Biopreservatives such as lactic acid bacteria (LAB) and their metabolites had been investigated by several authors (Buncic et al. 1997; Sakhare and Narasimha Rao, 2003). Considerable research has been done on the ability of LAB to inhibit growth of pathogenic microorganisms (Winkowski et al.1993; Minor-Pérez et al. 2004). The capability of these bacteria to control growth of spoilage microorganisms has not been investigateded to the same extent. To be successful in biopreservation, a bacteriocinogenic LAB culture must compete with the relatively high indigenous microbial loads of raw meat, to actively inhibit pathogenic and spoilage bacteria (Sakhare and Narasimha Rao, 2003; Minor-Pérez et al. 2004). Several studies have been carried out on the physical-chemical characteristics, sensory proprieties and nutritive values of camel meat (El-Faher et al. 1991; Elgasim and Al-Khanhal, 1992). Up to now, no data have been published on the preservation and extension of the shelf life of minced camel meat by using LAB or their metabolites except the use of some organic acid salts combined with Bifidobacterium by Al-Sheddy et al. (1999). The aim of this study was to investigate the possibility of extending the shelf life of fresh camel meat using Lactobacillus delbrueckii subsp. delbrueckii isolated from Moroccan foodstuffs, and selected for their inhibitory activity on Gram-negative and Gram-positive bacteria.

Ten samples of camel meat were collected from different butcher's shop in Morocco and transported to the laboratory using a refrigerated box (4ºC) and analyzed on arrival. LAB were isolated by homogenizing a 10 g subsample of each sample for 2 min in 90 ml of saline solution (8.5 g NaCl/l), and plating appropriate serial dilutions onto Man Rogosa Sharp (MRS) agar. LAB strains isolated from camel meat were selected on the basis of their inhibitory activities on various Gram – and Gram + bacteria to select the most efficient strain to be used in meat preservation. The antimicrobial activity of the isolated strains on Escherichia coli (4 strains), Pseudomonas aeruginosa (4 strains), Klebsiella pneumoniae (4 strains),Staphylococcus aureus (4 strains), Citrobacter freundii (2 strains), Bacillus subtilis (1 strain), Bacillus megaterium (1 strain), Bacillus cereus (3 strains) were determined by the well diffusion assay (Schillinger and Lücke, 1989). For the detection of antibacterial activity of the LAB strains, MRS broth was used. Ten ml of MRS broth was inoculated with each LAB strains and were incubated at 30ºC for 48 hrs. After incubation, a cell-free solution was obtained by centrifuging (6000×g for 15 min) the culture, followed by filtration of the supernatant through a 0.2 µm pore size filter thus obtaining cell-free filtrates. The pathogenic test bacteria were incubated in Brain Heart Infusion (BHI) broth at appropriate temperature for 24 hrs. Petri dishes with 20 ml of Muller Hinton agar were prepared, previously inoculated with 0.1 ml of a 24 hrs broth culture of pathogen microorganisms. Four wells (ø6 mm) were then made and filled using 100 µl of cell-free filtrate. The inoculated plates were incubated for 24 h at 37ºC, and the diameter of the inhibition zone was measured with calipers in mm. One of the most efficient strains was chosen to carry out this study. The isolate was Gram-stained and tested for catalase reaction. The isolate was then characterized by its growth at various temperatures (10, 15, 45ºC), tolerance of different salt levels (2%, 4%, and 6.5% NaCl), production of gas from glucose, dextran from sucrose and hydrolysis of arginine.The carbohydrate fermentation profiles of the selected isolate were investigated using API 50 CH strips according to manufacturer's instructions (API system, Bio-Merieux, France).

Camel meat was purchased from the retail in Rabat (Morocco). Amounts of 1 kg each were deboned and sliced. Fresh camel meat was minced with a sterile meat mincer in a sterile flask and the pH was checked before inoculating. The selected strain identified asL. delbrueckii subsp. delbrueckii was grown on MRS for 24 hrs at 30ºC. From this culture dilutions up to 10^-6 were plated on MRS to determine the cell concentration. The amount used to inoculate meat was around 10⁷ cfu/g. Glucose (5% w/w) was mixed with the minced meat. The inoculated meat was introduced in sterile plastic bags and stored at 22ºC. The control (minced meat without addition of glucose and lactic culture) was stored under the same conditions.

Samples (10 to 15 g), were cut into small pieces and blended in 90 ml of saline water to make the initial dilution (10^-1). Serial dilutions up to 10^-6 were then prepared. Standard plate count (SPC) was determined by plating appropriate dilutions on plate count agar. The plates were incubated at 30ºC for 48 hrs. LAB were plated on MRS, incubated at 30ºC for 48 hrs. Enterococci were determined on liquid media using 3 tubes per dilution. Appropriate dilutions were inoculated into Azide dextrose Broth, incubated at 30ºC for 24 hrs. Tubes that had shown growth were transferred on Ethyl Violet Azide Broth and incubated at 37ºC for 24 hrs. Tubes that had shown growth and formation of a violet precipitation in the bottom of tubes were counted. The number of positive tubes was reported to the table for the most probable number. Staphylococci were determined on Mannitol Salt Agar, the plates were incubated at 37ºC for 24 hrs. Coliforms were determined on Desoxycholate Lactose Agar, the plates were incubated at 37ºC for 24 hrs.

The strain of L. delbrueckii subsp. delbrueckii was first tested on different strains of Gram + and Gram – bacteria to study the inhibitory effects. The strains was inhibitory to all the strains tested which showed the almost the same sensitivity. All the diameters of the inhibition zones were in the range of 1.4 to 2.8 cm.

Results about the in vivo assay showed a pH drop from 5.5 to 4.0-4.2. This was achieved in 3 days incubation at ambient temperature. In the non-inoculated assay (control) the pH was around 7.2 after the same period. This may lead to a severe deterioration of meat leading to an off-odor appearance and many other changes in the meat. The pH decrease in the inoculated assay is due to the lactic acid formation by LAB. The pH decrease to 4-4.2 is related to the formation of the lactic acid in the medium. The microbial profiles including SPC, coliforms, staphylococci, enterococci and LAB for the inoculated assay and the control are reported in figure 1. SPC were decreased by approximately 2 log units during the first 24 hrs in the inoculated assay. The initial SPC in the mixture was around 1.4 x 10⁶ cfu/g, which decreased to 1.2 x 10⁴ cfu/g after 24 hrs and 8x10² cfu/g after 7 days of incubation at the ambient temperature (22ºC).

The same decrease pattern was also observed for the coliforms. Coliforms were reduced from 8 x 10² cfu/g to 10² cfu/g after 24 h and to less than 1 cfu/g after 7 days storage. It is interesting to note that the level of coliforms was reduced from the start of inoculation and development of an off-odour was prevented during storage of meat.

Fresh meat and fresh meat products are susceptible to biochemical changes due to the microbial growth at ambient temperatures, which are high in some countries (20 to 30ºC in Morocco). Minced meat is widely consumed in Morocco as a popular product called Kefta. This is exhibited at ambient temperature in retail and restaurants. Deterioration may occur fastly because of the high contamination, mincing and the high ambient temperature. It should be also assumed that all process used to extend the shelf life of fresh meat are based on low temperature (refrigerating or freezing). This may not be the case for fresh meat products exhibited in retail. Biological processes would be more efficient and safe to handle in this case such as the use of LAB to delay or to stop the spoilage microorganisms.

The desirable low pH (4.0-4.2) can be achieved faster for stabilization of meat incubated at ambient temperature using a LAB culture. Inhibition or reduction of spoilage and food poisoning microorganisms was demonstrated in meat during storage at high temperatures. The data generated in the present study is useful in hot countries like Morocco where refrigeration facilities are not available or inadequate especially to preserve camel meat or meat of other species for a long time. This procedure for preservation can also be used to improve sanitarian conditions in rural butcheries or popular restaurants when minced meat is exposed to sale at ambient temperature.

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