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PRACTICAL MANUAL CUM WORKBOOK on INDUSTRIAL MICROBIOLOGY

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 Practical Manual Cum Work Book
INDUSTRIAL MICROBIOLOGY

T.C.K. Sugitha
P. Raja
R. Rajesh
U. Sivakumar

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

DIRECTORATE OF NATURAL RESOURCE MANAGEMENT
TAMIL NADU AGRICULTURAL UNIVERSITY
2019
Authors : T.C.K. Sugitha,P. Raja,R. Rajesh, U. Sivakumar
ISBN No:

9 789387 443167
 Ex.No.1 Isolation of Yeast & Lactic acid bacteria

Industrial microorganisms are capable of producing industrially important products and

need to be selected carefully. To a greater extent, industrial microbes are metabolic specialists
capable of producing particular metabolites specifically and to high yield. Industrial microbes
may show many altered cellular and biochemical properties.

Criteria for selecting an industrially useful microorganism:

 It should be a high yielding strain.

 It should have stable biochemical characters.
 It should not produce undesirable/toxic substances.
 It should be easily cultivated on a large scale.

I) Isolation of yeast:

Yeast is a unicellular fungi mostly classified under Ascomycetes. Yeast cells are
usually spherical, oval or cylindrical and cell division generally takes place by budding.Yeasts
usually flourish in habitats where sugars are present such as fruits, flowers and bark of trees.
The well known industrial uses of yeast are the production of alcoholic beverage and baking.
Most important commercial yeasts are the bakers’ and brewers’ yeast which belong to the
genus Saccharomyces; because they are easily manipulable eukaryotic cells. Strains of
Saccharomyces cerevisiaeare of greatest technical importance among known types of yeasts.

Materials required:

 Grapes
 YPS medium (Yeast extract –Peptone-Sodium succinate)
 Sterile water blanks (99ml & 9ml)
 Sterile petriplates&pipettes.

YPS medium composition

Yeast extract :3g
Peptone :3g
Sodium succinate : 20 g
Calcium chloride : 0.02g
Magnesium sulphate : 0.02 g
pH : 6.8
Agar : 20 g
Distilled water : 1000 ml.
Procedure:
 Weigh one gram sample of grapes, crush and dilute it in a 250 ml flask containing 99 ml
of sterile water; this makes 10-2 dilution.
 Shake well for 10- 15 min at room temperature in order to bring the surface microbial
cells dissolved in water.
 Transfer 1 ml of the aliquot from 10-2 dilution to 9 ml sterile water blank using a sterile
pipette which gives 10-3 dilution.
 Make successive dilutions, in the same way, using 9 ml sterile water blanks so as to get
dilutions up to 10-5.

Transfer aseptically 1 ml of the sample dilution from 10-3 and 10-4 to sterile Petri plates.
Maintain three replications for each dilution in order to minimize handling error.

Now pour the melted and cooled media (YPS) into the Petri plates; immediately rotate
the plates gently both in clockwise and anti-clockwise direction in order to spread the
sample dilution evenly with the medium.

Incubate the plates in an upside-down position at room temperature for 2 to 7 days.

Note: Yeast colonies appear as large, smooth and glistening colonies compared to bacterial
colonies.

Yeast colony in plates Yeast cells under microscope

(Source: www.imperial.ac.uk)

Activity

 Observe the plates for colony formation.

 Count the number of colonies per plate and find out the mean cfu for each dilution.
 Observe the colony morphology in plates and make a diagram.
 Observe the cells under the microscope and make sketches.

Population count:

No. of yeast cells (CFU) per gram of the sample = Mean no.of CFUs’ x dilution factor

II) Isolation of Lactic acid bacteria:

 Lactic acid and acetic acid bacteria are the common groups of bacteria involved in
fermented foods and related products of fermentation. Many of the fermented products
include cheese, pickles, and fermented sausages. Bacterial fermentation improves the
aroma, flavor and nutritional qualities of the product. Lactic acid bacteria ferment
carbohydrates to lactic acid. The acidic environment thus created greatly alters the
undesirable microorganisms. These are responsible for the fermented vegetables (pickles),
meat, milk etc. They are widely distributed among plants and plant products.

Lactic acid bacteria (LAB) are gram-positive, usually non –motile and non
sporulating bacteria that produce lactic acid as the major or sole product of fermentative
metabolism. The cells often appear as chains under the microscope. Most lactic acid
bacteria obtain energy only from the metabolism of sugars and related fermentable
compounds and hence are usually restricted to habitats in which sugars are present. In
general, LAB has complex nutritional requirements and needs aminoacids, vitamins,
purines, and pyrimidines. The genera that comprise LAB are at its core Lactobacillus,
Leuconostoc, Pediococcus, Lactococcus, and Streptococcus.

Materials required:
 Any fermented product viz., (curd, cheese, idly batter, buttermilk, pickles etc.)
 MRS medium (de-Man,Rogosa and Sharpe’s Agar)
 Sterile water blanks ( 99ml & 9ml)
 Sterile petriplates& pipettes.

MRS medium composition

Glucose : 20 g
Peptone : 10 g
Beef extract : 10 g
Yeast extract :5g
K2 HPO4 :2g
Sodium acetate :5g
Triammonium citrate :2g
Magnesium sulphate : 0.2g
Manganese sulphate : 0.05g
Tween 80 : 1ml
Agar : 20 g
Distilled water : 1000 ml.
pH : 6.0

Procedure:

 Weigh one gram or one ml of the sample and dilute it in a 250 ml flask containing 99
ml of sterile water; this gives 10-2 dilution.
  Make successive dilutions in the same way using 9 ml sterile water blanks up to 10-4
dilution.

Transfer aseptically 1 ml of the sample dilution from 10-3 and 10-4 to sterile petriplates.
Maintain three replications for each dilution in order to minimize handling error.

Now pour the melted and cooled media (MRS) into the Petri plates; immediately rotate
the plates gently both in clockwise and anti-clockwise direction in order to spread the
sample dilution evenly with the medium.

Incubate the plates upside-down position at 30± 4ºC for 2 to 4 days.

Activity

 Observe the plates for colony formation.

 Count the no. of colonies per plate and find out the mean cfufor each dilution.
 Observe the colony morphology in plates and make a diagram.
 Observe the cells under the microscope and make suitable drawings.

Note:Lactobacillus sp. forms small, creamy, white colonies on MRS medium which are
generally distinct from other bacterial colonies.

LAB colonies inplates Different spp. of LAB cells under microscope

(Source: Todars, 2014)

Population count:

No. of yeast cells (cfu) per gram of the sample = Mean no.ofCFUs’ x dilution factor

Results:
Review Questions:

1. What are the prerequisites for a microbe to be used for the industrial purposes?
2. Can we isolate yeast from fermented pickles? Why?
3. What is the end product if the souring of wine occurs?
4. Mention the different species of Lactobacillus.
5. What are all the end products in the case of yeast and LAB fermentation?
6. What is the purpose of serial dilution?
 Ex.No.2 Estimation of Yeast biomass and Lactic acid
bacteria under different nutrient composition

The microbial population is affected by a number of factors, the control of which is

essential foroptimal activity. These factors include pH, temperature, nutrient availability, and
theconcentration of available nutrients. By determining most influencing factors, these
variables can be controlled in the fermentation process.While bacteria thrive on many different
types of food, yeasts require carbohydrates, such assugar and starch. From these, they produce
carbon dioxide (CO2) gas and alcohol. Thus,they have been useful to man for centuries in the
production of certain foods and beverages.

Some microbes are psychrotrophs, meaning that they can grow at relatively low
temperatures. The fermentation of wines and beer is often carried out at temperatures near 7oC.
This alsomeans that they can create a spoilage problem in meat coolers and other refrigerated
storageareas. Since temperature and pH are not in the scope, we deal with various nutrient
sources and how they affect the growth of yeast and lactic acid bacteria in this experiment.

Nutritional Requirements

Every organism must find in its environment all of the substances required for energy
generation and cellular biosynthesis. The chemicals and elements of this environment that are
utilized for microbial growth are referred to as nutrients or nutritional requirements. Many
microbes can be grown in culture media which are designed to provide all the essential
nutrients in solution.Microbes that are symbionts or obligate intracellular parasites of other
cells, usually eucaryotic cells, are (not unexpectedly) difficult to grow outside of their natural
host cells. Whether the microbe is a mutualist or parasite, the host cell must ultimately provide
the nutritional requirements of its residents.

The Major Elements

At an elementary level, the nutritional requirements of a bacterium and yeast revealed

by the cell's elemental composition, which consists of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and
traces of Zn, Co, Cu, and Mo. These elements are found in the form of water, inorganic ions,
small molecules, and macromolecules which serve either a structural or functional role in the
cells.

Trace Elements

Trace elements are metal ions required by certain cells in such small amounts that it is
difficult to detect (measure) them, and it is not necessary to add them to culture media as
nutrients. Trace elements are required in such small amounts that they are present as
"contaminants" of the water or other media components. As metal ions, the trace elements
usually act as cofactors for essential enzymatic reactions in the cell. One organism's trace
element may be another's required element and vice-versa, but the usual cations that qualify as
trace elements in bacterial nutrition are Mn, Co, Zn, Cu, and Mo.

Carbon and Energy Sources

Carbon is the structural backbone of the organic compounds that make up a living cell.
Based on their source of carbon,microbes can be classified as autotrophs or
heterotrophs.Industrial production aims at efficient conversion of sugar feedstock into yeast
biomass mainly in the later stages where biomass volume is high. Eg.Glucose, sucrose, fructose
etc.

Nitrogen source

Nitrogen is needed for the synthesis of amino acids, DNA, RNA, and ATP. Depending
on the organism, nitrogen, nitrates, ammonia, or organic nitrogen compounds may be used as a
nitrogen source.A few examples of organic sources are peptone, yeast extract, beef extract, etc.

Growth of Yeast and Lactic acid bacteria

Yeast is a unicellular eukaryote, whereas lactic acid bacteria are a prokaryotic probiotic.
Both of them differ in their nutritional requirements. The nutrient source for maximum growth
of yeast and lactic acid bacteria is assessed by their growth pattern in various carbon and
nitrogen sources. There are two methods for growth determination: a) Indirect b) direct

Growth is an increase in the number of the population rather than size. Yeast multiplies
by budding while lactic acid bacteria by binary fission. When growing exponentially by binary
fission, the increase in bacterial population is by geometric progression. The generation time is
the time interval required for the cells to divide. The time required for the population to double
is termed as doubling time.

No of generations (n)
ℎ –
=
0.301

Generation time =

a)Direct methods

It includes:
 Filtration
  Plate count
 Direct Microscopic count
 Most Probable number
b) Indirect Methods of Measurement

1. Turbidity: As bacteria multiply in media, it becomes turbid and % transmission or

absorbance determined by using a spectrophotometer.

Disadvantages:

 It cannot distinguish between live and dead bacteria.

 It requires a high concentration of bacteria (10 to 100 million cells/ml).

Advantages:: No incubation time required.

Fig 1. Typical growth curve of a bacteria (Source: www.microbialgrowth101.weebly.com)

2. Metabolic Activity:

Ass the microbe grows in the culture media, they produce certain metabolic products
such as carbon dioxide andacids. Quantification of these metabolic products provides details
about microbial growth. Inaddition to this, cellular protein can also be estimate
estimated and correlated
with turbidity and cell count.

Disadvantage: Measuring the metabolic products is expensive

3.Dry Weight/Biomass:

The microbial growth is determined on a weight basis. Bacteria or fungi in liquid media
are centrifuged and the resulting ccell pellet is weighed.

Disadvantage: Doesn’t distinguish live and dead cells.

Materials Required:

Yeast cells
Lactic acid bacteria culture
YPD broth
Peptic digest of animal tissue :20.0 g
Yeast extract : 10.0 g
Dextrose :20.0 g
Distilled water : 1000 ml.
pH : 6.5

MRS broth
Glucose : 20 g
Peptone : 10 g
Beef extract : 10 g
Yeast extract :5g
K2 HPO4 :2g
Sodium acetate :5g
Triammonium citrate :2g
Magnesium sulphate : 0.2g
Manganese sulphate : 0.05g
Tween 80 : 1ml
Distilled water : 1000 ml.
pH : 6.0

C sources: glucose, sucrose, fructose, maltose, mannitol

N sources : Peptone, Ammonium chloride

Procedure:

 Prepare YPD and MRS broth with given C and N sources

 Inoculate fresh culture of yeast and lactic acid bacteria (logarithmic phase) @ 2%
 Incubate at 30±2 oC for 24- 48 hours
 Harvest the cells by centrifugation at 10000 rpm for 5 min
 Transfer the cell pellet into a pre-weighed filter paper and observe the fresh weight
 Dry the pellet in a hot air oven at 105oC until a constant weight is achieved
 Observe the dry weight
 Express the biomass of yeast interms of mg/L or mg/ g of C source.

Note: Alternatively, the cells can be filtered through a sterile pre-weighed filter paper. The
fresh and dry weight is measured as mentioned above.
Growth pattern of Lactic acid bacteria

 Inoculate the given lactic acid bacteria (2% inoculum) into MRS broth prepared with
different C and N sources
 Incubate at 30±2 oC for 24- 48 hours
 Observe the growth by measuring the OD at 600 nm at every 2 h interval
 Plot the absorbance values against time in a semi log graph paper
 Plate count the corresponding population in the respective medium at every 2 hours
 Calculate the number of generations (n) and generation time (t).

Activity:

 Prepare YPD and MRS broth with the given C and N source
 Measure the absorbance at regular interval
 Determine the fresh and dry weight
 Plot graph based on OD value and cell population
 Work out the generation time
 Observe the most preferred C and N source for maximum growth

Results:

Review Questions:

1. What are the different phases of bacterial growth?

2. What happens when a microbial population is transferred from rich medium to poor or
minimal medium?
3. What is actually assessed in the turbidimetric method?
4. Differentiate autotrophs and heterotrophs?
5. What are the factors required for the successful cultivation of microbes?
6. If you are an entrepreneur, planning for yeast-based single-cell production, what are the
criteria for you consider for enhancing yeast biomass production?
 Ex.No.3 Screening of LAB for probiotic Characters –
PH, Bile salt

Probiotics are live microorganisms beneficial to host. According to FAO/WHO, probiotics

are termed as “Live microorganisms which when administered in adequate amounts confer a
health benefit on the hosts” eg. Yeast, Lactobacillus, Bifidobacteria, etc.Prebiotics are non-
digestible component which beneficially affects the host by selectively stimulating the growth
or activity of one or a limited number of colonic bacteria, thereby improving the health of the
host. It includes dietery fibre and carbohydrates. Strains
of Lactobacillus and Bifidobacterium have been extensively used as probiotic microorganisms
for humans. The mode of action of probiotics are:

i) Competitive exclusion
ii) Production of bacteriocin
iii) Production of orgaic acid
iv) Altered absorption of intestinal mucosa

Characteristics of effective probiotics:

An efficient probiotic microorganism should be able to:

a) survive the passage through digestive system

b) attach to intestinal epithelia and colonize
c) maintain good viability
d) utilize the nutrients and substances in normal diet
e) non pathogenic and non-toxic
f) capable of exerting a beneficial effect on host

In order to reach the colon in a viable state, they must cope with specific stress
challenges throughout the gastrointestinal tract, among which the presence of bile in the upper
parts of the small intestine is one of the main ones. The main components of bile are bile acids,
which are produced and conjugated with the amino acids glycine or taurine in the liver, to
generate conjugated bile salts. Bile is stored in the gall bladder and flows from there to the
duodenum during digestion, facilitating the solubilization and absorption of dietary fats. Thus,
under normal physiological conditions, our intestine holds a bile salt concentration gradient
ranging from more than 40 mM to less than 1 mM – equivalent to a range between 2% and
0.05% – which is responsible, among other factors, for shaping the microbial community
profile found in our gut.

Apart from its normal physiological function, bile is highly toxic for those
microorganisms unadapted to the intestinal conditions. Therefore, enteric bacteria, including
lactobacilli and bifidobacteria, must have evolved specific defense mechanisms to resist the
deleterious action caused by these compounds. The strong lipophilic nature of the steroid ring
makes the cell membrane the main target of these molecules, in which they disturb the lipid
packaging and disrupt the proton motive force, causing cell death. Furthermore, since the
unconjugated forms are weak acids, they can passively diffuse into the cell and, once inside,
they are dissociated producing acidification of the cytoplasm. Other side effects induced by
bile have been documented, including induction of oxidative stress and DNA repair
mechanisms, alterations of sugar metabolism, and protein misfolding. The pH of gastric acid is
1.5 to 3.5 in the human stomach lumen. Hence it is necessary to asses the pH and bile salt
tolerance of probiotic microorganisms prior to formulation development.

Materials required

MRS broth
Bilesalt
LAB culture
Oakridge tubes
Phosphate buffered saline
Procedure
1.Bile salt tolerance test (Al-Saleh et al., 2006)

Estimatehe ability of the selected lactic acid bacteria (LAB) strains to survive in bile
salt by determining their growth rate in MRS broth containing different levels of bile salt
(oxgall).

 Inoculate freshly prepared LAB strains at the rate of 1 per cent inoculum into sterile
MRS broth amended with bile salt (0.3%, 0.5%, and 0.7%)
 Incubate at 37˚C for 24 h under aerobic conditions in the incubator at 120 rpm.
 Measure the growth as optical density periodically at 0, 2, 4, 6, 8 and 24 h
spectrophotometrically at 620 nm.
pH tolerance (Buntin et al., 2008).

 Harvest LAB cells were grown in MRS broth at 37˚C under aerobic conditions
 Wash the cell pellets twice, and finally, resuspended in10 ml of phosphate-buffered
saline (PBS) in order to achieve 109 CFU ml-1
 Inoculate phosphate-buffered saline of pH values of 2, 3 and 7.0 and 9.0 (adjusted using
5M HCl and NaOH)
 Incubate at 37˚C, and determine the viability of the inoculated cells after exposure to
the acidic environment
 The plate on the MRS agar plates for 0, 1, 2, 3 and 24 h and incubate at 37˚C for 48 h
 Estimate the survival based on the number of colonies grown on the MRS agar plates
on comparison with the initial cell concentration

Activity

 Prepare MRS plate amended with different concentrations of bile salt ((0.3%, 0.5%, and
0.7%)
 Streak the given LAB culture and incubate
 Prepare MRS broth with different pH (2, 3 and 7.0 and 9.0)
 Inoculate the given LAB culture
 Measure the absorbance
 Observe the maximum tolerant concentration of bile and pH by the given LAB culture

Results:

Review Questions:

1.Why we should study the pH tolerance of LAB?

2.What is GRAS?

3.Name any two commercial formulations of probiotics?

4.Which is the largest group of probiotics in the human gut?

 Ex.No.4 Screening of LAB for probiotic Characters-
antimicrobial activity

Functional foods exert health benefits to the consumers and dominate the food market.
These functional foods targetedtowards improving the balance and activity of the intestinal
microflora. Consumption of food containing live bacteria is the oldest and still most widely
used way to increase the number of advantageous bacteria called "probiotics" in the intestinal
tract. Noteworthy, there are a large number of probiotic foods that date back to ancient times
which are mostly originated from fermented foods as well as cultured milk products. The quest
to find food ingredients with valuable bioactive properties has encouraged interest in lactic acid
bacteria (LAB) with probiotic attributes such as antimicrobial activity against pathogenic
microorganisms antiviral activity], anti-yeast property, antimutagenic, antiplatelet aggregation,
and antioxidant attributes etc. In general, it is believed that probiotics help keep up the balance
between harmful and beneficial bacteria in the gut thus maintaining a healthy digestive system.
The health benefits of probiotics have always been investigated with regard to their capability
to sustain their availability, viability, digestibility, and rendering of their health benefits to the
host without altering the safety and the organoleptic properties of the food in which they have
been incorporated. Many viable probiotic strains with beneficial functional properties are
available in the market as components of foods and beverages, in fermented dairy products like
yogurt or as probiotic fortified foods as well as food preservatives.

The health claims of probiotics range from regulation of bowel activity and well-being
to more specific actions such as the antagonistic effect on the gastroenteric pathogens like
Clostridium difficile, Campylobacter jejuni, Helicobacter pylori and Rotavirustargeted. The
mechanism of action of probiotics with anti-microbial properties is maybe due to:

 Production of bacteriocins such as nicin or lowering the pH by producing acidic

compounds like lactic acid.
 Compete with other infectious bacteria for nutrients and cell-surface and help toward
them off by inhibiting their colonization.
 Active enzymes which inhibit other pathogenic bacteria.
Materials Required:

 LAB strains
 MRS media
 LB media
 Pathogenic strains: E. coli, S. aureus, Y. enterocolitica and B. cereus
 Nutrient Agar medium

Procedure

I. Preparation of sample filtrate

 Inoculate LAB isolates from slants to fresh 250 ml MRS broth and incubate at 37oC for
48 h.
 Centrifuge the culture broth at 10,000 × g for 10 minutes.
 Collect the supernatant after centrifugation and pass through 0.2 µm sterile syringe
filter to obtain cell-free supernatant broths

II. Growth of pathogens

 Inoculate pure cultures of foodborne pathogens, namely Escherichia coli,

Staphylococcusaureus, Yersinia enterocolitica and Bacillus cereus from slants to brain
heart infusion broth (BHIB) and incubate at 37oC for 24 hrs.
 Suspend the cell pellet in 9 ml saline.

III. Antimicrobial activity test by agar well diffusion method

 Prepare a lawn of the indicator strain by spreading the cell suspension over the surface
of nutrient agar plates with a sterile cotton swab.
 Dry the plates and cut uniform wells using a sterile cork borer of diameter (5 mm)
 Fill each well with the cell-free filtrate obtained from the LAB (60 l) and incubate for
48 hours
 Observe for the zone of inhibition

Inference

Results are considered positive if the diameter (mm) of the ZOI was greater than 1mm.

Activity

 Plate the given pathogenic strains

 Fill cell-free culture filtrate of LAB in the wells
 Observe for the zone of inhibition and measure the diameter
Results:

Review Questions:

1.Why did you use the cell-free culture extract of LAB for antimicrobial activity?

2. What is bacteriocin and how does it help to maintain a healthy gut?

3. Do you think probiotics can mitigate lifestyle disorders? How?

4. Can you suggest a few methods/ food enrich the probiotic population in human beings?
 Ex.No.5 Leavening Properties of Yeast

Bread making by fermentation is an ancient process. Yeast, the most common one
being S. cerevisiae, is used in baking as a leavening agent, where it converts the fermentable
sugars present in the dough into carbon dioxide. This causes the dough to expand or rise as gas
forms pockets or bubbles. When the dough is baked, the yeast dies and the air pockets "set",
giving the baked product a soft and spongy texture. Most yeasts used in baking are of the same
species common in alcoholic fermentation. In addition, Saccharomyces exiguus (also known as
S. minor), a wild yeast found on plants, fruits, and grains, is occasionally used for baking. In
bread making, the yeast initially respires aerobically, producing carbon dioxide and water.
When the oxygen is depleted, anaerobic respiration begins, producing ethanol as a waste
product; however, this evaporates during baking. Thus the leavening property of yeast depends
on its metabolic activity to

a) Produce CO2
b) Sugar metabolism especially maltose

i) Incorporation of CO2:

Yeast utilizes the hexose sugars, especially maltose, to produce CO2, ethanol, and a
variety of secondary metabolites such as esters, aldehydes, and amino acids that contribute to
the development of flavor and aroma of the fermented food. The carbon dioxide thus produced
is responsible for not only increasing the volume of dough (leavening process) through gas
incorporation but also a valuable addition to the flavor and texture. Besides, the fermentation
products like vitamins and amino acids are responsible for the health and nutritional benefits
that are obtained from bread.

When yeast is used for making bread, it is mixed with flour, salt, and warm water or
milk. The dough is kneaded until it is smooth and then left to rise, sometimes until it has
doubled in size. Some bread doughs knock back after one rising and left to rise again by
kneading. Longer rising time gives better flavor, but if it is left for too long initially, the yeast
can fail to raise the bread in the final stages. The dough is then shaped into loaves, left to rise
until it is to the correct size, and then baked.

ii) Amylolytic activity

Wheat flour dough contains different sources of fermentable sugars. There are some
free saccharides naturally present in wheat flour. Their amounts are, however, relatively low
ranging from approximately 0.05% (for glucose, fructose, and maltose) to 0.2% to 0.3% (for
sucrose and raffinose). In wheat bran, however, relatively high sucrose concentrations (1.75%
to 3.0%) are present. Therefore, the wholemeal contains higher concentrations of free
saccharides than flour. Indeed, sucrose concentrations of 0.9% and 1.1% were reported for
whole meal dough.

Because of the relatively low amount of free saccharides in wheat flour, the majority of
fermentable sugars in the dough are generated by degradation of damaged starch by amylases.
Damaged starch refers to starch granules that are damaged during milling, and its amount
ranges from 5% to 8% (flour basis) in hard wheat flours obtained by roller milling. Damaged
starch is degraded to the fermentable sugar maltose by amylases that act on the α‐(1,4)‐ and/or
α‐(1,6)‐linkages of the starch polymers. Two types of amylases are present in wheat flour:
α‐amylases and β‐amylases. α‐Amylases are endo‐amylases that hydrolyze the α‐(1,4)‐linkages
inside the starch chain more or less randomly, thereby generating oligosaccharides and α‐limit
dextrins. β‐Amylases are exo‐acting enzymes that cleave maltose from the non‐reducing end of
the starch chain. The ability of a flour‐water suspension to produce maltose is known as the
amylolytic activity of the flour. In unyeasted dough samples, the enzymatic degradation of
damaged starch leads to a very fast increase of maltose levels during mixing and the first
minutes of incubation (0.1% in flour to 1% after mixing and 2% after 180 min of incubation).

Maltose levels in dough depend on the total damaged starch content and the α‐amylase
activity of the flour. Doughs prepared from flours with higher α‐amylase activities or higher
damaged starch contents will, therefore, contain higher maltose levels. Since α‐amylase activity
is often limited in wheat flour, fungal α‐amylase or α‐amylase from the malt is often added to
wheat flour to increase the level of fermentable sugars in the dough. Hence it is essential for
efficient amylase producing yeast strains for dough leavening.

Objective:

To affirm the leavening properties of yeast i) dough raising ii) amylolytic activity

Materials required:
Wheat flour - 1 kg
Dehydrated yeast - 30g
Salt - 15g
Sugar - 200g
Water - 500-600 ml
 Starch agar medium (pH 7.0)
Starch (soluble) 20.0g
Peptone 5.0g
Beef extract 3.0g
Agar 15.0g
Distilled water 1000.0 ml
 Gram’s iodine solution
 Sterile petriplates
 Dropper
 Inoculating loop
Procedure
Yeast cultivation and Dough Leavening
 Culture given yeast strains in conical flasks (250 ml media) containing yeast extract,
peptone, and sucrose.
 Incubate in the shaker at 30°C for 72 hours
 Collect yeast pellets after centrifugation at 10,000 rpm 5 min.
 Weigh 50-gram wheat flour and mix with 1% salt
 Dissolve 6% sugar in lukewarm water and add 0.6 g yeast pellets in the sugar solution
to allow its activation.
 Pour the activated yeast solution in the flour and mix well.
 Use the dough without any yeast as a negative control.
 Incubating the dough at 30°C for 2 hours for proofing.
 In order to assess the rise of the dough level, take 10 g of the dough mixture in
measuring cylinder.
 Incubate and note the level at every half hour (Karki et al., 2017).
 Bake in a hot air oven at 180°C for 20 minutes.

Amylolytic activity

 Melt the starch agar medium, cool to 450C and pour into sterile Petri plates
 Allow for solidification
 Label each of the starch agar plates with the name of the organism to be inoculated
 Make a single streak of each organism into the centre of the respective plate aseptically
 Incubate the bacterial plates for 48 hours and fungal plates for 72-96 hours in an
inverted position
 Flood the surface of the plates with iodine solution with a dropper for 30 seconds
 Pour off the excess iodine solution

Activity
 Knead the given wheat flour
 Compare the dough raising in yeasted and unyeasted wheatflour
 Check for the amylolytic activity of given yeast strain
 Examine the plates for the starch hydrolysis around the line of growth of each
organism, ie., the colour change of the medium.
 Record the results and discuss

Questions:

1. What is the other name for Saccharomyces cerevisiae?

2. Define Kneading and Panning.
3. What is amylase?
4. What is the function of amylase?
5. Name at least 5 microorganisms produce the enzyme amylase.
6. Mention the industrial applications of amylase.
7. What is the reaction of amylase with starch?
8. What is the role of Gram’s Iodine in the starch hydrolysis test?
Ex.No.6 Fermentor -components
components and functions

Fermentation is the process of conversion of organic substrates to the desirable

products by the action of microorganism viz., bacteria, molds, and yeasts. The fermentation
process consists of three stages.

Stage I Upstream processing which involv involves

es preparation of liquid medium,
separation of particulate & inhibitory chemicals from the medium,
sterilization, air purification.
Stage II Fermentation which involves the conversion of substrates to desired
product with the help of biological agents suc
suchh as microorganisms
Stage III Downstream processing involves separation of cells from the fermentation
broth, purification and concentration of desired product & waste disposal
or recycle.

Fermenter is the culture vessel where the fermentation process iiss carried out. The main
function of the fermenter is to provide a controlled environment with a particular nutrient
media for the growth of microorganisms to produce the target product that may be either cell
biomass, a metabolite or bioconversion product.

Substrate: Substrate may be solid, liquid or any waste material in which the
microorganisms grow and yield the product.

Parts of the fermenter:

.
 Fermenter consists of two major parts viz., basic, and monitoring/ controlling parts.
Basic parts are necessary for designing the fermenter unit whereas monitoring or control parts
are necessary for fermenter operation

a. Basic parts:
Fermentation Usually, the fermentation vessel is made out of copper / stainless
vessel: steel or mild steel coated with glass or epoxy material with ports
for various outputs, inputs and probes. The vessel should have a
smooth inner surface. The vessel should be strong enough to
withstand the internal pressure. It should be resistant to corrosion
and free from any toxic effect for the microbial culture. Based on
the capacity of the vessel it is grouped into three as a small, pilot,
and large-sized fermentor.

Top plate cover (made of steel)

Clamp top plate compressed onto the vessel using a clamp

Seal Separates the top plate from the vessel (glass) to prevent air
leakage. Sealing is an important criterion to maintain airtight
condition, aseptic and containment. Sealing ensures airtight
conditions despite the expansion of the vessel during
fermentation.
Drive motor used to drive the mixing shaft

Driveshaft mixes the medium evenly with its impeller

Marine impeller for plant tissue culture

Baffles prevent sedimentation on sides and imparts proper mixing

Sparger Provide adequate aeration and agitation to meet the metabolic

requirements of microorganisms. The agitators may be of a single
blade, multiple types of impeller type.
Exit gas cooler Acts as a condenser; removes as much moisture as possible from
exhaust
Inoculation port To add sterile inoculum

Feed pumps regulates the flow rates of additives (medium, nutrients) at

variable speed
Peristaltic pumps fixed speed pumps – used for continuous sampling

Syringe pump using a syringe – mostly used in batch fermentation

a. Monitoring/ controlling parts

Pt100 temperature sensor (platinum resistance electrode)

Foam probe kept above the medium level to sense foam formation

pH electrode senses pH

O2 sensor Monitors dissolved oxygen level

Heater pad directly heats the medium

Cold finger after direct heating – used to cool the vessel contents (closed
coil/pipe to pass cool water)

variable airflow meter attached to air sparger; indicates the rate of

Rotameter
airflow into the vessel

Pressure valve attached to rotameter for safer operation

Air pump supply of air

Peristaltic pump to pump in medium, acids, bases, antifoam etc.

Why is fermenter important for successful production?

 provides contamination-free environment

 specific temperature maintenance
 maintenance of agitation and aeration
 pH control
 proper monitoring
 Dissolved Oxygen (DO)
 ports for nutrient and reagent feeding
 ports for inoculation and sampling
 fittings and geometry for scale-up,
 minimize liquid loss and growth facility for a wide range of organisms
 Aseptic environment or contamination

Construction material used for designing the fermentor depends on the fermentation
process, type of the reactor and the product nature. Eg. Wooden tanks are preferred for lactic
acid production. Typically, fermentor is designed using copper, stainless steel, glass etc. Based
on the capacity of the fermentor, it is classified into three types.
 1. Small scale fermentor ranges from 1-2 lit with the maximum up to 12-15 lit.
2. Pilot-scale fermentor ranges from 25- gallons up to 2000 gallons.
3. Large scale fermentor ranges from 5000 – 10,000 gallon to 1,00,000 gallons.

Operation procedure:

 The fermenter is working under the principle of ‘steam under pressure.’

 The medium to be prepared is usually added directly to the fermenter and sterilized
(Normally, the working volume in a fermentor is always less than the actual volume).
 All the sampling/feed inlets and other ports are designed aseptically to ensure zero
levels of contamination.
 Necessary foam controls are provided in the fermenter (for that there should be
sufficient head space for aeration, splashing, and foaming of aqueous medium).
 Aerators and agitators are constantly stirred for constant aeration/agitation.
 After sterilization of the medium, allow it to cool to attain room temperature
(28±2ºC)
 Inoculate the required microbial inoculums @ 10 % to the fermenter through the
inoculation port (with proper sterilization)
 Allow it to grow till it attains the sufficient growth stage.
 The final products after fermentation are separated out by filtration, foam separation,
centrifugation or by membrane separation techniques.

Activity:

Observe the different parts of the fermenter and draw the diagram.

Observation:

Review Questions:

1. Why the fermenter is also called as ‘bioreactor’?

2. The fermenter vessel is mostly cylindrical. Why? What is its significance?
 Ex.No.7 Solid state fermentation – Mushroom
production

In contrast to submerged (SmF) fermentation, Solid State Fermentation (SSF) is defined

as any fermentation process allowing the growth of microorganisms on moist solid materials in
the absence of free-flowing water. The low moisture content means that fermentation can only
be carried out by a limited number of microorganisms, mainly yeasts and fungi some bacteria
also been used.

SSF promoted higher yields and better product characteristics than cultivation in the
liquid medium. In addition, costs are much lower due to the efficient utilization and value
addition of wastes. The main drawback of this type of cultivation concerns the scale-up of the
process, largely due to heat transfer and culture homogeneity problems.

SSF processes generally employ a natural raw material as carbon and energy source; the
nature of the solid substrate is one of the most important factor affecting SSF processes and its
selection depends on several factors mainly related factors cost and availability. The substrates
normally used are cellulose, sugars, and starch material nitrogen sources (ammonia, urea,
peptides) and mineral salts. Paddy straw is lignocellulosic waste material and the major
constituents of lignocellulose are cellulose, hemicelluloses, and lignin. Lignocellulosic waste
material yields a variety of products viz., enzymes, flavors, organic acids, single-cell protein,
and bioactive compounds.

Objective: Solid-state fermentation using cellulose waste material (paddy straw) for SCP
(Mushroom) production using Pleurotus sajor-caju.

Production of Mushroom by solid substrate fermentation

Paddy straw is a lignocellulosic material comprising roughly about 35-55% cellulose,

20-40% hemicellulose and 10-25% lignin. This by-product, rich in cellulose, can be effectively
utilized for the production of value-added products by the action of certain microorganisms
possessing the enzyme cellulase. One good example is the fermentation of cellulosic materials
present in paddy straw by the fungus Pleurotus sajar-caju resulting in the production of protein-
rich mushroom. Paddy straw serves as a suitable substrate for the growth of this mushroom
fungus.

Base materials required

 Pure culture of the mushroom fungus, Pleurotus sp.

 Potato Dextrose Agar medium (PDA)
Potato juice - 250g
Dextrose - 20g
Agar - 20g
Distilled water - 1000 ml
pH - 7.0
 Sorghum or wheat grains
 High density polypropylene bags (30x15cm size and 150 gauge thick) / saline bottles
(for spawn production)
 Calcium carbonate
 Non-absorbent cotton
 Polythene bags (60x30cm or 75x45cm) (for mushroom production)
 Processed paddy straw

Spawn production

i. Select healthy and whole sorghum grains

ii. Put the grains in water. Remove the chaffy and damaged grains which float on the top.
iii. Half cook the grains for 30 minutes in boiling water to soften them. Grains should not
be sticky but should slightly break
iv. Drain the excess water, spread the grains over a clean sac or cloth to dry them (approx.
30 min)
v. For every one kg of grains, mix 20g of calcium carbonate thoroughly (This is to adjust
the pH of the grains, to remove excess moisture and to prevent the grains from sticking
together)
vi. Fill the grains in clean glucose/saline bottles or in poly bags @ 250-300g, plug them
with non-absorbant cotton, cover with aluminum foil
vii. Autoclave the bottles/bags at 1.42 kg/cm2 pressure for 90 minutes.
viii. Cool them to room temperature and inoculate with a pure culture of Pleurotus
fungus aseptically
ix. Incubate the bags for 10-15 days to obtain complete growth of white mycelium
covering the entire spawn bag. This is known as the “Mother Spawn”
x. Use the mother spawn within 25-35 days for making further generations known as the
“Bed Spawn”. From each mother spawn bag (18-30 days old), 25-30 bed spawn bags
can be produced by transferring 10g of spawn from mother spawn bag to bed spawn
bag
Preparation of mushroom beds

1. Processing of substrate

i. Cut the raw paddy straw into bits of 3-5cm

ii. Soak them in water for 4-6 hours
iii. Drain the water and boil them at 800c for one hour (or) treat the straw with steam at
1.2kg/cm2 for 30-45 min
iv. Drain the water and spread the straw on a clean sac previously soaked in Carbendazim
solution or potassium permanganate solution @ 1g/lt
v. Shade dry to 65-70% moisture (water should not drip when squeezed by hand)
vi. 1 kg of straw when boiled and shade dried weighs about 4-5 Kg.

2. Spawning

i. Take a polythene bag of the given specification, tie it at the bottom to provide a flat
circular bottom
ii. Divide the spawn present in the spawn bag into four equal portions by spreading them
evenly in a clean disinfected plastic tray
iii. Spread the paddy straw bits uniformly into the bag to a height of 5cm. Sprinkle one
portion of spawn over this, evenly covering the entire surface. Gently press with
fingers
iv. Spread a second layer of straw to a height of about 10cm and sprinkle the second
portion of the spawn evenly. Give a mild pressing
v. Similarly, spread the third layer of straw to 10cm height and sprinkle the third portion
of spawn
vi. Spread the fourth layer of straw to 10cm height and sprinkle the fourth portion of
spawn
vii. Finally, spread paddy straw to 5cm height. Gently press with fingers and tie the bag
with a thread or rope
viii. For aeration, make 2-4 vents of one cm diameter at different places of the bag.

Filling mother spawn Cellulosic paddy straw Mushroom fruiting bodies

under SSF
3. Spawn running & cropping

 After preparation, arrange the cylindrical beds in racks in spawn running room
maintained at a humidity of around 80%
 Mycelium from the spawn slowly grows and covers the entire bed surface within 15-
25 days depending upon the species used. Until this stage, the water spray is not
needed. At the end of spawn run, small pinheads appear through the vents which is the
indication of fruiting
 Now transfer the beds to the cropping room with more ventilation and aeration. Here
open the beds carefully either fully or partly or in splits. Water spraying daily as fine
mist is necessary for the growing beds to develop into fruiting bodies (18–45 days).
The buds turn into mature mushrooms within 24-48 hours.
 Harvest the mushrooms before the margins roll upward or downwards
 The average yield of oyster mushroom would be approx. 635g/bed (500g of paddy
straw) with a good shelf life of up to 72 hours

Activity:

 Observe the bed for the emergence of fruiting bodies.

 Calculate the average yield per bed
 Estimate the product cost

Questions:

1. Mention some cellulose-rich substrates amenable for microbial fermentation.

2. Name a few microorganisms capable of degrading cellulose.

3. Mention the nutritive value of oyster mushroom.
4. Give a list of some value-added products produced by submerged and solid substrate
fermentation of cellulose.
5. Why fungi are more efficient in SSF than other yeast/bacteria? Substantiate.
 Ex.No.8 Sauerkraut fermentation

The word sauerkraut means Sour cabbage or acidic cabbage. Cabbage is the vegetable
used in Sauerkraut preparation, which is a good source of ascorbic acid, vitamins, and minerals
and possesses value for its salad and culinary properties. However, it is post-harvest losses are
as high as 60 %.Fermentation has been used since the early days of civilization to preserve
food materials and to develop newer products. Cabbage could be preserved as Sauerkraut,
which is an acid fermented product.
It is a result of natural fermentation by bacteria indigenous to cabbage in the presence
of 2-3 % salt. The fermentation yields lactic acid as the major product. The lactic acid, along
with other minor products of fermentation gives sauerkraut its characteristic flavor and texture.
Microflora involved
As no starter cultures are involved in this process, this is referred to as wild
fermentation. The normal flora of the cabbage leaves is relied upon to include the organisms
responsible for the desired fermentation, one that will enhance preservation and organoleptic
acceptability. The floral succession is governed mainly by the pH of the growth medium.
Initially, a coliform starts the fermentation. As acid is produced, an environment more
favorable for Leuconostoc is quickly formed.
The coliform population declines as the population of the strain of Lactobacillus builds.
As Leuconostoc is the heterofermentative lactic acid bacterium, much gas (CO2) accompanies
the acid production during this stage. The pH continues to drop, and a strain of Lactobacillus
succeeds in the Leuconostoc. (Sometimes Pediococcus arises instead of Lactobacillus). The
complete fermentation then involves the succession of three major groups of genera of bacteria,
a succession governed by the decreasing pH.
Addition of salt
Salting of the cabbage serves two major purposes. First, it causes as an osmotic
imbalance, which results in the release of water and nutrients from the cabbage leaves. The
fluid expelled is an excellent growth medium for the microorganisms involved in the
fermentation. It is rich in sugar and growth factors. Second, the salt concentration used inhibits
the growth of many spoilage organisms and pathogens. It does not obviously inhibit desired
floral succession.
Cabbage is approximately 90 % water, and the salt is dissolved entirely in the water.
The actual salt concentration (brine strength) experienced by the microorganisms in their
aquatic milieu is around 2.8 %.Thorough and even distribution of the salt is critical. Pockets of
low or high salt concentration would result in spoilage and /or lack of the desired fermentation.
Oxygen supply
Throughout the fermentation, it is critical that oxygen is excluded. The presence of
Oxygen would permit the growth of some spoilage organisms, particularly the acid-loving
molds, and yeasts.
Temperature
The time required for the fermentation depends on temperature. A temperature of 70°F
is preferred for the fermentation. If it is favorable, a period of 3-6 weeks will ordinarily
sufficient. At the end of this time, the following changes are marked.
 The product will have acquired its typical aroma.
 All the fermentable carbohydrates will have been consumed.
 The acid content will have risen to between 1.85 and 2.2 %
 The pH will be approximate to the range of 3.5 -3.7.
Aim
To understand the process of sauerkraut fermentation and to know the role of probiotics
in food fermentation.
Materials required
 Fresh heads of cabbage
 Large knives
 Cabbage shredders
 Containers for shredded cabbage
 Balance
 NaCl (Commercial grade)
 Airtight containers (Plastic/glass )
Procedure
 Trim the cabbage heads, removing the outer leaves and all bruised or soiled tissue.
 Wash the trimmed heads thoroughly with tap water.
 Cut the heads in half, removing the hard, central core.
 Shred the cabbage with the cabbage shredder.
 Weigh the shredded cabbage. Mix in the salt such that a final concentration of 2.5 % is
achieved. Complete, even mixing of the salt is highly critical.
 Pack the shredded cabbage into the containers, filling to approximately 75-80 % of total
volume. Compress the mixture moderately while avoiding, crushing or bruising the
cabbage tissue
 Close the container air tight
 Incubate at 240C for 5-6 weeks
Activity 1. Lactic acid bacteria, total bacteria, and total coliforms viable count
Do plating periodically in sauerkraut juice samples taken at 0,1,2,7,14 and 35 days of
incubation.(Take the 0 and 1-day samples undiluted, and the remaining samples in a 1/10
dilution which must be considered in the dilution/plating procedure ) for Lactic acid bacteria,
total bacteria, and total coliforms count
2. Acidity and pH determinations
Using pH paper, determine the pH of the undiluted juice sample. Add 10 ml undiluted
juice sample to the Erlenmeyer flask. Add 10 ml of distilled water. Boil the flask for 1 minute
to drive off the dissolved CO2. Cool and add 5 drops phenolphthalein. Titrate with 0.1 M
NaOH, until a light pink color persists. Using the following formula, calculate the percent
lactic acid (the predominant nonvolatile acid expected in the sauerkraut fermentation).

% Lactic acid = ml of 0.1 M NaOH x 0.9 / Sample volume

Titer value x Normality of alkali x Milli equivalent of lactic acid
% Lactic acid = ----------------------------------------------------------------------------------
Volume of sample taken for titration x wt. of the sample

Microbial dynamics during sauerkraut fermentation

Day Total aerobic plate Total coilforms /ml Total lactic acid % Lactic acid
count /ml bacteria /ml bacteria
R1 R2 Mean R1 R2 Mean R1 R2 Mean

Organoleptic test
 Evaluate Sauerkraut for its taste, color, flavor, texture and overall acceptability.
 Distribute the Sauerkraut to 10 individuals and evaluate after three weeks of preparation

The sensory evaluation card is given below

Character Grades
Colour 9 8 7 6 5 4 3 2 1
Flavor
Texture
Taste
Overall
acceptability
Grade 9 : very good acceptability
Grades 8-5 : Moderate acceptability
Grade 4-1 : low acceptability

Result
 Record the results of the plate count as well as sensory score

Review questions
1. What is the reason for the increased keeping quality of fermented cabbage?
2. Why inoculum is not added for sauerkraut fermentation?
3. What is the purpose of salt addition during sauerkraut fermentation?
 Ex.No.9 Production of Wine from Grapes

Introduction
Wine is a product of alcoholic fermentation of fruit juices that are rich in fermentable
sugars. It is a primary metabolite. Primary metabolites are the intermediary products produced
by microorganisms during the primary growth phase and these are necessarily required for the
growth of microbes. A typical microbial process in which the product is formed during the
primary growth phase is alcohol (ethanol) fermentation. Ethanol is a product of anoxic
metabolism of yeast and certain bacteria and is formed as part of energy metabolism.
Most fruit juices undergo natural fermentation caused by wild yeasts
(Saccharomyceesellipsoides) present on the fruit. From this, yeasts have been selected for more
controlled production, and today alcoholic beverage production is one of the largest industries
worldwide. The most important alcoholic beverages are wine, produced by fermentation of
fruit juice; beer, produced by fermentation of malted grains, and distilled beverages produced
by concentrating alcohol from fermentation by distillation.
Most wines are made from grapes only. Wines are divided into 1. Dry wine – in this all
sugars are perfectly fermented, 2. Sweet wines – in this some of the sugars are left unfermented
or additional sugar is added after fermentation, 3. Fortified wines – in this brandy / alcoholic
spirit is added after fermentation and 4. Sparkling wine – in this considerable CO2 is present
arising from final fermentation. Most of the wines have alcoholic content of 11-12 per cent.
Whereas, the dessert wines contain maximum alcoholic content of 19-21 per cent.

Materials required
o Graduated cylinder
o Test tubes
o Glass container
o Rubber stopper
o Tygon tubing
o Hydro meter
o Balance
o Ripened grape fruits
o Sucrose
o Active dry wine yeast, strains of Saccharomyces ellipsoides
Procedure
Preparation of starter culture (yeast)
Suspend 1 g dry wine yeast in 10 ml of warm water at about 35°C and allow the yeast
to grow in a loosely capped container at room temperature for 24 hours.
Primary fermentation
o Select fully ripened grapes with sweet taste and optimum flavor
o Remove Stalks of the berries since they contain tannin
o Wash the grapes with clean water
o Crush the cleaned grapes
o Crushed juice is called Must, which has a pH of 3-3.6. If the sweetness is low (Brix
value below 20 ) adjust the sugar content
o Inoculate with 20 ml of starter culture of the yeast.
o Pluck the bottle with the rubber stopper (A piece of tygon tubing is extended from the
stopper to provide a vent for the evolved carbon dioxide.The other end of the tubing is
dipped in water.The water prevents the entry of oxygen, which alters the metabolism of
the yeast and spoils the wine. At the same time, carbon dioxide can escape from the
bottle)
o Ferment at room temperature for one week

Secondary fermentation
o At the end of one week, decant the juice from the bottle to the clean container and
estimate the PA (Potential Alcohol scale) values with the hydrometer and alcohol
content.
o Pour the juice back in to the bottle,reassemble and continue the fermentation for
another 4-6 weeks

Composition of Must (approximately as follows (Haas, 1976)

Particulars Percentage
Sugars (glucose and fructose) 15-25
Acids (Mainly tartaric and malic) 0.9-1.5
Tannins (for white wine) 0.02 – 0.04
Tannins (for red wine) 0.1 – 0.25
Pectins 0.1 – 0.15
Ash 0.30 – 0.50
Aroma substances Trace
Nitrogenous substances Trace
Higher alcohols Trace
Aldehydes Trace
 Typical composition of a wine

Particulars Percentage
Extract (total solids) 2-3
Carbohydrates 0.03 – 0.5
Acids 0.5 –1
Ash 0.15-0.3
Amino acids Trace
Tannins Trace
Aroma substances Trace
Alcohol 6-9 wt.
8-13 vol

Flow chart for making wine

Grapes picking

Removal of stem, stalks etc.

Washing

Weighing

Crushing

Brix value below 20, need addition of sugar. Add sugar till bricks value reaches 20-250

Transfer to the large size flasks and add Yeast inoculums (Saccharomyces cervesiae var.
ellipsoideus) at 1- 3% level

Cover it with sterile cotton (allow aerobic fermentation for 2-3 days)

Remove cotton and plug it with rubber stopper (for anaerobic fermentation)

Decanting /clarification decanting (repeat)

 Ageing and maturation (8-10 months)

Filtration

Pasteurization

Bottling

Labelling

Observation
o Observe the wine for color, taste, flavor, pungency, clarity, etc.

Organoleptic Tests
Appearance: White wines that have become brown are usually oxidized and over-aged in
odor. A silky ‘wavy’ sheen in a hazy cloudy white or red dry wine accompanied by a
characteristic odor is unmistakable evidence of bacterial spoilage. If red wine is high in acid,
it will have a bright color. The older the wine, the more the red color shifts towards the brown
or purplish-red. The old port is apt to be tawny in color; young port red or purplish-red.
Angelica and Muscatel should be light gold or golden amber in color. Madeira is light amber
to brown Chablis and Riesling should be pale yellow, Sauterne more so. Sherry may range
from very pale to a dark amber, depending on the type.
Odour: Tasters should distinguish between bouquet and aroma. They designate bouquet as
the odors developed by the wine in normal aging through esterification, oxidation, etc. and
aroma as the odor derived from the fresh grapes. The age of the wine greatly affects the
bouquet and with experience, the taster can tell much about the age of the wine by its bouquet.
Slight vinegar souring or lactic souring can be detected.

Taste: Following factory examination, one proceeds to the evaluation of the taste. The four
aspects of taste are sour, sweet, salty and bitter. The sour or acid taste is of value for all wine
types, particularly table wines. A low acidity gives a wine of q flat or insipid taste. Sweetness
is the most important for sweet table and dessert wines but is also especially critical for
sparkling wines. Red wines all have some bitter taste.
Flavor: Flavour is made up, in part of bouquet and aroma and in part by taste evident chiefly
to the tongue.

Observation and Results

Review questions
1. What is difference between red and white wine?
2. Give one example of each of primary metabolites and a secondary metabolite?
3. What is the raw material and name the organisms used to make wine?
4. What is the purpose of doing inoculation for winemaking?
 Ex.No.10 Cell separation of yeast and LAB by
Centrifugation and Filtration

Several industrial microbial products are produced by culturing different microbial

cultures in fermentation broth under favorableconditions.Prior to the production of target
molecules like enzymes, antibiotics, organic acids, alcohols, etc., fermentation broth has to
filter to be free from the cell mass. Different techniques are available to separate the biomass
from fermentation broth. Techniques like centrifugation, filtration, sedimentation,
flocculation, spray drying, and freeze-drying are available. On the basis of the scale,
economics, product sensitivity and purity of the products required, any one or combination of
the above techniques are used.

Centrifugation involves the application of centrifugal force to separate particles from a

solution according to their size, shape, density, viscosity of the medium and rotor speed. This
method involves separating molecules of different densities by spinning them in solution
around a centrifugal axis (in a centrifuge rotor) at high speed. This process is used to separate
two miscible substances. More-dense components of the mixture migrate away from the axis
of the centrifuge (move to the outside), while less-dense components of the mixture migrate
towards the axis, i. e., move to the center.The firstcommercial scale centrifugewas designed in
1878 by the Swedish inventor De Laval to separate cream from milk. In 1923, Svedberg's
invented the analytical ultracentrifuge, operating at 10 000 rpm.

Centrifuges are used in different fields ranging from large-scale commercial

applications to laboratory-scale scientific research. In microbiology, this process is used to
separate microbial cells from the liquid media without cell disruption. This process also aids
in separating microbial products containing liquids from cell debris and other solid materials.

Filtration is a physical operation that separates solid particles from a suspended liquid
by adding a physical filter medium through which only the liquid can pass. Solid particles that
cannot pass through filter papers are retained and get separated on one side, while liquid
particles pass through the filter medium is collected on another side. A liquid medium that
passes through the filter medium is called filtrate. Solid particles that cannot pass through the
filter medium are described as oversize, and the fluid that passes through is called the filtrate.

Materials required:

1. Each two hundred ml broth cultures of Lactobacillus and Yeast in the late log
phase.
2. Laboratory centrifuge with 50 ml centrifuge tube loading capacity.
3. Filter paper with 0.45µ pore size and around 100 mm diameter.
4. Sterile glass funnels, Sterile 50 ml centrifuge tubes.
Procedure:

Cell separation using centrifuge technique:

1. Prepare two hundred ml nutrient broth for each culture of Lactobacillus and Yeast.

2. Inoculate the log phase cultures of Lactobacillus and Yeast in nutrient broth.

3. Incubate the cultures in a thermo-controlled orbital shaker at 160 rpm until the
culturesreach the late log phase.

4. Take one set culture broth, fill in centrifuge tube to ¾th of the capacity.

5. Place the centrifuge tubes in a centrifuge rotor, ensure proper weight balance.

6. Run the centrifuge at 8000 rpm for 10 min.

7. Discard the supernatant.

8. Harvest the precipitated cell using a spatula, transfer to a Petri plate.

9. Dry the biomass by placing under hot air oven at 72°C, until the consistent weight
is reached.

10. Calculate the yield of biomass per ml of the culture broth used.

Cell separation using filtration technique:

1. Prepare two hundred ml nutrient broth for each culture of Lactobacillus and Yeast.

2. Inoculate the log phase cultures of Lactobacillus and Yeast in nutrient broth.

3. Incubate the cultures in a thermo-controlled orbital shaker at 160 rpm until the
cultures reach the late log phase.

4. Filter the culture broth using a 0.45µ membrane filter using glass funnel and
conical flask.

5. Discard the culture filtrate. Dry the biomass present in the membrane filter by
placing under hot air oven at 72°C, until constant weight is reached.

6. Calculate the yield of biomass per ml of the culture broth used.

Review questions:

What are the techniques available for cell separation?

What is the principle behind the centrifugation technique?

What is Svedberg's unit?

What is filtrate and oversize?

Why are late log phase cultures preferred for cell harvesting?

References:

Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A

remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943.

Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental

laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p.
235. ISBN 9780470087664.
 Ex.No.11 Cell Disruption Technique by Sonication

Cell disruption is the process of obtaining intracellular biological materials that open
the cell wall. It is an essential preparative step for the purification of intracellular proteins,
enzymes, and nucleic acids and enhances the recovery. Biological products may be
extracellular, intracellular or periplasmic. Different cells have different structures; hence, they
require different methods for cell lysis. Cell disruption methods are categorized into:

i)Mechanical cell disruption: This method completely destruct the cell wall in a
non-specific manner. The cells are subjected to high stress via pressure, abrasion with rapid
agitation, with beads or ultrasound.Eg.high-pressure homogenizer (liquid shear), Bead mill
(solid shear).The demerits of beadmill methods are: high chance of contamination, poor scale-
up, and additional cooling systems required (temperature increases as the size of beads
increase).

ii)Non-mechanical cell disruption:It is more benign and comprises of a)physical b)

chemical c) and biological methods. Non-mechanical cell disruption methods perforate or
permeabilize cells rather than tearing them apart.

Physical cell disruption: Thermolysis, decompression, osmotic shock

Chemical cell disruption: detergents that damage the lipoprotein of microbial cell
membranes. The detergents are divided into cationic, anionic and non-ionic. Sodium dodecyl
sulphate (SDS) is the common anion detergent used to disturb the protein-protein interactions.
Triton X 100, an non-inonic detergent solubilizes membrane proteins. Cationic detergent,
ethyl trimethyl ammonium bromide acts on phospholipids, lipopolysaccharides and cell
membrane.Solvents such as alcohols, dimethyl sulfoxide, methyl ethyl ketone or toluene.

Biological cell disruption: usage of digestive enzymes. Eg. Lysozyme (hydrolyzes β 1-

4, linkages of peptidoglycan).

The ideal technology for cell disruption is based on the following criteria:

i) Maximum release of the product of interest

ii) No mechanical or thermal denaturation of the product during cell lysis
iii) Minimal release of proteases that may degrade the product
iv) Minimal releases of soluble contaminants and particulates that may
influence downstream process
v) Product stability
vi) Speed and cost of the method
Ultrasonication

Ultrasonic disruption is a mechanical cell disruption technique caused by ultrasonic

vibrator.It produces a high-frequency sound with a wave density of about 20kHz/s. A
transducer then converts the waves into mechanical oscillations through a titanium probe,
which is immersed into the cell suspension.

Principle

Sound is a wave of alternating high and low pressure.Sonication typically uses

ultrasound waves with frequencies of 20 kHz (20,000 cycles per second) or higherto agitate
particles in a solution. It converts an electrical signal into a physical vibration to break
substances apart. These disruptions can mix solutions, accelerate the dissolution of a solid
into a liquid, such as sugar into water, and remove dissolved gas from liquids. These
frequencies are above what is being heared, but ear protection is still recommended during
sonication because the process creates a loud screeching noise. The higher the frequency,
the stronger the agitation of particles.

Sonication Process

Compression Compression

Rarefaction Rarefaction

One Cycle

Bubble Bubble grows in successive Reaches unstable Violent Collapse

forms cycles by rectified diffusion size

Fig 1. Sound Propagation in a liquid showing cavitation bubble formation and

collapse

1. The ultrasonic sound waves on a liquid cause periodical compression and rarefaction.
This phenomenon is called cavitation
2. Microbubbles occur in rarefaction due to negative pressure
 3. The microbubbles contain vaporized
vaporized liquid and gas that was previously dissolved in
the liquid
4. As the wavefront propagates, the bubble size increases and finally collapses.

The energy from sound waves creates friction in the solution, which creates heat. To
stop a sample from heating upup and degrading, keep it on ice before, during, and after
sonication.If cells and proteins are too fragile to withstand sonication, a gentler
alternative is enzyme digestion or grinding with sand.

Sonicator Parts
A sonicator is a powerful piece of lab equipment
equipment with an ultrasonic electric
generator that creates a signal to power a transducer. The transducer converts the electric
signal using piezoelectric crystals – crystals that respond directly to electricity by creating a
mechanical vibration. The sonicator
sonicator preserves and amplifies the vibration until it passes to
the probe. The probe moves in time with the vibration to transmit it to the solution and
moves up and down quickly. The sonicator operator can control amplitude based on the
properties of the solution.
ution. A small probe tip produces a more intense reaction than a large
probe tip, but a large tip reaches more of the solution.

Not all sonicators have probes. Some sonicators produce sound waves in samples in
an ultrasonic water bath.In In an experimental setting,
setting, this is usually carried out using an
ultrasonic bath or ultrasonic probe generally referred to as sonication.

Probe Sonicator Bath Sonicator

(Source
Source : Nithyanantham et al., 2014, Bionanoscience
Bionanoscience)
Applications of sonication

 Sonication is used to disrupt cellular membranes and release the contents of the cell,
this process is generally referred to as sonoporation.
 It can also be used to fragment/shear DNA, preventing it from interfering with further
sample preparation.
 Other biological uses include the production of nanoparticles, liposomes, extraction of
anthocyanins, and antioxidants.

Depending on the cell type (bacterial or eukaryotic), it can be challenging to lyse certain
cells and placing them in a detergent buffer alone will not result in full cell lysis. It is also
required to lyse the cellular organelles besides the cell wall to release the cytosol. Sonication
of cells using a titanium probe can help lyse cells fully and help to extract DNA, RNA and
protein contents of cells.

Procedure

1. Centrifuge cells for 5 mins at 270 x g in a microcentrifuge.

2. Aspirate the remaining media and resuspendthe cells in 30 – 100 μL of phosphate
buffer.
3. Incubate the pellet on ice for 30 min.
4. Sonicate the samples as follows.
5. Place the sonicator probe at a frequency of 20 kHz.
6. Place the cells in a 1.5 mL microcentrifuge tube and gently move under the tip of the
sonicator probe.
7. The probe will begin to vibrate the buffer for 2 X 10 sec to reduce the viscosity (This
may result in foaming of the samples)
8. Depending on samples and viscosity of the samples, cells can be sonicated again for a
further 10 s.
9. Once the samples are sonicated incubate on ice for 5 min.
10. Centrifuge at 10,000 x g for 20 min to pellet debris (Debris may contain unlysed cells,
nuclei or unlysed organelles).
11. Transfer the supernatants to a new microcentrifuge tube and label.
12. Store at -20 °C.

Hints and Tips

1. Always keep samples on ice. The energy from the sonicator which causes the sample
to break apart also heats it. If the sample gets too hot, the protein will start to degrade.
In order to prevent this try to keep sample on the ice at all times, before, after and
during if possible.
 2. Reduce the temperature by pulsing. Most sonicators have a pulse mode, which reduces
the heating up of the sample during sonication. If pulsing is not an option/not
available, turn the sonicator on for 5 seconds and then off. Repeat as many times as
necessary.
3. Don't over-Sonicate. Sonicating sample for too long can degrade protein. Finding that
perfect balance may take some optimization and can vary for different cell/tissue types
and sample volumes.
4. Sample volume and probe size. The probe can vary depending on the sample size.
Each probe has a recommended sample volume range. Small tips (microtips) are
recommended for processing samples inside small, thin vessels and never samples
larger than 50ml. Microtips are made for short processing times. Microtips will
generate a considerable amount of heat in small volumes and therefore, should be used
in the pulse mode to prevent heat buildup.
5. Protect your ears. Although sonication is ultrasonic and out of the range of human
hearing the collapse of tiny cavitation bubbles created by the sonication creates a loud
screeching noise.
6. Try to limit foaming. If the probe isnot appropriately submerged in the sample it can
lead to foaming, however, if its too deep you won't get proper lysis
7. For sonication, a lower amplitude for longer will reduce heating of the sample – within
reason, you do have other experiments to get on with.Amplitude and intensity have a
direct relationship. In order to be able to reproduce results, the amplitude setting,
temperature, viscosity and volume of the sample are all parameters that need to remain
consistent.
8. Always clean the sonicator tip between samples. Cleaning the sonicator tip is critical
in limiting protein carryover. Wiping the sonicator probe with 70 %ethanol or
sonication of ethanol in a beaker is an effective way of cleaning the sonicator tip.
Activity

Sonication of the given bacterial cell sample

Results

Review Questions:

1. What is the principle of sonication?

2. Why is sonication an efficient method for cell lysis?
3. How can you minimize the heat generated during sonication?
4. What is the degassing of water?
5. Where is bath sonicator used?
6. Name two typical applications where the principle of sonication is employed?
 Ex.No.12 Separation of milk protein by Precipitation
using organic acids

Milk is considered wholesome and nutritionally completenutriotinal and often

considered as a primary source of proteins for humans. Milk is rich in vitamins, minerals,
proteins, carbohydrates, and lipids. Milk contains three kinds of proteins viz., caseins,
lactalbumins, and lactoglobulins. These proteins are globular and easily solubilized in water
as a colloidal suspension. Among the three proteins present in milk, casein is found in a
significant proportion. As an example, in cow’s milk, 82% of milk protein is casein and the
remaining 18% protein in serum or whey protein. The casein in milk is present as calcium
salt, calcium caseinate. Calcium caseinatecontains phosphorus, has an isoelectric point of pH
4.6. The pH of milk is about 6.6; therefore, casein has a negative charge at this pH and is
solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the
casein micelles are neutralized and the neutral protein precipitates, with the calcium ions
remaining in solution: Other groups of proteins found in milk are known as serum (whey)
proteins. Serum proteins do not contain phosphorus. These proteins remain in solution in milk
at pH 4.6.

The first developed technique to separate casein protein from milk is a physio-
cehmical one. This technique is based on the principle that lowering the pH of the milk to 4.6
leads to casein coagulation and precipitation. All types of acids, including Hydrochloric,
sulphuric, acetic, and citric acids, can be used to lower down the pH of the milk and to
separate the milk protein – casein.

The natural example of casein separation is the souring of curd formation of milk.
When Lactobacillus culture ferments carbohydrate lactose naturally found in sugar, lactic acid
is produced. This lowers down the pH of milk and leads to the curdling process, natural
separation of casein from milk.

This experiment is designed to learn the technique to isolate milk proteins using
organic acids.

Materials required:

1. Samples of milk.
2. Organic acids viz., Citric acid, acetic acid and lactic acid.
3. Hot plate, Thermometer, weighing balance, beakers and filtration set up.
Procedure:

1. Take 200 ml milk in a glass beaker.

2. Heat the milk sample to 55°C.
3. Add different 10% organic acids warm milk samples.
 4. Add slowly, drop by drop, and continue while the addition of acids.
5. Continued until the milk becomes clear like water or until the separation of
proteins is stopped.
6. Filter the processed milk samples using a clean muslin cloth.
7. Weight the separated milk proteins using laboratory balance.
8. Calculate the yield of milk proteins obtained using different organic acids.

Review questions:

What are all the different types of proteins present in milk?

What is the isoelectric point?

How proteins in milk gets separated during milk souring?

Which microbial culture is used in the curdling process?

References:

Maubois., J. L.. Separation, extraction and fractionation of milk protein components.

Le Lait, INRAEditions, 1984, 64 (645_646), pp.485-495.

Fox, P. F., and P. L. H. McSweeney. Dairy Chemistry and Biochemistry. 1998.

Blackie Academic & Professional, an imprint of Chapman & Hall, London.
Ex.No.13 Separation of Soy Protein by precipitation
using salt

Proteins obtained from plant origin play a vital role in meeting protein demand for
human consumption. Legume proteins stand second next to animal protein in meeting protein
demand for the human and animal populations. Soybeans are used as an essential source of
protein worldwide. Soy protein is generally regarded wholesome as being concentrated in
protein bodies, which are estimated to contain at least 60–70% of the total soybean protein.
Soy protein is used as a source of proteins since 1959 due to its nutritional properties. Soy
protein has been in use since 1936 for industrial uses like paper coating, pigment binding, and
other sticking agents.

Soybean is a leguminous crop that has originated from East Asia. Protein separated
from soybean is known as soy protein.Soy proteins consist ofglycinin and β-conglycinin
proteins. Soy protein is an easily digestible, widely available vegetable source that provides
allessential amino acids in the appropriate amounts. Soy protein is made from soybean meal
that has been dehulled and defatted. Processed soy protein is available in three major forms;
soy flour, soy protein isolates, and soy protein concentrate.

Principle of soy protein precipitation:

As the first step, proteins found in soybeans are made to solubilize in water by milling.
From the solution, solid residues other than soluble protein are removed by physical filtration.
Further, soluble proteins are precipitated from the dissolved form by coagulation. The use of
coagulantssuch as ammonium sulfateneutralizes the charges resulting in a decrease in
electrostaticrepulsion between protein particles triggering aggregation and precipitation.
Precipitated protein is purified and processed for further use. The process of precipitation of
proteins using salts is called salting out.

Materials required:

1. One kg Matured soybean grains.

2. Milling machine.
3. Magnesium sulfate.
4. Cheesecloth.
5. Weighing balance.
6. Beakers and glassware.

Procedure:
 Take one kg of matured soybean grains.
 Soak in water for 12 hours.
  Mill the soaked grains with water in 1:3 ratio.
 Remove the solids by filtering through double-layer cheesecloth, twice.
 Heat the soy milk at 85°C.
 Add Magnesium sulfate, drop by drop, stir well.
 Continue the addition of salt until soy milk becomes clear and protein coagulation is
over.
 Leave it as such for 10 min, separate the coagulated proteins using a strainer.

Results:
Weigh the protein and calculate the yield by using the formula,

Weight of the protein obtained

Yield of protein = X 100
Weight of the grains taken for milling

Review questions:

1. What is the principle behind protein precipitation?

2. What are all the different kinds of proteins present in soy?
3. What are all the industrial uses of soy protein?
4. What is salting-out?

References:

1. John Dzikunoo, George S. Ayernor, Firibu K. Saalia. Effect of Methods of Extraction

on Physicochemical Properties of Soy Proteins (Tofu). American Journal of Food
Science and Nutrition Research. Vol. 2, No. 5, 2015, pp. 138-144.

2. Pearson, A.M., “Soy proteins,” Nakamura, In: T. Yano, R. Matsuno, K. Nakamura,

Editors. Developments in food engineering. London, U.K. Blackie Academic &
Professional Publishing Co. 1994, pp. 67-104.
 Ex.No.14 Extraction of Biocolorants from Plant Sources

Attractive colors enhance the palatability of food. Food color is often seen as
synonymous with quality,and foods with special appeal and color are generally preferred.
Color is an essential element in enhancing foodstuffs, constituting one of the major dietary
additives. The food industry has therefore resorted to enhance the color of foods to increase
consumer acceptability. Consequently, over the centuries, color has come to play a prominent
role in things important to humans: food, medicine, and physical appearance. Concern over
the questionable safety of synthetic red food dyes as additives
has prompted research in the use of natural pigments as food colorants. Apart from this, the
natural colorants have its own role in textile industries also.

Food colors can be classified primarily into two types, viz., (i) natural colors and (ii)
synthetic colors (permitted and non-permitted colors). Those available in the market are
termed as nature-identical colors, where the colors are manufactured by chemical synthesis
are synthetic colors.

Limitations of natural food colorants

 low stability in the food processing procedures, formulation, and storage conditions
 impart undesirable odor or flavor to the food.

The selection of a food colorant is usually made after it is deemed satisfactory according
to the following criteria:

 target shade,
 the physical/chemical attributes of the food matrix,
 stability to processing and storage conditions
 regulatory issues

Biocolorants

Among the biological sources, many natural dyes/colors are obtained mainly from plants,
producing various colors such as red, yellow, blue, black, brown and a combination of these.
Green is the dominant pigment in plants, while the carotenoids are a large group of pigments
associated with chlorophyll and responsible for autumn leaf pigmentation. Many of the
intense colors in flowers and fruits are contributed by the flavonoid pigments and closely
related compounds with a diverse range of colors, which are the result of structural
differences between the compound and the relative concentration of specific pigments within
the cells. Betalains, a restricted group of pigments, get their name from the red-violet pigment
isolated for the first time in crystalline form from the root of the beet Beta vulgaris, L. Natural
colorants, and pigments from plants have used in coloring food, beverages, soft drinks,
confectionery, bakery products, etc. (Table 1).

Merits of biocolorants

 Environmentally friendly
 Non-toxic
 Antioxidants
 More preference over synthetic colors.

Information: Over 2000 pigments have been reported to be produced by various parts of the
plant. Of which, only a little more than 150 have been exploited commercially. In India alone,
about 450 plants are known to yield colors.

Sources of Biocolorants from Plant Origin

Colour Sources Applications/Uses

Chlorophylls/Chlorophyllin Grass, algae, alfalfa, celery, Pasta,confectionery
Natural green and green green beans, bell peppers, medicines, processed food,
broccoli, broccoli, green vegetable oils,ice cream, and
cabbage, barley, and other coloring materials
herbs
Carotenoids and Mushroom, turmeric, carrot,
Baby foods, sauces, fruit
Xanthophylls Marigold, Onion, Paprika,
drinks, candy and cake,
Orange, Yellow, Red to red pepper,Cocao, Ginger,
chocolate, biscuits, snack
reddish-orange, brown, etc Vegetables food seasoning,
pharmaceuticals
Betalains Beetroot and opuntia Beverages, frozen foods, fruit
Bluish red, red-violet and fillings,chewing gums,
yellow medicinal products, dairy
products
Flavonoids/anthocyanins Strawberries, Hibiscus, Confectionery, food
Red, purple, dark violet, Grapes skin, Indigo plant, products, and dessert
purplish red, bluish-purple Clerodendron, red cabbage, products, health care
blueberries, black grapes products, fruit base, sauces,
jelly, grape juice, tea-based
beverages, pie and cookie
fillings
Aim: To extract carotenoids from carrots, tomatoes, turmeric, and other vegetables and to
quantify the amount present per unit weight of the given sample

Materials required

· Carrots, tomato, turmeric and other vegetables

· Hexane: acetone (1:1)
· ethanol,
· 10% NaCl solution
· Silica gel and calcium sulfate hemihydrates (4:1)
· Mobile phase ( hexane and acetone in the ratio of 3:2).
· Separating funnel

Preparation of TLC plates

Ø Prepare simple TLC platesusing microscopic slides.

Ø Mix silica gel (finely powdered) and calcium sulfate hemihydrates together

Ø Form a paste and evenly distribute over the surface of the slide and keep aside to dry.
Prior to usage, activate the slides by baking in the oven at 120°C for 30-40 min

Extraction of carotenoids using solvents

 Cut the vegetables separately and weigh 2g of each vegetable

 Crush the given vegetable in a pestle and mortar.
 Add solvents (a mixture of hexane and acetone in the ratio of 1:1) into the mortar and
crush thoroughly
 Add 5ml of acetone slowly at regular intervals.
 Collect the solvents separately and repeat the process with the sample again for double
extraction.
 Filter the solvents containing carotenoids through a filter paper
 Transfer into a separating funnel and add 50ml of distilled water along with 50 ml of
10% NaCl solution.
 Shake the mixture vigorously and keep aside for the layers to separate.
 The upper layer contains carotenoids and collectsthem separately after the removal of
the water and NaCl solution.
 Read the absorbance of carotenoid at 630 nm against a standard chemical (Zexanthin,
lycopene, etc).
 Calculate the amount of carotenoid present in 100g of each food sample

Purification using thin-layer chromatography method

 Draw a thin line on the activated TLC plate about 1.5 cm above the bottom.
 Place a spot of the extract on the line and allowed it to dry. This can be followed by
repeated addition of the extract on the same spot.
  The developing chamber is a beaker containing a mixture of hexane and acetone in the
ratio of 3:2.
 Place the TLC plate inside the developing chamber and cover the top
 Allow the solvents to rise on the plate till it reaches 1.5 cm close to the top.
 Calculate the relative front (Rf) and compare with the control

Activity

 Extract carotenoids from the given samples

 Quantify carotenoids in UV-VISIBLE Spectrophotometer
 Prepare TLC plates
 Purify carotenoids and observe the RF values

Results

Review Questions

1. What is the FDA? Are there any specific FDA standards for synthetic colors?

2. What are the health hazards caused by chemical biocolorants?

3. Define type I and II biocolorants? What is their permissible limit in foods?

4. Do you think naturally sourced biocolorants are feasible? What are the ways you can
suggest to improve the recovery of plant and microbe based bicolorants?
 Ex.No.15 Microbial Cell Immobilization by Calcium Alginate
Method

Immobilization is a general term describing a wide variety of the cell or the particle
attachment or entrapment.The term ‘immobilization’ was first proposed at the first enzyme
engineering conference in 1971. Immobilization of bacterial cells into protective carriers
increases the survival of cells in a concrete matrix.The encapsulation of bacterial cells into a
polymeric matrix such as calcium alginate (Ca-alginate) is a promising example.

Types of immobilization techniques:

i)Active immobilization

 Entrapment in porous matrices

 Encapsulation
 Adsorption

ii) Passive immobilization

 Biofilms

Immobilization of Microbial cells

A medium for the entrapment of living microbial cells should cause as little trauma to
the cells as possible. There should, ideally, be no shock to the cells from change of
temperature, change of osmotic pressure, change of chemical environment, and chemical
reaction. Entrapment in calcium alginate fulfills these criteria, in general, and as a result has
proved to be one of the most widely used methods of cell immobilization.

Principle

Alginate reacts with most polyvalent ions (magnesium being an exception) to form
cross-linkages. As the content of the polyvalent ion increases, the solution of sodium alginate
becomes viscous, followed by gelling and precipitation. Uniform gels can be produced by
arranging a uniform distribution of calcium ions throughout the liquid phase before setting
commences.

Alginic acid is a constituent of marine algae produced by the brown algae of the
Phaeophyceae, which occur in intertidal zones and free-living, for example, in the Sargasso
Sea. There are various commercial sources of alginic acid, principally Macrocystispyrifera,
the giant kelp which is harvested off the Pacific coast of the United States, Laminariaspecies
(coasts of Europe, Japan, and North East America), and Ascophylum species which grow
around the British coast. Alginic acid is a copolymer of rnannuronic acid and α-L-glucuronic
acid linked by (1-4)glycosidic linkages.

Advantages

 High cell concentrations

 Cell reuse without the costly processes of cell recovery and cell recycle
 Eliminates cell washout at high dilution rates
 Combination of high cell concentrations and high flow rates allows high volumetric
productivities
 May provide favorable micro-environmental conditions for cells (i.e. cell-cell contact,
nutrient-product gradients, pH gradients), resulting in better performance of the
biocatalysts (higher yields, growth and production rates)
 Immobilization can improve genetic stability
 Protects against shear damage

Limitations

 The product must be secreted

 Diffusion becomes a significant factor in the system
 Growth and gas evolution can mechanically disrupt the immobilizing matrix
 Maintenance of immobilized cells is significantly more complex than the maintenance
of immobilized enzymes

Materials Required
 Yeast potato Dextrose (YPD) (for the cultivation of Yeast cells)
 Sodium alginate solution (2.5% w/v in 0.1% NaCl)
 Calcium Chloride (CaCl2 ) solution (0.05 N)
 Sucrose solution (1% w/v)
 Glutaraldehyde (2.5% v/v)

Procedure

 Grow the yeast cells in YPD/PDA medium

 Keep it in a shaker for 24hours at 100 rpm
 Filter out the yeast cells with the help of Whatman filter paper
 Take a 20 ml sodium alginate solution and add 3 ml yeast cell in it.
 Mix properly and incubate at room temperature for 30 minutes.
  Add 3 ml of glutaraldehyde solution and incubate at room temperature for 90
minutes
 With the help of 10 ml pipette, add this
this mixture into the beaker containing 100
ml CaCl2 solution dropwise
 Filter out the beads with the help of normal filter paper
 Wash the beads 2-3
2 times with sterile Distilled water
 Load these beads into thoroughly washed glass column/ beaker
 Add 50ml off sterile 1% sucrose solution
 After every 30 minutes interval collect the 1 ml of sample and estimate the
amount of glucose by using Dinitrosalilcylic acid method (DNSA) method

Fig 1. Steps involved in immobilization of yeast cells

Procedure for Estimation of Glucose by Dinitrosalilcylic acid method (DNSA) method.

 Prepare a standard solution of carbohydrate (here glucose) having a concentration of

1.0 mg/ml. Take different volumes of glucose solutions like 0.5, 1.0, 1.5 & 2.0 ml etc.
into various tubes previously labeled as S1, S2, S3, S4, etc. respectively.
 One tube should be labeled as blank.
  Pipette out 3 same volume of sucrose solution from reaction mixture from above
experiment Add distilled water in all tubes in such a way that the total volume will be
2.0 ml.
 Add 2.0 ml of DNSA reagent in all tubes. Mix it properly by reversing the tubes or by
using magnetic stirrer.
 Keep all the tubes in a boiling water bath for 10 minutes.
 Then allow it to cool down.
 Take absorbance at 540 nm (using a green filter) and plot a standard curve.
 Calculate out the concentration of glucose produced in the reaction mixture of the
above experiment

Observation

Increase in the concentration of glucose from the sucrose with respect totime by
Active yeast cell (Invertase) indicates that Yeast Cells immobilized and they are viable.

Activity

 Prepare YPD broth, CaCl2 (0.05 N) and Sodium alginate solution (2.5% w/v in
0.1% NaCl)
 Harvest yeast cells
 Prepare alginate beads immobilized with yeast cells
 Determine the viability of yeast cells based on invertase activity during time
course

Results:

Review Questions

1. How do you assess the viability of immobilized yeast cells?

2. What are the merits of immobilized cells over free cells during fermentation in a
bioreactor?
3. What is invertase?
4. Can we use the immobilization technique for enzymes? What are the advantages?
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