Advance Journal of Food Science and Technology 3(4): 289-293, 2011
ISSN: 2042-4876
© Maxwell Scientific Organization, 2011
Received: May 30, 2011
Accepted: July 18, 2011
Published: August 31, 2011
Effects of Packaging, Treatments, and Storage Conditions on the Survivability of
Aerobes and Anaerobes in Vacuum Packaged ‘Wara’ a Soft White Cheese
Victoria O. Adetunji
Department of Veterinary Public Health and Preventive Veterinary Medicine,
University of Ibadan, Ibadan, Nigeria
Abstract: This study assessed total aerobes and anaerobes in vacuum packaged ‘wara’ a West African soft
white cheese processed with lemon juice as coagulant. The cheese was treated with Carica papaya (Vcpc),
Terminalia cattapa (Vtcc) crude extracts, nisin (Vnc), and the combination of these three treatments (V+3) and
stored at 15 and 28ºC for a three weeks storage period. The total aerobes and anaerobes were suppressed to an
undetectable level (<1.00 logcfu/mL) in Vcpc at 15ºC in the first week of storage. In Vtcc the total aerobes
which was initially 6.24 logcfu/mL in the first week of storage was suppressed to 3.16 log cfu/mL by the 3rd
week of storage. Inhibition of microbes was not apparent in Vnc and V+3. Significant differences occurred in
the microbial count at the two storage temperatures and storage weeks (p<0.05). It can therefore be concluded
from this work that Vacuum packaging and addition of crude extracts (Carica papaya, Terminalia cattapa) in
soft cheese storage can suppress total aerobes and anaerobes) Therefore their use in extension of the shelf-life
of soft cheese is recommended.
Key words: Aerobes, anaerobes, Carica papaya, Terminalia cattapa, nisin, soft cheese
Vacuum packaging and modified atmospheric
packaging have been used for the preservation of many
products: fruits, vegetables, cheese among others (Kader
et al., 1989; Gorny, 1997; Pintado and Malcata, 2000).
These methods reduce the oxygen level which retards
browning and spoilage and maintain fresh appearance
(Ray, 2000). However, it can cause off-flavour and
flavour loss (Cameron and Smyth, 1997). In the case of
meats, up to 10-20% carbon (iv) oxide may develop
within 4 hours and the concentration may ultimately reach
30% from the respiratory activities of the aerobic biota
(Gardner et al., 1967).
These methods and others may be used
synergistically to further obtain higher microbial quality
of the preserved products. One of such is the inactivation
of spores of Bacillus cereus in cheese by high hydrostatic
pressure with the addition of nisin or lysozyme (LopezPedemonte et al., 2003).
Carica papaya a milk coagulant (Veringa, 1961) has
also been used for medicinal purposes (Tang et al., 1976;
Lal et al., 1976; Kermanshai et al., 2003). Antihelmintic
and antibacterial effect of extracts of Carica papaya has
been demonstrated (Adebiyi and Adaikan, 2005).
Terminalia cattapa is also of high medicinal value. It has
been shown to have anticarcinogenic potential,
aphrodisiac activity and also used in the treatment of
sickle cell disorders (Ratnasooriya and Dharmasi, 2000).
The wound healing potential of some species of
Terminalia grown in Mali and the antimicrobial activity
of extracts Terminalia cattapa root have been described
(Diallo et al., 2002; Pawar and Pal, 2002).
INTRODUCTION
The number of microorganisms found on produce can
vary according to a number of factors including product
quality, product characteristics, and normal microflora of
the product, handling, processing, storage, distribution
and retail display (Gonzalez-Fandos et al., 2000).
Microbial growth is influenced by the physiology of the
minimal processed product, as well as pre-harvest
management, pre- and post-processing treatments and
application of an appropriate packaging (Watada, 1997).
The utmost aim of preservation is to prevent food spoilage
of easily perishable products by extending their shelf- life
to meet market demands. Some of the modern
technologies employed include modified atmosphere
packaging, use of protective cultures, use of bacteriocins
and other culture products and enzymes (Gould, 1996).
Wara an unripened soft cheese has an average shelflife of 2-3 days when stored in whey at ambient
temperature (approximately 28oC) (Adegoke et al., 1992;
Belewu et al., 2005). Attempts have been made in the
recent past to include starter cultures or various
preservatives such as propionic acid, sodium benzoate,
and sorbic acid in the production of wara (Belewu et al.,
2005; Aworh and Egounlety, 1985). Some of these
preservatives have been shown to be effective in
inhibiting mesophilic and psychrotrophic bacteria as well
as coliforms. However, these preservatives may not be
easily accessible to the local cheese processors in West
Africa. Therefore, the need for improvement on the
methods of preservation of the soft cheese in this study.
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Adv. J. Food Sci. Technol., 3(4): 289-293, 2011
Nisin is a bacteriocin produced by Lactococcus lactis
sub-specie lactis. It has been shown to inhibit Listeria
monocytogenes in cottage cheese stored at 4ºC
(Benkerroum and Sandine, 1988). It was also observed
that bacteriocin (nisin) delayed the growth of
psychrotrophic organisms that cause cheese spoilage.
Non-sterilized cottage cheese samples without nisin
spoiled 1 week earlier than those with nisin (Benkerroum
and Sandine, 1988).
This work assesses total aerobes and anaerobes in
vacuum packaged lemon juice cheese treated with two
indigenous plants Carica papaya and Terminalia cattapa
crude extracts and the bacteriocin-like substance extracted
from Lactococcus lactis.
cheese processing. The milk described above was heated
to approximately 50ºC in about 30-40 min and stirred
gently during heating using magnetic stir bars. Freshly
squeezed lemon juice (49.5 mL) was added to the warm
milk, and the milk and lemon juice mixture was heated
with intermittent stirring until it reached the boiling point.
The milk with added lemon juice was kept at the boiling
point until it coagulated and the separation of curd and
whey became visible. The milk pots were then removed
from the heating source, and the curds and whey were
ladled into sterile egg separators (8 mm in diameter),
which facilitated whey drainage and gave the cheese its
characteristic shape and size. Yield from this process was
600 g of soft ripened cheese ready for treatment with
crude extracts.
MATERIALS AND METHODS
Preparation of crude extracts: Terminalia cattappa
(2.38 g) was grinded to powder and extracted in 10 mL of
70% ethanol. Carica papaya (4.5 g) was also grinded and
extracted in 20 mL ethanol to obtain between 0.2-0.25
g/mL concentration. After extraction the mixture was
sieved and the resulting liquid was concentrated in a
rotavapour (Drissa et al., 2002).
Preparation of the Nisin: A bacteriocin(nisin) producing
Lactococcus lactis strain was isolated from unpasteurized
raw milk according to Barrow and Feltham (1993). Test
for bacteriocin production was done according to Aslim
et al. (2005) with slight modification. 24 h fresh culture of
Lactococcus lactis strains showing inhibitory qualities
against test (Listeria monocytogenes and E. coli
(laboratory stock cultures from food) bacteria was grown
on M17 agar and inoculated into tryptose soy broth and
incubated at 37ºC for 24 h. This was then incubated at
37ºC for 24 h. The 24 h broth culture was then centrifuged
at 3000 rmp for 5 min. The superna- tant was decanted
into sterile test tubes, adjusted to pH 6.5-7.0 with NaOH
(40G/1000 mL) to remove organic acid effect. H2O2 was
neutralized by addition of catalase from bovine liver at
200 :/mL. The mixture of the supernatant of Lactococcus
lactis culture, NaOH and catalase was filtered and
sterilized with a 0.2 :m Millipore filter membrane.
Inhibitory effect of free bacteriocin (nisin) on test bacteria
was then determined by agar well diffusion method using
the filtrate from the mixture and the supernatant alone.
Mueller Hinton II agar plates were punched with 8 mm
diameter sterile clubs. The bottom of each well was
covered with viscous agar which was allowed to solidify
to prevent spreading of the mixture and supernatant
through the base. The wells were then filled with 20 :L of
the mixture and filtrate of supernatant alone thereafter was
refrigerated for 2 h to allow diffusion of test substrate,
followed by incubation for 24 h at 37ºC. The diameter of
inhibition was then measured with meter ruler. A zone of
inhibition of 18-20 mm was reported against the two
foodborne pathogens.
Sample preparation, packaging and storage: Wara
cheese from the above process was divided aseptically
into 60 10 g pieces. Then five sample groups of 8 from
these pieces were then subjected to treatment by
drenching with 1 mL.
Each of 1. Terminalia cattapa seed extracts 2. Carica
papaya seed extracts 3. Nisin 4. Treatment with all 3 (T.
cattapa and C. papaya extracts and nisin) 5. Untreated
control. All cheese samples after treatment were stored in
vacuum packaged bags. One of each pair treatment was
stored at 28ºC while the other pair was stored at 15ºC for
a 3 week storage period.
Microbiological sampling: Sampling was done on every
week for a 3 weeks storage period. A separate sample was
used representing the various weeks of storage on each
sampling day. The pH of the samples was taken using a
VWR scientific model 1800 electrode pH meter. The
samples were then homogenized within each bag with
10mls of 0.1% peptone buffer with Seward Stomacher
Lab. System on each sampling day. Serial dilution was
made with 0.1% peptone water for ease of counts.
Appropriate dilutions were then surface plated on tryptose
soy agar for total aerobic bacteria, and anaerobic bacteria
counts. Plates for aerobic bacteria were incubated
aerobically at 37ºC for 18hours while plates for anaerobic
bacteria were incubated anaerobically at 37ºC in a GasPak
system for 18 h. Discrete colonies were counted using a
darkfield colony counter. Counts were expressed in
cfu/ml and then converted to log10 cfu/mL.
Preparation of milk for wara processing: Four liters of
12% fat pasteurized whole milk was purchased from a
store. The milk was maintained at 4ºC in a cooler and
transported to the laboratory where it is stored at 4ºC until
use. This milk was put in a sterile pot for ‘wara’ soft
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Adv. J. Food Sci. Technol., 3(4): 289-293, 2011
Table 1: Microbial counts and pH of vacuum packaged lemon juice cheese treated with Carica papaya and Terminali cattapa crude extracts and nisin
Storage
Vcpc
Vcpc
Vtcc
Vtcc
Vnc
Vnc
V+3
V+3
Conc.
Conc.
Temperature
28ºC
15ºC
28ºC
15ºC
28ºC
15 ºC
28ºC
15ºC
28ºC
15 ºC
Total aerobe
Day 1
<1.0a
<1.0a
<1.0a
<1.0a
<1.0b
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
1stwk
7.61c
<1.0f
8.60b
6.24e
9.43a
7.39dc
8.65b
7.18d
7.50c
1.00f
2ndwk
7.59c
4.81g
6.08e
4.61g
9.45a
8.68b
6.99d
5.39f
7.24c
6.60d
3rdwk
*5.10d
5.3d
6.86b
3.16c
10.19a
10.02a
7.10b
5.48c
6.45b
4.73g
Anaerobes
Day 1
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
<1.0a
1stwk
*6.70c
<1.0g
7.39b
5.92d
9.55a
4.64e
7.41b
6.83c
7.65b
2.81f
2ndwk
6.68c
4.48e
*4.84e
4.78e
7.14a
6.12c
6.73b
5.39d
7.52a
6.00c
3rdwk
6.59d
5.28g
5.70f
5.70f
10.82a
10.5b
7.75c
6.49d
6.24e
4.68h
PH
Day1
5.75b
5.75b
5.75b
5.75b
5.75b
5.75b
5.75b
5.75b
5.65c
5.65c
1stwk
5.71b
5.64c
5.60d
5.50e
4.94g
5.07f
5.76a
5.74b
5.80b
5.71b
2ndwk
5.48c
5.70b
4.90d
5.71b
4.57e
4.92d
5.46c
5.81a
5.23c
5.66c
3rdwk
5.46c
5.45b
4.62d
5.48b
5.10c
4.61d
5.48b
5.85a
4.84d
5.70b
Vcpc = vacuum packaged Carica papaya treated cheesea; Vtcc = vacuum packaged Terminali cattapa treated cheeseb;
Vnc = vacuum packaged nisin treated cheese; V+3 = vacuum packaged cheese treated with a, b and c; Con. C = vacuum packaged cheese with no
treatment; Values followed by same lowercase letters in the same colum are not significantly different with respect to storage weeks; Values followed
by different lowercase letters in the same colum are significantly different with respect to storage weeks;
*: Values which are significantly different with respect to treatments and storage temperature
storage temperature. However, significant reductions from
7.39-4.84, 9.55-7.14 and 7.41-6.73 logcfu/mL were
obtained in Vtcc, VnC, and V+3 respectively at 28ºC
storage temperature in the 1st and 2nd week of storage. A
sharp increase in anaerobes from 4.84-5.7, 7.14-10.82 and
6.73-7.75 logcfu/mL was observed in Vtcc, VnC, and
V+3 respectively at 28ºC storage temperature in the 3rd
storage week. Counts were undetectable in Vcpc in the 1st
storage week at 15ºC storage temperature but increased
rapidly to 5.28 logcfu/ml in the 3rd week. The total
anaerobes which were 5.92 and 6.83 logcfu/mL in the 1st
storage week decreased to 5.7 and 6.49 logcfu/mL in Vtcc
and V+3 respectively in the 3rd week at 15ºC storage
temperature. However, counts increased from 4.64 and
2.81 logcfu/mL in the 1st storage week to 10.5 and 4.68
logcfu/mL in the 3rd week in VnC and Con.C,
respectively (Table 1).
The pH of the cheese in the different storage medium
also varied significantly with the storage week and
temperature. Decrease in pH from 5.71, 5.76, and 5.80 to
5.46, 5.48 and 4.84 logcfu/mL were observed in Vcpc,
V+3, and Con.C, respectively at 28ºC storage
temperature. Also a decrease occurred from 5.64 and 5.07
to 5.45 and 4.61 in Vcpc and Vnc respectively at 15ºC
storage temperature in the 1st to 3rd week of storage. On
the other hand, an increase in pH from 4.94 and 5.74 to
5.1 and 5.85 was obtained in Vnc and V+3 at 28 and
15ºC, respectively (Table 1).
Statistical analysis: The study was performed in two
replicates, each with appropriate duplications. All
microbiological data were transformed into Log10
CFU/mL or Log10 CFU/g before comparison of means.
Analysis of data was accomplished using the Fishers
least significant difference of means of bacterial
populations between storage weeks and treatment were
calculated with the General Linear Model (GLM)
procedure of SAS based on a 95% confidence level (SAS,
2000).
RESULTS
There were significant differences in the total aerobes
and anaerobes of the vacuum packaged lemon juice
cheese treated with Carica papaya (Vcpc), Terminalia
cattapa (Vtcc) crude extracts, nisin (Vnc), the
combination of the three (V+3), and the control
experiment (Con.C) at 15 and 28ºC, respectively
(Table 1). At 28ºC storage temperature, the total aerobes
in the 1st, 2nd and 3rd week were 7.61, 7.59 and 5.1; 8.6,
6.08 and 6.86; 9.43, 9.45 and 10.19; 8.65, 6.99 and 7.1;
7.50, 7.24 and 6.45 for Vcpc, Vtcc, Vnc, V+3 and Con.C,
respectively. The corresponding values at 15ºC storage
temperature were <1.00, 4.81 and 5.3; 6.24, 4.61 and
3.16; 7.39, 8.68 and 10.02; 7.18, 5.39 and 5.48; <1.00, 6.6
and 4.73, repectively for Vcpc, Vtcc, Vnc, V+3 and
Con.C. The total aerobes were undetectable (<1.00) in
Vcpc and Con.C at 15ºC storage temperature in the 1st
week but increased to 4.81 and 6.60, respectively by the
2nd week. However, a decrease in total aerobes was
observed in Vtcc and V+3 at 15ºC storage temperature in
the 2nd week. Also, significant reductions were observed
in the total aerobes in Vcpc at 28ºC storage temperature
(7.61-5.1) and in the control cheese (Con.C) (7.50-6.45)
by the 3rd week of storage (Table 1).
The total anaerobes reduced insignificantly from the
1st to the 3rd week (6.7-6.59 logcfu/mL) in Vcpc at 28ºC
DISCUSSION
The higher total aerobic counts in the weeks 1 and 2
in this study compared to anaerobes observed in this study
could be due to the retention of some oxygen within the
soft cheese for cellular respiration by the oxygen
dependent aerobic microbes. There have been reports on
the correlation between oxygen content and microbial
growth (Gould, 1989). The fluctuation in the total aerobic
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Adv. J. Food Sci. Technol., 3(4): 289-293, 2011
counts in different storage time could due to variations in
the oxygen content of the cheese samples. The lower
aerobic bacterial counts reported sometimes along the
storage week could be as a result of gradual oxygen
depletion within the cheese; being used up by the
microbes. Similar report on decline in total viable
bacterial counts of soft cheese during storage under
reduced oxygen has been given (Whitley et al., 2000).
The inhibition of bacteria in anaerobic conditions has
also been reported (Farber, 1999; Church and Parsons,
1995). The sharp increase in anaerobes in nisin treated
samples is expected because of increase in growth of
anaerobes like lactic acid bacteria.
The significant differences recorded in the microbial
loads at 15 and 28ºC storage temperatures are due to
change in the respiration rate influenced by temperature
changes. Higher temperature could favour the respiration
rate of microbes in the vacuum packaged cheese thus
favouring the microbial growth. This report corroborates
with the report that the respiration rates of carrots
increased by about 5-fold when temperature increased
from 0ºC to 10ºC, and the increase in microbial
population was about 100-fold greater at 10ºC than at 0ºC
(Izumi and Watada, 2004). Increase antimicrobial efficacy
of carbon iv oxide modified atmosphere packaging of
food at reduced temperature has been reported (GonzalezAguilar et al., 2004).
The inability of nisin substance to suppress microbial
growth despite its well known antimicrobial abilities
could be due to its limited spetrum of activity. Earlier
reports also showed the differences in efficacy of
bacteriocins produced by various strains of Lactococcus
spp. (Adetunji and Adegoke, 2007). The lower pH
recorded as a result of addition of nisin was inimical to
the microbial growth probably due to reduced efficacy.
Vacuum packaged lemon juice cheese treated with
Carica papaya (Vcpc) and Terminalia cattapa (Vtcc)
crude extract preserved cheese better than when only
vacuum packaging was used but their effectiveness in
cheese preservation is both time and temperature
dependent. The combination of the two plants crude
extracts and nisin to preserve the cheese did not improve
the microbial quality of cheese. Similar to this report in an
earlier study inhibition of enterobacteriacea was not
evident in vacuum packaged cheese treated with Carica
papaya and Terminalia cattapa crude extract (Adetunji,
2008). It is therefore evident in this study that much lower
temperature with the addition of the two vegetable
extracts can be suggested to give a much more lower
microbial count. It is recommended that modern
technologies should be employed in West African
countries soft cheese processing to completely evacuate
the air bubbles that might be retained within the cheese as
this can limit the packaging success. The use of purified
plant extracts and reduction in water activity can be
introduced in future research.
CONCLUSION
Arising from this study storage of soft cheese at
lowered temperature with the addition of the two
veg table extracts will give lower microbial count.
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