EFFECT OF STARTER CULTURES ON VARIOUS CLASSES OF FATTY ACIDS IN SUDANESE FERMENTED CAMEL MILK (CAMELUS DROMEDARIUS) GARISS

The objective of this research was to study the variation of classes of fatty acids in gariss (fermented camel milk) prepared under controlled conditions (starter cultures and time of fermentation). Inoculations of raw camel milk with selected LAB strains (E. duransR03, E.faecium NWL and L. plantarum BJ6 and their combination as well as the control fermentation without starter cultures) was performed at varying periods of time (zero, 3, 6, 9 and 12h) at ambient temperature, then the role of these conditions on fatty acids classes were studied. Camel milk fermented under starter-culture controlled conditions contained unsaturated fatty acids, including the essential fatty acids. Considerable amounts of omega3 and omega6 fatty acids and the absence or presence of low amounts of short chain fatty acids were found compared to cow milk.


INTRODUCTION
There are various traditional fermented camel milk products that are produced by camel herders in different parts of the world (Yagil, 1982;Abdelgadir et al., 1998;Lore et al., 2005;Hassan et al., 2008;Abdel Rahman et al., 2009;Konuspayeva and Faye, 2011). Fermented milk products such as suusac and gariss are produced from camel milk in Kenya, Somalia and Sudan (Abdelgadir et al., 1998;Lore et al., 2005).
Camel's milk is produced in certain areas of Sudan, under nomadic conditions. The camel's milk being abundant in remote localities, the camel herders have to prepare gariss, a fermented product, on which they sustain living for several months as the sole source of various nutrients (Dirar, 1993;Abdelgadir et al, 1998). Gariss is a special kind of fermented milk, prepared solely from camel milk under more or less shaking. Besides its use as food, camel milk has been used in many regions as a cure for certain diseases (Dirar, 1993;Abdelgadir et al., 1998;Suleiman et al., 2007). In the Horn of Africa, 10% of produced milk is derived from camels (Faye and Konuspayeva, 2012). However, most camel milk is produced in traditional farming or pastoral systems by hand milking that cannot provide consistent quantity and quality of raw milk for urban markets (Abeiderrahmane, 2005). The camel dairy industry, including machine milking, processing, and distribution, has been established in the last decade but it is still in an early stage of development (Nagy et al., 2013). Seifu et al. (2012) isolated and characterized lactic acid bacteria from Ethiopian traditional fermented camel milk, and they concluded that the isolated lactic acid bacteria species could be considered as potential candidates for development of starter cultures that can be used for the production of fermented camel milk products under controlled condition. Enterococcus species are known by their production of enterocins which exert different specific inhibition activity against pathogenic bacteria (Sabia et al., 2002).
The study of microflora in traditional fermented dairy products as gariss and preparation of starters is of a good concern. To obtain the gariss with better quality and to produce this traditionally fermented product at the industrial level with high quality, control starter cultures must be used. For many authors, the presence of enterococci is evidence of possible fecal contamination and therefore a risk to consumers because although these strains are known for their low virulence, they pose serious health problems due to the emergence of many antibiotic-resistant strains (Akhmetsadykova et al., 2014). The objective of the present work was to know the changes in the fatty acids classes of gariss prepared under controlled conditions in order to assess the influence of the strains used.

MATERIALS AND METHODS
Fermented camel milk (gariss) was prepared under controlled conditions. Inoculations of camel milk was performed for varying periods of time (zero, 3h, 6h, 9h and 12h) at ambient temperature with selected LAB strains (E. duransR03, E.faecium NWL and L. plantarum BJ6 and their combination as well as the control, fermentation without starter cultures). The preparation of the starter culture to be inoculated with the specific strain in nonsterilized camel milk prepared in last 24h (with the three strains i.e. E. duransR03, E.faecium NWL and L. plantarum BJ6 and their combination). That means we used pure strain inoculated in camel milk (non sterilized one) left for 24 h, then it used for gariss preparation. A 3% starter culture was used to prepare gariss, and then the fermentation was carried out according to the traditional gariss preparation methods. Five gariss batches(Batch A= E. duransR03, Batch B=E.faecium NW, Batch C= L. plantarum BJ6, Batch D= the combination and Batch F= the control -fermentation without starter culture) were prepared, and for each 500 ml of camel milk 3% batch, 24 hours starter cultures were inoculated. Batch one was inoculated with strain Enterococcus duransR03, batch two with strain E.faecium NWL, batch three with Lactobacillus plantarum BJ6, batch four consisted of a mixture of the strains at equal proportions and batch five was a control batch which was left uninoculated. The preparation was left to ferment for 12 hours at ambient temperature. Samples were withdrawn in 0, 3, 6, 9 and 12hours to perform the fatty acids classes of the produced gariss.

Fatty acid analysis
From the extracted lipids, stored at 4°C, the method of Konuspayeva et al. (2008) was used to prepare and quantify fatty acid methyl esters which were taken for analysis by GC system (Massy, France) mass spectrometry. The fatty acids were identified by comparison of retention time with known standards and were expressed as percentage of total fatty acids, and then the fatty acids classes were calculated.

Definition of the fatty acid classes
The fatty acids (FA) were grouped into different classes according to their carbon chain length or their carbon saturation status: Short chain fatty acids (SCFA): All FA with few Carbone atoms between C4 and C8. Medium chain fatty acids (MCFA): All fatty acids with medium carbon atoms between C10 and C15 Long chain fatty acids (LCFA): All fatty acids with many carbon atoms between C16 and C22:6 Saturated fatty acids (USFA): All fatty acids with saturated carbon atoms. Mono-unsaturated fatty-acids (MUSFA): All fatty acids with one unsaturated carbon atom. Poly-unstaurated fatty acids (PUSFA): All fatty acids with more than two unsaturated carbon atoms.

Statistical analysis
Statistical Packages for Social Sciences (SPSS 16.00) was used to analyze data using ANOVA and Duncan Multiple Range Test (DMRT) for mean separation.

RESULTS AND DISCUSSION
All the classes of fatty acids in gariss prepared by starter cultures strains as well as the combination had variable trends during the study period.

Short chains fatty acids (SCFA)
The proportion of SCFA in gariss varied from 0.26 to 0.41% at t 0 (milk) to 0.50-0.71 % at t 12h according to the type of starter (Table 1). However, there was some difference between the used starters. In gariss prepared by E. durans R03, E.faecium NWL, L. plantarum BJ6 and their combination, SFCA proportion significantly (P≤0.05) increased between t 0 and the end of fermentation (Table 1). The same trend was found in the control during the same period of fermentation. This indicated that fermentation with or without using starter cultures increased SCFA during fermentation process, contrary to that was reported by Abdelrahman (2007) for which SCFA were lowered by fermentation of camel milk using different starter cultures. Konuspayeva et al. (2008) reported that camel milk has low content of short-chain fatty acids but the proportion reported was slightly higher (1.16%).

Medium chains fatty acids (MCFA)
The proportion of MCFA in gariss varied from 14.99 to 15.36% at t 0 (milk) to 14.59-15.96 % at t 12h according to the type of starter (Table 1). However, there was some difference between the used starters. MCFA of gariss prepared by E. durans R03 and L. plantarum BJ6 decreased significantly (P≤0.05) between t 0 and t 12h , while in gariss prepared by E.faecium NWL and their combination it increased significantly (P≤0.05), similar trend being found in the control during the same period of fermentation (Table 1). Abdelrahman (2007)

Long chains fatty acids (LCFA)
For the LCFA the proportion in gariss varied from 84.29 to84.61% at t 0 (milk) to 83.54-84.74 % at t 12h according to the type of starter (Table 1). However, there was some difference between the used starters.
LCFA of gariss prepared by E. durans R03 and L. plantarum BJ6 significantly (P≤0.05) increased between t 0 and t 12h (Table 1), while LCFA in gariss prepared by E.faecium NWL and their combination it decreased significantly (P≤0.05) during the fermentation process, the same trend being found in the control. Abdelrahman (2007)  For SFA, the proportion is passing from 61.25to 66.57% at t 0 (milk) to 61.10-66.13 % at t 12h according to the type of starter (Table 2). However, there was some difference between the used starters.
SFA of gariss prepared by E. durans R03 and L. plantarum BJ6 significantly (P≤0.05) decreased between t 0 and t 12h

Unsaturated fatty acids (USFA)
The proportion of USFA is passing from 61.25to 66.57% at t 0 (milk) to 61.10-66.13 % at t 12h according to the type of starter (Table 2). However, there was some difference between the used starters.
USFA of gariss prepared by E. durans R03 significantly (P≤0.05) increased between zero time and the end of fermentation period; while the USFA of gariss prepared by E. faecium NWL and their combination significantly (P≤0.05) decreased (Table 1), the same trend being found in the control during the same period of fermentation. Gariss prepared by L. plantarum BJ6 single culture was not affected by the period of fermentation process. Abdelrahman (2007)

Mono-unsaturated fatty acids (MUSFA)
The proportion of MUSFA varied from 61.25to 66.57% at t 0 (milk) to 61.10-66.13 % at t 12h according to the type of starter (Table 2). However, there was some difference between the used starters.
The proportion of MUSFA of gariss prepared by E. durans R03 and L. plantarum BJ6 were not changing between t 0 and t 12h , while in gariss prepared by E.faecium NWL and their combination it decreased significantly (P≤0.05) at the same time ( Table 1). The MUSFA of the control significantly (P≤0.05) increased during the same period of fermentation. From his part, Abdelrahman (2007)

Poly unsaturated fatty acids (PUSFA)
The quantity in proportion of PUSFA is passing from 61.25to 66.57% at t 0 (milk) to 61.10-66.13 % at t 12h according to the type of starter (Table 2). However, there was some difference between the used starters.
PUSFA of gariss prepared by E. durans R03 and L. plantarum BJ6 significantly (P≤0.05) increased between t0 and t 12h while PUSFA in gariss prepared by E.faecium NWL significantly (P≤0.05) decreased, the same trend being found in the control (Table 1). The proportion of MUSFA of gariss prepared by the combination was not changing during all fermentation process. This result is reverse to that reported by Abdelrahman (2007)

Omega 3 and omega 6 fatty acids
The ω 3 and ω 6 fatty acids are in low proportion in milk and gariss. The range was 0.45-0.87 and 0.38-0.76% at t 0 and t 12h Guler and Gursoy-Balci (2011) reported that the type of cultures storage periods had no effect on long-chain free fatty acids in yogurts from goat milk. During the storage, short-chain free fatty acids were different according to culture type used and increased during storage, while the levels of medium-chain free fatty acids, except for decanoic acid, were unchanged and the amount of long-chain free fatty acids decreased during storage.
As far as we know, our paper is the first attempt to use E.faecium, E.durans and L. plantarum as starter culture to produce fermented camel milk (gariss) in the laboratory conditions. LAB distribution showed a high diversity of species that are dominant and was frequently described in various dairy products. In fermented camel milk from Kazakhstan called Shubat different microorganisms were identified as Enterococcus durans; Enterococcus faecalis; Enterococcus faecium and others (Akhmetsadykova et al., 2014). By using well identified starter cultures of LAB strains associated with gariss preparation and by controlling the conditions of product preparation, a wholes me product of consistent quality will be desired for the consumers of the urban areas. That will lead to the commercialization of gariss product where fresh milk is procured by nomads in the remote areas. Consequently, it could be one of the most income generating activities that can give great push to those people of the remote areas.

CONCLUSION
The fatty acids classes of fermented camel milk have various trends affected by starter cultures fermentation. The uses of some species such as E. duransR03, E.faecium NWL and L. plantarum BJ6 in production of laboratory scale fermented camel milk gariss, suggests their possible use as starter culture in the manufacture of commercially gariss products. However, more studies are needed to complete the isolation and characterization of new LAB strains that could be present in camel milk produced in Sudan and to compare the results with reports from other countries and regions. Also, the organoleptic analysis of the gariss produced under the above mentioned conditions are also recommended for further studies.