Lactic acid bacteria from “ Sheep ’ s Dhan ” , a traditional butter from sheep ’ s milk : Isolation , identification and major technological traits

Veintiséis bacterias lácticas fueron aisladas de “Sheep ́s Dhan”, una mantequilla tradicional hecha con leche de oveja en Jijel (al Este de Argelia). Estas cepas pertenecen a tres géneros: Lactococcus, Leuconostoc y Lactobacillus. Los resultados mostraron que Lactococcus lactis ssp diacetylactis fue la especie predominante en esta mantequilla tradicional. Los resultados de la evaluación de la aptitud tecnológica indican que la principal cepa tiene una buena aptitud de acidificación, algunas de ellas mostraron una buena actividad proteolítica y únicamente Leuconostoc mesenteroides ssp. dextranicum fue capaz de producir exopolisacárido.


INTRODUCTION
Milk fat is one of the major products in the dairy industry and has been primarily used in the production of butter (Arul et al., 1988).It is a complex fatty acid composition that results in a unique mixture of triglycerides with a wide range of molecular weights and degrees of saturation (deMan and deMan, 1983).Besides fats, butter contains small percentages of protein, milk sugar and water which make it a suitable substrate for microorganisms (Catsberg and Kempen-van Dommelen, 1990).
In many regions of East Algeria, the traditional butter is called "Sman", "Dhan" or "Zabda".The major traditional butter produced in Jijel is called "Dhan".Rural people still produce ''dhan'' by traditional methods using primitive utensils.The raw material is cow's milk, goat's milk and sheep's milk which, after spontaneous coagulation, is churned, and finally the "Dhan", "Dhan El Maaza" and "Dhan El Naadja" is, respectively, recovered.
To our knowledge there is still no information about the lactic acid bacteria of sheep's Dhan or Dhan El Naadja.The aim of this study was the isolation and identification of lactic acid bacteria from this traditional product and, then to determine its technological properties.

Sheep Dhan samples
The butter used in this study was the traditional "dhan'' made from raw sheeps' milk (Dhan El Naadja).In the traditional stages of the manufacturing of Dhan, raw sheep's milk is left to sour spontaneously at ambient temperature until coagulation occurs.On gelation, the product is called rayeb; however, by churning the fermentate, the product is separated into lben and raw butter called Dhan.Three samples of butter were collected from local retailers in the regions of Sayda-Zguiwartan and Beni Yadjis south of Jijel in East Algeria.The samples were aseptically placed into sterilized bottles.

Isolation of bacterial strains
Samples were heated at 45 °C and then centrifuged at 3000 rpm for 15min.The intermediate liquid phase was recovered and then six decimal dilutions were carried out (Leveau and Bourgeois, 1980).Dilutions of 10 Ϫ5 and 10 Ϫ6 were plated in duplicate onto MRS agar (de Man et al., 1960) and M17 agar (Terzaghi and Sandine, 1975).The plates were incubated at 32 °C for 24 h in anaerobic conditions.
Twenty-six colonies were picked out from the higher dilutions (10 Ϫ6 ) and sub-cultured in MRS and M17 broth.

Physiological and biochemical tests
The identification of the isolates was performed according to the criteria of Bergey's Manual of Determinative Bacteriology Holt et al. (1994) and using the methods and criteria of Sharpe (1979) and Kimoto et al., (2004).
The isolates were initially subjected to the Gram staining and the catalase tests (3% H 2 O 2 ).Only the Gram positive, catalase negative isolates were further identified.Growth at different temperatures was determined in MRS and M17 broths at 10 °C, 15 °C, 40 °C and 45 °C.NaCl tolerance (4% and 6.5%) was performed on MRS and M17 broths; reductase, the hydrolysis of arginine and Sherman tests (Milk with 0.1% and 0.3% of methylene blue) were also recorded.Acetoin production was determinate using the Voges-Proskauer test.
The ability of the isolated strains to produce acid from different carbohydrates was determined by API 50 CHL/CHS test kits (Bio Merieux, S.A., S.N 41.0014 A, France.).The API test strips were prepared as recommended by the kit supplier and scored after incubation for 24 and 48 hours at 37 °C.The results were loaded onto the API system software, which used the phenotypic data to predict a species identity (%) for each isolate.

Assessment of technological performance of some isolated strains
The acidifying property was performed on skim milk.Skim milk powder was weighed and dissolved in water to reconstitute 12% skim milk (w/v), then sterilized using an autoclave at 120 °C for 10min and then cooled at 37 °C.Sterilized milk was inoculated with active culture (1%) of each strain and incubated at 37 °C for 3h, 6h, 12h, 18h and 24 hours.
The determination of the total acidity (°D) was performed by titration with N/9 NaOH in the presence of phenolphtaleine (Accolas et al., 1977).Total acidity (°D) was determined using the following formula: Total Acidity (°D) ϭ V NaOH ϫ 10 , Where V NaOH is the NaOH volume used for titration lactic acid contained in 10 ml of fermented milk.
The exopolysaccharide production from sucrose was recorded in MRS agar (20 g.L Ϫ1 sucrose) for lactobacilli and in M17 agar for Lactococcus and Leuconostoc.After incubation for 16 to 24 h, mucoid colony formation on agar medium is related to exopolysaccharide production (Leveau et al., 1991).

Classification of the isolates
Twenty-six strains were isolated, purified and further identified from traditional dhan made from sheep's milk.All of the isolates were found to be Gram-positive, catalase-negative, non motile and mesophilic (Tables 1, 2 and 3).
Two isolates obtained from MRS agar were homofermentative bacilli, able to grow at 15 °C but not at 45 °C (Table 1).On the basis of physiological and biochemical tests, melibiose fermentation and arginine hydrolysis (Montel et al., 1991), these isolates were identified as Lactobacillus plantarum.
After a series of purification steps on M17 medium, thirteen isolates were found to be homofermentative cocci, producing gamma haemolysis, reductase positive and promoted growth in a Sherman medium (Table 2).Seven out of 13 of these isolates showed positive acetoin production.The standard physiological and not documented.Seven LAB species were found which were represented by Lb. plantarum, Lc. lactis ssp cremoris, Lc. lactis ssp lactis, Lc. lactis ssp diacetylactis, Ln. lactis, Ln. mesenteroides ssp lactis and Ln.mesenteroides ssp dextranicum.These results are not in accordance with those reported by Sagdic et al. (2002) who isolated LAB from traditional Turkish yayik butter and isolates are represented essentially by strains of Leuconostoc pseudomesenteroides, Leuconostoc gelidum, Lactobacillus delbrueckii ssp.bulgaricus and Enterococcus faecium.In a previous study, Kacem and Karam (2006)  biochemical tests led to identifying these isolates as follows: seven isolates of Lc. lactis ssp diacetylactis, three isolates of Lc. lactis ssp lactis and three isolates of Lc. lactis ssp cremoris.
Eleven isolates of cocci picked from M17 agar grew at 15 °C but not at 45 °C.They are heterofermentative; eight of them have the ability to use citrate and only three isolates produced exopolysaccharides (Table 3).All these characteristics, together with the profiles of carbohydrate fermentation, identified three isolates as Leuconostoc mesenteroides ssp dextranicum, five isolates as Leuconostoc lactis and three isolates as Leuconostoc mesenteroides ssp lactis.
In our study, LAB was isolated from sheep's dhan, a traditional butter made from sheep's milk.The microbiological study of this local product is  In the study by Haddadin (2005), Lb.plantarum was the fastest acid producing isolated strain.The same author has reported that one isolate of Lc.lactis was the fastest to coagulate milk with coagulation obtained after six hours of incubation at 30 °C.

Exopolysoccharides production
The type and character of starter organisms that are used in the production of fermented milks are two of the most important factors in determining the overall quality of the final product.
From the results presented here, it is clear that lactococci are the dominant LAB in sheep's dhan.These results are in accordance with various reports indicating that the predominant lactic acid bacteria in butter was represented by Lc. lactis ssp.cremoris and Lc.lactis ssp.diacetylactis (Guiraud, 1998;Beerens and Luquet, 1987).
In a previous study, Idoui and Karam (2008) reported that Lb.plantarum is the dominant isolate in Jijel's traditional butter, a traditional butter made from cows' milk.In the same way, Benkerroum and Tamime (2004) have reported that Lb.plantarum is the dominant LAB in Maroccan smen.
Lactococcus and Leuconostoc were the two genera found in the three dhan samples.They represented 50 % and 42.31% respectively of the total isolates, while Lc.lactis ssp.diacetylactis is the dominant strain (26.92%).This strain is very beneficial for traditional and industrial butter; it produces diacethyl which is the major flavoring component in the final product (Schieberle et al., 1993).

Acid production
The time it took for the milk to coagulate was used to classify the isolated strains as fast, intermediate and slow acidifying strains.The milk

Proteolytic activity
As shown in table 5, the values of the diameter of the proteolysis zone of LAB strains ranged from 5.5 and 7.0 mm after incubation for 24 hours.The isolates were able to grow on YMA media where bacterial proteolytic activity led to clear zones.Lc. lactis ssp lactis SB11 showed the highest activity (7mm) while Ln.mesenteroides ssp dextranicum SB20 strain showed the lowest activity (5mm).Twelve out of 26 isolates were able to grow in the same media (YMA) but they showed no proteolytic activity.These results are in agreement with those found by Idoui and Karam (2008) who reported that LAB isolated from Jijel's traditional butter made from cows' milk has proteolytic activity.Peterson et al. (1990) reported that important differences exist between species of LAB in terms of the types and quantities of peptidase activities.The results of the study conducted by Haddadin (2005) indicated that all isolated strains had an important nutritional request for easily assimilated nitrogen compounds.The weak proteolytic activities of the genus Lactobacillus do not permit protein hydrolysis, casein in particular, in order to obtain low molecular weight compounds.(1984) and Idoui and Karam (2008).In a previous study, Bouzar et al. (1996) reported that Lb.delbrueckii ssp.bulgaricusCNRZ 1187 and two variants from it produced different yields of neutral heteropolysaccharides when grown in milk.The production of exopolysaccharides has been documented by Zambou Ngoufack et al. ( 2004), who reported this feature in several gram-positive and gram-negative bacteria, including LABproduction.Broadbent et al. (2003) indicated that the incorporation of exopolysaccharide producing cultures into dairy foods can provide viscosifying, stabilizing and water binding functions.It was also reported that the strains producing EPS play beneficial roles in the rheological behavior and texture of the fermented milks (Sutherland, 1990).

CONCLUSION
Results obtained in this study showed that sheep's butter contained a diversity of LAB and twenty-six isolates were identified; Lactococcus and Leuconostoc were the predominant genus in this product.The microbiological study of this local product is not documented, so the results give an idea about the lactic flora in this traditional product.
The results of the assessment of the technological aptitude indicated that the acidification kinetic confirm the weak production of lactic acid by some isolates; some of them showed a good proteolysis activity and only Ln. mesenteroides ssp.dextranicum isolates were able to produce exopolysaccharide.For future applications, we would select the best combinations amongstrains which would permit the manufacturing of traditional or industrial butter.