Identification of the lactic microorganism responsible for milk coagulation in Abeche (Chad)

1 Laboratory of Hygiene and Food Industry of Animal Origin (HIDAOA). EISMV, P.O Box 5077, Dakar, Senegal. 2 Faculty of Human Health, University of N’Djamena, P. O. Box: 1117, N’Djamena, Chad. 3 Laboratory of Microbiology Immunology Infectious Pathology (MIPI). EISMV, P.O. Box 5077, Dakar, Senegal. 4 Department of Animal Biology, Faculty of Science and Technology, University Cheikh Anta Diop (UCAD), Senegal.


INTRODUCTION
Milk is the staple food in a domestic economy.In Chad, the local artisanal dairy industry has developed considerably based on micro-enterprise collection and dairy processing (Koussou, 2008).Milk is a highly nutriatious food for its richness in carbohydrates, fats, vitamins and minerals (Aggad et al., 2009;Ahmed et al., 2010).Indeed, the dairy product comes in many forms depending on the region and varied tastes.The curd is especially appreciated by the Chadian consumers.Fermented milk has always been traditionally consumed in many countries (Nakasaki et al., 2008).Most studies on traditional fermented products showed that mesophilic lactic acid bacteria are the main cause of these fermentations group (Ben Amor et al., 1998).Lactic acid bacteria also through their lipase can decompose fat and free fatty acids in milk, resulting in the appearance of rancid odor in the dairy product.When lipolysis is not controlled or exaggerated, there is the appearance of strong and rancid odors (Vignola, 2002).Among the lactic acid bacteria with habitat as milk, we have Streptococcus, Lactobacillus, Enterococcus, Leuconostoc and Aerococcus (Luquet and Corrieu, 2005).In Chad, especially in Abeche, no study has been done about this subject.This lack of scientific information led us to seek and identify these bacteria in different varieties of "Laban", which is very well consumed by the population.The objective of this study was therefore *Corresponding author.E-mail: abdelti@yahoo.fr or abdelti@gmail.com.
to identify some species of lactic acid bacteria present in "Laban".

Sampling
In the suburban area of Abeche, we collected aseptically and randomly 100 samples of 250 ml of milk curd in sterile plastic jars.Before introducing the curd in the jars, the technician washes hands first with soapy water and take precautions to avoid contact with the curds.After collection, the samples were quickly introduced into a cooler box containing bottles of dry ice and transported at a low temperature to the laboratory for analyses.

Isolation and Identification of lactic acid bacteria
de Man, Rogosa and Sharpe (MRS) and M17 as agar media and broth were used to isolate lactic acid bacteria after incubating at 30°C for 48 h.One milliliter of each milk sample was homogenized in 9 ml of sterile saline.Lactic acid bacteria were enumerated by serial dilutions in sterile saline that were placed on the appropriate agar medium.Gallery API 20 STREP and API 50 CH gallery-L with their reagents (Biomerieux, France) were used in the identification using software API Web TM (Biomerieux, France).

Isolation and identification of lactic flora
The Table 1 shows the results of biochemical analyses of isolated strains with API 20 STREP.The test showed that the positive and negative reactions tend to equilibrate.
The Table 2 shows the digital profile and identification of strains.It was observed that there was a predominance of Lacotococcus lactis subsp lactis.The results of analyzes based on the biochemical and physiological criteria of isolated bacteria are shown in Table 3. Captured in Table 4 are the biochemical analyses of isolated strains with API 50 STREP.

DISCUSSION
The results of this study have identified six strains of Lactococcus lactis subsp.lactis, two strains of Lactococcus lactis subsp.cremoris and one flora contamination represented by a strain of Enterococcus faecium and Enterococcus durans strain.Ben Amor et al. (1998) found similar results of 12 isolates.They showed that the mesophilic lactic flora is represented by three species of Lactococcus flora associated with contamination of the Enterococcus genus.Moreover, Ngassam's studies (2007) on 10 samples of curled have identified a single species in all samples (100%): Lactococcus lactis subsp.lactis with a percentage of identification of 98%; while El- Baradei et al. (2007) have identified in the Egyptian traditional curd milk (Domiati) the species Lactococcus lactis subsp.lactis, Lactococcus garvieae and Lactococcus raffinolactis.Previously Lafarge et al. (2004) have also determined the curd pre-dominant presence of Lactococcus lactis subsp.lactis species by molecular methods.Also, further more studies (Katinan et al., 2012;Conte, 2008;Badis et al., 2005) reported the prevalence of Lactococcus    The percentage of identification obtained by the API software web MT are the order of 99.9% for most strains except LBA2, LBA8 and LBA9 that are a little discriminating profile compared to Cornobacterium pisicola Identification with a percentage of 52.6% and 3 test against (ribose, melibiose and sorbitol).Our results are similar to those of Ben Amor et al. (1998).These authors on eight strains were able to identify seven strains of Lactobacillus plantarum and strain Cornabacterium pisicola with a percentage of 50.7% and identification of three tests against.Lactobacillus plantarum has also been isolated (16 isolates) in raw goat milk from two Algerian goat populations (Badis et al., 2005).Ngassam (2007) meanwhile isolated a variety of six species of the Lactobacillus genus.The differences in species and their diversity may be explained by the origins of milk.

Conclusion
This work has identified certain lactic acid bacteria in curd available in Abeche.The study identified some lactic acid bacteria in the "Laban" consumed in of Abeche town.These include Lactoccocus lactis subsp.lactis Lactoccocus lactis subsp.cremoris and Lactobacillus plantarum.But these results are far to be completed because the country is too large as well as the agro-ecological zones are varied.Knowledge of most lactic acid bacteria in Chad will help in the production of dairy products with longer shelf-life.Other studies, including the characterization of molecular biology of lactic acid bacteria is necessary to provide benefit information to improve the technological and sanitary quality of milk.

Table 1 .
Biochemical analysis of isolated strains with API 20 STREP.

Table 2 .
Digital profile and identification of strains of Lactococci by the API 20 STREP galleries.

Table 3 .
Biochemical analysis of isolated strains with API 50 STR.

Table 4 .
Biochemical analysis of isolated strains with API 50 STREP.