Detection of meat spoilage (decomposition)
Meat spoilage (decomposition) is the process by which organic
matter is broken down into a simpler form, primarily in meat protein
but also fats and carbohydrates, by the action of bacteria, molds and
yeasts, reducing the meat into a number of simpler chemical substances,
many of which are gaseous and foul smelling.
The spoilage process is initially fuelled by the breakdown of
carbohydrate. As time passes, however, protein molecules are broken up
into simpler substances by acids, alkalis, endogenous enzymes and
bacteria, the degree of decomposition varying greatly with the different
agencies. Of these agencies, the putrefactive bacteria carry the process
further, breaking up the protein molecule into proteoses; then peptones,
peptides and amino acids; and finally indole, skatole and phenol,
together with various gases including hydrogen sulphide, carbon
dioxide, methane, ammonia and biogenic amines such as tyramine,
putrescine and cadaverine. These end products are known as
spoilage or freshness indicators.
Methods used for detection of meat spoilage
Traditional methods:
1. Organoleptic examination
- Changes in color, consistency, odor and taste.
2. Microbiological examination
- Total or selective microbial plate counts
3. Physical examination
a. Monitoring of ultimate pH (pHu)
b. Extract-release volume (ERV)
4. Chemical examination
a. Eber's ammonia test
b. Estimation of total volatile basic nitrogen (TVB-N) using
Conway micro diffusion cell.
c. Lead acetate paper test for detection of H2S
d. Determination of fat rancidity (lipid oxidation):
Thiobarbituric Acid (TBA) test
Modern (rapid) methods:
1. Molecular
a. PCR
b. Quorum sensing signals
2. Biochemical
a. Fluorescein diacetate hydrolysis test
b. Resazurin reduction test
3. Spectroscopy
- Near infrared (NIR) spectroscopy
- Fourier transform infrared (FT-IR) spectroscopy
4. Nanotechnology
a. Carbon nanotubes sensor
b. Optoelectronic silicon chip
Traditional methods
Traditional methods to detect food (meat) spoilage are the
sensory evaluation based on panelists, accompanied by determination
of total or selective microbial plate counts. However, classical
microbiological methods are slow, while sensory analyses have also
the disadvantage to detect signals of spoilage with delay, even when
expert panels are employed. The less the expertise of the panelists, the
more delayed the detection of spoilage and the less the remaining
product shelf-life during which corrective action could be taken.
1.Organoleptic examination (Sensory evaluation based
on panelists): Decomposed meat characterized by:
- Changes in color into gray, yellow or green.
- Softening in consistency.
- Repulsive odor and unacceptable taste.
- Alkaline reaction
2. Microbiological examination (Total or selective microbial plate
counts).
The total viable count of bacteria
(TVC) expressed as colony forming
unit (cfu)/g on fresh meat or a meat
product sets a limit to its shelf-life.
Meat will “spoil” with TVC at 106
cfu/g because of off-odors. Slime and
discoloration appear at 108 cfu/g. The
main factors determining the time
taken for the TVC to reach these
levels are the initial count due to contamination during slaughtering and
processing, further contamination during storage, temperature, pH and
relative humidity.
Bacteria relevant to meat, meat products and other food are divided
into three groups according to the temperature range within which they
can grow: mesophiles 10– 45°C, psychrophiles 0–28°C and
psychrotrophs 10– 45°C, or slow growth at 0–10°C.
Some bacteria cause product spoilage, others cause food poisoning. The
former limit product shelf-life but the latter cause illness. Almost all
food poisoning bacteria are mesophiles so refrigeration below 10°C
offers good protection. Many mesophiles cause spoilage, but since meat
is refrigerated most spoilage is due to psychrophiles. Storing meat at
temperatures close to 0°C will inhibit the growth of pyschrotrophs.
Shelf-life will be extended by avoiding contamination through good
hygiene practices.
Recommended microbiological criteria for fresh meat
Good Critical Not acceptable
-
TVC cfu/g <104 >104 <105 >105
Enterobacteriaceae -
cfu/g <102 >102 <103 >103
3.Physical examination
a. Monitoring ultimate pH (pHu)
b. Extract-release volume (ERV)
Monitoring ultimate pH (pHu):
It is accepted that meat from fatigued animals spoils faster. The pH
of the meat from these animals, on the completion of rigor mortis, is in
the region of 6.5 rather than the lower normal value in a rested animal of
around 5.6. Such a low ultimate pH (pHu 5.5- 5.8) slows the growth of
bacteria, as this is outside their optimal pH range, thus slowing down the
functioning of the enzyme systems and the transport of nutrients into the
microbial cells.
Then pHu remains constant for sometimes depending on several
factors, later the pHu begins to rise slowly due to natural autolysis and
bacterial multiplication. When the pHu reaches 6.4, there is a
suspicion of presence of (incipient decomposition). And when the
pHu reaches 6.8 or over, objective signs of spoilage (apparent
decomposition).
Extract-release volume (ERV):
Extract release volume (ERV) appears to have considerable possibilities
for assessing the spoilage of beef. It also has a highly significant
correlation with WHC.
The procedure is based on measuring the volume of the aqueous filtrate
released from slurry of meat in a fixed time. The ERV decreases as
spoilage progresses and no filtrate is obtained from putrid meat.
Procedures:
1. Weigh 25 g minced sample into the 100 ml vortex beaker of a
laboratory homogenizer.
2. Add 100 ml buffer solution, pH 5.8 ( 50 ml 0.2 M K2PO4 and 3.72
ml M NaOH diluted to 200 ml with distilled water), blend for 2
minutes.
3. Pour the homogenate into a "Whatman filter paper No. 1" and
measure the volume collected in 15 min.
Result:
An ERV of 25 ml (obtained with 25 g meat with 100 ml buffer solution
of pH 5.8 and a filtration time of 15 min) has been recommended as a
rejection cut-off figure.
A major drawback is the fairly wide range of values given by fresh
meats (21-35 ml).
4. Chemical examination
a. Eber's ammonia test
b. Estimation of total volatile basic nitrogen (TVB-N) using
Conway micro diffusion cell.
c. Lead acetate paper test for detection of H2S
Eber's ammonia test (1896):
Eber proposed that with the addition of HCl to the decomposed
meat, a grey or white nebulae of (NH3)Cl is formed due to presence of
free ammonia in the decomposed meat.
Procedures:
Place a 2 ml of Eber's reagent (1 part HCl + 1part ether + 3 parts 96%
alcohol) in a test tube fitted with a tight cork had a fixed thin glass rod.
Quickly push the glass rod in the meat sample and rotate it so that a
small piece of the sample can be removed with the rod.
The rod is transferred into the tube provided that the meat sample is not
in contact with the reagent solution.
Result:
In a few seconds grey or white nebulae is formed if ammonia (more than
30 mg N/100
g)is present. These nebulae are better seen if black back ground is put
behind the test tube.
Disadvantage
Eber's test for free ammonia often gives negative results with meat
weeks old and even showing incipient putrefaction.
Estimation of total volatile basic nitrogen TVB-N using Conway
micro diffusion cell:
Total volatile basic nitrogen (TVB-N) has been used as an index of
decomposition in meats and fish since 1952 and is still widely used. It is
suggested that marine fish may be classified into three freshness groups
on the basis of TVBN concentration: class I <30 mg N/100 g, class II
30-40 mg N/100 g and class III >40 mg N/100 g. Earlier reports had set
the cut-off point at a maximum of 30 mg% volatile nitrogen bases for
human consumption.
In meat, TVB-N consists entirely of ammonia, with only traces of
Trimethylamine (TMA). TMA however, provides an accurate indication
of bacterial spoilage in fish species. Elasmobranch fish decomposition is
also characterized by large amounts of ammonia formed by the
breakdown of urea which is present in considerable quantities or by
bacterial attack, as in shark muscle.
Determination of TVB-N using Conway micro diffusion cell based on a
method consists of distillation of the volatile bases into a suitable system
such as boric acid or standard weak acid and then measuring the TVB-N.
Reagent:
1- Absorbent boric acid (H3BO4):
Dissolve 10 gm. of boric acid in 200 ml methyl alcohol. Then add 700ml
distilled water plus 30 ml of indicator solution mixture (0.033gm
Bromothymol blue+ 0.033gm Methyl red+ 100 ml Alcohol). The color
of the reagent is slightly brown.
2- K2CO3 50% solution (previously boiled for few minutes)
3- 1/50N HCl (for titration).
4- Trichloroacetic acid 20% solution (as a preservative).
Procedures:
1. Put 1 ml of the absorbent boric acid solution in the inner chamber of
the Conway micro diffusion cell.
2. Homogenize 5 gm. of meat with 45 ml distilled then filter. Put 1 ml of
the meat filtrate outer chamber of the Conway micro diffusion cell,
followed by addition of 1ml of K2-CO3 solution then quickly cover
the cop edges of the Conway micro diffusion cell with Vaseline and
fix the glass cover with metal clips.
3. Incubation of the plate at 37o C for 80 minutes (alternatively at 27o C
for 100 minutes, 20o C for 120 minutes, 16o C for 140 minutes or 10o
C for 160 minutes) to allow adsorption of the released volatile
nitrogen "TVB-N" from the sample in the outer chamber to the boric
acid mixture in the inner chamber.
4. Titration of the boric acid mixture against 1/50N HCl until the color
is changed into green color and record (R) which is the number of ml
used for titration.
Calculation:
TVB-N mg N/100 g = 28 X R X sample dilution (1/10)
Result:
Beef is generally considered to be fresh if TVB-N is less than 20 mg
N/100g fat free meat. This method is unsuitable for detecting incipient
spoilage. Ammonia contents of 3-10 mg N/100 g fresh beef have been
reported. On storage, the meat is not necessarily unpalatable until the
value reaches 30 mg%.
Lead acetate paper test (1966):
It is used for the detection of hydrogen sulfide (H2S)
Procedures:
Soak 2-inch x 1-inch strips of filter paper in hot 5% lead acetate
solution and heat at 70°C until dry.
Cut the suspected meat into small pieces and put them in a
conical flask or (McCartney bottle). Then cork the flask (or
bottle) with its cover which contains a suspended lead acetate
paper.
Put the flask (or bottle) in a water bath for 10-15 minutes.
N.B. If no blackening has occurred by the end of the incubation period,
add 0.5 ml of 2N hydrochloric acid and replace the plug and lead acetate
paper immediately. If any H2S has been produced but has remained in
sample, the addition of the acid will cause the liberation of H2S.
Result:
If the meat is decomposed H2S will be liberated and form lead sulfide
which changes the color of the paper into brown to blackish or black
color.
Determination of fat rancidity (lipid oxidation): Thiobarbituric
Acid (TBA) test
Principals:
Malondialdehyde (MDA), a secondary product from unsaturated
fatty acids oxidation, is a three-carbon dialdehyde with carbonyl groups
at the C1 and C3 positions.
The amount of MDA has been commonly used as an oxidation
index in meat and meat products and is commonly determined by the
Thiobarbituric Acid (TBA) assay as MDA is considered the major
TBA–reactive substance (TBARS). The TBA test is a colorimetric
technique in which the absorbance of a red chromogen formed between
TBA and MDA is measured at 538 nm.
Procedures:
1. Briefly, the TBA test can be performed by distillation of the
sample followed by reaction of the distillate with the TBA.
2. Transfer 5 ml of each TBARS distillate and 5 ml of TBA reagent
into screw-cap test tubes. After thorough mixing, heat the test,
tubes in vigorously boiling water bath for 45 minutes and cool
in tap water.
3. Determine absorbance of the solutions at 538 nm within 30 min of
cooling.
Result:
Meat products are generally considered to be not rancid if TBA is
less than 0.9 mg MDA/kg
Modern (rapid) methods
Traditional methods give retrospective results, and this can be a major
drawback within the modern meat industry as monitoring procedures,
such as HACCP systems, need to give results in real time to enable
corrective actions to be taken as soon as possible. Therefore, in recent
years, there has been an increasing interest in rapid methods for
detection of meat spoilage, and in effectively incorporating such data to
models aiming at providing accurate shelf-life predictions.
1. Molecular
a. Real-time PCR to quantify total or specific spoilage
microorganisms
b. Detection of quorum sensing signals N-acyl homoserine
lactones (AHLs) excreted by potential spoilage bacteria, such
as Enterobacteriaceae, during storage of meat.
2. Biochemical
a. Fluorescein diacetate hydrolysis test
b. Resazurin reduction test
3. Spectroscopy:
Near infrared spectroscopy (NIRS) is an analytical technique that
uses a source producing light of known wavelength pattern
(usually 800–2 500 nm) and that enables to obtain a complete
picture of the organic composition of the analyzed
substance/material.
It is based on the principle that different chemical bonds in organic
matter absorb or emit light of different wavelengths when the
sample is irradiated.
Additionally, Fourier transform infrared (FT-IR) spectroscopy is a
technique which is used to obtain an infrared spectrum of
absorption or emission of a solid, liquid
or gas. An FT-IR spectrometer simultaneously collects high
spectral resolution data over a wide spectral range. This confers a
significant advantage over a dispersive spectrometer which
measures intensity over a narrow range of wavelengths at a time.
NIR and FT-IR spectroscopies are recently proven to be used as a
rapid, reagent-less and non-destructive method for total volatile
basic nitrogen (TVB-N).
4. Nanotechnology
a. Carbon nanotubes sensor: The Massachusetts Institute of
Technology (MIT) researchers modified the carbon
nanotubes with a metalloporphyrin containing a central
cobalt atom bound to several nitrogen-containing rings.
Cobalt-containing metalloporphyrins bind strongly to
biogenic amines, such as putrescine and cadaverine,
produced by species of bacteria that cause meat spoilage and
this increases the electrical resistance of the carbon
nanotubes.
b. Optoelectronic silicon chip: An electronic nose for real-time
detection of spoilage detect TVB-N. Commercially available
sensor “FOODsniffer” is a miniaturized optoelectronic
silicon chip able to simultaneously detect in short time a
panel of harmful substances (such as ammonia) packaged
into a single-shot cartridge and a portable and easy-to-use
reader.
