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FoodAnlaysis.
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Beatriz13XO
ANÁLISIS BROMATOLÓGICO
3º Grado en Ciencia y Tecnología de los Alimentos
Facultad de Veterinaria
Universidad de Córdoba
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Food Analysis Beatriz Notario Ríos
Part 0: Quality management systems in

analytical laboratories
Contextualization of quality
It is the standard of something as measured against other things of a similar kind, or the
degree of excellence of something. It is “an improvement in product quality”.
• It is the general excellence of standard or level.
• It is a superior standard that can provide higher acceptance from the consumers
compared to other similar products.
Definition of quality
The quality of a product or service is the perception that the customer has of it, it is a
mental fixation of the consumer who assumes conformity with that product or service
and the ability of the same to meet their needs.
Analytical quality assurance

A laboratory quality control is a mechanism designed to detect, reduce, and correct
potential internal analytical deficiencies before issuing a result.
• Its purpose is to increase the quality and reliability of the reported results.
In an analytical quality assurance there are 4 parts:

• Quality manual: laboratory activities or processes are written here.
• Processes: these are the activities that the laboratory performs.
• Procedures: it is a synonym for “protocol”. It is how analytical techniques are
done.
• Reports: results are written in this document. It is also known as the “analytical
bulletin”.
The main supporting documents of a quality management system are the Quality
Manual and Standard Operating Procedures (SOP). The latter have been prepared by
the lab staff responsible for the different knowledge areas in which the lab is structured.
Quality management procedures: these documents describe the methodology for

carrying out activities related to the quality management system itself.
• They fulfill, among others, the general requirements of the UNE-EN ISO 17025:
2005 standard.
Ciencia y Tecnología de los Alimentos 1
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ISO 10725 standard

ISO/IEC 17025:2005 specifies the general requirements for the competence to carry out
tests and/or calibrations, including sampling. It covers testing and calibration performed
using standard methods, non-standard methods, and laboratory-developed methods.
If testing and calibration laboratories comply with the requirements of this International
Standard, they will operate a quality management system for their testing and
calibration activities that also meets the principles of ISO 9001.
Moreover, this International Standard covers technical competence requirements that

are not covered by ISO 9001.
Likewise, according to Regulation (EC) No. 2004/882/CE*, results coming from

accredited labs (those having the EN ISO/IEC 17025 standard) will be exclusively
considered as valid.
Its characteristics are:

• It’s voluntary.
• It’s transitory: it has to be renovated every year to know that they follow
correctly the standards.
• It’s partial/expandable: you are accredited for just one technique. However, you
can expand the number of techniques accredited.
• It has economical cost: around 6.000-10.000 euros for each accreditation.
• These rules are applied everywhere, no matter the country, which means that
they do not need to be accredited in the other countries as well.
The steps that you have to follow are:

• Plan:
o Disseminate the ISO 17025 standard.
o Diagnosis of the current system.
o Detailed implementation planning.
o Set quality policies and objectives.
o Write down the QM, procedures and reports.
o Select those methods to be accredited.
• Do:
o Implementation of the QMS.
o Set lab facilities.
o Use and validation of lab documents.
o Environmental controls.
o Calibration of equipment and apparatus.
o Purchase of CRMs.
o Validation of assays. Uncertainty calculations.
o Interlab comparison trials.
o Training of the internal audit staff.
• Verify:
o Perform internal audits.
o Review of the applied system.

• Act:
o Corrective and preventive measures.
o Improvement measures.
o External accreditation audit.
Accreditation
Accreditation is the international tool used to generate confidence in the correct
performance of a very specific type of activity called Conformity Assessment Activities,
which include testing, calibration, inspection, certification and verification, among
others.
ENAC evaluates the compliance of established requirements in the following standards:

• Testing laboratories UNE-EN ISO/IEC 17025. These labs perform analytical
techniques.
• Calibration laboratories UNE-EN ISO/IEC 17025. These labs calibrate

equipments.
ENAC accreditations are recognized in more than 50 countries as ENAC is a signatory of

the Mutual Recognition Agreements established internationally among accreditation
bodies worldwide (www.ilac.org).
• These agreements include virtually all of the EU, USA, Canada, Japan, China,
Australia, among other countries.
• The ILAC is the organism that coordinates accreditation entities of every country.
Unlike certification (ISO 9001), which is the confirmation that an organization has
established a quality management system according to certain requirements,
accreditation confirms the technical competence (provides trust to the consumer) and
guarantees the reliability of the results generated in the laboratory.
In addition, ENAC and the international accreditation organizations produce specific

documents where clarify the purpose of different standards and, in some cases,
individualize them to specific sectors.
The ISO, UNE and EN standards are private documents that are subject to the laws of
intellectual property protection, so they must be purchased from the owner. The
Spanish Standardization Association (UNE), the body responsible for standardization at
the national level.
Benefits of accreditation
For accredited organizations:
• Recognition.
• Competitive advantage.
• Access to markets.
• Ongoing improvement,
• Facilitates access to public procurement (where public companies compete).
For the administration:

• Cost reduction.
• Supervision of the compliance control system.
• Allows the Administration to participate, to the extent of their needs, in the

assessment and monitoring processes.
For the market:

• Cost and time reductions.
• Minimization of risks.
• Increase of confidence.
• Enables the selection of reliable providers.
• Protection from possible errors.
Validation of analytical methods

Validation is the process of determining the performance characteristics of a
method/procedure or process. It is a prerequisite for judgement of the suitability of
produced analytical data for the intended use.
• It is a comparison of your method to an established one.
This implies that a method may be valid in one situation and invalid in another.
Consequently, the requirements for data may, or rather must, decide which method is
to be used. When this is ill-considered, the analysis can be unnecessarily accurate (and
expensive), inadequate if the method is less accurate than required, or useless if the
accuracy is unknown.
• In-house: methods created in the lab that need to be validated.
The steps to do a validation are:

• Establishing a validation planning.
• Performance of validation parameters.
• Evaluation of validation results.
• Validation report.
The types of validations are:

• Verification: for a standard method.
• Complete validation:
o For a modified standard method.
o For an in-house method.
The person responsible for the validation, according to the characteristics of the
analytical method, applies the following validation concepts/parameters:
• Accuracy.
• Detection limit of the analytical method.
• Quantification limit of the analytical method.
• Linearity of the working range.
• Recovery percentage.
• Precision: repeatability and reproducibility.
• Robustness.
• Sensibility.
Horwitz trumpet
This means that, if we were going to measure samples in small

concentrations/measures, the possibility of an error is expanded, because we have to
be more accurate. Size matters.
Validation parameters
Quantification limit (of the method): Minimum analyte concentration that can be
detected following the procedure of the analytical method with an acceptable level of
confidence to affirm that the target concentration is greater than the blank.
Detection limit (of the method): Minimum analyte concentration that can be
determined with an acceptable level of accuracy and precision. It is established using an
appropriate sample or reference material.
Normally corresponds to the lower point of the calibration curve (excluding blanks). It
should not be determined by extrapolation.
Linearity
Linearity is the ability of the analytical method to produce results directly proportional
to the concentration or amount of analyte in a defined acceptance range.
To estimate the goodness of fit of the experimental data to a linear regression line, the
correlation coefficient (p) is calculated. The value of r is given by:
The best indicator of the linear model, replacing r is the tr-value with n-2 degrees of
freedom, which is compared to the tabulated t- value for the required confidence level.
The null hypothesis is "there is no correlation between X and Y", if the observed value
of tr is greater than t, the hypothesis is rejected and there is a significant linear
correlation with the calculated probability.
Trueness (accuracy)
According to the Codex alimentarius, trueness is the concordance between the results
of a test and the reference value.
Percentage of recovery: To determine the effectiveness of a method (and also of the

working range), recovery experiments can be carried out. Recovery can be defined as
the “fraction of the analyte determined after addition of a known amount of the analyte
to a sample”.
• Cx = average concentration of analytical results.

• Cm = sample concentration.
• Ca = amount of added analyte.
Precision
According to the Codex Alimentarius, precision is repeatability and reproducibility. The
degree of precision is usually expressed in terms of imprecision and is calculated as the
standard deviation of the results.
Repeatability: Degree of similarity obtained in simultaneous repetitions of the analytical

method:
• Same method.
• Same analyst.
• Same laboratory.
Reproducibility: Reflects the variability when applying a method at different conditions

(intercomparing exercises), like different analysts.
Rudggeness, robustness
An analytical method is rugged or robust if results are not (very) sensitive to variations
in the experimental conditions. Such conditions can be temperature, extraction or
shaking time, shaking technique, pH, purity of reagents, moisture content of sample,
sample size, etc.
A robust method is a good indicative of its confidence.
All these studies are carried out when changes are made to the method to improve its
efficiency, evaluate costs or when variations in environmental conditions occur.
Sensitivity
It is the slope of the response-concentration curve, or the response change per unit of
concentration. If the slope is steep the method has a high sensitivity and if the slope is
not steep, the method has a low sensitivity.
• < slope = < sensibility
In nutrient composition studies trace element analysis requires high sensitivity, which
in practice can be achieved by electronic amplification or by chemical concentration of
the analyte.
Selectivity, specificity
It is the ability of a method to respond exclusively to the substance to be analyzed.
However, some methods with poor specificity are sometimes accepted when the
purpose of the analysis is to capture similar compounds within a group (e.g, total fat,
ash).
Specificity refers to the method's property of producing a measurable signal due only to
the presence of the analyte, free from interference of other components in the sample
matrix.
Working range
The working range of a method is the concentration range at which accuracy and
precision suitable for the purpose of the method can be obtained.
• It is defined by the linearity of the method response.
• Normally it will be between the Limit of quantification and the maximum
theoretical value.
Part 1: Introduction and sampling

Introduction to food analysis
• Know from different point of views the composition (scientific, industrial,

administrative) of the food.
• Know if it complies with different nutritional claims.
• Data are processed and different measures will be adopted.
Problems when performing food analysis:

• Large variability of components, which are also not in fixed quantities in similar
products.
o It makes analysis difficult when looking for components that are present
in just one ingredient of the food.
§ Know the cholesterol content in pasta products to know the exact
amount of egg.
• A large number of components in the food, which causes a high number of
analytical interferences (component in food that is hiding the analyte that we
are looking for).
o They should be removed by extraction, purification, separation steps,
etc.
• Minority components (traces) are also of interest, by which purification and
concentration steps have to be performed using analytical techniques with
enough sensitivity.
• Additive effect: deviation in the result that always has the same magnitude. It is
directly proportional to the actual concentration.
• Multiplicative effect: deviation is not the same according to the magnitude. It is
not proportional but it is increasing as the concentration increases.
The application depends on: it is the selection of a specific method to analyze a

component or characteristic in the particular food.
• Purpose of the analysis.
• Characteristic of methods employed.
• Component of characteristic of interest.
The main food analytical techniques are:

o Physical techniques: spectrophotometric; chromatography; electrophoresis;
calorimetry; gravimetry.
o Biochemical techniques: enzymatic; immunological.
o Sensory analysis: triangular test; duo-trio test.
o Microbiological analysis: PCR; plate count methods.
o Nutritional analysis: RDI; nutrients density.
Steps to follow for the performance of food analysis

Sample selection and preparation:
• Sampling techniques.
• Transformation of “sample” into “analyte”.
• Samples analyses are normally performed by the company or through an
external lab.
Then it would be the development of the procedure, and, lastly, the calculation and
expression of results.
• Information management systems (LIMS). It is a software tool provides
standardization of everything carried out in the lab.
Questions to consider in a QMS
Official analytical methods: AOAC
AOAC (Association of Official Analytical Chemists) is a private scientific association

funded in 1884.
Its main purpose is the publication of official methods of analysis that can be used by
regulatory and research organizations worldwide:
• Development and validation of analytical methods through consensus between
intercomparing lab exercises.
• Improvement of quality assurance procedures in laboratories.
• Development and promotion of criteria to meet the accreditation standards of
analytical laboratories.
Performance and development of an AOAC official method:

• Selection of analytical methods published in the scientific literature or
development of novel methods by collaborating researchers belonging to AOAC.
• Multilateral comparative studies.
• Review of methods by experts worldwide and adoption as Official Analytical
Method.
• Publication of the method in the AOAC journal.
• Training courses.
General guidelines on food sampling

The objective is to accept or reject a food lot (gives the traceability) (batch). It will be
accepted if it complies with certain characteristics.
Sometimes it is important to monitor the food sampling,
The analyte is in the sample. The sample is in the batch. Lastly, the batch is in the
consignment.
Selection of sampling procedures

The bigger sampling size, the higher reliability of the sampling process.
Its limitations are:

• Information about how contamination is distributed in a sample.
• Required time.
• Cost.
• Sampling methods.
• Logistics.
• Analysis.
• Data processing.
The main purposes of food sampling are:

• Nutritional labelling.
• Detection of food contaminants and foreign materials.
• Food quality assurance: foundations and application of food quality control.
• Acceptability of raw materials, ingredients or products.
• Production of batches of finished products.
• Detection of food frauds.
• Microbiological hygiene and safety.
• Authenticity.
A sampling plan is a planned procedure which enables one to choose, or draw separate
samples from a lot, in order to get the information needed, such as a decision on
compliance status of the lot. It consists in the parameters n (nº of samples) and c (nº of
samples that can exceed the limit).
• You can either mix the samples and analyze them all, or not mix them and
analyze them separately.
Factors considered in the sampling process
Sampling plans
Attributes: they measure a given quality of a food to accept /reject a batch.
• Presence of Salmonella spp in whole eggs.
Variables: they measure a given value or concentration in a food in order to check

whether or not it conforms to a specification.
• Tin level in food packages, which cannot be exceeded.
Simple: they are based on taking a random sample from a batch. The batch is rejected
if the number of defective units is greater than a specified value.
Double: they are applied in case the results of the simple sampling plan are inconclusive.
The decision of acceptance or rejection of the lot is given by the result obtained from
the combination of two drawn samples.
Sequential: they are based on a continuous sampling where the number of units taken
vs. the number of defective ones is represented.
•
• If the defective units are falling in between the reject/accept part, it is needed
to keep analyzing.
• If the defectives units are too high and go to the “reject” part, the batch will be
rejected.
• If the defectives units are low and go to the “accept” part, the batch will be
accepted.
Stratified sampling: divide the sample into smaller samples.
Systematic sampling: random sampling process through an established time interval.
Cluster sampling: subgroups N2 and N4 are discarded for the analysis. This is used in
order to save money.
Composite sampling: pulling different subsamples into one big sample. Very common
for eggs and for pathogens. The result will be extrapolated to the subsamples.
Storage, transport and pre-treatment of samples

Errors of a non-statistical nature in sampling are associated with poor storage and
transport conditions of the samples.
Samples should be placed in containers that protect them from moisture or other
external factors:
• Light, air, heat etc.
If necessary, refrigeration or freezing should be applied:

• Samples containing unstable chemicals, perishable samples, etc.
During transport some preservatives may be used to stabilize samples, as long as they
do not interfere with the food matrix.
• Mercury Chloride, Chloroform etc.
Samples must be correctly labelled during storage and transport to ensure their
traceability.
Reduction of particle size: it will depend on the type of matrix to be analyzed.

• Meat products: particle size should not be too small to avoid excessive humidity
loss.
Enzymatic inactivation: Enzymes present in foods hydrolyze compounds of interest for

the analysis.
• The enzymatic activity must be controlled by heat destruction or by freezing the
samples (-20 or -30° C).
• Changes in pH, addition of salt.
Lipids oxidation:
• Very fatty foods should not be trimmed at high temperatures.
• High when unsaturated fatty acids are present à storage with nitrogen or in
vacuum – packaged.
• Addition of antioxidants that do not produce interference in the analysis.
• Recommendation: unsaturated fatty acids should be extracted just before
analysis.
Microbial growth and inactivation:

• Avoid cross – contamination.
• Handling and storage of samples under sterile conditions if subsequently
subjected to microbiological analyses.
Part 2: Moisture analysis

Introduction and concepts
The importance of moisture analysis:
• Legal and labelling requirements: they tell the maximum and minimum amount
of water that the food should have.
• Economic: to avoid frauds.
• Microbial stability: how this water is in the food (free or bonded).
• Sensory characteristics: the quantity of water will determine the texture, taste
and appearance of the food.
• Food processing operations: predictions of the behavior of foods during
processing such as mixing, drying, flow through a pipe, packaging, etc.
Definitions
The moisture content of a food material is defined through the following equation:
• % moisture = (mw/ms) x 100
o mw = mass of water.
§ It is related to the number of water molecules (nw) by the
following expression:
• mw = nwMw/NA
o mw is the molecular weight of water (18.0 g/mole).
o NA is the Avogadro’s number (6.02 x 1023
molecules/mole).
o ms = mass of the sample.
An appreciation for the principles, advantages and limitations of the analytical

techniques for moisture content depends on the understanding of the molecular
characteristics of water:
• 1 O atom with two lone pairs of electrons that each has a small negative charge.
It covalently bonds to 2 H atoms with a small positive charge.
• Consequently, water molecules are capable of forming relatively strong
hydrogen bonds with 4 neighbouring water molecules. The strength and
directionality of theses hydrogen bonds are the origin of many of the unique
physicochemical properties of water.
The analytical techniques should be able to distinguish water (the analyte) from the
other components in the food (the matrix). The characteristics of water that are most
commonly used to achieve this are:
• Its relatively low boiling point.
• Its high polarity.
• Its ability to undergo unique chemical reactions with certain reagents.
• Its unique electromagnetic absorption spectra.
• Its physical properties: density, compressibility, electrical conductivity and

refractive index.
Despite having the same chemical formula, the water molecules in a food may be
present in a variety of different molecular environments depending on their interaction
with the surrounding molecules. The water molecules in these different environments
normally have different physiochemical properties:
• Bulk/pure water: Bulk water is free from any other constituents, so that each
water molecule is surrounded only by other water molecules. It therefore has
physicochemical properties that are the same as those of pure water, e.g.,
melting point, boiling point, density, compressibility, heat of vaporization,
electromagnetic absorption spectra.
• Capillary or trapped water: Capillary water is held in narrow channels between
certain food components because of capillary forces. Trapped water is held
within spaces within a food that are surrounded by a physical barrier that
prevents the water molecules from easily escaping, e.g., an emulsion droplet or
a biological cell. The majority of this type of water is involved in normal water-
water bonding and so it has physicochemical properties similar to that of bulk
water.
• Physically bound water: A significant fraction of the water molecules in many
foods are in molecular contact with other constituents, like proteins,
carbohydrates or minerals. Different physicochemical properties than bulk
water.
• Chemically bound water: Some of the water molecules present in a food may be
chemically bonded to other molecules as water of crystallization or as hydrates,
e.g. NaSO4·10H2O. These bonds are much stronger than the normal water-water
bond and therefore chemically bound water has very different physicochemical
properties to bulk water
Challenges for moisture analysis:

• Because of the different microenvironments, which all have different
physicochemical properties.
• There could also be different proportions of bulk water, trapped water, etc.
• Water could be present in the three different states (liquid, gas, ice)
Sample preparation
It should be taken into account:
• Selection of a representative sample.
• Prevention of changes in the properties of the sample prior to analysis:
o To avoid water loss, by preventing foods from temperature changes, or
exposure to the atmosphere.
o Homogenize sample in the container before withdrawing a portion to be
analyzed.
• To weight the sample as soon as possible.
Analytical techniques
Evaporation methods
The sample is heated at specific conditions, and the water loss is used to calculate the
moisture content in the sample. The estimate of the moisture depends on the type of
oven, conditions and drying time/temperature. We obtain the result in an indirect way.
It is a very easy and simple method, so it is very extended among laboratories of food
analysis.
It can take from minutes to more than 24 hours.
There are specific temperatures for different foods in order to eliminate the totality of
water, which is normally higher than 100ºC.
The efficiency and speed in moisture evaporation depends on:

• Sample size.
• Particle size.
• Surface area during evaporation.
*The limitations and other issues are:

• Decomposition of other food components at very high temperatures/long time.
o Why?:
§ Because water can be released from the decomposition of these
components. This water will also be evaporated and the result will
be overestimated.
§ Other components, with heat, can bond to water,
underestimating the result of quantity of water in the food.
• In a lesser extent, although constant: changes in the sample weight due to the
loss of volatile components, such as acetic, propionic or butyric acid, alcohols,
esters, etc.
o You have to control the temperature and time of evaporation.
• Clumping and surface crust formation: it is more difficult for water to escape
from the food.
o To avoid this, samples are often mixed with dried sand, which does not
react with anything, and prevents the formation of crust in the surface.
• Type of sample pans (disposable) and the use of fiberglass lids (avoid spattering;
not metallic, but a lid that lets water evaporate but not spill sample).
• Avoid spattering, because if not, you are able to lose sample.
• Sample pans and lids manipulation.
o We can add weight with our hands. Every material used must be well
desiccated.
The moisture calculation follows these equations:

• % moisture (weight) =
!"#$%&'( *(#+,% #- %,( $.!/0( #-#%#.0 $.!/0( *(#+,%12'#(2 $.!/0( *(#+,%
o #-#%#.0 $.!/0( *(#+,%
𝑥 100 =
#-#%#.0 $.!/0( *(#+,%
𝑥 100
!"#$! &'()*$ +$#,-.

• % solids (weight) = #/#.#'* &'()*$ +$#,-. 𝑥 100
The evaporation devices are:

• Convection oven: high variation inside the oven (up to 10ºC).
• Forced draft ovens: Air is forced to circulate inside the oven, so as to achieve a
more uniform temperature distribution within the oven. The difference is not
larger than 1ºC.
o Extra: collectors for air distribution → air moves horizontally
• Vacuum oven: Weighted samples are placed under reduced pressure (typically
25-100 mm Hg).The boiling point of water is reduced when it is placed under
vacuum. Drying foods in a vacuum oven therefore has a number of advantages
over conventional oven drying techniques. If the sample is heated at the same
temperature, drying can be carried out much quicker. Alternatively, lower
temperatures can be used to remove the moisture (e.g. 70oC instead of 100oC),
and so problems associated with degradation of heat labile substances can be
reduced.
o Temperature can be lowered, so it is useful for more volatile foods.
Forced draft oven

Drying time: 0.75 – 24 h. Standardized time or until samples reach constant mass
Food samples that have high moisture contents are usually dried in two stages to
prevent "spattering" of the sample, and accumulation of moisture in the oven.
• For example, most of the moisture in milk is removed by heating on a steam bath
prior to completing the drying in an oven.
Drawbacks of high temperature treatments: sugars decomposition.
Vacuum oven
Reduced pressure: 20-100 mm Hg.
Advantage: a more complete removal of water and volatiles without decomposition

within a 3–6-h drying time.
Important points in the use of a vacuum drying oven:

• Temperature used depends on the product, such as 70ºC for fruits and other
high-sugar products. Even with reduced temperature, there can be some
decomposition.
• If the product to be assayed has a high concentration of volatiles → use of a
correction factor to compensate for the loss.
• In a vacuum, heat is not conducted well. Thus pans must be placed directly on
the metal shelves to conduct heat.
• Evaporation is an endothermic process; thus, when several samples are placed
in an oven of this type, temperature will drop, causing the cooling effect of
evaporation.
o Do not rise the temperature or the sample will be burned.
• The drying time is a function of the total moisture present, nature of the food,
surface area per unit weight of sample, whether sand is used as a dispersant, and
the relative concentration of sugars and other substances capable of retaining
moisture or decomposing. The drying interval is determined experimentally to
give reproducible results.
Microwave analyzer
1st precise and rapid technique that allowed to make in-process adjustment of the
moisture content in food products. The technique was introduced in the late 1970s.
Microwave drying provides a fast, accurate method to analyze many foods.

• Limitation: it tests only single samples, one sample at a time.
Considerations:
• Power settings are dependent upon the type of sample and the
recommendations of the manufacturer of the microwave moisture analyzer
• Sample must be of a uniform, appropriate size
• The sample must be centrally located and evenly distributed, so some portions
are not burned and other areas are underprocessed
• Sample pads should be considered. They should absorb liquids well and avoid
spattering. There are several different types, including fiberglass and quartz fiber
pads.
o For optimum results, the pads should not absorb microwave energy, as
this can cause the sample to burn, nor should they fray easily, as this
causes them to lose weight and can affect the analysis.
Vacuum microwave oven accommodates one sample in triplicate or three different

samples at one time.
• In 10 min, the results are reported to be similar to 5 h in a vacuum oven at 100°C.
• It is not as widely used as conventional microwave analyzers, but can be
beneficial in some applications.
Infrared drying
In this technology, heat penetrates into the sample being dried. Heat penetration to
evaporate moisture from the sample can significantly shorten the required drying time
to 10–25 min.
Factors that must be controlled include distance of the infrared source from the dried
material and thickness of the sample.
The analyst must be careful so that the sample does not burn or case harden while
drying.
Infrared drying ovens may be equipped with forced ventilation to remove moisture air,
and with an analytical balance to read moisture content directly.
No infrared drying moisture analysis techniques are approved by AOAC International

currently. However, because of the speed of analysis, this technique is suited for
qualitative in-process use.
Advantages and drawbacks

Advantages: precise, relatively cheap, easy to use, officially recognized for many
applications and, depending on the type of oven, several samples can be analyzed
simultaneously.
Drawbacks: destructive, not adequate for some foods; depending on the case, it may
take very long.
Distillation
Water is extracted by distillation is measured directly. Evaporation and condensation
are done in this process.
Distillation techniques involve codistilling the moisture in a food sample with a high
boiling point solvent that is immiscible in water, collecting the mixture that distills off,
and then measuring the volume of water.
Solvents used could be less dense than water (e.g., toluene or xylene) or more dense
than water (e.g., tetrachlorethylene).
• The advantage of using a more dense solvent is that material to be dried floats,
therefore it will not charr or burn. In addition, there is no fire hazard with this
solvent.
Practical considerations: 3 potential sources of error should be eliminated if observed:

• Formation of emulsions that will not break between water and the solvent,
which become stable, making it harder for us to extract water.
• Clinging of water droplets to dirty apparatus.
• Decomposition of the sample with production of water, such as carbohydrate
decomposition, generating water.
The advantages are:

• It is adequate for low-moisture foods.
o The distillation method is an AOAC- approved technique for moisture
analysis of spices, cheese and animal feeds.
• It also can give good accuracy and precision for nuts, oils, soaps, and waxes, with
a high content in short chain fatty acids, which remain solubilized with the
solvents.
• The equipment is relatively cheap, and it is easy to use and set up.
The drawbacks are:

• It is destructive.
• It is time consuming.
• It involves the use of flammable solvents.
• It is not applicable for all foodstuffs.
Chemical methods
Karl – Fisher titration
Basis: this method is based on the fundamental reaction described by Bunsen in 1853
involving the reduction of iodine by SO2 in the presence of water:
2H2O + SO2 + I2 à H2SO4 + 2HI
• Iodine reacts specifically with water, and not with any other component.
• Iodine and SO2 in the appropriate form are added to the sample in a closed
chamber protected from atmospheric moisture.
• HI is colourless, while I2 is dark purple. The excess of I2 that cannot react with
water can be determined visually. The endpoint colour is dark red-brown.
• Some instrumental systems are improved by the inclusion of a potentiometer
(conductometric method) to electronically determine the endpoint, which
increases the sensitivity and accuracy.
• This was modified to include methanol and pyridine in a four-component system
to dissolve the iodine and SO2.
The volumetric titration can be done manually or with an automated unit. The
automated volumetric titration units use a pump for mechanical addition of titrant and
use the conductometric method for endpoint determination.
This is the method of choice for determination of water in many low moisture foods
such as dried fruits and vegetables, candies, chocolate, roasted coffee, oils and fats or
any low-moisture food high in sugar or protein.
The Karl Fisher reagent is very unstable. We need to standardize it with a stable
compound, like Sodium tartrate dehydrate, which always has 15.66 % H2O.
• 100 g NaT·H2O à 1 mL K-F = 15.66 g H2O à 1 mL K-F
• (Ww/Ws) x 100 à to figure out Ww à (15.66/1 mL K-F) x 10 mL K-F = 156.6 g H2O
012$3 4 0&
Calculations: %H2O = 5!
𝑥 100
• KFReq = Karl Fisher reagent water (moisture) equivalence.
• Ks = mL used to tritate sample.
• Ws = weight of sample (mg).
Before the amount of water found in a food sample can be determined, a KFR water
(moisture) equivalence (KFReq) must be determined. The KFReq value represents the
equivalent amount of moisture that reacts with 1ml of KFR. Standardization must be
checked before each use because the KFReq will change with time.
Advantages: heat is not required, thus it is very adequate for thermally labile
substances, or heat-reactive such as high sugar concentration or high volatile
compounds. It is rapid, accurate and not expensive.
Major difficulties and sources of error:
• Incomplete moisture extraction. For this reason, fineness of grind (i.e., particle
size) is important in preparation of cereal grains and some foods.
• Atmospheric moisture: external air must not be allowed to infiltrate the reaction
chamber.
• Moisture adhering to walls of unit. All glassware and utensils must be carefully
dried.
• Interferences from certain food constituents. Ascorbic acid is oxidized by KFR to
dehydroascorbic acid to overestimate moisture content; carbonyl compounds
react with methanol to form acetals and release water to overestimate moisture
content (this reaction also may result in fading endpoints); unsaturated fatty
acids will react with iodine, so moisture content will be overestimated.
Gas production
Some specific reactions between certain reagents and water result in the production of
gases. For example, the mixture of a food sample and powdered calcium results in
acetylene production, thus the gas is directly proportional to moisture content.
CaC2 + H2O → C2H2 (gas) + Ca(OH)2
Gas can be measured as:

• Gas volume.
• Decrease of sample weight.
• Increase of the pressure in the vessel containing the mixture.
Physical methods
Research on physical methods is based on the fact that many physical characteristics of
water are different from those of the food matrix, such as density, electric conductivity
or refraction index.
Dielectric method
Basis: The electrical properties of water are used to determine the moisture content of
certain foods → measuring the change in capacitance or resistance to an electric
current passed through a sample.
The moisture determination in dielectric-type meters is based on the fact that the
dielectric constant of water 80.37 at 20oC is higher than that of most solvents. The
dielectric constant is measured as an index of capacitance.
• Example: used widely for cereal grains.
Requirements:
• Calibration against samples of known moisture content as determined by
standard methods.
• Important factors which should be controlled:
o Sample density or weight/volume relationships.
o Sample temperature.
Advantages: very useful techniques for process control measurement applications,

where continuous measurement is required.
Inconvenient: these methods are limited to food systems that contain no more than 30-
35% moisture.
Hydrometry
Hydrometry is the science of measuring specific gravity or density, which can be done
using several different principles and instruments.
• It is commonly used for routine testing of moisture (or solids) content of
numerous food products.
Advantages: with proper technique, it is highly accurate.
There are two types:

• Hydrometer.
• Pycnometer.
Hydrometer
Basis: A second approach to measuring specific gravity → Archimedes’ principle, which
states that a solid suspended in a liquid will be buoyed (float) by a force equal to the
weight of the liquid displaced.
A hydrometer is a standard weight on the end of a spindle, and it displaces a weight of

liquid equal to its own weight.
• For example, in a liquid of low density, the hydrometer will sink to a greater
depth, whereas in a liquid of high density, the hydrometer will not sink as far.
Hydrometers are available in narrow and wide ranges of specific gravity. The spindle of
the hydrometer is calibrated to read specific gravity directly at 15.5 or 20oC. The
accuracy of specific gravity measurements can be improved by using a hydrometer
calibrated in the desired range of specific gravities.
Depending on the fluid to e measured, we will find different devices:

• The Quevenne and New York Board of Health lactometer: used to determine
the density of milk. The Quevenne lactometer reads from 15 to 40 lactometer
units and corresponds to 1.015 to 1.040 specific gravity.
o Degree above 15.5oC, 0.1 lactometer unit is added to the reading.
o Degree below 15.5oC 0.1 lactometer unit is subtracted to the reading.
• The Baumé hydrometer: it was originally used to determine the density of salt
solutions (originally 10% salt). From the value obtained in the Baumé scale, you
can convert to specific gravity of liquids heavier (more dense) than water.
• The Brix hydrometer: is a type of saccharometer used for sugar solutions such
as fruit juices and syrups and one usually reads directly the percentage of sucrose
at 20oC. The terms Brix and Balling are interpreted as the weight percentage of
pure sucrose.
• Alcoholometers: used to estimate the alcohol content of beverages. Such

hydrometers are calibrated in 0.1 or 0.2o to determine the percentage of alcohol
in distilled liquors (AOAC Method 957.03).
Pycnometer
It is one approach to measure specific gravity by comparing the weights of equal
volumes of a liquid and water in standardized glassware. This will yield density of the
liquid compared to water.
• You put the same volume of sample and water, so you are only going to take into
account the weight.
The density of the sample is calculated with the following formula:
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑝𝑦𝑐𝑛𝑜𝑚𝑒𝑡𝑒𝑟

= 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑝𝑦𝑐𝑛𝑜𝑚𝑒𝑡𝑒𝑟
This method is used for determining alcohol content in alcoholic beverages, solids in
sugar and solids in milk.
Refractometry
The refractometry can determine moisture in liquid sugar products and condensed
milks.
Advantages: If it is performed correctly and no crystalline solids are evident, the

refractometer procedure is rapid and surprisingly accurate. It has been valuable in
determining the soluble solids in fruits and fruit products.
The refractive index (RI) of an oil, syrup, or other liquid is a dimensionless constant that
can be used to describe the nature of the food. When a beam of light is passed from one
medium to another and the density of the two differs, then the beam of light is bent or
refracted.
The (RI) (η) is a ratio of the sines of the angles:
𝑠𝑖𝑛𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑡 𝑟𝑎𝑦 𝑎𝑛𝑔𝑙𝑒

𝜂=
𝑠𝑖𝑛𝑒 𝑟𝑒𝑓𝑟𝑎𝑐𝑡𝑒𝑑 𝑟𝑎𝑦 𝑎𝑛𝑔𝑙𝑒
All chemical compounds have an index of refraction. Therefore, this measurement can
be used for the qualitative identification of an unknown compound by comparing its RI
with literature values.
RI varies with concentration of the compound, temperature (because we are measuring

in some way the density), and wavelength of light. Instruments are designed to give a
reading by passing a light beam of a specific wavelength through a glass prism into a
liquid, the sample.
Bench-top or hand-held units use Amici prisms to obtain the D line of the sodium
spectrum or 589 nm from white light.
Whenever refractive indices of standard fluids are given, these are prefaced with:
𝜂678 = 𝑎
• Value from 1.3000 to 1.7000.
• The Greek letter η is the symbol for RI.
• The 20 refers to temperature in C.
• D is the wavelength of the light beam (D line of the sodium spectrum) = 589 nm
The fact that the RI of a solution increases with concentration has been exploited in the
analysis of total soluble solids of carbohydrate-based foods such as sugar syrups, fruit
products, and tomato products.
Infrared analysis
Infrared spectroscopy has attained a primary position in monitoring the composition of
food products before, during, and following processing. It has a wide range of food
applications and has proven successful in the laboratory, at-line, and on-line.
Infrared spectroscopy measures the absorption of radiation (near- or mid-infrared) by

molecules in foods. For water, near-infrared (NIR) bands (1400–1450; 1920–1950nm)
are characteristic of the bond –OH stretch/movement of the water molecule and can be
used to determine the moisture content of a food. NIR has been applied to moisture
analysis of a wide variety of food commodities.
The only disadvantage is the long time that it takes to do calibration curves and robust
calibrations.
Freezing point
Freezing point decreases when adding solutes to water.
Example: freezing point of milk is its most constant physical property.

• It varies within narrow limits around -0.503oC and -0.541oC (average =
-0.517oC).
The principal utility of freezing point is to measure for added water application of
measuring the freezing point in milk is to detect milk watering.
• FDA (Food & Drug Administration) will reject all milks with freezing points above
-0.503oC.
% added water=0.517–PF x 100 0.517

• Where PF is the freezing point of the sample
Water activity
Water content alone is not a reliable indicator of food stability, but water activity, which
is calculated as:
Aw = P P0
= ERH 100
• P is the partial pressure of water above the sample.
• P0 is the vapor pressure of pure water at the same temperature.
• ERH is the equilibrium relative humidity surrounding the product.
Samples are introduced in a small closed chamber at constant temperature, and a

relative humidity sensor is used to measure ERH of the sample atmosphere after
equilibration.
Unit 2: Ash analysis

Applications
Nutritional evaluation.
Further analysis of specific inorganic elements: once you get the ashes, you can continue
to analyze the inorganic matter.
Usually, we expect a constant content of ash in animal food products. However, in foods
of plant origin, ash content may vary.
Ash content of selected foods

In dry dairy products, the concentration of ash is higher because water has been
eliminated, so the solutes are being concentrated.
Definitions
Ash refers to the inorganic residue remaining after either ignition or complete oxidation
of organic matter in a foodstuff.
It is needed to know the basic knowledge of the characteristic of various ashing

procedures and types of equipment in order to know if the results are reliable.
Dry ashing
Use of a muffle furnace capable of maintaining temperatures of 500-600ºC.
Water and volatile compounds are vaporized.
Organic substances + atmospheric O2 à CO2 + nitrogen oxides.
Most minerals are converted to oxides, sulfates, phosphates, chlorides and silicates.
Elements such as Fe, Se, Pb and Hg may partially volatilize, so there can be an
underestimation when you want to know the value.
Wet ashing
Organic substances are oxidized by using acids and oxidizing agents of their
combinations.
Minerals are solubilized without volatilization. Wet ashing often is preferable to dry
ashing as a preparation for specific elemental analysis.
If perchloric acid is used in the combination of acids, a special perchloric acid hood is
required.
Sample preparation
The sample size can vary from 2 g to 10 g.
Operations like milling, grinding, and the like probably will not alter the ash content
much; however, if this ash is a preparatory step for specific mineral analyses,
contamination by microelements is of potential concern. Remember, most grinders and
mincers are of steel construction.
• Repeated use of glassware can be a source of contaminants as well.
• The water source used in dilutions also may contain contaminants of some
microelements.
o Distilled-deionized water always should be used.
Plant materials are generally dried by routine methods prior to grinding. The
temperature of drying is of little consequence for ashing. Plant material with 15% or less
moisture may be ashed without prior drying.
Animal products, syrups, and spices require treatments prior to ashing because of high
fat, moisture (spattering, swelling), or high sugar content (foaming) that may result in
loss of sample. Meats, sugars, and syrups need to be evaporated to dryness on a steam
bath or with an infrared (IR) lamp. One or two drops of olive oil (which contains no ash)
are added to allow steam to escape as a crust is formed on the product.
Smoking and burning may occur upon ashing for some products (e.g., cheese, seafood,
spices). Allow this smoking and burning to finish slowly by keeping the muffle door open
prior to the normal procedure.
A sample may be ashed after drying and fat extraction. In most cases, mineral loss is
minimal during drying and fat extraction. Under no circumstances should fat-extracted
samples be heated until all the ether has been evaporated.
Analytical techniques
Dry ashing
During dry ashing, temperatures between 500-600oC are reached. Crucible selection
becomes critical in ashing ,and the type depends on the specific use:
• Quartz crucibles: resistant to acids and halogens, but not alkali.
• Porcelain crucibles resemble quartz crucibles in their properties, but will crack
with rapid temperature changes.
o Porcelain crucibles are relatively inexpensive and usually the crucible of
choice.
• Steel crucibles are resistant to both acids and alkalis and are inexpensive, but
they are composed of chromium and nickel, which are possible sources of
contamination.
• Platinum crucibles are very inert and are probably the best crucibles, but they
are currently far too expensive for routine use for large numbers of samples.
• Quartz fiber crucibles are disposable, unbreakable, and can withstand
temperatures up to 1000◦C. They are porous, allowing air to circulate around the
sample and speed combustion.
o This reduces ashing times significantly and makes them ideal for solids
and viscous liquids.
o Quartz fiber also cools in seconds, virtually eliminating the risk of burns.
• All crucibles should be marked for identification.
Advantages:
• Safe method.
• It requires no added reagent.
• It requires no blank subtraction.
• Once ignition begins, little attention is needed.
• Usually a large number of crucibles can be handled at once, and the resultant
ash can be used additionally in other anaylses for most individual elements, acid-
insoluble ash, and water-soluble and insoluble ash.
Disadvantages:
• Length of time required (12-18 h or overnight).
• Expensive equipment.
• Loss of volatile elements: As, B, Cd, Cr, Cu, Fe, Pb, Hg, Ni, P, V and Zn.
• Possible interactions between mineral components and crucibles.
Procedures
AOAC International has several dry ashing procedures for certain individual foodstuffs.
The general procedure includes the following steps:

• Weight a 5–10-g sample into a tared crucible. Predry if the sample is very moist.
• Place crucibles in a cool muffle furnace. Ignite 12–18 h (or overnight) at about
550◦C.
• Turn off muffle furnace and wait to open it until the temperature has dropped
to at least 250 C
̊ , preferably lower. Open door carefully to avoid losing ash.
• Transfer crucibles to a desiccator with a porcelain plate and desiccant.
• Cover crucibles, close desiccator, and allow crucibles to cool prior to weighting.
Some observations/cautions are:

• Warm crucibles will heat air within the desiccator.
• With hot samples, a cover may bump to allow air to escape.
• A vacuum may form on cooling. At the end of the cooling period, the desiccator
cover should be removed gradually by sliding to one side to prevent a sudden
inrush of air.
• Cover with a ground glass sleeve or fitted for a rubber stopper allow for slow
release of a vacuum.
The calculations are:

'&- +$#,-.
% ash (dry basis) = !"9 ('..$" +$#,-. 𝑥 100 =
+$#,-. ':.$" '&-#/,;.'"$ +$#,-. <: =">=#?*$
𝑥 100
<"#,#/'* &'()*$ +$#,-. 4 !"9 ('..$" =<$::#=#$/.
Dry matter coefficient = % solids/100
Suggestions that may be helpful and accelerate incineration:

• High-fat samples should be extracted either by using the crude fat determination
procedure or by burning off prior to closing the muffle furnace.
• Glycerin, alcohol, and hydrogen will accelerate ashing.
• Samples such as jellies will spatter and can be mixed with cotton wool.
• An alcoholic solution of magnesium acetate can be added to accelerate ashing
of cereals. An appropriate blank determination is necessary.
Wet ashing
It is sometimes called wet oxidation or wet digestion.
Its primary use is preparation for specific mineral analysis and metallic poisons.
Advantages:
• Minerals will usually stay in solution, and there is little or no loss from
volatilization because of the lower temperature
• Oxidation time is short.
Disadvantages:
• It takes constant operator attention.
• Corrosive reagents are necessary.
• Only small numbers of samples can be handled at any one time.
• It requires a hood, hot plate, and long tongs, plus safety equipment.
• If the wet digestion utilizes perchloric acid, all work needs to be carried out in an
expensive special fume hood called a perchloric acid hood.
o Wet oxidation with perchloric acid is extremely dangerous since the
perchloric acid has a tendency to explode.
• While perchloric acid does not interfere with atomic absorption spectroscopy, it
does interfere in the traditional colorimetric assay for iron.
• Unfortunately, a single acid used in wet ashing does not give complete and rapid
oxidation of organic material, so a mixture of acids often is used. Combinations
of the following acid solutions are used most often: (1) nitric acid, (2) sulfuric
acid-hydrogen peroxide, and (3) perchloric acid.
o Different combinations are recommended for different types of samples.
The nitric–perchloric combination is generally faster than the sulfuric–
nitric procedure.
• While wet digestion with perchloric acid is an AOAC procedure, many analytical
laboratories avoid if possible the use of perchloric acid in wet ashing and instead
use a combination of nitric acid with either sulfuric acid, hydrogen peroxide, or
hydrochloric acid.
Procedure
A wet ash procedure using concentrated nitric and sufuric acids is detailed below:
• Weigh a dried, ground 1-g sample in a 125-ml Erlenmeyer flask.
• Prepare a blank (3 mL of sulfuric acid + 5 mL of nitric acid). Blank is to be run with
every set of samples.
• Add 3 mL of sulfuric acid followed by 5 mL of nitric acid to the sample in the flask
• Heat the sample on a hot plate at ≈200 oC Brown-yellow fumes Will be observed.
o Once these fumes cease and white fumes from decomposing sulfuric acid
are observed, the sample will become darker.
o Remove the flask from the hot plate.
• Slowly add 3-5 mL of nitric acid.
• Put the flask back on the hot plate and allow the nitric acid to boil off. Proceed
to the next step when all the nitric acid is removed and the color is clear to straw
yellow. If the solution is still dark in color, add another 3-5 mL of nitric acid and
boil. Repeat the process until the solution is clear to straw yellow.
• Allow the sample to cool to room temperature, then transfer the sample to a
sized volumetric flask.
• Dilute the sample to volume with ultrapure water, and mix well.
The following procedure for a modified dry–wet ash sample destruction may be used.
• It is listed under “Minerals in Infant Formula, Enteral Products, and Pet Foods”
• Dry-Wet-dry:
o After evaporating moist from the sample, ash dry at 525oC followed by
wet with deionized distilled water plus nitric acid (0.5-3 mL) and dry on a
hot plate or steam bath, and then return to a 525oC furnace for 1-2 h.
Repeat the process if carbon persists.
o Dissolve the ash in nitric acid.
Microwave ashing
Both wet ashing and dry ashing can be done using microwave instrumentation.
Sample preparation time can be reduced to minutes. This advantage has led to
widespread use of microwave ashing, especially for wet ashing.
Microwave dry ashing: Microwave muffle furnaces can reach temperatures of up to

1200oC, and can ash samples in minutes. They are equipped with exhaust systems. Any
crucible that may be used is a conventional muffle furnace may be used in a microwave
furnace. Some systems can process up to 15 crucibles at a time. Time, temperature
parameters and a step-by-step (ramping) format can be set.
Microwave wet ashing: Open- and closed-vessel microwave systems are available.
These equipments allow the input of time, temperature in a step-by-step (ramping)
format. Additionally, in the case of closed-vessel microwaves, pressure and power can
be set by the user. The choice of the system depends on the amount of sample and the
temperatures required for digesting; open-vessel microwaves are recommended when
working with large samples (and can process up to 6 samples), and at atmospheric
pressure; closed-vessels microwaves are used for high-temperature/high-pressure
reactions, and can process up to 40 samples at a time. These instruments do not require
the use of a fume hood, because a vapor containment system contains and neutralizes
harmful fumes.
Inorganic compounds analysis

Once ash is obtained, minerals can be individually analyzed by methods such as titration
for chlorine, colorimetry for phosphorus, or atomic absorption spectrometry for many
minerals.
Unit 3: Fat analysis

Its applications are:
• Nutritional, because it is important to know the % of fat needed in a diet.
• For quality standards.
• For manufacturing specifications.
Analytical methods
Water insolubility is the essential analytical property used as the basis for the
separation of lipids from proteins, water, and carbohydrates in foods.
However, this “general rule” is not always met: for example, some glycolipids are soluble
in alcohols and have a low solubility in hexane.
The wide range of relative hydrophobicity of different lipids makes the selection of a
single universal solvent impossible for lipid extraction of foods.
Also, for a successful fat extraction, it is necessary that bonds between lipids and
proteins or CH be broken so that the lipids can be freed and solubilized in the extracting
organic solvents.
Solvent extraction methods

Sample preparation
For the best results, sample preparation should be carried out under an inert
atmosphere of nitrogen at low temperature to minimize chemical reactions such as lipid
oxidation.
Preparatory steps common in lipid analysis:

• Predrying simple.
o Why? Because many common organic solvent used are either
hydrophobic or hygroscopic, lipids extraction cannot be effectively
extracted.
o High temperature is not desirable for drying as some lipids become
bound to proteins and CH, and bound lipids are not easily extracted with
organic solvents. Vacuum oven drying at low temperatures or
lyophilization are preferable.
o Also, predrying makes the sample easier to grind for better extraction ad
breaks fat-water emulsions.
• Particle size reduction.
o For better extraction, the sample and solvent are mixed in a high-speed
comminuting device such as a blender.
• Acid hydrolysis.
o Acid hydrolysis can break both covalently and ionically bound lipids to
proteins and carbohydrates into easily extractable lipid forms.
o It is necessary in those foods with significant proportion of these lipids,

such as dairy, bread, flour and animal products.
o The sample can be predigested by refluxing for 1 hour with 3N
hydrochloric acid (HCl). Ethanol and solid hexametaphosphate may be
added to facilitate separation of lipids from other components before
food lipids are extracted with solvents.
Solvent selection
Ideal solvent for fat extraction:
• High solvent power for lipids and low or no solvent power for proteins, amino
acids, and carbohydrates.
• They should evaporate readily and leave no residue.
• They should have a relatively low boiling point.
• They should be nonflammable and nontoxic in both liquid and vapor states.
• They should penetrate sample particles readily, be in single component form to
avoid fractionation.
• Nonhygroscopic.
• Inexpensive.
However, it is very difficult to meet all of these requirements.
Ethyl ether and petroleum ether are the most commonly used solvents.
• Ethyl ether:
o Boiling point = 34.6ºC.
o Better solvent for fat than petroleum ether.
o Expensive compared with other solvents.
o Greater danger of explosion and fire hazards.
o It is hygroscopic and forms peroxides.
• Petroleum ether:
o It is mainly composed of pentane and hexane
o Boiling point = 35-38ºC.
o It is more hydrophobic than ethyl ether.
o Cheaper, less hygroscopic, and less flammable than ethyl ether.
A combination of 2 or 3 solvents is frequently used.
The solvents should be purified and peroxide free and the proper solvent-to-solute ratio
must be used to obtain the best extraction of lipids from foods.
Continuous solvent extraction method: Goldfish method

A solvent from a boiling flask continuously flows over the sample held in a ceramic
thimble.
Fat weight= (W beaker + W fat) – W beaker
Incovenients: channeling, which results in incomplete extraction.
Semicontinuous solvent extraction method: Soxhlet method

It is an AOAC method for cereal fat and meat fat.
For semicontinuous solvent extraction, the solvent builds up in the extraction chamber
for 5–10 min and completely surrounds the sample and then siphons back to the boiling
flask. This method provides a soaking effect of the sample and does not cause
channeling.
This method requires more time than the continuous method

• Often, this method is considered as reference for evaluation of other methods.
• Procedure:
o If the sample contains more than 10%H2O, dry the sample to constant
weight.
o Place the predried sample into a predried extraction thimble, with
porosity permitting a rapid flow of ethyl ether.
o Cover sample in thimble with glass wool.
o Weigh predried boiling flask and put anhydrous ether in the boiling flask.
o Assemble boiling flask, Soxhlet flask, and condenser.
o Extract in a Soxhlet extractor at a rate of 5-6 drops per second by
condensation for about 4 h, or for 16 h at a rate of 2-3 drops per second
by heating solvent in boiling flask.
o Dry boiling flask with extracted fat in an air oven at 100◦C for 30 min, cool
in desiccator, and weigh
o Calculation: % fat on dry weight basis = (W fat in sample / W dried
sample) x 100
Semicontinuous solvent extraction method: Mojonnier method

It can be applied to both liquid and solid samples.
It does not require removal of moisture from the sample.
Procedure for analysis of milk fat:

• Bring the sample to about 20ºC.
• Weigh the sample into a Mojonnier fat extraction flask.
• Add NH4OH (it neutralizes the acidic sample and dissolves protein) and shake
vigorously; add ethanol and shake (it prevents possible gel formation); add ethyl
ether and shake (it dissolves the lipid); add petroleum ether and shake (removes
moisture from the ethyl ether extract and dissolves more nonpolar lipid).
• Centrifuge for 30s at 600 rpm and decant ether solution from the Mojonnier flask
into the previously weighed Mojonnier fat dish.
• Perform 2nd and 3rd extractions in the same manner as for the 1st extraction
described above.
• Evaporate the solvent in the dish on the electric hot plate at ≤100◦C in a hood.
• Dry the dish and fat to a constant weight in a forced air oven at 100◦C ± 1◦C.
• Cool the dish to room temperature and weigh
• Calculations:
• A pair of reagent blanks must be prepared every day. For reagent blank
determination, use distilled water instead of milk sample
Nonsolvent wet extraction methods

Babcock method for milk fat
Basis: H2SO4 is added to a known amount of milk in the Babcock bottle. The sulfuric acid
digests protein, generates heat, and releases the fat. Centrifugation and hot water
addition isolate fat for quantification in the graduated portion of the test bottle.
The fat is measured volumetrically, but the result is expressed as percent fat by weight.
It takes 45 min aprox.
Duplicate tests should agree within 0.1%.
The Babcock method does not determine the phospholipids in the milk products.
It is not applicable to products containing chocolate or added sugar without

modification because of charring of chocolate and sugars by sulfuric acid.
Gerber method for milk fat analysis

The principle of the Gerber method is similar to that of the Babcock method, but it uses
sulfuric acid and amyl alcohol.
It is simpler and faster than Babcock method and has wider application to a variety of
dairy products.
The isoamyl alcohol generally prevents the charring of sugar found with the regular
Babcock method.
Instrumental methods
Advantages: they are rapid, nondestructive, and require minimal sample preparation
and chemical consumption.
Disadvantages: the equipment can be expensive and measurements often require the
establishment of calibration curves specific to various compositions.
NMR (Nuclear Magnetic Resonance)

Basis:
Many nuclei possess an angular momentum, which means that they have a
characteristic spin quantum number (I), and may be analyzed using NMR.
These nuclei are also charged, and a spinning charge generates a magnetic field. The
nuclei behave like tiny magnets that interact with an applied, external magnetic field.
Once the nuclei are placed within a strong external magnetic field (B0), the spin of the
nuclei will align with that field.
However, after a pulse of radio frequency energy is applied to the system, the nuclei
absorb energy and shift to a higher energy state. The pulse is applied by a transmitter
coil. The most common pulse is the 90◦ pulse.
Once the net magnetization has been tilted by a 90◦ pulse, the magnetization begins to
decay back to the original state.
During this process, termed NMR relaxation, the nuclei begin to relax back to the
equilibrium state.
The instrument consists of a superconducting cryomagnet (the NMR magnet), an

electronics console, and a data/ work station that also controls all the functions of the
instrument.
High-resolution pulsed NMR: it requires an homogeneous magnetic field, and it

provides structural information of molecules.
Low-resolution pulsed NMR: systems designed to correct non homogeneous zones of

the magnetic field. The following information can be obtained: total solid fat, liquid fat,
crystallization mechanisms of fat blends.
X-ray absorption
X-ray absorption of lean meats his higher tan that of high-fat meat. A calibration curve
is required.
Dielectric method
Lean meat have dielectric constants higher than those of high-fat meats. A calibration
curve is required.
Infrared analysis
It is based on the absorption of IR energy by fat at a wavelength of 5.73 μm.
The higher the absorption, the higher the fat content of foods. A calibration curve is
required.
Ultrasonic method
It is based on the acoustic properties of the different food compounds.
This method is based on the speed of sound passing thorough the food at different
temperatures.
A calibration curve is required.
Colorimetric method
For example, the measure of the color developed after reaction of milk fat and
hydroxamic acid, and other reagents. A calibration curve is required.
Density measure method

Food density is inversely proportional to its fat content.
Milko-tester equipment measures the turbidity of light scattering caused by fat globules
in milk.
Fat characterization
Methods for bulk oils and fats: iodine value
The iodine value (or iodine number) is a measure of degree of unsaturation, which is the
number of carbon–carbon double bonds in relation to the amount of fat or oil.
Iodine value is defined as the grams of iodine absorbed per 100 g of sample. The higher
the amount of unsaturation, the more iodine is absorbed and the higher the iodine
value.
Iodine value is used to characterize oils, to follow the hydrogenation process in refining,
and as an indication of lipid oxidation, since there is a decline in unsaturation during
oxidation.
• ICl: reagent.
• R-CH=CH-R: fat
• B – S: B has all the remaining because there is no fatty acid for it to react.
High values of iodine value → large difference (B – S) → short amount of iodine

remaining in the sample → higher number of insaturations in the sample.
Methods for bulk oils and fats: Polar components in frying facts
Physical and chemical changes that occur during frying include:
• Viscosity.
• Foaming.
• Free fatty acids (FFA).
• Degree of saturation.
• Hydroxyl and carbonyl formation.

• Saponification value.
They all increase their value.
Deterioration of used frying oils and fats can be monitored by measuring the polar
components, which include monoacylglycerols, diacylglycerols, FFAs, and oxidation
products formed during heating of foodstuffs. Nonpolar compounds are primarily
unaltered triacylglycerols.
The polar compounds in a sample can be separated from nonpolar compounds using
chromatographic techniques.
Polar components are measured by dissolving the fat sample in organic solvent, then
applying the solution to a silica gel column. Polar compounds are adsorbed onto the
column. Nonpolar compounds are eluted, the solvent evaporated, the residue weighed,
and the total polar components estimated by difference.
The Spanish Standard for heated Oils and Fats establishes a limit of 25% polar
components in frying oils and fats. You measure it with trips.
Lipids oxidation
Rancidity refers to the off odors and flavors resulting from lipolysis (hydrolytic rancidity)
or lipid oxidation (oxidative rancidity).
Changes in quantities of lipid oxidation reactants and products over time:
The peroxide value is defined as the milliequivalents (mEq) of peroxide per kilogram of
sample. It is a redox titrimetric determination.
Peroxide value measures a transient product of oxidation, (i.e., after forming, peroxides
and hydroperoxides break down to form other products). A low value may represent
either the beginning of oxidation or advanced oxidation.
Thiobarbituric acid reactive substances (TBARS) test

This test measures secondary products of lipid oxidation, primarily malonaldehyde. It
involves reaction of malonaldehyde (or malonaldehyde- type products including
unsaturated carbonyls) with TBA to yield a colored compound that is measured
spectrophotometrically at 530 nm.
The food sample may be reacted directly with TBA, but is often distilled to eliminate
interfering substances, and then the distillate is reacted with TBA.
A calibration curve is requires.
The TBA test correlates better with sensory evaluation of rancidity than does peroxide
value.
The TBA test with minor modifications is frequently used to measure lipid oxidation,
especially y meat products.
An alternative to the spectrophotometric method described is to determine the actual

content of malonaldehyde using HPLC analysis of the distillate.
Unit 4: Protein analysis in foods

Importance of analysis
Nutrition labeling: proteins content, proteins composition...
Pricing: the cost of certain commodities is based on the protein content as measured by
nitrogen content (e.g., cereal grains; milk for making certain dairy products, e.g. cheese).
Functional properties research: for example, gliadin and glutenin in wheat flour for
breadmaking, casein in milk for coagulation into cheese products, egg albumen for
foaming...
Biological activity determination: enzymes or enzyme inhibitors, for example,

proteolytic enzymes involved in the tenderization of meats, pectinases in the ripening
of fruits...
Analytical methods
Kjeldahl*
Proteins and other organic food components in a sample are digested with sulfuric acid
in the presence of catalysts. The total organic nitrogen is converted to ammonium
sulfate. The digest is neutralized with alkali and distilled into a boric acid solution. The
borate anions formed are titrated with standardized acid (HCl), which is converted to
nitrogen in the sample. The result of the titration (volume of titrating agent) is
proportional to the nitrogen content.
Procedure
Sample preparation. Samples should be homogeneous à solids should be ground.
Digestion. Place sample (accurately weighed) in a Kjeldahl flask. Add acid and catalyst
(usually, metallic compounds such as Cu, Se or Hg); digest until clear to get complete
breakdown of all organic matter. During digestion, protein nitrogen is liberated to form
ammonium ions; sulfuric acid combines with these ammonium ions (forming sulfate
ammonium, which is non-volatile), and oxidizes organic matter; and carbon and
hydrogen elements are converted to CO2 and H2O.
Neutralization and distillation. Alkali (sodium hydroxide) is added to neutralize the

sulfuric acid. The ammonia formed is distilled into a boric acid solution containing the
indicators methylene blue and methyl red.
Tritation. Borate anion (proportional to the amount of nitrogen) is titrated with

standardized HCl.
Calculations:
Moles of HCl = moles of NH3 = moles of N in the sample
A reagent blank should be run to subtract reagent nitrogen from the sample nitrogen.
% N = N HCl x (corrected acid volume/g of sample) x (14g N/mol) x 100
• N HCl = normality of HCl in mol/L.

• Correccted acid volumen (mL) = volumen std. acid for sample – volumen std. acid
for blank.
A factor is used to convert percent N to percent crude protein. Most proteins contain
16% N, so the conversion factor is 6.25 (100/16 = 6.25).
or
Nitrogen to protein conversion factors for various foods:
Alternate procedures
In place of distillation and titration with acid, ammonia or nitrogen can be quantitated
by:
• Nesslerization:
This method is rapid and sensitive, but the ammonium dimercuric iodide is colloidal and
color is not stable.
• pH measurement after distillation into known volumen of boric acid.

• Direct measurement of ammonia, using ion chromatographic method.
Advantages and disadvantages

Advantages:
• Applicable to all types of foods.
• Inexpensive (if not using an automated system).
• Accurate: an official method for crude protein content.
• Has been modified (micro Kjeldahl method) to measure microgram quantities of
proteins.
Disadvantages:
• Measures total organic nitrogen, not just protein nitrogen.
• Time consuming (at least 2 h to complete).
• Poorer precision than the biuret method.
• Corrosive reagent.
DUMAS
Samples are combusted at high temperatures (700–1000◦C) with a flow of pure oxygen.
All carbon in the sample is converted to carbon dioxide during the flash combustion.
Nitrogen- containing components produced include N2 and nitrogen oxides.
The nitrogen oxides are reduced to nitrogen in a copper reduction column at a high
temperature (600◦C). The total nitrogen (including inorganic fraction, i.e., including
nitrate and nitrite) released is carried by pure helium and quantitated by gas
chromatography using a thermal conductivity detector (TCD).
Procedure
Samples are weighed into a tin capsule and introduced to a combustion reactor in
automated equipment. The nitrogen released is measured by a built-in gas
chromatograph.
You have to calculate the quantity of proteins with the same calculations as in the
Kjeldahl method.

Advantages:
• Applicable to all kind of foods.
• Requires no hazardous chemicals.
• Can be accomplished in 3 min.
• Recent automated instruments can analyze up to 150 samples without attention.
Disadvantages:
• Expensive equipment is required.
• Measures total organic nitrogen, not just protein nitrogen.
Infrared spectroscopy
Infrared spectroscopy measures the absorption of radiation (near- or mid-infrared
regions) by molecules in food or other substances. Different functional groups in a food
absorb different frequencies of radiation.
For proteins and peptides, various mid-infrared bands (6.47 μm) and near-infrared (NIR)
bands (e.g., 3300–3500 nm; 2080–2220 nm; 1560–1670 nm) characteristic of the
peptide bond can be used to estimate the protein content of a food.
Advantages:
• Applicable to a wide range of food products.
• Samples can be analyzed rapidly: 30 s – 2 min.
• Minimal training of analysts.
Disadvantages:
• Expensive.
• Instruments must be calibrated properly.
*Biuret
A violet-purplish color is produced when cupric ions are complexed with peptide bonds
(substances containing at least two peptide bonds, i.e., biuret, large peptides, and all
proteins) under alkaline conditions. The absorbance of the color produced is read at 540
nm. The color intensity (absorbance) is proportional to the protein content of the
sample.
Reaction of peptide bonds with cupric ions
Procedure
A 5-ml biuret reagent is mixed with a 1-ml portion of protein solution (1–10mg
protein/ml). The reagent includes copper sulfate, NaOH, and potassium sodium tartrate,
which is used to stabilize the cupric ion in the alkaline solution.
After the reaction mix is allowed to stand at room temperature for 15 or 30 min, the
absorbance is read at 540nm against a reagent blank (if the reaction mixture is not clear,
filtration or centrifugation before reading absorbance is required)
A standard curve of concentration versus absorbance is constructed using bovine serum

albumin (BSA). This is a standard protein that you can buy if you don’t have the protein
that you are investigating.

Advantages:
• The biuret method has been used to determine proteins in cereal, meat, soybean
proteins, as well as protein content of isolated proteins.
• Rapid (less than 30 min), simple and cheap (less expensive than Kjeldahl
method).
• Color deviations are encountered less frequently than with Lowry, ultraviolet
(UV) absorption, or turbudimetric methods.
• Very few substances other than proteins in foods interfere with the biuret
reaction.
• It does not detect nitrogen from nonpeptide or nonprotein sources.
Disadvantages:
• Not very sensitive as compared to the Lowry method.
• Absorbance could be contributed from bile pigments if present.
• High concentration of ammonium salts interfere with the reaction.
• Color varies with different proteins, this is why it is important to work with the
same protein.
• Opalescence could occur in the final solution if high levels of lipid or CH are
present
• Color must be standardized against known protein (e.g., BSA) (i.e. a calibration
curve is needed) or against the Kjeldahl nitrogen method.
Lowry
The Lowry method combines the biuret reaction with the reduction of the Folin–
Ciocalteau phenol reagent (phosphomolybdic-phosphotungstic acid) by tyrosine and
tryptophan residues in the proteins. The bluish color developed is read at 750 nm (high
sensitivity for low protein concentration) or 500 nm (low sensitivity for high protein
concentration).
Procedure
Proteins to be analyzed are diluted to an appropriate range (20–100 μg).
K Na Tartrate-Na2CO3 solution is added after cooling and incubated at room

temperature for 10 min.
Freshly prepared Folin reagent is added and then the reaction mixture is mixed and
incubated at 50°C for 10 min.
Absorbance is read at 650 nm, and a standard curve of BSA is carefully constructed.

Advantages:
• Very sensitive, (50-500 times more tan Biuret) and with a low detection limit.
• Less affected by turbidity of the sample.
• More specific than many other methods.
• Relatively simple.
Disadvantages:
• Because if its high sensitivity, it has not been widely used to determine proteins
in food systems without first extracting the protein from the food mixture.
• For the following reasons, the Lowry procedure requires careful standardization
for particular applications:
o Color varies with different proteins to a greater extent than the biuret
method.
o Color is not strictly proportional to protein concentration.
o The reaction is interfered with to varying degrees by sucrose, lipids,
phosphate buffers, monosaccharides, hexoamines, and with high
concentrations of reducing sugars, ammonium sulfate, and sulfhydryl
compounds interfere with the reaction.
Bradford
When Coomassie Brilliant Blue G-250 binds to protein, the dye changes color from
reddish to bluish, and the absorption maximum of the dye is shifted from 465 to 595
nm. The change in the absorbance at 595nm is proportional to the protein concentration
of the sample. Like other dye-binding methods, the Bradford relies on the amphoteric
nature of proteins. When the protein containing solution is acidified to a pH less than
the isoelectric point of the protein(s) of interest, the dye added binds electrostatically.
Binding efficiency is enhanced by hydrophobic interaction of the dye molecule with the
polypeptide backbone adjoining positively charged residues in the protein.
Procedure
Coomassie Brilliant Blue G-250 is dissolved in 95% ethanol and acidified with 85%
phosphoric acid.
Samples containing proteins (1-100 μg/mL) y and standard BSA solutions are mixed with
the Bradford reagent.
Absorbance at 595 nm is read against a reagent blank.
Protein concentration in the sample is estimated from the BSA standard curve.

Advantages:
• Rapid (2 min), reproducible, sensitive (several folds more sensitive tan the Lowry
method).
• No interferences from ammonium sulfate, polyphenols, carbohydrates such as
sucrose or cations such as K+, Na+, and Mg+2.
• Measures proteins or peptides with molecular mass approximately equal to or
greater than 4000 Da.
Disadvantages:
• Interfered with both nonionic and ionic detergents.
• The protein–dye complex can bind to quartz cuvettes. The analyst must use glass
or plastic cuvettes.
• Color varies with different types of proteins. The standard protein must be
selected carefully.
Bicinchoninic acid (BCA)

Proteins and peptides (as short as dipeptides) reduce cupric ions to cuprous ions under
alkaline conditions, (similar in principle to that of the Biuret reaction). The cuprous ion
then reacts with the apple-greenish bicinchoninic acid (BCA) reagent to form a purplish
complex (one cuprous ion is chelated by two BCA molecules). The color measured at 562
nm is near linearly proportional to protein concentration.
Procedure
Mix (one step) the protein solution with the BCA reagent, which contains BCA sodium
salt, sodium carbonate, NaOH, and copper sulfate, at pH 11.25.
Incubate at 37°C for 30 min
Read the solution at 562 nm against a reagent blank.
Construct a standard curve using BSA.

Advantages:
• Sensitivity is comparable to that of the Lowry method; has been used in protein
isolation and purification, while its suitability in complex food systems has not
been reported.
• One-step mixing (easier than in the Lowry method)
• The reagent is more stable than for the Lowry reagent.
• Neither nonionic detergent and buffer salts nor denaturing reagents interfere
with the reaction.
Disadvantages:
• Color is not stable with time. The analyst needs to carefully control the time for
reading absorbance.
• Any compound capable of reducing Cu+2 to Cu+ will lead to color formation.
• Reducing sugars interfere to a greater extent than in the Lowry method, as well
as high concentrations of ammonium sulfate.
• Color variations among proteins are similar to those in the Lowry method.
Ultraviolet 280nm absorption method

Proteins show strong absorption in the region at ultraviolet (UV) 280 nm, primarily due
to tryptophan and tyrosine residues in the proteins. Because the content of tryptophan
and tyrosine in proteins from each food source is fairly constant, the absorbance at 280
nm could be used to estimate the concentration of proteins, using Beer’s law (A = E280 l
c).
• I = constant
• c = concentration of the protein.
Since each protein has a unique aromatic amino acid composition, the extinction
coefficient (E280) must be determined for individual proteins for protein content
estimation.
Procedure
Proteins are solubilized in buffer or alkali.
Absorbance of protein solution is read at 280 nm against a reagent blank.
Protein concentration is calculated according to the Beer’s law.

Advantages:
• The UV 280-nm method has been used to determine the protein contents of milk
and meat products.
• Rapid and relatively sensitive.
• No interference from ammonium sulfate and other buffer salts
• Nondestructive; samples can be used for other analyses after protein
determination; used very widely in postcolumn detection of proteins.
Disadvantages:
• Nucleic acids also absorb at 280 nm. The absorption 280 nm/260 nm ratios for
pure protein and nucleic acids are 1.75 and 0.5, respectively. One can correct the
absorption of nucleic acids at 280 nm if the ratio of the absorption of 280 nm/260
nm is known.
• Aromatic aminoacid contents in the proteins from various food sources differ
considerably.
• The solution must be clear and colorless (centrifugation & filtration), as turbidity
could increase absorbance falsely.
Special considerations
Nonprotein nitrogen is present in practically all foods.
To determine protein nitrogen, the samples usually are extracted under alkaline
conditions then precipitated with trichloroacetic acid or sulfosalicylic acid. Heating could
be used to aid protein precipitation by acid, alcohol, or other organic solvents.
In addition to acid precipitation methods used for nonprotein nitrogen determination,

other methods such as dialysis and ultrafiltration and column chromatography could be
used to separate proteins from small nonprotein substances.
Unit 5: Carbohydrates analysis

Nutritional: main source of energy.
Physiological regulators: dietetic fiber.
Functional properties: contribute other attributes, including bulk, body, viscosity,

stability to emulsions and foams, water-holding capacity, freeze-thaw stability,
browning, flavors, and a range of desirable textures (from crispiness to smooth soft
gels). They also provide satiety.
Prebiotics: nondigestible polysaccharides. They are going to pass through the small
intestine, to reach the large intestine and help with the microbiota.
Sample preparation
First, drying. Most food commodities.
Second, grinding. It is recommended.
Lastly, lipids extraction, for example, with CHCl3-MeOH by means of Soxhlet extractor.
This step makes CH extraction more complete.
The residue is the sample ready for carbohydrates analysis.
Analytical methods
Monosaccharides and oligosaccharides

Extraction
For determination of any mono- other oligosaccharides present, the dried, lipid-free
sample is extracted with hot 80% ethanol, as this solution solubilizes mono-, di- and
oligosaccharides (due to its polarity). However, much of the composition of a food (other
than water) is in the form of polymers, and almost all polysaccharides and proteins are
insoluble in hot 80% ethanol. Thus, this extraction is rather specific.
Procedure: refluxing for 1 h, cooling, and filtering. Extraction should be done at least
twice. If the foodstuff or food product is particularly acidic, for example a low-pH fruit,
neutralization before extraction may be necessary to prevent hydrolysis of sucrose,
which is particularly acid labile; thus, precipitated calcium carbonate is routinely added.
The 80% ethanol extract will contain components other than carbohydrates, in
particular ash, pigments, organic acids, and perhaps free amino acids and low molecular-
weight peptides. Because the mono- and oligosaccharides are neutral and the
contaminants are charged, the contaminants can be removed by ion-exchange
techniques.
Finally, the aqueous alcohol is removed using a rotary evaporator and a temperature of
45- 50oC.
Sometimes, for extraction, refining, filtration or a final passage through a hydrophobic

column is used as a final cleanup step.
Analysis: Total carbohydrates à Phenol-Sulfuric Acid method

Principle:
• Destruction of carbohydrates with strong acids and/or high temperatures
(monosaccharides).
• Complex reactions after a simple dehydration reaction.
• Furan derivatives production.
• Condensation of furan derivatives with phenolic compounds, like phenol,
resorcinol, etc.
• Colored compounds are useful for the analysis.
• Absorbance measurement at 490nm.
• Standard curve with mixtures of the same sugars present of D-glucose.
Advantages:
• Simple, rapid, sensitive, accurate.
• Specific for carbohydrates.

• Inexpensive and stable reagents.
• Sugars, sugar derivatives and oligo- and polysaccharides can be determined.
Drawbacks:
• Phenols are corrosive and toxic.
• Corrosiveness nature of sulphuric acid.
Total reducing sugars: Somogyi – Nelson method

Principle:
• Method used to determine reducing sugars (giving up of electrons).
Cu2+ + reducing sugar à Cu+ + arsenomolybdate complex à Cu2+= stable blue color
• Absorbance measurement at 520nm.
• Standard curve of the sugar(s) being determined or D-glucose.
Specific analyses for mono- and oligosaccharides à Enzymic methods

Principle:
• Methods for the determination of carbohydrates.
• Specific or non for the substance being measured.
• There are many different kits which contain specific enzymes, other required
reagents, buffer salts, detailed instruction.
Advantages:
• Low detection limits.
• Quite specific method.
Sample preparation:
• Carrez treatment: it breaks emulsions, precipitates proteins and absorbs some
colours. It should be apply to food products prior to determination of CH by
enzymic methods.
• Example: determination of glucose with glucose oxidase.
Other methods
Thin – layer chromatography.
Liquid chromatography.
Gas chromatography.
Capillary electrophoresis.
Unit 6: Vitamin analysis

They have a critical role in determining animal and human nutritional requirements.
To assess diet adequacy and improve human nutrition worldwide.
To ensure accuracy of food labeling.
Analytical methods
The factors to decide the method are:
• Accuracy and precision.
• Economic factors.
• Sample load to be handled.
• Applicability in certain food matrices.
o Many official methods presented by regulatory agencies are limited in
their applicability to certain matrices, such as vitamin concentrates, milk
or cereals.
Bioassays
We use living organisms that need vitamins to grow.
It requires plenty of time, up to weeks because you need to use experimental animals,
but it does not require extract preparation.
This type of assays is limited in to those instances in which no satisfactory alternative

method is available.
At the present, bioassays are used only for the analysis of vitamins B12 and D.
• Specifically, the determination of vitamin D, based on bone calcification, involves
deficiency studies as well as sacrificing the test organisms at the end of the test
in order to examine the calcification of the bones.
o You look for staining of the proximal end of the tibia or distal end of the
radius or ulna.
Microbiological assays
We use living organisms that need vitamins to grow.
Advantages and disadvantages:

• It requires extract preparation, although it is not so exigent as in the case of
instrumental and physicochemical methods.
• It is limited to water-soluble vitamins.
• They are not time-consuming, but it takes up to 16 hours.
The sample preparation may need an extraction:

• This generally includes one or several of the following treatments: heat, acid,
alkali, solvents and enzymes.
• In general, extraction procedures are specific for each vitamin and designed to
stabilize the vitamin.
• In some instances, some procedures are applicable to the combined extraction
of more than one vitamin. For example, for vitamins A, E or D, organic solvents
are added for extraction, saponification and re-extraction with organic solvents.
For unstable vitamins such as these, antioxidants are routinely added to inhibit
oxidation.
Some considerations:
• They are limited to the analysis of water-soluble vitamins.
• Very sensitive and specific for each vitamin.
• The methods are somewhat time consuming, and strict adherence to the
analytical protocol is critical for accurate results.
Principle:
• The growth of microorganisms is proportional to their requirement for a specific
vitamin.
• You use a microorganisms in order to know the quantity of vitamin in the food
based on the growth of the microorganism, as well as to know the growth of the
microorganism in the presence of known quantities of the vitamin.
How to measure the growth:

• Turbidity à bacteria and yeast.
• Acid production.
• Gravimetry.
• Respiration.
Example: Niacin (Vit. B3): Nicotinic acid and nicotinamide

Niacin is chemically synonymous with nicotinic acid, although the term is also used for
its amide (nicotinamide). The free acid is converted into nicotinamide in the body. It is
water-soluble Some synonyms used such as vitamin PP or PP factor stand for its well
known preventive effect of pellagra. Nicotinamide is found in all cells, generally bound
as prosthetic group to NAD and NADP coenzymes. Liver and meats from meat-producing
animals are important sources of this vitamin, as well as corn and other cereals.
Example: Folate (Vit. B9)

Folate is the general term including folic acid (pteroylglutamate, PteGln) and poly-γ-
glutamyl conjugates with the biological activity of folic acid. Folates are necessary for
maintenance and production of new cells. This is specially important during cell division
and proliferation periods such us the infancy and pregnancy. Folates are essential for
DNA replication. Folic acid is naturally present in nature in their conjugate forms
(folates), and are found in the liver, kidney, muscles, milk, cheese, leafy greens, legumes,
cauliflower.
Folates present a diverse array of compounds that vary by oxidation state of the
pteridine ring structure, one-carbon group carried by the specific folate, and the number
of conjugated Gln residues on the folate.
Additionally, folates are labile to oxidation, light, termal losses, and leaching when foods
are processed
• Analytical problem: Dietary Folate Equivalent.
Microbiological assay:
• Based on the extraction with 3 enzymes:
o a-amilase and protease: they free macromolecularly bound folates.
o Conjugase: it cleaves poly-g-glutamyl folates to PteGln3 or lower.
• After extraction with the 3 enzymes, they are deactivated by boiling for 5 min.
• Reducing agents including ascorbic acid or b-mercaptoethanol are added in
order to prevent oxidation.
• Lactobacillus rhamnosus is used in the assay.
• Incubation at 37ºC for 20-24 hours and absorbance measure at 550 nm.
Instrumental and physicochemical methods

Advantages and disadvantages:
• It requires extract preparation.
• It is simple, accurate and precise, specially the chromatographic methods (HPLC).
• It allows for the analysis of many vitamins and their isomeric.
• It is rapid.
Chromatography
It is a technique that aims at separating the components of a mixture on the basis of
their speed when they are carried by a fluid called the mobile phase passing through a
structure holding another material called the stationary phase.
What do chromatography techniques have in common?

Mobile and stationary phase.
Types of chromatography?
Paper chromatography, TLC, column chromatography, size-exchange chromatography,
ion-exchange chromatography, affinity chromatography, HPLC, gas chromatography,
Paper chromatography
Retention/retardation factor (Rf): ratio of distance travelled by the solute to the
distance travelled by the solvent front.
• As long as experimental conditions are constant, Rf of individual compound is
reproducible.
HPLC: High Performance Liquid Chromatography

Detector features:
• Universal.
• High sensitivity and reproducibility.
• Nondestructive of samples.
• A linear response to solutes.
In normal phase chromatography, the stationary bed is strongly polar in nature (e.g.,
silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar
samples are thus retained on the polar surface of the column packing longer than less
polar materials.
Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar

(hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of
water and methanol or acetonitrile. Here the more nonpolar the material is, the longer
it will be retained.
There are two modes depending on the flow of the mobile phase:
• Isocratic: composition of mobile phase remains constant throughout the run.
• Gradient: composition of mobile phase (polarity of mobile phase) varies
throughout the run.
Results:
• Qualitative, based on the Retention time (tr).
• Quantitative, based on the integration of the chromatographic peaks.
• tm: column’s void time. Time required to elute non retained solutes.
• W: baseline width. We measure W by extending tangent lines from the inflection
points on either side of the chromatographic peak through the baseline.
Vitamin A
Tips:
• All work must be performed in subdued artificial light. Care must be taken to
avoid oxidation of the retinol throughout the entire procedure.
• Solvent evaporation should be completed under nitrogen, and hexadecane is
added to prevent destruction during and after solvent evaporation.
The area of the peaks is what is taken into account to know the quantity of the
substance.
Vitamin E
Tips:
• Vitamin E is subject to oxidation. Therefore, saponification is completed under
reflux, in the presence of the antioxidant, pyrogallol, with the reaction vessel
protected from light.
Vitamin C (L-ascorbic acid & L-dehydroascorbic acid)

Tips:
• It is very susceptible to oxidative deterioration, which is enhanced by high pH
and the presence of ferric and cupric ions. For these reasons, the entire analytical
procedure needs to be performed at low pH and, if necessary, in the presence of
a chelating agent.
• Mild oxidation of ascorbic acid results in the formation of dehydroascorbic acid,
which is also biologically active and is reconvertible to ascorbic acid by treatment
with reducing agents such as b-mercaptoethanol.
Questions:
• The principle of the method is to titrate the sample.
• The volume I measure is the volume of the titrate.
• Quantity of Vit.C = (Volume vit.C x Quantity of titrate)/Volume of titrate = mg
o Concentration of Vit. C = Quantity of vit C/mL of Vit. C.
• In order to analyze the concentration of L-dehydroascorbic in a food, add a
reducing agent to turn the dehydroascorbic into ascorbic; then do another
titration.
Other methods: Microfluorometric method

• Principle:
o Ascorbic acid, following oxidation to dehydroascorbic acid, is reacted
with o-phenylenediamine to produce a fluorescent quinoxaline
compound.
o This method measures both ascorbic acid and dehydroascorbic acid.
• How to determine the concentration of vitamin C as a result of fluorescence
measurements?
o The ratio of influence of the sample to the fluorescence to the standard
is linearly proportional to the concentration of vitamin C in the standard.
• Vitamins B1 (Thiamin) and B2 (Riboflavin) are also determined by fluorometric
methods.
Unit 8: Phytochemicals
Functional and nutraceutical foods
There is an interest in investigating bioactive compounds in the prevention of diseases.
Nutraceuticals: chemical substances that are found naturally in food or other non-
digestible forms, which have been determined to benefit health, as they prevent one or
more diseases or improve the physiological state of individuals.
• Example: antioxidant properties of vitamins C and E.
Functional food: natural or formulated food that exerts a beneficial effect on consumer
health and/or reduces the risk of illness.
• Phytochemicals: they are bioactive compounds present in vegetable foods.
Phytochemical compounds
Etymology is derived from Greek language “phytos” = plant. Taking this into
consideration, phytochemicals are “chemical compounds of plants”.
They have a physiological role in the plant as protection.
They have a physiological function in the human body as a health promotor.
They are a very large group of compounds. It is known that there are hundreds or even
thousands of these elements, although until now, only the health properties of some of
them have been investigated.
Their molecular bases and their interactions with other components of the diet are not
well known.
Classification of phytochemicals
Basing on biological protection properties and physico-chemical properties:
• Carotenoids: they are pigments responsible for the color. They are a
polymerization of the isoprene.
o Examples: beta carotene, lycopene, xanthophylls (lutein, zeaxanthin).
o Reactive oxygen capture compounds and have a preventive effect
against certain types of cancer.
• Flavonoids: they are phenolic compounds based on the flavone nucleus.
They are present as glycosides.
o Examples: anthocyanins, flavones, luteolin, flavonols (quercetin),
tannins (catechin, proanthocyanidin).
o They inhibit platelet aggregation and have antibacterial, anti-
inflammatory and antimutagenic properties.
• Glucosinolates: Brassica vegetables. They are glycosides that contain sulfur.

They are degraded by the enzyme myrosinase into isothiocyanates, nitriles
and glucobrassicin.
• Phytostrogens: they are compounds similar to the hormone 17-b-estradiol.
They suppress the oestrogen response. They favor the reduction of serum
cholesterol levels and act as protectors against certain types of cancer.
o Examples: isoflavones, lignans, cumestans.
Some flavonoids of interest in foods

More than 5,000 flavonoids have been already identified, such as:
• Citroflavonoids: quercetin, hesperidin, rutin, or limonene.
o Quercetin is a yellow-greenish flavonoid present in onions or apples.
o They are present in foods like broccoli, cherries, grapes or red cabbage.
o Hesperidin is found in the skins of oranges and lemons.
• Soy flavonoids or isoflavonoids: they are present in foods with soybeans such
as beans, tofu, tempeh, milk, textured vegetable protein, flour, miso. The two
best known are genistein and daidzein.
• Proanthocyanidins: they are located in grape seeds, red wine and extract of bark
of the marine pine.
• Anthocyanidins: they are vegetable pigments responsible for the red and blue-
red colors of cherries.
• Ellagic acid: it is a flavonoid found in fruits such as grapes and vegetables.
• Catechin: green and black tea are good sources.
• Kaempferol: it appears in leeks, broccoli, radish, endives and red beet.
Flavonoids
They are phenolic compounds that play an important role in plant biology.
They are antioxidants, have cell growth regulation, have bactericidal action and have
iron and copper fixation.
They are constituents of the non-energetic part of the human diet but our organism
cannot synthesize them and must, therefore, be supplied by diet.
• The intake should be of 23 mg/day.
o Quercetin = 16 mg/day.
o Beta-carotene = 2-3 mg/day.
o Vitamin E = 7-10 mg/day.
• They all share a common structure of diphenyl-pyran rings, composed of two
rings of phenyls linked through a pyran ring C (heterocyclic) with different
numbers of hydroxyl groups attached to the structures of the rings.
The chemical structure varies:
• Flavonols: catechin and quercetin.

• Flavones: diosmetin.
• Anthocyanidins: anthocyanins.
• Glycone = soluble part of flavonoids

o HPLC Chromatography is used to separate glycone from aglycone.
• Aglycone = hydrophobic
Origin and structure of flavonoids compounds

Phenolic compounds are secondary products of the catabolism of sugars.
Coumaric and cinnamic acids, as well as aromatic amino acids (phenylalanine and
tyrosine) are produced through the shikimate acid pathway.
Universal metabolite of higher plants. It was initially isolated from the Asian plant
"Shikiminoki" illilcium sp.
The condensation of a cinnamic acid molecule with a benzene ring formed by the
condensation of three molecules of acetyl coenzyme A (Krebs cycle), produces a series
of molecules named as flavonoids.
Phenolic compounds are defined as secondary products of sugars catabolism.
Through the shikimate pathway, coumaric and cinnamic acids are generated as well as
aromatic aminoacids (phenylalanine and tyrosine)
Flavonoids are a series of substances produced by a condensation of a cinnamic acid

molecule with a benzene ring which is formed by condensation of three acetyl-CoA
molecules (Krebs cycle).
These molecules are composed by two benzene rings joint by a cationic unsaturated and
oxygenated heterocycle. By means of diverse transformations (hydroxylation,
methoxylation, esterification and glucosidification) the different family compounds are
obtained.
Single or additional sugars are joint to the aglycone molecule, which can be esterified at
the same time with different organic acids. Glycosylation occurs normally in 3, 5 and 7
bounds of A and C rings.
Analysis of flavonoid compounds

Chromatography is the main technique in food analysis.
First, HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detector).
• For the identification and quantification of flavonoids according to their
solubility and their molecular weight.
Second, LC-MS (Liquid Chromatography connected with Mass Spectrometry).

• For the confirmation of molecular structure. Because some may have the same
molecular weight but different molecular structure.
Reverse-phase liquid chromatography:

• Stationary phase is non-polar, being frequently an hydrocarbon.
• Elution is carried out with a mobile phase of high polarity such as with an
aqueous solution containing diverse concentrations of solvents like methanol,
acetonitrile or tetrahydrofuran.
• Analytes are soluble in the polar phase.
UV-vis Detector (DAD)

It is a universal detector that is based on the absorption of chromophore compounds of
analytes in a wavelength range between 190-600nm.
The sensitivity of the technique is limited by the narrow optical path that the capillary
offers for the measurement of absorbance.
The tungsten lamp emits light in the visible range and enters the deuterium lamp, which
adds UV (and visible) light. The polychromatic beam passes the flow cell. The grating
splits up the polychromatic beam to different wavelengths, the intensities of which are
measured by an array of photodiodes. As substances can be identified by their spectra,
the DAD has a higher selectivity.
• Visualization of the UV-vis spectra throughout the analysis.
• Determination of the maximum absorbance.
• Identification of compounds.
The intensity of the light is

Polyphenols extraction
Extractable polyphenols (EP): they are present at the external part of the food and are
easily separated by mechanical procedures. They are mainly soluble substances.
• 2,5 g of frozen sample + 100 mL ethanol 80%.
• Homogenization.
• Filtration and storage at -20ºC until HPLC analysis.
• Other procedures include centrifugation and extraction by organic solvents such
as methanol, acetone, etc.
Non-extractable polyphenols (NEP): they are inside the cell wall, to break it we can do
it mechanically or chemically. They are mainly non-soluble substances.
• Acid hydrolysis: the residue (about 0,2 g) is subjected to a hydrolysis with
methanol: H2SO4 (90:10, v:v) by stirring in a bath at 85ºC and for 20 h. After this
time, centrifugate 15 minutes at 3000 rpm and collect the supernatant.
• Base hydrolysis: the residue (0,2 g) is subjected to alkaline hydrolysis with 15 ml
of 2M NaOH at room temperature for 4 hours with stirring. The supernatant is
then centrifuged and taken to pH 4 and a final volume of 20 ml.
Determination of polyphenols by HPLC-DAD-MS

To increase the concentration we can use distillation (rotavapor at 25ºC) in order to
obtain a concentration of 10x. then we filtrate it and inject 20 microliters in the HPLC.
Isocratic (uses the same solvent, the same composition at all time) and non-isocratic
(different solutions in different times) gradients.
The flow rate is of 1 mL/min. The stationary phase is non-polar. However the mobile
phase is:
• A: 95 % water (acetic acid, 1%) + 5% methanol (acetic acid, 1%)
• B: 88 % water (acetic acid, 1%)+ 12% methanol (acetic acid, 1%)
• C: 20 % water (acetic acid, 1%)+ 80% methanol (acetic acid, 1%)
• D: 100 % methanol (acetic acid, 1%)
DAD wavelengths:
• 280nm: purplish colours.

• 320 nm.
• 360 nm.
• 510nm: reddish colours.
Characterization of flavonoids by HPLC-DAD-MS

The limiting factor for the development of the LC-MS coupling has been the obtaining
of interfaces that allow the direct introduction of the LC flow in the MS detector.
Problems to be solved:
• High flow rate of the mobile phase (1 ml/min), to be vaporised in MS.
o It does not happen in the gas chromatography.
• Characteristics of the mobile phase (organic solvents, aqueous, non-volatile
inorganic salts etc.) incompatible with MS.
o All these substances are interfering with the MS, so they are emitting
some noise and should be removed.
• Characteristics of eluted compounds: complex molecules of high molecular
weight, low volatility, high polarity , or thermolabile.
o This heterogeneity does not facilitate the MS.
However, it was observed that the presence of solvent did not seem to disturb the
analysis by MS and could even help the ionization of the analytes. The most commonly
used interfaces nowadays are thermal fogging (thermospray), Atmospheric Pressure
Chemical Ionization (APCI), and electrospray ionization (ESI).
Thermospray: The sample is heated and a steam jet is produced through a nebulizer
that is driven under vacuum. The drops formed reduce their size by desolvation and a
static charge occurs. Charged particles enter into the MS, creating repulsive forces which
can end in a coulomb explosion. After this explosion we create atoms (solvation).
Electrospray ionization: The flow coming from the LC, passes through a capillary at
atmospheric pressure, maintaining a high voltage that disperses the liquid current,
forming highly charged droplets (nebulization) that are desolvated.
• The size of droplets decreases until reaching a point at which the repulsive forces
between the charges on the surface of the drops are strong enough to overcome
the cohesive forces of surface tension. Afterwards, smaller drops are produced
by explosion until the ions become to the gas phase, being transferred through
a series of focusing lenses to the analyzer of the mass spectrometer.
The MS spectrum analysis of negative ions provides structural information that allows
to differentiate and determine different positions of glycosylation
It also allows identification of isomers
Quercetin à Product ions Y0-CO-H
The analysis of the MS spectrum of positive ions provides complementary information

on structural characteristics related to the position of sugars.
Analytical techniques for anthocyanins

It is a flavonoid. Derived from the Greek, "Antho“ = flower; "Kyano" = blue. Coined by
Marquart in 1835 to designate the blue pigments of flowers.
At the end of 1990 it was proved that not only the blues, but practically all the red, blue
and violet tones of flowers, fruits, stems, leaves and roots were attributable to pigments
of this nature.
They are found in some fruits with red-blue-purple color, such as blueberries,
raspberries, cherries, red cabbage, plums or grapes.
Its function in plants is to attract predators to consume their fruits, and thus help the
dispersion of seeds.
In humans, anthocyanins have anti-aging, antiviral, and protective properties of the eyes
and the cardiovascular system.
Anthocyanins, as well as procyanidins (also called condensed tannins or

proanthocyanidins), represent some of the most studied groups of chemical compounds
in wines.
In 1958 it was used thin layer separation chromatography. Nowadays, an appropriate

system for the qualitative analysis of anthocyanins requires firstly extraction, followed
by purification and identification.
For extraction, various types of solvents have been used in the maceration process: HCl,
mixtures of HCl and MeOH, mixtures of trifluoracetic acid and MeOH.
For separation and isolation, the development of HPLC techniques have improved the
investigation of anthocyanins. In a reverse phase column, most extracts require the
application of an elution gradient due to the polarity and complexity of the molecule,
while in some simple cases the isocratic elution may be sufficient to separate
anthocyanins. Most of the gradients used for anthocyanins are composed of methanol
and an acid (frequently perchloric or formic acids).
In reverse phase HPLC, the order of elution follows that of polarity: delphinidin <cyanidin
<petunidin <pelargonidin <peonidin <malvidin.
The above findings confirm the characterization rules in reverse phase HPLC:
• The retention time of the aglycone is correlated with the hydrophobicity of the
molecule and elution depends on the polarity of the solvent;
• Monoglycosylated anthocyanins elute earlier;
• The addition of a second carbohydrate increases the polarity, resulting in a

decrease in its retention time.
For identification, UV-Vis Spectrophotometry is used.

• Maximum absorption (Vis) between 510-550 nm (band I). Anthocyanins also
have a maximum absorption in the UV region around 270-280 nm (band II), due
to phenolic groupings.
• The groupings of molecules or the addition of sugars entail deviations of the
maximum absorption wavelengths of about 10-15 nm (hypsochromic (deviation
into shorter wave lengths, shift to the blue side) and bathochromic (deviation
into longer wave length, shift to the red side) shifts). However, no displacements
occur at the wavelengths of maximum absorption in the UV region.
In the Vis spectrum, hydroxyl groups increase the maximum absorption wavelength
(λmax). Thus, derived compounds of delphinidin have higher λmax (525-528 nm) than
those of cyanidin (λmax = 516-520).
Also methoxy groups produce some displacement of the color towards the red zone;
methoxy groups are important for the stability of the molecule
The acylated molecules usually present a maximum absorbance value in the range 310-
335 nm corresponding to the acyl group.
Disadvantage of UV-Vis spectrophotometry:

• Not enough to discriminate pigments with similar retention times or spectral
characteristics, such as the presence of isomeric molecules of the same pigment,
for which mass spectrometry or nuclear magnetic resonance is necessary.
These techniques are very demanding because they have to work with very pure
compounds, free contaminated, so the previous isolation of the anthocyanins must be
very precise.
• For quantitative analysis of anthocyanins, absorbance measurements are used,
taking advantage of the ability of anthocyanins to change color as a function of
pH
Estimation of anthocyanins absorbance:

• Absorbance of anthocyanins = (A λmax pH 1.0 – A 700 nm pH 1.0) - (A λmax pH
4.5 – A 700 nm pH 4.5) This differential pH method aims to eliminate
interferences. The λmax ~ 510-540 nm.
• Concentration of anthocyanins (mg / 100 g) is estimated using a known
absorptivity value for a solution of the pure pigment; this value is based on the
predominant anthocyanin present in the plant material and in the extractant
solvent used.
The Beer-Lambert law relates the intensity of incoming light (I0) in a medium with the
outgoing intensity (I1) after absorption occurs. The relationship between both
intensities can be expressed as:
This is used when the concentration is high. When the concentration is low we use Mass
Spectrometry.
Unit 9: Other food contaminants

Introduction of contaminants in the food chain
Substance that could be chemical or biological origin that can be intentionally or
unintentionally added.
There can be an introduction of substances in the food production and distribution

chain.
• Pesticides, chemical substances for plants and animal diseases treatment,
agricultural treatments, packaging conditions, etc.
• It can cause a possibility of contamination or introduction of harmful substances.
Hazard:
Severity: potential effect of the hazard in human health.
Dose: related to the concentration of the substance in the food. Amount ingested in
order to have a disease.
The chemical contaminants mostly notified:

• Allergens (histamine).
• Pesticides.
• Antibiotic residues (tetracycline in beef).
• Heavy metals (mercury, lead, cadmium).
Some Novel contaminants:

• Packaging materials (bisphenol A).
• Degrading compounds (acrylamide).
• GMO.
The tolerance levels are related to the MRL (Maximum Residue Limit).
• There is a need to establishing accurate methods to analyze these constituents:
o Low levels (increase detection limit of the techniques).
o Not completely developed.
Analytical procedure of food contaminants

In order to determinate antibiotic residues, allergenic compounds and GMO.
The analytical approach is:

• Choice of the analytical method (quantitative (most expensive), semi-
quantitative or qualitative).
• Sample preparation:
o Homogenization.
o Extraction and cleanliness.

o Derivatization: step in which we transform the chemical structure of the
molecule in order to be detected by the equipment.
o Analysis: chromatographic techniques, immunomagnetic techniques,
electrophoresis, enzyme inhibition, etc.
We should take into consideration some factors like the time, effort, cost and reliability.
• Single contaminant detection à qualitative (screening) methods.
• Estimation of concentrationà quantitative methods.
They should all consider the food matrix, analyte characteristics, polarity, volatility,
stability and reactivity.
Analysis of antibiotic residues

Animal antibiotics supply:
• They reduce incidence of infectious diseases.
• They increase the growing rate.
• They produce a reduction of feed to obtain the same weight gain.
Beta-lactams, cephalosporins, tetracyclines, chloramphenicol etc.
Factors to be considered:
• Doses employed and duration.
• Resting periods (time elapsed between the last dose and the entry of the animal
to the slaughterhouse or its subsequent use for the production of eggs and / or
milk).
o They are mandatory.
• Tolerance levels allowed.
Hazards:
• Food allergenic compounds.
• Antibiotic resistance to sublethal doses .
Types:
• Anti-thyroid: bociogenic agents or growth promoters.

• Anabolic: therapeutic purposes, improvement of production.
• Inhibitory substances and chloramphenicol.
• Clenbuterol and beta-agonists.
Purification techniques and concentration of the analyte of interest (degreasing, protein

hydrolysis, protein precipitation, washing, etc.)
Evaluation methods:
• Inhibition of microbial growth.
• Use of receptors.
• Enzymatic assays.
• Immunoassays.
1.Microbial growth inhibition: Creation of inhibition halos on agar plates, turbidity

tests, organic acid production tests (based on color changes).
2. Utilization of receptors:
• They are based on the use of antibiotics contained in a kit (A) that compete with
those existing in the sample to be analyzed (B). Antibiotics bind to specific sites
in the cell wall of bacteria.
• The sample is incubated for a certain time, centrifuged and placed in a reader.
The higher the concentration of A, the lower the antibiotic residue
Food allergens
Allergen risk management

Food allergies affect between 2 and 4% of the population in Europe and approximately
5- 8% of children. ~ 10-20 million people in the EU
Inadequate immune response to a constituent of the food (in most cases proteins), so
that the food causes an allergic reaction when ingested.
Those having the highest impact are those in which the immune system produces IgE
antibodies against food proteins.
Food allergy should not be confused with food intolerance, such as lactose intolerance,
in which the immune system does not intervene.
Analytical techniques may vary:

• The "visually and physically clean" standard should be used as a starting point
for allergen risk management.
• Analytical techniques in development, although there are methods designed for
different allergenic compounds by ELISA (www.moniqa.org).
• The type of sample taken for analysis will ultimately depend on the specific
activity being monitored and the manufacturing environment.
o Environmental Swabs – monitoring residual allergens on food contact

surfaces.
o Purge Materials/ Flushing Mass – monitoring system used in those
equipments where wet cleaning is not appropriate.
o Air Samples/ Settle Plates – used to monitor dusting.
o CIP Rinsate – used to monitor effectiveness of clean-in-place systems.
o Finished product – used to monitor effectiveness of cleaning following
cleaning in conjunction with other samples listed above.
Analysis of food allergens

Protein/peptides detection methods are preferable to DNA detection methods (PCR)
since it is possible that the presence of DNA does not indicate the presence of allergenic
proteins and that a negative PCR result does not indicate the absence of proteins.
• Validation of cleaning processes, product test: ELISA
• Routine verification checks: LFD (Lateral Flow Devices) although they require
confirmation by ELISA.
• If the results in ELISA are above 10-20 ppm- PCR.
• MS in case quantitative results are required and conventional techniques are not
available.
Protein-based detection methods

As all the food allergens listed in Annex III of Directive 2007/68 / EC are proteins, with
the exception of sulphur dioxide and sulphites, proteins constitute the primary analyte
object of the detection.
Immunological methods (ELISA, LFD) and protein separation (MS)

• ELISA assays: it can be direct, indirect (more sensible à lower concentrations),
Sandwich (mostly used because it is easy to perform and has the same sensibility
as the indirect) or Competitive (low concentration or complex molecular
structure).
o Low detection and quantification limits (ppm).
o Official method for gluten detection.
o It has been validated for certain foods: dairy, nuts, cookies etc.
o Disadvantages: they can only detect a single target allergen per test
Biased results depending on the kit used (different sensitivity).
• Lateral Flow Devices (LFD):
o Immunochromatographic technique.
o Qualitative determination by means of a colorimetric test.
o Used for process control samples (in situ).
o They require subsequent confirmation by ELISA
Mass spectrometry (MS):

• It has the ability to directly detect proteins or peptides at low levels, similar to
those detected by ELISA and PCR.
• Ability to detect multiple allergens in a single analysis.
• The accurate detection of the allergen lies in the identification of peptide
fragments that are cleaved by the enzyme trypsin during the sample extraction.
• Disadvantages: high cost and need for specialized lab staff
PCR and RT PCR:

• They do not detect the target allergen but the DNA marker, which may or may
not be related to the amount of allergens in the food product.
• An advantage of PCR technology compared to ELISA is that all test components
are commercially available and easy to develop.
• Disadvantages: highly unstable in acid media (e.g tomato sauce) Possibility of
cross- contamination.
• Laboratories must have 4 physically separated areas:
o Preparation of the simple.
o Preparation of PCR mixtures.
o PCR treatment.
o PCR post-treatment (gel electrophoresis).
Genetic Modified Organisms (GMO)

GMOs are those organisms that have been modified by the application of recombinant
DNA technology or genetic engineering, a technique used for altering a living organism’s
genetic material
• Tolerance or resistance to herbicides.
• Production improvement.
• Better nutritive and organoleptic characteristics.
Prevalence of GMO in corn and soybeans in the US> 80% (2006)

Need to evaluate the safety of the products before launching them on the market
• Immunoassay techniques.
• PCR.
DNA extraction: it depends on the matrix to be treated. Basically it is fragmenting or

crushing a sample of the food to be treated. The remains of organic matter are removed
to extract the DNA (lipid-detergent, protein-protease). The DNA precipitates with the
addition of alcohol (ethanol or isopropanol).
PCR-amplification: it is a cyclic method in which millions of copies of the identified DNA

are made.
• DNA polymerase or dnTP (polymerize new DNA).
• Primers.
• Buffer solution.
Analysis of the DNA fragments by agarose gel electrophoresis.
Unit 10: Determination of trace

elements and heavy metals in food
Definitions, current legislation and contamination sources
Environmental contamination:
• Water pollution by anthropogenic and natural way drastically affects food
security and public health
Food contaminants:
• “Any substance not intentionally added to food or feed for food producing
animals, which is present in such food or feed as a result of the production
(including operations carried out in crop husbandry, animal husbandry and
veterinary medicine), manufacture, processing, preparation, treatment, packing,
packaging, transport or holding of such food or feed, or as a result of
environmental contamination”
Heavy metals are taking part of contaminants present in foods (Cd, Li, Hg).
The distribution, mobility, biological availability and toxicity of chemical elements is not
a function of their concentration but of the chemical form in which they are found.
It is necessary to know the chemical species of the elements to understand the chemical
and biochemical reactions they participate, and therefore, to obtain information
regarding the essential and toxic nature of the chemical elements.
Speciation analysis: Environmental legislation based on the maximum allowable

concentration of chemical species.
• Ex. Chromium (III) is an essential element with a role in glucose and lipid
metabolism while chromium (VI) is highly toxic à speciation analysis to
distinguish the species of the same metal.
• Thus, food safety measures cannot be decided without speciation.
Current food legislation

COMMISSION REGULATION (EC) No 333/2007 of 28 March 2007 laying down the
methods of sampling and analysis for the official control of the levels of lead, cadmium,
mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs
Commission Regulation (EC) No. 1881/2006 setting maximum levels for certain
contaminants in foodstuffs.
General requirements: The methods of analysis used for the control of food must
comply with the provisions of points 1 and 2 of Annex III to Regulation (EC) No 882/2004.
The methods of analysis for total tin are appropriate for the official control of inorganic
tin levels. Chapter 35 of the Annex to Regulation (EEC) No. 2676/90 of the Commission
[6] establishes the method for the analysis of lead in wine.
"Where no specific method for determining the presence of contaminants in food

products has been prescribed at EC level, laboratories may choose any validated method
of analysis (if possible, validation will include a certified reference material), provided
that the selected method meets the specific performance criteria".
Classification of metals
Essential metals: They must be in the diet in sufficient quantities. If they are not
provided by the diet, health alterations may occur. For example: sodium, potassium,
calcium, copper, zinc and manganese.
• Minerals and electrolytes: calcium, phosphorus, magnesium, sodium, potassium,
chlorine
• Trace elements: iron, selenium, copper, zinc, cobalt, molybdenum, fluorine,
chromium
Non-essential metals: such as lead, cadmium, mercury, aluminum etc.

When these metals are absorbed in small quantities there is the possibility of excreting
through urine, gastric juices, etc.
The increase in the concentration of heavy metals in food can cause a toxic effect to the
user, the severity of this effect will depend on the nature, quantity and chemical form
of the metals, the concentration of the metal in the food and the resistance of the
organism to the synergistic or antagonistic effects to other chemical contaminants.
Analysis
Sample preparation
There are some regulations:
• Standard UNE-EN 13804 "Determination of trace elements. Use criteria, general
considerations and sample preparation".
• Sampling protocol, sample preparation and analysis of heavy metals in fishery
products. Prepared by the Spanish Food Safety Agency (AESA).
It is recommended to have a sample mass of at least 200 g (Fresh products, Canned

foods, Salted and smoked foods, Fish**, Cephalopods, Crustaceans, Meats, Juices, etc.).
Homogenization of samples
Homogenization in a closed plastic bag using a laboratory Stomacher blender is often
used to homogenize fresh food samples.
Advantages:
• There is no direct contact between the sample and the equipment, thus reducing
the risk of contamination.
• There is no need to clean the pieces of the equipment.
A less expensive alternative are laboratory mixers with titanium sheets or homemade
mixers (e.g. coffee grinder). They are more difficult to clean and increases the risk of
contamination with Fe, Cr and Ni if stainless steel blades are used.
Drying of samples
It is carried out through a muffle furnace at 60-120° C
Lyophilization, what is lyophilization?.

• To sublimate the sample from solid to gas + vacuum to eliminate the most part
of water from the food.
Drying does not generally produce losses with exceptions as in the case of Se in the
muffle drying.
Digestion of samples
Objective: to eliminate the organic matter from the sample (mineralization)
• By calcination (or dry ashing through a muffle furnace). The sample is subjected
to temperatures of up to 550 ° C in platinum or quartz crucibles. This technique
is not suitable for volatile elements such as Se and Hg.
• Wet digestion: It is carried out by heating with acids for long periods of time.
Acids used: HNO3 / H2O2, HNO3 / H2SO4 / HClO4 and HNO3 / HClO4. They reduce the
loss of volatile elements such as Se.
Wet digestion in closed system: The sample is subjected to high pressures in closed
containers. This method reduces the use of acids and the heating time. It also reduces
the loss of volatile elements.
Analytical methods
Instrumental methods:
• Atomic Absorption Spectroscopy.
• Atomic Emission Spectroscopy.
• Colorimetric methods.
Foundations of absorption and emission spectroscopy methods:

• Atomic Absorption Spectroscopy is an instrumental method of analytical
chemistry that allows to measure the specific concentrations of a material in a
mixture and to determine a great variety of elements.
• Atomic Emission Spectroscopy is an analytical method that uses the intensity of
light emitted from a flame, plasma, arc or spark at a particular wavelength to
determine the concentration of an element in a sample.
Some other methods are: Spectrophotometry (not sensible enough), Fluorometry (not
sensible enough), Flame Atomic Absorption Spectroscopy (FAAS), Graphite Furnace
Atomic Absorption Spectroscopy (GFAAS), Hydride Generation Atomic Absorption
Spectroscopy (HGAAS), Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-

AES), Inductively Coupled Plasma Mass Spectroscopy (ICP-MS).
Flame atomic absorption spectroscopy
The free atoms produced in an atomizer from a sample (flame or electrically heated
graphite furnace) can absorb radiation at a specific wavelength of resonance generated
by an external source (for example a hollow cathode lamp).
The radiation generated passes through the atomizer containing the atomic vapor of the
element. Part of the light will be absorbed, and the degree of absorption will be
proportional to the density of atoms.
It is very versatile, more than 60 metallic elements can be determined, in a wide range
of concentrations by this method with a good sensitivity and precision. Different forms
of sample injection and atomization have been developed which can be easily
automated for routine analysis. It is also not very expensive.
The sensitivity is not enough according to the other techniques. The working range is
not very high. It is used for the determination of elements that are in high
concentrations.
Graphite furnace atomic absorption spectroscopy

Electrothermal atomization (ETA) in different steps to produce a vaporized analyte:
• Drying step to vaporize the solvent (70-120°C).
• Burning step (calcination) to remove the organic matter or volatile components
from the matrix (350-1250°C).
• Atomization step (2000-3000°C).
• Cleaning step at maximum temperature to burn the remaining analyte.
• Depending on the following of these steps, the accuracy can change.
Sensitivity of the technique is approx. 1000 times higher than FAAS
Careful optimization of all heating parameters is required during the method

development to obtain reproducible and accurate results.
It can produce higher interference than in FAAS. It is needed to introduce background

noise correctors (e.g. Deuterium and Zeeman background correction methods).
Some analytes can be lost due to volatility (Se), especially in the presence of halides, to
avoid matrix modifiers that reduce the volatility of the analyte. To avoid that matrix
modifiers that increase the volatility of other elements that interfere with the volatility
of an analyte are added.
Hydride generation atomic absorption spectroscopy

HGAAS can determine elements forming volatile hydrides such as As, Bi, Sn and Te. In
this technique, the analyte is reduced to its volatile hydride, transferred by a jet of gas
to a hot quartz cell, decomposed and atomized.
The separation of the analyte greatly reduces the interference of the matrix in such a
way that concentration levels of μg / kg can be determined.
Flow injection to analyze sample volumes as small as 100 μl.
A special treatment is required after sample digestion to generate a specific oxidation

state (e.g. Se + 4) for the hydride formation and certain metals (for example Cu, Fe) can
interfere with the formation of the hydride (Hidrogen+element).
It is a very high specific technique, which reduces interferences and facilitates the
analysis.
The preliminary step (generation of hydrid) should be carefully done, because the
sample can be easily cross-contaminated.
ICP-AES spectroscopy
The Inductively Coupled Plasma Atomic Emission Spectroscopy is a type of emission
spectroscopy that uses the inductively coupled plasma to produce excited atoms and
ions that emit electromagnetic radiation at wavelengths characteristic of a particular
element.
The amount of energy emitted will depend on the amount of atoms present in the
studied metal.
Atomization of the sample: To excite the atoms, argon plasma is used at 10,000 °K,
constituted by a gaseous conductive mixture of argon, electrons and cations of the
sample to be analyzed.
The ICP-AES is composed of two parts: the atomization source or ICP and a detector
(optical spectrometer).
The sample is nebulized to get an aerosol of particles and an atomizer (plasma or flame)
produces independent atoms or ions.
ICP-MS
The Inductively Coupling Plasma (ICP) is a source of ionization at atmospheric pressure
which, together with a vacuum mass spectrometer (MS), constitutes the ICP-MS
equipment.
In this technique, a plasma ion source at high temperature and at atmospheric pressure
is combined with a vacuum mass spectrometer as a sensitive detector.
• It allows multi-element determination
• Isotope separation
• Low detection limit à 0.1 ppb.
• Great performance.
• Great complexity: a person should be properly formed in order to maintain and
use the technique.
Comparison between techniques

Sensibility:
Costs:
Working range:
Unit 11: Sensory analysis

Concept
Sensory evaluation is a scientific discipline used to evoke, measure, analyse and
interpret those responses to products as perceived through the senses of sight, smell,
touch, taste and hearing.
Applications
Market studies (preferences and acceptability).
Market claims (eg.: «new creamier», «best ever»...).
Product development.
Impact evaluation of changes of ingredients or process modification on sensory quality

and/or product acceptability.
Quality assurance programme.
Product description: sensory profile.
Shelf-life establishment.
For each application you have to choose the test method as well as the judges. The test
method can have different data analysis. The judges and the test method both require
sensory testing.
Senses and sensory properties

Sensory properties are perceived when our sensory organs interact with stimuli in the
world around us. There are five senses:
• Gustation/taste.
• Smell.
• Touch.
• Vision.
• Audition.
Vision
Vision is the first “barrier” for food evaluation. The sensory properties evaluated are
colour, appearance, shape, surface, size and brightness.
Colour is the property possessed by an object of producing different sensations on the

eye as a result of the way it reflects or emits light. It has 3 primary qualities:
• Hue: describes the wavelength of the colour. It is the base colour.

• Saturation/chroma: it refers to the purity and intensity of a hue.
• Lightness/value: measures the degree of light reflected.
Colour evaluation: colour is the unique property which can be measured by

instrumental analysis more effectively than by visual analysis.
• Lab equipment: spectrophotometer, specific colorimeters for foods.
• Sensory evaluation of colour: colour scales, colour atlas, etc.
Colour could determine judges response with respect to other sensory properties. To
overcome this problem, sometimes it is necessary to hide the colour, for example, by
using a red/orange bulb in the sensory booths, or by using coloured glass for the sample
container.
Olfactory system
Smell is detected by the olfactory epithelium. Following colour, smell is the property
that mostly affects acceptance of foods by consumers.
Odorants can reach the olfactory epithelium by two routes:

• Orthonasal olfaction: the detection of an odor through the nostrils by sniffing or
inhalation.
• Retronasal olfaction: the detection of an odorant when it is released from food
in your mouth during chewing, exhalation, or swallowing. During this process,
the odorant passes through the posterior nares of the nasopharynx.
Smell is detected by the olfactory epithelium. Following color, smell is the property that
mostly affects acceptance of foods by consumers.
Smell vs. flavor

Smell of food not only goes through our nostrils (nasal odor) but also up to the back of
the throat (retronasal odor) to the nose. These smells help us sense flavor.
It is said that flavor is the perception of the taste and smell together.
Characteristics related to odor and time:

• Persistence: odor is still perceived in the absence of the food/substance tested.
• Adaption to odor after a certain period of time: decrease in the response under
conditions of constant stimulation occurs. This process prevents the brain from
going into sensory overload.
• Fatigue.
Odor testing should be fast, with a deep sniff, and the tested food should be moved
away from the nose immediately.
Testing facilities should be free of odors.
Avoid “contamination” between odors. Food samples should be kept is hermetically

closed containers.
Taste
There are five basic tastes: sweet, sour, salty, bitter and umami.
• The Japanese word “umami” translates as “pleasant to the taste, agreeable,
good, mild, savory, delicious”.
• Sources of the taste include monosodium glutamate, broth and shiitake
mushrooms.
The sensation produced by spicy foods or alcohols are not tastes. They are sensations.
The former, a burn sensation, and the second, a numbing sensation of the tongue.
The nose and mouth are vastly innervated by the trigeminal nerve, including fungiform
papillae.
Many food components stimulate these nerve endings and have irritative aspects:
• Sting from horseradish and mustards.
• Burn of chili peppers.
• Tingle from carbon dioxide.
• Numbing from menthol.
These compounds have long-lasting time properties. They can cause defensive physical
reactions, like salivation or tearing. They have desensitizing properties, meaning that
they reduce the sensitivity to stimuli and result in a delayed ability to recover full
sensitivity.
Factors that affect the perception capacity of flavor:

• Persistence: after food is swallowed, stimuli are still perceived during a certain
period of time. It could be complex, associating flavors, tastes and trigeminal
sensations.
• Adaptation: physiological changes in taste cells when they are repeatedly
exposed to a certain stimulus. It makes the taste intensity decrease with time,
while increasing the intensity of another different taste.
• Fatigue: issue related to that above. Fatigue establishes the maximum number
of products that can be evaluated before information quality provided by judges
turns poor.
• Aftertaste: it is the taste intensity of a food that is perceived immediately after
that food is removed from the mouth.
Trigeminal sensations (irritative sensations) are: spicy, astringent (drying and puckering
sensation of mouth and gums), alcoholic (numbing sensation), acrid (irritative sensation
of the mucosa of the posterior part of the oral cavity).
Touch
Merkel cells or mechanoreceptors are located in the nerves terminals under the
epidermis of the skin.
In the sensory evaluation of foods, texture perceptions through fingers, hands, lips,
tongue, gums, cheeks and soft palate are specially important.
Through touch, a variety of sensations are detected, such as temperature, weight,

surface characteristics and texture.
Auditory system
Through the ear, we are capable to detect sounds, that are the result of the vibrations
of the air. These vibrations are generated by vocal cords, lips and tongue of persons
when speaking, by objects when falling down, breaking, tearing, etc.
The vibrations are first localized by the pinna and then travel down the auditory canal
toward the eardrum (or tympanic membrane) at the end of the canal. The eardrum then
vibrates and the ossicles transmit these vibrations through the middle ear, producing a
change in the pressure of the cochlear fluids (inner ear), and then, a movement of the
basilar membrane of the cochlea. Finally, this movement is converted into a neural
response through the release of neurotransmitters.
The auditory system participates in the detection of food texture, as not only vibrations
through the air are transmitted, but also the vibrations produced by the mastication of
foods.
Texture
Texture is the sensory property detected by the senses of touch, vision and audition,
when a food is submitted to a deformation.
It is incorrect to consider “food texture” as a unique characteristic. Texture must be

described as a number of texture attributes or texture characteristics.
Texture attributes can be classified in 3 categories:

• Related to the mechanical properties of foods.
• Related to the geometrical properties of foods.
• Related to food composition
Texture
Mechanical characteristics
Hardness:
• Physical: force necessary to attain a given deformation.
• Sensory: force required to compress a substance between mola teeth (in the
case of solids) or between tongue and palate (in the case of semi-solids).
Cohesiveness: brittleness, chewiness, gumminess.

• Physical: extent to which a material can be deformed before it ruptures.
• Sensory: degree to which a substance is compressed between the teeth before
it breaks.
• Brittleness/fracturability:
o Physical: force with which a material fractures. A product of high degree
of hardness and low degree of cohesiveness.
o Sensory: force with which a sample crumbles, cracks or shatters.
• Chewiness:
o Physical: energy required to masticate a solid food to a state ready for
swallowing. A product of hardness, cohesiveness and springiness.
o Sensory: length of time (in sec) required to masticate the sample, at a
constant rate of force application, to reduce it to a consistency suitable
for swallowing.
• Gumminess:
o Physical: energy required to disintegrate a semi-solid food to a state
ready for swallowing. A product of a low degree of hardness and a high
degree of cohesiveness.
o Sensory: denseness that persists throughout mastication. Energy
required to disintegrate a semi-solid food to a state ready for swallowing.
Viscosity:
• Physical: rate of flow per unit of force.
• Sensory: force required to draw a liquid from a spoon over the tongue.
Springiness/elasticity:
• Physical: rate at which a deformed material goes back to its undeformed
condition after the deforming force is removed.
• Sensory: degree to which a product returns to its original shape once it has been
compressed between the teeth.
Adhesiveness:
• Physical: work necessary to overcome the attractive forces between the surface
of the food and the surface of the other materials with which the food comes in
contact.
• Sensory: force required to remove the material that adheres to the mouth
(generally the palate) during the normal eating process.
Geometrical characteristics: particle size and shape; particle size and orientation.
Other characteristics: moisture and fat content (oiliness, greasiness) of a product.
Instrumental analysis
There are different models of texturometers are available nowadays, although all of
them have the following parts:
• A device to contact the sample (probe).
• A motorized armature that compresses or stretches the sample (movement

source).
• A strain gauge to measure the stresses being applied (registration element).
The type of movement applied to foods could be compression, cut, puncture, extrusion,
flexion, tension, etc.
TPA (texture profile analysis): we are going to simulate 2 bites/compressions. You can
set the instrument in different parameters (degree of deformation, constant velocity of
the probe, etc.). Sometimes, the settings of the parameters are already provided
because they are standardized.
Thanks to the graphics we can determine adhesiveness (A3), cohesiveness (A2/A1),

hardness (F2), elasticity/springiness (D2/D1), fracturability (F1).
• The more cohesive, the ratio between the areas will be closer to 1. The same
happens to elasticity.
• Some internal structure of the food can break, having no opposite force.
• Hardness depends on the settings.
• Adhesiveness happens when the food sticks to the probe and the force is
opposite because of the gravity.
Unit 12: Sensory evaluation methods

Introduction
Depending on the application you will select the method, the judge. Depending on the
test and judges you will use
Discrimination: Are these products different?
Affective or hedonic: what is the acceptability of a product? Is one product preferred

over another?
Descriptive: in case of different product, how are they?
Discrimination tests
Different tests:
• Pair comparison test: A vs. B à equal or different?
• Duo-trio test: A: A vs. B à which is the same as the control (A), A or B?
• Triangle test: B A B à which is the different one, A or B?
o You present two equal and one different. You have to identify the
different one of the three.
• Two out of five test: A A B B B à identify the 2 series.
• Ranking test: A, B, C, D, E (no more than 5) à rank according to an attribute.
It has a basic question: are these products different?
Basic setup:
• 25-50 panelists.
• Screened for acuity (keenness or sharpness of perception) à can they smell and
taste well?
• Given triangle, duo-trio or paired comparison tests.
• Analysis is done using tables which compare results to chance. This analysis
ensures that the difference was real and not because people chose the correct
sample by luck/chance.
Advantage: quick and simple.
Limitations: limited results – only “yes” they are different or “no” they are no.
Affective or hedonic test

What is the acceptability of a product? Is one product preferred over another?
The aim is to know the satisfaction or acceptability degree of a product.
The basic question is “are products liked?”
The basic setup is:

• 75-100 consumers per test.
• Screened for product use (Do they buy the product? And how often?)
• Asked degree of liking (how much do they like it) and/or preference questions.
The advantage is that it provides essential information (do they like it or not?)
The disadvantage is that it may be difficult to get a representative sample of consumers.
Acceptance test
Between three products (A, B, C), which is the acceptance degree?
There are several types of scales that can be used:

• Sample Ballot: taste each product is the order listed. Circle how much you like
the product using a 9-point hedonic (liking) scale, which is the most common, or
a smiley scale, which is used with kids.
Preference test
We have two types:
• Duo test: A vs. B à A or B? Why?
• Trio test: A vs. B vs. C à A or B or C? Why?
It is used to determine which product is preferred, although people have the option to
choose “no preference”.
It is used the Sample Ballot, where you have to taste each product in the order that they
are listed, and then circle the number of the product that you prefer, all things
considered.
Descriptive test
“How are products from a sensory perspective?”
They describe and measure differences between products.
Most food companies have a panel that is trained on each of their products. To train a
panel takes several weeks to months. There are several different methods of training:
• Quantitative Descriptive Analysis (QDA method).
• Flavor Profile.
• Texture Profile.
• Spectrum Descriptive Analysis.
• Free-Choice Profiling.
Trained means that the panelists are trained to evaluate products similar to how any
instrument would give a reading.
• In essence, the panelists are calibrated so that they have an understanding of
each attribute and the range of intensity.
• For example, a trained panel would be given a sample of grape juice and would
be able to rate the level of turbidity, colour, viscosity, etc.
Mean attribute ratings are calculates, statistics are used to determine if the means are
significantly different.
The data can be plotted onto graphs – such as the spider plot – to easily compare
samples.
There are two necessary stages for the Flavor Profile, the QDA method and the Free-
Choice Profiling:
• Identification of attributes (qualitative).
• Scoring attributes (quantitative).
The basic question is “How do products differ in all sensory attributes?”.
The basic set up is:

• 4-10 panelists.
• Screened for acuity.
• Trained.
• Asked to rate intensity for all sensory attributes.
• Analysis is done using a t-test to determine if means are statistically different.
The advantage is that is gives detailed quantitative information.
The limitation is that it is time consuming.
Flavor profile
The panel used is of 4-6 trained panelists. These panelists undertake the stages 1 and 2
collectively.
The panel sit around a round table and evaluate one sample at a time and record the
ratings. Panels the discusses ratings and arrives at a consensus.
The advantage is that it is a small panel.
The disadvantages are:

• Consensus method means risk of bias from dominant personality.
• Danger of lack of consistency and reproducibility.
QDA method
The panel used is of 6-10 trained panelists. These panelists develop agreed terminology
beforehand (stage 1). They then evaluate products one a t a time in separate booths
(stage 2). Sometimes, a reference product is used at the beginning of the session to
remember the agreed terminology.
Panelists are discouraged from discussing results afterwards.
Scoring is by marking on a line. The results are analyzed statistically. It could lead to
inconsistency of results.
Free-Choice profiling
The panel used is of 6-10 trained panelists. These are allowed to invent their own terms
to describe the sensory attributes of a set of samples.
Samples are from the same category of products.
Panelists develop their own scoresheets. Stages 1 and 2 are carried out individually.
Panel training requirements are not so exigent. Panel is closer to a consumer panel.
The disadvantage is that the data analysis and interpretation is very difficult.
Unit 13: Conditions and errors in

sensory evaluation
General conditions related to food samples
Temperature: regular temperature of food consumption. For example:
• Fruits, confectionary, snacks, biscuits, bread: ambient temperature.
• Cooked or fried vegetables/meats/fish: they must be warmed until 80ºC, and
after, the must be placed in a water bath at a constant temperature: 57 ± 1ºC.
• Hot drinks and soups should be served at 60-66ºC.
• Cold beverages such as milk, juices... should be served at 4-10ºC.
• Ice-cream and smoothies: at -1ºC. Generally, these products must be drawn from
the freezer around 5 minutes before being served.
Time for sensory evaluation: 11:00-12:00; 17:00-18:00.
Limit the number of food samples to 5 as maximum.
Sample size: for liquids, a minimum of 50 mL; for solids, a minimum of 25 g.
Characteristics of judges of a trained panel

Personality:
• Work as a team
• Have cosmopolitan preferences
• Be positive but not overbearing
• Be a good listener and communicator
• Be committed
• Be flexible
Health:
• Good general health; any physiological or health restrictions must be
documented, e.g. allergies, false teeth, migraines, as these may affect their
participation in certain tests.
Sensory acuity: Assessor should have at least normal sensory acuity with regard to:
• Detecting stimuli.
• Discriminating stimuli.
• Recognizing and describing stimuli.
Errors that should be avoided
Expectation error
Knowledge of experimental objectives, or the samples to be evaluated, can influence an
assessor’s judgement. People tend to find what they expect to find. For example, codes
such as ‘A’, ‘1’ or round numbers (e.g. 100, 250) can be associated with a higher score.
Other numbers can have particular associations, e.g. 999 or 911 and danger.
• Do not include people with product knowledge on the panel.
• Provide assessors with the minimum amount of information required to perform
the test.
• Do not disclose information regarding the samples unless it is necessary for
ethical procedures, e.g. use of novel ingredients.
• Code samples. Use codes such as random three or four-digit numbers and not
letters or colours.
Suggestion effect
Comments or noises made out loud, e.g. urghh! or Mmmm! can influence sensory
judgements.
• Isolate assessors during sample evaluation, e.g. use of sensory booths.
• Discourage assessors from discussing samples before or after evaluation unless
instructed to do so.
Distraction error
Assessors can be easily distracted from the task in hand, either by stimuli in the test
environment, e.g. radios and other conversations, or by personal preoccupations, e.g.
time pressure or domestic issues.
• Ensure test area is quiet.
• Create an environment that encourages professionalism amongst the assessors.
• Prohibit the use of electronic devices, e.g. mobile phones during testing.
Stimulus and logical error

Stimulus error occurs when assessors use additional information to make a judgement
about the samples under assessment. When this stimulus is also logically associated
with one or more of the characteristics under evaluation, it is called logical error.
An obvious examples is when products of a deeper colour are presumed to be more

flavor intense. There are also other less obvious stimuli that may be exploited by
assessors, such as cues regarding product branding; running a panel at an unusual time,
which may prompt assessors to think there is a production problem; using more
luxurious containers may lead assessors to think products are of higher quality.
• Ensure sample characteristics are consistent and/or mask irrelevant differences
where possible, e.g. use of coloured lighting, blindfolds...
Halo effect and proximity error

Judgements concerning the rating of one attribute (often, the most highlighted
attribute) may influence the ratings of other attributes when assessors are asked to
judge several attributes at once. This is more likely with untrained assessors.
For example, a sweeter sample may be rated as softer, or stickier, than it would actually
have, had these ratings been made on separate occasions. Furthermore, when rating
several attributes at a time, the ratings of attributes following on from one another tend
to be related.
• Where possible, evaluate one, or at least a limited number of attributes, at a
time.
• Where possible and appropriate, use trained assessors.
• Where appropriate, randomize the order of attribute evaluation if several
attributes have to be rated at once.
Attribute dumping
If assessors are not given the opportunity to rate all the attributes they perceive as
changing in the products under evaluation, they may still record this observation using
the scales available.
For example, if products are changing in terms of sweetness but no sweetness scale
exists, they may register these changes on a flavour intensity scale such as strawberry
flavour. This is known as ‘attribute dumping’.
• Enable assessors to score all attributes which vary or indicate that opportunities
to rate all varying attributes will be given.
Habituation
When assessors score similar products on a regular basis, e.g. on quality panels, they
can develop a habit of assigning similar scores each time rather than scores which truly
represent the samples.
• Vary products or introduce spiked samples from time to time.
Order effect
The score assigned to a sample can be influenced by the sensory character of the
preceding product. For example, a sample may be rated as less sweet if it follows one of
greater intensity. In addition, some sample positions are often favoured, e.g. products
in position one are often scored higher in hedonic tests.
• Randomise or balance the order of presentation of samples.
• For affective tests, use a dummy sample in position one.
Contrast and convergence effects

If two products in the sample set are strikingly different, assessors may exaggerate their
ratings of this difference (contrast). If similar products are rated as part of a widely
varying sample set, then the difference between them may be rated smaller than it
actually is (convergence).
• Randomise or balance the order of presentation of samples.
• Consider removing outlying samples from the sample set.
Central tendency error

When using scales, assessors tend to avoid the extremes and confine their ratings to the
middle of the scale. This is more likely to occur with untrained assessors or when
assessors are not familiar with the product range.
• Train assessors in the use of the scale and expose them to a wide product range
where possible.
• Use a large enough scale to differentiate between the products, particularly with
untrained assessors.
Motivation error
A motivated panellist will learn better and, ultimately, perform more reliably. If
assessors do not respect the panel leader or product manufacturer, they may rate
samples based on how they feel. This can be an issue when using employee panels.
• Respect assessors.
• Give regular feedback to assessors.
• Carry out sessions in a professional manner.
Unit 14: Sensory analysis. Data treatment

Statistics concepts
Consumers sensory evaluation à target: consumer
• Draw conclusions of the population that are consumers.
Experts sensory evaluation à target: food product

• Not interested in the population of experts, we are interested in the product and
its characteristics.
• We want to characterize the food.
Descriptive statistics
Is summarizing the data at hand through certain numbers like mean, median, etc. so as
to make the understanding of the data easier.
It does not involve any generalization or inference beyond what is available. This means
that the descriptive statistics are just the representation of the data (sample) available
and not based on any theory of probability.
Measures used:
• Mean (average), median (50% percentile, 50% of the data is below this value),
mode (most repeated value).
• Variance, standard deviation.
• Absolute and relative frequency (times that a value is repeated). Cumulative
frequencies (add frequencies from the lowest to the highest value). Frequency
histogram.
• Probability distributions: specifies the relative likelihood of all possible
outcomes.
o Y = probability to find that variable.
o X = random variable.
o Tipes: all distributions describe population characteristics/phenomena
or relationships between them.
§ Discrete variables à mass function.
• There are no values between any pair of values, like the
number of bowls that are in a box.
• Binomial, negative binomial, poisson, geometric,
hypergeometric, discrete uniform
§ Continuous variables à density function.
• Infinite values between any pair of values, like the height.
• X2, exponential, t-Student, normal, gamma, beta, F,
continuous uniform, Weibull.
The distribution function provides the probability that the realization of a random
variable will be less, than or equal to a certain outcome. It is an alternative
representation, which can be ascendent or descendent.
If supposed a normal distribution, all values of the variables in the tails have low
probability.
Inferential statistics
It uses measurements from the sample of subjects in the experiment to compare the
treatment groups and make generalizations about the larger population of subjects.
Hypothesis tests
Null hypothesis H0: randomness/by chance explains results.
Alternative hypothesis H1: randomness does not explain results, there is something
more than the randomness.
Procedure:
• To know the distribution governing the phenomena under study.
• To know the probability of outcomes at randomness.
• Probability limits or p-values; 1 or 2-tails test.
• Reject or not reject H0
Binomial process
It is a random counting system where there are n independent trials, each one of which
has the same probability of success p, which produces a successes from those trials
(where 0<s<n and n>0).
3 values: n, p, s.
• Classical example: to toss a coin à n + p = s
• Problem: I’d like to know the probability of getting “head” when tossing a coin
three times.
o Each trial can be thought of as a random variable that returns either
“head” with probability p or a “cross” with probability (1-p). Such a trial
is often known as a Bernoulli trial, and the probability (1-p) is often given
the label q.
Ways of presentation of an event:

/ /!
• Bernouilli coefficient: nCs = ( & ) = &!(/;&)!
C474D C474D
o n=3; s=0 à(C4C) 4 (D;8) = (C4C)4 (D474C) = 1
Probability of each way of presentation of an event: ps·(1-p)n-s

• P = 0.5
Probability of observing s heads in n random experiments = pBin(s) = (sn) x ps·(1-p)n-s

• Mass function of the Binomial distribution (n, p).
N=5, S= number of consumers getting the right answer (triangle test à P = 0,3)
• N=5, S=0 à1 àprob of observing (P=0,3) à0,168
• N=5, S=1 à5 à 0,361
• N=5, S=2 à10 à 0,309
• N=5, S=3 à10 à 0,132
• N=5, S=4 à5 à 0,028
• N=5, S=5 à1 à 0,002
Are any sensory tests explained by the binomial process?
Discrimination test
Are these products different?:
• Pair comparison test: A vs. B à equal or different? à P=0,5
• Duo-trio tes: A: A vs. B à which is the same as the control (A), A or B? à P=0,5
• Triangle test: B A B à which is the different one, A or B? à P=0,3
• Two out of five test: A A B B B à identify the 2 series. à P=0,2 à there are two
probabilities, to either get it right or not.
• Ranking test: A, B, C, D, E (no more than 5) à rank according to an attribute
àyou can’t apply the binomial test here
Affective or hedonic test

The aim is to know the satisfaction or acceptability degree of a product.
• Acceptance test: A, B, C à acceptance degree?
• Preference test:
o Duo test à A vs B à A or B? Why? à it is a binomial process
o Trio test à A vs B vs C à A or B or C? Why? à it is a trinomial process
Descriptive test
It is not a binomial process.
Unit 15: Microbiological food analysis (I)

Introduction
Role of microbiologists in food industries:
• Staff training on hygienic and handling practices as well as appropriate cleaning
and disinfection procedures.
• Elaboration of GHP codes.
• Development and/or modifications of novel processes and products.
• Surveillance of the microbiological conditions of all those raw materials and
foods that are produced.
• Process control.
• Product traceability.
• Research and resolution of problems of microbiological nature.
Microbiological specifications for foods

They are useful to evaluate the potential deterioration of foods and quality / safety
maintenance of the product.
To be able to list the microbiological results and relate them to hygienic production
standards.
Verify compliance with microbiological standards and criteria --- Reg. EC- 1441/2007.
Conventional techniques: Result in a longer period (24-72h). They do not allow to verify
in situ the microbial concentration in a sample.
Rapid techniques: based on chromogenic media or molecular techniques. They allow

obtaining results in 12-24h.
Microbiological analyses for monitoring QAS and HACCP

Microbiological analyses of final products, intermediate products, environment and
surfaces are relevant within a HACCP system.
Identification of potential hazards of microbiological origin.
Evaluation of the effectiveness of the control of critical points (CCPs).

• E.g: Analysis of peroxidase and alkaline phosphatase in milk to verify thermal
treatment (pasteurization).
EC regulation for microbiological criteria in foods
Microbiological criteria also give guidance on the acceptability of foodstuffs and their
manufacturing, handling and distribution processes. The use of microbiological criteria
should form an integral part of the implementation of HACCP-based procedures and
other hygiene control measures.
‘Microbiological criterion’ means a criterion defining the acceptability of a product, a

batch of foodstuffs or a process, based on the absence, presence or number of
microorganisms, and/or on the quantity of their toxins/metabolites, per unit(s) of mass,
volume, area or batch.
‘food safety criterion’ : defines the acceptability of a product or a batch of foodstuff

applicable to products placed on the market. Pathogens.
‘process hygiene criterion’ : indicates the acceptable functioning of the production

process. Such a criterion is not applicable to products placed on the market. Pathogens
and spoilage microorganisms.
Information included in the regulation:

• Food category.
• Sampling plan: n, c.
• Microbiological limits: m y M.
• Reference analytical method.
• The point or points of the food chain in which the criterion is applied; - any
measure that must be adopted when that criterion is not met.
A microbiological criterion consists of:

• A description of the spoilage microorganisms / pathogens and / or their toxins /
metabolites.
• The analytical methods for their detection and / or quantification;
• A plan defining the number of samples to be taken and the sample size;
• The microbiological limits that are considered appropriate for the food at the
specified points in the food chain;
• The number of analytical units that must comply to these limits.
Criteria selection for the establishment of a sampling plan:

• They can differ in accordance to their objective and application.
• The need to establish a sampling plan may evaluate:
o Food-hazard combination.
o Producer/consumer level assumed.

o Economical cost.
o Potential benefits resulting from its application.
For sampling plan optimization, previous information should be compiled to assure

acceptance/rejection of a determined lot at a certain (selected) confidence level.
Sampling, transport and storage of samples for analysis

It is necessary to take a sufficiently representative number of samples, depending on
the type of contamination.
Sampling tools must be sterile and aseptically packaged. External contamination should
be avoided during sampling.
As far as possible, physical, chemical and microbiological changes in the sample should
be avoided during storage and transport until analysis.
Carbon dioxide, ice, refrigerated containers etc.
The samples should NOT be frozen before analysis (except those that have been
previously frozen) since the microbial load may differ from that actually present in the
original simple.
Sample storage without analyzing for a period longer than three days at refrigeration
can lead to a higher microbial load (multiplication of psychrotrophic microorganisms).
Liquid samples
The sample is placed in a container where it must be mixed before taking the simple.
100-500 ml of sample are placed in a sterile container and transported to the laboratory
under refrigeration (~ 4° C).
Solid samples
Use of sterile equipment (tweezers, scissors, teaspoons) depending on the nature of the
simple.
The sample must be taken at random from different locations.
Environmental control samples
Sedimentation technique: exposure of petri dishes during a given time (cfu / cm2 / h).
Collection in liquid medium: Consists in passing a certain volume of air in the form of
bubbles through a culture broth or isotonic solution, in which, theoretically, most of the
microorganisms are retained. Media: peptone water, Ringer solution.
Filtration: A determined volume of air is passed through a filter in which the carrier
particles of microorganisms are retained. Means: cellulose or gelatin membranes.
Impaction: A determined volume of air is impacted on a solid culture medium.
Culture media:
• PCA: aerobic mesophilic bacteria.
• Rose Bengal Agar / Saboraud: molds and yeasts.
• VRBG: Enterobacteriaceae.
• Baird Parker: Staphylococcus spp.
Surface control samples

Contact plates (RODAC, Replicate Organisms Direct Agar Contact):
• Several studies have concluded that a contact plate only collects 0.1% of the
microbiological contamination of surfaces (it is considered that a Rodac plate has
25 cm2), therefore it is recommended that the count be divided by 25 and
multiply by 1000 to be able to express the result in cfu / cm2 of the studied
surface.
Laminocultures:
• Sampling of liquid foods (cfu / ml): laminocultures with two or three culture
media.
• Surface control or solid foods (cfu / cm2): In this case they have a flexible
support that with the simple hand pressure to an angle of 90° with respect to the
stopper, thus allowing a perfectly parallel position with the surface to be
sampled.
Microbial count is carried out by counting the colonies and dividing by 10.
Laminocultures (Sampling of liquid foods):

• Microbial count in liquids of the table can be achieved by visual comparison with
the figure below. If the count obtained is higher than 100,000 cfu / ml, it is
recommended to repeat the process from a dilution of the sample in Peptone
Water and then multiply the results by the dilution factor. In this way, more
reliable results are obtained.
Swabbing method:
• It is mostly used for irregular surfaces.

It is performed with sterile templates with an area of 10x10 cm.
• The plates should be counted with a number of colonies between 30-300.
• The arithmetic average is multiplied by the inverse of the dilution and by the
inverse of the inoculum volume (surface plating). Pour plating (1 ml) does not
need this transformation.
• Surface plating = 0.1 ml.
• Nº. of microorganisms / cm2 = (number of cfu / plate / 100) x 10.
Quality assurance of culture media for microbiological analysis (ISO

11133:2014)
Assurance and quality management of culture media.
Requirements for microorganisms that are used to test culture media.
Physico-chemical characteristics and monitoring procedures for culture media:

• Agar.
• Broth cultures.
• Transport media.
Requirements and information provided by the culture media suppliers.
Culture medium: formulation of substances, in liquid, semi-solid or solid form, which

contain natural and/or synthetic constituents intended to support the multiplication,
(with or without inhibition of certain microorganisms), identification or preservation of
viability of microorganisms.
Classification of culture media (as a function of their use).
Transport medium: medium designed to preserve and maintain the viability of

microorganisms whilst minimizing numerical change in the time period between sample
collection and laboratory processing of the sample.
Preservation medium: medium designed to preserve and maintain the viability of

microorganisms over an extended period, to protect them against the adverse
influences which may occur during long-term storage and to allow recovery after this
period.
Diluent medium & Suspension medium: medium designed to separate microorganisms

from a solid test product into a liquid phase and/or to reduce their concentration by
dilution without multiplication or inhibition during the time of contact.
Resuscitation or pre-enrichment 103edium: medium enabling stressed and damaged

microorganisms to repair and recover their capacity for normal growth without
necessarily promoting their multiplication.
Selective enrichment 104edium: enrichment medium that allows the multiplication of

specific microorganisms whilst partially or totally inhibiting the growth of other
microorganisms.
Non-selective enrichment medium: enrichment medium that allows the growth of a

wide variety of microorganisms.
Isolation culture media: they are used for microbial identification.

• Differential media.
• Chromogenic media.
• Confirmation media.
Testing of the performance of solid culture media (quantitative tests)

They are based on the productivity (PR) and selectivity (SR) ratios:
• PR: proportion of the number of colonies counted in the culture medium of
interest (Ns) in relation to the existing number (N0) in a reference medium.
PR = Ns/N0
• SF: it is evaluated by inoculating different dilutions of a problem culture medium
compared to a reference medium.
o D0: highest dilution where growth is observed in the reference medium.
o Ds: highest dilution where growth is observed in the testes medium.
o SF, D0 y Ds are expressed in logarithmic units.
S F = D0 – DS
Testing the performance of liquid culture media (quantitative tests)
From both tests, those dilutions with a number between 10 and 100 cfu are chosen.
Productivity is considered satisfactory if the dilutions obtained from the tested medium
and the reference medium match.
Selectivity (SF) = D0 – DF
• D0 = lowest dilution showing no growth in the reference medium.
• DF = highest dilution showing growth in the problem medium (at least 10 cfu).
• SF> 2
Unit 15: Microbiological food analysis (I)

Enumeration and detection of foodborne pathogens
Listeria monocytogenes (ISO 11290-1 and 11290-2)

It can grow in refrigeration temperatures, low pH and high solid concentrations.
Fraser broth base: it is a medium used for the selective enrichment of L. monocytogenes
in food
• Listeria hydrolyses the middle aesculin in glucose + esculetin, which forms a black
complex with ferric ions from ferric citrate.
• Its high salt concentration increases its selectivity.
• Lithium chloride --- inhibits growth of Gram-negative bacteria.
• Supplement --- nalidixic acid (antibiotic).
Oxford and Palcam agar: selective agar for the isolation of L. monocytogenes in food
• Listeria forms small colonies of black or grey-green color.
• After 48h of incubation they can form a central depression.
• Contains a chromogenic substrate that causes typical colonies of L.

monocytogenes to turn green.
Salmonella (ISO 6579)

Rappaport Vassiliadis broth: Medium used for selective enrichment of species of
Salmonella spp. (apart from Salmonella Typhimurium) from food.
• Nutritious culture medium that contains a phosphate buffer system, high
concentration of magnesium and sodium salts and the presence of green
malachite (oxalate) dye.
• The selective enrichment is due to the ability of Salmonella to survive and
multiply at relatively high osmotic pressures (due to the high concentration of
magnesium chloride in the medium), at relatively low pH.
• The toxic effect of malachite green to Salmonella is suppressed due to the
presence of magnesium chloride.
• Incubation at 41-42 ° C enhances selectivity towards Salmonella isolation.
Agar Xylose Lysine Deoxycholate (XLD): differentiation of typical colonies of Salmonella

Sodium deoxycholate inhibits the growth of the Gram-positive contaminant flora.
• The acid produced by the fermentation of D (+) - Xylose, lactose or sucrose
produces a turn to yellow of the Phenol Red content in the medium. The typical
colonies of Salmonella are recognized as having red-orange colonies with a black
center due to the increase in pH that they have caused in the medium and the
consequent turn of the Phenol Red.
• In addition, by the presence of Sodium Thiosulfate and Ammonium Iron (III)
Citrate, the hydrogen sulfide producing bacteria provide a black coloration of the
medium at high pH.
Staphylococci (ISO 6888-1 and 2)

Baird Parker agar base: agar used for the differentiation of S. aureus colonies
• Egg yolk emulsion and potassium tellurite must be added to the agar base.
• The growth of undesirable microorganisms is inhibited by the presence of lithium
chloride and potassium tellurite.
• Due to the presence of sodium pyruvate and glycine, the growth of Staphylococci
is favoured.
• S. aureus gives rise to black colonies, by the reduction of potassium tellurite in
combination with the egg yolk, surrounded by a halo of transparency due to
lecithinase activity.
• For the direct demonstration of coagulase-positive S. aureus, the medium can be
supplemented with rabbit plasma instead of egg yolk.
Escherichia coli O157:H7 (IDO 16654-2002)

Mc Conkey Sorbitol agar:
• This medium replaces the lactose from the McConkey Agar medium with sorbitol
as an energy source.
• E. coli O157: H7 not fermenting sorbitol grows on this colorless medium unlike
the rest of E. coli that will appear pink.
• Colonies of E. coli O157 are negative for beta-glucuronidase.
• Both the violet crystal and the bile salts act as inhibitors of Gram-positive
bacteria and the neutral red is the pH indicator.
• For characterization of serotype O157 a supplement of cefixime + tellurite is
added (CT-SMAC).
Bacillus cereus (ISO 7932-2004)

MYP AGAR (Mannitol Egg Yolk Polymyxin Agar):
• The addition of Polymyxin-B Sulfate to this agar base inhibits the growth of
secondary flora.
• Is negative mannitol, which allows its distinction from the accompanying
mannitol-positive flora, which causes Bromotimol Blue to turn yellow.
• Being a microorganism with lecithinase activity a precipitation white halo is
formed around the colony as a result of the degradation of egg lecithin.

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3º Grado en Ciencia y Tecnología de los Alimentos

Reservados todos los derechos.

Part 0: Quality management systems in

Analytical quality assurance

In an analytical quality assurance there are 4 parts:

Quality management procedures: these documents describe the methodology for

Ciencia y Tecnología de los Alimentos 1

ISO 10725 standard

Moreover, this International Standard covers technical competence requirements that

Likewise, according to Regulation (EC) No. 2004/882/CE*, results coming from

Its characteristics are:

The steps that you have to follow are:

Ciencia y Tecnología de los Alimentos 2

o Review of the applied system.

ENAC evaluates the compliance of established requirements in the following standards:

Ciencia y Tecnología de los Alimentos 3

• Calibration laboratories UNE-EN ISO/IEC 17025. These labs calibrate

ENAC accreditations are recognized in more than 50 countries as ENAC is a signatory of

In addition, ENAC and the international accreditation organizations produce specific

For the administration:

Ciencia y Tecnología de los Alimentos 4

• Allows the Administration to participate, to the extent of their needs, in the

For the market:

Validation of analytical methods

• In-house: methods created in the lab that need to be validated.

The steps to do a validation are:

The types of validations are:

Ciencia y Tecnología de los Alimentos 5

This means that, if we were going to measure samples in small

Ciencia y Tecnología de los Alimentos 6

Percentage of recovery: To determine the effectiveness of a method (and also of the

• Cx = average concentration of analytical results.

Repeatability: Degree of similarity obtained in simultaneous repetitions of the analytical

Reproducibility: Reflects the variability when applying a method at different conditions

Ciencia y Tecnología de los Alimentos 7

A robust method is a good indicative of its confidence.

Ciencia y Tecnología de los Alimentos 8

Part 1: Introduction and sampling

• Know from different point of views the composition (scientific, industrial,

Problems when performing food analysis:

Ciencia y Tecnología de los Alimentos 9

The application depends on: it is the selection of a specific method to analyze a

The main food analytical techniques are:

Steps to follow for the performance of food analysis

Questions to consider in a QMS

Ciencia y Tecnología de los Alimentos 10

Official analytical methods: AOAC

AOAC (Association of Official Analytical Chemists) is a private scientific association

Performance and development of an AOAC official method:

General guidelines on food sampling

Sometimes it is important to monitor the food sampling,

Ciencia y Tecnología de los Alimentos 11

Selection of sampling procedures

Its limitations are:

The main purposes of food sampling are:

Ciencia y Tecnología de los Alimentos 12

Factors considered in the sampling process

Variables: they measure a given value or concentration in a food in order to check

Stratified sampling: divide the sample into smaller samples.

Ciencia y Tecnología de los Alimentos 13

Systematic sampling: random sampling process through an established time interval.

Storage, transport and pre-treatment of samples

If necessary, refrigeration or freezing should be applied: