DOI: https://doi.org/10.52973/rcfcv-e32121
Received: 17/03/2022 Accepted: 19/07/2022 Published: 24/11/2022
1 of 6
Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32121, 1 - 6
ABSTRACT
Meat is an excellent medium for bacterial growth due to its high water
and nutrient content. The nitrogenous compounds (NC) are derived
through decarboxylation of amino acids due to microbial enzymes. The
objective of this study was to evaluate the concentration of 3 NC and
the proliferation of some microorganisms in rabbit meat with three
treatments (T) , classied by three types of packaging for 21 days (d)
in rabbit meat stored cold. The meat samples were obtained of the
Longissimus thoracis et lumborum muscle. Each sample was divided
and two groups were formed. The rst group was used to measure the
physicochemical characteristics of the meat, and the second group
was used to quantify NC and bacterial isolation. The pH in the meat
decreased from 0 to 21 d in the three T. The brightness (L*) decreased
(P<0.05), while the variables a* and b* increased (P<0.05) to 21 d for
all groups. Histamine and cadaverine remained low and were similar
in the three T (P>0.05). Putrescine (PU) increased (P<0.05) from 7 to
21 d in the Control-Plastic (CP) and Semi-permeable plastic lm (SP)
groups vs. Vacuum packing (VP). The Enterobacteriaceae remained
constant throughout the experimental period in the three T, compared
to the aerobic mesophilic, which was higher (P<0.05) until 21 d of
the evaluation in CP and SP. The type of packaging and cooling time
inuenced the concentration of NC. The VP had the lowest level of
PU and mesophilic bacteria until 21 d of storage.
Key words: Bacteria contamination; meat; packaging; rabbit
RESUMEN
La carne es un excelente medio para el crecimiento bacteriano
debido a su alto contenido de agua y nutrientes. Los compuestos
nitrogenados (NC) se obtienen a través de la descarboxilación de
aminoácidos debido a las enzimas bacterianas. El objetivo de este
estudio fue evaluar la concentración de 3 NC y la proliferación de
algunos microorganismos en carne de conejo con tres tratamientos
(T), clasificados por los tipos de empaques almacenada en frío
durante 21 días (d). Las muestras de carne se obtuvieron del músculo
Longissimus thoracis et lumborum. Cada muestra se dividió y se
formaron dos grupos. El primer grupo se utilizó para medir las
características sicoquímicas de la carne, y el segundo grupo se
utilizó para cuanticar los CN y el aislamiento bacteriano. El pH en la
carne disminuyó de 0 a 21 d en los tres T. La luminosidad (L*) disminuyó
(P<0,05), mientras que las variables a* y b* aumentaron (P<0,05) a
los 21 d para todos los T. La histamina y cadaverina se mantuvieron
bajas y fueron similares en los tres T (P>0,05). La putrecina (PU)
aumentó (P<0,05) de 7 a 21 d en los grupos Control-plástico (CP) y
Película plástica semipermeable (SP) vs. Empaque al vacío (VP). Las
bacterias Enterobacteraceae se mantuvieron constantes durante
todo el período experimental en los tres T, en comparación con las
bacterias aerobias mesólas, las cuales fueron superiores (P<0,05)
hasta los 21 d de evaluación en CP y SP. El tipo de empaque y el
tiempo de enfriamiento inuyeron en la concentración de NC. El VP
tuvo el nivel más bajo de PU y bacterias mesólas hasta los 21 d de
almacenamiento.
Palabras clave: Contaminación bacteriana; carne; empaque; conejo
Prole of Nitrogenous compounds and Bacterial proliferation in Rabbit
meat stored cold with three types of packaging
Perl de compuestos nitrogenados y proliferación bacteriana en carne de conejo almacenada en frio
con tres tipos de empaques
Technical note
Verónica Reséndiz-Cruz
1
, Esperanza García-López
2
, Jacinto Efrén Ramírez-Bribiesca
3
, David Hernández-Sánchez
3
,
Isabel Guerrero-Legarreta
4
, Daniel Mota-Rojas
5
, Juan Edrei Sánchez-Torres
6
and Rosy Gabriela Cruz-Monterrosa
1
*
1
Universidad Autónoma Metropolitana, Departamento de Ciencias de los Alimentos. Unidad Lerma, Lerma de Villada, México.
2
Universidad Nacional Autónoma
de México, Facultad de Estudios Superiores Cuautitlán, Laboratorio de Medicina Preventiva. Cuautitlán Izcalli, México.
3
Colegio de Postgraduados, Programa
de Ganadería. Montecillo, México.
4
Universidad Autónoma Metropolitana, Departamento de Biotecnología. Unidad Iztapalapa, México.
5
Universidad Autónoma
Metropolitana, Medicina Veterinaria y Zootecnia. Unidad Xochimilco, México.
6
Universidad Autónoma del Estado de México, Facultad de Medicina Veterinaria y
Zootecnia. Toluca, México. *Email: r.cruz@correo.ler.uam.mx
Nitrogen and Bacterial proliferation in Rabbit meat stored cold / Reséndiz-Cruz et al. __________________________________________________
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INTRODUCTION
Meat is one of the most perishable foods due to its high-water
content and available nutrients. Still, when there are physical changes
in color, smell, texture, oxidation, and growth of microorganisms,
there is a rejection of meat from the consumers. A bacterial load
greater than 10
7
colony forming units· square centimeter
-1
(CFU·cm
-2
) in
the meat packets causes a bad smell, and an amount of 10
9
CFU·cm
-2
the smell was putrid, causing decarboxylation of the free amino
acids [8]. During the decomposition of the meat, there is a formation
and accumulation of nitrogenous compounds (NC) [22, 31]. The
quantication of the NC in the meat indicates the beginning of the
microbial activity and its deterioration in nutritional value [21, 22].
There are two standard packaging systems; these are the
conventional polystyrene foam tray and vacuum packaging [7].
Specically, there is no information on rabbit (Oryctolagus cuniculus)
meat quality over shelf life. The objective of the study was to identify NC
production, microbial contamination, and some quality parameters in
rabbit meat, using three types of packaging at different storage times.
MATERIAL AND METHODS
Study place
The animals were obtained from the rabbit farm of the Chapingo
University, Mexico, located at 19
o
29' North and 98
o
54' West [17].
Physicochemical analyses of the meat and the identification of
bacteria were carried out in the Facultad de Estudios Superiores,
Cuautitlán of the National University.
Selection and slaughter of rabbits
Sixty adult male rabbits, New Zealand breed, were divided into
three experimental treatments (T), homogeneous in weight (2.35 ±
0.25 kilograms (kg). Each T was housed in three-place cages (30×42
centimeters -cm-, height 30 cm). All the animals were slaughtered
not longer than 2 minutes (min) after each animal was removed from
its cage. The procedure was bled by cutting the jugular vein and the
carotid artery (less than 30 seconds –s – ) and then the skin, genitals,
urinary bladder, gastrointestinal tract and distal parts of legs were
removed [14].
Obtaining samples
Previously, a refrigerator (Imbera, VR19, Koblens, USA) was assigned
to preserve the meat samples, and it was disinfected with 10% of the
nitric acid solution and washed with distilled and deionized water.
Then, 30 grams (g) of the Longissimus thoracis et lumborum muscle of
each animal was obtained. Each sample was divided into six portions
of 5 g and two groups were formed (3 sub-samples in each group). The
rst group was used to measure the physicochemical characteristics
of the meat, and the second group was used to quantify NC and
bacterial isolation.
All samples were identied inside plastic bags and refrigerated
(4°C) at times zero (less than 12 cooling hours -h-: 0, 7, 14, and 21 days
-d-, 45 samples for each T-time). Subsequently, the samples stored
for each time were randomly divided into 15 samples for each type
of packaging, classied into the following Ts:
1.
Control-plastic (CP), samples covered with a transparent
plastic bag (polyethylene). 15×25 cm. Caliber: 18 milimicra (µm).
2.
Polyethylene tray with semi-permeable plastic lm (SP): Unicel
tray (Reyma®) with 11 cm diameter and a exible food-grade
lm (12 µm).
3. Vacuum packing (VP): Vacuum bags of 15×20 cm and 90 μ·2.8
mililiters
-1
-mL- (Torrey® packer model-Evd20); the vacuum
time was 40 s and the sealing of 2 s.
Physical measurements of meat
The pH and colorimeter electrodes were previously disinfected
between each sample with a 10% nitric acid solution and washed
with deionized water.
Meat pH
A portable potentiometer (HANNA model-HI99163, USA) was used.
The electrode was pressed moderately on the surface of each sample,
and the pH reading was recorded for four consecutive times.
Meat color
A Hunter Lab portable colorimeter (CR-410, Konica-Minolta,
Inc. Japan) was used to measure the variables L* (luminosity), a*
(red-green) and b* (yellow-blue). It was calibrated with the tile to the
reference coordinates: L* = 94.7, a* = 0.3130, b* = 0.3191); subsequently,
three points were measured on the surface area of the meat sample.
Bacteriological tests
Samples of 5 g of meat were mixed with 45 mL of sterile physiological
saline; serial dilutions were prepared up to 1:1000. Two agar media
Standard (Bioxon®) was used for the quantify Enterobacteriaceae
(Violet Red Bile Glucose Agar: MH581) and total aerobic mesophilic
(Plate Count Agar: ICMSF – 2000). The incubation was carried out from
24 to 48 h at 35°C, in a Felisa® equipment (Felisa: FE-500, Feligneo,
México). The volume spread calculated the number of colonies that
arose on a pour plate at the end of the designed time.
Quantication of nitrogenous compounds
The samples were removed from the assigned packing and they
were macerated to measure NC through a 3-phase procedure [22]:
First phase. Preparation of solution and individual standards of
the NC: In each tube, putrescine (PU), cadaverine (CA), histamine
(HI) standards (Sulpenco, Merck, USA) were weighed to have a
concentration of 10 milligrams·mL
-1
(mg·mL
-1
). Each standard was
diluted in 1 mL of hydrochloric acid and homogenized (base solution).
Second phase. Extraction: 5 g of each meat sample was weighed
into test tubes, and 5 mL of perchloric acid (6%) was added,
homogenizing in the vortex (Scientic Industries, SI-0236, USA) and
allowed to stand for 1 h in refrigeration. All samples were centrifuged
(Universal 320/320R Hettich® ,México) at 4950G-force (10 min at 4°C),
and the solutions were ltered, adding 1 mL of 2 Molar (M) NaOH. The
pH was maintained at 6 and the tubes were kept in ice water for 20
min. Derivatization: All tubes were removed from the cold water, and
20 microlliters (μL) of benzoyl chloride was added and homogenized.
They were left at rest for a further 20 min and 2 mL of 5 M sodium
chloride was added, homogenizing in a vortex, and one mL of ethyl
ether was added. Then, the ether sample was evaporated with a
ow of nitrogen (10 min) and 500 μL of Milli Q water and 500 μL of
acetonitrile were added and vortexed.
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High-performance liquid chromatography
Third phase: The ltered sample (1 mL) was injected into the High-
performance liquid chromatography (HPLC) with a diode detector array
(Model 1100 Horse Power(HP) Agilent Technologies. Wilmington, USA).
An elution gradient program was used with a 50:50 mixture, acetonitrile
as solvent A and puried ultrapure water as solvent B. The ow rate
used was 1 mL·min
-1
. The temperature of the column was 40°C, and the
euent from the column was analyzed at 254 nanometers.
Statistical analysis
All rabbits were randomly distributed with a 4×3 factorial arrangement.
The normal distribution in the number of bacterial colonies was
determined with the Kolmogorov-Smirnov test. All the variables were
analyzed with a PROC MIXED design [28] and the mean comparison was
made with the PDIFF test, using the statistical package [28].
RESULTS AND DISCUSSION
pH and bacterial count
TABLE I shows all results. The pH in the CP, SP, and VP Ts decreased
0.74, 0.85, and 1.08 units (u) from 0 to 21 d, respectively. There were no
differences (P>0.05) in pH between the three Ts at storage times of 0,
7, and 14 d, the value of pH=6.7 was within the range reported by other
authors [4, 23]. Except for 21 d, the pH of the CP T was higher (6.28,
P<0.05) and decreased 0.21 and 0.45 u for the SP and VP Ts (P<0.05),
respectively [9]. Possibly, this effect is due to the accumulation of
lactic acid, which could cause protein denaturation and water retention
decreased [15]. The lower concentration of pH in the VP T was possibly
due to the higher content of lactic acid accumulated in this type of
packaging and a lower bacterial proliferation of aerobic mesophiles
and Enterobacteriaceae (TABLE I), due to the vacuum process (EVAC-
8-RHI, Inox, NH_Rhino, China). Other authors report that contamination
with bacteria and some fungi could induce the formation of alkaline
compounds [2,16], causing the decomposition of meat and increase
of pH and ammonia [4, 30].
Color
The L* value decreased (P<0.05) 2.52, 3.99, 4.96 u from 0 to 21 d
for the CP, SP and VP Ts (TABLE I ). Contrarily the values of a* and b*
increased (P<0.05) 2.85, 3.07, 3.27, and 5.96, 7.77, 4.06 u from 0 to 21 d
for the CP, SP and VP Ts, respectively. The L* and a* indices between
the CP, SP and VP Ts did not show signicant differences (P>0.05)
from the times 0 to 21 d; except fora decrease of 2.4 u in L* of SP T
compared with VP T. The b* index was similar in all Ts at 0 d (P>0.05),
although from 7 to 21 d of the SP T (average: 11.7) was increased
1.63 u, while the VP T (average 7.25) decreased 2.82 u (P<0.05), both
compared to the CP T (average 10.07).
The L* decreased with the cooling time, but the index a* and b*
increased (P<0.05) in the three types of packages evaluated. There
is no published data on rabbit meat, but color values were similar to
bovine meat [10, 25]. L* value was inuenced by the concentration
of reduced myoglobin, oxymyoglobin, and metmyoglobin [13] and the
index a* and b* increase with the maturity time of meat due to the
greater passage of light in the meat tissue. Maybe it was associated
with the opening of the packages, and the meat had contact with
oxygen due to which myoglobin was transformed into oxymyoglobin
and, it was intensifying the brightness and red color [24].
Nitrogenous compounds and bacterial contamination
The PU content did not show significant differences (P> 0.05)
between the three T during the rst 7 d storage (TABLE II ). Then, PU
increased drastically (P<0.05) from 7 to 21 d of storage. CA and HI had
signicant differences (P<0.05) from 14 to 21 d between the SP and
VP T. The storage time and the type of packaging mainly inuenced
the production of PU, and a high level of PU is associated with the
proliferation of Pseudomonas spp. in aerobic conditions at 37°C [1]. The
PU was the main NC formed; its value was similar to another report in
chicken(Gallus gallus familiaris) (45.2 mg·kg
-1
[22]) during 17 d of storage.
In contrast, the average concentration of CA in the three T was lower
(2.54 mg·kg
-1
) than reported (5.7 mg·kg
-1
) in chickens at the end of the
evaluation. CA and HI at 7 d of storage had a higher concentration than
in bovine (Bos taurus) meat (1.85 and 2.11 mg·kg
-1
), stored for 7 d in trays
packing with trays of polystyrene [12].
The production of CA in rabbit meat is possibly associated with lysine;
this amino acid was the precursor of NC [5]. Enterobacteriaceae also
induced the highest CA content [6]. However, the number of these
bacteria was lower than that reported by other authors [1, 12], which
suggests that a smaller amount of Enterobacteriaceae is associated
TABLE I
Physico-chemical variables and bacterial count (mean ± sd) in three
of packaging at dierent days of refrigeration in rabbit meat
(muscle Longissimus thoracis et lumborum)
Refrigeration days
0 7 14 21
Control Plastic (CP)
pH
6.65 ± 0.05cA 5.70 ± 0.01aA 5.74 ± 0.05aA 6.28 ± 0.14bC
Meat colour
L*
58.97 ± 1.03cA 57.60 ± 0.48bcA 56.44 ± 0.47abA 55.30 ± 0.53aAB
a*
14.62 ± 0.86aA 17.34 ± 0.39bA 17.16 ± 0.46bA 17.93 ± 0.85bA
b*
4.11 ± 0.80aA 9.57 ± 0.23bB 10.54 ± 0.27bB 10.11 ± 0.31bB
Semi-permeable (SP) plastic lm)
pH
6.69 ± 0.05cA 5.74 ± 0.04aA 5.71 ± 0.03aA 6.07 ± 0.11bB
Meat color
L*
60.05 ±1.26cA 57.55 ± 0.53bA 56.98 ±0.66bA 53.64 ± 0.73aA
a*
15.06 ± 1.14aA 17.97 ± 0.43bA 17.96 ± 0.42bA 18.47 ± 0.47bA
b*
3.98 ± 0.88aA 11.45 ± 0.35bC 11.53 ± 0.27bB 12.29 ± 0.39bC
Vacuum packing (VP)
pH
6.70 ± 0.05bA 5.55 ± 0.03aA 5.58 ± 0.03aA 5.73 ± 0.03aA
Meat color
L*
60.39 ± 0.78cA 58.20 ± 0.51bA 56.50 ± 0.78abA 56.08 ± 0.45aBC
a*
13.71 ± 0.67aA 17.00 ± 0.36bA 16.95 ± 0.37bA 17.01 ± 0.29bA
b*
3.19 ± 0.41aA 7.06 ± 0.21bA 7.45 ± 0.21bA 7.26 ± 0.16bA
CP: Polyethylene plastic bag, SP: Polyethylene tray with semipermeable
plastic film, VP: Vacuum packed (vacuum time 40 seg and sealing 2
seg), s.d.: standard deviation, L*: luminosity, a*: index color red-green,
b*: index color yellow-blue. a-c: Dierent lowercase letters in the same
rows indicate signicant dierences between storage days (P<0.05). A-C:
Dierent capital letters in the same column indicate signicant dierences
between packages (P<0.05)
Nitrogen and Bacterial proliferation in Rabbit meat stored cold / Reséndiz-Cruz et al. __________________________________________________
4 of 6
TABLE II
Concentration of nitrogenous compounds (mg·kg
-1
= mean ± s.d.)
in three types of packaging on dierent days of refrigeration
in rabbit meat (Longissimus thoracis et lumborum)
Day 0 7 14 21
PU 1.47 ± 0.23aA 1.13 ± 0.18aA 20.36 ± 9.3bB 29.08 ± 7.4bB
CA 2.89 ± 0.45aA 2.75 ± 0.39aA 2.84 ± 0.47aA 2.20 ± 0.60aA
HI 6.09 ± 0.85aA 5.49 ± 0.26aA 8.47 ± 0.99bA 10.62 ± 1.59bA
En Un 3.69 ± 0.89aA 3.83 ± 1.23aAB 6.02 ± 0.83aB
Mes 2.24 ± 0.57aA 4.07 ± 0.24abA 5.66 ± 0.49bB 4.95 ± 0.77bA
PU 1.31 ± 0.20aA 0.67 ± 0.04aA 18.30 ± 7.34bB 32.12 ± 9.8cB
CA 2.84 ± 0.52aA 3.49 ± 0.44aA 2.03 ± 0.21aA 2.23 ± 0.20aA
HI 5.83 ± 0.64aA 6.60 ± 0.46aA 8.79 ± 0.91bA 8.87 ± 1.41bA
En 3.33 ± 0.70a Un 6.05 ± 0.64aB 3.99 ± 1.3aAB
Mes 3.54 ± 0.58bcA 2.53 ± 0.62abA 4.29 ±1.2bcAB 4.87 ± 0.7cA
PU 1.40 ± 0.20aA 0.44 ± 0.03aA 1.19 ± 0.16aA 15.93 ± 2.1bB
CA 2.71 ± 0.83aA 3.62 ± 0.64aA 5.59 ± 1.13bB 3.20 ± 1.3aA
HI 5.94 ± 0.5abA 4.86 ± 0.46aA 6.83 ± 0.6bcB 8.42 ± 0.1bcA
En Un 3.30 ± 0.10aA 1.98 ± 0.08aA 2.32 ± 0.34aA
Mes 3.19 ± 0.5aA 3.36 ± 0.3aA 2.99 ± 0.8aA 4.34 ± 1.1aA
CP: Polyethylene plastic bag, SP: Polyethylene tray with semipermeable plastic
lm, VP: Vacuum packed (vacuum time 40 seg and sealing 2 seg). s.d.: standard
deviation, En:
Enterobacteriaceae, Mes: Mesophiles, Un: Undetermined, a-c:
Dierent lowercase letters in the same rows indicate signicant dierences
between storage days (
P<0.05), A-C: Different capital letters in the same
column indicate signicant dierences between packages (
P<0.05)
with a lower amount of CA. On the other hand, the PU content compared
to CA was similar to other studies [19, 29]. Also, NC can be formed
by the degradation of glutamine, arginine, and agmatine. Arginine is
easily converted into agmatine by the decarboxylation of arginine by
agmatine-deiminase and transformed to PU [6,18].
Although, HI concentration was greater than the CA [12, 19] and
CA concentration was not a risk of intoxication in this study, the
permissible CA limit was about 40 mg [18, 27]. The increase of NC
was given with the cooling time [1, 20]. In this regard, Vinci and
Antonelli [32] evaluated the amount of NC in bovine meat and chicken
meat; nding that NC production differs between two types of meat
up to 15 d of storage. The proteolytic enzymes easily attack meat
chicken, and consequently, there is better availability of amino acid
precursors for NC. In the case, rabbit meat has small bers, but the
collagen content of the Longissimus muscle is higher (7.0 mg·g
-1
) when
compared to the collagen of pigs (Sus scrofa domesticus) (5.9 mg·g
-1
),
bovines (3.4-5.8 mg·g
-1
) and chickens (3-4 mg·g
-1
). The collagen causes
greater hardness of the meat and possibly prevents the attack of the
proteolytic enzymes, consequently there may be less availability of
amino acid precursors for the formation of NC [26]. So, the lower
content of NC in rabbit meat can be due to this effect explained and,
the activity amino acid-decarboxylase inuenced by bacteria [1, 12] .
Temperature, oxygen availability, redox potential, and pH [11, 30] are
other factors that also participate in the NC formation.
The detection of HI in chicken meat was reported from 11 d of storage
[1] and in sh (Cyprinus carpio) meat on the 3
th.
d, with a maximum
peak at 12 d of storage [4]. In this study, the three types of packaging
showed an increase in HI with storage time. In reference, PU in the CP
and SP packages was related to the presence of high oxygen and less
carbon dioxide levels, unlike the VP generating selective microbial
proliferation [29]. The most common bacteria in VP were aerobic
mesophiles and Enterobacteriaceae [1]. The aerobic mesophiles were
higher at the end of the evaluation in the CP and SP (4.7), but the VP
T was similar throughout this experiment (TABLE II ). It is dicult to
have a direct correlation between the counts of microorganisms and
NC [6]; although in this study was observed that the PU content and
the aerobic mesophiles count were higher in the CP and SP. The results
were similar with beef vacuum packed, where the count of aerobic
mesophiles [8] and Enterobacteriaceae [3, 8, 9] increased in 14 d of
storage. Currently, there is interest in Enterobacteriaceae detection
in food samples, because these microorganisms are a public health
risk by toxicological effects.
CONCLUSION
The storage time changed the variables of color, pH, NC content,
and bacterial count. At the end of the evaluation (21 d), there were
differences in conservation time and the packaging type. The number
of Enterobacteriaceae was not altered by the storage time. The type
of packaging and the cooling time inuenced the concentration of
NC; mainly the VP had the lowest concentration of PU and mesophilic
bacteria until 21 d of storage.
Conict of interest
The authors declare no potential conicts of interest.
Control plastic (CP)
Semi-permeable (SP)
Vacuum packing (VP)
mg·Kg
-1
mg·Kg
-1
mg·Kg
-1
________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32121, 1 - 6
5 of 6
ACKNOWLEDGEMENTS
This research was supported by the Universidad Autónoma
Metropolitana Lerma and Colegio of Postgraduados of Mexico.
Ethics Approval
The study was approved and conducted in accordance with the
guidelines of the Institutional Animal Care of Colegio de Postgraduados.
México. (Approval ID: 09-2019).
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