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PRACTICAL MANUAL BOOK 1
FOOD SCIENCE AND TECHNOLOGY
DEPARTMENT,
FEDERAL UNIVERSITY OF TECHNOLOGY,
AKURE
Compiled by:
Prof. T. N. Fagbemi
Prof. (Mrs.) O. F. Osundaunsi
Dr. (Mrs.) A. I. Akinyede
Mr. S. O. Oguntuase
Mrs. E. T. Oyebode
Mr. L. O. Alatise
Mrs E. R. Olukanye-David
Miss C.Y. Babatuyi
TABLE OF CONTENT
CHAPTER ONE: FST 309 (FOOD MICROBIOLOGY) 2
CHAPTER TWO: FST 311 (FOOD PROCESS ENGINEERING)
2.1 Particle Size Reduction Using Various Mills. 33
2.2 Determination of Rate of Drying 34
CHAPTER THREE: FST 313 (HUMAN NUTRITION)
3.1 Assessment of Nutritional Status 37
3.2 Measuring Malnutrition with Shakir Strip or Tape 38
3.3 The Use of Local Events Calendar to Determine a Child’s Age 39
3.4 Estimation of Total Energy Requirements Using Factorial Method. 40
3.5 Use of Ballistic Bomb Calorimetry to Measure the Energy Value of A Foodstuff 42
CHAPTER FOUR: FST 316 (FOOD ANALYSIS I)
4.1 Proximate Composition 46
4.1.1 Determination of Moisture Content 46
4.1.2 Determination of Ash 48
4.1.3 Determination of Fat Using Soxhlet Extraction Method 50
4.1.4 Determination of Crude Fibre 50
4.1.5 Determination of Protein 52
4.1.6 Determination of Crude Protein 54
4.1.7 Determination of Total Carbohydrate 54
4.1.8 Carbohydrate Determination Using Polarimeter 56
4.2 Determination of Fat in Milk Using the Rose-Gottlieb Process 57
4.3 Determination of Iron in Beverage By Atomic Absorption Spectrophotometer 57
4.4 Determination of Riboflavin In Powdered Skim Milk Using Flourimeter 58
4.5 Water Analysis 60
4.5.1 Determination of Chloride in Water-Mohr’s Method 60
4.5.2 Determination of Alkalinity of Water 60
4.5.3 Determination of Ca2+ and Mg2+ in Water 61
4.5.4 Determination of Total Hardness 63
4.6 Analysis of Milk and Milk Products 65
4.6.1 Determination of Total Solids 65
4.6.2 Lactometer (Rapid) Method for Total Solids 66
4.6.3 Crude Isolation of Casein From Fresh and Fermented Milk 66
4.6.4 Acidity and Ph of Milk 67
4.6.5 Determination of Total Acidity 67
4.8.1 Determination of pH 68
4.8. Analysis of Beverage/Juice 68
4.8.2 Total Sugars (Soluble Solids) By Refractometric Method 68
4.8.3 Total Titratable Acidity 68
4.8.4 Determination of Diacetyl Value For Citrus Fruits 69
4.8.5 Determination of Ascorbic Acid (Indophenol Method) 69
4.8.6 Vitamin C Determination Using The Absorptiometric or Spectrophonmetric
Method 70
CHAPTER FIVE: FST 317 (FOOD CHEMISTRY I)
5.1 Determination of Smoke, Flash and Fire Points of Oil 73
5.2 Determination of the Melting Point of Oil 73
5.3 Phosphate Test in Milk 74
5.4 Turbidity Test for Sterilized Milk 75
5.5 Determination of Peroxidase 75
CHAPTER SIX: FST 318 (FOOD STORAGE)
6.1 Determination of Moisture Content of Grain and Flour using Moisture Meter 78
6.2 Determination of Insect Infestation of Grain by Counting and Weighing Method 78
CHAPTER SEVEN: FST 320 (CEREAL, ROOT & TUBER TECHNOLOGY)
7.1 Determination of Particle Size Analysis and Interpretation Using Graph 80
7.2 Determination of Particle Size Analysis and Interpretation Using Calculation 80
7.3 Determination of Total Ash of Flour 81
7.4 Determination of Acid Insoluble Ash 81
7.5 Functional Properties 82
7.5.1 Bulk Density: (BD) 82
7.5.2 Water/Oil Absorption Capacity 83
7.5.3 Foam Capacity (FC) and Foam Stability 83
7.5.4 Emulsification Capacity (EC) 84
7.5.5 Wettability 84
7.5.6 Gelation Capacity 85
7.5.7 Gelatinization Temperature 85
7.5.8 Viscosity 85
7.6 pH Measurement 86
7.7 Loaf Volume Determination by Seed Displacement Method 86
7.8 Amylose Contents of Starch 87
7.9 Determination of Gluten in Flour 88
7.9 Determination of Bromate in Flour 88
CHAPTER EIGHT: FST 322 (FOOD PROCESSING & PRESERVATION)
8.1 Canning of Pineapple 90
8.2 Laboratory Production of Burukutu 90
8.3 Canning of Mango Pieces 91
8.4 Freezing of Selected Vegetables.
8.4.1 Freezing of Okro 91
8.4.2 Freezing of Bitter Leaves 92
8.4.3 Freezing of Sweet Corn 93
8.5 Dehydration of Vegetables. 94
8.5.1 Dehydration of Okra 94
8.5.2 Dehydration of Peppers 95
8.5.3 Dehydration of Onion 95
8.6 Production of Tomato Powder 96
8.7 Production of Corn Dough Product 96
8.8 Production of Flour from Yam, Cocoyam 98
COURSE CODE: FST 309
COURSE TITLE: FOOD MICROBIOLOGY
PRACTICAL 1
Title: Introduction to the Microbiology Laboratory
Aim: To introduce students to the basic equipment in a standard food microbiology laboratory,
their uses and modes of operation.
Introduction: Microbiology is the study of living things that are too small to be seen by the
naked eye and can be seen, viewed and studied with the aid of a microscope. This food serve as a
carrier of chemical and biological substances even added or acquired as contaminants from soil,
water, air, food handlers, equipment. Food is what gives the body good health and can be plant
or animal origin. Thus, food microbiology refers to that aspect of applied science that deals with
the interactions between foods and microorganisms. The microorganisms can be grouped into
two (2) categories of prokaryotic (bacteria, rickets is as single cellular and Eukaryotic
(multicellular, without cell wall e.g. algae, fungi, protozoa and higher plants and animals
Microbiology is concerned with the study of their form, structure, reproduction,
identification, physiology and metabolism. The microscopic size of these organisms makes them
easily ubiquitous, occurring virtually in everywhere including the intestinal tract of man other
animals and their body surfaces.
Standard equipment in microbiology laboratory
1 Optical Equipment
i. Microscopes, Spectrophotometer, Fluoremeter, Particle counter
1. Laminar air flow cabinet: means air inflow of 0.4mls
2. Centrifuges: for separation of lighter portion of solution mixture or suspension from the
heavier portion by centrifugal force with different resolution per mill (rpm)
3. Heating units
i. Bunsen Burner, Hot plate, Immersion heater, Muffle furnace, Oven, Incubator, Water
bath, Metal block heaters, Retorts (Autoclaves): Autoclaves are manufactured to
stand a capacity heating of 30Ib pressure (30 psi or 0.3mpa).
Autoclaving pressure:
101b pressure (0.10mpa or 10psi) at 115.50c for 30mins,
151b pressure (0.15mpa or 15psi) at 121.50c of 15mins,
201b pressure (0.2mpa or 20psi) at 126.60c of 10mins
4. Balance
5. Petri dishes
6. Heamocytometer (Neubauer counting chamber): ussually employed in counting blood
cells, also used in counting other cells ranging for 3μm to 4μm in diameter and larger.
7. Glasswares
i. Bottles,beakers, burettes/Pipettes, cylinders, dessicators, flasks, funnels, Test
tubes
8. Inoculating/Wire loop and straight wire.
PRECAUTIONARY MEASURES
- Do not suck anything with the mouth, use rubber teats and for sucking.
- Always wear a laboratory coat.
- Avoid hand to mouth operation, while in the laboratory.
- Keep fingernails short and loose should be tied up
- Always wear shoe
- Swab the bench with disinfectant before and after working
- Thoroughly wash your hands with soap and water before and working.
- Treat all microbial cultures as if they are pathogens
- Autoclave materials to be disposed off.
ASSIGNMENT
1. Draw and label the equipment used in a standard microbiology laboratory; stating their
functions/uses and modes of operation.
2. Define the following terms.
- Mesophilic organisms
- Thermophilic organism
- Thermoduric organisms
- Psychotrophic organisms
PRACTICAL 2
Title: Microbiological media
Aims: To know different culture media, and
To know some terminologies in microbiology
Apparatus/Materials: Autoclave, petri dishes, incubator, inoculating chamber, agar media, etc.
Introduction: There are lots of different culture media used in food microbiology laboratory.
TERMINOLOGIES
- Colony: macroscopically visible growth of microorganisms on a solid culture medium
representing a colony.
- Culture: it’s a population of growth of microorganisms either on solid, in liquid or both
or in a sloppy medium.
- Cultivation: Growth of microbial populations in an artificial environment such as the
culture media under certain laboratory conditions. It can be carried out in the test tubes,
conical flasks, petri dishes and bottles.
- Incubation: The placement and conditioning of an already incubated culture medium in a
specific environment that favours the growth of microorganisms in the laboratory i.e. Act
of creating an enabling environment for microorganisms, infective materials.
- Inoculation: Process that involves the introduction of microorganisms, infective material
or serum into culture media or living animal or plant tissues.
- Inoculum: Microorganisms/Microbial material used to seed or inoculate a culture vessel
or culture medium.
- Isolation: Separation of a particular microorganisms from the mixed population that
exists in nature
- Sterile: A culture vessel free of any living organisms or microorganisms. It allows an
objective and realistic results to be obtained with regard to the study of any
microorganisms.
- Aseptic transfer: Transfer or movement of microorganisms from one place to another or
from one culture medium to another without contamination.
- Culture Medium: Any substrate or material that can adequately support growth of
microorganisms.
- NATURE OF CULTURE MEDIUM
- Can be in liquid or broth, solid or semi-solid (sloppy) form.
TYPES OF CULTURE MEDIUM
(1) Basic Media/General purpose medium/Routine Laboratory Media: Medium that
supports the growth of microorganisms that do not need or have special nutritional
requirements. It is used for sub culturing microorganisms from differential or selective
media prior to performing biochemical and serological identification tests. Example
Nutrient agar (NA), Plate Count agar (PCA)
(2) Differential Media: Media which contain or involve the incorporation of certain
reagents or chemical substrate such as dye as in indicator to permit differentiate between
types of bacteria in a sample base on their morphological appearance e.g. Eosin
Methylene Blue agar (EMB) to different Escherichia Coilform coliforms showing
metallic sheen.
(3) Selective Media: Allows the addition of certain specific chemical substances to the
nutrient agar which prevent/inhibiting/supposing the growth of one group of bacteria
from the rest. It could be devoid of essential nutrients e.g. EMB and MCA contain dye
that inhibit the growth of gram positive bacteria but allow gram negative bacteria, also
Deoxychocolate Citrate agar (DCA) or Salmonella and Shigella agar (SSA) for isolation
of only salmonella and shigella.
(4) Enrichment Media: Addition of nutrients such as blood serum or plant extracts or
animal tissue to nutrients broth/agar to support the growth of fastidious organisms such as
Selenite-F-broth for salmonella typhi
(5) Assay Media: prescribed composition used for the assay of vitamins, amino acids and
antibiotics. It is used to test or assaying the potency/efficacy of particular product/drugs
against certain microorganisms e.g. incorporation of paper disc of various antibiotics into
the nutrient agar.
(6) Enumeration Media: Allows the determination of the quantity of microbial load of any
given materials/samples e.g. milk and milk products, alcoholic and non alcoholic, water.
(7) Characterization Media: To characterize of microbes particularly bacteria. It reveals the
growth pattern
(8) Maintenance Media: Maintaining the viability and stability of microorganisms.
(9) Transport Media: Formulated and used for the temporary storage of species while they
are being transported or transfer from the field to the laboratory for analysis. It contains
only buffer and salt, lack carbon, nitrogen, organic compounds and growth factors to
prevent microbial multiplication e.g. Ringer solution and Stuarts.
Types of culture:
i. Pure culture: Only a single specie of microorganisms in a cultured plate or broth
ii. Mixed culture: several species of microorganisms in a cultured plate or broth
Procedure:
All the media will be shown to the student and what the type of microorganisms they are
used to isolate with different incubation period
PRACTICAL 3
Title: Aseptic Techniques
Aim: To know different aseptic techniques, and
How to employ aseptic condition during microbiological analysis
Introduction: Aseptic technique is a way of ensuring safety and Good Manufacturing Practice
(GMP) of analysis to prevent or limit microbial contamination from environment.
Procedures:
(a) Dry Heat: use of dry heat in sterilization of materials e.g.
Steps:
I. Wash the glasswares, put in the hot air oven, set at 1600C for 2hours. This destroys
microbes by dehydrating the cells thereby inhibiting protein coagulation
(b) Moist Heat: Use of air or steam generated when liquid suspension as water is subjected
too high temperature.
(i) Boiling: Boiling (at1000C) helps to reduce the number of vegetative cells. Total
sterility cannot be achieved by the use of boiling water but being used as an
alternative method where other suitable or better methods are not available.
(ii) Pasteurization: immerse the sample in hot water maintain ateither flash temperature
i.e. high temperature short time (HTST) at 700C for 2 sec or Batch temperature i.e.
low temperature long time (LTLT) at 620C for 3 minutes. The temperature and the
time can be adjusted as required.
(iii) Autoclave (steam under pressure): It work on the principle of release of heat of
vaporization through condensation of steam on organisms responsible for the
coagulate of protein, hence the rapid killing of microorganism on materials
1320C (at 27 pound per square inch) for 12mins
1210C (at 15 pound per square inch) for 15mins
1150C (at 10 pound per square inch) for 10mins
How to operate:
Put enough water into the inner container of the autoclave usually at the lower level
Load the materials to be sterilized into the inner container, close the lid and tighten the bolts
Plug the autoclave and open the escape valve to allow air inside the autoclave to escape for about
2-5 minutes
Close the valve and allow the pressure to build up to the required temperature
Watch out for a sound to indicate the desired pressure has built up
Start timing for the required period of time
Disconnect from the mains then allow the autoclave to cool down before you open
Chemical disinfection and sterilization
The use of chemical substances helps to achieve disinfection and sterilization. Chemical
disinfectants are not reliable sterilization agents as they as they affect some biological materials.
Only ethylene oxide; a toxic gas can be used to sterilize materials that cannot be autoclaved and
which would not be effect on biological material. Some common laboratory disinfectants
include: 3% Lysol, 75% Alcoholic (ethanol), 1% Domestos, 0.5% Phenol, 1% Mercuric chloride,
40% Formation (40% w/v formaldehyde + 10% methanol in water).
PRACTICAL 4
Title: Isolation of microorganisms from food sample for microbiological analysis
Aim: To determine the total viable count of microorganisms, using different isolation techniques
Introduction: Microorganisms are everywhere and it usually isolated from materials such a food
samples, running water, air etc. to know and identify the possible microorganisms present.
Microorganisms form discrete colonies on solid media and this may be obtained by modification
of the plating method. This method involves separation and immobilization of individual
organisms on or in a nutrient medium solidified with agar or some other appropriate solidifying
or jelling agent. After separation/inoculation and incubation by subculturing, each viable
organism gives rise through growth, to a colony from which subsequent transfer through aseptic
technique can be readily made. This operation tends to prevent any possible contamination from
any source, resulting in the emergence of the pure culture. The variants of such as pour-plating
with serial dilution method, spread plating, stabbing method, dropping method, dilution shake
culture/spread plate method etc.
Sampling: The procedure used to draw and constitute a sample. The adequacy and condition of
the sample or specimen received for examination are of primary importance. If samples are
improperly collected and mishandled or are not representative of the sampled lot, the laboratory
results will be meaningless. Because interpretations about a large consignment of food are based
on a relatively small sample of the lot, established sampling procedures must be applied
uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed
within the food or when disposal of a food shipment depends on the demonstrated bacterial
content in relation to a legal standard.
Sampling plan: Is a systematic way to assess the microbiological quality of food lots.
Lots: Refers to a batch of products manufactured under the conditions at the same time.
A comprehensive sampling plan includes:
(a) The microbe or group of microbes of concern or interest.
(b) Number of samples to be tested (n)
(c) Testing methods
(d) Microbiological limit (s), m and M
Acceptable (≤m)
Marginally acceptable (>m and ≤M)
Unacceptable (>M)
(e) Number of samples which falls into each category of microbiological limit (i.e.
acceptable/marginal/unacceptable).
Types of sampling plan: commonly used in food Microbiology
(a) Two-Class attributes plan: Sample (s) is (are) taken from the lot and tested. Only one
microbiological limit “m”, is involved. Two classes of attributes ≤m and >m could be
identified. Maximum allowable number of samples (s) that yielded unsatisfactory test
results is represented by “c”.
(b) Three-class attributes plan: Two microbiological limits, m and M are set. “m: reflects
the upper limit of a Good manufacturing practice (GMP) and “M” mark the limit beyond
which the level of contamination in hazardous or unacceptable.
Method of collection
Tap water:
i. Remove any internal and external fittings such as hosing.
iii. Clean the end of the tap thoroughly with a clean disposable cloth (and detergent if
necessary). Sterilise with sodium hypochlorite solution (sufficient to give 1% available
chlorine) made up on the day of use, or chlorine dioxide foam. Sterilisation can be
carried out by preparing a hypochlorite solution in a measuring jug and suspending it
under the tap, such that the end of the tap is immersed in the solution for 2 to 3 minutes.
Alternatively, use a wash bottle to spray hypochlorite solution onto the outside and
inside of the tap spout. Leave for 2-3 minutes before rinsing.
Safety Note: Sodium hypochlorite is highly corrosive and should be handled with care.
Nitrite gloves and goggles should be worn, and if contact with skin, eyes or clothes occur,
wash the affected area immediately with copious amounts of water. Contact with clothes
may result in a bleaching effect.
iv. Turn on the tap gently to avoid unnecessary aerosol production and run water to waste for 2-3
minutes.
iv. Aseptically open a labelled sterile bottle (1 litre or 500 ml bottle containing neutraliser;
fill almost to the brim with water, replace and tighten the lid and shake the bottle to
distribute the sodium thiosulphate.
vi. Water samples should be stored between 2 and 80C. They should be submitted to the
laboratory to ensure that they can be examined promptly, ideally the same day, but always within
24 hours of collection.
Air:
Aseptically expose the cultured labelled plate to the sampling environment for 5-10 minutes and
quickly cover and for incubation at required temperature and time.
Swab Samples:
Wear gloves
Select a sampling area of about 10 cm X 10 cm (or 20 cm x 20 cm)
Break the seal round the tube containing the swab
Remove the swab from the tube and rub and roll it firmly several times across the
sampling area.
Return the swab into the tube and label the sample
Send the sample to the laboratory for analysis.
If one is sampling a dry surface, it is recommended that a wet or moistened swab is used. The
swab test method has proved a popular testing method with flood damage insurance claims,
where there may be sewage contamination. If swab samples are collected for culture analysis,
they should be sent to the laboratory within 24 hours after collection. If the analysis of the swab
samples involves enumeration of the microbial contaminants, the size of the area sampled should
be provided to the laboratory.
Food sample:
i. The sampling procedure may vary depending on the type of food, and the reason for
sampling. For example, if food-handling practices within a catering unit are being
investigated, it may be appropriate to sample the food using the utensils that would normally
be used for handling or serving the food. However, if the intact food is to be examined as
supplied by the producer to the hospital catering department, the sample should be collected
using sterile utensils.
v. Put on a new pair of disposable gloves for each food sample, taking care not to
contaminate the outer surface of the gloves.
vi. Using appropriate sterile utensils, take a portion of the food. This will normally be a
representative portion of all components but may be a specific portion such as a core
sample, surface sample, filling etc. Place the food sample into a sterile food-grade bag or
plastic honey jar, taking care not to allow the sample to make contact with the outside or
top edge of the container. Label the container with the location and sample details,
sender’s reference, sampling officer and date and time of sampling. When a secure chain
of evidence is required, place the container into another sterile bag & seal with a tamper
evident tag.
iv. At least 100 grams of food is usually required, unless an alternative quantity has
previously been agreed with laboratory staff. Where intact foods are to be examined, take
the whole sample and place inside a food-grade bag in its original wrapping.
vii. Record any relevant information such as the place of sampling, temperature of storage,
type of packaging and type of sample if not representative.
vi. Store samples in a cold-box, preferably between 0 and 50C (taking care to keep raw foods
in a separate box from ready-to-eat foods, if possible, and hot food separate from cold), and
return to the laboratory as soon as possible (normally the same day, unless there is a particular
reason for a delay such as sampling late in the evening).
viii. If necessary, samples can be left in a cold-box overnight, provided that it is
properly packed with an adequate number of ice blocks (10% of the total volume of the
cold-box should be taken up by ice blocks), or transferred to a secure fridge or cold-
room, and submitted to the laboratory as early as possible on the following day.
(a) Pour plating: Serial dilution is usually carried out before the pouring of the plate.
Preparation of diluent: i. To make the initial dilution, taken 1ml or 1g of the original
material with a sterile pipette and addto 9ml of sterile diluent (usually physiological
saline: 0.85g Sodium Chloride with 100ml of distilled water or 0.5g of Peptone) and
thoroughly mixed. This is 10-1 dilution
ix. To make further dilutions, dispense 1ml from dilution 10-1 into another bottle of a fresh
sterile 9ml diluent to make it 10-2. This is continued until a desired dilution is obtained.
x. Transfer 1ml of the desired dilution of the sample into a sterile petri dish.
xi. Pour sterile molten (at 450C) agar into the petri dish. For aseptic pouring, hold the
Erlenmeyer flask in the right hand; remove the plug with a combination of the thumb and
the little finger of the left hand.
xii. Flame the mouth of the flask immediately.
xiii. Carefully raise the lid of the petri dish at an angle with the left hand until just
sufficient to allow the mouth of the flask pass under it.
xiv. Pour the molten agar gently into the dish.
xv. Carefully but thoroughly rotate the dish in clockwise and anticlockwise directions so that
agar form a uniform lager.
xvi. When the agar has cooled and hardened, invert the petri dish to prevent
condensation dropping from the lid into the agar.
(b) Streaking plate method: sterilize the wire loop by passing it over blue flame until it
turns reddish.. Use the sterile wire loop to pick loopful of microbial suspension and used
it to make series of parallel, non overlapping streaks on the surface of an already
solidified agar plate. The final streaks should not touch the source of the inoculums on
the agar plate. In this event, the inoculum is progressively diluted with the successive
streaks, such that even if the initial streaks results into confluent growth well isolated
discrete colonies develop along the later streaks on the plate, which are pure cultures.
(c) Spreading plate method: Use specialized sterile glass rod to evenly tin out the microbial
suspension on the already solidified agar plate, in which a sterile loop have firstly been
used to inoculate the plate. Commonly used on liquid microbial suspension.
(d) Stabbing plate method: Use a sterile wire loop or pin to cut the solidified agar medium
in such a way that the medium breaks. This method of inoculation allows the
development of the organisms inside the medium of growth, thus suitable for the isolation
and cultivation of anaerobic microorganism.
(e) Dropping method: This involves the use of sterilized pipette. The sterile pipette is used
in taking known volume (0.5-1ml) of the inoculum culture in liquid suspension into either
molten but cool agar medium or broth medium in test tubes plugged with cotton wool.
This method allows the development of the microbial growth on the surface of the broth
medium. The homogenization of the broth medium continuously allows not only the even
growth of the microbes but also the equal accessibility of the microbes to the nutrients
contained in the medium of growth.
(f) Dilution shake culture method: It’s a modification of the pour plate method employed
for strict anaerobes (oxygen sensitive anaerobes). It involves the use of seven (7) to ten
(10) test tubes containing molten but cool agar medium .The first test tube is inoculated
and mixed thoroughly, followed by the transfer of one-tenth of its content to the second
until the last tube. The test tubes are rapidly cooled and sealed with layers of sterile
petroleum jelly and paraffin on the surface to prevent access of air to the agar column.
The discrete colonies develop deep in the agar medium. The major problem associated
with this method is that of the difficulty associated with the subsequent transfer of the
colonies embedded in the agar medium.
Total viable count = Number of colony (N)
Volume used for dilution (Aliquot) (V) × dilution factor (D)
Colony Forming Unit = cfu1/ml or g; Spore Forming Unit = (sfu1/ml or g)
Incubation period: Lactobacilli at 37±20C for 24 to 48hours under anaerobic condition by using
Anaerobic jar with gas packs; Other bacteria at 37±20C for 24hours and Fungal at 250C for
24hours
ASSIGNMENT
Advantages and disadvantages of serial dilution
Advantages of pour plate over streaking techniques
PRACTICAL 5
Title: Isolation of microorganisms from food sample for microbiological analysis
Aim: To determine the spoilage and pathogenic microorganisms using isolation technique such
as pour-plating method.
Introduction: Spoilage and pathogenic microorganisms occur in food as a result of inadequate
heat treatment and inefficient sampling method used or given to such foods especially the
processed and packaged foods. Likewise, raw food samples also get spoilt as a result of storage
temperature and time being exposed to. There are some factors that contribute to the spoilage and
pathogen detection in food samples such as pH, temperature, water activity, humidity,
atmospheric oxygen, condition of storage/preservation and as well as processing operations. This
can also, when the critical control point analysis is inadequate and faulty.
PROCEDURE:
(g) Pour plating: Serial dilution is usually carried out before the pouring of the plate.
Preparation of diluent:
(h) i. To make the initial dilution, 1ml or 1g of the original material is taken with a sterile
pipette and added to 9ml of sterile diluent (usually physiological saline:0.85g Sodium
Chloride with 100ml of distilled water or 0.5g of Peptone) and thoroughly mixed. This is
10-1 dilution
.To make further dilutions, 1ml from dilution 10-1 is dispensed into another bottle of a fresh
sterile 9ml diluent to make it 10-2. This is continued until a desired dilution is obtained.
ii. Transfer 1ml of the desired dilution of the sample into a sterile petri dish.
iii. Pour sterile molten (at 450C) agar into the petri dish as described in practical 3A
iv.When the agar has cooled and hardened, invert the petri dish to prevent condensation
dropping from the lid into the agar and incubated at desired temperature and time.
PRACTICAL 6
Title: Identification and Characterization of Microorganisms
Aim: to observe colonies of microorganisms on cultured media and their characteristics by
macroscopic morphology
Apparatus/Materials: Petri-dishes, incubator, spirit lamp, ethanol etc.
Introduction: Microorganisms can be observed by direct morphology Growth of bacteria cells
on solid medium results in colony formation, which is visible to the naked eyes. The appearance
of these colonies is fairly constant for a given species. It is therefore of taxonomic value. The
direct observation of microorganisms involves observing the colonial characteristics of a
particular species of microorganisms on a solid agar surface.
Procedure:
Different cultured plates with various discrete colony morphologies will be used to identify
various microorganisms. These characteristic include:
i. Shape: it can be circular, irregular, rhizoid, filamentous, puntiform or spindle.
ii. Chromogenesis
iii. Opacity: this shows the appearance of colony in light. It can be translucent,
transparent or opaque.
iv. Elevation: it can be flat, raised, umbonate, convex, low convex or convex
papillate.
v. Surface: can be smooth or rough, dull or glistering.
vi. Edge: it can be entire, lobate dentate, fimbriate, undulate, crenated, lobate with
radial striations or rhizoid.
vii. Consistency: this is about the texture of the colony from how they appear. It can
be butyrous (covering the plate and butter-like), friable (dry and easy break) and
viscous (slimy-drawing).
viii. Emulsifiability: ability to dissolve in water and grow in water
ix. Size: there are different sizes of colonies, ranging from very tiny to very large
x. Colour: it can be red, greenish, creamy, whitish, black, grey, yellow, etc.
PRACTICAL 7
Title: Staining Techniques, Identification and Characterization of Microorganisms
Aim: To observe colonies of microorganisms on cultured media and their characteristics by
microscopic morphology
To know the different types of staining techniques
Apparatus/Materials: Petri-dishes, incubator, spirit lamp, ethanol, distilled water, microscopic
slides, staining reagents etc.
Introduction: Staining techniques are employed in microbiology to enable and aids in for
characterization of bacteria, yeasts and moulds. There are three major types of stain. They are:
i. Acid stain: these are stains which posses their colouring matter in the acidic radical thus,
rendering the basic radical colourless. Examples are: Acid fuchsin for detection of the
Acid-fast Bacillus (AFB), Malachite green for spores staining, Nigrosin.
ii. Basic stain: possess their colouring matter in the basic radical, with the acid radical
remaining colourless. Examples are Crystal violet for gram’s reaction, Methylene blue
that readily stain bacterial cell, Neutral red and Safranin O for counter stain in gram’s
reaction.
Neutral stain: it consist of equal volumes of solutions of specific acid and basic dyes.
Examples are Giesma for malaria staining, Leishmen and Wright stains.
Procedure:
Gram staining technique
i. Prepare a heat fixed smear from an 18-24 hour old culture.
ii. Stain with crystal violet solution for 1-2 minutes and pour off the solution.
iii. Rinse with Gram’s iodine solution and allow the iodine to stand for 1 minute
iv. Pour off the iodine solution and decolorize with 95% alcohol unit no more violet
runs from the slide.
v. Rinse under gently running tap water and counterstained with Safranin o for 30
secs.
vi. Wash with water, air dry and examine microscopically using oil immersion
objective lens (x100).
vii. Report your observations
Gram-positive bacteria retain the crystal violet stain while the gram negatives take on the
red stain.
iii.
Microorganisms can be observed by microscopic morphology
Microscopic Observation
Microorganisms can be observed in the stained and unstained conditions using a
microscope. The morphological and staining reaction of individual organisms serves as
preliminary criteria for placing an unknown species in its appropriate biological group.
A gram-stained smear is first examined. This reveals the gram reaction of the individual
cells, whether positive or negative, shape and size, as well as the position of endospore if present.
Other staining procedures include:
i. Negative staining: for capsule determination
ii. Flagella staining
iii. Lactophenol cotton blue staining: for staining the cytoplasm of fungi.
ASSIGNMENT
1. What is a colony?
2. What is the basis of the gram reaction?
3. Why is iodine used in gram staining?
PRACTICAL 8
S/No Characteristics Gram positive Gram negative
1. Lipid content of the cell wall 1-5% 12-25%
2. Amino sugar content of the cell wall 10-22% 2-8%
3. Thickness of the cell wall 20-80 mm 15-20 mm
4. Cell wall digestion by lysosome Many species Require pre-treatment
5. Digestion by trypsin or pepsin (dead cells) Resistant Susceptible
6. Susceptibility to Penicillin &sulphonamide Marked Much less
7. Resistance of physical disruption More resistant Less resistant
8. Inhibition by basic dyes e.g. crystal violet Marked Much less
Title: Identification of microorganisms
Aim: To identify microorganisms by their biochemical characterization examination.
Introduction:
CLASSIFICATION
Classification of microorganisms requires knowledge about their characteristics features,
which is obtained by some techniques. The eukaryotic organisms such as protozoa, algae and
fungi possess distinct morphological features, which may by use for identification purpose.
Owing to their small size and the paucity of morphological characteristics bacteria and viruses
are more difficult to identify than the other groups of organisms. Hence they are identified and
subsequently classified on the basis of biochemical, physiological and in recent times, genetic
properties in addition to their morphological features. The various biochemical and physiological
test that are performed for identification purpose may be divided into 3 main parts.
1. Reaction involving nitrogen compounds: These are biochemical reaction in which the
microorganisms use nitrogen compounds either as substrate of enzymatic activity or as
source of nitrogen. They include gelatin hydrolysis, casein hydrolysis, production of
indole, production of ammonia from peptone, decarboxylation of amino acids, production
of hydrogen sulphide, urease activity, reduction of nitrates, action of litmus milk, LV
(lecithovitellin) test.
Procedure
1. Gelatin hydrolysis: ability of microorganism to produce enzymes (proteolytic) to
breakdown gelatin by losing its gelling qualities.
Frazier gelatin agar
i. Nutrient agar plus 0.4%
ii. Sterilize by autoclaving for 15mins at 1210C.
Test Reagent: Mercury chloride solution
Mercury chloride -15.0g
Conc. Hydrochloric acid -20ml
Distilled water – 100ml
Technique
i. Inoculate a poured, dried plate of the medium by streaking once across the surface,
ii. Incubate at optimum temp for 2-7 days.
iii. Flood the plates with a solution of the test reagent
Observation
1. Unhyrolysed gelatin form white opaque precipitate with the reagent.
2. Hydrolysed gelatin appears as a clear zone.
2. Casein hydrolysis: Ability of the microorganism to utilise casein as a source of nitrogen to
produce protease enzymes which breakdown casein into amino acids.
Medium
i. Milk agar (nutrient agar plus 10% skim milk)
ii. Autoclave at 1210C for 15 minutes at 0.15mpa
Test Reagents
Same as for Frazier’s gelatin medium
Technique
i. Inoculate poured plates of the medium by streaking once across the surface.
ii. Incubate at the optimum temperature for 2-7cubate at the optimum temperature for 2-7
days
Observation
i. Clear zones indicate casein hydrolysis
ii. Flood plates with test reagent. Unhyrolysed casein forms white opaque precipitate;
hydrolysis is indicated a clear zone.
3. Production of indole: Differentiation of coliforms
Medium
I. Tryptone water (1-2% tryptone and 0.5% NaCl in water)
Test reagent
Kovac’sindole.
Para – dimethyl aminobenzaldehyde _ 10g
Pure –amyl alcohol 150ml
Pure amyl alcohol 50ml
Conc. Pure HCL %50ml
Technique
i. Inoculates a tube of tryptone water with a loopful of a broth culture of the
organisms under study.
ii. Incubate for 5-7 days at the optimum temperature gently and then allow to stand.
A deep red colour develops in the presence of indole which separates out in the
alcohol layer.
4. Production of ammonia from peptone: Ability of bacteria to produce deaminase
enzymes capable to breakdown peptone or other proteins to release ammonia.
Deaminase Medium
Peptone water (1% of peptone and 0.5% of NaCl in water).
Technique
i. Inoculate a tube of peptone water with a culture of organism under study.
ii. incubate this and also a sterile control tube which consists of uninoculated
peptone water, at the optimum temperature for 7 days.
iii. Add a loopful of the culture to a loopful of Nessler’s reagent on a slide.
Alternatively, add 1ml of the culture to 1ml of Nessler’s reagent in a clean test
tube. Do the same of the control tube.
5. Urease activity: To distinguish between members of EnterobacteriaceaeProteus vulgaris,
Eschericheria coli and Salmonella
Urease medium
Peptone 0.1g
NaCl 0.5g
KH2p04 0.2g
D (4) glucose 0.1g
Phenol red (0.2% in 50% ethanol) 0.6ml
Agar -2.0g
Distilled water - 100ml
Technique
i. Prepare the above medium, distribute into bottles or test tubes, sterilize at 1210C
for 15 minutes and cool to 45-500C.
ii. Urea is added to the basal medium to give a final concentration of 2% urea. The
bottles are slanted and the contents allowed cooling and setting in that position.
iii. Streak a little of the culture of the organism over the surface of the urea agar
medium. Also inoculate a control of the basal medium containing no added urea.
iv. Incubate at the optimum temperature
v. Examine daily (for up to 7days) for the production of alkali (red colouration).
6. Nitrate reduction: Ability of the microorganism to be capable of reducing nitrate to
nitrite or even ammonia or nitrogen.
Nitrate medium
Composition and preparation
i. Peptone water plus 0.1% potassium nitrate
ii. Distribute the medium into tubes, each with an inverted Durham tube and sterilize at
1210C for 15 mins.
Reagents
Gries-llovary’s reagent
i. 8g sulphanilic acid in 100ml of acetic acid
ii. 5g naphthylamine in100ml acetic acid
Techniques
i. Inoculate a tube of nitrate medium with a loopful of a broth culture of the
bacterium
ii. Incubate for 5 days at the optimum temperature
iii. Examine for the presence of gas in the inverted Durham tube
iv. Test for presence of nitrate using Griesss-llosvay’s reagent.
Observation
i. Pink, redormaroncolour indicates positive reaction.
ii. No colour change indicate negative reaction
iii. If negative, add a pinch of zinc dust as stated above.
7. Production of hydrogen sulphide: Ability of microorganism to decompose organic
sulphur such as cysteine and cystine or reduce inorganicsulphur to produce hydrogen
sulphide.
Hydrogen sulphide medium
Either
i. Cysteine broth: Peptone water or nutrient broth plus 0.01% cysteine or
ii. Thiosulphate broth: peptone water or nutrient broth plus 0.01% thiosulphate.
Indicator paper
Soak strips of filter paper in saturated solution of lead acetate. Dry the paper strips and
then sterilize at 1210c for 15mins.
Techniques
i. Inoculate a tube of cysteine or thiosulphate broth with loopful of a broth culture of
the organism.
ii. Remove the plug from the tube places the indicator paper strip in the mouth of the
tube. The strip must be placed in such a way that its lower end is above the
medium but below the inner end of the plug and do it for an uninoculated control
broth.
iii. Incubate for 3-5 days at the optimum growth temperature.
iv. Examine and record your observation
Observation
i. Production and liberation of hydrogen sulphide causes blackening of the lead
acetate paper strip.
ii. If no blackening has occurred, add 0.5ml of a 2ml HCl acid and replace plug and
the paper strip immediately. If any sulphide has been produced but has remained
in solution, the addition of the acid will cause the release of hydrogen sulphide.
The uninoculated control broth should be treated in like manner.
8. Decarboxylation of amino acids:
Decarboxylase medium
Amino acid - 1.0g
Peptone - 0.5g
Beef extract - 0.5g
Dextrose - 50mg
Bromocresol purple (0.2%) – 0.5ml
Cresol red (0.2%) – 0.25ml
Distilled water -100ml
Technique
i. Use a straight inoculating needle to inoculate a tube of decarboxylase medium.
ii. Layer the tube with 2ml sterile liquid paraffin.
iii. Incubate at the optimum temperature.
iv. Examine daily for about 5 days.
OBSERVATION
i. Yellow coloration indicates production of acid by the dextrose metabolism
ii. If decarboxylation occurs, the medium becomes violet. The control (i.e.
uninoculated broth tube should remain yellow.
CLASS WORK
Demonstrate each method to student and divide class into 8 groups, with each group
taking up each group up each method.
PRACTICAL 9
Title: Identification of microorganisms
Aim: To identify microorganisms by their biochemical characteristics
Introduction:
1. Reactions involving carbon compounds: In these reactions, carbon compounds serve as
substrates for biochemical activities which involve the production of specific enzymes
that degrade the compounds in characteristic manner. Some of these tests include
hydrolysis of starch, oxidation/fermentation test (o/f), methyl red/vogesproskauer test
(MRVP) , utilization of citrate, fermentation of sugars, ONPG test, Gluconate
2. Miscellaneous test: Besides the reaction involving carbon and nitrogen compounds,
other tests of paramount importance are carried out for the purpose of bacterial
identification. These tests are mostly also based on enzyme reactions. They include
catalase test, oxidase test Haemolysis of blood, Coagulase test, growth on MacConkey
medium, Ejikman test, Phosphatase activity and Pigment formation
Procedures:
Hydrolysis of starch
Reagent
Gram’s iodine (as employed previously for Gram staining)
Medium
Starch Agar (Nutrient agar plus 1% solute starch
Techniques
i. Pour 20ml of molten starch agar at 450C into a sterile petri dish and allow to cool
and set-invert in an incubator at 370C.
ii. Streak the organism once across the surface of the plate.
iii. Incubate the plate at optimum growth temperature for 3-5 days
iv. Flood the plate with some quantity of Gram’s iodine
Observation
i. Unhydrolysed starch forms a blue or blue black colour with the iodine.
ii. Hydrolysed starch appears as a clear zone which results from amylase activity.
iii. Reddish brown zones around the colony indicate partial hydrolysis of starch (to
dextrin’s), which results from amylase activity.
2. Fermentation of sugars
Medium
Peptone - 1.0%
NaCl - 0.1%
Fermentable sugar - 1.0%
Indicators
Any of the following indicators in the concentration given may be used.
i. Andrade’s indicator - 1.0%
ii. Phenol red - 0.1%
iii. Bromocersol purple - 0.0025%
Preparation of medium
i. To the above medium add an indicator of choice and discharge 5-10ml into test
tubes.
ii. Include an inverted Durham tube in each tube
iii. Sterilize at 1210C for 15mins at 0.15mpa
Techniques
i. Incubate one tube of each carbon type medium with the same organism.
ii. Incubate at the optimum temperature. Also incubate auninoculated control.
iii. Examine daily and record the results.
Observation
Acid production is shown by a change in the colour of the indicator. If gas is produced, it
accumulates in the Durham tube. Gas production is dependent on acid production, but acid may
be produced without gas being produced.
Suggested recording of observation
Ab – acid full colour change
As-small amount of acid, i.e. indicator not fully changed
Gb- small (Durham tube more than ¼ full)
3. Oxidation – fermentation (O/F) test: Ability of the microorganism to breakdown
carbohydrate either by aerobic (oxidatively) or anaerobic (fermentatively)
Medium – Hugh and Laifson’s medium
Peptone 0.2g
NaCl 0.5g
K2HP04 30mg
Bromothymol blue 0.1ml
Agar 1.5g
Distilled water 100ml
Techniques
i. Add carbohydrate (usually dextrose) to the basal medium above (1%).
ii. Stabs inoculate two tubes each for each organism under study.
iii. Cover the surface of the medium in one tube with sterile paraffin.
iv. Incubate at optimum temperature for up to 14 days and examine.
Observation
i. Utilization of dextrose or any other carbohydrate results in the production of acid.
Acid production is indicated by a change in the colour of the medium from blue to
yellow.
ii. Fermentative organisms produce acid in both tubes
iii. Oxidative organisms produce acid only in the open tube i.e. without paraffin
3. MRVP test: To distinguish Eschericheria coli from Aerogenes
Medium
Dextrose 0.5g
KH2p04 0.5g
Peptone 0.5g
Distilled water 100ml
Dissolve ingredients in the water and sterilize at 1210C for 15mins at 0.15mpa.
Reagents
a. 0’mearra’s creatine and 40% KOH
b. Barrit’s 5% ethanol solution of – naphthol and 4% KOH
c. Methyl red
Technique
i. Inoculate a tube of MRVP broth with the organism
ii. Incubate the culture at optimum growth temperature
iii. At the end of incubation, aseptically divide the content of the tube into two portions
and label M and V respectively.
Treat M as follows
i. Add 5 drops of methyl red solution and examine the colour. Red colour indicates
position reaction. i.e. acid produced and yellow colour indicates negative reaction.
Treat V as follows:
i. Barritt’s method
a. Pipette 1ml portion from the culture and place on the rack
b. To the 1ml of culture, add 0.5ml of 6% -naphthol solution and 0.5ml KOH and
shake the tube.
c. Development of a red coloration, usually within 5 minutes constitutes a positive
reaction.
ii. 0’Meara’s method
a. To the remaining culture, add a pin-point of creatine, followed by 5ml of 40%
KOH and shake.
b. Barritt’s 5% ethanolic solution of-naphthol and 40%.
c. The development of a pink in the medium, usually within 30 minutes indicates a
positive reaction
4. Utilization of citrate as the sole source of carbon Koser’s citrate medium: To
differentiate Escherichria coli by their ability to utilize citrate as sole carbon source.
Composition
Sodium ammonium hydrogen phosphate 0.15g
Potassium di-hydrogen phosphate 0.10g
Magnesium sulphate 0.20g
Sodium citrate 0.20
Bromothymol blue 0.16ml
Distilled water 100ml
Techniques
i. Inoculate Koser’s citrate medium with a straight inoculating wire from a peptone
water culture or from a saline suspension prepared from a young agar slants
culture. Wire loop should be avoided because besides making the medium
unacceptably turbid. It may carry appreciable quantities of organic compounds to
the medium.
ii. Incubate at the optimum growth temperature for 2-6 days and examine for change
in the colour of the indicator.
Observation
A change in the indicator from green to blue indicates utilization of the citrate. All
positive reaction should be confirmed by subculturing into fresh media.
Miscellaneous
1. Catalase test: Two pairs of H+ combined with oxygen to give water to bring about
complete oxidation of acetyl group.
4Fe2+ + 4H+ +02 4Fe3+ + 2H20
2Fe2+ + 2H+ +02 2F3++H202
H202 2H20 + 02
Medium
Nutrient agar in plates or slopes
Reagent
Hydrogen peroxide (3% volume concentration)
Technique and observation
i. Pour 1ml of hydrogen peroxide over the surface of an agar culture (slope or
plate). Alternatively a loopful of growth may be emulsified with a loopful of
hydrogen peroxide on a slide.
Examine for the formation of oxygen bubbles which is indicative of the presence of
catalase. The hanging drops methods
i. Place a little Vaseline around the edge of the hollow in a clean cavity slide.
ii. Transfer a loopful of the culture to the center of a clean cover slip lay on the
bench. The drop culture should not be spread.
iii. Carefully invert the cavity slide over cover slip in such a way that the drop is in
the centre of the cavity. Now press down the slide gently but firmly enough so
that the Vaseline seals to the cover slip in position.
iv. Invert the slide quickly but smoothly so that the drop of culture lies in a hanging
position.
v. Examine the preparation immediately avoiding excessive illumination, which
could quickly cause the organism under study to loose motility.
vi. Report your observation
Observation: Mobile bacteria will be seen to dart across the microscopic field. This could be
distinguished from the Brownian movement characteristics of all small particles.
2. Oxidase test: Separation of Neisseria in mixed culture and in differentiating
Pseudomonas from Enterobacteria
Medium
Nutrient agar in petri-dish
Reagent
1% aqueous solution of tetramethyl-p-phenlendiamine hydrogen chloride.
Technique 1 and observation
i. Streak the culture onto the dry surface of a nutrient agar plate and incubate at
optimum growth condition until reasonable growth has been obtained.
ii. Pour the reagent over the surface of the agar growth.
iii. Oxidase positive colonies develop a pink colour which becomes successively
dark-red, purple and black within 10-30minutes.
Techniques 2 and observation
i. Add a few drops reagent to a piece of No 1 whatman filter paper in a petri dish.
ii. With a platinum loop or glass rod, smear some bacterial growth onto the
impregnated filter paper.
iii. A purple coloration is produced within 5-10 seconds by indicated by a purple
colouration within 10-16 seconds, any later reaction being taken as negative.
3. Coagulase test: To differentiate pathogenic from non-pathogenic staphylococci
Technique
This test is performed usually with 18-24 hours culture to obtain best results.
i. Slide method
a. Mark a slide into two sections place a loopful of normal saline (0.85% NaCl
in aqueous solution) in each section and emulsify a small amount of an 18-24
hour agar culture in each drop until a homogenous suspension is obtained.
b. Add a drop of human or rabbit plasma to one of the suspension and stir for
seconds and leave the other as control.
c. A coagulase positive result is indicated by clumping, which will not re-
emulsify.
ii. Tube method
a. Place 0.5ml of diluted plasma in each of two small test tubes.
b. To one tube add 0.5ml of an 18-24 hours broth culture and sterile saline to the
other as control.
c. Incubate both tubes at 370C and examine after 1hour and at intervals of up to
24hours.
d. Clotting indicates that the organism under study is coagulase-positive.
Coagulase normally takes place within 1-4 hours. The second tube serves as
control and should show no coagulation.
4. Heamolysis: Ability of some microorganisms such as streptococci and staphylococci
to produce toxin known as haemolysins to cause lysis of red blood cell
Technique
i. Melt 20 ml of nutrient agar or any suitable (solid) medium and cool to 450c.
ii. Add 1-2 ml of blood aspetically to the molten agar and mix thoroughly by gentle
rotation between the hands.
iii. Pour the contents of the tube or bottle into a sterile petri dish, allow it to
solidify and dry.
iv. In case of Staphylococci, streak a dried plate of the medium so as to produce
separate distinct colonies. If Streptococci are being examined, either prepare
pour plate or incubate streaked plates anaerobically.
v. Incubate at the optimum temperature for examine for 24-48hours.
Observation
Clear zone around the colonies indicate haemolytic activity
Class work
The best demonstrated to the students and the class divided into 9 groups with each
groups with each group taking one test.
PRACTICAL 10
Title: Microbiological Examination of Foods
Aim: To analyses water microbiologically by the multiple tube fermentation technique (most
probable number MPN test).
Materials: Durham’ tubes, MacConkey broth (MCB), test tubes/bottles, autoclave etc
Introduction: The multiple tube fermentation technique is a method is a method usually
employed in estimating the number of coli form bacteria in water, sewage, milk and milk
products. Water purity is usually tested by sing indicator organisms. These are organisms
associated with the intestinal tracts whose presence in water indicates fecal contamination of
water. The presence of the indicator is presumed to warn of the possible presence of pathogens.
The coliform group of organisms is the most widely used microbial indicator of water quality.
They are gram negative, non-spore forming, aerobic or facultative anaerobic bacteria that
ferment lactose and produce acid and gas.
Coliforms can either be fecal (such as Escherichia coli which indicates a possibility of
sewage contamination) or non-fecal. The non-fecal occur naturally in the soil, water or
vegetation indicating contamination from airborne sources or product contact surfaces. The
microorganisms of importance in the microbiological analysis of packaged drinking water such
as Aerobic mesophilic bacteria, Coliforms and Escherichia coli. The aerobic bacterial counts
provide an estimate of the number of viable microorganisms in the packaged water. Counts
greater than 100cfu/ ml indicator poor GMP (good manufacturing practice) in the multiple tube
technique for estimation of coli forms, many sample of different quantities of the liquid are
examined in order to obtain a mean result.
Many important human pathogens are maintained in association with living organisms
other than humans including many wild animals and bird. Some of these bacterial and protozoan
pathogens can survive in water and infect humans water purification is a critical link in
controlling disease transmission in waters.
CRITERIA FOR AN INDICATOR ORGANISM
1. Indicator bacterium should be suitable for the analysis of all types of water: tap, river,
ground, impounded, recreational, estuary, sea and waste.
2. Indicator bacterium should be present whenever enteric pathogens are present.
3. Indicator bacterium should survive longer than the hardiest enteric pathogen.
4. Indicator bacterium should not reproduce in the contaminated water and produce an
inflated value.
5. Assay procedure for the indicator should be great specificity; other bacteria should not
give positive result. The produce should have high sensitivity and detect low levels of
the indicator.
6. The testing method should be easy to perform.
7. Indicator should be harmless to humans.
8. The level of the indicator bacterium in contaminated water should have direct
relationship to the degree of fecal pollution.
The original tests for coliform are presumptive, confirmed and completed test.
Presumptive Step: This is carried out by means of tubes inoculated with three different sample
volumes to give an estimate of the MOST PROBLEM NUMBER (MPN) of coliforms in the
water. Complete and confirmed tests which complete the tests requires at least 4 days of
incubation and transfers.
Also, membrane filtration technique has become common and often preferred method of
evaluating the microbiological characteristics of water. The water sample is passed through a
membrane filter and the filter with its trapped bacteria is transferred to the surface of a solid
medium or to an absorptive pad containing the desired liquid medium. Use of proper medium
enables the rapid detection of total coliforms, fecal coliforms, or fecal Enterococci by their
presence of their characteristics colonies.
Molecular Techniques: Are now used routinely to detect coliforms in waters and other
environments, including foods. 16SrRNA gene-targeted primers for coliforms enable the
detection of 1cfu of Eschericheria coil per 100ml of water. It is short enrichment step precedes
the use of the PCR amplification allows the differentiation of nonpathogenic and enteroxigenic
strains, including the shiga-like toxin producing Eschericheria coil 0157:H7.
PROCEDURE
1. Preparation of medium
i. Single strength MacConkey ( bile –salt lactose peptone water)broth
ii. Double strength medium
Same as above but with each of the components double except water
Techniques
1. Dispense 50ml and 10ml of the double strength, as well as 1ml and 0.1ml and 0.1ml
of the single strength as well as 1ml into screw capped bottle of above medium into
screw capped bottles of appropriate sizes. Each bottle should contain an inverted
Durham tube to collect gas (if produced)
2. Using sterile pipettes, discharge the liquid samples into the medium as follows:
a. One 50ml sample to 50ml double strength medium
b. Five 10ml sample to 10ml double strength medium
c. Three 10ml sample to 10ml double strength medium
d. Five 1ml sample to 5ml single strength medium
e. Three 1ml sample to 5ml single strength medium
f. Five 0.1ml sample to 5ml single strength medium
g. Three 0.1ml sample to 5ml single strength medium
iii. Incubate the bottles at 370C and examine after 24 – 48 hours.
iv. Observation for growth, acid and gas production. Tabulate the numbers of positive
and negative tubes and obtain the most probable number from the McCray table
Confirmed test/confirmation test
Tubes that are positive for gas production are inoculated into brilliant green lactose bile
broth in the confirmed test, and positive tubes are used to calculate the most probable number
(MPN) value. Incubate for 48± 3hrs at 350C.
Completed test
Plate the tube on EMB agar and incubate at 370C for 24± hrs. It is used to establish that
coliform bacteria are present. The coliform colonies are inoculated on nutrient agar slant and a
broth tube. After 24hrs of incubation, make a gram’s stained slide from the slant. If the bacteria
are gram negative non-sporing rods and produce gas lactose, the completed test is positive.
Note: Although this method is used mainly for estimating coliform density it may be applied to
any other microorganisms in which growth can be easily observed by such feature as turbidity,
acid and gas production. It can be employed for yeasts and moulds in fruits juices and beverages
as well as for Clostria in food emulsion.
Advantage and disadvantages of the membrane filter technique for evaluation of the microbial
quality of water.
ASSIGNMENT
What is responsible for the colour change and gas observed in the positive tubes?
COURSE CODE: FST 311
COURSE TITLE: FOOD PROCESSING ENGINEERING
CHAPTER TWO
2.1 PARTICLE SIZE REDUCTION USING VARIOUS MILLS.
This is one of the size reduction operations which involve the breakdown of solid
materials through the application of mechanical forces. Milling is favoured in some food
industries especially those who are involved in flour production
Milling may aid the extraction of a desired constituent from a composite structure e.g. flour from
Wheat. Milling to a definite size range may be a specific product requirement. A reduction in
particle size of a material leads to an increase in surface of solids which may assist in many rate
processes.
The aim of this practical is to carry out milling of some certain food materials using (1) Pestle
and mortar, (ii) Kenwood grinder and (iii) Electric mill; to evaluate its efficiency, effectiveness
and thereby carry out the analysis of the final products.
1. Pestle and Mortar.
Find its average size. Transfer it into the pestle and mortar and continue to grind for about 30
minutes. Remove the milled products, weigh and carry out the particle size analysis using sieves
of 50-300 µm (about 5 sieves are enough).
2. Kenwood Grinder
Take about 200g of the food sample. Find the average size. Transfer it to the grinder. Grind for
30 minutes with constant stirring to ensure grinding. Remove milled sample, weigh and carry out
particle size analysis as in 1 above.
3. Electric Mill
Take about 200g of the sample. Determine its average size. Transfer to the Electric mill and mill
for 30 minutes. Remove the milled products, weigh and find the particle size analysis as in 1
above.
(a) For each milling operation calculate the reduction ratio.
(b) Plot the graph of percentage of sample versus the sieve size.
(c) Plot the graph of the weight versus sieve size
(d) Calculate the percentage loss in the sample for each process.
(e) Assuming the kicks constant to be 3.175 x 102 for each mill. Calculate the energy use for each
milling operation.
Questions
1. What method of milling do you prefer most for the operation?
2. What can you infer from your graphs?
3. Why is it necessary to classify products during milling operations?
4 State the basic principle behind the milling operations employed in this work.
5. State other milling devices used in the laboratory
2.2 DETERMINATION OF RATE OF DRYING
Drying can be defined as the application of heat under controlled conditions to remove
the majority of moisture/water which are normally present in food and this can be by evaporation
or sublimation (in case of freeze drying). It is necessary to improve the keeping quality of food
material through reduction of microbial water activity.
As much as dehydration or dying is very important, it also hasits disadvantages which
include deterioration in taste, flavour and texture of food. The structure of food can also be
disrupted as a result of drying as well as loss of nutrients. The main idea or concept of behind
dehydration or drying is that hot air is blown over a wet food, the heat is then transferred to the
surface of inform of latent of latent heat of vaporization which leads to evaporation of water. The
vapor diffuses through a boundary film and the water vapour is carried away by the moving air..
Generally, when food sample is placed in a dryer there is a short critical down period as
the surface heats up to the wet bulb temperature, dehydration then commences with the fact that
the moisture moves from the food interior at equal rate, the phenomenon centimes until a critical
moisture content is reached, because after heat application, the moisture contents continues to
drop, i.e. the falling rate period. Actual drying is achieved at the stage. Where the weight
obtained after about three consecutive reaching, or more the weight remains constants and this
period is referred to as constant dying period.
Apparatus/Materials: Oven, Knife, Tray, Desiccators, Yam sample, weighing balance, water
Procedure:Peel off the yam sample, cut into equal sizes and thicknesses of about 5 slices and
label each of the sliced yam A-E, take the initial weight of each slice, transfer them into a pre-
heated oven of about 700C. After a specified time, i.e. 30mins or 1hr, transfer the sliced yam
sample into a desiccator to cool down. After cooling, weigh each of the yam samples, ensuring
that they are not exposed to the atmosphere to prevent reabsorption. Transfer back to the oven
and allow it to dry for the specified period above. Continue the process until repetitive weights of
about 3 or more processes are obtained to confirm the drying process.
QUESTION
1. Draw a graph depicting the rate of drying of the yam samples by plotting a graph of
moisture loss against time?
2. Calculate the percentage moisture loss at each interval of time?
3. Determine the wet bulb, temperature, critical drying and constant drying periods on your
plotted graph?
4. Determine the slope of your graph?
5. State clearly your precaution, observation and conclusion?
COURSE CODE: FST 313
COURSE TITLE: HUMAN NUTRITION
CHAPTER THREE
3.1 ASSESSMENT OF NUTRITIONAL STATUS
Anthropometrics indexes provide an approximate reflection of nutritional status. The
indicators used most often are body weight and height, in relation to a subject’s age and sex.
Others include arm, head and thigh circumferences and skin-fold thickness. The main
anthropometrical indexes used are: weight-for-height, height-for-age, weight-for-age and body
mass index (BMI). National Centre for Health Statistics (NCHS) data is used as a standard since
many studies have shown that the growth of normal, healthy and adequately nourished children
almost, always approximates these references values. Other acceptable reference values can also
be used.
Procedures for measuring weight
- Scales should be platform/level type if possible.
- Scale should be set at zero.
- The subject should stand or lie still while being weighed.
- The subject should not be weighed with a full bladder.
- The subject should wear a minimum of clothing.
% Weight-for-height
(Or height-for-age) = observed weight (or height) x 100
Reference weight for patient’s height
(Or reference height for patients’ age)
- Record weights immediately after weighing
- Weight distorting factor (e.g. occurrence of diarrhea) should be discovered through
conversation
- Subsequent weights should be taken at the same time of the day.
- Scale should be maintained regularly.
Procedure for measuring height
- Measurement scale should not be of material, which stretches.
- Scale should be placed on flat surface (for children < 3yrs) or on a wall.
- Children < 3yrs should be measured while lying flat and those above 3 years are measure
while standing upright in bare feet.
- The horizontal rod (or board) should be exactly horizontal, on the crown the head (not the
hair) and the subjects should look straight.
- The height should be recorded immediately after measurement and checked at the time.
Information obtained can be used to classify individual as either normal wasted stunted or
stunted and wasted. The intensity of wasting and stunting can be calculated as follows.
Note: The present deviations can be compared with the medium NCHS standard to find the
actual classification (i.e. Mild, Moderate or Severe).
3.2 MEASURING MALNUTRITION WITH SHAKIR STRIP OR TAPE
Malnutrition bears hardest on small children, contributing to a massive over 50%
mortality rate amongst the under fives in some parts of Nigeria today. We can help to overcome
this problem by making a Shakir strip and using it to measure the mid arm circumference (on the
left side) midway between the acromion and the olecranon with the arm hanging loosely by the
side of small children (1 to 5 years old) in the community. This is because mid upper arm
circumference is the same between these ages.
Materials: Old X-ray films, strong washing soda or diluted bleach, spirit felt pen or marker.
Procedure
a. Making Shakir strip
Most hospitals or clinics have old X-ray films. Remove the emulsion from the film by
soaking it all night in strong washing soda or diluted bleach. You will then have a sheet of strong
clear plastic. (A strong wire or rope could be used instead).Scratch the plastic or rope with a
sharp point in the three places representing 0, 12.5cm and 13.5cm.Cut out the strip or tape ½ cm
wide and colour from 0cm-12.5cm with red spirit felt pen, 12.5cm -13.5cm with yellow and
13.5cm and above should be given.
b. Using Strip or Tape
Make sure the child is more than one and less than five years old. Let the child’s left arm
hang loosely by his side and place the tape round the middle of the upper arm.
Result
When the tape is wrapped around the arm, the measuring point overlaps one of the
coloured zones towards the other end of the tape: Green (>13.5cm) mark indicates normal or
acceptable nutritional status; yellow (>12.5-13.5cm) indicates borderline nutrition or probably
malnutrition; Red (<12.5cm) indicates malnutrition.
Note: The tape is useful in screening programmes but does not distinguish between loss of fat
and loss of muscle and more importantly, may be misleading, children with severe kwashiorkor
and oedematous upper arms should be recognized unwell and abnormal, without resort to the
strip.
Questions
1. If the mid upper arm circumference of most of the children you are assessing came in the
red and yellow colours, what would be your advice to improve their nutritional status?
2. Explain the advantages of using arm circumference to measure child’s nutritional status
in under fives clinic.
3. Why is it not possible to use this method in children under one year?
3.3 THE USE OF LOCAL EVENTS CALENDAR TO DETERMINE A CHILD’S AGE
A local-events calendar is used to determine the age of a child in the assessment of
nutritional status.
Materials: Calendar, biro, government bulletin, atlas of Nigeria, diary of events (both local and
national), arts and culture, and vegetations.
Procedure
Make a separate calendar for each place, as the people in the different places remember
different things. Make it with the help of the people in the village, the clinic, or the local
government offices.Get the times of planting, weeding, harvesting and selling; the seasons and
the holidays and feasts. Find out when important things happened in that place; such as visit of
the President, a census, a flood or famine, the opening of a new school, or a change of headman.
Use your calendar, to cross check what a mother tells you.
Note: A calendar is very necessary in every clinic and recording the most important events such
each month for the last three to five years to ascertain the true age of the child.
Table: Sample design of Local Events Calendar
SEASON ANNUAL EVENT MONTH EVENTS IN THE
YEAR(S)
January
February
March
April
May
June
July
August
September
October
November
December
Questions
1. Explain why every under fives clinic should have a local events calendar.
2. List the importance of the calendar in assessing nutritional status of children in primary
health care clinics.
3. Discuss the (pros and cons) advantages and disadvantages if any, in using the local events
calendar in clinical practice.
3.4 ESTIMATION OF TOTAL ENERGY REQUIREMENTS USING FACTORIAL
METHOD.
The human body’s total energy requirement can be subdivided into three separate
categories of need, each of which can be estimated separately, depending on the person’s
circumstances and behavior. The three categories of energy requirement are for the maintenance
of basal metabolism, to power physical activity and to release energy from food (TEF).
The factorial method of measuring total energy expenditure involves calculation of each of the
three categories of energy need as follows:
Calculation of basal metabolism using one of the following methods:
Method A: Kcal/kg/hr method in which BMR for
Males = body weight (kg) x 1.0 x 24
Female = body weight (kg) x 0.9 x 24
Method B: Harris-Benedict method formula in which BMR for
Males = 66.4 (13.7 x weight (kg)) + (5.0 x Height (cm)) – (6.8 x age)
Females = 655.0+(9.6 x weight(kg)+(1.8 x Height(cm) – (4.7 x age)
Method C: Metabolic body size method in which BMR = 70 x (weight (kg)¾
Method D: FAO/WHO/UNU equating method in which BMR for male = 11.6 x weight (kg) +
879
Calculation of activity costs based on the record of all activities over a 24-hour period.
The energy required for activity is derived from table below:
Energy cost
Activity Time (hr) Kcal/Kg/Hr Kcal/Kg
(A) (B) (A) X (B)
Dressing 1.5 0.7 1.05
Sitting 6.0 0.4 2.4
Dish washing 0.5 1.0 0.5
Walking (3mph) 2.0 2.0 4.0
Standing 1.0 0.5 0.5
Typing 4.0 1.0 4.0
Sleeping 8.0 - -
Playing piano 0.5 2.0 1.0
Eating 0.5 0.4 0.2
Total 24 13.65
Energy cost for activity = body weight (kg) x 13.65 for this example.
Calculation of the thermic effect of food
This equals 10% of basal energy cost (calculated in step 1, above) + activity energy cost
(calculated in step 2 above), that is 0.1 (basal needs + activity needs).
Calculation of total energy expenditure
This is done by adding the basal energy needs (step 1) + activity needs (step 2) + thermic
effect of food (step 3)
Questions
1. Explain the distinction between the kilocalorie and kilo joule.
2. What are the difference between Basal Metabolic Rate (BMR) and Resting Energy
Expenditure (REE)?
3. Record your activities for a day and use it to calculate your energy requirement for that
particular day.
3.5 USE OF BALLISTIC BOMB CALORIMETRY TO MEASURE THE ENERGY
VALUE OF A FOODSTUFF
Many foodstuffs supply energy and if the food is burned the heat energy produced can be
measured.
The ballistic bomb calorimeter involves the principle of direct calorimetry in determining
the energy content of various foodstuffs. The measurement is expressed in calories/g
(kilocalories/g). 1 calorie/g = 4.2. kilojoules/g. It is interesting to compare the energy value of
certain foods. The method can also be used to determine the gross energy, digestible energy and
metabolisable energy of different foods.
Materials: Ballistic bomb calorimeter (complete unit with O2 cylinder), Cotton loop (with
correction value = 0.2), Sucrose standard 3.94 kcal/g (or benzoic acid), Different
Foodstuffs:Sago flour,Lupark butter, Bread, Peas.
Reference Schedule (B) for details of calculation.
Procedure
The method involves applying a spark to burn a known quantity of food exposed to a
current of O2 at 25atm in the bomb or chamber. The temperature rise is related to the heat that
comes out of food.
The set-up of the experiment is as follows:
i. The foodstuff is weighed out, put into a small crucible them mount on the stand
provided for it in the calorimeter unit
ii. A cotton loop (2” with correction value = 0.2) is attached to the fuse and then passed
into the food sample. (N/B: Loss of food sample should be avoided)
iii. The bomb is inserted into position with its collar tightened up by anticlockwise
movement
iv. Attach the thermocouple to the bomb and measure the potential difference as shown
by the galvanometer
v. The knob of the O2 cylinder is tapped up gently to open for O2 to flow into the bomb
until the pressure gauge achieves 25atm.
vi. Zero the reading of the equipment and turn off O2 supply
vii. Press the knob on the front panel of the calorimeter in order to apply a spark
viii. Record the highest point of deflection on the galvanometer
ix. Release O2 by anticlockwise turning off the knob on the right side below the collar of
the stem on which the bomb rests
x. Loosen the collar by clockwise movement
xi. Remove the bomb to tap water to cool from inside
xii. Repeat aforementioned procedure for subsequent samples.
Note:The ballistic bomb calorimeter is a very safe mechanism but it can blow up if care is not
taken during use.
Questions
1. Do all the foodstuff/samples give the same energy value?
2. Compare the results with (a) each other, (b) accepted value and comment. Which samples
have higher/lower values?
3. Why is O2 instead of air in this experiment?
4. Explain the significance of energy value of foods.
5. Sketch the main features of a Ballistic Bomb Calorimeter.
References
Adams, F.A. and Cockett, R.G. (1975): Experimental Catering Science Workbook, Edward
Arnold, pp. 22-25
Davidson, S. and Passmore, R. (1979): Human Nutrition and Dietetics. 8 th Ed ELBS London, pp
202-50
Dialogue on Diarrhoea, (1986): Health Basics – Growth Monitoring, AHRTAG, 85 Marylebone
London, U.K
Hollis, C. (1986): Using Communication to solve Nutrition Problems: International Nutrition
Communication Services (ICNS), pp. 140-89.
Maurice, K. and David, M. (1976): Nutrition for Developing Countries, Oxford University Press
London, 4th Ed. Pp 1, 6
Miller and Payne (1959): Brit J. Nutrition pp. 13, 507
Rees, A.M. (1976): Experiments in Home Economics, Part III: FoodScience. Blackwell
Scientific Publications, London, pp. 97 and 98
Posit, E.M.E. (1988):Paediatric Nutrition, Rutterworth and Co., Publishers Ltd., Liverpool.
Institute of Child Health, London, U.K. Child-to-child Programme
COURSE CODE: FST 316
COURSE TITLE: FOOD ANALYSIS I
CHAPTER FOUR
4.2 PROXIMATE COMPOSITION
4.2.1 DETERMINATION OF MOISTURE CONTENT
A. USING THE OVEN METHOD
The determination of moisture content is one of the most important and widely used
measurements in samples that absorb and retain water. Chemical analyses are normally made on
dry matter basis. Moisture content determination look very simple in concept, but in practice the
accurate determination is complicated by number of factors which vary considerably from one
sample to another. Among the factors are the relative amounts of water available and the ease
with which the moisture can be removed. Methods are based upon the removal of water from the
sample and its measurement by loss of weight or the amount of water separated. Air or vacuum
oven drying at 70° – 80°C are considered to be reliable sample and water is the only volatile
constituent removed. Sample should be dried to a constant weight.
Procedure
(1) Weigh a clean and well labeled dish that has been oven dried (W1)
(2) Add enough sample into the dish and weigh (W2)
(3) Transfer the dish and content to the thermo setting oven at about 1050C for about 2-4
hours.
(4) Transfer dish from oven to desecrator, cool for about one hour and weigh. Repeat
step3- 4 to obtain constant weight W3.
(5) Calculate % moisture content.
(6) In the case of hygroscopic substances a dish with a cover must be used.
(7) Experiment must be performed at least in duplicate.
% Moisture= Loss of weightWt . of samplebefore drying
x100
¿W 2−W 3
W 2−W 1x 100
%Total Solid=W 2−W 3
W 2−W 1x 100∨% Dry matter
B. USING THE VACUUM OVEN METHOD (INDIRECT METHOD)
Vacuum oven methods of moisture determination are generally the standard and most
accurate procedure of moisture determination for most foods. This method gives a very close
estimate of true moisture content. It also enables the fast removal of residual water in food
substance without changing the organic compounds than other drying methods.
Apparatus:
1. Metal or platinum dish (with tight cover fitting)
2. Air tight desiccators
3. Vacuum Oven, connected with pump capable of maintaining partial vacuum in oven with
pressure equivalent to -25mm Mg and provided with a thermometer.
Connect H2SO4 gas drying bottle with oven to admit dry air when releasing vacuum.
Materials: Wheat flour, Corn flour, Yam flour. Mix sample very well and store in air tight
container.
Method: Accurately weight 2-3g of the sample in covered dish previously dried at 98-1100c,
cool in descanter and weight soon after reaching room temperature.
Loosen cover (do not removed) and heat at 98-1100c to constant weight by subsequent heating,
cooling and weighting. Admit dry air into oven to bring to atmospheric pressure. Immediately
tighten cover on dish and transfer to desiccator and weigh soon after reaching room temperature.
Report flour residue as total solids and loss in weight as moisture calculating moisture in both
wet and dry basis.
C. USING THE DEAN AND STARK METHOD
This is a very useful method of moisture determination for samples which cannot easily
be heated in an oven, for example waxed packaging materials or foods containing volatile oils
which would be lost on oven heating and estimated as moisture.
The method involves distilling cut and measuring the water from a weighted sample,
using a liquid completely immiscible with and lighter than water and having a B.P. at about
1000C (e.g. heptanes B.P. 980C or toluene B.P. 1110C.)
Method: Thoroughly clean all glass-ware and dry by baking in an oven. Select the capacity of
the graduated receiver according to the weight and expected moisture content of the weigh and
expected moisture content of the sample, e.g. 7.5cm3receiver for sample up to 21%. 2.0cm3
receiver for sample up to 10g with less than 20% moisture.
While the apparatus is still warm from the oven wet the inner surface with the small
quantity of the distillation. Then ¼ fill the flask. Weigh out a representative sample of the
material under test ensuring minimum exposure to the air before and after weighing, and transfer
as quickly as possible to the distillation flask.
Ensure that the sample is quickly covered by the liquid. Gently boil until no further water
collects in the receiver (may take up to two hours). Read off the volume of water collected in the
graduated receiver using a stout copper wire or a policeman to remove globules of water which
stick to the ides of the condenser. Report % moisture (Dean and Stark).
4.2.2 DETERMINATION OF ASH
A. TOTAL ASH
The ash of a biological material is an analytical term for the inorganic residue that
remains after the organic matter has burnt away. The ash is not usually the same as the inorganic
matter present in the original material since there may be losses due to the volatilization or
chemical interaction between the constituents. The importance of the ash content is that it gives
an idea of the amount of mineral elements present and the content quantitative constituents of
proteins, lipid or fat, carbohydrate, plus nucleic acid. Sample rich in organic matter can be
preheated on the flame or hot plate.
Procedure
(a) Place silica dish or crucible into muffle furnace for about 15 minutes at 3500C.
(b) Remove after one hour and cool to room temperature, weight the crucible (W1).
(c) Add enough sample into the crucible (0.5 – 2g) the quantity will depend on texture and
source of sample) and weigh content (W2).
(d) If sample is wet or fresh plant sample, it should be pre-dried. Increase the temperature
from 2000C – 4500C this is to avoid incomplete ashing. Ash sample until is become
whitish in colour). If ashing is incomplete (evidence of black particles) within a
reasonable period remove crucible, cool, bath and return to the furnace.
(e) Remove from furnace to desiccators and allow to cool to room temperature.
(f) Reweigh the crucible and content (W3).
% Ash=W 2−W 3
W 2−W 1x100
% organicmatter=100−% Ash
Note: The ash should be preserved for mineral analysis
B. ACID INSOLUBLE ASH
Materials: 6N HCl, Silver nitrate solution, Whatman filter paper No 42, Water bath,Bunsen
flame oven at 5500C, Desiccator.
Procedure
i. Use the residue at the end of ash content determination
ii. To it, add 23ml of 6N HCl and heat for 10min in a water bath
iii. Add the content and filter with an ash less Whatman filter paper No 42
iv. Wash filter paper and its contents with distilled water until the filtrate becomes free
from acid, which can be detected by the use of silver nitrate.
v. Return filter paper with its contents to the crucible and dry using a Bunsen flame
before igniting in oven at 550°C for 1 hour
vi. Cool sample in the desiccator and record the weight
vii. Continue to dry to a constant weight
Calculation:
N=W 1
W
Where;
N = % of acid insoluble ash (dry weight basis)
W1 = weight of the insoluble ash
W = the dry weight of sample
Question
1. What is the significance of determining the ash insoluble content of flour? Comment on
your results.
4.1.3 DETERMINATION OF FAT USING SOXHLET EXTRACTION METHOD
By definition, fats are mixtures of various glycerides of fatty acids, which are soluble in
certain organic solvents. Extraction is carried out with soxhlet apparatus with N-Hexane or
petroleum ether. The usual procedure is to continuously extract the fat content with 40/600C
Petroleum ether/ N-Hexane in a (Soxhlet extractor. The ether extraction method is based on the
principle that non-polar components of the sample are easily extracted into organic solvents.
Direct extraction gives the proportion of free fat but gives no clue to the particular fatty acids.
The soxhlet extractor is mostly suitable for dried samples.
Procedure
1. Weigh thimble previously dried (W1) it should be fat free.
2. Add enough samples into the thimble and weigh again (W2).
3. Weigh the 500ml round bottom flask (fat free) W3.
4. Fill the flask with petroleum ether up to 2/3 of the 500ml flask.
5. Fit up the extractor with a reflux condenser. Adjust the heat source so that the solvent
boils gently, leave it to siphon over several hours (5-8 hours).
6. Finally wait until the petroleum ether has just siphoned over the barrel. Detach the
condenser and remove the thimble of filter paper. Distill petroleum ether from the flask.
7. Dry the flask containing the fat residue in an air oven at 1000C for 5 minutes or on water
bath. Cool in a dedicator and weight (W4).
8. Place the thimble in the beaker in an oven at 500C and dry to constant weight with
sample. Cool in dedicator and weight (W5). The % of extracted lipid can be given by
either.
4.1.4 DETERMINATION OF CRUDE FIBRE
Crude fibre is that portion of the plant material which is not ash or dissolves in boiling
solutions of 1.25% H2SO4 or 1.25% NaOH. Crude fibre was originally thought to be indigestible
portion of any main food. It is known however that fibre consists of cellulose which can be
digested to a considerable extent by both ruminants and non-ruminants.
The interest in fibre in food and feed has increases, based on the noticed number of serious
illnesses with diet low in fibre. Fibres swell and form gelatinous mass with high water retention
capacity with the digestive system. Findings show that fibre products can absorb cholesterol;
toxic agents raise the excretion of bile acids and sterols.
The following diseases are associated with diets low in fibre content: constipation, Appendicitis,
hemorrhoid, cancer of the large bowel, Diabetes mellitus, obesity and coronary heart disease as a
result of increase fibre intake which result into increase blood-cholesterol level. (Reference
Appendix to fibertec Manual by Finn Alstin).
Determination of fibre content of plant tissue is relatively simple. The method is essentially
conventional, and if rigidly adhered to will provide a distinction between the most digestible and
least digestible carbohydrates. The starch and the protein are dissolved by boiling the sample
with acid and then with NaOH. The residue of cellulose and lignin is washed, dried and weighed.
The residue is ashed and the weight of the ash subtracted from the weight of the residue.
Procedure
1. Transfer about 3.5 – 5g sample into 500ml conical flask.
2. Add 200ml of boiling 1.25% H2SO4 and bring to boiling within one minute and allow to
boil gently for 30 minutes exactly using cooling finger to maintain constant volume.
3. Filter through poplin cloth or filter paper by suction using Buchner funnel, rinse well
with hot distilled water, and separate material back into the flask with spatula.
4. Add 200ml of boiling 1.25% NaOH and few drops of antifoaming agent, bring to boiling
within one minute and boil gently for 30 minutes using cooling finger (KOH) can be used
in the place of NaOH) and vegetable oil as antifoaming agent)
5. Filter through poplin cloth and wash with hot distilled water. Rinse four times with hot
distilled water, and once with 10% HCl, four times again with hot water, twice with
methylated spirit and three times with petroleum ether (where methylated spirit is not
available). Ethanol could be used as a substitute for methylated spirit.
6. Salvage the residue into crucible after drain, dry in the oven at 1050C, cool in desiccators
and weight W2.
7. Place in muffle furnace at about 3000C for about 30 minutes.
8. Remove into desiccators and allow cooling to room temperature, weighing again W3.
%Crude Fibre=W 2−W 3
W 1x 100
4.1.5 DETERMINATION OF PROTEIN
The accepted standard method for the determination of nitrogen in sample involves the
complete digestion of sample in hot concentrated acid, and in the presence of an appropriate
metal ion catalyst. The catalyst is to convert all nitrogen in the nitrogenous materials in the
sample into ammonium ion. Upon the addition of alkali in the digest, ammonia is released which
may then either be distilled out of the sample and determined by simple acid-base titration, or the
ammonia can be hypochlorite, to give a coloured derivative which can be measured with
colorimeter or spectrophotometer.
The Kjeldahl digestion is usually performed by heating the sample with H2SO4 –
containing substances which promote oxidation of organic matter by increasing by boiling point
of the acid (K2SO4 or Na2SO4) and Se or Cu which increase the state of oxidation of organic
matter. These reagents here referred to as digestion catalyst.
It is necessary to digest the sample for certain period until you obtain a clear solution to
ensure accurate results.
Procedure
Stage I – Digestion
1. a. Weigh about 0.2g wet sample into 500ml Kjeldah flask, and 10ml conc. H2SO4 with
one Kjeldah catalyst tablet.
b. Weigh about 0.5g dry sample into 500ml micro kjeldah flask, and 5ml conc. H2SO4
with half kjeldah catalyst tablet. Let the weight be (W1).
2. Heat on a heater start with a low heat for about 15 minutes, increase to medium heat for
about 30 minutes again and finally at high heating until digested. Rotate the flask at
intervals until the digest is clear (light green or grey white) continue heating for few
minutes after that to ascertain complete digestion.
3. Allow to cool, wash sample residue if any and filter, make up the digest up to 50, 100ml
or as appropriate (V1).
Note: Catalyst can be formulated when tablet is not available 100g K2SO4 + 10g CuSO4. 5H2O +
1g selenium or 60g K2SO4 + 6.5g H2O
Digestion- the organic matter is oxidized by concentrated, H2SO4 in the presence of
catalyst such as copper, mercury, selenium oxide and the nitrogen in converted to ammonium
sulphate.
N2 + H2SO4 catalyst NH4SO4
Stage II – Distillation
1. Place 5ml of 2% boric acid (H3BO3) into 100ml conical flask (as receiving flask),
H3BO3as an acid will trap down the ammonia vapour from the digest. 2% is 2g made up
to 100ml distilled water it requires hot water to dissolve.
2. Add 3 drops of mix indicator. (H3BO3) and the indicator can be prepared together. Mix
indicator 0.198g bromocresol green plus 0.132g Methyl red in 200ml alcohol.
3. Place the receiving flask so that the tip of the condenser tube is below the surface of the
boric acid.
4. Pipette 5ml for samples rich in nitrogen and 10ml for sample low in nitrogen into the
markham distiller or any available distiller that have similar operation.
5. Add 10ml of 40% NaOH, tighten the joints and distill about 50ml into the receiving flask
(V2) (40% 10m is 40g made up to 100ml).
Distillation- the ammonium sulphate is treated with concentrated sodium hydroxide.
Ammonia is liberated and distilled into a standard quantity of dilute acid (H2SO4 or HCl) or weak
acid (boric acid).
(NH4)2SO4 + 2NaOH Na2SO4 + 2NH3 + H2O
Stage III – Titration and Calculation
Titrate the distillate with standard mineral acid (0.01M HCl or 0.02M H2SO4). Titrate the
blank with the acid as well.
HCl + NH3 + H3BO3
4.1.6 DETERMINATION OF CRUDE PROTEIN
The amount of crude protein contained in seed, roots, tubers and other food stuff can be
obtained by multiplying the nitrogen content of the food by 6.25. The factor 6.25 owes its origin
to be assumption that all food protein contains 16% nitrogen, and that all nitrogen in a feed is
present as protein. Although these assumptions are not entirely valid.
The protein contained in plant tissue or feed may vary in terms of nitrogen content- from
13 – 18%. In many cases, some relevant factors are as shown below:
Product Factor Product Factor
Egg Whole 6.25 Oil seed 5.4
Egg Albumin 6.38 Wheat 6.70
Egg Vitellin 6.22 Rice and Rice Flour 5.70
Casein 6.40 Oats 5.83
Milk and Milk Products 6.39 Millet 6.30
Animal and Fish 6.26 Legume 6.25
Cereals 5.90 Sorghum 6.25
Plant leaf protein 6.6 Groundnut 5.4
Soya beans 5.70
The crude protein value is obtained by multiplying the nitrogen value by 6.25 and the
value for apparent total meat is obtained by converting the protein to the relevant factor. The
value of total meat is based on the assumption that the nitrogen is derived from meat protein
however this analysis does not distinguish between meat protein and other non-meat source of
protein.
Questions
1. Comment on your results.
2. Why is the protein of meat said to be of high biological value?
4.1.7 DETERMINATION OF TOTAL CARBOHYDRATE
Carbohydrates are the important components of storage and structural materials in the
plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates are the
monosaccharaides which cannot be split by hydrolysis into more simpler sugars. The
carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars by
acid hydrolysis and estimating the resultant monosaccharaides.
The Principle is that Carbohydrates are first hydrolysed into simple sugars using dilute
hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This
compound forms with anthrone a green colored product with an absorption maximum at 630nm.
Materials
1 2.5 N‐HCl
2 Anthrone Reagent: Dissolve 200mg anthrone in 100mL of ice cold 95% H2SO4. Prepare
fresh before use.
3 Standard Glucose: Stock – Dissolve 100mg in 100mL water. Working standard – 10mL
of stock diluted to 100mL with distilled water. Store refrigerated after adding a few drops of
toluene.
Procedure
1. Weigh 100mg of the sample into a boiling tube.
2. Hydrolyse by keeping it in boiling water bath for 3 hours with 5mL of 2.5 N‐HCl and cool to
room temperature.
3. Neutralise it with solid sodium carbonate until the effervescence ceases.
4. Make up the volume to 100mL and centrifuge.
5. Collect the supernatant and take 0.5 and 1mL aliquots for analysis.
6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard. ‘0’
serves as blank.
7. Make up the volume to 1mL in all the tubes including the sample tubes by adding distilled
water.
8. Then add 4mL of anthrone reagent.
9. Heat for eight minutes in a boiling water bath.
10. Cool rapidly and read the green to dark green color at 630nm.
11. Draw a standard graph by plotting concentration of the standard on the X‐axis versus
absorbance on the Y‐axis.
12. From the graph calculate the amount of carbohydrate present in the sample tube.
Note: Cool the contents of all the tubes on ice before adding ice‐cold anthrone reagent.
Calculation
Amount of carbohydrate present in 100mg of the sample =mg of glucoseVolume of test X 100
4.1.8 CARBOHYDRATE DETERMINATION USING POLARIMETER
Optical activity and Mutarotation of glucose
Weigh 10gm glucose on the torsion balance to nearest – 0.5g. Transfer the sugar to a
100ml volumetric flask, dissolved in distilled water with agitation to secure rapid solution, make
up to the flask at once and mix thoroughly. Note the time (this will be zero time on the graph
which you will subsequently construct). Without losing time, pour roughly half of the solution
into a conical flask, add two drops of Conc. Ammonia solution, mix and label in “neutral glucose
solution”. At once fill a 2dm polarimeter tube with the neutral solution, read the optical rotation
and note the time. Return the solution to the graduated flask.
Next read the optical rotation of the ammonia solution, and note the time. Discard the
contents of the tube and wash in with distilled water. Now return to the neutral solution, and
proceed as before to read its optical rotation and to note the time. Repeat this first at interval of
15 or 2 minutes and then at interval of around 30 minutes. (It should be possible to secure about
six observations in the course of the class period).
Towards the end of the second reading on the ammonia solution. This reading should be
differed appreciably from the first reading on this solution.
For the neutral solution, tabulate the calculated values of D against time of observation.
From the tabulated results plot a graph showing the relation of D to time that has clasped since
making the solution.
Calculate also the D values secured from the readings on the readings on the ammonia
solution.
The specific rotation D 20 of pure anhydrous glucose is +52.5
Questions:
1. How would you avoid errors from mutarotation when measuring the concentration of a
glucose solution by polarimetry?
4.2 DETERMINATION OF FAT IN MILK USING THE ROSE-GOTTLIEB
PROCESS
Weigh out 10g of milk in to the test tube add 1 ml of 0.88 ammonia solution and mix.
Then add 10 ml of alcohol (95%) and again mix well. Add 25 ml peroxide – free diethyl ether,
cork the tube and shake vigorously for 1 min. Add 25 ml of recently distilled light petroleum
(B.P. 40-600C) and shake vigorously for 30 sec. After separation is complete, transfer the fat
solution into a suitable flask (previously dried at 1000C, cooled and weighed). To the tube add
two successive lots of 5ml of mixed ether and transfer (without shaking) to the flask. Then repeat
the extraction (with 15ml of ether and 15ml of light petroleum) and the subsequent operations
twice more. Distil off the solvents from the flask, dry the fat for 1hr at 100 0C, cool and weigh. If
any non-fatty matter appears to be present, wash out the fat from the flask with light petroleum,
dry, reweigh and correct the result accordingly.
4.3 DETERMINATION OF IRON IN BEVERAGE BY ATOMIC ABSORPTION
SPECTROPHOTOMETER (AAS)
In this experiment Atomic Absorption Spectrophotometer is used to determine the iron
content of a drink. The method can be used for all foodstuffs, but these normally require ashing
in a muffle furnace and then dissolving the residue in acid before analysis. For beverages, this
preliminary step is not required. As fruit juices contain considerable quantities of citric acid is
added to prevent the suppression of iron absorption.
Materials: Stock iron solution, Ferric chloride solution containing 25 mg/l of iron, AnalaR
hydrochloric acid (sp. Gr. 1.16), Orthophosphoric acid 1% v/v (2.5ml of orthophosphoric acid
(sp. Gr. 1.75) diluted to 250ml with water).
Procedure
Calibration: Prepare standard iron solutions as follows:
i. Into 100ml volumetric flasks pipette 0, 4.0, 8.0, 12.0, 16.0, 20.0ml of stock iron
solution.
ii. To each flask add 10ml of hydrochloric acid (sp. gr. 1.16) and 1.0ml of
orthophosphoric acid (1% v/v) and make up to the mark with water. These solutions
contain 0, 1.0, 2.0, 3.0, 4.0, 5.0 µg/ml iron in 10% v/v hydrochloric acid and 0.01%
orthophosphoric acids.
Preparation of Sample
i. De-gas the drink by repeated pouring a sample between two beakers.
ii. To a 100ml volumetric flask pipette 10ml of sample, 10ml of hydrochloric acid (sp.
gr. 1.16) and 1.0ml of orthophosphoric acid (1% v/v) and make up to the mark with
water.
Analysis by atomic absorption spectrophotometer
i. Aspirate the standard solutions followed by the sample solutions. Check that the
absorbance of the sample comes within the absorbance range of the standards; adjust
the volume of the drink used in making up the sample if necessary
ii. Check the absorption of the most concentrated standard solution after the last sample
solution has been run
iii. Plot a calibration graph of iron concentration of the standards against absorption
iv. Read off the iron concentrations in the sample
v. Calculate the iron concentration of the drink (µg/ml).
Standard addition method
i. Take 4 samples of 5ml from the degassed drink
ii. Add 0ml, x ml, 2x ml, 3x ml of the stock iron solution, where x ml is the volume
required to increase the iron content of the solution by about 50% i.e. if you found the
sample contained y µg/ml of iron form the first part of this experiment, then in each
5ml aliquout of sample there will be 5y µg of iron. Then you need to calculate the
volume, x, of standard that contains 5y/2µg of rion.
iii. Dilute each aliquout with water to the same fixed volume in each case.
iv. Determine the absorption of each sample and calculate the iron content of the drink.
Compare your results with those obtained by the external standard method and
comment on the results.
Account for differences between replicate data in your discussion.
4.4 DETERMINATION OF RIBOFLAVIN IN POWDERED SKIM MILK USING
FLOURIMETER
Riboflavin is a water-soluble vitamin found in a variety of plant and animal tissue.The
ribitol group of riboflavin is completely split off by light in neutral solution, which accounts for
the destruction of riboflavin in milk left exposed to sunlight. After the ribitol group has split off,
the molecule no longer has the ability to fluoresce, as does riboflavin.
Materials: Sulphuric acid 0.2M, Sodium acetate 2.5M, Potassium permanganate (KMnO4) 4%,
Hydrogen peroxide 3%, Riboflavin standard solution 10µg/ml, Working solution: 1µg/ml, Dilute
10ml of standard solution to 100ml with water. Prepare fresh for easy assay.
Procedure
i. Analyse two replicate samples. For each sample, suspend 3g of sample in 75ml 0.2M
H2SO4 and heat for 45 minutes over a boiling water bath.
ii. While the solution is boiling prepare a series of standard solutions containing 0.2, 0.4,
0.6, 0.8 and 1.0µg/ml riboflavin and measure the fluorescence at excitation 480nm
and emission 516nm
iii. Cool the Sulphuricacid extract and adjust to pH 4.3 using 5ml (approx) 2.5M Sodium
Acetate.
iv. Dilute the suspension to 100ml and then filter on a Buchner funnel through a
Whatman No 1 filter paper, discarding the first 15ml
v. Oxidize 60ml, of the filtrate with 2ml 4% KMnO4 solution and 3 minutes later
discharge the colour with 2ml 3% H2O2 solution
vi. Add water to make a final volume of 65ml, mix and filter, place 15ml of this into
each of two test tubes, add 1ml water to one and 1ml standard riboflavin
(intermediate solution) to the other. Measure the fluorescence using, 480nm
excitation, and 515nm emission.
A = fluorescence reading sample + water
B = fluorescence reading sample + riboflavin
The later measurement is an internal standard. To A add a small pinch of sodium hydrosulphite
(do not add too much or solution will go cloudy) to break down the riboflavin, then read the
fluorescence C.
Then A – C = fluorescence due to sample
And B – A = fluorescence due to added riboflavin.
Making the appropriate adjustment for dilution of extract and sample weight, calculate the
riboflavin content of your sample, express the results as mg/100g sample. Compare your results
with the literature and in your discussion account for any differences you have found between
the two milk samples.
4.5 WATER ANALYSIS
4.5.1 DETERMINATION OF CHLORIDE IN WATER-MOHR’S METHOD
The determination of chloride ion concentration is by titration using silver nitrate. This
method is only for water containing 5-150ppm of chloride. Silver ions react with chloride ions to
form practically un-dissociated silver chloride. Excess ion together with potassium chromate as
indicator form a reddish brown complex compound silver chromate.
Ag+ + Cl- Ag+Cl-
2(Ag+ + CrO4- Ag2CrO4
Materials: 0.01N silver nitrate, 0.1N nitric acid, 10% potassium chromate, 1% phenolphthalein.
Procedure
i. Pipette 100ml water sample into a 250ml conical flask
ii. Add a crop of phenolphthalein (if a red colour is produced add 0.1N nitric acid until
the solution becomes colourless: if no red colour is produced, add 0.1 CaCO3 and
wait for some minutes)
iii. Add 2 to 3 drops of K2Cr2O4
iv. Titrate with 0.01N silver nitrate until colour changes from yellow to reddish brown.
Calculation
ppmCl = 3.33x
Where x = vol of 0.01N AgNO3 used in the titration.
4.5.2 DETERMINATION OF ALKALINITY OF WATER
The alkalinity of water is determined by titration with a standard solution of an acid to
end points with pH value at 8.3 and 4.5. These end points are usually detected with
phenolphthalein (pH 8.3) and methyl orange (pH 4.5) indicator.
Materials: Water sample, Sulphuric acid 0.02N, phenolphthalein indicator, methyl orange
indicator.
Procedure
a. Alkalinity to phenolphthalein (P)
i. Measure 100cm3 of sample and transfer to porcelain basin
ii. Add 3-4 drops of phenolphthalein indicator- if the sample turns pink, run in 0.02N
H2SO4, slowly stirring at the same time with a glass rod until the colour just
disappears
P=Vol of 0.02 N H 2 SO4
Vol of sampleusedx 1000
= ppm in terms of CaCO3
b. Alkalinity to methyl orange (M)
i. Measure 100cm3 of the sample and transfer to a porcelain basin
ii. Add 1cm3 of Methyl orange indicator and titrate slowly with 0.02N H2SO4until the
colour shows the first change from yellow to orange.
M=Vol of 0.02 N H2 SO4
Vol of sample usedx 1000
= ppm in terms of CaCO3
Questions
1. Why is it necessary to determine the chloride content of water?
2. What is the economic importance of using hard water in food processing?
3. Compare your results with standards and comment on quality of the water.
4. If the water sample is not good for food processing explain a method you could use to
make it adequate for processing.
4.5.3 DETERMINATION OF Ca2+ AND Mg2+ IN WATER
The object of the experiment is to determine the percentage of magnesium and calcium
by titration with EDTA using Eriochrome black T as indicator. The titration is carried out at pH
10 (by adding a pH 10 buffer solution made of 570ml conc. Ammonia; 70g ammonium chloride
and made up to 1litre with distilled water as the mg-EDTA complex is unstable in acid).
Materials
Buffer (pH 10)- Add 142 ml of 28% wt/wt aqueous NH3 to 17.5g of NH44 and dilute to 250ml
with water.
Erichrome black T indicator – dissolve 0.2g of solid indicator in 15ml of triethanoalmine plus
5ml of absolute ethanol.
Prepare an approx 0.1M solution of disodium ethylene-diamine tetra acetate dehydrate Na2H2
C10H12O8N2. 2H2O (Mwt = 372.25) by dissolving 9.5g in distilled water and make up to 250ml in
a graduated flask.
Procedure
i. Pipette a sample into 250ml flask (A 1000ml sample sea water or 500ml sample tap
water). If you use 1.00ml seawater add 50ml of distilled water to each sample and 3ml of
pH 10-buffer and 6 drops Erichrome Black T indicator. Titrate with EDTA from a 50ml
burette and note when the colour changes from wine red to blue. You may need to find
the end point by titrating several times with EDTA.
ii. Repeat titration with three samples to find accurate values of Ca2+ and Mg2+
concentration. Perform a blank titration of 50ml of distilled water and subtract blank from
each result.
iii. To determine Ca2+ pipette four samples of unknown into a clean flask and add 50ml of
distilled water or 100ml of sea water, add 30 drops 50% NaOH to each solution and swirl
of r2 min to precipitate Mg (OH)2, which may not be visible. Add 0.1g of solid hydroxyl
naphthol blue to each flask. This indicator remains blue at higher pH than Erichrome
Black T. Titrate until end point.
Titrate other 3 samples of after reaching blue end point allow each sample to remain for 5 min
with swirling occasionally so that Ca(OH)2ppt may re-dissolve. Then tirate back to blue end
point. (Repeat until blue turns red). Performance blank with 50ml of distilled water).Calculate
the harness of your diluted tap water expressed in parts per million (ppm) of CaC03 using this
equation:
Hardness = MEDTA x VEDTA x RMMCaCO3 x 40, 000
= ppm CaCO3
Assuming 1cm3 of water = 1g of water
Where M = molarity, V = volume,
RMM – Relative Molecular Mass.
Questions
1. Write the structure of Ethylenediaminetetra acetic acid EDTA.
2. What mass of CaCO3 in glass of drinking water of 250ppm?
3. Why do we use Erichrome Black T as indicator in determining hardness in water?
4.5.4 DETERMINATION OF TOTAL HARDNESS
Hardness is determined by titrating a sample at the correct pH value with the
disodium salt of EDTA in the presence of a suitable indicator. Under these conditions, the
indicator has a red colour in the presence of Ca and Mg. On addition of EDTA to the red colour
solution the Ca and Mg are converted to their EDTA complexes and when this process is
completed the colour of the indicator reverts to blue.
a. Total Hardness
Materials: Ammonia buffer solution, EDTA solution 0.02N, Total hardness and Ca hardness
indicator tablets (B.D.H.)
Procedure
i. Measure 10cm3 of the sample and transfer to a white porcelain basin
ii. Add 2cm3 ammonia buffer solution and total hardness tablets
iii. Crush the tablets and stir well
iv. Titrate with 0.02N EDTA until the solution in the dish has lost all traces of real colour
(the final colour at the end point is usually a clear blue, but with some it may be clear
grey).
P=Vol of 0.02 N EDTAVolof sampleused
x1000
= ppm in terms of CaCO3
b. Calcium Hardness
Materials: 4N Sodium Hydroxide, Calcium Hardness indicator tablet, EDTA 0.02N.
Procedure
i. Measure 100cm3 of the sample and transfer to a white porcelain basin
ii. Add 1cm3 of 4N NaOH solution calcium hardness indicator tablet
iii. Crush the tablet and stir well
iv. Titrate with 0.02N EDTA solution when the addition of 0.1cm3 of 0.02N EDTA produces
no further colour change.
Ca Hardness=Vol of 0.02 N EDTAVolof sample
x1000
= ppm in terms of CaCO3
Questions
1. Why is it necessary to determine the chloride content of water?
2. What is the economic importance of using hard water in food processing?
3. Compare your results with standards and comment on quality of the water.
4. If the water sample is not good for food processing explain a method you could use to
make it adequate for processing.
4.6 ANALYSIS OF MILK AND MILK PRODUCTS
4.6.1 DETERMINATION OF TOTAL SOLIDS
Instead of determining the percentage of moisture, it is customary in the case of fluid
whole milk to determine the percentage of dry matter (total solids). Bysubtracting the percentage
of total solids from 100, the percentage of water present in the sample can be obtained.
Weight method
Milk is dried in the oven under standard conditions and the residue weighed.
Materials: Dish and lid, flat bottom, (porcelain dish), Oven at 1000C, Desiccator.
Procedure
i. Heat the clean dry empty dish and lid in the oven, cool in a desiccators and weight
ii. Add 3-4g milk, replace the lid and weigh again
iii. Place the dish without the lid on a boiling water- bath until the water is evaporated
from the sample, wipe the under surface of the dish and place in the oven at 1020C for
2 ½ hours.
iv. Put the lid on the dish, cool in the desiccator and weigh
v. Continue heating and weighing at hourly intervals until successive weighing do not
vary by more than 0.5mg.
Calculation
%Total Solid=Weight oresidueWeight of milk
x100
4.6.2 LACTOMETER (RAPID) METHOD FOR TOTAL SOLIDS
The density of the milk is measure using a milk hydrometer (lactometer) and the total
solids are calculated.
Materials: Milk hydrometer, cylinder at least 4mm greater in diameter than the bulb of the
hydrometer
Procedure
i. Warm the milk to and keep at 40-500C for 5 minutes. Carefully but thoroughly mix
avoiding inclusion of air bubbles and then cool to 20 ± 20C
ii. Immediately fill the cylinder to overflowing by pouring the sample over the
lactometer bulb
iii. Allow the bulb to float in the milk and then meniscus, the eye being horizontal to it.
iv. Calculate the total solids at 200C from the formula
MSNF = 0.25D + 0.22F + 0.72
TS = L + 1.2F + 0.14
Where L = Lactometer reading (scale/4)
F = % Fat milk
D = Density of milk from the Lactometer reading (Sp. gr. x 1000)
4.6.3 CRUDE ISOLATION OF CASEIN FROM FRESH AND FERMENTED MILK
Casein is the principal protein compound in milk. It contains carbon, hydrogen, oxygen,
nitrogen, sulphur and phosphorus. Casein can be precipitated by the action of acids, alcohol,
rennet, heavy metals heat and acid. The higher the temperature the less the acid required for
coagulation.
Materials: Fresh and fermented milk, Dilute solution of acetic acid, Beakers, Dropper, pH
meter.
Procedure
i. Pour the milk into a clean beaker and note the initial pH.
ii. Add the dilute acid solution drip by drop and monitor the pH change as dropping
continues until the pH reaches 4.7
iii. Record your observation.
Enzymic method:
The enzymes rennin found in calves stomach coagulates milk without decrease in pH.
This phenomenon has become the basis of traditional cheese making process.
Procedure
i. Add rennet to the milk sample (Approx. 1g of rennet is used for every 5kg of milk.)
ii. Allowed to stand for about 20-30min
iii. Record your observation.
Questions
1. What are whey proteins?
2. What is the appropriate name of major whey proteins>
4.6.4 ACIDITY AND pH OF MILK
Cow’s milk is acid to phenolphthalein, alkaline to methyl orange, but amphoteric to
litmus due to the presence of phosphates. The pH is usually between 6.4 and 6.6. The total
acidity of freshly drawn milk is usually about 0.14% (as lactic acid). On storage, the acidity
increases due to the action of microorganisms, and a sour taste is perceptible when this reaches
about 0.3%. Milk is decidedly sour at 0.4% and when the acidity reaches 0.6% it curdles at
ordinary temperatures.
4.6.5 DETERMINATION OF TOTAL ACIDITY
The sample is titrated with sodium hydroxide to a phenolphthalein endpoint using milk
containing rosasinine as a comparison standard.
Materials: Two 100ml porcelain-evaporating dishes, Burette, 5ml, 0.1N NaOH solution,
Rosalinine acetate solution, Phenolphthalein.
Procedure
i. Pipette 10ml of milk into each evaporating dish (2 in number)
ii. To one dish add 1ml of the dilute rosalinine solution and stir with a glass rod.
iii. To the other, add 1ml of phenolphthalein solution and titrate with 0.1N NaOH,
stirring the sample until the colour is the same as that of the rosalinine comparison
standard.
Calculation
% Lactic acid = ml NaOH x 0.09
4.7 DETERMINATION OF pH
The term pH is used to measure the amount of hydrogen ion concentration (H+) of a
solution. It is therefore described as a measure of the acidity or alkalinity of the solution.
pH=Log10 {H+}-1
Materials: pH meter, weighing scale, water bath, filter paper.
Procedure
i. Weigh 5g flour sample into 50ml of water
ii. Allow it to stand for 30 minutes in 400C water
iii. Filter solution and determine pH using a pH meter
4.8 ANALYSIS OF BEVERAGE/JUICE
4.8.1 TOTAL SUGARS (SOLUBLE SOLIDS) BY REFRACTOMETRIC METHOD)
Materials: Refractometer, thermometer, juice.
Procedure
i. Determine the refractive index of the juice at 200C
ii. Use the table given for refractive index and % sucrose
4.8.2 TOTAL TITRATABLE ACIDITY
Materials: Phenolphthalein indicator, glass wares, 0.1N NaOH, juice (fruit or veg.)
Procedure
i. Titrate 10ml of the sample against 0.1N NaOH using 1 or 2 drops of phenolphthalein
as indicator.
Calculation
1ml 0.1M NaOH = 0.007g citric acid
4.8.3 DETERMINATION OF DIACETYL VALUE FOR CITRUS FRUITS
The ripening process of fruits increases the susceptibility of the fruits to invasion. Bruises
and damages are added source of inoculums. Fruits have a low pH that inhibits most bacteria.
The spoilage organisms are the acid tolerant bacteria mainly gram positive Lactobacilli and
leuconostoc. Diacetyl is produced from the degradation of citric acid by Leuconostoccremoris
and lactobacilli. The presence of diacetyl is determined using colorimeter.
Materials: Sterile flask, pipettes and test tubes, colorimeter, orange.
Procedure
i. Squeeze out fruit juice in a sterile flask (matured fruit)
ii. Using a sterile pipette transfer 10ml of the fruit juice sample into a sterile test tube.
iii. Prepare a blank using a sterile pipette and transfer from stock 10mls of buffered
peptone water into a sterile test tube.
iv. Insert test tube containing samples into a colorimeter and allow warning for 15 min
v. Select a suitable wave length and set at zero absorbance using the blank sample
vi. Remove blank and insert the sample test tube
vii. Record result.
Question
What is the implication of low or high pH in terms of microbial invasion of fruits?
4.8.4 DETERMINATION OF ASCORBIC ACID (INDOPHENOL METHOD)
This method was designed got fruit juices but can be applied successfully to most liquid
samples, or samples that can be easily dissolved. The exception is blackcurrant juice, which is
too densely coloured.
Materials: Standard indophenols solution (freshly prepared), Standard ascorbic acid
solution,20% metaphosphoric acid or 4% oxalic acid, Acetone
Procedure
a. Standardization of indophenols
i. Pipette 10ml of standard ascorbic acid solution into a small flask and titrate with
indophenols solution until a faint pink colour persists for 15 sec.
ii. Express the concentration as mg ascorbic acid equivalent to 1ml of the dye solution.
As the endpoint vml dye solution will be equivalent to 0.002g (smg).
Therefore the concentration of the standard ascorbic acid per ml of dye will be 0.002g/v ml
Procedure
i. Pipette 50ml of un-concentrated juice (or the equivalent of the concentrated juice)
into a 100ml volumetric flask.
ii. Add 25ml of 20% metaphosphoric acid or 4% oxalic acid as stabilizing agent and
dilute to volume
iii. Pipette 10ml into a small flask and add 2.5ml acetone
iv. Titrate with indophenols solution until a faint pink colour persists for 15 sec. (The
acetone may be omitted if SO2 is known to be absent)
Calculation
Ascorbic acid (mg/100ml juice) = 20 (V) (C)
Where V = ml of indophenol solution in titration
C = mg ascorbic acid/ml indophenols.
Questions
1. Do results conform to the standards for each product? What are the likely results for the
variation in the results?
2. Describe the relationship between optical rotation and the amount of total soluble solid in
a fruit
3. What is the relationship between the specific gravity and the brix of a fruit juice?
4. Why must the indophenols be standardized be using it?
4.8.5 VITAMIN C DETERMINATION USING THE ABSORPTIOMETRIC OR
SPECTROPHONMETRIC METHOD
Reagents:
Stock ascorbic acid solution prepares 0.1% solution of ascorbic acid in 0.4% oxalic acid
solution. Working Standards (W.S.) Take 5, 10, 15, 20 and 25ml of stock ascorbic acid solution
and make up each to 500ml with 0.4% oxalic acid solution. These solution numbered 1 to 5
contain 1, 2, 3, 4 and 5 mg ascorbic acid per 100ml respectively. Standard dye solution 12mg of
2, 6 dichlorophenol indophenols per litre.Standard curve. To four ascorbictiometer tubes add the
following:-
a) D.W – 10ml water
b) No 1 – 1ml 0.4% oxalic acid
c) S – 1ml WS No. 1 + 9ml water
d) No 2 – 1ml WS No 1.
Adjust the absorptiometric to zero using water (D.W.) and a green filter (approx. 520ml. To tube
no 1, add 9ml standard dye solution (0.0012%) mix and record to reading (L1) exactly 15
seconds. After adding they dye then adjust the instrument to zero with tube S in the
absorptionmeter. To tube No. 2, add 9ml dye, mix and read after 15 seconds. (L2). Record L1 and
L2 for Each working standard and construct the standard curve with concentrations of ascorbic
acid (mg/100ml) as absissea and L1 and L2 for each working standard as ordinates.
Procedures:
Macerate 50g of the food sample for 3mins.In a blender with 350ml of 0.4% oxalic acid
solution and filter. Obtain L1 as described above. To tubes add 1ml filtrated + 9ml water and
adjust the instrument to zero. Then to tube No 2 and 1ml filtrated + 9ml dye and record L2 after
15 seconds. Calculate L1 and L2 and obtain the concentration of ascorbic acid from the standard
curve.
References
Bolaji, P. T. (1979): The Quality of Water before and after use in the Food Industry, HND
Dissertation, Department of Science and Food Technology, Grimsby College of
Technology, p. 33
Daniel C.H. (1977). Qualitative Chemical Analysis pp. 358.
Dikko S.B. Fodeke, B.A. and Trywianshi, C. IJMB Chemistry Laboratory Manual pp. 137-138
James M. J. (1991) Modern Food Microbiology, 4 th ed. Published by Van Nostrand Reinhold,
New York.
Lee, F.A. (1987): Basic Food Chemistry, CBS publishers and distributors, New Delhi.
Mayer, L.H. (1987): Food Biochemistry CBS publishers and distributors, New Delhi.
COURSE CODE: FST 317
COURSE TITLE: FOOD CHEMISTRY I
CHAPTER FIVE
5.1 DETERMINATION OF SMOKE, FLASH AND FIRE POINTS OF OIL
The smoke, flash and fire points of fatty material are measures of its thermal stability.
The smoke point is the temperature at which smoking is first detected in a laboratory apparatus
protected from drafts and provided with special illumination, The flash point' refers to the
temperature at which volatiles evolving from the heated oil' will flash, but not support
combustion. The temperature at which the substance supports combustion is the fire point.
Procedure:
(i) Pour l0ml volume of the oil into an evaporating dish.
(ii) Suspend a thermometer at the centre of the dish ensuring that the bulb just dips inside
the oil 'without touching the bottom of the dish.
(iii) Gradually raise the temperature of oil using a kerosene stove or electric stove.
(iv) The temperature at which the oil sample gives off a thin bluish smoke continuously is
noted as the smoke point.
(v) Similarly, the temperature at which the oil started flashing (when flame was applied)
without supporting combustion is equally noted as the flash point.
(vi) The temperature at which the oil starts supporting combustion is recorded as the fire
point.
5.2 DETERMINATION OF THE MELTING POINT OF OIL
Commercial fats are heterogeneous mixtures and do not a sharp melting point. The
melting point can be determined using Fisher-John melting apparatus.
Procedure:
i. Make a little smear of the frozen oil on the heating plate of the fisher-John melting
apparatus and cover with the observation lens.
ii. Switch on the apparatus
Observe the temperatures at which the oil just began to melt and when the little smear
completely melts through an inserted thermometer
5.3 PHOSPHATE TEST IN MILK
The basic principle behind phosphate test is that any phosphate present in milk splits the
substrate, p-nitrophenyl phosphates to give p-nitrophenol, which is highly coloured in alkaline
solution. The enzyme phosphatase is secreted by the mammary gland of the cow and is always
present in raw milk. Its normal action is the hydrolysis of phosphoric acid esters. Phosphatase is
only slightly more resistant to heat than Mycobacterium tuberculosis throughout the entire range
of pasteurization conditions, either by the holding method or by the flash method.
Materials: Lovibond comparator and its disc APTW OR aptw7, 2 fused glass cells of 25mm
depth, Water bath, Incubator, 5ml pipette, 1000ml-graduated flask,Measuring
cylinder,150/15mm test tubes with rubber stopper to fit,Buffer solution,Substrate-p-nitrophenyl
phosphate.
Procedure
i. Pipette 5ml of the buffer substrate solution into a test-tube containing stopper and
bring to a temperature of 370C
ii. Add 1ml of the milk sample to the test tube and mix the contents well by shaking.
iii. Incubate for 2hours at 370C. Do it for a blank from boiled milk (e.g. homogenized
milk)
iv. Remove the test tube from the water bath and mix the content again very well.
v. Place blank on the left hand side of the comparator and the test sample on the right
vi. Take reading of reflected light by looking down on the two apparatus, with the
comparator, facing a god source of day light- If artificial light is needed for matching,
use a “day light” type of illumination. Revolve the disc until the test sample is
matched.
vii. Record readings falling between two standards by affixing X-plus or minus sign to
the figure for the nearest standard.
Note: Test is considered satisfied when milk gives a reading of 10ug or less of p-nitrophenol/ml
of milk. Properly pasteurized milk will give no discernible colour.
Questions
1. What is the important of phosphotase test in milk?
2. What are the precautions to note when carrying out this experiment?
5.4 TURBIDITY TEST FOR STERILISED MILK
When milk is heated to 800C or above, the albumin in the milk becomes denatured. If the
solution of in organic salts or acid are added the denature albumin separates with the casein. In
the case of sterilized milk, the heat treatment should be sufficient to denature the albumin. Thus,
if a properly sterilized milk is treated with Ammonia Sulphate, filtered and the filtrate heated,
there should not be any turbidity. In the case of UHT milk however, a slight turbidity is given
due to the fact that it has been heated only for a short time interval.
Materials: Ammonium Sulphate, conical flask, test tube, filter paper, water bath.
Procedure
i. Weigh 4.0g Ammonium Sulphate (ANALAR GRADE) into a 50ml conical flask
ii. Add 20ml of the sample milk and shake for 1 min to dissolve the salt
iii. After 5 min, filter the sample and collect the filtrate in a test tube
iv. When not less than 5ml of filtrate has collected, place the tube in boiling water for
5min
v. Cool the test tube and examine for turbidity, if there is no turbidity, the milk sample
has been sufficiently sterilized.
Questions
1. What advantage has the use of a Rosaline acetate solution during the determination of
acidity of milk?
2. Briefly describe the conversion of lactose to lactic acid during fermentation of milk.
3. Mention three products that could result from the fermentation of milk
4. What observations did you make during the test for adequacy of sterilization?
5. Which other test would you carry out to determine the adequacy of sterilization?
6. Which method would you use to test for adequacy of pasteurization?
5.5 DETERMINATION OF PEROXIDASE
Procedure
i. Add 5ml of distilled water and 1ml each of guicol or catechol and 0.5% H202 to
blanched potatoes
ii. Shake the test tube and allow for 2-5 minutes. Record your observation for each of
the sample and comment on the result.
Table: Determination of peroxidase
Sample Control 2 3 4 5 6
Time of blanching (minute)
Observation
Remark
Result Interpretation:
A reddish brown colour is considered for presence of peroxidase. If no color develops it
is negative. The result may also be received as a trace. Any strong reaction is positive. Both
negative and trace reaction may be regarded as satisfactory evidence of effective blanching.
Questions
1. Briefly explain the types of browning reactions.
2. Which type of browning is most prevalent in fruits, vegetables, root and tubers?
3. What is peroxidase?
4. Explain any method other than balancing that can be used to arrest browning in fruits and
vegetables
5. What is the most effective time/temperature to blanch potatoes according to your result?
REFERENCES
Hobart H.W. et al (1988): Instrumental method of analysis, 7 th ed. Wadsworth publishing
company. Belmont, California.
Woods, A. E. and Aurand, L. W. (1977): Laboratory manual in food chemistry, AVI publishing
Co. Westport Connecticut.
COURSE CODE: FST 318
COURSE TITLE: FOOD STORAGE
CHAPTER SIX
6.1 DETERMINATION OF MOISTURE CONTENT OF GRAIN AND FLOUR
USING MOISTURE METER
The percentage moisture of a grain determines its stability, the lower the moisture content
the higher its shelf life. The accurate determination of moisture content is complicated by a
number of factors that varies from one sample to another. Among the factors are relatively
amount of water available and ease with which the moisture can be removed. Moisture content
can be determined by direct heating method, chemical method, vacuum oven method, immiscible
solvent, distillation method, dielectric method.
Procedure: The moisture content of grain is determined using the grain moisture meter and the
percentage moisture content of the grain is recorded and noted.
6.2 DETERMINATION OF INSECT INFESTATION OF GRAIN BY COUNTING
AND WEIGHING METHOD
Grain quality may vary with the variety or type of grain selected by the farmer. It will be
influenced by climate and soil conditions during the growing season, cultivation practices,
weather condition at harvest and by harvesting techniques. Apart from short term aging or
maturation immediately after harvest, quality cannot be improved during storage, handling and
processing.
Procedure: A quantity of sample was taken from a storage bag and was stored. The bulk sample
was counted and the number of bulk was recorded. Also, the grain infested was sorted out from
the non-infested ones and the number of infested grain and non-infested were also counted and
recorded. The percentage infestation was calculated.
Calculation
% Infestation= Number of infe stedNumber of bulk
x100
COURSE CODE: FST 320
COURSE TITLE: CEREAL, ROOT AND TUBER TECHNOLOGY
CHAPTER SEVEN
7.1 DETERMINATION OF PARTICLE SIZE ANALYSIS AND INTERPRETATION
USING GRAPH
Procedure: The mesh is properly cleaned, weighed and arranged according to their sizes. Pour
500g of the sample on the sieve as arranged on the shaker, set for 15 minutes and start it. The
sieves were then re-weighed after the set time to know the weight of material retained by each
sieve. Plot a graph of Cumulative %MR against sieve size and Graph of % Frequency against
sieve size. From the graph, deduce the average particle size.
Calculation
% MR= MRTM
x100
Where, MR = Mass Retained
TM = Total Mass
To convert the sieve size in BSS to mm
Sieve¿ (mm¿)= 2.54Sieve¿ BSS ¿
¿ x 10
7.2 DETERMINATION OF PARTICLE SIZE ANALYSIS AND INTERPRETATION
USING CALCULATION
Procedure: The mesh is properly cleaned, weighed and arranged according to their sizes. Pour
500g of the sample on the sieve as arranged on the shaker, set for 15 minutes and start it. The
sieves were then re-weighed after the set time to know the weight of material retained by each
sieve. Calculate the mean diameter and the average particle size.
Calculation
% MR= MRTM
x100
Where, MR = Mass Retained
TM = Total Mass
To convert the sieve size in BSS to mm
Sieve¿ (mm¿)= 2.54Sieve¿ BSS ¿
¿ x 10
7.3 DETERMINATION OF TOTAL ASH OF FLOUR
The ash of a biological material is an analytical term for the inorganic residue that
remains after the organic matter has burnt away. The ash is not usually the same as the inorganic
matter present in the original material since there may be losses due to the volatilization or
chemical interaction between the constituents. The importance of the ash content is that it gives
an idea of the amount of mineral elements present and the content quantitative constituents of
proteins, lipid or fat, carbohydrate, plus nucleic acid. Sample rich in organic matter can be
preheated on the flame or hot plate.
Procedure
(g) Place silica dish or crucible into muffle furnace for about 15 minutes at 3500C.
(h) Remove after one hour and cool to room temperature, weight the crucible (W1).
(i) Add enough sample into the crucible (0.5 – 2g) the quantity will depend on texture and
source of sample) and weigh content (W2).
(j) If sample is wet or fresh plant sample, it should be pre-dried. Increase the temperature
from 2000C – 4500C this is to avoid incomplete ashing. Ash sample until is become
whitish in colour). If ashing is incomplete (evidence of black particles) within a
reasonable period remove crucible, cool, bath and return to the furnace.
(k) Remove from furnace to desiccators and allow to cool to room temperature.
(l) Reweigh the crucible and content (W3).
% Ash=W 2−W 3
W 2−W 1x100
% organicmatter=100−% Ash
Note: The ash should be preserved for mineral analysis
7.4 DETERMINATION OF ACID INSOLUBLE ASH
Materials: 6N HCl, Silver nitrate solution, Whatman filter paper No 42, Water bath, Bunsen
flame oven at 5500C, Desiccator
Procedure
i. Use the residue at the end of ash content determination
ii. To it, add 23ml of 6N HCl and heat for 10min in a water bath
iii. Add the content and filter with an ash less Whatman filter paper No 42
iv. Wash filter paper and its contents with distilled water until the filtrate becomes free
from acid, which can be detected by the use of silver nitrate.
v. Return filter paper with its contents to the crucible and dry using a Bunsen flame
before igniting in oven at 550°C for 1 hour
vi. Cool sample in the desiccator and record the weight
vii. Continue to dry to a constant weight
Calculation:
N=W 1
W
Where;
N = % of acid insoluble ash (dry weight basis)
W1 = weight of the insoluble ash
W = the dry weight of sample
Question
1. What is the significance of determining the ash insoluble content of flour? Comment on
your results.
7.5 FUNCTIONAL PROPERTIES
Functional properties have been defined by Matil (1971) as those characteristics that
govern the behaviour of nutrients in food during processing, storage and preparation as they
affect food quality and acceptability. Some important functional properties that influence the
utility of certain foods are water absorption capacity, oil absorption capacity, emulsion capacity,
whip ability, foam stability, viscosity, swelling capacity etc. The practical determination of some
of these functional properties shall be considered.
7.5.1 BULK DENSITY: (BD)
Procedure:
(i) Weigh l0ml capacity graduated measuring cylinder
(ii) Gently fill the cylinder with the sample
(iii) Tap gently the bottom of the cylinder on the laboratory bench several times until there is
no further diminution of the sample level after filling to the 10ml mark.
Calculation:
The bulk density (g/ml) = weight of sample (g)
Volume of sample (ml)
7.5.2 WATER/OIL ABSORPTION CAPACITY
Procedure:
(i) Weigh 1 g of sample into a conical graduated centrifuge tube
(ii) Using a waring whirl mixer, mix. thoroughly the sample and l0ml distilled water or oil
for 30 seconds.
(iii) The sample is allowed to stand for 30 minutes at room temperature and then centrifuged
at 5,000 x g for 30 minutes.
(iv) The volume of free water or oil (the supernatant) IS read directly from the graduated
centrifuge tube.
Note: Absorption capacity is' expressed as grams of oil or water absorbed (or retained) per gram
of sample.
Calculation: The amount of oil or water absorbed (total minus free) is multiplied by its density
for conversion to grams. Density of water is l g/rnl that of oil will vary depending on the type of
oil (which can be determined). Bleached palm oil for example has a density of 0.88g/ l ml
7.5.3 FOAM CAPACITY (FC) AND FOAM STABILITY
Procedure:
i. Blend 2g of flour sample with 100mi distilled water in a warring Blender (the suspension
should be whipped at 1600 rpm for 5 minutes).
ii. Pour the mixture into a 250ml measuring cylinder and record the volume after 30
seconds. Foam capacity is expressed as percent increase in volume using the formula' it
of Abbey and Ibeh (1988).
Foam Capacity = Volume afterwhioping - Volume before whipping X 100
Volume before whipping
(% volume increase or
% whippability)
(iii) Record the foam volume at 15, 30, 60, and 120 after whipping to determine the foam
stability (FS) according to Ahmed and Schmidt (1979).
Foam stability = Foam volume after time’t’ x 100
Initial foam volume
7.5.4 EMULSIFICATION CAPACITY (EC)
Procedure:
i. Blend 2g flour sample with 25ml distilled water at room temperature for 30 seconds
in a waning blender at 1600 rpm
ii. After complete dispersion, add gradually 25ml vegetable oil (groundnut oil) and
continue the blending for another 30 seconds.
iii. Transfer into a centrifuge tube and centrifuge at 1,600 rpm for 5 minutes. The volume
of oil separated from the sample after centrifuge is read directly from the tube.
Calculation:
Emulsion capacity is expressed as the amount of oil emulsified and held per gram of sample
(Padmashree et al.1987)
Emulsion Capacity == Xx100
y1
Where X = height of emulsified layer
Y = height of whole solution in the centrifuge tube.
7.5.5 WETTABILITY
Procedure:
i. Into a 25 ml graduated cylinder with a diameter of 1 cm add .Ig sample
ii. Placing a finger over the open end of the cylinder invert it and clamp at a height of l0cm
from the surface of a 600ml beaker containing 500ml of distilled water
iii. Remove the finger to allow the test material to be dumped.
The wettability is the time required for the sample to become completely wet.
7.5.6 GELATION CAPACITY
Procedure:
i. Prepare sample suspensions of 2-20% (W/V) in 5ml distilled water in test tubes
ii. Heat the sample test tubes for 1 hr in a boiling water bath followed by rapid cooling
under running cold tap water
iii. Cool the test tubes further for 2 hrs at 4 C.
The gelation capacity is the least gelation concentration determined the concentration when the
sample from the inverted test tube will not fall or slip.
7.5.7 GELATINIZATION TEMPERATURE
Procedure:
i. Prepare a 10% suspension of the flour sample in a test tube.
ii. Heat the aqueous suspension in a boiling water bath with continuous stirring.
iii. Record the temperature 30 seconds after gelatinization isvisually noticed as the
gelatinization temperature.
7.5.8 VISCOSITY
Procedure:
i. Suspend 10% flour in distilled water and mechanically stir for 2 hours at room
temperature.
ii. Using Oswald type viscometer measure the viscosity.
7.6 pH MEASUREMENT
Procedure:
i Prepare a 10% W/V suspension of the sample in distilled water
ii. Mix thoroughly in a warring micro-blender, then measure the pH with a good pH meter
(Figure 3.9)
7.7 LOAF VOLUME DETERMINATION BY SEED DISPLACEMENT METHOD
Loaf volume is one of the quality indices used to evaluate the performance of baked
loaves and is often employed in determining flours that have good baking potentials. Higher loaf
volume is an indicator that the flour used in baking the loaf has enough gluten capable of
sustaining the structural fabric of the dough which will prevent it from collapsing after rising.
The loaf volume of baked loaves that come out of the oven with regular shapes can be
determined by simply determining the volume of the loaf from the dimension. For example the
volume of a rectangular shaped loaf can be determined by multiplying the length by the width
and by the height (Odland and Davis, 1982).
Seed displacement method is commonly used to determine the volume of pastry products
especially loaves [more importantly when the shape of the loaves are very irregular]. Usually a
special seed displacement apparatus are employed. However in the absence of that a good
sizeable container 4-5 volume of the product to be used to measure the amount of the seed that
can be displaced by the product.
Procedure;
i Weigh the loaf samples in a suitable laboratory balance.
ii Fill a container 4-5 volume of the loaf sample with the seeds [usually grape seeds or any
other small sized seeds], until the seeds dropping from a height of 1/2foot above the
container are over flowing.
iii Using a straight ruler edge cut off all seed above the container rim such that the seeds
from a plateau with the rim of the container.
iv Pour out the seeds and weigh them, the weight of the seeds that filled the container is
equivalent to the total weigh of seed that completely occupied the volume of the
container.
v Where the volume of the container is not predetermined and graduated on the `container ,
use water and measuring cylinder to determine the actual volume of the container as
filled to the brim.
vi Then filling 1/3 volume of the container with the seeds lay the loaf flat at the centre of the
container and fill up the container to over flow from ½ foot above the container. Again
using ruler to cut off the seeds above the rim of the container as the seeds form plateau
with the container rim.
vii Collect all the seeds displaced by the loaf sample and weigh [This weight of seeds
correspond to the volume of space displaced by the loaf sample placed in the container)
Loaf Volume (cm3) = W2 x W1
W1
Where:
W1 = Weight of seeds that filled the container.
W2 = Weight of seeds displaced by the loaf sample.
V1 = Volume capacity of the container.
Specific volume (cm3/g) = V
W
Where:W = Weight of loaf sample.
V = Volume of loaf sample.
7.8 AMYLOSE CONTENTS OF STARCH
Procedure:
i. To 10mg of the starch in 100ml beaker add 10ml 0.5N KOH and disperse uniformly
using a stirring rod.
ii. Transfer the dispersed sample into a 50ml flash and dilute to the mark with distilled
water.
iii. Taking 5ml of the test sample solution in a 50ml flask add 5ml 0.1N HCl followed by
0.5ml iodine reagent before diluting to 50ml.
iv. Read the absorbance at 625mm in a spectrophotometer after standing for 5minutes.
v. Prepare a standard curve using difference concentrations of pure amylase (2mg-
10mg)
vi. The amylase concentration of the test sample is extrapolated from the standard curve
using the absorbance value.
7.9 DETERMINATION OF GLUTEN IN FLOUR
Procedure: Weigh 20g of flour sample into a beaker and add 10ml of water and mix carefully.
After mixing, take the dough formed under running water and wash the dough. Allow the starch
in the dough wash away so there won’t be any traces of starch found in the dough. Weigh an
empty petri dish, transfer the dough in the petri dish, oven dry the dough and weigh after oven
drying.
Calculation
%Gluten=W 2−W 1
Initial weight of samplex100
Where
W1 = weight of empty petri dish
W2 = weight of petri dish + sample after drying
7.9 DETERMINATION OF BROMATE IN FLOUR
2% of Potassium Iodide and 10% of Sulphuric acid were prepared. 10mls of Potassium
Iodide was taken and mixed with 3mls of Sulphuric acid in a beaker. Take a little flour sample
and spread on a platform/slab and then sprinkle the mixed chemical in the beaker on the spread
flour. If there is a change in colour, then there is presence of bromated but if there is no change
in colour then bromated is absent.
COURSE CODE: FST 322
COURSE TITLE: FOOD PROCESSING AND PRESERVATION
CHAPTER EIGHT
8.1 CANNING OF PINEAPPLE
Select sound, mature not over ripe fruit. Wash the pineapple selected and soak in 1%
potassium permanganate for about 5minutes. Rinse very well with clean tap water. Peel the
pineapple core (remove) the eyes cut into small pieces of about ½ inch thick.
Weigh the pineapple pieces and note the weight. Prepare simple syrup of 10% sugar m/v
in which 0.1% citric acid and sodium benzoate. Fill the pineapple pieces into the already
sterilized can to about ¾ full. Top with the syrup to cover the pineapple pieces. Exhaust the filled
cans in a water bath at a temperature of 85oc for about 20mins. Seal the cans immediately with
the seaming machine. Heat process sealed cans in the boiling water at 100oc for 30 minutes.
Remove heat processed cans and cool immediately in clean water to about 30oc. Dry, and shelf.
Questions:
1 Why is 100oC chosen as the sterilizing temperature?
2 Why is sodium benzoate chosen as the preservative?
3 Name two organisms that can cause spoilage of this canned product.
8.2 LABORATORY PRODUCTION OF BURUKUTU
Burukutu is a locally fermented cereal product produced mainly from the alcoholic
fermentation of guinea corn some amount of coarse gari is added to increase the starch level for
increased starch - sugar - alcohol conversion.
It has a low alcohol content and usually taken for its thirst quenching ability.
Procedure:
i. Weigh a known amount of guinea corn and clean very well by sifting off the chaff and
removing heavier foreign material by hand picking.
ii. Steep in water for about 3 days, remove from steep water and keep in a humid place until
there is clear evidence of germination.
iii. Dry the malted grains in the oven at a temperature of 40-500F until it is well dried.4. Dry
mill
iv. Mix part of the milled guinea corn with coarse gari flour in ratio 4:1 by volume
v. . Add enough water to adequately make mixture into Slurry of medium consistency and
cook until it gelatinizes.
vi. Disperse gelatinized mixture with adequate amount of water and add fresh guinea corn,
maize flour mixture (4:1) to the gelatinized mixture in ratio 1:10
vii. Mix thoroughly and allow to ferment for 24 hours.
viii. Filter the mixture using muslin cloth.
ix. Bottle and store on the shelf.
8.3 CANNING OF MANGO PIECES
Select, disease free, mature, not over ripe (sliceable) mango fruits. Wash selected fruits in
water to remove filth. Soak in 1% potassium permanganate for 5 minutes. Rinse very well with
clean tap water to remove traces of the permanganate dip. Peel manually using a sharp stainless
kitchen knife. Slice the flesh leaving the seed. Cut slice into pieces of not more than ½ inch thick
prepare 10% sugar syrup and add some pulped mango flesh to give flavour and increase the
sugar content. Add 0.1% citric acid and 0.1% sodium benzoate to the syrup. Fill mango pieces
into already sterilized cans to about ¾ full. Top with the prepared syrup to cover the mango
pieces. Exhaust in water bath at 800c for 20 minutes. Seal immediately after exhaustion. Heat
process in water at 100oc for 30 minutes. Cool cans in water to ambient temperature. Dry , label
and shelf.
8.4 FREEZING OF SELECTED VEGETABLES.
8.4.1 FREEZING OF OKRO
Sort okro provided for size and unwanted materials removed. Grade for quality which
includescolour, tenderness and freedom from defects.
Wash sorted and graded samples thoroughly with tap water to remove dirt and other
adhering foreign materials and drain. Trim okro pods with knife to remove apical end and stalks.
Divide okro into two halves. Redivide each half into four parts. For the first four parts treat as
follows:
- No treatment, pack in double gauge polythene
- Dip in sodium metabisuphite at a concentration 0.2% (2000ppm) for one minute, remove
and allow to stand for 2 hours for effective penetration of the chemical. Package in
double guage polythene.
- Blanch in boiling water for half a minute. Cool immediately and pack in the double
guagepolythene bag.
- Blanch in sulphited boiling water at a concentration of 2000ppm for half a minute. Cool
quickly in suphited water at the same concentration as the blanching water. Package in a
secondary package and freeze in the contact deep freezer.
- Package in double gauge polythene.
For the second half, re-devived into four parts and treat as follow:
- Shred okro, (as for home soup prep.) and package in double guage polythene and freeze.
- Shred okro, prepare sodium metabisuphite at a concentration of 0.2% (2000ppm). Take
some of the sulphite solution and mix with the shredded okro and allow to stand for 10
minutes for effective penetration. Package and freeze.
- Shred okro. Bring water to 1000C. Add some of the boiling water to the shredded okro.
Place on heat source, and add sodium metabisuphite to make a 0.2% (2000ppm) solution.
Take some of the boiling water and add to the shredded okro. Place on heat source and
heat for half a minute. Cool quickly in a cold water bath. Package and freeze.
Question
Evaluate the samples made into soups sensorily for colour retention (bring green,
mucilage content (drawness) and flavour after 30days, 60 days and 80days frozen storage
respectively.
8.4.2 FREEZING OF BITTER LEAVES
Select defect free leaves, not over matured. Wash in tap water many times to removed
sand and silt. Divide washed bitterleaves into two halves. For the street half rub between the
palms and discard the coloured water using a sieve, rinse, until it comes to your taste (there
should however remain some tolerable bitter taste). Press out water. Re-divide the first half into
four parts and treat as follows:-
- Package into double gauge polythene and freeze.
- Soak in 0.2% (2000pm) sulphited ambient temp. Water and allow to stand for
30mimutes. Press out water, package and freeze.
- Bag pressed bitter leaves in a muslin bag blench and boiling water for 1 min. remove and
cool quickly in cold water. Press out water and package in double gauge polythene,
freeze.
- Blanch in boiling sulphited water at a concentration of 2000ppm for 1 minute. Cool in
sulphited water. Press out water, package and freeze leafy.
For the second half, shred in washed bitter leaves do not rub between the palms. Carry
out the treatment described for the first half; before packaging and freezing.
N.B: During soup preparation the second half samples, needed to be rubbed between the
palms to reduce the bitter taste.
Question
Evaluate the samples sensorily for desirable bitter leaf green colour, texture
(tough or tender) and tolerable bitter taste, after a frozen storage period of 60 days.
Which do you prefer?. “Green” leafy vegetable and the fluted pumpkin leafy vegetable
provided should be treated as in the case of the second half of the bitter leaf, that is,
without rubbing between the palms. Evaluation should also be carried out sensorily on
the samples for their peculiar green colour and flavour after frozen storage for a period of
60 days.
8.4.3 FREEZING OF SWEET CORN
Dehusk and desilk just harvested, fresh, mature, sweet corn. Wash in clean cool water.
Divide corn into two halves. Soakone half in very cold water. Meanwhile blanch the second
water for two minutes (allow cob to be totally covered by blanching water. Calculate blanching
time from the time blanching water reboils) quickly remove blanched corn on the cob from
blanch water and cool rapidly in cold water. Decob blanched corn – kernels using a sharp
stainless knife. Rinse and drain corn – kernels. Pack into double gauge polythene bags and
freeze.
Question
Carry out sensory evaluation of the frozen stored corn samples for sweetness after 15
days, 30 days respectively.
8.5 DEHYDRATION OF VEGETABLES.
The modern process of food dehydration refers to virtually complete water removal from
foods by the application of heat usually in the presence of a controlled flow of air with minimum
changes in food properties. Preservation is the principal reason for dehydrating foods; however
other reasons are to decrease weight and bulk, to reduce cost and also to produce convenient
items.
A major criterion of quality dehydrated foods is that on rehydration they be very close to
or virtually undistinguishable from the original fresh food material used in their preparation.
Dehydrated foods under appropriate conditions can remain stable for periods of a year or more.
Dehydration of selected vegetable shall be carried out in these practical.
8.5.1 DEHYDRATION OF OKRA
Select fresh tender okra fingers (using the ability to break tip) easily with finger at the
selection task. Wash selected okra fingers in water to remove adhering soil particles; and drain in
plastic colander. Trim washed samples with knife to remove tips and stalks. Slice okra into
pieces of 3.0mm thickness. Blanch the okra slices bagged in muslin bag to batches of 100g in
boiling water to which sodium metabisuphite had been added when boiling to give 1g/3kg of
sliced okra. The blanching time shall be for period of 30seconds and 60seconds. Transfer the
blanched slices into cold water for cooling and drain in colanders.
The cooled blanched slices were loaded in trays about25mg/cm2. The slices given
different blanching times were dehydrated simultaneously in an air oven set at 600C for about
12hours.
Question
1. Determine the moisture contents of the fresh okra and dehydrated samples.
N.B: reconstitute the dehydrated okra by milling 20g into a particle size of about 0.4mm and mix
with the amount of water lost during the dehydration plus water added to the fresh sample 20g of
the fresh okra sample shall be finely shredded and 10mls of hot water added to it. With the use of
a fork homogenize the fresh and reconstituted dehydrated samples.
2. Carry out sensory evaluation ofthe colour and drawing ability of the samples presented
using the scoring system in terms of the bright green colour and drawiness respectively.
Rate the samples for their overall acceptability.
3. Report observation on the effect of shelf and refrigerator storage of the dehydrated
samples on the sensory proprieties earlier evaluated.
8.5.2 DEHYDRATION OF PEPPERS
Sort the different peppers provided (Tatase, Rodo, Shombo, Atawewe) for ripeness (red
colour), disease free samples.
Wash in water to remove dirt attached to the surface of the peppers and drain. Slice or split the
peppers. Bag differently in muslin bag and blanch in boiling water for 3minutes and 5minutes
respectively and drain. Dehydrate the samples in air oven at 580C for 24hours mill and sieve
dehydrated samples. Package in polythene bags or plastic containers and store on the shelf for
storage studies.
Question
1. Rank the samples for their colour retention and pungency.
2. Investigate the following parameters on the shelf life of the samples namely colour,
pungency and ‘caking’ after 8 weeks.
8.5.3 DEHYDRATION OF ONION
Select mature and unspoilt onions. Wash in water to remove dirt. Hand peel using knives
to remove scales. With the use of sharp stainless knife blade cut to a thickness of about 1/8 times
in rings. (Cut horizontally along the vertical axis) spread thinly perforated trays load trays into
drier already set at 650C for about twelve hours. Reduce drier temperature to about 450C-500C for
two hours. Pack in airtight containers.
Question
1. Calculate the present weight of the fresh edible portion and dehydrated anions to the
weight received.
2. Determine the moisture content of the onions on wet dry basis.
3. Carry out packaging and storage, Studies (plastic containers, heavy duty cellophane bags;
stored on the shelf under ambient temperature and in cold storage in the refrigerator) for
three months, making monthly observations.
8.6 PRODUCTION OF TOMATO POWDER
Tomato powder is a product resulting from dehydration of tomato slices, concentrated or
paste. As with all dried products, tomato powder should disperse readily in water and produce a
reconstituted product of food flavour, colour, physical stricture and chemical composition. In
addition, the powder should possess good keeping qualities when stored under suitable
conditions. Dehydrate tomatoes have a longer shelf life when compared to the fresh product
dehydration may be accomplished by tunnel, kiln or tray, cabinet, vacuum and spray drying.
Materials: Fresh tomatoes, 1.8% Na2S203, Hammer mill, Sieve, Cabinet dryer, Sharp knife,
Packaging films, Bowls, Heat source
Method
i. Take about 500g of graded fresh tomatoes
ii. Wash the tomatoes and slice to a thickness of 2-3mm
iii. Blanch at 600C for 10 minutess or sulphite using 1.8% Na2S203
iv. Drain the blanched or suhlphited tomatoes slices
v. Dehydrate in a cabinet dryer at 52+30C for 2 days
vi. After the drying, remove from the cabinet dryer and comminute into powder using
hammer mill
vii. Sieve and package using high density polythene.
Questions
1. Compare the quality of the blanched and dehydrated tomatoes with the sulphited ones,
which one has a better organoleptic qualities?
2. Why should the tomatoes be dehydrated at 520C?
3. Which method of dehydrating tomatoes is the best? explain your answer
8.7 PRODUCTION OF CORN DOUGH PRODUCT
Corn like other cereal belongs to the family Gramineal.Corn servers as a main food
product in many parts of the world. Industrially corn processing can be classified into three
namely: wet milling dry milling and fermentation. The wet millers use it for starches, corn sugar,
corn oil, while the dry millers produces corn flours, 'cereals' and also corn oil. The fermentation
processors produce neutral spirits and alcoholic beverages from them.
In the home, foods have been prepared from corn through the above mentioned processes - wet
milling, dry milling and fermentation. One of the fermented food made from corn dough will be
produced in this practical.
Method:
1. Clean the corn provided by removing undesirable materials such as stones, pieces of cobs,
chaff, etc.
2. Weigh the given weight of cleaned dried corn
3. Wash and leave in soak (steep) for four days under room temperature.
4. Discard soaking water, rinse auto Note the weight of soaked corn.
5. Mill the soaked corn, Take a known weight of the freshly milled corn, cook, Taste and note
(sweet, bitter, sour or bland)
6. Keep remaining milled corn under room temperature overnight
7. Cook the overnight milled corn stirring constantly to avoid lumps and burning. Add a little
water at a time until desirable texture is attained
8. Shape as desired, weigh and note the taste.
Questions:
1. Account for the increases/decreases in the weights of the corn, milled corn and product,
substantiating with figures.
2. Comment on the pH value of the products from the freshly milled corn and overnight milled
corn.
3. What could be responsible for the taste of the products?
Exercise:
1. Record the pH measurements of the freshly milled corn, milled corn kept overnight and their
products respectively.
2. Record temperatures of the freshly milled corn and milled corn kept overnight, just before
cooking, including the environmental temperatures.
3. Evaluate the products for taste.
8.8 PRODUCTION OF FLOUR FROM YAM, COCOYAM
Production of Yam Flour
Thickness of
yam
Sodium
metabisulphaite
concentration
Drying
temperature
Drying time Parboiling or
Blanching time
0.5cm
1.0cm
1.5cm
300ppm
400ppm
500ppm
600C
650C
700C
Sun drying
With the understated parboiling (blanching) formula by Ige et al (1981)
Y =3.5 +8t, find the blanching time for the various yam thickness before starting this
practical.
In the formula state above Y = time (in minutes) required for the yam piece to attain the
required flabbiness.
t = thickness (in centrimetres, and of the yam pieces)
The white yam variety is provided. Brush off all sand used to increase the dirth of the
yam tubers.
Weigh the yam tuber(s), note the weight. Wash in running tap water to remove adhering
soil, etc. manually peel yam tuber(s) under water. Weigh peeled tuber(s) for calculation of waste
in processing of yam tuber (s) into flour. Cut peeled yam still under water into thickness of
0.5cm and 1.5cm. transfer yam pieces into plastic basin containing previously prepared sulphite
solutions of concentrations 300ppm, 400rpm, and 500ppm. Allow yam pieces to remain in
sulphite solution for 30 minutes. Using the approximate time in minutes (from calculation using
the process time formula, given). Blanch the 0,5cm, 1.0cm and 1.5cm thick yam pieces.
Resulphite yam cuts after blanching for 30 minutes and drain. Dehydrate each sample of yam
thickness at 600C, 650C and 700C for twelve (12) hours in air ovens and sundry some
respectively the untreated yam pieces. Mill the sundried and dehydrated samples. Sieve with the
mechanical sieve filter mesh size. Package yam flour produced in high density poly ethylene
bags and seal. Label carefully and store.
Activity: reconstitute the yam flour in boiling water with constant stirring until a desirable
consistency is obtained. Note the ratio of flour to hot water by weight used to obtain the desirable
consistency.
References
Luh, B.S and Woodroof, J. G (1975): Commercial Vegetables processing AVI Pub,
Westport, Connecticut.
MacCarthy, D. (1985): Concentration and Drying of Foods. Elsevier Applied Sci. Pub,
London.
