
CHO CHO HC=N-Enzyme
E
E E E Immobilization using glutaraldehyde
E E Matrix
E
E E E
Covalent bonding
Fig. 20.2 Different immobilization techniques
110
Practical 21
DETECTION OF RDMs IN BREWING YEAST
Background
Approximately 10% of the yeast genome is located in mitochondria.

The wild type mitochondrial phenotype is denoted ρ+ (rho+). Sub-lethal
mutation in mitochondrial DNA can occur spontaneously or due to
stresses of repitching, handling, etc., in individual brewing yeast cells.
This event produces Respiratory Deficient Mutants (RDMs) that are
unable to metabolize non-fermentable carbon sources such as glycerol,
lactate, or ethanol. These mutants are also unable to oxidize glucose to
CO2 and water because they lack many components of the electron
transport chain and are therefore, respiratory deficient. When plate-cultured
in special media (such as YEP-lactate agar), the mutant cells produce
small colonies – hence the term petite. Such respiratory deficient
mutation can range from point mutation (mit–), through deletion
mutation (ρ–) to complete elimination of mictochondrial DNA (ρo).
Petite mutation results in changes in metabolic and physiological
responses of brewing yeast. They yield low biomass, increase diacetyl
levels in beer (this in undesirable), and cause inadequate attenuation of
beer. A healthy yeast culture should have less than 2% petite colonies in
the population. In practice, however, a 10% level is generally taken as
the maximum limit.
Objectives
1. To detect RDMs in brewer’s yeast

2. To determine the percentage of RDMs
3. To compare the colony characteristics of RDMs and normal
cells
Principle
Respiratory deficient cells are unable to reduce Triphenyl

Tetrazolium Chloride (TTC, a colorless salt) to a colored form. Thus,
when the colonies on plate are overlaid with TTC agar and incubated for
a few hours, normal cells reduce the salt to give red-pink colonies while
the petite colonies remain unchanged.
111
Requirements
1. Brewer’s yeast broth (actively growing)

2. YPD agar (also written YEPD, see composition later) – 100 ml.
You may also use molasses agar or MYGP agar (Practical 4)
3. TTC agar (see composition later) – 50 ml
4. Routine microbiology equipment and glassware
Procedure
1. Prepare 4-5 YPD plates (you may also use molasses agar).
2. Transfer a loopful of yeast broth to the first YPD plate.
3. Spread serially on the remaining (3 to 4) YPD plates (as
described in Practical 4).
4. Incubate at 28-30°C in inverted position for 3 days.
5. Select plates that have 30 to 100 well-isolated colonies.
6. Observe the colony characteristics.
7. On top of the colonies, add a layer (10 ml) of melted (~ 50°C)
TTC agar.
8. Incubate the plates at 28-30°C for 1-3 hrs.
9. Observe for change in color of the colonies immediately. Pink to
red colonies are Respiratory Sufficient while rest (atypical colonies)
are Respiratory Deficient Mutants (RDMs).
10. Count the colonies and report the number of RDMs (if present)
in percentage.
Observation
1. Reaction of colonies to TTC agar overlay

2. Colony characteristics of RDMs and normal cells

2. Color of RDMs and normal colonies on TTC agar overlay
3. Percentage of RDMs
4. Suitability for repitching
5. Morphology of normal and RDMs
112
Questions
1. Does excessive repitching give RDMs? Why?

2. What is the Hayflick limit of yeast cells?
3. Why are the colony characteristics of normal and RDM cells
different in YEP-lactate agar?
4. Can you replace sodium lactate in YEP-lactate agar with glycerol,
ethanol, etc.? Why?
Media composition
1. YPD agar (Yeast extract-Peptone-Dextrose agar)

Yeast extract : 1%
Biological peptone : 2%
Glucose : 2%
Agar-agar : 2%
Water : Required amount to make 100 ml
Sterilize by autoclaving and 121°C for 15 min
2. TTC agar (Triphenyl tetrazolium chloride agar)
Solution A
Place in a 100-ml conical flask 0.325 g NaH2PO4, 0.29 g

Na2HPO4, 0.75 g agar and 25 ml distilled water. Swirl to mix, heat
to dissolve, and sterilize in autoclave at 121°C for 15 min.
Solution B
Place in another 100-ml conical flask 0.05 g TTC, add 25 ml of

distilled water, mix, and sterilize in autoclave at 121°C.
Mix solution A and B when the temperature has reached about

50°C, just before layering on the plates. Keep Solution A in water
bath to avoid solidification before mixing.
Note
Comparison of the morphologies of normal cells and RDMs can be

carried out by negative staining. However, it is difficult to compare the
cell morphologies from separate slides (and therefore separate
microscopic observation). To this end, the juxtaposition technique (devised
113
by the authors) in which smears of two or more cell types can be
juxtaposed in the same slide, can be used for the direct comparison.
The process, which is very simple and effective, is outlined as

follows:
1. Prepare a negative stain using yeast A (see Practical 1 for

discussion on negative staining).
2. Air-dry the smear and then stick a cellotape over the dry smear.
3. Run a sharp razor blade across the fixed tape and peel off one of
the portions.
4. With a moist cotton, wipe the smear in the exposed portion
repeatedly to remove the smear. Wipe once more with 70%
alcohol and allow the slide to air-dry.
5. Follow step 1 using yeast B on the cleaned part of the slide. Let
the smear juxtapose the taped part.
6. Follow step 2, taking care not to overlap the tapes.
7. Wipe the taped surface once more.
8. Put a drop of immersion oil over the juxtaposed portion and
view under oil-immersion objective. You should now be able to
see the two types of yeast cells together, neatly separated by a
boundary
9. You can now record the cell shape, size and organization (see
Practical 1 for micrometry).
10. Take photographs of the cells with an inbuilt camera (or an
external camera). You can also use your smartphone for the
same (take the help of your instructor).
114
Practical 22
DEMONSTRATE UV RADIATION LETHALITY IN YEASTS
Background
Short wavelengths of light are toxic to most microorganisms. There

are some exceptions, though, e.g., photosynthetic bacteria that can use
UV light for energy. Short wavelengths of light include both ionizing
radiations (e.g., γ-, X-, and cathode rays) and somewhat longer
electromagnetic radiations called UV rays (100-400 nm, Fig. 22.1).
The UV spectrum is divided into three bands (photobiological

designations):
1. UVA (315-400 nm): Tanning rays

2. UVB (280-315 nm): Burning rays
3. UVC (100-280 nm): Dangerous rays
UV rays around 260 nm have the greatest lethality. They work by

forming covalent bond between adjacent pyrimidine bases (thymine
and/or cytosine) of nucleic acids to form dimers. If a cell tries to replicate
UV-damaged DNA with dimers present, it usually dies.
Ultraviolet Visible Infrared
200 300 400500 600 700 800 900 1000

Wavelength (nm)
Fig. 22.1 Absorption of UV radiation by DNA
Fortunately, cells have evolved some protective mechanisms against

this damage. One of such mechanisms is photoreactivation, in which a
specific photoreactivating enzyme (photolyase, Fig. 22.2), using energy
from visible light, restores the DNA by splitting the pyrimidine dimers.
In another repair mechanism, termed excision repair mechanism, enzymes
115
remove the dimers and then patch the affected DNA. Because the repair
processes are so effective, only a source of high energy UV-C (such as
germicidal lamp) will produce substantial killing and mutation in normal
yeast cells.
O O O O
CH3 H3C
HN CH3 HN CH3 HN NH
+
UV-radiation
O N O N Photolyase O N N O
H H H H
Thymine Thymine Thymine dimer
Fig. 22.2 Thymine dimer formation and repair
An advantage of using UV light for killing (and also mutation) is its

ease of application. Hence it is widely used in such places as hospital
operating rooms, sterile pharmaceutical filling rooms, and inoculation
rooms. UV light, however, cannot penetrate deep into the surface. An
important point to be noted in the use of UV light is that it is damaging
to all tissues alike. Precaution should therefore be taken not to get
exposed to this ray in any event.
Objectives
1. To determine the rates of yeast cell death after exposure to UV

light for varying lengths of time
2. Observe abnormalities/mutations in yeast cells
Principle
When a freshly seeded yeast plate is briefly exposed to UV light of

wavelength at 254 nm, many cells are killed due to the genotoxic nature
of the light. The extent of death of the cells is a function of length of
exposure to UV light as well as the wavelength of light chosen. Those
that survive the UV treatment may or may not have undergone
mutation. By varying exposure time, a survival curve (or death curve)
can be obtained to demonstrate the ultraviolet lethality in yeasts, similar
to the hypothetical survival curve shown in Fig. 22.3.
Requirements
1. Actively growing yeast culture (S. cerevisiae)

116
2. UV-cabinet (254 nm wavelength)
3. YPD agar plates (molasses agar can also be used) - 10 plates
4. Glass spreader (dallying rod)
5. Test tubes for serial dilution
6. Aluminum foil
7. Incubator
8. Stop watch
9. Dark box
Procedure
1. Obtain an actively growing yeast culture (S. cerevisiae).

2. Carry out serial dilution in sterile water until the cell
concentration is ~103 cells/ml. Carry out process as follows:
i. Take a standard test tube with sterile water (diluent).
ii. Add a small amount of actively growing yeast cells (from
broth) and mix the contents well in a vortex mixer.
iii. Observe the turbidity in the test tube. Adjust the
turbidity (by adding sterile water) such that the turbidity
is barely noticeable with naked eye. This approximates to
cell concentration of ~ 106 cells/ml.
iv. Carry out further dilution in test tubes to give ~ 103
cells/ml as follows:
a. Take 1 drop (~ 0.03 ml) of culture from the dilute
broth and transfer to a flask containing 30 ml
sterile water to give ~ 103 cells/ml.
b. Agitate thoroughly to obtain a uniform distribution
of the cells.
3. Take 10 YPD plates (previously plated, and with dry agar
surface).
4. From the last dilution, transfer with the help of sterile, graduated
1-ml pipette, 0.1 ml of broth to all the YPD plates.
5. Spread the inoculum uniformly on the agar surface with the help
of a sterile, bent glass spreader (see Practical 5). You can use
either inoculation turntable or spread manually.
6. Repeat the process (spreading) for all the remaining YED plates.
Between each spreading, sterilize the glass rod by immersing it in
70% ethanol and brief flaming. Allow the spreader to cool in
sterile zone. The sterilization is not actually for asepsis. Rather, it is
an attempt to maintain equal cell number in the plates by avoiding
carry-over from the dallying rod. Following points must be
117
noted during spread plating: (i) Avoid spreading the inoculum all
the way to the edge of the agar (ii) Carry out spread plating as
soon as possible otherwise some cells may strongly stick to the
agar surface (iii) Avoid disturbing plates for 10 to 20 min after
spreading. Drying time varies with room temperature and
humidity.
7. Prepare the plates for UV irradiation as follows:
i. Cut out 5 circles of aluminum foil. Make sure that the
diameter is slightly greater (~ 2 cm) than the diameter of
the base plate (plate containing the medium).
ii. Neatly cut the circles into halves so that you get 10 half-
circles of aluminum foil.
iii. Remove the lid of the plates and cover the base plates
with the half-circle foils so that exactly half the base
plates are covered. The exposed part will receive the
radiation while the covered part will serve as a control
for each plate (Fig. 22.3).
8. Now take the plates for irradiation at λ = 254 nm for varying
time periods (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 sec). Label
the plates 4, 10, 15, etc., to indicates exposure times.
9. Turn on the UV cabinet for 30 min before the actual irradiation.
Do not proceed without your instructor’s guidance.
10. Turn off the lamp. Be careful not to expose yourself (skins, eyes,
etc.) to UV light. It is carcinogenic!
11. Place the plate labeled “5”, “10” and “15” directly below the
lamp (at a distance of 10-12 cm), with the lid removed.
Assumption has been made here that you have general UV
cabinet with 8W tube (and can cover 3 Petri plates, lengthwise).
During the placement of the plates, be sure to arrange the plates
as shown in Fig. 22.5 to avoid the stray light (and therefore
diffused demarcation of the radiation).
12. Close the chamber and turn on the lamp (careful!). You should
have a stopwatch or similar gadget ready before doing this. The
lamp may not light up as soon as you switch on. Assuming a
delay of 1 s each time you switch on, you can add 1 s more in
your time (for example 6 s instead of 5 s) for correcting the
exposure duration. Use a stopwatch or similar gadget for noting
the time. Do no forget to remove the lid before UV exposure.
13. Turn off the light, cover the plate with lid, take out the plate and
put it in a dark box (you can use suitable carton box for this).
118
14. Repeat the process for other plates. Remember that you have
already irradiated the second plate for 5 s! Now you must expose
it to extra 5 s + 1 s (for correction) to make 10 s. Take the
instructor’s help to calculate and manage the exposure times.
15. After you have finished irradiating, invert all the plates and
incubate at 30°C in dark for at least 24 hrs. If the incubator has a
glass window, paste a paper to block the visible light.
16. Observe the plates. You will probably see something like given
in Fig. 22.5. Take photographs of all the plates kept serially in
ascending order of exposure time.
17. Count the colonies in plates for % survival as follows:
i. For control population (number of colonies if the plates
had not been irradiated), count the colonies in the
control (covered portion).
ii. For survived population (number of colonies surviving
the irradiation), count the colonies in the irradiated
(exposed portion).
18. Calculate the % cell surviving on each plate (= each duration of
irradiation) at a given radiation intensity as follows:
Colonies in irradiated portion
i. % Cell survived = × 100
Colonies in control portion
Wattage of lamp
ii. Radiation intensity = Wm −2 .
Area
In this case, the area refers to the surface area of the sphere
(4 πr2) having radius equal to the distance between the plate and
the lamp (see Fig. 22.6 for explanation).
19. Plot the graph as shown in Fig. 22.7.
Observation
1. Colony distribution
2. Colony characteristics
3. Visibility of UV light (when viewed through the screen)
1. Success or failure, along with plausible reasons

2. Minimum exposure time needed to exhibit the death effect
3. Discussion on the survival curve obtained
119
Questions
1. Do you think the survived colonies are mutants? Why?

2. What type of mutation does UV radiation cause?
3. Does UV mutation have relevance with RDMs?
Helpful diagrams
Base plate
Alminum foil
Plate lid
Base plate with

the medium
Fig. 22.3 Wrapping of the spread-plated base plate with Al foil
Al-foil cover
UV lamp
Seeded base plate

Correct method of exposure
Incorrect method of exposure

Fig. 22.4 Irradiation of seeded plates
Control Irradiated
5s 10 s 15 s 20 s 25 s
Fig. 22.5 Effect of UV radiation duration on yeast cells
120
UV lamp
8W, λ=254 nm
r = 12 cm
Plate
(side view)
Plate
(top view)
Al foil
Fig. 22.6 Calculation of radiation intensity
From Fig. 22.6, the radiation intensity is calculated as follows:
8W
I= 2
= 44.21 Wm −2 ( Js−1m −2 )
4π ( 0.12 ) m 2
100
Cell survival (%)
10
0.1
0 20 40 60 80 100 120 140
Exposure time (sec) in UV light (λ = 254 nm)
Fig. 22.7 A hypothetical survival curve after UV irradiation of yeast cells
121
Practical 23
DETERMINATION OF AMYLASE ACTIVITY OF
AMYLOLYTIC STARTER
Background
General
Hydrolysis of starch by acids or enzymes into simple sugars is called

amylolysis. The enzymes responsible for starch hydrolysis are called
(collectively) amylases. Amylases for the commercial uses (e.g., in
fermentation, detergents, High-Fructose Corn Syrup production, etc.)
come from fungi, bacteria or plants. Malt used in beer and whisky
production contains plant amylases. Oriental alcoholic cereal fermented
beverages utilize fungal amylases. Molds, notably species of Aspergillus,
Rhizopus (Amylomyces) and Mucor, are very good amylase producers.
Types of amylases
Molds present in the amylolytic starters can produce α-, β-, as well
as glucoamylase (γ-amylase). However, α-amylase is the dominant (and
also the most important) amylase in both fungal and plant amylases.
Amylases act differently on starch molecules. α-amylases hydrolyze the
starch molecule at random from the interior part of the starch (Fig.
23.1) and are therefore termed endoamylases. They rapidly decrease the
viscosity of the starch solution. β-amylases sequentially hydrolyze starch
molecule from the non-reducing end to produce maltose units. The
viscosity is not reduced rapidly but the hydrolyzed product is sweet in
taste. Glucoamylase (α-1,6-glucosidase) breaks down α-1,6 as well as α-
1,4-glycosidic linkage in the branched portion of the starch molecules.
Both α- and β-amylases cannot attack the α-1,6 linkages. They also
cannot attack a few α-1,4-linkages in the vicinity of the α-1,6 branch
points. This inability to attack branch points of starch by amylases
results in the formation of short, highly branched fragments (residues)
of glucose polymer called limit dextrin.
Before amylases can act on starch, the latter must be converted into
susceptible, gelatinized form. This is where the role of cooking cereals
(in the homes and in fermentation industries) comes in.
122
Assay of amylase
A number of methods have been developed for the assay of

amylase activity on starch. Quantitative procedures may involve the
measurement of new reducing groups, hemiacetal or aldehyde groups
that result from the hydrolysis of the glycosidic acetal linkages. The
colorimetric measurement of the formation of reducing groups has
mostly been by the use of alkaline copper, alkaline ferricyanide or
alkaline 3,5-dinitrosalicylic acid (DNS). A semi-quantitative
determination of starch hydrolysis by α-amylase involves measurement
of the decrease in the blue iodine color. This method is especially useful
in survey of biological samples for α-amylolytic activity. The procedure
reflects the endo cleavage of relatively large starch chains and cannot be
used to assay exo-acting amylases. Measurement of the decrease in the
viscosity of a starch solution has also been used to measure α-amylase
activity. This method, like the iodine procedure only measures α-
amylase activity and the results cannot be readily expressed in
international units.
Some workers have used a modification of agar diffusion method

for the assay of amylolytic activity. Clear radial diffusion zones are
measure after 4 hrs of incubation at 20°C. A linear relationship between
the logarithm of enzyme activities and the area of clear zones is
obtained.
Other tests include use of DNS reagent. Maltose (a reducing sugar)

reacts with and reduces the pale yellow colored alkaline DNS to the
orange-red colored, 3-amino, 5-nitrosalicylic acid after being heated for
5 min. Essentially, the intensity of the color (at λ = 546 nm) is
proportional to the concentration of maltose present in the solution.
Thus, increased amylase activity produces more maltose, which reduces
more DNS, which then turns the solution a darker orange-red.
The α-amylase activity has been variously defined. According to

Moulin and Galzy (1978), one α-amylase unit is defined as the amount
of enzyme that hydrolyzes 10 mg starch in 30 min under the given
conditions. Mulimani and Lalitha (1996) define one unit of enzyme
activity as the amount of enzyme that liberates 1 μM of maltose
equivalent in 1 min, which is more generally used.
123
Assay of amylolytic activity is a routine test in fermentation-,
pharmaceutical- and allied industries. However, there is no standard
protocol for the determination of amylolytic activity of amylolytic
starters, murcha for instance. Modifications of the aforementioned
methods have been used by some workers.
Relevance of amylase analysis in amylolytic starters
Amylolytic starters obviously contain small amounts of pre-formed

amylases, produced during the fermentation. Unlike malt, the primary
purpose of the starter is not to supply amylase but to include sufficient
inoculum for the main fermentation wherein growth of microorganism,
production of enzymes, and breakdown of starch proceed in parallel. It
is thus clear that the pre-formed enzymes have only negligible role
(unless the starter is used in very large amounts!) in the fermentation.
The assay of amylolytic activity vis-à-vis pre-formed amylase, therefore,
does not seem relevant for quality tests of starters. In breweries and
malting industries, however, the quality of malt is judged by analyzing
amylolytic power in terms of diastatic power or degree Lintner. For
amylolytic starters, of more relevance could the assessment for
amylolytic potential of the starter flora. However, literature on this
aspect is almost nonexistent.
Objectives
1. To prepare murcha samples for analysis

2. To determine out amylolytic activity of murcha sample
Principle
Amylases are extracted from a known quantity of murcha sample in

malate buffer and the rate of release of reducing sugar (as maltose) by
the extract from starch solution under standard conditions of incubation
(for reaction) is determined by Lane and Eynon (Fehling’s solution)
method. The result is expressed as 1 μM of maltose equivalent formed
in 1 min.
Requirements
1. Murcha sample – 5 g
2. Sodium malate buffer (pH 5.4, 0.5M) – 50 ml (see composition
later)
124
3. 1% starch solution – 100 ml (dissolve by boiling in distilled
water and cool to ~ 40°C)
4. Fehling’s solution A and B – Mix in the ration 1:1 just before
titration
5. Conical flask – 100-ml capacity
6. Incubator or water bath
8. Titration arrangement (pipette, burette, etc.)
9. Weighing- and volume-measuring arrangement
10. Maltose standard – 2 mg/ml (the concentration may need
adjustment), 100 ml
11. Porcelain crucibles (for titration)
12. 1% methylene blue solution
13. pH meter
14. Centrifuge – standard laboratory centrifuge
15. 1N NaOH – 100 ml
16. Distilled water – warm (~ 40°C)
Procedure
1. Macerate 5 g murcha sample in 50 ml malate buffer (pH 5.4).

2. Hold for 20 min at 40°C for enzyme extraction.
3. Centrifuge at 1000 rpm for 10 min and collect as much
supernatant as possible.
4. Prepare 3 conical flasks (50 to 100-ml capacity), 2 for control
and 1 for sample. Label them properly (C1, C2 and S, for
example) to help identify the flasks.
5. Add 5 ml of distilled water in C1 and 5 ml each of extract in C2
and S.
6. Add 20 ml of starch solution and 25 ml of warm water (~ 40°C)
in all the three flasks and mix well.
7. Add 5 ml each of 1N NaOH in C1 and C2 and bring to boil.
Next, rapidly cool the flasks to ~ 40°C and, along with flask S,
incubate at 40°C for 15 min.
8. Now, add 5 ml of 1N NaOH in flask S, bring to boil, and cool
to ~ 40°C. The purpose of NaOH addition and boiling is to
inactivate the enzymes.
9. Take 2 ml of standard Fehling’s mixture (see Notes for
standardization) in a clean porcelain crucible and add ~ 25 ml of
distilled water.
125
10. Bring the Fehling’s mixture to boil and while still boiling, add the
contents of C1 through a pipette (or a microburette). The
addition should be steady and boiling should continue. Continue
addition until the Fehling solution fades to yellowish tinge. It is
possible that you will not get the near-end point even after you
have emptied all the contents. In that case, assume absence of
reducing sugars in the contents. In case the color changes, add 1
ml of methylene blue, without stopping the boiling. If the dye
turns blue, continue addition of the contents until the blue color
vanishes and you get a brick red precipitate. Here also, you may
not come to the end point, in which case you must assume
absence of reducing sugar in the contents.
In case you observe the end point (the brick red colored
residue), record the titer (volume of contents needed) to reach
the end point, say v1 ml.
Do the same with C2 and S to get the titers v2 ml and vS ml,

respectively.
11. For the standardization of Fehling’s mixture, follow the

procedure given in step 10, but taking 10 ml Fehling’s mixture in
the crucible and standard maltose (2 mg/ml) in the pipette (or a
microburette). Let the titer be vM ml.
12. Carry out calculation for the standardization of Fehling mixture
as follows:
mg maltose 2×vM
Fehling factor = = = 0.2v M
ml of Fehling mixture 10
13. This implies that 2 ml of Fehling’s mixture used in titration of

the contents of flasks C1, C2 and S is equivalent to 2 × 0.2 vM mg
of maltose. In other words, each titer (whatever the value) is
always equivalent to 2 × 0.2 vM mg of maltose, provided you
reach the end point.
14. Having obtained the titers, use unitary method to calculate mg of
maltose as follows:
First, determine the mg of maltose present in the sample after

enzyme reaction, say the value is S.
126
Second, determine the mg of maltose present in C1 after
incubation, say the value is C1.
Third, determine the mg of maltose present in C2 after

incubation, say the value is C2.
Finally, subtract (C1+C2) from S to get the actual quantity of

maltose released. That is, S – (C1+C2) = mg of maltose liberated
by the enzyme extract.
15. Back-calculate to obtain mg of maltose that could be obtained

had all the extract (50 ml in place of 5 ml aliquot) been used.
You can easily calculate it by multiplying with dilution factor, in
this case, 10.
16. Since you have carried out the reaction for 15 min, divide the
result with 15 to get mg of maltose formed in 1 min.
17. Calculate μM of maltose formed per minute from the relation:
1 mg maltose is 2.92 μM ( 1 mole = 342 g of maltose)
18. Finally, obtain the amylase activity of murcha by dividing the

amount of murcha sample taken initially (i.e., 5 g) by the result
obtained in step 17. This gives 1 μM of maltose equivalent
formed from starch in 1 min at 40°C.
Observation
1. Titration data for C1, C2, and sample S
1. Amylase activity of murcha sample

2. Amount of reducing sugar (maltose equivalent) in control
samples C1 and C2
3. Theoretical soundness of the method (hint: only β-amylase
produces maltose)
4. Comparison with data from similar studies (if available)
127
Questions
1. What do you know about γ-amylase?

2. How can you selectively determine α- and β-amylase in a
mixture of amylases?
3. How does α-amylase reduce the viscosity of starch solution?
4. Why was the amount of Fehling’s solution reduced during
titration with the enzyme extract?
5. What is the purpose of constant boiling during titration?
6. Why do you think Fehling’s solution was diluted with water
before titration?
7. How does methylene blue confirm the end point of the titration?
Preparation of reagents
Sodium malate buffer (0.5M)
Dissolve 6.7 g malic acid, 3.5 g NaOH, and 2.92 g NaCl in 90 ml

deionized water. Add 0.3 g of CaCl2.2H2O and dissolve. Adjust to pH
5.4 by drop-wise addition of 2M NaOH.
Add 0.05 g of sodium azide as preservative (sodium azide is a

poisonous chemical and must be used according to supplier’s safety
instructions). In presence of sodium azide, this reagent is stable at room
temperature for > 1 year. If sodium azide is not added, the reagent is
stable at 4°C for 2 weeks.
1. Fehling’s solution
Mix Fehling A and Fehling B in equal volumes to give a

predetermined volume (i.e., not more than the volume needed
for the test). Use only fresh mixture and discard the leftover.
Fehling A: Dissolve 69.28 g CuSO4.5H2O in water, dilute to 1,000

ml and, if necessary, filter through Whatman filter No. 4
Fehling B: Dissolve 346 g of Rochelle salt (potassium sodium

tartrate, KNaC4H4O6.4H2O) and 100 g NaOH in water and
make up to 1000 ml
128
2. 2M NaOH (2N)
Dissolve 8 g NaOH pellets and make up the volume to 100 ml
3. Maltose standard (2 mg/ml)
Dissolve 2 g maltose in distilled water to make 100 ml. Dilute 10

ml of this solution to 100 ml in distilled water to give 2 mg
maltose per 100 ml. Use graduated pipette and volumetric flask
(not measuring cylinder).
Helpful diagrams
Branched region
(resistant to both amylases)
Action of β-amylase Action of α-amylase
Fig. 23.1 Action of β- and α-amylase on starch molecule
129
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Glossary
Active Dry Yeast (ADY): Dry form of yeast used in bread making
(baker’s yeast), Saccharomyces cerevisiae.
Adjuncts: Non-malted carbohydrate ingredients used in beer making,
e.g., sugar, rice, corn.
Aeration: To supply air to the growth of microorganism.
Agar (agar-agar): A polysaccharide obtained from red sea-weed. It is
used as a solidifying agent in the preparation of culture media. Most
organisms cannot decompose this material.
Aging: Bringing about maturity in flavor quality of food and beverage.
Normally, it takes extended storage periods to obtain this quality
(hence aging). In alcoholic beverages, slow oxidation is the major
reaction that occurs during aging.
Ale: Beer produced by using top-yeast (strains of Saccharomyces cerevisiae
that collect on the surface after fermentation has completed).
Alginate: Polysaccharide produced by certain bacteria and sea-weeds. It
is used in laboratories and food and pharmaceutical industries for
preparing gels.
Amelioration: Manipulation of sugar and acid content of grape juice
(intended for wine production) to bring consistency in the
composition.
Ammoniacal: Having smell of ammonia (pungent).
Amylolytic: Having ability to break down (hydrolyze) starch.
Anaerobic: That requires oxygen-free condition for growth.
ANOVA: Analysis of Variance (a statistical test for the comparison of
more than two means).
Anthocyanins: A class of purple or red coloring matter (pigment)
found in fruits and vegetables.
Antibacterial: That acts against bacteria.
Antibiotic: Chemical compounds used to fight against disease-causing
microorganisms (pathogens). Most of them are produced by
microorganisms, a few are produced by chemical synthesis.
Antibiotic spectrum: The range of action of antibiotics. An antibiotic
that acts against only one pathogen is said to have ‘narrow
spectrum’. An antibiotic that can acts against more than one
pathogen type is referred to as broad spectrum.
Asepsis: A condition free of contamination. Procedure performed
under such condition ‘aseptic technique’.
Aspergillus: A type of mold (see Fig. 4.1).
134
Assimilate: To utilize (digest, metabolize) for deriving energy needed by
the cell.
Autoclave: A closed, pressure vessel for sterilizing culture media. It
works on the principle of pressure cooker. Sterilization is usually
done at 121°C for 15 min. The equivalent pressure inside the vessel
is 15 psig (pound per square inch gauge).
Azeotrope: Mixture of two or more liquids that will boil off together at
a given temperature, like a single liquid. Mixture of two liquids with a
single boiling point is called binary azeotrope. Similarly, mixture of
three liquids with a single boiling point is called ternary azeotrope.
Baker’s yeast: Saccharomyces cerevisiae that is used by bakers for preparing
leavened breads.
Batch process: In the context of fermentation, it is a process in which
the nutrients/substrates are supplied in the fermenter only once. The
product is recovered after the fermentation has completed.
Thereafter, the vessel is cleaned, filled again, and the next round of
new fermentation carried out. This cycle of substrate input to
recovery constitutes a batch.
Beer: Undistilled alcoholic malt beverage that has been preserved and
flavored with hops (dried blossoms or product thereof of female
hop plant Humulus lupulus). Hops give the characteristic bitterness of
beer (Fig. 11.1 and 11.2).
Beverage: A drink of any type, e.g., soft drinks, tea/coffee, wine, etc.
Binning: Aging of wine in corked bottles. Constant contact of wine
with the wooden cork helps the wine to age.
Blackstrap molasses: A by-product of sugar refinery. After sugar has
been extracted by concentration and crystallization from cane juice,
the non-crystallizable fraction (called mother liquor) is concentrated
to give a thick mass called molasses. It is dark in color, has a total
soluble substance of ~ 80% and fermentable sugar content ~ 50%.
Blackstrap molasses is an important medium for many fermentation
industries. The familiar distilled alcoholic beverage ‘rum’ is obtained
by fermentation of molasses.
Blending: Judicious mixing or combining of different ingredients to
develop a unique type of product. The blending operation is a very
delicate work.
Bulk density: It is the mass of material (particles, grains, seeds, etc.)
that occupies a unit volume of the container. Bulk density is an
important physical property of grains, flours and powders because it
135
plays an important role in storage, packaging, transportation, and
marketing.
Caramel: Burnt sugar used to give flavor and brown color to foods and
beverages.
Caramelize: Burning of sugar to produce caramel.
Carbohydrate: Polyhydroxy aldehyde or polyhydroxy ketone
compounds. Starch, glucose, lactose, etc., are some examples of
simple carbohydrates. Pectins, gums, hemicellulose, etc., are
examples of complex carbohydrates.
Cell build-up: Increase in number of cells.
Chaptalization: Addition of sugar in the grape juice (must) to adjust
sugar concentration. Also see amelioration.
Characteristic: A typical or noticeable quality of someone or
something.
Chemotherapeutic agent: Agent used for therapy (the treatment of
illness / disease).
Colony: Aggregate of microbial cells visible as clumps on the surface of
solid growth medium. Although microbial cells are microscopic,
when billions of them grow together they are visible as colonies.
Colony characteristics: The highly characteristic, distinct type of
colony shown by each type of microorganism.
Congener: The flavor principles produced by microorganisms during
fermentation. The term has greater relevance to alcoholic
fermentation. Beers have over 1000 flavor components arising from
fermentation. Rum, whiskey, brandy, etc., are congeneric alcoholic
beverages whereas vodka, gin, etc., are non-congeneric alcoholic
beverages.
Conidiospore: Also called conidia (singular = conidium) are free spores
not enclosed in a spore-bearing sac (see Fig. 4.1).
Crabtree effect: Also called glucose effect, refers to shifting of microbial
metabolism to fermentative mode when readily fermentable sugar
(e.g., glucose) level in the medium is above 3-5%. This has relevance
to cell economy. That is, when the cell can access plenty of readily
assimilable sugar it will not use pathways that produce unnecessary
energy. However, when the sugar level becomes very low, provided
aeration is done, the organism will exhibit metabolic shift to
respiratory mode whereby it will obtain very large amount of energy.
This latter metabolic dominance is called Pasteur effect.
Crowded-plate: An agar plate with very large number of colonies
(around 300).
136
Dallying rod: L-shaped, bent glass rod used for spreading inoculum on
the surface of agar plate (see Fig. 5.2).
Decoction: A method of extracting malt and adjunct constituents for
beer production. This method is used for lager beer production.
Warm water is added to the milled barley malt + cereal adjuncts and
enzymatic reaction carried out. The process as a whole is called
mashing.
Distillation: Separation of relatively volatile component by heating to
form vapor, followed by condensation. The condensate is called
distillate.
Dry wine: Although this can be subject to controversy, dry wines are in
general wines that contain less than 0.5% sugar.
EMP pathway: Embden-Meyerhof-Parnas pathway, a metabolic
pathway that describes the conversion of glucose to pyruvate /
lactate.
Enrichment: In distillation, enrichment means ‘become richer and
richer in ethanol’. This occurs in the distillation tower where the
condensed dilute ethanol meets with the rising ethanol-water vapor.
Enzyme: A biomolecule that does the job of a chemical catalyst.
Enzymes are basically globular proteins. They transform substrates
into products with very high degree of specificity and at a rate
compatible to the cell. Some examples of enzymes are: invertase that
hydrolyzes sucrose (table sugar) to glucose and fructose; amylase that
hydrolyzes starch into simple sugars (glucose, maltose, etc.).
Extracellular: Formed inside the cell and subsequently secreted out of
the cell. Many microorganisms produce extracellular enzymes. For
example, molds produce extracellular amylase (enzyme that breaks
down starch). These enzymes act on the substrate and hydrolyze
them into small units, which are later transported inside the cell for
utilization.
Fermentation: Any large-scale microbial and/or enzymatic process.
Fermentation kinetics: Study of rate processes in fermentation. The
three important rate processes dealt in fermentation kinetics are (i)
rate of utilization of substrate, (ii) rate of cell growth, and (iii) rate of
product formation.
Fermenter (fermentor): A vessel in which fermentation is carried out.
A typical batch fermenter is cylindro-conical in shape, has a nominal
capacity of 50,000 L, is equipped with an impeller (mixing ancillary),
aerator (for supplying air/oxygen), heat exchange coils, and probes
for measuring pH, temperature, etc.
137
Flocculation: Reversible aggregation of cells, resulting in the formation
of clumps (flocs).
Flora: In biotechnology, flora implies the microorganisms (they are
plants!).
Fortification: In wine industry, fortification means addition of spirit to
wine to make it stronger or ‘fortified’. Fortified wines have 17-21%
alcohol by volume.
Free run wine: Wine prepared from the siphonable portion of the
liquid from primary fermentation. Such wines are considered to be
of superior quality. The sludge or sediment (called lees) that remains
after siphoning can be pressed and the resulting extract fermented to
produce what is called ‘press wine’. Press wine is considered to be of
inferior quality because it contains excess tannins.
Fusel oil: Also called higher alcohols, they collectively represent amyl
alcohol, butyl alcohol and propanol formed during alcoholic
fermentation. They are oily and insoluble in water and have a boiling
point range of 125-140°C. Their presence in alcoholic beverages in
excess of 0.5 g/L is undesirable. Higher alcohols are formed by the
metabolism of amino acids like valine, leucine, etc.
Gallization: Addition of sugar and water in the grape juice (must) to
adjust sugar concentration. Also see ‘amelioration’.
Genus: The seventh of eight major taxa, or groups, in the taxonomic
system: domain → kingdom → phylum or division → class → order
→ family → genus → species. The binomial system of nomenclature
describes each living organism by two names: genus and species. For
example, in Saccharomyces cerevisiae, Saccharomyces is the genus and
cerevisiae is the species. The binomial nomenclature is always written
in italics. The genus should always have the first letter capitalized
whereas the species should always be written in lowercase.
Glucose effect: Another term for Crabtree effect.
Gundruk: A traditional fermented vegetable pickle indigenous to Nepal.
It is taken as side-dish, soup, etc.
Hayflick limit: The maximum number times a cell will multiply before
dying. The Hayfilck limit of yeast is about 10.
Heads: The initial portion of alcohol distillation. This portion contains
methanol, aldehydes, esters and similar compounds that are more
volatile than ethanol. Because of very bad smell and taste, this
portion is separated from the main fraction in alcohol distillation.
Hedonic rating: Term used in tasting panels where the judges indicate
the extent of their like or dislike for the food.
138
Heterofermentative: Organisms that use glucose through pentose
phosphate pathway (HMP pathway) and produce lactic acid, CO2
and ethanol in equal proportion. The designation is relevant to lactic
acid bacteria (LAB) such as species of Leuconostoc, Oenococcus and
Weisella. See homofermentative also.
Higher alcohol: see Fusel oil.
Homofermentative: Also called homolactic, this group of microorganism
utilizes glucose via EMP pathway and produces lactic acid as the
principle metabolite. Examples are Lactobacillus plantarum and species
of Lactococcus, Enterococcus, Pediococcus and Streptococcus. See also
heterofermentative.
Hypheal tip: The radiating tip of mycelia / hyphae.
Immobilization: Restriction of movement of enzymes and cells by
confining them in a carrier material by covalent bonding,
entrapment, encapsulation, etc.
Incubation: Providing favorable temperature, humidity, atmosphere,
etc., to allow growth of relevant microorganisms.
Infusion: A method of mashing. This technique is used for preparing
ales (see decoction).
Inoculate: To introduce microorganism into a medium. Inoculation is
usually done using inoculating loop or inoculating needle.
Inoculating loop: A thin nichrome wire with a small wire loop for
picking up and transferring, streaking or inoculating microorganisms
(see Fig. 6.1). It has a handle at the opposite end of the loop.
Inoculating needle: It is similar to inoculating loop but has no loop
(and therefore has a needle-shaped end).
Invert sugar: Sucrose (table sugar) that has undergone hydrolysis to
form a mixture of glucose and fructose.
Isolate: To segregate, to separate.
Jand: Cereal-based, undistilled, sour-sweet alcoholic beverage
indigenous to Nepal. It is similar to the African opaque beer.
Kimchi: A Korean fermented vegetable (cabbage) dish similar to
Nepalese gundruk.
Kinema: Traditional fermented soybean food indigenous to Nepal. It is
mostly prepared and eaten by Limbu people (an ethnic group of
Nepal).
Koji: A fungal (mold) starter culture used by Japanese for the
preparation of sake (Japanese rice wine), soysauce, miso, etc.
Krausen: The stage in beer fermentation when the growth of yeast is
vigorous and a thick layer of foam is produced.
139
Lager: One of two major categories of beer. It is produced using the
‘bottom yeast’ Saccharomyces carlsbergensis (synonym: uvarum). See also
ale.
Lexicon: A list of words used in particular language, or subject or a
dictionary. The sensory quality of traditional foods is often very
difficult to describe. In such cases development of lexicon becomes
very important.
Liquefaction: Random breakdown (hydrolysis) of starch by amylase
enzyme to render it liquid.
Maceration: The act of leaving food in a liquid so that it absorbs the
liquid and becomes soft. In red wine making, the crushed grapes are
inoculated with yeast and kept for primary fermentation for about a
week, which is an example of maceration.
Malo-lactic fermentation: Also called ML-fermentation in short, this is
an extended fermentation with lactic acid bacteria (Leuconostoc oenus,
in particular) to remove the excess acidity of wine. ML-fermentation
also gives mellowness / maturity to wine.
Malt: Cereal grain, usually barley, which has been allowed to germinate
for a given time period and the germination terminated by drying.
The process of preparing malt is called ‘malting’ and the expert
preparing it is called‘malster. Malt is used in many cereal-based
alcoholic fermentations. It provides enzymes for hydrolyzing the
substrate, imparts characteristic flavor, and serves as a substrate for
fermentation. In beer, it also imparts color.
Manapu: A traditional amylolytic starter culture for the production of
cereal-based alcoholic beverages, e.g., jand and raksi.
Medium: Food for in vitro (artificial environment) cultivation of
microorganisms, plants, etc.
Mesu: A traditional fermented food prepared from bamboo shoot. Mesu
is indigenous to Nepal.
Metabolite: Biomolecules resulting from the cellular reactions.
Micrometry: Measurement of size of microscopic objects. 1 micrometer
(µm) equals 10-6 meter.
Microscope: Equipment used for observing microscopic objects. It has
the dual function of magnification and resolution.
Mitochondria: Membrane-bound organelle found in most eukaryotic
cells. They are described as cellular power plants because they supply
most of the energy needed by the cell.
Molasses: See blackstrap molasses.
140
Mold: A fungus that grows in the form of multicellular filaments called
hyphae. Bread mold is the most common form of mold found in
food (see Fig. 4.1).
Morphology: The form and structure of plants, animals, and
microorganisms.
Mother culture: A starter culture taken from the previous batch. For
example, dahi (Nepalese yoghurt) requires mother culture termed
joran.
Mucor: A genus of mold.
Murcha: Starter culture similar to manapu.
Murcha plant: Term used to collectively refer to plants used in the
traditional preparation of murcha.
Must: The freshly pressed grape juice, along with skins, seeds and pulps,
intended for fermentation into wine.
Mycelium: Thread-like filaments (hyphae) of fungus (molds and
mushrooms).
MYGP: Malt Yeast-extract Glucose Peptone medium, generally used for
the growth of yeasts.
Natto: A Japanese fermented soybean food similar to kinema of Nepal.
Nigaar: Nearly limpid alcoholic extract that builds up during prolonged
fermentation of jand.
Overproof: Containing a greater proportion of alcohol than ‘proof
spirit’, especially containing more than 50% alcohol by volume.
Parasite: An organism that grows, feeds, and is sheltered on or in a
different organism, while contributing nothing to the survival of its
host.
Pasteurization: Heat treatment of liquid foods at a specified time-
temperature regime such that all the pathogens are destroyed,
without radically altering the taste and nutritional quality.
Pearson square: A quick, simple method of calculating the amount of a
supplement required to achieve the desired composition.
Peepa: A bamboo tubing used to suck in extract from tongba (see Fig.
15.1).
Petite mutants: Microorganisms with mitochondrial mutation, resulting
in respiratory-deficient strains. Such colonies are smaller (hence the
term petite) than the normal parent strains.
pH: Negative logarithm (to base 10) of hydrogen ion concentration
[H+], given by pH = -log[H+]. It represents the active acidity of a
solution/slurry. A pH of 7 is taken to be neutral. Solutions become
progressively alkaline as the pH rises from 7 to 14 and becomes
141
progressively acidic as the pH decreases from 7 to 0. A change pH
by 1 unit implies 10-fold change in acidity.
Pharmaceutical: Related to drugs used in medical treatment.
Physicochemical properties: Physical and chemical properties studied
together.
Pitch: To add inoculum (usually yeast) for alcoholic fermentation
Plate count agar (PCA): A solid medium used for enumerating
microorganism. It contains agar as the solidifying agent.
Pomace: The residue of fruits and vegetables after extracting the juice.
Press wine: Wine prepared from the clear portion of grape juice
obtained after primary fermentation (also see free-run wine).
Preservative: Any agent that arrests the spoilage (generally of food and
beverages).
Primary fermentation: The first 4-5 days of fermentation of purple
grapes in red wine making. This stage is needed for extracting the
purple color of grapes. During this stage, most of the sugar will be
utilized for alcohol formation and yeast biomass will increase by
several folds.
Proof: Unit of measurement of alcohol concentration. Generally, 1%
alcohol by volume at 60°F = 2°proof.
Proteolytic: Having the ability to break down (hydrolyze) proteins, e.g.,
proteolytic enzymes like pepsin, trypsin, etc.
Pycnometer: Glass apparatus for determining specific gravity and
density (see Fig. 7.2).
Quantitative: Related to the determination of concentration / amount
of substance
Racking: Refers to separation of the clear liquid portion from the
sediments (lees). In winery, this is done by siphoning with a flexible
pipe.
Raksi: The distillate obtained by pot-distillation of jand. The alcohol
content of raksi ranges from 20% to 50%.
Reboiler: Heat exchangers typically used to provide heat to the bottom
of industrial distillation. They boil the liquid from the bottom of a
distillation column to generate vapors which are returned to the
column to drive the distillation separation. The heat supplied to the
column by the reboiler at the bottom of the column is removed by
the condenser at the top of the column (see Fig. 8.1).
Refractometer: A device for measuring refractive index and total
soluble solids (TSS) of a solution.
Respiratory deficient mutants (RDMs): see petite mutants.
142
Restructured foods: These are products in which pieces of foodstuffs
are bound together to make it resemble the original product.
Rhizoid: A filamentous root-like outgrowth on the underside of the
thallus in mosses, molds, etc. (see Fig. 4.1).
Rhizopus: A type of mold (Fig. 4.1). Some Rhizopus strains are used for
the production of fermented foods, e.g., tempeh (a soybean food) of
Indonesia.
Ruh beer: Almost completely fermented beer, ready for lagering with
some yeast present following the completion of primary
fermentation (~ green beer).
Rum: Congeneric, distilled alcoholic beverage produced from sugarcane
byproducts (usually blackstrap molasses).
Saccharify: Convert starch by enzymatic hydrolysis into simple sugars.
Saccharine materials: Sugary materials, e.g., table sugar, dextrose,
lactose, etc.
Saccharomyces: A very common type of yeast. Its species are used in
alcoholic fermentation and as baker’s yeast.
Saprophyte: An organism, such as a fungus or a bacterium, that lives
and feeds on dead and decaying plant and animal matter.
Sauerkraut: Lactic fermented cabbage, a very common German dish.
Schizosaccharomyces: A type of yeast that reproduces by binary
fission like bacteria. Also dubbed fission yeast.
Screening: Use of selective procedures to discover microorganisms or
metabolites of interest.
Secondary fermentation: The second part of two-stage fermentation.
Has relevance to red wine and beer fermentation.
Selective medium: Microbial medium containing components that
favor the growth a given group of microorganism only. For example,
SS agar allows selective growth of Salmonella and Shigella only.
Selective pressure: The selective effect of selective medium or
condition. For instance, succession by anaerobes with the depletion
of oxygen in the medium during fermentation. Here, the lack of
oxygen favors the growth of anaerobes.
Serial dilution: Stepwise dilution of a substance in solution. Usually the
dilution factor at each step is constant, resulting in a geometric
progression of the concentration in a logarithmic fashion. A 10-fold
serial dilution could be 1M, 0.1M, 0.01M, 0.001M, etc. Serial
dilutions are used to accurately create highly diluted solutions. In
microbiology, this is done to reduce the population of the cells so
that the colonies that grow in the agar plate can be easily counted
(e.g., 30-300 colonies).
143
Shelf-life: The length of time that a commodity may be stored at
ambient condition without becoming unfit for use or consumption.
It applies to foods, beverages, pharmaceutical drugs, chemicals, and
many other perishable items. It is also defined as the recommended
maximum time for which products of fresh (harvested) produce can be stored,
during which the defined proportion of the goods remains acceptable under
expected (or specified) conditions of distribution, storage and display.
Short-circuit: In continuous fermentation, this refers to passing out of
the feed medium without proper mixing in the fermenter for
complete reaction.
Sinki: A traditional, lactic fermented radish dish.
Slant: Also called slope, an agar medium set in an inclined glass tube for
growing and preserving microbial cultures. The slanted surface
provides greater area for growth, is easier to streak, and helps in
avoiding contamination during streaking (because the tube is not
held upright).
Species: In microbiology, species refers to collection of strains that
have many stable properties in common and differ significantly from
other groups of strains (see also genus)
Specific gravity: Also called relative density, is the ratio of the density
(mass of a unit volume) of a substance to the density of a given
reference material (usually water). In simple terms, specific gravity is
the ratio of weights of equal volumes of the test substance and water
at a given temperature. It is a dimensionless quantity.
Specimen: Sample (material), a limited quantity of something which is
intended to be similar to and represent a larger amount of that
thing(s).
Spent wash: The sludge material that remains after the volatile material
(e.g., alcohol) has been removed by distillation.
Sporangiospore: An asexual spore formed within a sporangium or sac-
like structure following the division of the cytoplasm (see also
‘conidiospore’). Mucor and Rhizopus species of molds produce
sporangiospore whereas Penicillium and Aspergillus species produce
conidiospore (see Fig. 4.1).
Spore: A minute propagule lacking a preformed embryo, the smallest
being a single cell. May be formed following sexual or asexual
processes. Spores are formed in all divisions of fungi. Similar
structures formed by bacteria are called endospores.
Spot culture: A microbial growth on an agar surface prepared by
placing or planting (and not streaking) the source microorganism.
When the culture is inserted deep into a vertical agar tube with an
144
inoculating needle, the resulting growth or culture is called ‘stab
culture’.
Staining: Application of dye (or stain) to microbial or other specimen
for microscopic examination. Staining renders the otherwise-
invisible cells visible.
Starter culture: Also called starters, refers to microbial inoculum used to
begin the fermentation process in the preparation of various
fermented foods and beverages. These starters usually consist of a
cultivation medium, such as grains, seeds, flour, or nutrient liquids
that have been well colonized by the microorganisms used for
fermentation. For instance, a small amount of yoghurt from an
earlier batch can be used as starter for the next batch.
Statistical test: A tool for the treatment of research data to obtain
objective inference.
Sterilize: To remove all life forms. In the laboratory, this is done to kill
contaminants in media, glassware, air, etc. Autoclaving of media,
flaming of inoculating loop before streaking, etc., are examples of
sterilization.
Subculture: Transfer and maintenance of cultures in a fresh medium.
Support material: Material that serves as a physical carrier of
microorganisms, enzymes, etc.
Tails: The higher-boiling fraction in distillation (see fusel oils).
Tape culture: Microscopic slide of mold culture prepared by sticking
the colonies in cellotape followed by fixing on the slide (see Fig.
4.2).
Thermostable: Not negatively affected by high temperatures.
Tongba: A variation in serving jaand (see Fig. 15.1).
Triphenyl tetrazolium chloride (TTC): A colorless salt used in
differentiating petite mutant yeasts and normal yeasts.
TSS: Total soluble solids expressed in percentage by weight. The
measurement can be directly taken using an optical instrument called
refractometer (see refractometer).
Underproof: Strength of alcohol below 50% alcohol by volume (see
proof).
Wash: Fermented ethanolic solution, ready for distillation (see also
‘spent wash’).
Wash-out: A condition in continuous fermentation where the cells are
removed from the outlet at a rate greater than the rate of its
formation in the fermenter, eventually leading to complete removal
of the cells from the fermenter.
145
Wine: Alcoholic beverage produced by partial or complete fermentation
of grape juice.
Wort: Liquid extracts of malt and adjuncts ready for fermentation to
produce beer.
Yeast: A unicellular fungus.
YEP-lactate agar: Yeast extract peptone lactate agar medium. The
medium is used to confirm whether or not the test yeast is a
respiratory deficient mutant.
YPD agar: Yeast peptone dextrose agar medium. The medium is used
as a basic growth medium for yeast before overlaying the plates with
TTC agar for detecting the petite mutants.
Zone of inhibition: A clear region around the colony arising due to
inhibition of growth of the test organism by a test compound or
metabolite (e.g., antibiotic).
Zygospores: A diploid reproductive stage in the life cycle of many fungi
and protists. Zygospores are created by the nuclear fusion of hyphae
of different mating types. Zygospores remain dormant until the
condition becomes favorable for germination.
Zymase: An enzyme complex that catalyzes the fermentation of sugar
into ethanol and CO2. It naturally occurs in yeast.
146

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Basic Practical Manual on Industrial Microbiology

First Edition: 2016

This practical manual is basically meant for Food Technology

Each practical described in this manual consists of following

Although the experiments given in this manual follow some sort of

Students should be encouraged to find answers to the thought-

This practical manual has been prepared for students of the

The manual includes many non-standard methods also, some of

Central Campus of Technology Basanta K. Rai

Note to the users iii

SECTION II: Screening and culture preservation

SECTION III: Fermentation and fermented products

SECTION IV: Amylolytic starters

SECTION V: Traditional fermented foods

SECTION VI: Miscellaneous practicals

Fig. 1.1 Calibration of ocular micrometer for a given objective 6

Fig. 10.1 Equipment needed for wine making 59

In microscopy, micrometry refers to the measurement of the size of

A light microscope cannot resolve sizes less than 0.2 µ. At best, it

The graduation inscribed in ocular micrometer is arbitrary while

The ocular micrometer must be recalibrated every time micrometric

1. Cells appear less shriveled and distorted because no heat-fixing is

1. To become familiar with micrometry and related calculations

Measurement of dimensions of microorganisms is done under

1. Stage and ocular micrometer

1. Calibrate the ocular micrometer (for the oil immersion objective

1. End-to-end or center-to-center superimposed readings of ocular

1. Calculation for calibration of the ocular division

1. Can you use micrometry as an aid in the determination of

Cell size measurement

Average length ± sample standard deviation = 6 ± 1.63 μ

Proceed in similar way for breadth of the yeast cells.

Fig. 1.1 Calibration of ocular micrometer for a given objective lens

Cellotape Mineral oil

Fig. 1.3 Fixing of cellotape on smear

Fig. 1.4 Appearance of cells through ocular micrometer

The standard method of determining microbial population is serial

1. To carry out micrometric calculations

A uniform smear of the test organism on a standard area (on the

1. Stage and ocular micrometer

1. Take 1 ml of the actively growing culture (yeast) in a clean test

Results and discussion

1. Calculation of area of the microscopic field

1. Using this method, is it possible to count only the viable cells?

Let us assume that we got following data:

The dilution performed before preparing the slide is 1000 folds

Area of the field = (π×2002/4) μ2 = ~ 31416 μ2

Trim this portion with Bounded area for

Fig. 2.1 Preparation of slide for DMC

Fig. 2.2 Vortex mixing of cells

Replica plating is a microbiological technique in which one or more

The purpose of replica plating is to be able to compare the master

1. To study the working principle of replica plating

A culture plate (master plate) containing well developed colonies is

1. Master plate (containing well-developed, 30-300 colonies)

1. Assemble the replica plating unit. Maintain asepsis during fixing

1. Imprint on the velveteen

1. How can you carry out test of sugar assimilation (auxanography)