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INTERNSHIP REPORT

ONONONON

A practical experience

BY

WASEM TAHIR M.Sc. (Hons)

DEPARTME�T OF FOOD SCIE�CE & TECH�OLOGY FACULTY OF �UTRITIO� SCIE�CES �WFP AGRICULTURAL U�IVERSITY

PESHAWAR, PAKISTA�. 2000

INTERNSHIP REPORT

ONONONON

BY

WASEEM TAHIR

M.Sc. (Hons)

A Internship Report submitted to the National Foods Ltd., Karachi in partial fulfillment of

the requirement for the internship program Approved By:

Dr Rafiullah Khan Chairman

DEPARTME�T OF FOOD SCIE�CE & TECH�OLOGY

FACULTY OF �UTRITIO� SCIE�CES

�WFP AGRICULTURAL U�IVERSITY

PESHAWAR, PAKISTA�.

CONTENTS i

CO�TE�TS Acknowledgements

I. I�TRODUCTIO�

1.1 Responsibilities of the Quality Control Department. ........................................ 4

1.2 Relationship of Quality Control with other Departments. ................................ 6

1.3 ISO-9000 Series (Theory & Practice). .............................................................. 8

1.4 Some General Principles. ................................................................................... 9

1.5 Spices .............................................................................................................. 10

II. MICROBIOLOGICAL TESTS

2.1 Preparation of Culture Media. ......................................................................... 16

2.2 Total Plate Count. ............................................................................................. 18

2.3 Enumeration of Total Coliforms. ..................................................................... 20

2.4 Enumeration of Fecal Coliforms. .................................................................... 21

2.5 Yeast count. ...................................................................................................... 22

2.6 Mold Count ...................................................................................................... 23

2.7 Determination of Salmonella. .......................................................................... 24

2.8 Gram Staining. .................................................................................................. 26

III. CHEMICAL / PHYSICAL TESTS

3.1 Determination of pH/Soluble solids/Bostwick value. ..................................... 27

3.2 Determination of %purity of Acetic Acid. ...................................................... 29

3.3 Determination of %purity of Salt sample. ....................................................... 31

3.4 Determination of Iodine in fortified salt. ......................................................... 32

3.5 Determination of Insect fragment extraneous matter. ..................................... 33

3.6 Determination of Acid value of oil. ................................................................. 34

3.7 Determination of Free Acidity. ........................................................................ 35

3.8 Determination of Moisture content.................................................................. 37

3.9 Determination of %Total Ash. ......................................................................... 38

3.10 Determination of Acid Insoluble Ash. ............................................................. 38

3.11 Pungency Test. ................................................................................................. 39

3.12 Chromatography Test. ...................................................................................... 42

3.13 Determination of Particle size. ......................................................................... 44

3.14 Determination of Aflatoxin contamination. .................................................... 45

3.15 Determination of Density. ................................................................................ 46

IV. RESEARCH A�D DEVELOPME�T PROJECT

4.1 Preparation of Low Salt Brine Medium. ......................................................... 47

4.2 Treatments. ....................................................................................................... 48

4.3 Observations ..................................................................................................... 48

V. VISITS TO PLA�TS/SALT LAKE

5.1 Salt Plant. .......................................................................................................... 50

5.2 Paste Plant. ........................................................................................................ 51

5.3 Packing Plant. ................................................................................................... 51

CONTENTS

ii

5.4 Pickle Plant. ...................................................................................................... 52

5.5 Dehydration Plant. ............................................................................................ 53

5.6 Grinding Plant. ................................................................................................. 54

5.7 Visit to Salt lake, Khipro .................................................................................. 55

INTRODUCTION 3

I. I�TRODUCTIO�

Contacted National Foods Limited, Karachi, Quality Control department. Internship

program was scheduled as

1. General Orientation 2-Weeks

2. Microbiological testing 2-Weeks

3. Processing Plants Study 2-Week

4. Project 2-Week

Quality control department of National Foods Ltd. comprises a laboratory,

employing 17 staff members. In series the laboratory is divided in different sections:

1. Microbiology Lab.

2. Physical lab.

3. Instrumental Lab.

4. Chemical Lab.

5. Sensory Evaluation Lab.

1. MICROBIOLOGY SECTIO�

Microbiology section is equipped with testing equipment, necessary apparatus and

glassware for microbiological testing. Main equipment are incubator (oven), Water bath,

Refrigerator, Microscope, Spectrophotometer, chemicals (Media, Indicators) etc.

2. PHYSICAL SECTIO�

Physical section is equipped with instruments necessary for physical testing i.e. pH

meter, Aflatoxin instrument etc.

3. I�STRUME�TAL SECTIO�

Instrumental lab is equipped with different instruments necessary for measurement of

packaging materials.

4. CHEMICAL SECTIO�

Chemical section consists of chemicals required for chemical testing of food

materials. It’s equipped with cooking site, chemicals, titration equipment (Burette, Pipettes,

Flasks, Beakers and glassware etc.,) preservatives, flavor and color materials.

5. SE�SORY EVALUATIO� SECTIO�

Sensory evaluation lab is equipped with separate desks for judges to evaluate the

food product through their senses.

INTRODUCTION 4

1.1 RESPO�SIBILITIES OF THE QUALITY CO�TROL

DEPARTME�T

Some of the specific responsibilities of the quality control department may be listed

as follows:

1. Inspection of supplies and materials.

2. Inspection of raw products.

3. Scheduling of operations.

4. Measurement of production efficiency.

5. Measurement of equipment efficiency.

6. Inspection of the finished product.

7. Warehousing controls.

8. Shipping and storage controls.

9. Preparation of specifications and procedures in written handbook form.

10. Preparation of statistical procedures and schedules.

11. Sanitation inspections.

12. Conformances to local and federal regulations.

13. Waste disposal control.

14. Basis for pricing policy.

15. Basis for inventory policy.

16. Basis for budget policy.

17. Basis of evaluating individual personal performances.

All these functions may be discussed under the following headings:

1. Establishment of specifications.

2. Development of test procedures.

3. Development of sampling procedures.

4. Recording and reporting.

5. Trouble shooting.

6. Special problem.

7. Training of personnel.

Establishment of Specifications

Specifications for raw materials, supplies, in-plant processes, containers, and the

finished product including shelf life, must be available in clear, unambiguous language so

that everyone involved can appreciate exactly what is wanted. Such specifications should be

established with the assistance of sales and production personnel since they are in position to

know customer requirements, and production capabilities. The primary responsibility for

preparing these specifications, however, rests with the quality control department.

Development of Test Procedures

Quality levels and production variables must be tested on some scale. Hence, it is up

INTRODUCTION 5

to the quality control department to define or to develop for any specific purpose, a means of

measuring every quality attribute and production variable of importance at every step in the

process from the raw material until the product is consumed. These tests may be developed

for specific purposes with the aid of research and development personnel, or they may be

adopted in whole or in part from existing grades and standards developed by Trade

Associations or Government Agencies.

Development of Sampling Schedules

Since 100 per cent inspection is rarely feasible or desirable, it is the function of the

quality control department to establish efficient procedures for handling samples, and for

determining the number of units and frequency of sampling, so that quality may be evaluated

with maximum reliability at minimum cost.

Recording and Reporting

The quality control department is responsible for setting up the necessary forms so

that results may be recorded easily and transmitted promptly to the proper personnel, usually

production, who are in position to take action when and as soon as necessary. Quality control

charts may frequently replace the usual tabulating forms, the major advantage of the control

chart being that an out of control situation may be noted at a glance, and is not lost in a mass

of numerical values.

Trouble Shooting

When a situation is found to be out of control, the quality control person should see

to it that the situation is corrected immediately. If that is impossible, further work should be

initiated promptly with co-operation of the research department to correct the problem.

Special Problems

When special problems arise in any part of the organization, whether originating in

sales as a result of customer complaints, in purchasing as a result of poor raw materials, or in

production as a result of equipment or personnel problems, the quality control department

should in all instances serve as the "channel of communications" among departments in

order to facilitate solution of the problem.

Training of Personnel

Line personnel may or may not be under the direct supervision of, and responsible to,

a central quality control department. There is no doubt, however, that line personnel should

be instructed by the quality control department in the sampling, testing, and reporting

procedures established by quality control. Another educational function of the quality control

department, together with the support of top management, is to promote a spirit of quality

mindedness among all personnel.

INTRODUCTION 6

1.2 RELATIO�SHIP OF QUALITY CO�TROL WITH OTHER

DEPARTME�TS

1. Relation to management.

2. Relation to sales and purchasing.

3. Relation to research and development.

4. Relation to production.

RELATIO� TO MA�AGEME�T

Organizationally, there must be a direct line from the quality control department to

top management, so that specific contribution of quality control may have an equal voice

with other departments. From management standpoint, quality control may be considered as

management's tool for delegating authority and responsibility for product quality. While

relieving itself of the burdensome detail, management still retains the means of assuring

satisfactory results. The only way to accomplish this is for the quality control department to

report directly to top management.

Quality control reports provide management not only with information on whether

the production operation is in control, but with the basic information for decision making on

such basic problems as inventory, pricing, and budget policies.

A quality control program must have the complete support of top management. It

should be clearly understood throughout the organization that quality control reports and

indications for action must be acted upon unhesitatingly.

Quality control should provide a channel of communication and a means for

participation in the quality program by all concerned employees and groups.

RELATIO� TO SALES A�D PURCHASI�G

Since the sales department is the primary contact between the processor and

customer, it is the salesman who should be in the position to appreciate most fully exactly

what the buyer is looking for in the product. Sales, therefore, may be considered as the

buyer's representative, in developing specifications. It appears to be self-evident, and yet it

needs to be stressed repeatedly that a product can be marketed successfully only if it meets

the customer's requirements, not the manufacturer's opinion of what these requirements

should be.

In return for their assistance in establishing the specifications, sales and purchasing

departments should be provided with the assurance that the sampling, testing, and reporting

provided by quality control insures the presence of the quality claimed. A customer-

complaint service may be another function performed by quality control department in co-

operation with all departments, but primarily for the benefit of the sales department. Here,

too, quality control may serve as a channel of communication between sales and purchasing,

and production.

INTRODUCTION 7

RELATIO� TO RESEARCH A�D DEVELOPME�T

Since quality control and research and development are so closely related, it is

advisable first to distinguish between the two before discussing their relationship.

Essentially, quality control is the continuing assessment of a current operation, while

research and development is a search for something new or different. Thus, quality control,

in finding or pinpointing a situation that is out of control, may indicate where research and

development is needed. Research and development, on the other hand, in developing a new

product, process, or equipment may elicit a change in quality control procedures. Thus, the

two functions can be clearly distinguished so that each can be organized in a separate

department. Whether they should be organized separately or not depends largely on the size

of the organization and on personnel.

Once buyer and vendor specifications are clearly understood and defined, it is the

function of the quality control department to establish means for measuring these quality

attributes. Unless such test methods are available in useful an practical forms, it becomes

necessary to create new methods for specific purposes. This developmental work can be

accomplished by the co-operative effort of the two departments. If it is assigned entirely to

research and development, the work should be done under the guidance of quality control

and any resultant methods should then be applied first on a tentative basis by quality control.

RELATIO� TO PRODUCTIO�

The quality control should not be subordinated to production but should report

directly to top management is one law in the organizational scheme that cannot be

compromised. The purposes and attitudes of the production supervisor, no matter how

quality conscious he may be, are, and should be, different from those of the quality control

inspector. The responsibility of production department is to pack maximum quantities in

terms of product yield and plant capacity; whereas, the responsibility of the quality control

department is to insure that this objective is achieved without impairment to quality and with

a maximum of profit.

It follows that a quality control department must be independent of the production

department, and would, therefore, imply that the quality control inspector on the production

line, as well as in the laboratory, should be directed by, and report to, the central quality

authority.

In many cases, it may be impractical to have anyone but the production employee

handle quality control, as in the case of an automatic machine operator, whose major

responsibility is simply to see that the equipment is operating properly. It may appear that the

operator himself is the person best qualified to perform the tests and maintain the records by

which the performance of the equipment in his charge may be gauged, and controlled. He

should certainly be afforded the opportunity to do so. Yet, the nature of tests for evaluating

performance and the method of recording and evaluating the data should be established by

quality control.

INTRODUCTION 8

All line inspectors and certainly all laboratory personnel, regardless of whether they

are quality control or production employees, should receive their instructions and training

from the quality control department.

1.3 ISO-9000 SERIES (THEORY A�D PRACTICE)

The ISO-9000 series consists of 5 documents whose focus is quality assurance.

ISO-9000 : Quality management and quality assurance standards, Guidelines for

selection and use.

ISO-9001 : Quality system, Models for quality assurance in design/development,

production, and servicing.

ISO-9002 : Quality systems, Models for quality assurance in production, installation,

and servicing.

ISO-9003 : Quality systems, Model for quality assurance in final inspection and test.

ISO-9004 : Quality management and quality system elements.

Quality System elements defined in ISO-9004

1. Management responsibility.

2. Quality System.

3. Contract Review.

4. Design Control.

5. Document and Data Control.

6. Purchasing.

7. Control of Customer Supplied product.

8. Product Identification and Traceability.

9. Process Control.

10. Inspection and Testing.

11. Inspection Measuring and Test Equipment.

12. Inspection and Test status.

13. Control of non-conforming product.

14. Corrective and Preventive action.

15. Handling, Storage, Packing, Preservation and Delivery.

16. Quality Records.

17. Internal Quality Audits.

18. Training.

19. Servicing.

20. Statistical Techniques.

ISO-9001 requires 20 points.

ISO-9002 requires 19 points. (Except 4)

ISO-9003 requires 16 points. (Except 4,6,9,19)

National Foods Limited is an ISO-9001 certified company which is the most comprehensive

INTRODUCTION 9

quality assurance package.

1.4 SOME GE�ERAL PRI�CIPLES

A Definition of Quality and Quality Control

Quality of foods may be defined as the composite of those characteristics that

differentiate individual units of a product, and have significance in determining the degree of

acceptability of that unit by the buyer.

Quality is commonly thought of as degree of excellence. In the broader sense in

which it is used here, it may be considered as a specification or set of specifications, which

are to be met within given tolerances or limits.

Quality control may be defined as the maintenance of quality at levels and tolerances

acceptable to the buyer while minimizing costs for the vendor.

Quality Specifications for the Buyer

The quality control system, as well as the production system, is properly geared to

the buyer’s specifications. The buyer is not necessarily the ultimate consumer, but he may be

a broker, wholesaler, distributor, or another food manufacturer using the particular product

as a raw material. Thus, a quality control cycle should begin and end with the customer’s

specifications. The first step is to determine as precisely as possible, in terms of

specifications, just exactly what the customer is looking for.

Fig I. QUALITY CO�TROL CYCLE

REPORTING

CONTROL CHARTS

The next step is to set up instruments and procedures by which these characteristics

of interest to the customer may be measured. These methods should be as precise and

accurate as possible, while at the same time as rapid, simple, and inexpensive as possible.

Objective methods should be used where available.

CUSTOMER SPECIFICATIONS

FOR EACH

QUALITY FACTOR

TESTING

METHODS

CONTROL STATIONS

ACTION WHEN

NEEDED

INTRODUCTION 10

Once the methods have been selected, a sampling schedule must be worked out

which will provide maximum information at minimum cost, and control stations set up

preferably, in the plant rather than in a central laboratory. Results obtained at the control

stations should be posted immediately at the control charts so that action may be taken as

soon as a quality characteristic goes out of control. A final inspection of the finished product,

as well as the reactions of the accounting department and finally the customer himself, will

provide a measure of the success of the control program, and suggestions for modifications.

1.5 SPICES

pices and herbs are similar to other agricultural commodities in that they are

classified as raw agricultural commodities although they are normally received by

the processor in a partially processed or dehydrated form. Unlike fresh agricultural

commodities, they do not have to be protected from microbiological spoilage but,

like grain crops, (wheat, corn, etc.), they are susceptible to insect infestation.

For this reason and since their functional properties are highly sensitive to storage

and handling conditions, the quality control program on the part of the processor must

begin as early in the distribution chain as possible. For practical reasons, this generally

begins at the port of entry. The functional properties of spices provide different

characteristics depending on the item, e.g., black pepper and cinnamon impart a

distinctive flavor while paprika and turmeric are used principally for their coloring

characteristics. Other items, such as parsley and saffron, are used both for their coloring

and flavor contribution. For these reasons, the analytical methods for these commodities

require the measurement of a broad range of quality attributes.

PREPURCHASE CO�SIDERATIOI�S

The spice trade relies heavily on samples submitted in advance by the shipper,

frequently form long distances. Thus, quality control usually begins prior to purchase.

Even before examination of the prepurchase sample, certain factors must be weighed to

determine whether a given lot of raw material should be brought into the plant.

Foremost among attributes by which to judge the acceptability of a spice is its

value as a sensory quality enhancer. The most common analytical quality indicator for

spices is the quantity of steam distillable volatile oil which in the trade is considered as

having a good correlation with spice flavor. In products, which have characteristic throat

or mouth heat (pepper, capsicum), the nonvolatile extract is of more importance.

Appearance and acid insoluble ash also used to evaluate spice quality.

Similarly to the quality factors but perhaps more important economically are the

“output” or minimum requirement standards. These are the analytical values below which

a product cannot be accepted. Higher than minimum standard products can be reclassified

once brought into the building, but lower than minimum lots are worthless. In the spice

S

INTRODUCTION 11

trade, such standards are written for chemical (volatile oil, color, piperine) and physical

characteristics (extraneous matter). Sanitary analyses always have been an important

influencing factor but are becoming increasingly more important in this time of growing

consumer and government concern over food safety.

A�ALYTICAL METHODS

Sample Preparation

Before any analyses are performed, the proper sample must be drawn and

prepared. The sample should be drawn in such a manner that it is truly representative of

the entire lot, a factor which cannot be controlled in prepurchase samples.

Moisture

One of the most important analyses from both the quality and economic points of

view is moisture content. An excessively moisture content in spices is conducive to

development of mold on storage. Ground spices too high in moisture tend to become

lumpy in their containers. Too dry a product is dusty. For economic reasons, the buyer

usually prefers the lowest moisture content since he does not want to be in the position of

buying water at the price of pepper or cassia, for example.

There are two official methods of moisture analysis (ASTA 1968; AOAC 1970).

The method used most often for higher moisture spices (>4%) is azeotropic distillation

with toluene. The method requires distilling sufficient spice to yield 2-5 ml of water with

toluene, collecting the distillate in a Bidwell-Sterling type trap. For high sugar spices

such as red peppers, paprika, and chili powder, which may decompose at 100oC to form

water, benzene is used in place of toluene. The procedure is relatively fast, requires a

minimum of preparation and attention, and is of adequate accuracy. Apparatus

requirements, however, may become unwieldy if many samples are to be analyzed.

For most low moisture products, the vacuum oven method is the method of

choice. The method requires the weighing of moisture lost from product maintained in an

oven at 70oC under a pressure of not more than 100mm of mercury for 6hr.

Ash

The total ash determination of a spice is used as an indicator of cleanliness. Each

spice has a small residual of non-ignitable inorganic material, but excessive residue

indicates that the sample may be contaminated with dirt, stones, or other non-ignitable

material foreign to the spice. A more significant indication of inorganic contamination is

the acid insoluble ash. It is a measure of the silicone dioxide present and a more specific

indicator of extrinsic dirt.

Steam-volatile Oil

INTRODUCTION 12

The steam-volatile oil analysis is one of the most meaningful and frequently used

analytical procedures for most spices, since much of the flavor of many spices is

considered to be contained in the steam-distillable oil. Thus, it is usually considered that

the lot of spice with a higher steam-distillable oil content will have the stronger flavor.

Most specifications are properly based on the steam-distillable oil content of a spice.

Color Power

ASTA method describes an analytical procedure of the color power of turmeric.

The method involves an alcoholic extraction of ground turmeric and a spectrophotometer

analysis for curcumin, the coloring constituent of turmeric. Like ASTA method for

extractable color in paprika, other capsicums and their oleoresins, it is an accurate

spectrophotometric procedure. The use of pure chemical standards for color units also

adds to the precision of these procedures.

Crude Fiber

The crude fiber determination method is a measure of the organic matter in the

dried residue remaining after digestion of the sample with dilute sulfuric acid and sodium

hydroxide. The analysis is used to determine adulteration in ground spices. For example,

a ground black pepper which was adultered with pepper or other grain hulls would have

an abnormally high crude fiber analysis.

Starch

A similar function is served by ASTA and AOAC method for starch analysis.

This method detects adulteration by the measurement of reducing sugars which are

produced by acid hydrolysis. The sugar (starch) content is determined by precipitating

the sugars from the hydrolysate with a modified Fehling’s solution, weighing the dried

Cu2O precipitate. An alternate approach could be a spectrophotometric sugar analysis of

the hydrolysate. Somogyi (1945) has developed a spectrophotometric determination of

reducing sugars which, with slight modifications should be applicable to spices.

Alcohol Extract

As the name implies ASTA and AOAC method determines the amount of alcohol

soluble materials in spices. The procedure is taken after a similar U.S.P. Standard used as

an empirical indicator of quality. For all practical purposes, this procedure has been

replaced by the volatile oil determination.

Particle Size

Screen analyses are important on all spice products. The range of particle sizes of

a product can influence its appearance, its stratification when mixed with other

ingredients, and even its flavor intensity. The analyses are performed using U.S. Standard

or Tyler equivalent sieves. Metal mesh sieves are used for all products with the exception

INTRODUCTION 13

of high oil spices such as mustard flour which require the use of Standard Testing Silk

Screens. Sometimes it is necessary to defat the mustard flour by extraction with

petroleum ether before analysis.

Methylene Chloride Extract

The nonvolatile methylene chloride extract analysis, while originally introduced

as a method of determining the piperine content of black pepper, is applied to other heat-

containing spies, e.g., red peppers and ginger. Obviously, other extractable materials

interfere with the accuracy of this determination as a “heat” indicator. Although the

methylene chloride extraction is most applicable to heat-containing spices, the method is

gaining more popularity in the detection of adulteration, e.g., some suppliers add

cottonseed oils to ground spices to improve appearance. This practice is detectable by

methylene chloride extraction.

The Scoville Heat Test

The Scoville Heat Test is an organoleptic method for measuring pepper pungency

by determining the heat threshold of a product through organoleptic judgements of

varying dilutions of the product. In this way, number values can be assigned to various

lots of red pepper for more accurate quality grading.

Light Berries and Extraneous Matter

The objective of ASTA method is to determine the quantity of light berries, dust

chaff, pickings, and other extraneous matter present in black or white pepper. The

economics and sanitary implications of excessive extraneous matter content of pepper is

obvious. Such analyses are done first by screening small particle adulterants, then hand

picking the sifted sample for any sticks, stones, stems, foreign seeds, and other larger

pieces of extraneous matter. Formerly, there were only “in-house” standards for an

accept-reject decision. Now, there are industry acceptable standards published.

Light berries are those that float to the top of an alcohol-water solution. Their

presence in excess is an indication of overripe or infested berries since some insects eat

the inside of the berry, thereby creating a hollow, light berry.

Cleanliness

Spice cleanliness is of growing concern to spice importers. To control and ensure

the reasonable cleanliness of all unprocessed foreign and domestically-produced spices,

seeds and herbs, the American Spice Trade Association (ASTA) has initiated cleanliness

specifications which are recognized as an integral part of any import contract.

ASTA’S Cleanliness Specifications for Unprocessed Spices, Seeds and Herbs

(Foreign and Domestically Produced) list first cleanliness specifications for extraneous

matter is defined as “everything foreign to the product and includes but is not restricted

INTRODUCTION 14

to: stones, dirt, wire, string, stems, sticks, nontoxic foreign seeds, excreta, manure, and

animal contamination”. The following lists ASTA’s criteria for cleanliness of various

products. The product is considered as clean if the percentage of the product as it is

commonly known, is free from extraneous matter. A lot may not be brought into the

country unless it meets the following specifications:

Cleanliness Specifications

For purposes of these Specifications, extraneous matter is defined as everything foreign to the product

itself and includes but is not restricted to: stones, dirt, wire, string, stems, sticks, nontoxic foreign seeds,

excreta, manure, and animal contamination.

Cleanliness under these Specifications is indicated in the following table which shows the percentage of

the product, as it is commonly known, that is free from extraneous matter. A lot must be reconditioned1

TABLE-1 Spices Cleanliness Specifications

SPICES % SEEDS %

Black pepper 99.0 Cardamom seed, decor., green and

bleached

99.5

White pepper 99.0 Anise seed 99.0

Cassia 99.0 Coriander seed 98.0

Cinnamon 99.0 Cumin seed 98.0

Capsicums (chilies) 99.0 Dill seed, natural 99.0

Nutmeg, whole and broken 99.5 Dill seed, dewhiskered 99.0

Mace 99.0 Fennel seed 98.0

Ginger, peeled 99.0

Ginger, unpeeled 99.0 Herbs

Cloves 99.0

Allspice (pimento) 99.0 Sage leaves 99.0

Turmeric 99.0 Oregano leaves 99.0

Basil leaves 99.0

Seeds Laurel (bay) leaves 98.0

Thyme leaves 98.0

Celery seed 99.0 Rosemary leaves 98.0

Mustard seed 99.0 Tarragon leaves 98.0

Poppy seed 99.5 Marjoram leaves 99.0

Sesame seed, natural and hulled 99.5 Chervil leaves 99.0

Caraway seed 99.0 Savory leaves 99.0

A lot must be reconditioned if it does not meet the following specifications.

Rodent Excreta: - If more than 2 of the total number of subsamples show the presence of rodent pellets or

any 1 subsample contains more than 2 pellets.

Exception: - In the case of fennel seed, if 20% or more of the subsamples contain any excreta, the lot

must be reconditioned. (For example, if 2 subsamples of a 10-unit sample each contain any animal excreta,

the lot must be reconditioned.)

1 “Reconditioned” means the product must be recleaned to meet the specifications prior to importation.

INTRODUCTION 15

Other Excreta: - If more than 2 of the total number of these same subsamples show the presence of 50 mg

or more excreta, or and average of 10 mg or more per pound for all subdivisions of the sample.

Exception: - In the case of fennel seed, if 20% or more of the subsamples contain any animal excreta,

the lot must be reconditioned.

Insects: - If more than 2 dead insects are found in each of 2 of the total number of subsamples. In

no case shall there be more than four dead insects in the total number of these subsamples.

Exception: - In the case of fennel seed, if 20% or more of the subsamples containing any whole

insects, the lot must be reconditioned. (For example, if 2 subsamples of the 10-unit sample each contain 1

whole insect, the lot must be reconditioned.

Whenever a live insect or insects are found, the lot must be fumigated, sifted and blown. After

fumigating sifting and blowing, samples shall be drawn in accordance with sampling procedures, and

reanalyzed, and a new Certificate of Analysis issued.

Mites and Psocids: - If it appears to the unaided eye that 50 or more mites and psocids are present

and this is confirmed by the flotation test. Mites and/or psocids are not to be counted as insects.

Mold: - If mold is present in excess of 5% by weight of the samples (except nutmegs, thin quill

cassia and black and white pepper).

In the extent of mold and/or insect infestation by count in whole nutmegs is in excess of 10%.

With regard to broken and /or reconditioned nutmegs, not more than 5% by weight may show evidence of

mold and/or insect infestation. Nutmegs, whole or broken, with mold and/or insect infestation in excess of

25% may not be reconditioned.

If the extent of mold in thin quill cassia is in excess of 10 % by weight. The cassia must be

sampled and examined as received in its original imported form-it may not be altered in shape or form prior

to being analyzed.

If the mold on whole white and black pepper is in excess of 1% by weight. A pepper berry is

classified as moldy it contains any mold visible to the naked eye.

Insect Defiled: - If the total sample quantity reveals the presence of 5% or more weight or insect bored or

otherwise defiled seeds, leaves, or roots. A lot is considered defiled whenever a sample

shows visible evidence of webbing or insect feeding.

Light Berries: - Black pepper: if the light berries, though not considered extraneous matter, exceed 4% by

weight.

MICROBIOLOGICAL TESTS 16

II. MICROBIOLOGICAL TESTS

1. Preparation of culture media.

2. Determination of Total Plate Count.

3. Enumeration of Total Coliforms.

4. Enumeration of fecal coliforms.

5. Determination of Yeast.

6. Determination of Mold Count.

7. Determination of Salmonella.

8. Gram staining.

2.1 EXPERIME�T �O. 1

PREPARATIO� OF CULTURE MEDIA

ulture media routinely used in the modern food-testing laboratory may be purchased

in the form of commercially dehydrated powdered formulations. These mixtures are

capable of supporting growth of a large variety of pathogenic and saprophytic

microorganisms. The preparation of the completed laboratory medium from the dried

powdered mixtures is a relatively simple task for the laboratory technician. The

resulting media are very uniform and quite stable and can be stored under refrigeration for

approximately two weeks before use in the laboratory. All prepared culture media to be used

in the microbiology laboratory must be in the sterile state unless otherwise specified.

The label on each bottle of culture medium lists the directions for its preparation, the

recommended method of sterilization, the primary purpose for the medium, and the list of

ingredients constituting the mixture. The concentrations of ingredients are expressed in g per

liter (1000 ml). High quality distilled water should always be used to rehydrate dry culture

media. Tap water is not recommended for the preparation of culture media or reagent

solutions because of the possible presence of toxic chemical elements which are likely to

interfere with microbial activity and growth.

It is a relatively simple procedure to prepare 1000 ml of a given culture medium by

determining the quantity of powdered medium (in grams) specified in the directions for use.

Larger or smaller volumes of a given culture medium may be prepared by determining the

proportionate quantity of the following simple proportion, the amount of powder in grams

may be determined:

Example

X/Y = 24/1000ml

X = amount of powdered medium required

Y = volume of finished medium desired

24g/1000 ml = proportion indicated in directions for use

Best results are obtained by measuring the desired amount of distilled water into a

clean Pyrex glass or stainless steel container. Then add the correct amount of powdered

C


medium slowly while stirring. Stir mixture until all lumps, if any are dissolved.

Broth media (with agar-agar or gelatin) readily dissolve in distilled water at room

temperature. Generally, heating of these mixtures is not required before dispensing the

medium into suitable containers. Culture media containing agar (in any amount) require

heating to the boiling point with constant stirring to ensure solublization of the agar particles

in the mixture. Agar particles will not dissolve unless the mixture reaches an overall

temperature of 98oC or more for several minutes. Do not allow agar particles to settle to the

bottom of the container while heating. This could cause burning and charring of the agar

particles. Media containing gelatin are best dissolved by heating to 50oC(preferable in a 50

oC

waterbath). Agar and gelatin culture media must be in complete solution before being

dispensed into individual containers to be sterilized.

During the preparation of culture media, care should be taken to avoid extraneous

chemical contamination during the rehydration process. Chemically clean containers,

spatulas, and weighing pans should be used during the preparation procedure.

All culture media reaching the laboratory to be used in bacteriological investigations

must be sterile (microorganism-free). Culture media are routinely sterilized by the autoclave

method employing the conditions of 121oC for 15 minutes. Steri-tap and other sterilization

indicators may be used to determine that adequate sterilization of the materials so treated is

taking place. Containers of culture media to be sterilized should never be more than 2/3 full

and should be placed in the autoclave chamber loosely or far enough apart to allow for

adequate steam circulation and heat transfer. Prolonging the sterilizing cycle may destroy the

nutritional value of the medium. Repeated melting of agar media is a poor laboratory

practice because of the destruction of the gelling properties of the agar and the resulting

decrease in the nutritional value of the medium.

Prepared culture media should always be stored in a cool moist environment to retard

evaporation of moisture from the medium. Prolonged storage of culture media is not

recommended. Generally, only enough media should be prepared and stored as will be used

in a 2 to 3 week period. If at all possible, culture media should be prepared fresh, sterilized,

stored overnight under incubation conditions to detect possible contamination, and used the

following day.

Culture media routinely used in the food microbiology laboratory are listed in the

following sections.

1. Eosin Methylene Blue Agar

2. Lactose Broth

3. Total Plate Count Agar

4. Selinite Broth

5. Bismuth Sulphite Agar

6. Brilliant Green Bile Broth

7. Trypton

8. Mackonkey Broth


9. Standard Plate Count Agar

10. Rose Bengal Chloramphenicol Agar Base

11. Salmosyst

12. Triple Sugar Iron Agar

13. Salmonella Shigella Agar

14. XLT4

15. Sabouraud Dextrose Agar


DETERMI�ATIO� OF TOTAL PLATE COU�T

Purpose & Scope

To determine total number of organisms present per gram of sample. Applies to incoming

materials/In-process and Final products, wherever required.

Procedure

1. Apparatus being used must be cleaned and sterilized.

2. Equipment

are pipettes, petri dishes, spreader, test tubes, conical flask, cotton plug, incubator,

autoclave, water bath, burner, glass marker and match box.

3. Reagents being used are Ethanol, Nacl, Peptone, Plate Count Agar (medium).

Preparation of Diluent

4. Prepare diluent according to the following ratio.

Nacl: Peptone

0.85g: 0.1g dissolved in 100 ml distilled water, and shake well.

5. Distribute 90ml of diluent in 250ml conical flask and 09 ml in each 25ml test tube.

6. Plug it and autoclave it, at 121°C for 15min.

Preparation of Medium

7. Prepare the medium as directed in the brand according to the requirement.

8. Heat in water bath till dissolved.

9. Autoclave at 121°C for 15min.

10. Cool it to approximately 45°C.

11. Pour suitable amount of agar medium in each sterilized petri plate aseptically.

12. Allow the agar medium to solidify.

Sampling

13. Aseptically weigh 10g of given sample if the sample is not powdered then grind it.

14. Mix the sample in 90ml diluent to make 1:10 dilution and shake it thoroughly.

15. Subsequently make serial dilution (depending upon the nature of sample). 1:10,


1:100 by taking one (1) ml from previous dilution and add in 9ml sterile diluent.

16. Mark the plates and tubes, take 0.1ml of diluted sample from every dilution

separately and transfer in to three (3) different petri plates of media and spread with

sterile spreader.

17. Incubate at 35-37°C for 24 hrs in an inverted position.

18. Count the colonies developed on these plates and multiply by its dilution factor.

Total Plate Count = No. of colonies x Dilution factor x Volume factor.



E�UMERAIO� OF TOTAL COLIFORMS

Purpose

To determine the total number of Coliform present per gram of sample. Applies to Incoming

materials/In-process and Final products.

Procedure

1. Before performing this test make sure all-following apparatus must be sterilized.

2. Apparatus being used are Pipettes, Spreader, Incubator, Autoclave, Water bath, Test

tubes, burner, conical flask, glass marker, Durham tubes and Test tubes stand.

3. Reagents being used are Nacl, Peptone, MacConkey Broth/MacConkey Agar.


4. Same as EXP/2


5. Prepare the medium as directed in the brand according to the requirement. Heat in

water bath till dissolved.

6. In case of Agar follow all the steps of EXP/2. In case of broth distribute 9ml of

prepared broth in each test tube, introduce durham tubes in it and then plug it with

cotton.

7. Autoclave at 121°C for 15min.

8. Then cool at room temperature till the temperature of the medium reaches 40°C to

45°C; setup a series of 9 tubes of broth in a test tube rack.

Label the tubes properly.

Sampling

9. Aseptically weigh 10g of given sample, if the sample is solid then grind it.

10. Transfer the sample to 90ml diluent (sterilized) to 1:10 dilution.

11. Shake it thoroughly.

12. Subsequently make serial dilution 1:100, 1:1000 by taking 1 ml previous dilution and

add in 09ml sterilized diluent.

13. Take 1ml of the sample separately from every dilution and transfer into three

different test tubes set of MacConkey's broth, shake it.

14. Incubate at 37°C for 24hrs in water bath.

15. Observe the color and gas; record the result with the help of MPN table.



E�UMERATIO� OF FECAL COLOFOMS BY MP� METHOD

Purpose

To determine the total number of Fecal Coliforms present per gram of sample. Applies to

Incoming materials/In-Process and final products.

Procedure

1. Before performing this test make sure that all following apparatus must be sterilized.

2. Apparatus used are Pipettes, Petri dishes, Conical flask, Spreader, Incubator,

autoclave, water bath, test tubes, Durham tubes, glass market, test tube stand, Burner,

wire loop, match box and cotton plugs.

3. Reagents used are Nacl, Peptone, Trypton Broth, Positive tubes of MacConkey's

Broth, ethanol, Brilliant Green Bile Broth and Kovac's reagent.


4. Same as EXP/2

Preparation of Media

5. Prepare the Brilliant Green Bile Broth medium as directed in the brand according to

the requirement.

6. Boil in water bath till dissolved. Distribute 9 ml of Broth in 25ml. Test tubes and

introduce Durham's tubes and plug it.

7. Prepare 1% of Trypton Broth in distilled water.

8. Distribute 3ml of Trypton Broth in 10ml test tubes and plug the test tubes with

cotton.

9. Autoclave all tubes at 121°C for 15 min.

10. Cool at room temperature toll the temperature of the medium reaches 40oc to 45oC.

Sampling

11. Inoculate 1 wire loop from each positive tube of MacConkey's Broth into Brilliant

Bile Broth and Trypton Broth.

12. Incubate at 45°C ± 0.55 for 24 hours in water bath.

13. After incubation transfer 1-2 drops of Kovac's reagent into the Trypton Broth and

observe the formation of red ring at the top of the medium and color and gas of

Brilliant Green Bile Broth observe and record result with the help of MPN table.



DETERMI�ATIO� OF YEAST COU�T

Purpose

To determine the number of yeast cells present per gram of sample. Applies to Incoming


Procedure

1. Make sure that all equipment is clean and sterilized.

2. Apparatus used are Pipettes, Petri plates, glass marker, conical flak, spreader,

Incubator, autoclave, water bath, test tubes.

3. Reagents used are Nacl, Peptone, Reagents of Gram's staining, Sabouraud dextrose

agar.


4. Same as EXP/2.


5. Same as EXP/2.

Sampling

Step 1-4 same as EXP/2.

6. Incubate at room temperature for 48 hours.

7. Count the colonies developed on these plates and multiply by its dilution factor and

Confirm suspect yeast by Gram's stain.



DETERMI�ATIO� OF MOLD COU�T

Purpose

To determine the number of mold cells present per gram of sample. Applies to Incoming


Procedure

1. Make sure that all equipment is clean and sterilized.

2. Apparatus used are Pipettes, Petri plates, glass marker, conical flask, spreader,

Incubator, autoclave, water bath, test tubes.

3. Reagents used are Nacl, Peptone, Rose Bengal Chloramphenicol agar Base,

Chloramphenicol vial as directed in brand.


4. Same as EXP/2.


5. Same as EXP/2. One step is different as after cooling add Chloramphenicol vial as

directed on brand.

Sampling

6. Step 1-4 same as EXP/2.

7. Incubate at room temperature for 48 hours.

8. Same as EXP/2.



DETERMI�ATIO� OF SALMO�ELLA

Purpose

To determine the number of salmonella organisms present per gram of sample. Applies to

Incoming materials/In-Process and Final products.

Procedure

1. Make sure that the apparatus be cleaned and sterilized.

2. Apparatus used are Pipettes, Petri dishes, Spreader, glass marker, Incubator,

autoclave, water bath, cotton plugs, wire loop, match box, burner and test tubes.

3. Reagents used are Nacl, Peptone, Ethanol and media used Lactose broth, Bismuth

Sulphite agar, Triple sugar iron agar, Selinite enrichment broth.

Pre-Enrichment

4. Dissolve 1.3gm of lactose broth in 100ml of distilled water or 2.925 gm in 225 ml

distilled water.

5. Autoclave at 121°C for 15 min.

6. Cool at room temperature at 40 to 45°C then aseptically transfer 25 gm of sample in

prepared lactose broth. Incubate at 37°C for 24 hours in water bath.

Enrichment

7. Prepare the Selinite enrichment broth in 50ml of distilled water as directed in the

brand.

8. Boil to dissolve don't autoclave.

9. Then add 50ml of Pre-enrichment broth (LACTOSE BROTH) into

ENRICHMENT BROTH to make it double strength.

10. Incubate to 43-44°C for 24 hours in water bath.

Isolation by Selective media

11. Prepare the Bismuth Sulphite agar as directed in the brand according to requirement,

boil to dissolve in a water bath for 1-2 min avoid over heating, don't autoclave.

12. Pour into sterile petri dishes. Let all the plates solidify.

13. Inoculation is done by two different procedures.

14. Spreading on Bismuth Sulphite agar.

15. Streaking on Bismuth Sulphite agar.

16. Inoculate one wire loop of Selinite broth and streak on the bismuth Sulphite agar, or

Transfer 0.1ml of Selinite enrichment broth in bismuth Sulphite agar and spread it

with the spreader.

17. Incubate the plates at 37°C for 24hours.

18. Typical colonies of Salmonella may appear as Brown, Gray, Black, Green,


surrounded by Metallic sheen.

Identification of different Salmonella species

19. Prepare the Triple sugar iron agar as directed in the brand according to requirement.

20. Heat in water bath till dissolved.

21. Take 4-5ml of prepared TSIA in test tubes and sterile in an autoclave at 121°C for 15

min.

22. After sterilization allow the media to solidify so as to obtain a deep layer of BUTT &

SLANT, both about 4-5cm long.

23. Stab the TSIA BUTT with the culture from BSA, using a sterile inoculating needle.

24. Streak the same culture on TSIA slant.

25. Incubate the tubes at 37°C for 24 hours.

26. Examine the tubes.

TABLE-2 OBSERVATIO�S

SALMONELLA SPECIES SLANT BUTT GAS H2S

S.typhi Alkaline Acidic - +

S.typhi.P.A. Alkaline Acidic + -

S.typhi.P.B. Alkaline Acidic + +++



GRAM STAI�I�G

Purpose

Staining to determine the gram + or gram - organisms.

Procedure

1. Apparatus used are slides, burner, needle, dropper, microscope.

2. Reagents used are Crystal violet, Iodine, Alcohol, Saffranin.

3. Make smear.

4. Dry and heat fix the slide.

5. Pour crystal violet for 1-2min.

6. Wash with water and pour Iodine for 1min.

7. Wash with Alcohol through dropper.

8. Pour Saffranin and wash with water.

Gram + will show Purple color under microscope.

Gram - will show Pink color under microscope.

STA�DARDS

TABLE-3 STA�DARDS FOR MICROBIOLOGICAL TESTS

S.NO PRODUCTS Total Plate Count Total Coliform Mold/Yeast

1. Wet product

Target value

(Ketchup, Pickle,

Sauces, Pastes, chilly)

5000

< 1000

< 11

< 3

100

< 50

2. Pappadom < 5000 < 3 100

3. Spices 1000,000 10,000 10,000

E.Coli & Salmonella

absent

4. Past (Export) < 5000 < 3 100

5. Sauces < 5000 < 3 50

6. Chutney < 3000 < 3 < 100

CHEMICAL / PHYSICAL TESTS 27

III. CHEMICAL / PHYSICAL TESTS


DETERMI�ATIO� OF pH/TOTAL SOLUBLE SOLIDS/BOSTWICK VALUE

Purpose

To determine the pH of sample.

Procedure

1. Apparatus used are pH meter, Standard buffer solutions of pH 4, pH 7

pH Meter

2. Standardize the pH meter with pH 4.0 and 7.0 buffers.

3. Pour the sample (juice) in 50ml beaker and insert the electrode or electrodes to

determine the pH.

4. For Semi-solid foods take 10-15% of sample and dilute in 50ml distilled water.

5. Note the reading after 20-25 seconds, wash the electrodes with distilled water and

wipe with a small piece of cotton soaked in distilled water.

6. Record the reading.

7. Dry with a piece of filter paper and continue with the next determination.

pH Paper

1. Wet about 0.5mm length of the pH paper with the sample solution.

2. Note the color.

3. Record the pH from the chart provided with the pH paper.

DETERMI�AIO� OF SOLUBLE SOLIDS

Purpose

To determine the total soluble solids with the help of hand refractometer.

Procedure

1. Take representative sample of a well-mixed portion of sugar syrup, squash, jam or

jelly from seed and fiber.

2. Place a drop on absolutely dry refractometer prism.

3. Close the lid and read directly at 20°C.

4. If the correction is necessary, use the correction factor.


DETERMI�ATIO� OF BOSTWICK VALUE

Purpose

To determine the viscosity of sample.

Procedure

1. Place the Bostwick instrument on a smooth surface and balance it (Make sure that

the Bostwick instrument is dry).

2. Fill the chamber of Bostwick instrument uniformly.

3. Start the stopwatch with releasing the clip.

4. Note the reading after 10 seconds.



DETERMI�ATIO� OF %PURITY OF ACETIC ACID

Purpose

To determine the % purity of Acetic Acid.

Procedure

1. Apparatus used are Pipettes, Burette, Conical flasks, beakers.

2. Reagents are Acetic Acid (Sample and Standard), NaOH 0.25M, Phenolpthaline.

Preparation of 0.25M �aOH solution

3. 2.5g of NaOH pellets were dissolved in 250ml of distilled water and thoroughly

mixed.

Preparation of 0.5% Acetic Acid solution

4. 0.5ml Acetic Acid was taken by 1ml pipette and dissolved in 100ml distilled water in

volumetric flask.

Titration

5. Fill burette with 0.25M NaOH solution.

6. Noted initial reading.

7. Took 10ml of Acetic Acid in a conical flask.

8. Add 2-3 drops of indicator Phenolpthaline.

9. Titrate against 0.25M NaOH solution.

10. End point was appearance of pink color.

11. Note the reading.

12. Take 3 consecutive readings.

13. Calculate the difference as percent purity to the standard Acetic Acid.

Percent Purity = Sample reading / Standard reading x 100


TABLE-4

S.NO SAMPLE NO. PERCENTAGE PURITY

1 STANDARD 100

2 PICKLE 94.26

3 ONE 92.65

4 TWO 100

5 THREE 95.7

6 FOUR 94.26

7 FIVE 97.14

8 SIX 98.5

9 SEVEN 91.8

10 EIGHT 94.26

11 NINE -

12 TEN 97.1



DETERMI�ATIO� OF SALT PURITY

Purpose

To determine the percent purity of salt sample.

Procedure

1. Apparatus used are Pipettes, Burette, Conical flasks, beakers.

2. Chemicals: AgNO3 (0.05M)

Potassium Chromate (Indicator)

Standard Salt Solution (0.10M)

Preparation of 0.10M salt solution

3. Dissolve 0.585g salt in 100ml-distillled water.

Nacl (23 + 35.5) = 58.5g/1000ml

4. To prepare 0.1M or 1/10 M in 100ml dissolve 0.585/100ml.

Titration

5. Take 10ml of (0.10%) salt solution (standard and sample) separately in titration flask

6. Add 2-3 drops of Phenolpthaline indicator.

7. Titrate against AgNO3 (0.05M).

8. end point is the appearance of brown ppt.

9. Reaction

AgNO3 + Nacl NaNO3 + Agcl (Brown ppt.)


S.NO SAMPLE INITIAL

READING

FINAL

READING

DIFFERENCE %PURITY

1 Standard 13.8ml 33.1ml 19.3ml 100%

0 19.3ml 19.3ml

2. Sample-1 0 18.8ml 18.8ml

18.8ml 37.7ml 18.9ml

Mean 18.5ml 97.18%



DETERMI�ATIO� OF THE CO�CE�TRATIO� OF FORTIFIED IODI�E I�

IODIZED SALT

Purpose

To determine the concentration of fortified Iodine in Iodized salt sample.

Chemicals

1. Starch Indicator (Freshly Prepared)

0.5g/50ml: 0.5g/50ml

Starch: Standard salt to get 100ml of starch indicator.

2. H2SO4 (2M)

3. Potassium Iodide (KI) 10% solution.

4. Sodium Thiosulphate.

Procedure

1. Take 40-50g sample after quartering.

2. 50g salt was grinded to obtain powder form.

3. 10g sample of powdered form was dissolved in 50ml distilled water in conical flask.

4. 2ml H2SO4 (2M) was added. (To liberate iodine).

5. Add 5ml KI (10%) sol. producing yellow color of solution and immediately capped.

6. Place in dark place for 15 minutes.

7. Titrate the sample solution against Sodium thiosulphate.

8. After 5-10 drops titration add starch indicator.

9. End point is the colorless solution.

10. Match with standard titration values from table.


S.NO Initial Reading Final Reading Difference

Sample-1 9.5ml 16.5ml 7.00ml

16.5ml 23.5ml 7.00ml

23.5ml 30.5ml 7.00ml

Mean 7.00ml

The table value showed the concentration to be 74.1ppm/g.



DETERMI�AITO� OF EXTRA�EOUS MATTER/I�SECT FRAGME�T TEST

Purpose

To determine the insect fragments in material.

Procedure

1. Weigh 25g (Chili Powder) sample.

2. Dissolve 400-500ml tap water in 1000ml beaker.

3. Add 5ml of concentrated Hcl.

4. Boil for 45min to 1 hrs.

5. Add 30-40 ml Mineral oil.

6. Stir for 20-25min.

7. Pour the sol. in to Buchner funnel.

8. Wash till it becomes clear.

9. Filter through filter paper.

10. Examine under microscope.

Observation

Insect matter will break into two pieces.



DETERMI�ATIO� OF RA�CIDITY / ACID VALUE OF OIL

Purpose

To determine the Rancidity/Acid value in a sample of oil.

Applies to incoming materials/In-process and Final products.

Procedure

1. Apparatus/Chemicals required, two conical flasks 300ml, Electronic balance of

accuracy 0.1mg, Burette, Beaker, Ethanol, NaOH (0.05N), Oxalic acid (0.05) &

Phenolphthalein.

2. Make sure that all apparatus is present in clean form.

3. Weigh 1gm of oil in on conical flask and left the other empty.

4. Add 15ml of ethanol in both the flasks.

5. Left both flasks for 15min.

6. In the mean time standardize NaOH with oxalic acid (C2H2O4) 0.05N.

7. After 15min add 2 drops of Phenolphthalein indicator in both the flasks.

8. Titrate against standardized NaOH.

9. From difference in value calculate the acid value in the given oil.

CALCULATIO�

i. Weight of Oil = 1g.

ii. Volume of NaOH consumed for blank flask = a ml.

iii. Volume of NaOH consumed for sample flask = b ml.

iv. Difference in volume = b-a = c ml.

v. Total no. of gram of Alkali required for 1g oil

= [Normality of NaOH x 40 x c (ml)]/1000

= X g.

1g of oil sample has = Xg of free acid.

100g of sample have X * 100 = Yg free fatty acid.

Free acid value or rancidity of oil sample = Yg.



DETERMI�ATIO� OF FREE ACIDITY

Purpose

To determine the amount of free Acid in a sample. Applies to Incoming materials/In-process

and final products.

Procedure

1. Apparatus/Chemicals/other requirements, 100ml volumetric flask, conical flask

250ml, Beaker, Pipettes, glass rods and Burette, (0.25M) NaOH & Phenolpthaline

indicator.

2. Weigh 2-3g of sample and dissolve in 100ml (approx.) water in a beaker. (If sample

have oil first separate oil and blend in blender then dissolve in distilled water).

3. Do the experiment in triplets.

4. Heat the beaker on low flame for 1-2min (approx.). Filter (not necessary for all

samples). Collect the filtrate in a conical flask.

5. Fill the burette with 0.25M standardized NaOH solution.

6. Add 2-3 drops of phenolphthalein indicator in the flask and titrate against NaOH till

light pink color appears.

7. Note the reading and calculate the value of free acid as acetic or citric acid.

CALCULATIO�S

1. Weight of Sample = Xg (5g).

2. Molarity of NaOH = 0.25M.

3. Volume of NaOH (Consumed against sample solution) = a ml.

4. Number of moles of NaOH = (0.25 x a) / 1000

= b ml.

5. Percentage as Acetic Acid = b/x x 100

= c ml.

Weight of Acetic Acid in 100g of sample = c x 60

= e %.

6. Percentage as Citric Acid = b / 3 x 100 / x

= Y moles.

Weight of Citric Acid in 100g of sample = Y x 192

= Z%.



S.NO Initial Reading Final Reading Difference

1. 30.8ml 44.9ml 14.1ml

2. 10.3ml 23.3ml 13ml

3. 22.4ml 36.6ml 14.2ml

4. 36.6ml 50.2ml 13.6ml

Mean 13.725ml

Volume of NaOH = 13.725ml.

No.of moles of NaOH = (0.25 x 13.725) / 1000

= 0.00343 moles. (b).

Acetic Acid = b/x x 100 = C.

= 0.00343 / 5 x 100

= 0.0686.

Weight of Acetic Acid in 100g= c x 60

= e%.

= 0.0686 x 60

= 4.1175 %.



DETERMI�I�G MOISTURE

Purpose

To determine the moisture content of food.

Procedure

1. Weigh an empty flat-bottomed dish.

2. Place the sample in the weighed dish.

3. Weigh the dish with the sample.

4. Place the dish in an oven at 100-102°C or vacuum oven at 70°C.

5. Remove the dish after 3hrs, cool in a desiccator and weigh.

CALCULATIO�S

1. Weight of empty Petri dish (P) = 46.5526g

2. Weight of sample (A) = 2.0026g

3. Weight of dish + sample (Balance) = 48.5563

Kept for 3 hrs in oven at 102°C.

4. Weight of dish + (Dried sample) B = 48.3612g

5. Weight of Dried sample (B) = 4 - 1

= 48.3612 - 46.5526

= 1.8076g

6. Moisture loss = A - B

= 2.0026 - 1.8086

= 0.194

% Moisture = [Wt. loss (g)/Initial sample weight] / 100

= 0.194 / 2.0026 x 100

= 9.68 %.

% moisture standard up to 12 % moisture for grinding.


3.9 & 3.10EXPERIME�T �O. 9 & 10

TOTAL ASH / ACID I�SOLUBLE ASH

CALCULATIO�S

1. Weight of empty crucible = 115.5413g.

2. Weight of sample = 3.0012.

3. Weight of crucible + sample (A) = 1805421g.

Kept for 2 hrs in furnace at 500-600°C.

4. Weight of Ash + Crucible (B) = 15.7092.

5. Weight of Ash = B - (1)

= 15.7092 - 1505413

= 0.1679.

%Ash = [Weight of Ash (g)] / Weight of sample x 100

= 5.59%.

Ash standard = 5-9%.

Ash dissolved in 2% Hcl solution and heated for 10min on a weighed crucible, filtered and

the filter (neutral) from filter paper discarded while filter paper (ashless) with acid insoluble

ash is transferred to a weighed crucible and kept in furnace for 1 hr at 500-600°C.

6. Weight of empty crucible = 16.5412g

7. Weight of crucible + Ash =16.5419g

= 00.0007g

8. % Acid Insoluble Ash = Weight of acid insoluble ash (g) x 100

Weight of sample (g)

= [0.0007/3.0012] / x 100

= 0.0233%.



PU�GE�CY TEST

Purpose

To determine the pungency in chilies.

Procedure

Apparatus

i. Erlenmeyer flask, narrow neck, 125ml.

ii. Pipette, 1ml, 0.01ml graduations.

iii. Pipettes, volumetric, 2ml and 5ml.

iv. Volumetric flasks, stopered, 50ml and 100ml.

v. Funnel.

vi. Filter paper, Whatman no.1.

Reagents

i. Ethanol.

ii. 5% sucrose in tap water.

Procedure

1. Make an alcoholic extract of the sample according to schedule A based on

anticipated pungency. Chilies should be extracted for a period of 16 hours. Shake

occasionally.

2. Decant or filter to get a clear extract.

3. For the material to be tested, dilute the quantity indicated in schedule B to 50ml with

5% sucrose solution using pipettes and volumetric flask. In any given test start with

an amount of alcoholic extract considered to be too small so that negative response

will be obtained and increase the amount using the schedule B until 3 out of 5 tasters

report positive results. Record individual response to each dilution.

4. Before the first tasting and between each tasting have the individual sip or rinse their

mouth with water at 90-100 oF.

5. For tasting, 5ml of the solutions prepared as indicated in step 3 are to be swallowed

one at a time from small cups. The judgement as to whether or not heat is present is

to be made between 20-30 seconds. After swallowing. The minimum interval

between tasting the solutions should be 5min.

6. Tasters are to continue through the sequence of solutions until each reports a burn

sensation. Report the heat units for the first solution for which 3 out of 5 panelists

report positive using values found in schedule B.

Note: For schedule A & B please see appendix 1.


APPE�DIX-1

PU�GE�CY OF CHILLIES:

SCHEDULE A

Make an extract according to anticipated pungency. Schedule B indicates range in Scoville

units for each of the alcohol extracts indicated below.

A1 10g + 50ml ethanol

B1 5.0g + 50ml ethanol

C1 2.0g + 50ml ethanol

D1 1.0g + 50ml ethanol

A 0.5g + 50ml ethanol

B 0.25g + 50ml ethanol

C 0.1g + 50mlethanol

D 0.05g + 50mlethanol

E 5.0ml of D made to 50ml.

F 5.0ml of D made to 100ml.

TABLE-8 For Chili B ml is selected

M Units A ml B ml C ml D ml

100 0.25 0.50

95 0.26 0.53

90 0.28 0.56

85 0.29 0.59

80 0.31 0.63

75 0.33 0.67

70 0.35 0.72

65 0.38 0.77

60 0.42 0.83

55 0.45 0.91

50 0.50 1.00

45 0.555

40 0.63

37 0.68


M Units A ml B ml C ml D ml

34 0.74

31 0.80

28 0.89

26 0.38 0.96

25 0.40 1.00

24 0.42

22 0.46*

20 0.50

18 0.56

16 0.63

14 0.72

12 0.83

10 0.50 1.00

9.5 0.53 1.11

9.0 0.56 1.25

8.5 0.59

8.0 0.63

7.5 0.67

7.0 0.72

6.5 0.77

6.0 0.83

5.5 0.91

* Pungency noted.



CHROMATOGRAPHY TECH�IQUE

Theory of chromatography

Chromatographic separations are dependent upon two major processes namely:

i) Multiple partition ii) Adsorption process.

Paper used in Chromatography

The majority of paper chromatography has been carried out on standard filter paper.

Paper is a ransom pile of cellulose fibers.

The paper is to act as a support for the stationary phase. In general, Whatman No 1 or

Whatman No 3 filter papers are used for analytical work and Whatman No 3 or Whatman

3MM for preparative work.

TABLE-9 CHRACTERISTICS OF WHATMA� CHROMATOGRAPHY

PAPERS

Rate of flow

Fast Medium Slow

Thin Papers No.4

No.54

No.540

No.7

No.1

No.2

No.20

Thick Papers No.31

No.17

No.3

No.3 MM

Purpose

To determine the qualitative composition of sample. Separation of inks by ascending and

descending chromatography.

Procedure

Inks required:

i Blue-black

ii Black

iii Green

iv Red

v Brown

vi Royal blue


Solvent:

n-Butyl alcohol 3 volumes, ethyl alcohol 1 volume, 2N-ammonia 1 volume.

1. Make the sample solutions and clearly define them.

2. Mark the filter paper used in paper chromatography as for samples. Draw a straight

line to represent the base.

3. Apply the samples through micropipette as dots on marked points.

4. Dry the doted points.

5. Put one end of the paper in solvent trough.

6. Wait for the development.

7. Measure the front and calculate the Rf (Relative front) value.



DETERMI�ATIO� OF PARTICLE SIZE

Purpose

To determine the particle size of a sample.

Procedure

1. Take 50g of the sample.

2. Pass through the required size of mesh sieve.

3. Collect the passed material or the remaining material.

4. Weigh the passed and remaining material.

5. Multiply by x 2. To find in 100g.

6. The passed (powder) or the remaining (big size) should not be more than 10%.



DETERMI�ATIO� OF AFLATOXI� I� CHILLI

Purpose

To determine the aflatoxin in the sample.

Procedure

1. Apparatus/Chemicals required are Viscom Aflatoxin apparatus, Methanol sol.

(80%), Fluted filter paper, Microbial filter paper, Aflatoxin column, Aflatoxin

developer.

2. Grind (whole) raw chili to powdered form.

3. Take 50g sample.

4. Weigh 5g Nacl table salt fortified and mix with above sample.

5. Blend (chili + salt) mixture with 200ml of 80% methanol solution for 1min.

6. Filter through fluted paper.

7. Take 10ml of filtrate and dilute it by adding 40ml distilled water making up to the

mark up of 50ml.

8. Again filter through special microbial filter paper.

9. Take 10ml of filtrate.

10. Attach aflatoxin column to syringe.

11. Add 10ml of filtrate through syringe and wash.

12. Wash 2 times with distilled water and discard the washings.

13. Wash with 1ml methanol (80%) and collect washing in a test tube.

14. Add 1ml of Aflatoxin developer.

15. Shake vigorously.

16. Calibrate Viscom Aflatoxin apparatus.

17. Warm up for 30 seconds.

18. Insert test tube.

19. Note the reading.

READING: 82 PPB.

The result is significant as compared to the standard of 20PPB.



DETERMI�ATIO� OF DE�SITY

Purpose

To determine the density of solid samples.

Procedure

1. Apparatus used are graduated cylinder, electronic balance.

2. Fill the specified volume of graduated cylinder with the sample.

3. Weigh the sample.

4. Calculate the density as

Density = Mass/Volume

RESEARCH AND DEVELOPMENT PROJECT 47

IV. RESEARCH A�D DEVELOPME�T PROJECT

4.1 PREPARTIO� OF LOW BRI�E SALT MEDIUM

Low Salt Brine Medium was prepared by mixing the following formulation.

TABLE-10 FORMULATIO�

S.No Ingredient Percentage By weight or volume

1 Refined Salt @ 3% 150g / 5l

2 Lactic acid @ 1% 50ml

3 Citric acid @ 0.3% 15g

4 Acetic acid @ 0.2% 10g

5 Cacl2 @ 0.1% 5g

6 Potassium metabisulphite @ 0.1% 5g

7 EDTA @ 50ppm 0.25g

8 Potassium Sorbate @ 0.1% 5g

9 Sodium acid pyrophosphate @ 0.2% 10g

Total 250.25g / 5l

4.2 TREATME�TS

Vegetables used for the preparation of pickle are conventionally stored in silos of

salt, a layer of salt is dispersed in the silos made of concrete wall enameled with suitable type

of enamel to prevent the concrete to contaminate the vegetables. On the bed of salt vegetable

is spreaded again it is covered with salt. This is done repeatedly up to the top of silos.

A lot of salt is used and wasted during this process, salt once used for one product is

not used for another time. It was felt to develop a Brine medium to preserve the vegetables

for pickling for a long time but using low salt concentration and some preservatives.

The first formulation involves a number of preservatives which will be reduced

gradually in amount and number. Following vegetables were selected to be kept in this low

salt brine medium.

Cucumbers, Tomatoes, Green Chili and Papaya.


The treatments were as follows:

TABLE-11 TREATME�TS

S.No Vegetables Treatments

T1 T2 Packing (Plastic bags)

1 Cucumber Unblanched and

Sliced

Blanched and Sliced 250g kept in 500ml

sol.

2 Tomato Blanched and

Whole


sol.

3 Green Chili Blanched and

Whole


sol.

4 Papaya Blanched and

Diced

Blanched/Diced

/Flavored

250g kept in 500ml

sol.

4.3 OBSERVATIO�S

The treatments were sensory evaluated for Color, Taste and Smell. The pH change

was also recorded for 2 weeks.

TABLE-12 CHA�GE I� COLOR, TASTE A�D SMELL AFTER 2 WEEKS

S. No Vegetable Treatment Color Taste Texture Odor/Smell

1 Cucumber T1 Transparent Salty Soft Smelled

bad

T2 Transparent Salty Soft -

2 Tomato T1 White Bright Salty Soft -

T2 Bright Salty Very Soft -

3 Green Chili T1 Darkened/

Blacked

Salty Soft -

T2 Darkened/

Blacked

Salty Soft -

4 Papaya

T1 Bright Salty Same as

placed

-

T2 Bright Salty Same as

placed

-


TABLE-13 VEGETABLES pH I� 2 WEEKS / BRI�E SOLUTIO� PREPARED

O� 14-9-2000 WAS AT pH 2.96

Date Cucumber Tomato Green Chili Papaya

T1 T2 T1 T2 T1 T2 T1 T2 T3

15-9-2000 3.27 3.22 2.98 3.21 2.98 3.42 3.20 3.19 3.22

16-9-2000 3.29 3.24 3.01 3.22 3.08 3.46 3.24 3.22 3.25

18-9-2000 3.20 3.18 3.00 3.15 3.08 3.39 3.15 3.14 3.16

19-9-2000 3.21 3.18 3.03 3.15 3.12 3.41 3.14 3.14 3.16

20-9-2000 3.28 3.26 3.14 3.23 3.18 3.45 3.18 3.18 3.23

21-9-2000 3.28 3.26 3.15 3.23 3.21 3.46 3.19 3.19 3.26

22-9-2000 3.19 3.16 3.06 3.15 3.14 3.38 3.13 3.13 3.16

23-9-2000 3.23 3.20 3.12 3.18 3.22 3.43 3.14 3.15 3.17

25-9-2000 3.19 3.17 3.11 3.15 3.21 3.38 3.11 3.11 3.14

VISITS TO PLANTS / SALT LAKE 50

V. VISITS TO PLA�TS

5.1 SALT PLA�T

Flow Charts (Unit Operations)

1.RAW MATERIAL (Unloaded, checked for quality)

2.ROLLER CRUSHER (Grinding, Washing with brine)

3.Cyclone (Washing, Settling)

4.Centifuged (Separate brine)

5.Screw Conveyor

6.Rotary Drier (Temp above 250°C for 1-2 minutes)

7.Sieving (Through standard mesh size)

2-Sieves to separate undesired larger and powdered form.

8.Packing (Bulk)

9.Mixer (Iodine mixed to 76ppm and silicone dioxide kept)

10.Packing (Consumer size and final)

11.Sealing.


5.2 Paste Plant

1.Bottle Washing (With bleach water and plain water)

2.Boiler (Supply steam from soft water have feed water tank)

3.Cold Mixer (To mix cold ingredients)

4.Three (3) Cooking Vessels

(JAMS/JELLIES)(INTERMEDIATE)(HOT & SPICY)

5.Filling Machine

6.Capping

7.Sealing

8.Labelling

9.Packing

5.3 Packing Plant

1.Raw Material

2.Weighing (According to recipe)

3.Primary Packing

4.Secondary Packing


5.4 Pickle Plant

1.Recieving

2.Cutting

3.Brinning

4.Cutting

5.Washing

6.Sorting

7.Weighing (37 Kg)

8.Cooking (15-20 min)

9.Maturation (24 hrs)

10.Filling (Manually)

11.Packaging

12.Packing/Sealing


5.5 Dehydration Plant

1.Receiving

2.Shredding

3.Filling of trays

4.Loading trays to Tunnel Drier (Specific Time & Temperature)

5.Changing trays to opposite tunnels

6.Loading trays to Cabinet Drier (For finishing specific time & temperature)

7.Packing


5.6 Grinding Plant

1.Receiving

2.Elevator (To elevate by aspirator)

3.Sifter (With 20 & 6 number mesh screens)

4.Picking Conveyor

5.Crusher

6.Hammer

7.Seiving (Great Western model 34 mesh 12 sieves)

8.Screening (Rotex Screener 16 mesh)


Visit to Salt Lake, Khipro, Mir Purkhas, Sindh, Pakistan.

Author with National Foods Laboratory staff examining Chili carpeted for sun drying,

Khipro.

Author standing in Salt Lake, Khipro.

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