Rapid Detection Methods

Conventional Methods

• based on traditional culture methods

• identification is based on the morphological, biochemical,

physiological (phenotype), and/or genetic characteristics

(genotype) of the microorganism using a variety of approaches

• still regarded as the gold standard due to their reliability,

efficiency, sensitivity, and range of applications

Dry Weight Methods

• indirect method of measurement of

microbial growth

• simple method used for biomass estimation

• widely used for the assessment of biomass

in fermentation culture samples

Viable Count Method

• used to estimate microbial populations

• relies on the growth of the cells in

either a liquid culture medium, on an

agar media, or on membrane filters

Membrane Filtration

• technique for testing water samples

• water is drawn through a special

porous membrane designed to trap

microorganisms larger than 0.45 μm

• the filter is applied to the surface of

Endo agar plates and incubated for 24

hours

Turbidity Test

also widely used for the

estimation of cells in suspensions by

using a spectrophotometer

Gram Stain

⚬ common technique used to differentiate two large groups

of bacteria based on their different cell wall constituents

Rapid Detection Methods

• able to detect the presence of pathogens in

raw and processed foods immediately

• sensitive enough to detect pathogens that

present in low numbers in the food

• more time-efficient, labor-saving and able

to reduce human errors

Immunological Methods

• based on antibody-antigen interactions

• have been used for the detection of E. coli, Salmonella

species, L. monocytogenes, Bacillus cereus,

staphylococcal enterotoxins, and Campylobacter

species, among others

Enzyme-Lined Immunosorbent Assay (ELISA)

• analytical technique to detect the presence of

an antigen or antibody in a given sample

• color development is measured

spectrophotometrically

Lateral Flow Immunoassay

diagnostic device used to confirm the presence or

absence of a target analyte, such as pathogens or

biomarkers in humans or animals, or contaminants in

water supplies, foodstuffs, or animal feeds

Nucleic acid-based methods

• operate by detecting specific DNA or RNA sequences in the

target pathogen

• detect the specific genes in the target pathogens, therefore

preventing ambiguous or wrongly interpreted results

Polymerase Chain Reaction

• allows the detection of a single bacterial pathogen that is present in the food by

detecting a specific target DNA sequence

• operates by amplifying a specific target DNA sequence in a cyclic three steps process

Biosensors

systems containing a biological detection material

combined with a chemical or physical transducer

• can convert biological, chemical, or biochemical

signals into measurable electrical signals

Bioreceptor

■ responsible for recognizing the target analyte

■ can be any of the following:

• biological material: enzymes, antibodies, nucleic acids,

and cell receptors

• biologically-derived material: aptamers and recombinant

antibodies

• biomimic: imprinted polymers and synthetic catalysts

Transducer

■ converts the biological interactions into a measurable

electrical signal

■ can be optical, electrochemical, mass-based, thermometric,

micromechanical or magnetic

Optical biosensors

• characterized by high sensitivity, simple handling, and rapid

detection

• contains a biodegradable polymer by analytical enzymes

secreted by microorganisms during the deterioration of the

natural product

• as the number of bacteria increases, there is increased

secretion of enzymes that cause food degradation, which

will be visible with the degradation of the polymer

Electrochemical biosensors

• used for the identification and measurement of foodborne pathogens

• measure an electrochemical response

• classifications:

⚬ amperometric biosensors

⚬ impedimetric biosensors

⚬ potentiometric biosensors

⚬ conductometric biosensors

amperometric biosensors

■ used to examine electrochemical reactions while measuring the

current change in a constant potential

■ analyte concentration in a solution is proportional to the

response of the biosensors

potentiometric biosensors

■ based on the measurement of oxidation and reduction potential

of an electrochemical reaction

■ include the use of ion-selective electrodes to convert the

biological reaction into an electrical signal

■ antibody-antigen binding causes a small change in charge of

proteins that can be determined potentiometrically

impedimetric biosensors

■ based on changes in the conductivity of the environment

through microbial metabolism of electrically charged ionic

compounds and inert substrates of acidic products (i.e. amino

acid, lactic acid, and acetic acid)

■ allows the detection of only the viable cells, which is a major

concern in food safety

conductimetric biosensors

■ many microbe-catalyzed reactions involve a change in ionic

species which is associated with a net change in the

conductivity of the reaction solution

■ conductance measurement is non-specific, but extremely

sensitive

Mass-based biosensors

• operate based on the detection of small changes in mass

• involve the use of piezoelectric crystal which will vibrate at a certain

frequency when induced by an electrical signal of a certain frequency

• bioreceptors (e.g., antibodies) for the detection of pathogens (e.g.,

antigens) are immobilized on this crystal

Electronic Nose

• comprises of sophisticated hardware with sensors, electronics, pumps, flow controllers, software's, data preprocessing and statistical analyzer

Food additive

"any compound not typically consumed as a food by

itself and not normally used as an ingredient in the

food but is intentionally added in the manufacture,

processing, preparation, treatment, packing, packaging,

transport and holding of the food, to perform a

technological function (including organoleptic)"

Instrumenental Techniques

- Spectroscopic

- Chromatographic

- Electroanalytical

Spectroscopic

based on electromagnetic radiation measurements

• common spectroscopic methods:

⚬ Ultraviolet/visible spectroscopy

⚬ Near-infrared spectroscopy

⚬ Fourier transform infrared spectroscopy

⚬ Fluorescence spectroscopy

Chromatographic

based on the displacement of the food additives between

a mobile and stationary phase

Electroanalytical Techniques

measure the electrical

properties of the food additive

UV-Vis Spectrometry

• measures the fraction of radiation

transmitted through the sample

• efficient, low cost, non-destructive and

environmentally friendly, simple

Near-Infrared (NIR) Spectrometry

• useful for qualitative analysis, such as identifying specific functional groups present in substances

• enables measuring the amounts of various food additives in a variety of foodstuffs

Fourier transform infrared spectroscopy

• uses an interferometer

• radiation is not dispersed, but rather all wavelengths arrive at the detector simultaneously

• provides a rapid and accurate data acquisition and is a non-destructive procedure

Fluorescence Spectroscopy

• based on the emission of the electromagnetic radiation by chemical compounds

• analytical results are 1-3 orders of magnitude more sensitive than UV/Vis

• only used for rigid chemical structures analysis

Chromatographic

• analytical techniques employed to separate, identify and quantify

inorganic and organic compounds, based on the partitioning or

distribution of a sample between a mobile and a stationary phase

• 2 types:

⚬ gas chromatography (GC)

⚬ liquid chromatography (LC)

Gas Chromatography

• separates and identifies complex mixtures of volatile compounds and thermally stable organic compounds

• mobile phase --> inert gas

• stationary phase --> immobilised liquid or a solid packed in a

closed tube

• sample is converted to the vapor state and injected into a column

with a controlled temperature gradient to separate the molecules

according to several properties

Liquid Chromatography

• mobile phase - liquid; stationary phase - solid

• based on the adequate choice of a mobile phase, composed by

organic solvents and/or buffer solutions

• HPLC - most useful and widely applied chromatographic method

for analysis of additives and certain vitamins in food products

Electroanalytical techniques

• measure the electrical properties to identify and quantify organic

compounds as the electrical properties are proportional to the

target compounds

Voltammetric techniques

Ø electroanalytical techniques used to identify and quantify food

additives in complex samples

Ø involve the application of an electrical potential difference to

promote the reaction of electron transfer, called redox reaction