Physicochemical and microbiological study of “ shmen ” , a traditional butter made from camel milk in the Sahara ( Algeria ) : isolation and identification of lactic acid bacteria and yeasts

Se aislaron los microorganismos (bacterias aeróbicas, coliformes, bacterias acido lácticas, bacterias lipolíticas y levaduras) de 20 muestras de “shmen”, una matequilla tradicional del Sahara argelino hecha a partir de leche de camella. Los valores de pH, acidez, libre, Nacl, solidos totales, humedad y grasa oscilaron entre 3,11-4,97, 0,190,36%, 1.04-2,15%, 64,03-65,11%, 34,40-34,99% y 49,9056,00%, respectivamente. Entre los 181 cultivos puros de bacterias lácticas se identificaron Lactobacillus plantarum (40 cepas), Lactobacillus delbrueckii ssp. bulgaricus (35 cepas), Lactococcus lactis ssp. lactis biovar diacetylacti (22 cepas), Lactococcus lactis ssp. cremoris (18 cepas), Lactobacillus paracasei ssp. paracasei (10 cepas), Leuconostoc pseudomesenteroides (9 cepas) and Leuconostoc gelidum (12cepas) Enterococcus faecium (35 cepas). Asimismo, se detectaron levaduras en todas las muestras (55 cultivos puros). De estos, 40 se identificaron como Saccharomyces cerevisiae y 15 como Saccharomyces sp.

In the Algerian Sahara, there is a popular butter made from camel milk and is called shmen or semma (FAO, 1990).In this region, fresh camel milk is difficult to preserve because it usually contains a lot of impurities (sand, hair…) and rapidly becomes rancid.The Touaregs (nomad tribe of Sahara) improve its preservation quality by transforming it into a clarified butter (shmen).This product has played a major role in the diet of Touareg communities in the Sahara and today, there is a special demand for this product among consumers.
Shmen, can be preserved for up to one year depending on the moisture, humidity and room temperature of the storage place.This product is eaten as butter, used as oil for food preparation or for cooking, or also used as a hairdressing and as a skin cosmetic by both sexes.Also, shmen is used for roasting coffe beans in special Touareg traditional ceremonies.
In many regions of Sahara (Ain-Safra, Mograr, Bechar and Saida) (south-western Algeria), camel milk is fermented in a manner similar to that mentioned by Gast et al. (1969).The milk is stored in containers made of goat skin and allowed to ferment for 24 to 48 hours at room temperature.
GRASAS Y ACEITES, 57 (2), ABRIL-JUNIO, 198-204, 2006,  ISSN: 0017-3495     Churning is done when the container is half filled with sour milk.Some cold water is added into the goat skin before the end of churning in order to improve the firmness of the butter.It is then placed in a kettle and melted at 100°C for 30 minutes.A clarifying agent is added to hot butter and it is stirred with a wooden spoon.This agent can be crushed dates or a grated, roasted piece of ram horn, or also leaves of certain plants or seeds.Heating destroys most of the bacteria and the clarifying agent collects the dirt and floats to the top, where it can be skimmed off.
Many Algerian studies have been carried out to isolate lactic acid bacteria from cow, sheep and goat's milk (Karam and Zadi, 1994;Karam, 1995, Kacem et al., 2003, and Kacem, 2005), camel's milk (Zadi Karam, 1998) or from traditional butter (Idoui, 1994 andKacem, 2005).However, no information exists on indigenous lactic acid bacteria of shmen.The microflora may be affected by the chemical composition of camel milk, differences in traditional processing methods, packaging material and storage conditions.Therefore, the knowledge of bacterial microflora involved in butter fermentation is of prime importance in predicting and determining final shmen quality.
In this study, we aimed to know the biochemical and microbiological characteristics of shmen and to isolate lactic acid bacteria and yeasts from this traditional product.

Samples Collection
Samples of shmen were obtained from the Ain-Safra (four samples), Mograr (five samples), Bechar (five samples) and Saida (six samples) Sahara regions in south-western Algeria.Thus, a total of 20 samples of shmen were aseptically taken and placed into sterilized bottles.

Physicochemical analyses
The pH of butter was determined electrometrically with a pH-meter (Micro pH 2002, Crison, Barcelona, Spain).Titratable acidity (as lactic acid) was measured as suggested by James (1995).Moisture and solid content were determined by heating the sample in an oven at 201°C until a constant weight was obtained (Anon. 1960).NaCl concentration was obtained using the method of the International Dairy Federation (Anon., 1969).The fat content was determined by the method of Anon.(1977).All analyses were performed in duplicate.

Microbiological analyses
Ten grams of butter sample were homogenized with 90 ml of 0.85% (w/v) sterile saline solution at 45°C for microbial analysis.Appropriate dilutions were spread-plated on the culture media indicated below.
Total lactic acid bacteria were enumerated in MRS agar (Merck, Darmstadt, Germany) (de Man et al., 1960) after 72 h at 30°C.Lactobacilli were counted in MRS agar adjusted to pH 5.4 with sodium acetate so that the growth of other organisms could be supressed (Garcia et al., 1987).Plates were incubated under anaerobic conditions (Gas Pak System, Becton Dickinson) at 30°C for 48 to 72 h until growth was observed.Lactococci were counted in M17 agar (Merck, Darmstadt, Germany) (Terzaghi & Sandine, 1975) after incubation for 48 h at 30°C.Total aerobic counts were made on plate count agar (Oxoid Ltd., UK) after incubation at 30°C for 72 h, and coliform counts on violet red bile agar (Oxoid) after incubation at 30°C for 24 h.Leuconostocs were enumerated on Sodium Azide Leuconostoc agar (SALA) (Merck, Darmstadt, Germany) (Harrigan and McCance, 1966) after incubation at 21°C for 72 h.Enterococci were enumerated on citrate azide agar (CAA) (Merck, Darmstadt, Germany) after incubation at 37°C for 72 h, yeasts on potato dextrose agar (PDA) (Oxoid) after incubation at 22°C for 5 days, psychrotrophs on plate count agar incubated at 7°C for 10 days, and lipolytic bacteria on tributyrin agar (Oxoid) after incubation at 30°C for 72 h.

Lactic acid bacteria
After bacterial counts, 10 colonies belonging to different types were randomly picked out from each 30-50 colony count plate of MRS, M17, Sodium Azide Leuconostoc agar or Citrate Azide agar and purified through three cycles of single colony cultures.
Lactobacillus plantarum ATCC 14917, Lactococcus lactis ATCC 11454, and Leuconostoc mesenteroides ATCC 23386 have been used as reference strains for lactic acid bacteria characterization and identification purposes.
Cell shape, cell arrangements, Gram-staining, catalyst ase activity (3% H 2 O 2 ), production of gas from glucose, temperature requirement (15, 40 and 45°C), NaCl tolerance (4, 6.5, 8 and 10% NaCl) and growth at pH 3.9 and 9.6 were performed in M17 or MRS broth.L-and D-lactic acid were analyzed enzymatically by the L-Lactic acid/D-Lactic acid kit (Roche diagnostic, Mannheim, Germany) according to the manufacturer's instructions.Homofermentative cocci which were capable of growing at 15 and 40°C but not at 45°C nor at pH 9.6 were considered as lactococci according to the methods and criteria of Mundt (1986).Homofermentative cocci, grouped in pairs or short chains, which grew at 15, 40 and 45°C, survived after heating at 60°C after 30 min, and grew at a pH 9.6 were considered as enterococci (Devriese et al., 1987).Heterofermentative cocci (leuconostoc) were identified according to Garvie (1986).
Lactobacilli isolates were characterized according to the criteria of Kandler and Weiss (1986) and Schillinger and Lüke (1989).Arginine hydrolysis was tested in MRS broth containing 3 g/l arginine and 2 g/l sodium citrate replacing ammonium citrate.Ammonia was detected using Nessler's reagent.Acetoïn production was determined in MRS broth using the Voges-Proskauer test.
Lactobacilli isolates were tested for production of acids from carbohydrates and related compounds by using the API 50 CH strips and API 50 CHL medium (API-System, S.A., La Balme Les Grottes, Montalieu-Vercieu, France).The tests were done according to the manufacturer's instructions and the results were read after incubation at 37°C for 48 h and 72 h.Identification of the isolates was done by the computerized database program provided by the manufacturer.

Yeasts
Yeasts were enumerated and the isolates distinguished separately according to their different morphological appearances upon growth in potato dextrose agar.Pure cultures of the yeast isolates were identified on the basis of the morphological and physiological criteria described by Kreger-van Rij (1984) as well as the criteria described by Barnett et al. (1990).The other biochemical tests were done with an API 20C AUX system (BioMerieux, S.A.) at 30°C, and readings were made at 24, 48 and 72 h.
Microorganisms were maintained in sterile reconstituted skim milk (10% w/v) at 4°C or at -20°C in MRS broth supplemented with 20% glycerol.Working cultures were also kept on MRS agar, M17 agar or potato dextrose agar slants at 4°C and streaked every four weeks.

RESULTS
Table I shows the values of pH, titratable acidity (TA), NaCl, total solids (TS), moisture, and fat obtained from shmen.Samples collected from the Mograr region had the lowest pH values (3.11) while shmen from Bechar region had the highest (4.97).Samples collected from Ain-Safra region had the highest mean TA value (0.36%) and the lowest mean TA was from samples collected from Saida (0.20%).While mean salt content in shmen from Ain-Safra region was the highest (2.15%), the samples from Mograr region had the lowest value (1.04%).The total solid, moisture, and fat contents of shmen samples ranged from 64. 03 and 65.11, 34.40 and 34.99, and 49.90 and 56.20 in all regions, respectively.
Table II shows the counts of aerobic bacteria, coliforms, lactic acid bacteria, lactobacilli, lactococci, enterococci, yeasts, psychrotrophs and lipolytic bacteria recorded in shmen samples from the four regions of Algerian Sahara studied.
High counts of aerobic bacteria (mean log counts 2.76 to 3.88), lactic acid bacteria (mean log counts 3.8 to 3.96) and yeasts (mean log counts 2.08 to 3.88) were recorded in all samples.However low incidences of coliforms (mean log counts 0.90 to 1.66) as well as psychrotrophs (mean log counts 1.05 to 1.53) were detected.Lipolytic bacteria were detected in all samples (mean log counts 2.10 to 2.56).
Among lactic acid bacteria, 181 isolates were examined for identification.Table III shows the species of lactic acid bacteria and number of strains isolated from shmen.The lactic acid bacteria were identified as Lactococcus (40 isolates).They grew in 4% NaCl but not in 6.5% NaCl nor at pH 9.6.All isolates grew at 15°C and 40°C but not at 45°C.A total of 35 isolates obtained from Citrate Azide agar (CAA) were identified as Enterococcus.They did not produce gas from glucose fermentation, produced L-lactic acid, grew at 15, 40 or 45°C, survived at 60°C after 30 min., grew in 6.5% salt or at pH 9.6.Using API 20 STREP pattern of carbohydrate fermentation, the 35 isolates were identified as E. faecium.
Isolates picked from MRS agar were identified as Lactobacillus (85 isolates).They grew at 15°C but not at 45°C nor at 10% NaCl.They produced L-Lactic acid with no gas production from glucose and have the ability to hydrolyze arginine.All of these characteristics, together with the API 50 CH pattern of carbohydrate fermentation, identified the isolates as L. plantarum (40 stains), L. delbrueckii ssp.bulgaricus (35 strains), and L. paracasei ssp.paracasei (10 strains).
A total of 55 isolates of yeasts were isolated from samples on potato dextrose agar plates.Of these, 40 were identified as belonging to the Saccharomyces because they reproduced by multilateral budding, formed pseudohyphae and asci containing one to four globose ascospores, fermented glucose, galactose and maltose, did not assimilate lactose and nitrate, and their cells were globose to subglobose or ellipsoidal to cylindrical.All of these characteristics, together with the API 20C AUX system (Bio Merieux, S.A.) pattern of biochemical tests, identified the 40 isolates as S. cerevisiae.The other 15 isolates were not identified as species level.

DISCUSSION
The results reported here constitute part of the study focusing on isolation and partial identification of lactic acid bacteria and yeasts from a food product hitherto not well examined: traditional butter (shmen) produced from camel milk in the Sahara regions GRASAS Y ACEITES, 57 (2), ABRIL-JUNIO, [198][199][200][201][202][203][204]2006  (Algeria).The biochemical and microbiological characteristics of this butter are not documented.Therefore, our results are compared to those obtained with traditional fermented camel milk or with butter made from different milk type.
The physicochemical study of shmen indicated that, the values of pH varied in all samples (3.11-4.97).Similar results were reported in earlier studies on butters (Filkensen, 1987 andSagdic et al., 2002).In all samples of shmen, the values of titratable acidity TA (0.19-0.36%) were higher than values found by Bilgin (1996) and Hayaloglu (1999).On the other hand, the mean NaCl content (1.04 and 2.15%) in all samples of butter was lower than the values found by El Sadek et al. (1975) and Hayaloglu (1999).In addition, the total solid, moisture, and fat contents, of shmen samples varied according to the samples of butter and regions.These results agreed with those obtained by Sagdic et al. (2002).
L. plantarum (22.1%),L. delbrueckii ssp.bulgaricus (19.3%),E. faecium (19.3%) and S. cerevisiae (63%) were the major species of lactic acid bacteria and yeasts isolated from this product.Results on the species composition of the lactic microflora of shmen were similar to those reported by Sagdic et al. (2002) who isolated lactic acid bacteria from traditional butter represented essencially by strains of Ln. pseudomesenteroides and Ln.gelidum, L. delbrueckii ssp.bulgaricus and E. faecium.In the same study the isolation of L. lactis ssp.cremoris, L. lactis ssp.lactis biovar diacetylactis and L. plantarum was not reported.However, in previous studies (Idoui, 1994;Benkerroum and Tamine, 2004;and Kacem, 2005), L. plantarum as well as the other mesophilic lactobacilli have been frequently reported to be the dominant microorganisms among the non-starter lactic acid bacteria in long ripening butters or cheeses due to their unique ability to grow in highly hostile environments and to possessing of a wide range of hydrolytic enzymes, including lipases and proteases (Wouters et al., 2002).Therefore, it is not surprising that these bacteria would be dominant and one of the responsible agents for the maturation of shmen.
Enterococci represent an important part of the bacterial microflora of shmen: mean log plate counts of enterococci in different samples range from 1.47 to 2.69 (Table II) and a total of 35 E. faecium strains were isolated (Table III).This group of bacteria play a major role in the ripening and aroma development in butter and many types of traditional cheeses (Centeno et al., 1996).In addition, E. faecium was selected together with other lactic acid bacteria for use in starter culture preparation (Coppola et al., 1988 andParente et al., 1989).A food company sought clearance from the British "Advisory Committee on Novel Foods and Processes" (ACNFP) for the use of E. faecium strains as a starter culture in fermented dairy products (ACNFP, 1996), and the Committee decided that the culture was acceptable for such use.Clearly, the enterococci play an important role in the manufacturing of butters typical to some regions, and their use has a major impact on this part of artisan dairy products.
L. delbrueckii ssp.bulgaricus was found in all butter samples; this microorganism represented 19.3% of the isolates.Similar results were reported by Dave and Shah (1997).E. faecium species was also isolated in this study.This genus has been isolated from butters (Hukari and Rautavaara, 1972;Ozalp et al., 1980 andSagdic et al., 2002) and isolates have been identified, characterized and utilized as starter cultures.Other bacteria such as Ln.pseudomesenteroides and L. lactis ssp.lactis biovar diacetylactis were also found in shmen.Generally, the conventional method for obtaining butter involves the ripening of cream through the addition of a starter culture mixture containing L. lactis ssp.lactis biovar diacetylactis and Ln.pseudomesenteroides to produce sufficient quantities of lactic acid to reduce the pH to around 4.6 to 4.4 (Kornacki and Flowers, 1998).Also, L. lactis ssp.lactis biovar diacetylactis strains produce a sufficient quantity of diacetyl which is the major flavoring component and antimicrobial property of cultured butter (Schieberle et al., 1993).Probably, the presence of this type of bacteria was responsible for the pleasant taste and smell of shmen.
On the other hand, analysis of shmen showed that mean log counts of yeast exceeded 3.88.Their high counts and well-known high lipolytic activity suggest that they may play an active role in shmen ripening.

CONCLUSION
The results of this study indicate that the natural Algerian butter from camel milk revealed a diversity of microflora.This diversity could be linked to the quality of camel milk (composition of milk during lactation period), pH, moisture, humidity, room temperature of the manufacturing place, etc.
"Shmen" samples from four regions were characterized by low incidences of opportunistic bacteria, including coliforms, psychrotrophs and lipolytic bacteria.This is important for some algerian populations who live in the Sahara.In addition, different species of lactic acid bacteria and yeasts present in this product were isolated and identified.Probably, more emphasis should be placed in our laboratory on the role of lactic acid bacteria and yeasts, which have not been fully investigated in shmen so far.We think this type of study will contribute to standardize the production methods of butter from camel milk and to improve its safety.

Table I Physicochemical characteristics of butter from camel milk (shmen)* collected in four regions of Algerian Sahara
as L. lactis ssp.lactis biovar diacetylactis and the 18 isolates as L. lactis ssp.cremoris.