A very good study material for food technology students Food testing and rapid detection methods by Manisha Dabral

1. FOOD TESTING &
RAPID DETECTION
METHODS
BY MANISHA DABRAL
SR.NO - 55

2. Food testing and analysis is an essential part of the food safety ecosystem to assure that the food is safe to
consume. This includes strengthening the network of food testing laboratories, assuring quality of food testing,
investing in human resources, carrying out surveillance activities and educating consumers.
INTRODUCTION
FOOD TESTING: Food test a method by which
certain components of a food mixture can be
detected.
RAPID DETECTION METHOD: Any method that
detects a food-borne pathogen in a shorter
period of time, as compared with conventional
methods is considered as a rapid method.

3. The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally.
Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food
supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming
and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required
in many food analyses.
FOOD TESTING WITH RAPID TESTS
METHODS:
1. Biosensors,
• Bioluminescence biosensor
• Fiber optic biosensor
• Electrical impedance biosensor
• Piezoelectic biosensors
2. molecular detection method
• Fluorescent in situ hybridization
• PCR
1. Biosensors:
• Defined as the indicator of biological compound,
• Biosensing methods for pathogen detection are centered on four basic pysiological or genetic properties of
micsroorganism.
i. Metabolic pattern of substrate utillization
ii. Phenotypic expression analysis of signature molecules by antibodies
iii. Nucleic acid analysis
iv. Analysis of interaction of pathogens with eukaryotic cells
3.Immunological detection methods
• LFD
• ELISA
4.Microscopic methods
• Direct epifluorescent Filter technique
• Flow Cytometry
• Solid-phase cytoometry

4. 2.Bacterial bioluminescence:
• The gene resposible for luminescenece in bacteria is call lux gene.
• The gene is introduced into the host specific bacteria.
• Detectors are then used to measure the emitted light.
• Capable of detecting 100cells/hr.
 Fiber optic biosensor:
• Basic principal of this is that when light propagates through the core of the optical fiber i.e.waveguide.
• It generates an evanescent field outside the surface of the waveguide
• When fluoresceent labeled analytics such as pathogens or toxins bound to the surfacee of the waveguide,are excited
by evanescent wave generated by laser (635nm) & emit fluorescent signal
• Signal travels back through the wavwguide in high
 Bioluminescence biosensor: Measures changes in luminescence emitted by living microorganisms.
There are two types of bioluminescence found in microbes.
1. ATP bioluminescence& 2.Bacterial biolumnescence.
1.ATP bioluminescence:
• Used to measure the effectiveness of cleaning surfaces and utencils
• Take a swab sample and combine it with a mixture of luciferas.
Following reaction takes place;
Luciferin + ATP= Luciferyl adenylate +PPi
Luciferyl adenylate +O2 = oxyluciferin+ AMP + light

5.  Electrical impedance biosensor:
• Detects microbs directly due to production of ions from metabolic end products or indirectly from liberation CO2
• Microbs metabolism results in an increase in impedance.
a bridge circuit measures impedance.
METHOD:
• Population of microbs provided with nutrients(no electrolyte) like lactose.
• Microbes utilize it, and convert into lactic acid (ionic forms) ,thus changing the impedance.
• Impedance is measured over 20 hours after inoculation in specific media .
Advantages:
• Simple to perform
• faster than agar plate count
• Capable of analyzing hundreds of sample at the same time since the instrument (bactometer) is computer
driven.
Disadvantages:
• Applicable for samples with high no. of microorganisms
• Food matrix may interfere with the analysis.

6. • Following pre-enrichment to reach these detection levels,the results can be obtained quickly (in about 3 hrs.)
Fish in combination with flow cytometry has been used afor rapid culture-independent detection of Salmonella
spp.in the products of tomatoes and other fresh products.
ii. Polymerase chain reaction(PCR):
One of the most commonly used molecular-based method for the detection of foodborne bacterial pathogens is polymerase
chain reaction (PCR).There re three processing steps in PCR:
i.Denaturation, ii.annealing, iii.Extension
• In the 1st step of the PCRmethod,DNA to 95-98oC,at which point double stranded DNA separates into two single strands.
• In the annealing step,synthetic oligonucleotide primers are added and bind to the single strands at a temperature of 55oC

7.  Piezoelectric biosensors:
• General principle is based on coating the surface of piezoelectric sensor with a sensitive binding substances.
• e.g. antibodies to bacteria and then it in a solution containing bacteria.
• The bacteria will bind to the antibodies and the mass of the crystal will increase while the resonance will decrease
propotionally.
2.Molecular detection methods:
here has been an explosion in the past 15 years in the introduction of nucleic acid-based assays for the detection & identification
of foodborne pathogens. There are many DNA-based assay formats,but only probes and nucleic acid amplification techniques
have been developed commercially for detecting foodborne pathogens .
Types:
i. Fluorescent In situ Hybridization (FISH),
ii. Polymerassee chain reaction(PCR)
 Fluorescent In Situ Hybridization(FISH):
• FlSH with oligonucliotide probes directed at rRNA is the most common method among molecular techniques not based on PCR.
• The probes used by FISH tend to be 15-25 nucleotides in length,and are covalently labeled at their 5c end with fluorescent lables
.
• After hybridization ,the specifically stained cells are detected using epifluorescence microscopy.
• The detection limit of this technique is around 104CFU/g

8. • Finally in the last step,the primer extended by DNA polymerase in the presence of adenine,guanine,cytosine and thymine
• The extension by polymerase occurs at a temperature near 72oC
 PCR METHODS: i. Simple PCR, ii. Multiplex PCR, iii. Real time PCR
PCR have been used in the detection of numerous foodborne pathogens like Listeria monocytogenes, Escherichia coli O157:H7,
Staphylococcus aureus, Campylobacter jejuni, Salmonella spp. and Shigella spp.
 Multiplex PCR (mPCR):
Multiplex PCR offers a more rapid detection as compared to simple PCR through the simultaneous amplification of multiple gene
targets. The basic principle of mPCR is similar to conventional PCR.Previously, mPCR was used to detect around two to three
pathogens only. Now, mPCR is more advanced and it can detect up to five or more pathogens simultaneously.

9.  Real-time or quantitative PCR (qPCR)
• Real-time PCR or quantitative PCR is different from simple PCR whereby it does not require agarose gel
electrophoresis for the detection of PCR products. This method is able to monitor the PCR products formation
continuously in the entire reaction by measuring the fluorescent signal produced by specific dual-labeled probes or
intercalating dyes.
• The increase in fluorescence can be monitored in real time,which allows accurate quantification over several orders of
magnitude of the DNA target sequence.
• There are two general techniques used to obtain a fluorescent signal from the amplification of product in PCR
• The first technique uses the inherent properties of flurescent dyes such as SYBR Green I.
• As the dyes bind to dsDNA & undergo a change in shape ,it increases their fluorescence.
• The 2nd appproach uses fluorescent energy transfer(FRET).
• FRET relies on the presence of two molecules that interact with one another,where at least one of the molecules must
have flurescent properties.
• The fluorescent molecules is known as the donor,while the non-fluorenscent molecule is known as the acceptor.
• During fluorescent resonance energy transfer,the donor molecules is excited by an external source.
• It emits light at a shifted, longer wavelength,which is then used to excite the acceptor molecules.
• It is not necessary for the acceptor molecule to emit light.
• The signal emanating from the acceptor molecules will be detected using the real- time instrument.
Advantages: Real- time PCR collects data in the exponential growth pphase as opposed to the plateau phase.
Results can be obtained in an hour or less,which is considerably faster than conventional PCR.
PCR requires only about 20-90 mins.
The limit of quantification of real time PCR with food samples is around 103- 104 CFU/g

10. 3. Immunological methods:
• The antibody-based system has facilitated the design of variety of assays and formats.
• Incubation times are usually very shhort for methods such as agglutification reactions commonly used for rapid
identification of microorganisms.
• Normally,the antibody is labeled with a fluorescent reagent or with an enzyme so that the antigen-antibody interaction may be
visualised more easily when it occurs.
• In some cases the antigen-antibody complex formed is directly measurable or even visible.
TYPES: i. LFD, ii.ELISA
 Lateral Flow Devices(LFD):
• Lateral flow devices (LFD) are typically comprised of a simple dipstick made of a porous membrane that contains colored latex
beads or collooidal gold particles coated with detection anibodies targeted toward a specific microorganism.
• The principle are found on the base of the dipstick which is put in contacted with the enrichment medium.
• if the target organism is present ,then it will bind with the colored particles.
• This conjugated cell/particle moves by capillary action untill it finds the immobilized capture antibodies.
• Upon binding with these, it forms a colored line that is clearly visible in the device window,indicating a positive result.
• As with other immunoassays, LFD also require previous enrichment.
• The technique is extremely simple to use and easy to interpret,requires no washing or manipulation,and can be completed
within 10 mins after culture enrichment.

11. There are various LFD on the market that have been validated for detecting different foof borne pathogens.
 Lateral Flow test types:
• Sandwich assay format: Used for
large analytes (proteins) with
multiple antigenic determinants.
• Competetive assay format: Used
for small molecules
(hormones,drugs,etc).
 Enzyme-linked Immunosorbent Assay(ELISA)
• The enzyme-linked immunosorbent assay(ELISA) is a biotechnical technique.
• That combines an immunoassay with an enzymatic assay.As LFD,it is a “sandwich” assay.
• An antibody bound to a solid matrix is used to capture the antigen fron enrichment cultures and a second antibody
conjugated to a enzyme is used for detection.

12. • The enzyme is capable of generating a product detectable
by a change in color,or in the case of enzyme-linked
fluorescence,which allows for indirect measurement using
spectrophotometry of the antigen present in the sample.
• Detection using automated and robotic ELISA is widely
used since they can reduce detection times after
enrichment to as low as 1-3h.
• Thus ,the results can be obtained in 2-3 days instead of the
3-5 days needed by conventional method.

13. 4.Microscopic methods:
 Flow cytometry
• Flow cytometry quantitavely measures optical characteristics of cells when they are forced to
pass individually through a beam of lght.
• Fluorescent dyes can be used to test the viability and metabolic state of microorgasnism.
• Samples are injected into a fluid(dye),which passes through a sensing medium in a flow cell.
• The cells are carried by the laminar flow of water through a focus of light,each
cell emits a pulse of fluorescence,and the scattered
• light is collected by lenses and directed onto selective detectors (photomultiplier tubes).
• This techniques is fast,automatic,and potentially very specific,as long as apporopriate dyes
are available for selectively labeling specific types of microorganism and appropriate methods
for separating cells from food are utilizd so as not to interefere with detection.
• The sensitivity of flow cytometry is low.
• The detection limit with food sample is around 105-107 CFU/g .
• Currently,there are various flow cytometry methods developed for foods,especially for liquid
samples such as dairy products,water,and other beverages.

14.  Solid -Phase Cytometry:
• Solid-phase cytometry(SPC) is a technique that combines aspects of flow
cytometry and epifluorescence microscopy.
• After filtration of the sample ,the retained microorganism are fluorescently labeled
with argon laser excitable dyes on the membrane filter and automatically counted
by a laser scanning device.
• Each fluorescent spot can be vidually inspected with an epifluorescence
microscope connected to a scanning device by a computer -driven moving stage.
• Depending on thefluorogenic labels used ,information on the identify and
physiological status of the microorganis can be obtained within a few hours.
• Only applicable if the no. of bacteria present is high.
• Recommended or the determination of the total viable mmicrobial count in liquid
samples,and enumeration of pathogens in food samples.

15.  Direct Epifluorescent Filter Techniques
• The direct epifluorescent filter technique(DEFT) is a microscopic method for
the enumeration of viable cells in a sample.
• Based on the binding properties of the fluoro-chrome acridine orange.
• Once treated with detergents and proteolytic enzymes ,the samples are
filtered through a polycarbonate membrane.
• The cells are stained on this same filter and examined under an
epifluorescent microscope , a process that.
• DEFT is a very labor-intensive technique.
• Does not have the capability of processing a large no. of samples.
• Only applicable if the no of bacteria is high.
• Additionally ,fluorescent food material can be trapped on the filter ,and the
technique can be used with raw food and usually for enumering totalviable
microorganism.
• DEFT may be used for the detection and enumeration of specific bacteria in
food samples.

16. Rapid Food Testing Devices & Kits
Country’s Food Regulator, the Food Safety and Standards Authority of India (FSSAI) has made a quick progress towards the use of advanced technologies
for food testing in the year 2019. Several new rapid food testing devices/kits for detection of food-borne pathogens and toxins have been introduced during 2019.
These devices ensure “faster, better, cheaper” real-time testing of food. These are expected to become an integral part of quality assurance/quality control
programs in the food industry and also for regulatory and surveillance purposes in 2020.

20. Accuracy & precision in food testing
Accuracy is defined as the closeness of the value obtained by the method in relation to the “true value” for
the concentration of the constituent. It is often expressed as percentage accuracy. Inaccuracy is, as a
corollary, the difference between the measured value and the “true value”.
The concept of a “true value” is, of course, hypothetical because the “true value” for a nutrient in a food is not
known. All analytical values are therefore estimates of that value.
Büttner et al. (1975) take the view that there exists a true value for all constituents in a sample of food. This is
fundamental to the analysts' art; it is not true that the value for a defined analytical sample of a food is the
“true value” for all samples of that food. The sampling error and the analytical errors for any specific method
determine the confidence limits for all determined values.
The accuracy of a method is usually determined by reference to standard amounts of the analyte and
preferably by the analysis of standard reference materials (SRMs) or certified reference materials (CRMs) that
have been analysed, often using several compatible methods, by a group of skilled analysts to provide
certified values together with the confidence limits of that value.
This is a difficult attribute to measure because its true value is unknown. The first stage is to analyse
standard amounts of the pure analyte. Recovery studies of standards added to the foods are useful,
especially if a series of different amounts is used and then a comparison made of the sensitivity of the method
for pure standards and the added standards.

21. Precision is a measure of the closeness of replicated analyses of a nutrient in a sample of food. It is a
quantitative measurement of “scatter” or analytical variability. Strictly speaking, it is imprecision that is
measured by carrying out replicate analyses on the same sample (which must be homogeneous and stable).
The measurements may be made by one analyst within one laboratory when the assessment is designated
“repeatability” (that is, within-laboratory precision) or by several analysts in different laboratories when it is
designated “reproducibility” (that is, between-laboratory precision). Comparisons can also be made among
different analysts in one laboratory (called “concordance”), and by one analyst on different occasions.
In each case the standard deviation (SD) of the analytical values is calculated (which means that there must
be a sufficient number of replications). The SD is customarily divided by the mean value to give a relative
standard deviation (RSD), or multiplied by 100 to give the coefficient of variation (CV). In analytical literature,
the RSD is used for reproducibility and rsd for repeatability.
It is important to recognize the distinction between accuracy (see the definition above) and precision. One
can have very high precision (a low RSD) and poor accuracy and, conversely, have high accuracy with poor
precision where the confidence limits of the value obtained will be wide. The ideal is to combine high
precision (low RSD) with high accuracy (as judged by the value obtained with an SRM).

22. Food quality can be assessed, evaluated, and controlled using destructive and nondestructive methods.Generally, the
food sample is lost after evaluation by the destructive methods. Using nondestructive quality evaluation techniques, food
samples can be analyzed without affecting the food system.
Advantages of non-destructive techniques:
Non-destructive food quality evaluation methods
Limitations of NDT :
 Expensive installation
 Requires experts to work with instruments
 Proper maintenance of equipment is required
 Used for internal quality assessment of all the items.
 Assessment is fast,user friendly,cheaper & accurate
 Increases product safety and reliability during operation
 Enhances the reputation of manufacturer as a producer
of quality goods
 Boost sale of product and provide economic benefits to
manufacturer.

24. 1.Optics:
1.1 Imaging analysis
• Imaging techniques
• Provide spatial information from
electromagnetic spectrum
• Determine quality and detect safety
• Classification and sorting of agriculture
produces
1.1.1 Hyper spectral imaging
system
 Principle:
Difference in the chemical
composition and inherent physical
structure, reflect, scatter, absorb
and emit electromagnetic energy in
distinctive patterns at specific
wavelengths

25. • Similar spectral patterns will indicates similarity in chemical
composition.
• Different concentrations of the major chemical compositions make the
difference in reflectance (absorbance) values at some key wavelengths.
1.1.2 Multispectral imaging system
 Langreo et al. (2014) determine the efficiency of MRI by detecting the foreign material present in powdered food by
incorporating peanut powder into wheat flour.

26.  Eight sample of peanut adulterated in flour were prepared ranging from 10 to 0.01 % by weight

27. 1.2 Spectroscopy
• Simple, accurate and expeditiousness
• Require minimum sample preparation
1.2.1 NIR (Near Infrared Resonance):
 Principle
Based on optical properties that respond of matter to near-infrared (0.7-25 μm) where the light either get
absorbed, reflected or transmitted

29. • Restricted to fruits with homogeneous pulp and thin skin
• Assess soluble solid content (SSC), titratable acidity and/or other physiological
properties in intact fruits such as in prune, stonefruit and apricot
• High penetration in range of 0.7-0.9 μm

31.  Slaughter et al. (1996) determine the soluble solid content of tomatoes of 30 popular varieties including
Arletta, Better Bush, Celebrity, Early girl, Heat wave, Jackpot, Sunny, Tango, etc were harvested in stage
of maturity from green to ripe red fruit.
 The optical absorption spectrum from 400 to 110 nm was measured at five different location in a
sequential manner (first four at equator and fifth at blossom end)

32.  Slaughter and Crisosto, 1998 determining the intemal quality of intact kiwifruit by
measuring fructose content (r = 0.96, SEC = 1.96%), glucose content (r = 0.97, SEC =
1.68%), soluble solids content (r = 0.99, SEC = 0.78"Brix), and dry weight (r = 0.97, SEC
= 0.61%) of kiwifruit.

33. 2. X-ray and Computed tomography:
 Principle:
X-rays are generated by bombarding electrons on a metallic anode (X-ray tube).
Traditional CT (Computed Tomography) is an imaging modality where an x-ray tube is rotated
around an object or objects and the attenuation is recorded on a detector
 Uses and application:
• Wavelength ranges from 0.1 to 10 nm with corresponding energies of about 0.12 to 12 keV
• Low penetration power
• Detect internal density changes and structural discontinuities caused by voids and cracks
variation.
• Determine bruising, water content of apples, select mature lettuce heads and detect hollow
potatoes and split pit in peaches etc.
• Barcelon et al. (1999) studied the internal changes occur in peaches with time when stored
weeks at 25°C, 75 % RH and suggest that

34. • Presence of voids, lower moisture content and
lower density fruit tissue lead to low X-ray
absorbed (dark) images, corresponding to a low
CT number.
• Moisture and density reduced significantly from
84.6 % to 62.6 % and from 1.082 to 0.955 g/cm³
and soluble solids increased from 11.5 to 19.23
brix

35. 3. Mechanics:
Ultrasonic waves (above the range of human hearing i.e. 20 KHz to 1 MHz) and acoustic sound waves (in the range of
human hearing i.e. 20 Hz to 20 KHz) are used to evaluate the quality of fresh vegetables non-destructively
3.1 Ultrasonic waves:
 Principle:
Ultrasound is generated by a transducer containing a ceramic crystal which is excited by a short electrical pulse having
several sine cycles is converted into mechanical wave that propagate as a short sonic pulse
 Uses and application:
Used to quantify relation with the ripening,
maturity, firmness and other internal quality of
fruit and vegetables
Meftah and Azimin (2012) demonstrate the
potential of ultrasonic for detecting foreign
bodies in canned products by mounting a
transducer to the side of can.

36. Ultrasonic inspection of cans
Oil was used to couple the ultrasonic pulse to the specimen. A TD Scan Advanced Ultrasonic Inspection System was used to
drive the transducer and to receive the echo signal. The result was displayed on the TD Scan monitor in real time.
Three types of foreign bodies were deliberately introduced i.e.
1.FB1: a rock of dimension 62 x 38 mm,
2.FB2: an aluminium plate of dimension 30 x 23 x 4 mm
3.FB3: an aluminium plate of dimension 122 x 21 x 1 mm

37. 3.2 Acoustic Principle:
In acoustic sound, a device used to lightly tap or thump the commodity to create a sound wave that pass through the
product tissue which is then scattered, transmitted or reflected from the object.
• Acoustic emission (AE) is an important attributed quality of food texture specially snack type food
• Crunchiness and crispness, which are noticed as signs of freshness, are other aspects of acoustic properties.
• Alteration of food texture can be measured by analysis of the emitted sound.
4. Electromagnetic:
4.1 Nuclear magnetic resonance Principle:
When nuclei with a magnetic moment, like protons, are placed in an external magnetic field, it align along the magnetic field
direction and rotate about their own axes at a specific frequency Ѡ₀ and develops a net macroscopic magnetization M
 Properties of MRI:
Number of a particular nucleus in the sample
such as the protons in water or oil
∝
Energy absorbed by the
nuclei

38. Now, NMR is well known as magnetic resonance imaging (MRI) MRI measure the NMR properties of a sample
as a function of spatial position.
Pulsed linear magnetic field gradients are used to produce frequency variations across the sample which are
converted into spatial coordinates.
Application:
 (Chen et al., 1993) find a desirable features for high speed sorting
using a surface-coil NMR probe that determine the oil/water
resonance peak ratio of the signal from one region in an intact
fruit.
 (Chen et al., 1993) use MRI based sensor for on-line grading of
cherries
 A set of 39 cherries were used with spin-echo pulse sequence
was used to acquire one-dimensional images.
 The cherries(both whole and pitted) were placed on the sample
holder, the pitting direction must be perpendicular to the moving
direction in order to detect clear distinctions
 Results were classified based on valley to peak ratio.
 If a valley to peak ratio is greater than or equal to the threshold
value it is classified as a whole cherry.
Sorting of cherries using NMR

39. 5. Chemical
5.1 E-nose:
A device intended to detect, identify and quantify specific
component of odors and flavors.
 Principle:
Sample delivery system:Generates headspace and
injects sample into detection system.
Detection system: React to presence of volatile
compounds, undergo chemical change and produce useable
signal.
Computing system: Combines the responses of
individual sensor elements and generated sample information
 Barbri et al. (2008) develop an electronic nose for the
quality control of beef and sheep meat which were stored
at 4°C for up to 15 days.
 The electronic nose system was successful in
discriminating between unspoiled/ spoiled beef or sheep
meats at rate of 98.81 or 96.43 %, respectively

40. 5.2 E-tongue:
An array ofsensorsthat are immersed in liquids, in order to identify their different physical-chemical characteristics, for
example, “tastes”.
 Principle:
Working Electrode: Serves as a surface where electrochemical redox reaction takes place. Surface area should very less
(few mm²) to limit current flow.
Reference Electrode: Measure the working electrode potential.
Auxiliary electrode: It completes the cell circuit. It is generally inert conductor. The current flow into the solution via the
working electrode leaves the solution via the counter electrode.

41. Safety issues of processed foods available in market
PROCESSED FOOD:
Any food that is altered during processing to make it more convenievnt,shelf stable or flavorful.
Highly Processed Food are chemically treated with additives or preservatives to improve their taste,texture
or to extend the shelf life.
Ultra Processed Food contains an unevenly high ratio of calories to nutrient may be considered unhealthy.
These product contain increased amount of salt,sugar and salt.
WHY PROCESSED FOODS ARE HARMING PEOPLE ?
 High in sugar and high-Fructose corn syrup.
 Engineered for overconsumption.
 Contain artifial ingredients.
 People can become addicted to junk food.
 Often high in refined carbohydrate.

42.  Low in nutrients.
 Low in fiber.
 Often high in trans fat.
EXAMPLE: canned goods, cookies,chips,soda,candy,bacon,sausages,hot dog,cheese slice etc.
 HEALTH RISK OF HEAVILY PROCESSED FOODS:
 Obesity,
 Anxiety & depression,
 Metabolic Syndrome-defined as a group of risk factors that can
lead to heart disease and type 2 diabetes,
 Colorectal cancer- When we eat processed meat which include
lunch meat, bacon ,sausage,hot,dog etc.
 Inflammatory Bowel diseases-Due to consumption chemicals
additives called emulsifiers found nearly in every processed food
product.

43. HOW TO REDUCE ROCESSED FOOD IN
YOUR DIET ?
 Check the label- the longer the ingredients list the more processed a
 food is .
 Opt for minimally processed meat-choose meatsa that have been minimally
processed.
 Cook more meals at home-avoid out at retsurants make your own meal
at home.
 Avoid junk food as much as possible.