Effectiveness of Commercial Organic Acids Mixture (AcetolacTM) to Extend the Shelf Life and Enhance the Microbiological Quality of Merguez Sausages
Bouchra El Ayachi,
The effect of the addition of organic acids on the shelf life and on the hygienic quality of Merguez sausages was studied. A commercial organic acid; AcetolacTM consisting of a mixture of sodium lactate (90%) and sodium acetate (10%) was added at different concentrations (0, 5, 10, 15 or 20 g kg-1) to the butter mixture for sausage making. Microbiological enumerations (total aerobic count, coliforms, fecal streptococci, Staphylococcus aureus and sulfite reducing clostridia) along with the pH and Total Volatile Basic nitrogen (TBV-N) were monitored during storage at refrigeration temperature (~8°C) for 15 days. The results showed that the addition of organic acids reduced significantly (p<0.05) the microbiological counts during the first three days of storage and such an effect increased with the concentration of added AcetolacTM. Thereafter, a significant (p<0.05) increase in the colony forming units (cfus) of all the bacterial groups enumerated was recorded with the exception of S. aureus whose cfus remained practically constant. Concomitantly, the pH and TBV-N content have increased throughout the whole period of storage. As for the shelf life, control samples (without added AcetolacTM) were altered at day 5 of cold storage as judged by a sensory panel on the basis of perceivable sensory attributes. However, such perceivable alterations were delayed at least 5 days in samples treated AcetolacTM at levels ranging between 10 and 20 g kg-1.
Merguez is a raw sausage of North Africa origin widely consumed in different countries around the world either fried, barbecued or as an ingredient in some typical traditional dishes. In Morocco, it is usually prepared by butchers to be sold within two days because of its highly perishable nature. This shelf life may be shorter when merguez sausages are produced in poor hygienic conditions and exposed for sale at ambient temperature as is usually the case in the traditional butcher shops. Furthermore, merguez is a potential vehicle for serious food borne pathogens including Listeria monocytogenes, Escherichia coli O157 (Benkerroum et al., 2003; Benkerroum et al., 2004).
Many strategies have long been used to enhance the safety and keeping quality
of meat products. Curing with a combination of sodium chloride and Nitrites
salts is probably the oldest and the most widely used method for the preservation
of meat products (Davidson and Harrison, 2002). However, the inability of nitrites
to efficiently control some meat-born pathogen (Incze, 1998; Cleveland et
al., 2001; Garriga et al., 2002) in addition to their potential carcinogenicity
(Hartman, 1983; Wary and Sharan, 1991) have stimulated the interest of researchers
and meat producers to search for alternative means to minimize or avoid their
use in meat preservation. Therefore, the efficacy of natural food grade additives
such as bacteriocins (Scannell et al., 2000; Benkerroum et al.,
2003; Guinane et al., 2005; Ghalfi et al., 2006), essential oils
of plant origin (Skandamis and Nychas, 2001; Cutter 2000; Tsigarida and Nychas,
2001), organic acids or their salts (Osthold et al., 1984; Unda et
al., 1990; Brinkmann, 2001), adjunct starter cultures (Luke, 2000; Hugas
and Montfort, 1997) alone or in combination with each other or with conventional
treatments in controlling specific pathogens and/or extending the shelf life
of meat products has been investigated according to the multiple hurdles technology
The present study aimed to evaluate the effectiveness of a commercial mixture of sodium lactate and sodium acetate (9:1) to enhance the hygienic quality and the shelf life of merguez sausages.
MATERIALS AND METHODS
Acetolac™, a commercial organic acid preparation consisting
of a mixture of sodium lactate (90%) and sodium acetate (10%) was added at different
concentrations to the butter mixture for merguez sausages making. The butter
mixture contained: lean beef, 80%; fat beef, 20%; sugar, 5 (g kg-1);
salt, 18 (g kg-1); red pepper, 10 (g kg-1); cumin, 5 (g
kg-1); black pepper, 5 (g kg-1); ginger, 5 (g kg-1);
olive oil (10 mL). The meat lean and the fat were cut up with a Kilia cutter
(Germany) then mixed to the other ingredients. Five trials were conducted simultaneously.
The same basal formulation described above was used for all trials; however,
acetolac™ was added to four test trials at a concentration of 5,
10, 15 or 20 g kg-1. No Acetolac™ was added to one trial
to serve as a control. The sausage mixture of each trial was stuffed into natural
casing (bovine small intestine) and stored in the refrigerator at approximately
Sampling and Analyses
Samples (80 g of intact merguez segments) were withdrawn from each trial
at regular intervals (0, 3, 7, 15 days) for sensory, chemical and bacteriological
analyses. The merguez segments were aseptically cut with a knife into small
pieces of about 0.5 cm each and kept in a sterile glass container for analysis.
The sensory evaluation was conducted by a trained sensory panel of four
persons who were asked to judge whether or not samples were altered on the basis
of the physical appearance and the smell.
The pH was measured using an E250 pH-meter (Metrhom Ltd., Heriseau, Switzerland)
on a 10 g sample minced and homogenized to 90 mL of distilled water. Total Volatile
Basic nitrogen (TVB-N) was determined according to the micro-diffusion test
of Conway (Conway, 1958)
A 25 g sample was pre-comminuted and mixed to 225 mL of a peptone solution
with an Ultra Turrax (IKA Type T25, Jante and Kunkel, Germany) under aseptic
conditions. The suspension was then decimally diluted and bacterial enumerations
were carried out by the standard pour-plate technique or by the Most Probable
Number (MPN) as appropriate. All media used were purchased from Biokar (France).
The total Aerobic Count (TAC) was carried out on plate count agar (BK 098) after
incubation at 30°C for 48 h; coliforms were enumerated on Desoxycholate
Citrate Lactose agar (DCL) after incubation at 37°C for 24 h; Fecal streptococci
counts were determined by the MPN using Rothe broth (BK 060) and Litsky broth
(BK 061) as the presumptive and confirmatory media, respectively and in each
step the incubation was carried out at 37°C for 24 h; Staphylococcus
aureus was enumerated on Baird Parker agar (BK 097) after incubation at
37°C for 24 h and the spores of sulphite reducing clostridia on meat liver
agar (BK 097) after incubation at 44°C for 72 h.
Each trial was repeated twice and each determination was done in duplicate.
Statistical analysis was done by analysis of variance α = 0.05% and Student
RESULTS AND DISCUSSION
Figure 1 summarizes the results of the trend of the pH and
AVB-N in merguez samples during storage at 8°C. The results show that the
pH has increased in all samples with a concomitant increase in the TVB-N content
(Fig. 2) indicating that the pH increase resulted from a proteolytic
activity that took place in merguez during conservation at refrigeration temperature.
Such an activity may result from microbial proteases mainly produced by psychrotrophic
bacteria (e.g., Pseudomonas sp., Proteus sp. or other species
of the Enterobacteriaceae family) known to be the main spoilage microorganisms
in meat products or by indigenous proteases naturally occurring in meat and
in the intestine used as a casing to portion the sausages. The Fig.
1 and 2 show also that the increase in pH and TVB-N was
significantly slower in presence of acetolac™ and that such an effect
was more pronounced as the concentration of added Acetolac™ increased.
||pH evolution during storage of merguez sausages treated with
different levels of organic acids
||Total Volatile Basic Nitrogen (TVB) evolution during storage
of merguez sausages treated with different levels of organic acids. Symbols
are as in Fig. 1
||Bacterial counts evolution as function of time in merguez
sausages treated with organic acids (Acetolac™) at different
concentrations: Total aerobic count (A), Coliforms (B), Fecal streptococci
(C) and Staphylococcus aureus (D). Symbols are the same as in Fig.
1. The counts of S. aureus in samples treated with 15 g kg-1
were the same as in those treated with 5 g kg-1 in D
At the highest concentration of added Acetolac™ (2% w/w), no significant
(p>0.05) changes (increase or decrease) in the pH were noted during the first
7 days of cold storage (Fig. 1). The fact that no decrease
in the pH was observed suggests that the acidifying bacteria naturally occurring
in the sausages were also inhibited by the organic acid mixture resulting in
a stabilisation of the pH during this period. It is worthwhile to mention that
the acidification of merguez sausages during storage is also regarded as a sensory
alteration by consumers.
Figure 3 showes the evolution of the viable counts of various
bacterial groups of health or spoilage significance as function of time during
storage at 8°C. A dramatic decrease in the colony forming units (cfu) of
all of the bacterial groups enumerated was recorded within the first three days
of storage except for the TAC in the control samples and in samples treated
with 5 g kg-1 where the cfus continue
to increase. However, after the third day a significant increase (p<0.05)
in the counts of all the bacterial groups was observed, with the exception of
Staphylococcus aureus (Fig. 3D) whose counts remained
practically constant throughout the whole period of storage after reaching a
minimum level at day 3. Growth re-initiation of microbial groups after the third
day of storage for all samples may be explained either by the pH increase or
by the depletion of the organic acids used as a carbon and energy source by
some microorganisms (e.g., yeasts and moulds). Adaptation of microorgnaisms
to antimicrobial agents upon extended exposure has also been reported to account
for such a rebound phenomenon (Davidson and Harrison, 2002). The counts of sulphite
reducing clostridia were below the detectable limit of 10 cfu g-1,
which is a good indication regarding the safety of the product, as the presence
of pathogenic clostridia have long been know to represent a major health risk
associated with the consumption of meat products.
As for the keeping quality of merguez sausages, the sensory analysis revealed that the growth of molds, surface mucoidness or discoloration, off-odours and other perceivable alterations were delayed by at least 5 days in samples with added organic acids at the concentrations ranging between 10 and 20% (w/w) as compared to control samples (without added Acetolac™). The latter samples were altered within 5 days of storage. Therefore, the treatment of merguez sausages with Acetolac™ has extended the shelf life of the sausages by five days at the refrigeration temperature which is regarded as very significant considering the highly perishable character of merguez.
The results of the present study suggest that the addition of Acetolac™ at levels ranging between 10 and 20 g kg-1 of butter mixture appears to effectively enhance the hygienic quality Merguez sausages and extend its shelf life by 5 days at refrigeration temperature. The use of Acetolac™ in Merguez preservation is even more appropriate as the organic acids that it contains are safe to consumers. However, this measure does not represent a substitute for the scrupulous application of the good manufacture practices, but rather add up a safety factor.
The authors are grateful to the Societe Anonyme des Palmeraies Koutoubia, SAPAK
(Mohammedia Morocco) for supporting part of the present research.
1: Benkerroum, N., A. Daoudi and M. Kamal, 2003. Behavior of a Listeria monocytogenes in a raw sausage (merguez) in presence of a bacteriocin-producing strain as a protective culture. Meat Sci., 63: 53-58.
2: Benkerroum, N., Y. Bouhlal, A. El Attar and A. Marhaben, 2004. Occurrence of shiga toxin-producing Escherichia coli O157 in selected dairy and meat products marketed in the city of Rabat, Morocco. J. Food Prot., 67: 1234-1237.
PubMed | Direct Link |
3: Brinkmann, W., 2001. Haltbarkeit und sicherheit verbessert. Milchsäure und ihre salze entfalten positive effekte auf fleischwaren. Fleischwirtsch, 6: 26-29.
4: Cleveland, J., T.J. Montville, I.F. Nes and M.L. Chikindas, 2001. Bacteriocins: Safe, natural antimicrobials for food preservation. Int. J. Food Microbiol., 71: 1-20.
CrossRef | PubMed | Direct Link |
5: Conway, E.J., 1958. Microdiffusion Analysis and Volumetric Error. The MacMillan Compagny, New York, pp: 199.
6: Cutter, C.N., 2000. Antimicrobial effect of herb extracts against Escherichia coli O157: H7, Listeria monocytogenes and Salmonella typhimurium associated with beef. J. Food Prot., 63: 601-607.
PubMed | Direct Link |
7: Davidson, P.M. and M.A. Harrison, 2002. Resistance and adaptation to food antimicrobials, sanitizers and other process controls. Food Technol., 56: 60-78.
Direct Link |
8: Garriga, M., M.T. Aymerich, S. Costa, J.M. Monfort and M. Hugas, 2002. Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage. Food Microbiol., 19: 509-518.
Direct Link |
9: Ghalfi, H., P. Kouakou, M. Duroy, A. Daoudi, N. Benkerroum and P. Thonart, 2006. Application of an antilisterial bacteriocin-producing strain of Lactobacillus curvatus CWBI-B28 as a protective culture in bacon meat and effect of fat and sodium nitrites on bacteriocin production and activity. Food Sci. Technol. Int., 12: 325-333.
CrossRef | Direct Link |
10: Guinane, C.M., P.D. Cotter, C. Hill and R.P. Ross, 2005. Microbial solutions to microbial problems: Lactococcal bacteriocins for the control of undesirable biota in food. J. Applied Microbiol., 98: 1316-1325.
PubMed | Direct Link |
11: Hartman, P.E., 1983. Review: Putative mutagens and carcinogens in foods.I. Nitrate/nitrite ingestion and gastric cancer mortality. Environ. Mut., 5: 111-121.
12: Hugas, M. and J.M. Monfort, 1997. Bacterial starter cultures for meat fermentation. Food Chem., 59: 547-554.
Direct Link |
13: Incze, K., 1998. Dry fermented sausages. Meat Sci., 49: 169-177.
14: Leistner, L., 1996. Food protection by hurdle technology. J. Food Prot., 2: 2-27.
15: Lucke, F.K., 2000. Utilization of microbes to process and preserve meat. Meat Sci., 56: 105-115.
Direct Link |
16: Osthold, W., H.K. Shin, J. Dressel and L. Leistner, 1984. Improving the storage life of carcasse by treating their surfaces with an acid spray. Fleischwirtsch, 64: 828-830.
17: Scannell, A.G.M., R.P. Ross, C. Hill and E.K. Arendt, 2000. An effective lacticin biopreservative in fresh pork sausage. J. Food Prot., 63: 370-375.
PubMed | Direct Link |
18: Skandamis, P.N. and G.J.E. Nychas, 2001. Effect of oregano essential oil on microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres. J. Applied Microbiol., 91: 1011-1022.
Direct Link |
19: Tsigarida, E. and G.J.E. Nychas, 2001. Ecophysiological attributes of a Lactobacillus sp. and a Pseudomonas sp. on sterile beef fillets in relation to storage temperature and film permeability. J. Applied Microbiol., 90: 696-705.
Direct Link |
20: Unda, J.R., R.A. Molins and H.W. Walker, 1990. Microbiological and some physical and chemical changes in vacuum packaged beef streaks treated with combination of potassium sorbate, phosphate, sodium chloride and sodium acetate. J. Food Sci., 55: 323-326.
21: Wary, K.K. and R.N. Sharan, 1991. Cytotoxic and cytostatic effects of arecoline and sodium nitrite on human cells in vitro. Int. J. Cancer, 47: 396-400.